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FACULTÉ DE MÉDECINE
GIGA-Neurosciences
Unité de Neuroendocrinologie du Développement
(Pr J.-P. Bourguignon)
Contribution à l’Etude des Effets Précoces
de Perturbateurs Endocriniens
sur
ur l’Axe Hypothalamo-Hypophysaire
Hypothalamo Hypophysaire
et la Maturation
aturation Sexuelle
S
de la Rate:
Rate
Aspects Mécanistiques
M
in Vivo et in Vitro
Travail présenté en vue de l’obtention du grade de
Docteur en Sciences Biomédicales et Pharmaceutiques
Pharmaceutiques
Grégory RASIER
Licencié en Sciences Biologiques
DEA en Sciences Biomédicales
Année académique 2011-2012
REMERCIEMENTS
Tout d’abord, je voudrais remercier mon promoteur, le Professeur JeanPierre Bourguignon, pour m’avoir sollicité et accueilli au sein de son Unité de
Neuro-Endocrinologie du Développement. Le projet de recherche et votre
enthousiasme m’ont rapidement convaincu. Votre esprit critique et vos idées
mûrement réfléchies m’ont accompagné tout au long de ce travail. Par votre souci
pédagogique, mais également votre rigueur scientifique, nos discussions m’ont
toujours enrichi et souvent permis de clarifier les choses. Par ailleurs, vous
m’avez permis d’assister à de nombreuses réunions scientifiques internationales
de haut niveau durant lesquelles j’ai pu rencontrer et discuter avec les «Papes de
la discipline», et ainsi m’épanouir davantage scientifiquement et acquérir une
expérience (à tous niveaux!) significative. Votre confiance et votre infinie
patience m’ont permis, en toutes circonstances, de travailler dans un climat de
sérénité. Pour tout cela, soyez assuré, Monsieur Bourguignon, de ma profonde
reconnaissance.
Ma gratitude va également au Professeur Vincent Seutin qui m’a ouvert les
portes de son Unité de Physiologie et Pharmacologie des Canaux Potassiques dans
le Système Nerveux Central. Pour votre disponibilité, mais également votre
conciliante compréhension en tant que Président du comité de thèse, soyez,
Monsieur Seutin, sincèrement remercié.
A diverses occasions, j’ai également bénéficié des hautes compétences et
des suggestions pertinentes des Professeurs Jacques Balthazart, Jean-Pierre
Thomé, Jean-Michel Foidart et Guy Plomteux. Je tiens à leur exprimer ma
reconnaissance.
Ce travail n’existerait pas sans l’aide, ni l’appui de Madame Arlette Gérard,
dévouée «sans compter» et référence de premier ordre. Comment te remercier
pour tout le travail accompli ainsi que pour ton accueil, ton soutien, ton oreille
attentive et ta disponibilité tout au long de cette aventure? Tout simplement,
MERCI! Arlette, ta modestie est à la hauteur de tes compétences techniques et
de ton érudition impressionnante…
Mes remerciements vont au Docteur Anne-Simone Parent dont la
discrétion va de pair avec ses compétences. Merci pour ton aide et ton soutien
sans réserve, que ce soit au laboratoire ou lors de congrès au cours desquels tu
m’as épaulé à maintes reprises!
Merci aussi au Docteur Marie-Christine Lebrethon dont les
encouragements, mais également les conseils, m’ont toujours aidé à garder le
moral.
Madame Josiane Laurent dont l’immense gentillesse et l’attention
généreuse a permis la connexion «laboratoire - bureau de Monsieur Bourguignon»
en toutes circonstances et avec une bonne humeur permanente.
Qu’il me soit également permis de remercier le Professeur Vincent Geenen
qui m’a accueilli au sein de son Unité de Neuro-Immuno-Endocrinologie et
Embryologie afin d’y réaliser mon mémoire de licences, et un peu plus… Il m’a
donné le goût pour la recherche et l’envie d’y persévérer…
Au Docteur Isabelle Hansenne avec qui j’ai partagé notre espace de travail
durant une grande partie de cette aventure. Toujours présente, prête à
soumettre un conseil avisé et pertinent, mais aussi à proposer son aide précieuse
et sans faille.
A Gaby (nos routes professionnelles se croisent régulièrement), Micke,
Henri, Chantal, Marie-Thérèse, Christel, Miguel, Andrée, Céline, Olivier,
Catherine, Marie, Fabienne, … Tous ceux avec qui j’ai partagé mes repas de midi,
discussions «politico-philosopho-sportives» ou autres activités. Soyez tous
sincèrement remerciés pour vos richesses, tant d’esprit que de coeur, que vous
m’avez apportées...
A ceux que j’ai rencontrés au sein de l’Unité de Recherche Thématique
GIGA-Neurosciences. Tout particulièrement à Renaud, Pierre, Morgan et
Jérôme.
Au cours de ces années, il m’a également été donné l’occasion de
rencontrer d’autres personnes remarquables. Je les remercie pour ces nombreux
moments partagés ainsi que leur soutien et leur amitié. Merci à vous tous!
Mention spéciale pour Schmoutz et Mike.
Il faudrait tous les mots, et bien plus, pour remercier les membres de ma
famille: mon épouse Anne-Pascale, pour son immense conciliance (je suis bien
conscient que ce terme n’existe pas mais, à mes yeux, il est le plus important et
j’en mesure tous les jours sa portée) dont elle a fait preuve durant mes
nombreuses crises d’angoisse consécutives aux craintes de ne pouvoir clôturer
positivement cette thèse. Pour tous ces moments que tu as endurés
silencieusement, je te dédie ce travail… Mes parents, mes trois frères, mes
beaux-parents, mes beaux-frères et belles-soeurs. De mille façons, ils ont
permis de repousser mes limites toujours un peu plus loin et de m’élever un peu
plus haut… Leur générosité, leur enthousiasme à toute épreuve ainsi que leur
confiance sans limite m’ont rassuré dans les moments difficiles. Un mot: MERCI…
Ma pensée va également à toutes les personnes que j’aurais oubliées de
citer ici! Qu’elles me pardonnent…
Ce travail a été réalisé grâce au soutien de l’Union Européenne (projet
EDEN), de la Fondation Léon Frédéricq, du Groupe d’Etude Belge des Pédiatres
Endocrinologues ainsi qu’à l’appui du Fond National de la Recherche Scientifique.
TABLE DES MATIERES
I. Introduction
1
A. Axe hypothalamo-hypophyso-gonadique
B. Sécrétion de la gonadolibérine (GnRH)
C. Maturation sexuelle femelle: comparaison entre l’humain et le rat
D. Effets des oestrogènes sur les neurones à GnRH
1. Nature et origine des oestrogènes et leurs récepteurs
2. Action de l’oestradiol sur le système à GnRH et le contrôle neuroendocrinien
de l’axe hypothalamo-hypophyso-gonadique
3. Effets rapides / non génomiques du 17β-oestradiol (E2) sur les neurones
à GnRH
E. Facteurs du mécanisme neuroendocrinien de la puberté
1. Facteurs centraux
2. Facteurs périphériques
1
5
10
14
15
19
20
20
28
II. Mise en situation du problème
31
A. Différenciation sexuelle, programmation foetale et perturbation endocrinienne
B. Historique de la découverte des perturbateurs endocriniens (PEs) et questions
actuelles
C. Exemple du dichlorodiphényltrichloroéthane (DDT)
1. Structure et propriétés
2. Effets chez l’Homme
3. Effets d’insecticides chez les rongeurs
D. Relations entre exposition pré/post-natale aux PEs et timing pubertaire
chez l’Homme
E. Implication des facteurs environnementaux, autres que les PEs, dans la puberté
précoce
31
17
32
36
36
38
38
39
44
III. Objectifs du travail
IV. Méthodologie
46
49
A. Incubation d’explants hypothalamiques
B. Dosage de la GnRH
C. Dosage de l’hormone lutéinisante
D. Dosage des isomères du DDT et ses métabolites
E. Détermination des phases du cycle oestral
F. Justification du choix de l’o,p’-DDT
G. Statistiques
49
53
54
54
55
55
56
V. Maturation avancée de la sécrétion de GnRH et précocité
sexuelle après exposition de rats femelles immatures à l’E2 ou
au DDT (cfr.: annexes IV & VI)
58
A. Objectifs de l’étude
B. Résultats
C. Discussion
58
59
65
VI. Mécanismes d’interaction des PEs sur la sécrétion de GnRH
induite par le glutamate (cfr.: annexes IV & VI)
70
A. Objectifs de l’étude
B. Résultats
C. Discussion
70
70
76
VII. Discussion générale et perspectives
80
Table des matières
Bibliographie
Annexes
95
Publication I:
Estradiol stimulation of pulsatile gonadotropin-releasing hormone secretion in vitro:
Correlation with perinatal exposure to sex steroids and induction of sexual precocity in
vivo.
Matagne V., Rasier G., Lebrethon M.-C., Gérard A. and Bourguignon J.-P.
Endocrinology 145 (6): 2775-2783 (2004)
Publication II:
Early onset of puberty: Tracking genetic and environmental factors.
Parent A.-S., Rasier G., Gérard A., Heger S., Roth C., Mastronardi C., Jung H., Ojeda S.
and Bourguignon J.-P.
Hormone Research 64 (2): 41-47 (2005)
Publication III:
Female sexual maturation and reproduction after prepubertal exposure to estrogens and
endocrine disrupting chemicals: A review of rodent and human data.
Rasier G., Toppari J., Parent A.-S. and Bourguignon J.-P.
Molecular and Cellular Endocrinology 254-255: 187-201 (2006)
Publication IV:
Early maturation of gonadotropin-releasing hormone secretion and sexual precocity after
exposure of infant female rats to estradiol or dichlorodiphenyltrichloroethane.
Rasier G., Parent A.-S., Gérard A., Lebrethon M.-C. and Bourguignon J.-P.
Biology of Reproduction 77 (4): 734-742 (2007)
Publication V:
Oxytocin facilitates female sexual maturation through a glia-to-neuron signaling pathway.
Parent A.-S., Rasier G., Matagne V., Lomniczi A., Lebrethon M.-C., Gérard A., Ojeda S.
and Bourguignon J.-P.
Endocrinology 149 (3): 1358-1365 (2008)
Publication VI:
Mechanisms of interaction of endocrine disrupting chemicals with glutamate-evoked
secretion of gonadotropin-releasing hormone.
Rasier G., Parent A.-S., Gérard A., Denooz R., Lebrethon M.-C., Charlier C. and
Bourguignon J.-P.
Toxicological Sciences 102 (1): 33-41 (2008)
Publication VII:
Neuroendocrine disruption of pubertal timing and interactions between homeostasis of
reproduction and energy balance.
Bourguignon J.-P., Rasier G., Lebrethon M.-C., Gérard A., Naveau E. and Parent A.-S.
Molecular and Cellular Endocrinology 324 (1-2): 110-120 (2010)
Table des matières
I. Introduction
I. INTRODUCTION
Résumé: Dans ce premier chapitre, nous décrirons d’abord l’axe hypothalamo-hypophyso-gonadique
ainsi que le mécanisme d’initiation de la puberté (centrale et périphérique) et le rôle des stéroïdes. La
genèse de la sécrétion de la gonadolibérine (GnRH), à l’origine du déclenchement pubertaire, sera
également revue et nous comparerons ensuite la maturation sexuelle chez la femme et le rat femelle.
Après avoir décrit la nature des oestrogènes, en particulier l’oestradiol, et leurs récepteurs, nous
distinguerons leurs effets lents/rapides (génomiques / non génomiques) sur le neurone à GnRH. Enfin,
nous terminerons cette introduction générale par une description des principaux facteurs centraux et
périphériques qui influencent le mécanisme neuroendocrinien de la puberté.
A. Axe hypothalamo-hypophyso-gonadique
Le système nerveux central (SNC) et les tissus endocriniens sont anatomiquement et
fonctionnellement reliés par l’axe hypothalamo-hypophysaire. La tige pituitaire connecte
l’hypothalamus à l’hypophyse, cette dernière étant constituée de deux parties distinctes:
l’antéhypophyse (adénohypophyse), sous le contrôle des hormones hypothalamiques; la posthypophyse
(neurohypophyse),
constituée
de
terminaisons
axonales
des
cellules
neurosécrétrices de l’hypothalamus. Différents axes hypothalamo-hypophysaires se
distinguent sur le plan fonctionnel parmi lesquels l’axe hypothalamo-hypophyso-gonadique.
Dans ce travail, nous nous sommes exclusivement focalisés sur ce dernier, en privilégiant
l’individu/animal féminin/femelle.
La puberté, initiée au niveau du SNC, est la période de vie durant laquelle une
(ré)activation de l’axe hypothalamo-hypophyso-gonadique conduit à une série de
changements physiques aboutissant à la maturation et au maintien d’un système de
reproduction fonctionnel. Classiquement, les manifestations d’activité des stéroïdes sexuels et
la fertilité résultent d’une activation en cascade de cet axe (Fig. 1). L’évènement central de
l’établissement de la puberté est l’augmentation de la fréquence et de l’amplitude de la
sécrétion de gonadolibérine (GnRH) hypothalamique, contrôlée par des mécanismes
redondants, inhibiteurs et stimulateurs, qui, respectivement, disparaissent ou apparaissent
(Bourguignon, 2004). Il est généralement admis que des variations pubertaires temporelles,
endéans une période physiologique de cinq ans, sont majoritairement déterminées par des
facteurs génétiques alors que les facteurs environnementaux jouent plutôt un rôle mineur
(Parent et al., 2003). L’influence de ces derniers sur la période pubertaire a suscité un réel
intérêt suite au constat d’une avance séculaire (entre les moitiés des 19ème et 20ème siècles) de
1
Introduction
l’âge à la ménarche, à la fois aux Etats-Unis et en Europe de l’Ouest, et également dans des
pays en voie de développement (Parent et al., 2003).
Une des premières expériences démontrant le rôle des stéroïdes dans l’apparition de la
puberté a consisté en l’observation des effets promoteurs d’extraits de follicules ovariens sur
le développement sexuel du rat femelle (Allen et Doisy, 1924). Un an plus tard, l’équipe de
Gustavson induisait une puberté chez des rates immatures après avoir injecté des extraits de
placenta (Frank et al., 1925). En 1929, chez deux rates immatures disposées en circulation
croisée, Kallas observait que l’ovariectomie de l’une provoquait une puberté précoce chez
l’autre. Ces expériences suggéraient que des facteurs sécrétés après ovariectomie, identifiés
plus tard comme les gonadotrophines, l’hormone folliculo-stimulante («follicle-stimulating
hormone», FSH) et l’hormone lutéinisante («luteinizing hormone», LH), responsables de
l’apparition de la puberté, pouvaient provenir d’un système différent des gonades. Cette
hypothèse fut confirmée par les expériences menées en 1952 par Harris et Jacobson, qui
montrèrent que la greffe d’hypophyses issues de rates immatures restaurait la fonction de
reproduction chez des femelles adultes hypophysectomisées. La restauration du cycle oestral
chez les femelles hôtes ayant reçu une hypophyse immature indiquait également que la
maturation de la fonction de reproduction n’était pas directement liée à l’hypophyse ellemême.
2
Introduction
INFLUX CEREBRAUX
INHIBITION
AA inhibiteur: GABA
STIMULATION
AA excitateur: glutamate
Neurones sécrétant kisspeptine
Facteurs de croissance
Neurones à GnRH
Cellules astro-gliales
FREQUENCE ET AMPLITUDE DE LA SECRETION DE GnRH
SYNTHESE ET SECRETION
DES GONADOTROPHINES HYPOPHYSAIRES FSH/LH
GONADES
Maturation
des cellules germinales
Synthèse et sécrétion
des stéroïdes sexuels
RETRO-CONTRÔLES PERIPHERIQUES
SIGNAUX ENVIRONNEMENTAUX
GENERATEUR HYPOTHALAMIQUE DE SECRETION PULSATILE DE GnRH
AUTRES SYSTEMES CELLULAIRES PERIPHERIQUES
Caractères sex. sec., croissance, …
Leptine, IGF-1, …
Figure 1: Représentation schématique des interactions hypothalamo-hypophyso-gonadiques au cours du
développement. AA: acide aminé; caractères sex. sec.: caractères sexuels secondaires; FSH: «follicle stimulating
hormone», hormone folliculo-stimulante; GABA: «γ-amino-butyric acid», acide γ-amino-butyrique; GnRH:
«gonadotrophin-releasing hormone», gonadolibérine; IGF-I: «insulin-like growth factor I», facteur de croissance
I similaire à l’insuline; LH: «luteinizing hormone», hormone lutéinisante.
Le concept suggérant que l’hypothalamus est la structure responsable de l’apparition
de la puberté, par la levée d’une inhibition, a été suggéré il y a une soixantaine d’années
(Harris et Jacobson, 1952; Donovan et van der Werff ten Bosch, 1956) en montrant que la
destruction de certaines régions de l’hypothalamus par électrolyse induisait une puberté
précoce chez le rat femelle. En 1962, McCann et ses collègues rapportaient l’existence d’un
facteur hypothalamique stimulant la libération de LH. Des études ultérieures permirent de
mettre en évidence et de caractériser la GnRH («gonadotrophin-releasing hormone»),
hormone peptidique stimulant la libération des gonadotrophines (Amoss et al., 1971; Matsuo
et al., 1971; Schally et al., 1971). Comme mentionné ci-avant, la sécrétion des
gonadotrophines hypophysaires dépend du signal stimulant de la GnRH et est modulée par le
rétrocontrôle des stéroïdes et peptides gonadiques. Au niveau des cellules gonadotropes de
l’hypophyse, la GnRH interagit avec des récepteurs membranaires couplés aux protéines G et
détermine la synthèse et la sécrétion de FSH et LH, laquelle est initiée par une mobilisation
3
Introduction
calcique, successivement intracellulaire et extracellulaire, avec la mise en jeu des systèmes
calcium-calmoduline et inositol-triphosphate comme seconds messagers (Reichlin, 1998).
Le mécanisme d’établissement de la puberté femelle a été revu par Ojeda et Terasawa
en 2002. Chez l’animal comme chez l’Homme, deux mécanismes différents peuvent exister:
dans la puberté centrale (physiologique), les oestrogènes sont produits grâce à la stimulation
ovarienne par le système hypothalamo-hypophysaire (Fig. 2) tandis que dans la puberté
périphérique (pathologique), les oestrogènes endogènes, sont produits indépendamment de la
sécrétion des gonadotrophines (Fig. 2) ou proviennent de sources extérieures. L’étude des
tissus sensibles aux stéroïdes sexuels tels l’utérus, le vagin et les seins ou des paramètres
variant à la suite d’effets oestrogéniques comme l’âge à l’ouverture vaginale (OV) chez le
rongeur ou en fonction du ratio d’effets oestrogéniques/androgéniques telle la distance anogénitale (murins et humains), peut fournir un éclaircissement dans la compréhension des
mécanismes des pubertés centrale et périphérique. Cependant, les signes de développement
pubertaire (à l’exception de la croissance testiculaire) ne permettent pas d’établir si la
maturation est conduite centralement en impliquant le système hypothalamo-hypophysaire ou
s’il s’agit d’une interaction périphérique directe au niveau des tissus cibles par les stéroïdes
sexuels, ou les deux mécanismes à la fois.
4
Introduction
Figure 2: Illustration de la fonction de l’axe hypothalamo-hypophyso-gonadique. A: stimulation en cascade
caractérisant l’apparition de la puberté centrale ou physiologique; B: puberté périphérique due à des oestrogènes
secrétés par l’ovaire de manière autonome ou d’origine extra-gonadique. Dans ce cas, le système hypothalamohypophysaire est inhibé en raison du rétrocontrôle inhibiteur.
B. Sécrétion de la GnRH
La GnRH a successivement été isolée à partir d’hypothalamus de porc (Matsuo et al.,
1971), de rat (Schally et al., 1971) et de mouton (Amoss et al., 1971). En 1984, Seeburg et
Adelman décrivaient la séquence nucléotidique du gène codant pour la GnRH, identifiée à
partir de l’acide désoxyribonucléique (ADN) complémentaire d’origine humaine. Deux ans
plus tard, la séquence nucléotidique du gène GnRH de rat était caractérisée (Adelman et al.,
1986). Ces premiers résultats, confirmés plus tard par Fernald et White (1999), indiquent que
le gène GnRH présente une structure similaire quelles que soient l’espèce et l’isoforme
considérées. Il comporte quatre exons: l’exon 1 code pour une région non traduite ou région
5’UTR («untranslated region»); l’exon 2 code pour un peptide signal, la GnRH, un site de
clivage protéolytique ainsi que la partie amino-terminale du «GnRH associated peptide»
(GAP); la partie centrale du GAP est codée par l’exon 3 tandis que l’exon 4 code pour sa
partie carboxy-terminale ainsi qu’une région non traduite (3’UTR). En outre, le gène exprime
des éléments de réponse pour l’acide rétinoïque, les oestrogènes et les hormones
thyroïdiennes. La protéine résultant de la traduction du gène codant pour la GnRH est appelée
5
Introduction
pré-pro-GnRH et aboutit à la formation de la GnRH mature après clivage du peptide signal et
ensuite du GAP (Wetsel et Srinivasan, 2002). Il s’agit d’un décapeptide de structure pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-amide, dont les extrémités carboxy- et aminoterminales sont respectivement modifiées en pyro-glutamate et glycine-amide (Matsuo et al.,
1971). On recense actuellement 15 isoformes différentes de GnRH, pouvant être regroupées
selon leur composition et leur localisation au niveau du cerveau (Parhar, 2002; Wetsel et
Srinivasan, 2002). Il a ainsi été découvert que, chez les rongeurs, les neurones à GnRH (plus
précisément GnRH-I) pouvaient également exprimer la GnRH-III ou GnRH de lamproie
(Hiney et al., 2002) tandis que la GnRH-II (ou GnRH de poulet) est principalement exprimée
dans les structures extra-hypothalamiques comme le mésencéphale (Gestrin et al., 1999).
Comme l’a décrit l’équipe de Kordon, la GnRH peut également subir des modifications posttraductionnelles telles que l’hydroxylation d’un de ses résidus (Gautron et al., 1992), ceci
étant le cas pour la proline en 9ème position. Son rôle n’est pas encore totalement élucidé mais
cette modification pourrait avoir une fonction au cours du développement, puisque la
libération de GnRH est maximale chez l’animal immature et diminue progressivement au
cours du développement (Rochdi et al., 2000).
Après sa libération au niveau de l’éminence médiane, la GnRH est dégradée par deux
enzymes différentes: la prolyl-endopeptidase (PEP) et une métallo-endopeptidase (EP 24.15).
La PEP clive la GnRH du côté amino-terminal au niveau de son résidu glycine, ce qui
engendre la production de la GnRH1-9. Cette dernière peut ensuite être clivée au niveau de sa
liaison Tyr5-Gly6 par l’EP 24.15 (Lew et al., 1994). La métallo-endopeptidase EP 24.15 a été
localisée au niveau de l’éminence médiane chez le rat (Wu et al., 1997) et son inhibition
provoque une augmentation de la sécrétion des gonadotrophines in vivo, lui suggérant un rôle
physiologique (Lasdun et Orlowski, 1990; Wu et al., 1997). Des études menées au sein de
notre laboratoire montrent également que les transcrits de la PEP sont présents au niveau de
l’hypothalamus et qu’ils sont traduits en une enzyme fonctionnelle capable de dégrader la
GnRH lors d’expériences in vitro (Yamanaka et al., 1999). Avec l’EP 24.15, la PEP est
impliquée dans la régulation de la sécrétion pulsatile de GnRH via les effets inhibiteurs des
produits de dégradation qu’elle génère, en particulier la GnRH1-5. En effet, l’inhibition de la
PEP par la bacitracine ou un antagoniste non compétitif (Fmoc-Pro-PyrrCN) accélère la
fréquence de sécrétion pulsatile de GnRH observée à partir d’explants hypothalamiques de rat
(Yamanaka et al., 1999). L’importance physiologique des endopeptidases dans la régulation
de la sécrétion de GnRH semble cependant varier selon les espèces puisque l’inhibition de la
6
Introduction
PEP ou de l’EP 24.15 chez la brebis ne modifie pas la sécrétion des gonadotrophines (Lew et
al., 1997).
A la suite de la caractérisation de la GnRH (Amoss et al., 1971; Matsuo et al., 1971;
Schally et al., 1971) et de la localisation, par immunohistochimie, des neurones qui la
produisent chez les rongeurs (Barry et al., 1973) et les primates (Barry et Carette, 1975), il a
été démontré que ce peptide est libéré à partir de l’hypothalamus de manière pulsatile dans la
circulation portale (Knobil et al., 1980). La sécrétion de GnRH présente donc des pics de
sécrétion d’une certaine amplitude et d’une fréquence donnée, détectables à intervalles
réguliers (Knobil, 1974). Cette pulsatilité est essentielle à la synthèse et à la sécrétion des
gonadotrophines hypophysaires, et donc à la maturation et au maintien des fonctions de
reproduction. Le concept ainsi proposé par Knobil, et aujourd’hui généralement admis, est
que la sécrétion de GnRH est sous le contrôle d’un générateur de pulsatilité. Il pourrait même
en exister différents, qui comprendraient les neurones à GnRH eux-mêmes (composante
intrinsèque) et l’appareil neurono-glial environnant (composante extrinsèque). Cette idée que
les neurones à GnRH puissent former le générateur de pulsatilité provient d’études réalisées
sur des neurones à GnRH immortalisés qui ont montré qu’ils sécrètent la GnRH de manière
pulsatile et impliquant une augmentation des taux calciques intracellulaires (Krsmanovic et
al., 1992; Nunez et al., 2000). Il a également été montré que des cultures primaires de
neurones à GnRH issus de placodes olfactives pouvaient sécréter la GnRH de façon pulsatile
(Terasawa et al., 1999; Duittoz et Batailler, 2000; Funabashi et al., 2000). Chez le rat,
l’existence d’un générateur de pulsatilité extrinsèque est également soutenue par les
observations suivantes: la suppression des connexions entre l’hypothalamus antérieur, où sont
localisés les corps cellulaires des neurones à GnRH, et l’hypothalamus postérieur au niveau
duquel se situent leurs terminaisons (qui contiennent la GnRH mature pouvant être libérée au
niveau de l’éminence médiane), n’affecte pas nécessairement ou pas immédiatement la
sécrétion pulsatile des gonadotrophines (Blake et al., 1973) tandis qu’une déafférentation
altère le niveau de celle-ci (Silverman et al., 1994). En outre, la sécrétion pulsatile de GnRH
peut être observée in vitro à partir d’explants hypothalamiques de rats dépourvus d’aire
préoptique, c’est-à-dire privés de la très grande majorité des péricaryons des neurones à
GnRH comme il fut montré par le modèle utilisé dans notre laboratoire (Purnelle et al., 1997),
renforçant encore davantage l’hypothèse de l’existence d’un générateur de pulsatilité
extrinsèque. Des investigations sur des rats transgéniques dont les neurones à GnRH
expriment la «green fluorescent protein» permettront peut-être l’identification des
composantes et/ou des générateurs de pulsatilité impliqués in vivo (Kato et al., 2003).
7
Introduction
En réponse à la GnRH libérée dans la circulation porte, l’hypophyse sécrète, à son
tour, la FSH et la LH. Il a été montré, in vitro chez le rat et in vivo chez le mouton, que la
sécrétion de ces gonadotrophines suivait parallèlement la sécrétion de GnRH, c’est-à-dire
qu’elle était également pulsatile (Clarke et Cummins, 1982; Levine et Ramirez, 1982; Clarke
et al., 1984). Ainsi, elle permet d’étudier indirectement la sécrétion pulsatile de GnRH in vivo.
Suite à leur libération dans la circulation périphérique, les gonadotrophines agissent au niveau
des gonades (ovaires ou testicules) afin de promouvoir la folliculogenèse ou la
spermatogenèse ainsi que la production des stéroïdes gonadiques, à savoir respectivement
l’oestradiol et la progestérone par les ovaires ou la testostérone par les testicules. Les
oestrogènes et la testostérone peuvent agir de façon inhibitrice au niveau de l’hypothalamus et
de l’hypophyse afin de réduire la sécrétion de GnRH et des gonadotrophines. Cet
enchaînement d’évènements en boucle porte le nom de rétrocontrôle négatif (Ramirez et
McCann, 1963; Kulin et al., 1969) et a lieu dans les deux espèces. A l’inverse, il existe aussi
un effet stimulant des stéroïdes, appellé rétrocontrôle positif, uniquement chez la femelle. Ce
dernier permet l’activation de l’axe hypothalamo-hypophyso-gonadique suite à une
augmentation graduelle de la concentration en oestrogènes durant la période précédant le pic
préovulatoire des gonadotrophines (Freeman, 1994). Différentes études ont montré qu’une
exposition aux oestrogènes supérieure à 15 h était nécessaire pour induire un pic préovulatoire
de LH, tant chez le rat (Sarkar et Fink, 1980) que chez le mouton (Moenter et al., 1990) ou le
singe (Xia et al., 1992). Ce pic préovulatoire de LH implique notamment une augmentation de
la sécrétion de GnRH au niveau de l’éminence médiane aussi bien chez le rat (Sarkar et al.,
1976) que chez le singe (Xia et al., 1992). Toutefois, il est à noter que chez la guenon dont
l’hypothalamus a été lésé, l’administration de GnRH synthétique, à une fréquence constante et
invariable, permet de restaurer un cycle menstruel avec pic préovulatoire. Ces résultats
indiquent que l’accroissement de la sécrétion de GnRH n’est pas nécessaire et que la genèse
du pic préovulatoire de LH peut impliquer directement l’hypophyse chez les primates (Knobil
et al., 1980).
La modulation de l’activité du générateur de sécrétion pulsatile de GnRH par les
stéroïdes a été démontrée par des expériences d'orchidectomie et d’administration de
testostérone chez le rat (Steiner et al., 1982) et chez le singe (Plant, 1982). Drouva et son
équipe (1984) ont montré que le 17β-oestradiol (E2) est spécifiquement impliqué dans la
sécrétion de GnRH et que son effet est modulé par des récepteurs qui ne requièrent pas une
translocation nucléaire du stéroïde. Une décennie plus tard, Trudeau et ses collègues (1993)
ont montré que les stéroïdes sexuels augmentent la sensibilité de l’hypophyse en réponse à la
8
Introduction
GnRH chez le poisson rouge. En 2002, il a été montré que l’action directe de l’E2 sur
l’hypophyse antérieure de rates ovariectomisées est requise pour déclencher le rétrocontrôle
négatif ou positif sur la sécrétion de LH (Yin et al.). Ainsi, les stéroïdes sexuels peuvent avoir
des effets soit directement sur la sécrétion de GnRH hypothalamique (Lee et al., 2004), soit
directement sur les cellules hypophysaires sécrétant les gonadotrophines (Saito et al., 2003).
Cependant, l’importance de l’E2 pour l’amplification de la sensibilité hypophysaire en
réponse à la GnRH durant la période préovulatoire a été remise en cause par l’augmentation
du taux d’acide ribonucléique messager (ARNm) du récepteur à la GnRH lors de la période
qui précède le pic préovulatoire d’oestradiol circulant. En effet, il apparaît que l’amplification
de cette sensibilité hypophysaire est le résultat d’une diminution de la concentration de
progestérone plutôt qu’une augmentation du taux circulant d’oestradiol.
Dans les noyaux arqué et périventriculaire antéro-ventral, les stéroïdes sexuels (E2 et
testostérone) modulent de façon opposée l’expression de la kisspeptine, un peptide de 54
acides aminés qui interagit avec le récepteur «G protein-coupled receptor» (GPR54): les deux
stéroïdes réduisent le taux de transcrits de kiss-1 (gène de la kisspeptine) dans le noyau arqué
et augmentent son expression dans le noyau périventriculaire antéro-ventral. Les neurones à
kisspeptine localisés dans le noyau arqué participent ainsi à la régulation du contrôle négatif
de la sécrétion des gonadotrophines hypophysaires tandis que ceux présents au sein du noyau
périventriculaire antéro-ventral contribuent à l’induction du pic préovulatoire de la LH chez la
femelle, déclenchant l’ovulation, ou jouent un rôle essentiel dans le comportement sexuel
chez le mâle (Couzinet et Schaison, 1993; Smith et al., 2007). Très récemment, il a été
observé chez des femmes saines post-ménopausées que les sécrétions de FSH et LH, en
réponse à la GnRH, diminuaient après une administration d’oestrogènes, avec une plus grande
sensibilité de la FSH que la LH (Shaw et al., 2010). Ceci suggère donc que l’hypophyse est
directement rétrocontrôlée négativement par les oestrogènes en réponse à la GnRH chez les
femmes et que cette inhibition contribuerait à la régulation différentielle des sécrétions de
FSH et LH (Couzinet et Schaison, 1993; McNeilly et al., 2003).
La nature des facteurs qui participent à la synchronisation des pics sécrétoires de
GnRH n’a pas encore été complètement élucidée, mais la plupart des études rapportent une
implication importante des flux calciques (Moore et al., 2002; Richter et al., 2002). Par
ailleurs, il existe des différences entre la fréquence de sécrétion pulsatile de GnRH obtenue à
partir de neurones isolés (Terasawa et al., 1999; Duittoz et Batailler, 2000; Funabashi et al.,
2000) ou d’explants hypothalamiques (Bourguignon et Franchimont, 1984; Bourguignon et
al., 1989a et 1989b; Bourguignon et al., 1990) in vitro et la fréquence observée in vivo (Sisk
9
Introduction
et al., 2001; Harris et Levine, 2003). Les types de cellule impliqués dans le phénomène de
synchronisation semblent aussi dépendre de l’espèce étudiée puisque, dans des cultures de
placodes olfactives issues de singes, il a été montré que des cellules non neuronales
participaient à ce processus (Richter et al., 2002) tandis que chez la souris, les cellules
impliquées sont de type neuronal (Moore et al., 2002), ces résultats plaidant en faveur d’un
rôle modulateur de l’appareil neurono-glial afférent aux neurones à GnRH. En effet, il devient
de plus en plus évident que l’activité sécrétoire des neurones à GnRH est régulée non
seulement par des influx trans-synaptiques, mais également via la sécrétion de molécules
trophiques («basic fibroblast growth factor», facteur de croissance des fibroblastes bFGF et
«transforming growth factor α», facteur de croissance de transformation α TGFα
principalement) d’origine neuronale et/ou astrogliale (Voigt et al., 1996; Dziedzic et al.,
2003). Par ailleurs, Prevot et son équipe ont suggéré que les tanycytes, des cellules
ependymo-gliales présentes dans l’éminence médiane, régulent la sécrétion de GnRH durant
le cycle oestral au travers de changements de plasticité qui permettent ou empêchent l’accès
direct des terminaisons nerveuses des neurones à GnRH vers la circulation porte (2003). Bien
qu’il soit établi que les neurones à GnRH et les astrocytes maintiennent un contact étroit, une
interaction davantage renforcée entre les axones des neurones à GnRH et les astrocytes a lieu
durant le développement sexuel femelle (Parent et al., 2007).
La sécrétion de GnRH est caractérisée par une augmentation de sa fréquence au cours
du développement chez le rat. Elle se stabilise chez le mâle adulte tandis que chez la femelle,
son amplitude in vivo et celle des à-coups sécrétoires in vitro sont augmentées au moment du
pic préovulatoire de GnRH (Levine et Ramirez, 1982; Parent et al., 2000). Chez la brebis, on
constate une augmentation de la fréquence et une diminution de l’amplitude de sécrétion de
GnRH in vivo au moment du pic préovulatoire de LH (Evans et al., 1995). Ces divergences
résultent des variations entre les espèces, mais aussi des conditions expérimentales in vivo et
in vitro.
C. Maturation sexuelle femelle: comparaison entre l’humain et le rat
Comme chez l’humain, la sécrétion de LH est également pulsatile chez le rat (Ojeda et
Urbanski, 1994). La fréquence et l’amplitude des à-coups sécrétoires augmentent à l’approche
de la puberté. Chez la femelle, apparaissent des mini-pics l’après-midi, qui ont été interprétés
comme des ébauches de pic préovulatoire (Urbanski et Ojeda, 1986). Chez l’humain comme
chez le rongeur, les ovaires sont loin d’être quiescents avant la puberté. Le principal stéroïde
10
Introduction
sécrété par les gonades féminines prépubertaires est l’oestradiol, bien qu’elles produisent
aussi de l’oestrone, de l’androstènedione et de la testostérone (Rosenfield, 1996). Les ovaires
humains foetaux produisent déjà des quantités substantielles d’oestradiol (Kaplan et
Grumbach, 1990). Les concentrations plasmatiques d’oestrogènes diminuent ensuite dès la
première semaine de vie post-natale jusqu’à la puberté, puis augmentent à cet évènement pour
atteindre un plateau en fin de puberté, à l’apparition des règles (ménarche) où elles seront
modulées selon les phases du cycle menstruel. L’oestradiol est responsable du développement
des caractères sexuels secondaires féminins (en ce compris le développement des seins, la
maturation du tractus génital, la distribution du pannicule adipeux et la croissance) ainsi que
de la minéralisation osseuse. La production d’inhibine, par contre, est négligeable durant la
vie foetale et le développement prépubertaire, mais lorsque les concentrations de FSH
augmentent au moment de la puberté, les taux circulants d’inhibine A et B augmentent
significativement (Kaplan et Grumbach, 1990). Ainsi, bien que des différences significatives
existent (Tableau 1), les mécanismes régulant la maturation ovarienne, l’activité sécrétoire et
le contrôle par les gonadotrophines sont relativement comparables chez le rat et l’Homme.
11
Introduction
Tableau 1: Différences de maturation sexuelle femelle chez l’Homme et le rat.
Espèces
Paramètres
Humain
Rat
A mi-gestation, puis diminue
Sécrétion pulsatile
à cause du rétrocontrôle
/
de GnRH
négatif exercé
durant la gestation
par les oestrogènes
Développement du SNC
à la naissance
Très mature:
Peu mature:
les neurones à GnRH migrent les neurones à GnRH migrent
dans l’hypothalamus
dans l’hypothalamus
avant la fin du premier quart
au cours du dernier quart
de gestation
de gestation
(Schwantzel-Fukuda et al.,
(Daikoku et Koide, 1998)
1996; Verney et al., 1996)
Juste après la naissance,
puis longue quiescence
juvénile et
réactivation pubertaire
Augmentation progressive
de la fréquence
dès la naissance,
avec une accélération
au moment de la puberté
Premier signe de
développement pubertaire
Début des seins (stade B2):
assez distant de la naissance
(10 ans soit 1/8 de la vie)
OV: assez proche de la
naissance (1 mois soit 1/48
de la vie)
Variations du timing
pubertaire chez des
individus partageant des
conditions de vie similaires
Très importantes:
5 ans soit 1/16 de la vie)
(Tanner, 1962)
Peu marquées:
4 jours soit 1/180 de la vie
(Ojeda et Urbanski, 1994)
Sécrétion pulsatile
de la GnRH post-natale
Puisque le système reproducteur des rongeurs femelles (principalement le rat et la
souris) partage un certain nombre de caractéristiques avec l’Homme, ces animaux ont été
largement utilisés en recherche et sont fréquemment employés dans des études de laboratoire
désignées pour élucider les mécanismes des fonctions reproductrices chez les mammifères.
Chez les souris femelles, la puberté est un processus transitoire qui comprend l’OV et la
première ovulation: la première est le signe initial du pic oestrogénique qui accompagne
l’établissement de la puberté et la seconde, suivie par une cyclicité oestrale, est le signal d’une
conduite centrale de l’activité ovarienne (Ramirez et Sawyer, 1965). Les évènements
correspondant chez l’humain sont, respectivement, le début du développement des seins
(stade B2) et la ménarche. Chez le rat, l’âge moyen à l’OV varie suivant les souches et les
conditions d’élevage, mais elle a lieu habituellement à ± 35 jours et l’intervalle moyen entre
l’OV et le premier oestrus dure 4,2 ± 1,1 jours. Chez les rates de laboratoire, la maturation
sexuelle débute très tôt, comparée à celle de la femme, et les variations individuelles des
12
Introduction
évènements pubertaires sont moins perceptibles que chez l’espèce humaine (Fig. 3).
L’apparition du premier épisode ovulatoire est une réponse ovarienne au pic sécrétoire massif
des gonadotrophines. Chez les rongeurs, la programmation du mécanisme central d’ovulation
prend place entre la dernière semaine de vie prénatale et la première semaine de vie postnatale (Naftolin, 1994) et une exposition précoce aux stéroïdes sexuels cause des
perturbations dans ce processus (Matagne et al., 2004).
Figure 3: Représentation schématique des principaux évènements pubertaires chez la rate femelle et chez la
femme. La comparaison est basée sur une échelle d’espérance de vie similaire. N: naissance; OV: ouverture
vaginale; SNC: système nerveux central.
Au cours du développement, une accélération de la fréquence et une augmentation de
l’amplitude de la sécrétion pulsatile de GnRH ont lieu. Ce changement développemental a été
mis en évidence par des études in vivo chez le singe (Watanabe et Terasawa, 1989) ainsi que
chez le rat (Sisk et al., 2001; Harris et Levine, 2003) et a pu être observé in vitro par
l’incubation d’explants hypothalamiques (Bourguignon et Franchimont, 1984). Il s’agit d’un
évènement majeur permettant l’apparition de la puberté. L’importance physiologique de la
sécrétion pulsatile de GnRH a été établie chez le singe où une lésion de l’hypothalamus abolit
13
Introduction
le générateur de pulsatilité et inhibe ainsi la sécrétion pulsatile des gonadotrophines.
Cependant, celle-ci peut être rétablie par des injections répétées de GnRH synthétique, ce qui
n’est pas observé si elle est administrée de manière continue (Belchetz et al., 1978; Knobil et
al., 1980). En outre, l’administration de GnRH synthétique à faible fréquence entraîne un
mode sécrétoire de type prépubère, où la sécrétion de FSH prédomine sur celle de LH
(Knobil, 1980) alors que l’administration du peptide hypothalamique à une fréquence
similaire à celle de l’âge adulte, à des singes immatures, induit une puberté précoce (Wildt et
al., 1980). De manière générale, les résultats issus des nombreuses études qui ont tenté
d’identifier les facteurs impliqués dans la maturation pubertaire (Ojeda et Urbanski, 1994;
Ojeda et Terasawa, 2002) indiquent qu’une modification de l’équilibre entre les effets
stimulants et inhibiteurs des deux neurotransmetteurs majeurs, à savoir respectivement le
glutamate et l’acide γ-aminobutyrique (GABA), au niveau du neurone à GnRH et/ou de ses
systèmes afférents, sont responsables de l’accélération de la fréquence de sécrétion pulsatile
de GnRH (Bourguignon et al., 1987; Ojeda et Urbanski, 1994; Bourguignon et al., 1997;
Ojeda et Terasawa, 2002).
D. Effets des oestrogènes sur les neurones à GnRH
La freination de l’axe hypothalamo-hypophyso-gonadique est réalisée par les
hormones périphériques (notamment gonadiques) qui exercent un rétrocontrôle inhibiteur sur
l'hypothalamus et parfois sur l'hypophyse. Ce rétrocontrôle est déclenché lorsque la
concentration en hormones périphériques dans le sang dépasse une valeur critique (spécifique
à chaque type d'hormones). Cette concentration atteinte, les hormones périphériques sont
détectées par les neurones hypothalamiques qui possèdent des récepteurs spécifiques aux
hormones périphériques. Ces derniers provoquent l’inhibition des neurones qui les portent et
ainsi un rétrocontrôle peut s'exercer: l’inhibition de l'activité des neurones sécréteurs est alors
répercutée en aval de tout le système neuroendocrinien, ce qui résulte en une réduction de la
concentration sanguine des hormones périphériques jusqu’à un niveau hormonal de base.
Ainsi, il s’opère un véritable retour à l’état initial et l’ensemble du système est prêt à
fonctionner à nouveau.
Dans les années ‘70, deux théories furent proposées pour expliquer le déclenchement
pubertaire. La première, l’hypothèse du «gonadostat», est basée sur des changements de
sensibilité hypothalamo-hypophysaire aux stéroïdes sexuels au cours du développement
(Ramirez et McCann, 1963; Kulin et al., 1969). Selon ces auteurs, l’apparition de la puberté
14
Introduction
est sous le contrôle des gonades et implique une diminution de la sensibilité hypothalamique
au rétrocontrôle négatif par les stéroïdes sexuels. Ainsi, les taux d’oestrogènes requis pour
annihiler les changements morphologiques des cellules gonadotropes, induits par castration,
étaient plus faibles chez le rat immature que chez l’adulte. La deuxième hypothèse qui peut
coexister avec la première, repose sur l’existence d’un mécanisme indépendant des gonades.
Elle fut émise sur base des changements développementaux de la sécrétion des
gonadotrophines, qui augmente à l’âge où la puberté devrait survenir chez le patient ou le
singe agonadique (Conte et al., 1980; Plant, 1986). A l’époque où ces deux théories ont été
établies, une augmentation de la sécrétion pulsatile de LH, associée au sommeil et spécifique
de la puberté humaine, fut mise en évidence (Boyar et al., 1972). Durant ces vingt dernières
années, les avancées majeures ont porté davantage sur les facteurs hypothalamiques et
périphériques impliqués dans la régulation de la sécrétion de GnRH (Plant et al., 1989;
Mitsushima et al., 1994; Ahima et al., 1997; Chehab et al., 1997; Cheung et al., 1997; Ma et
al., 1999; Ojeda et al., 1999).
1. Nature et origine des oestrogènes et leurs récepteurs
D’une importance primordiale dans le déroulement du cycle reproducteur des
mammifères femelles, les oestrogènes apparaissent de plus en plus comme des facteurs
pleiotropiques agissant dans de nombreux systèmes, et particulièrement au niveau du SNC. Ils
jouent ainsi un rôle crucial dans des phénomènes aussi divers que la neuroprotection ou la
mémorisation (McEwen, 2002; Balthazart et Ball, 2006). Ils sont principalement produits par
les ovaires et, outre 1’oestradiol, ils comprennent également l’oestrone, un produit dérivé du
premier. Par l’intermédiaire des cellules de la granulosa et des cellules thécales, les ovaires
produisent aussi la progestérone et ses dérivés (17α- et 20α-hydroxyprogestérone) ainsi que
des androgènes et leurs métabolites 5α-réduits, à savoir la testostérone et la 5αdihydrotestostérone (Mellon et al., 2001; Ojeda, 2004). La synthèse de tous ces stéroïdes
débute toujours par la transformation du cholestérol en prégnenolone qui peut ensuite être
transformée en progestérone.
Au cours de la dernière décennie, il a été montré que le cerveau peut également
produire localement des neurostéroïdes. Contrairement à l’ovaire, les enzymes nécessaires à
leur production sont localisées dans différentes zones du cerveau et/ou dans divers types
cellulaires (Mellon et al., 2001). Dans le cas d’une action au niveau du SNC, plus
spécifiquement au niveau de l’hypothalamus, l’oestradiol peut être soit d’origine
périphérique, soit provenir d’une synthèse de novo à partir de testostérone. La localisation de
15
Introduction
l’aromatase et de ses transcrits au niveau de l’hypothalamus (Wagner et Morrell, 1997;
Roselli et al., 1998; Naftolin et al., 2001) confirme l’hypothèse selon laquelle l’oestradiol est
synthétisé in situ. Ce mécanisme est d’ailleurs impliqué dans la différenciation sexuelle du
cerveau chez le rat où une augmentation de l’activité aromatasique est observée chez le mâle
durant les premiers jours de vie post-natale (Tsuruo et al., 1994; Roselli et al., 1998; Amateau
et al., 2004).
L’aromatase est une enzyme importante qui catalyse la conversion des androgènes en
oestrogènes. Elle appartient à la famille du cytochrome p450 et forme un complexe avec la
nicotinamide-adénine-dinucléotide-phosphate-(forme réduite)-réductase (Hong et al., 2009).
La formation d’oestradiol, qui résulte de l’aromatisation de la testostérone par la p450
aromatase, a lieu aussi bien dans des aires spécifiques du cerveau que dans les gonades
(essentiellement les testicules). Durant le développement du SNC, les oestrogènes locaux
régulent la différenciation sexuelle des structures neurales et modulent les fonctions
neuroendocriniennes et reproductrices ainsi que le comportement sexuel (Naftolin et Brawer,
1978), l’inhibition de l’aromatase diminuant d’ailleurs le comportement sexuel mâle chez la
caille (Cornil et al., 2006). Chez le poisson zèbre, l’activité élevée de l’aromatase B (AroB),
produit du gène cyp19b, est exclusivement exprimée par les celules gliales radiales (et non par
les neurones) principalement dans le bulbe olfactif, le télencéphale, l’aire préoptique et
l’hypothalamus, l’oestradiol y régulant son expression exclusivement dans l’aire préoptique et
l’hypothalamus médio-basal, régions majeures de production locale du stéroïde, tôt après la
fécondation (Tong et al., 2001; Goto-Kazeto et al., 2004). Cette régulation oestradioldépendante implique une action transcriptionnelle directe des récepteurs aux oestrogènes
(ERs) qui nécessite également la contribution du «estrogen responsive element» (ERE).
L’expression de l’AroB dans les cellules gliales radiales, connues pour leur rôle dans la
neurogenèse et considérées comme progénitrices cellulaires (contrairement aux autres
vertébrés qui présentent une neurogenèse limitée à l’âge adulte), suggère que la production
locale d’oestradiol dans ces cellules pourrait influencer la capacité du cerveau des poissons
téléostéens à croître durant l’âge adulte (Pellegrini et al., 2005). Ainsi, l’oestradiol, en
permettant l’établissement d’un environnement favorable, serait impliqué dans le maintien
et/ou l’activation des cellules progénitrices chez les poissons téléostéens (Kah et al., 2009).
Toutes ces observations mentionnées ci-avant confirment l’importance de l’aromatase dans le
développement et le fonctionnement du SNC rapportée par Lephart en 1996. En outre,
Balthazart a montré, dans l’hypothalamus de caille et du poisson zèbre, que l’activité de
l’aromatase est plus importante chez le mâle que chez la femelle. Elle diminue après
16
Introduction
castration et augmente à la suite d’administrations de testostérone, ce qui amène à penser que
les oestrogènes produits agissent par aromatisation centrale de la testostérone (1991). Chez
des cailles castrées, traitées ensuite par testostérone et des inhibiteurs de l’aromatase, une
inhibition des effets de l’androgène au niveau de l’hypothalamus a été observée (Foidart et
al., 1994), ce qui corrobore les résultats précédents.
L’activité de l’aromatase du cerveau est contrôlée de façon génomique par des
stéroïdes qui augmentent la transcription de l’enzyme, mais l’équipe de Balthazart a montré
récemment que son activité peut également être modulée en quelques minutes par des
phosphorylations
calcium–dépendantes
et
déclenchées
par
des
variations
de
neurotransmission glutamatergique (Balthazart et al., 2003; Balthazart et al., 2009). Ainsi, le
comportement sexuel mâle serait activé de façon non génomique, partiellement par la
testostérone, via sa conversion en oestradiol par l’aromatase dans l’aire préoptique. Par
ailleurs, la localisation de l’aromatase dans les boutons présynaptiques suggère que la
régulation des oestrogènes est, au moins en partie, trans-synaptique (Balthazart et Ball, 1998).
2. Action de l’oestradiol sur le système à GnRH et le contrôle neuroendocrinien de l’axe
hypothalamo-hypophyso-gonadique
Comme indiqué précédemment, les oestrogènes constituent un des facteurs principaux
influençant le système à GnRH. Toutefois, les mécanismes qui sous-tendent ces effets sont
complexes et non encore élucidés (Herbison, 1998; Matagne et al., 2005): les effets des
oestrogènes sur les neurones à GnRH in vitro ont fait l’objet d’une revue en 2003 (Matagne et
al.). Chez le rat mâle, une augmentation rapide des taux circulants de testostérone d’origine
testiculaire est observée à la naissance (Weisz et Ward, 1980). Ce pic natal de testostérone,
agissant au niveau de l’hypothalamus après avoir été aromatisé en oestradiol (Roselli et
Resko, 2001), est essentiel pour la différenciation sexuelle de la fonction de reproduction. Il a,
en effet, été montré que la suppression du pic d’androgènes, par castration (Corbier et al.,
1983; Corbier, 1985) ou par inhibition de l’aromatase (Swaab et al., 1995; Gerardin et
Pereira, 2002), provoquait l’apparition de comportements sexuels femelles ainsi qu’un pic
préovulatoire de LH après administration d’oestrogènes (Corbier, 1985) chez des animaux
génétiquement mâles.
L’E2 agit principalement via deux isoformes du ER réparties dans différents tissus de
l’organisme: les ERα et β (Kuiper et al., 1996; Mosselman et al., 1996). Il y a une dizaine
d’années, un troisième type de récepteur (ERγ) a été isolé chez les poissons téléostéens
(Hawkins et al., 2000) et n’a, jusqu’à l’heure actuelle, pas encore été mis en évidence chez les
17
Introduction
mammifères. Après la liaison de l’E2 à son type de récepteur dans le cytoplasme, le complexe
ligand-récepteur ainsi formé est internalisé au niveau du noyau cellulaire, où il se lie à une
séquence génomique spécifique ERE, qui activera ou réprimera la transcription de gènes
cibles. Ce sont ces effets génomiques qui sont responsables du rétrocontrôle hypothalamohypophysaire par les oestrogènes périphériques (Kalra, 1985; Xia et al., 1992). Les effets
rapides ou non génomiques de l’oestradiol seront rapportés plus loin dans ce chapitre.
Les deux isoformes ERα et β sont abondamment exprimées au niveau de
l’hypothalamus (Mitra et al., 2003; Perz et al., 2003). Cependant, la plupart des premières
études ont montré que les neurones à GnRH ne fixaient pas l’oestradiol (Shivers et al., 1983)
et n’exprimaient pas non plus ses récepteurs (Lehman et Karsch, 1993; Herbison et al., 1995),
bien que ceux issus de lignées immortalisées les expriment (Roy et al., 1999). Ces
observations ont conduit à l’idée que les oestrogènes agiraient de manière indirecte sur le
système à GnRH (Shen et al., 1998). Plus récemment, l’amélioration des techniques de
détection a redynamisé les recherches: Butler et ses collègues (1999) ont rapporté l’expression
des ERα et β sur les neurones à GnRH du rat. Ces résultats ont rapidement été confirmés par
l’équipe de Skynner (1999) et suivis par d’autres études menant aux mêmes observations
(Hrabovszky et al., 2000; Hrabovszky et al., 2001). Désormais, il semble même que la
majorité des neurones à GnRH expriment les deux isoformes (Herbison et Pape, 2001).
Chez les femelles, les taux d’oestradiol disponibles au niveau du cerveau sont
maintenus à un niveau plus faible que chez les mâles durant la période périnatale (Amateau et
al., 2004), probablement grâce à la présence de l’α-foetoprotéine (AFP) plasmatique
(MacLusky et Naftolin, 1981; Bakker et al., 2006). Similairement aux expériences réalisées
chez le mâle, l’injection de testostérone ou d’E2 à des femelles durant les premiers jours postnatals induit une masculinisation résultant en l’absence de cycle oestral chez l’adulte (Gorski,
1968; Gogan et al., 1980) ainsi qu’en la diminution de comportements sexuels spécifiquement
femelles (Diaz et al., 1995). Un même effet est observé chez les souris déficientes pour le
gène AFP, ce qui démontre l’importance de cette protéine foetale dans la différenciation
sexuelle (Gabant et al., 2002; Bakker et Baum, 2008). Les mécanismes par lesquels
l’oestradiol agit au niveau du cerveau en période périnatale semblent également varier selon
les types cellulaires puisque la masculinisation de cellules gliales hypothalamiques implique
une augmentation des taux de GABA (Mong et al., 2002) tandis que la différenciation
sexuelle des neurones hypothalamiques de l’aire préoptique, induite par l’E2, est modulée par
la prostaglandine E2 (PGE2), dont les effets masculinisants jouent un rôle important dans la
18
Introduction
détermination des comportements sexuels chez l’animal adulte (Amateau et McCarthy, 2002
et 2004).
3. Effets rapides / non génomiques de l’E2 sur les neurones à GnRH
Le mécanisme d’action des effets rapides de l’E2 a retenu l’attention des chercheurs, il
y a une dizaine d’années (Revelli et al., 1998; Falkenstein et Wehling, 2000; McEwen, 2002),
bien que de tels effets aient déjà été rapportés dans les années ‘70 (Sherwood et al., 1976;
Dufy et al., 1979). L’action de l’E2 peut moduler les propriétés électrophysiologiques des
neurones (Dufy et al., 1979; Nabekura et al., 1986) et activer ou inhiber les cascades de
seconds messagers intracellulaires tels que l’adénosine monophosphate cyclique (Gu et Moss,
1996; Zanassi et al., 2001) ou les kinases (Lagrange et al., 1999; Doolan et al., 2000). En
outre, les oestrogènes peuvent également agir sur les voies de neurotransmission les plus
importantes comme celles du glutamate (Smith et al., 1987; Gu et Moss, 1996) et du GABA
(Lagrange et al., 1995; Lagrange et al., 1999). Ces effets, considérés comme rapides donc non
génomiques, ont lieu endéans quelques secondes à quelques minutes, mais ils peuvent
également avoir des conséquences à long terme en stimulant ou réprimant la transcription de
gènes cibles (Zanassi et al., 2001).
L’étude des effets rapides des oestrogènes a également mis en lumière la nécessité de
rechercher des récepteurs additionnels puisque le mécanisme d’action des ERα et β est, par
définition, un mécanisme d’action lent et, de prime abord, ne correspond pas aux
caractéristiques des effets rapides des oestrogènes. Néanmoins, des études récentes ont montré
que les ERα et β pouvaient activer des évènements rapides tels que la phosphorylation des
«mitogen activated protein kinases» (MAPK), les kinases activant les agents mitogènes
(Wade et al., 2001), ou la production de monoxyde d’azote (NO) via l’activation de la
cascade de l’inositol triphosphate (Simoncini et al., 2000). Or, l’E2 couplé à la
serumalbumine bovine, est incapable de traverser la membrane plasmique. Cependant, il a été
rapporté que ce complexe, dans certains cas, pouvait agir de manière rapide et de façon
similaire à l’E2 (Lagrange et al., 1999; Prevot et al., 1999), indiquant que l’ER responsable
des effets rapides de l’E2 est probablement localisé au niveau de la membrane cellulaire. Ces
études n’excluent pas l’hypothèse des effets passant par les récepteurs classiques puisqu’il a
été rapporté que l’ERα pouvait se trouver au niveau de la membrane des neurones
hippocampaux (Clarke et al., 2000) ou des cellules tumorales pituitaires (Watson et al., 1999).
Par ailleurs, il apparaît que certains effets rapides des oestrogènes impliquent également
d’autres récepteurs membranaires couplés à des protéines G comme cela a été proposé par
19
Introduction
Kelly et ses collègues en 2002. D’autres études ont abouti à la caractérisation d’un ER
membranaire, à savoir le ERx, présentant une affinité plus grande pour l’α-isomère de
l’oestradiol que pour la forme β. La structure de son gène n’a pas encore été caractérisée, mais
il semblerait que ce récepteur puisse activer les MAPK (Toran-Allerand et al., 2002).
Il y a 6 ans, les effets génomiques et non génomiques des oestrogènes sur le contrôle
hypothalamique de l’axe gonadotrope ont fait l’objet d’une thèse de doctorat en sciences
biomédicales au sein de notre Unité de Recherche (Matagne, 2005). Il y a été montré que
l’incubation continue d’explants hypothalamiques en présence d’E2 entraîne une accélération
de la fréquence de sécrétion pulsatile de GnRH chez le rat femelle immature âgé de 5 et 15
jours (Matagne et al., 2004) et que cet effet implique une participation spécifique des
récepteurs au kaïnate ainsi que celle des ERα et β. Matagne et ses collègues ont également
observé que l’E2 peut rapidement augmenter la réponse sécrétoire de GnRH induite par le
glutamate ou le NO. Cet effet, probablement non génomique et impliquant les récepteurs au
kaïnate et les deux isoformes classiques d’ERs, dépend d’une augmentation des taux de
calcium intracellulaires ainsi que d’une activation de différents types de kinases (Matagne et
al., 2005; Moenter et Chu, 2012).
E. Facteurs du mécanisme neuroendocrinien de la puberté
Toute activation sécrétoire de GnRH au cours du développement (apparition de la
sécrétion de GnRH, puberté, pic préovulatoire, …) résulte potentiellement de deux
mécanismes différents. Le premier est la diminution d’un tonus inhibiteur qui limite la
sécrétion pulsatile de GnRH tandis que le second est l’augmentation d’un tonus stimulant qui
facilite sa sécrétion (Ojeda et Terasawa, 2002). Les neurones à GnRH peuvent être régulés
directement ou indirectement par les neurotransmetteurs et les neuropeptides sécrétés par les
cellules avoisinantes, neuronales ou gliales. Il existe de nombreux facteurs centraux et/ou
périphériques qui ont un effet stimulant et/ou inhibiteur sur le système à GnRH. Dans ce
travail, nous ne les décrirons pas de façon exhaustive, mais nous nous limiterons seulement
aux plus importants et à ceux démontrés comme impliqués dans nos conditions
expérimentales.
1. Facteurs centraux
- La stimulation du récepteur au N-méthyl-D-aspartate (NMDA) par le glutamate induit une
puberté précoce chez le rat (Urbanski et Ojeda, 1987; Smyth et Wilkinson, 1994) et le singe
20
Introduction
(Plant et al., 1989) alors que l’administration d’antagonistes tels que le MK-801 ou l’acide 2amino-5-phosphonovalérique retarde l’apparition de la puberté chez le rat (MacDonald et
Wilkinson, 1990; Urbanski et Ojeda, 1990; Meijs-Roelofs et al., 1991). La réponse sécrétoire
de LH en réponse à l’administration de NMDA est faible à 10 et 15 jours, atteint un niveau
maximal à 25 jours et diminue finalement à 40 jours chez le rat (Cicero et al., 1988). La
sécrétion in vitro de GnRH, évoquée par le glutamate, le NMDA ou le kaïnate, suit le même
profil avec une sensibilité maximale à 25 jours (Bourguignon et al., 1992). L’existence de
cette diminution de sensibilité de la sécrétion de GnRH au glutamate est surprenante: elle
pourrait faire suite à la diminution du tonus inhibiteur par le GABA qui survient avant la
puberté. Dès lors, durant cette dernière, le tonus glutamatergique facilitateur resterait actif tout
en diminuant, puisque le puissant tonus inhibiteur du GABA ne lui est plus opposé. En plus
de son action au moment de la maturation sexuelle, le glutamate intervient également dans le
déroulement du pic préovulatoire de LH, puisque ce dernier est bloqué par des antagonistes du
récepteur au NMDA (Lopez et al., 1990; MacDonald et Wilkinson, 1990; Urbanski et Ojeda,
1990) et à l’α-amino-3-hydroxy-5-méthylisoazol-4-propionate (AMPA)/kaïnate (Lopez et al.,
1990; Ping et al., 1997). Des études utilisant le marqueur c-fos comme témoin de l’activation
des neurones à GnRH indiquent qu’une grande proportion (>50 %) des neurones à GnRH,
activés au moment du pic préovulatoire, expriment des récepteurs kaïniques (Eyigor et
Jennes, 2000), ce qui confirme leur implication dans ce phénomène. Des travaux similaires
étudiant l’expression du récepteur au NMDA ont montré qu’il n’y avait pas d’augmentation
du nombre de neurones à GnRH exprimant ce récepteur et le marqueur c-fos au moment du
pic préovulatoire (Ottem et al., 2002).
- L’acide aminé GABA est un facteur-clé impliqué dans les changements développementaux
de la sécrétion de GnRH au niveau du SNC. Certaines études ont mis en évidence un rôle
inhibiteur du GABA alors que d’autres ont souligné son rôle stimulant. Par ailleurs, certains
auteurs rapportent une augmentation de l’excitabilité des neurones à GnRH adultes par le
blocage spécifique du récepteur GABAA au moyen de bicuculline (Han et al., 2004) tandis
que d’autres montrent que les neurones à GnRH sont excités suite à une activation de ce
même récepteur, tout au long du développement, suggérant que l’effet inhibiteur modulé par
le récepteur GABAA serait exercé sur un système neuronal autre que le neurone à GnRH luimême (DeFazio et al., 2002). La composante ontogénétique est sans doute capitale car la
majorité des études utilisant des neurones foetaux ou néonatals montrent un effet stimulant
alors que celui-ci est clairement inhibiteur par la suite, particulièrement avant le
déclenchement pubertaire (Matagne et al., 2003; Parent et al., 2005). Malgré l’effet inhibiteur
21
Introduction
du GABA établi, son rôle au niveau de l’axe endocrinien GnRH/LH semble moins
catégorique et inclut des effets stimulants sur la sécrétion de GnRH (Masotto et al., 1989;
Martinez de la Escalera et al., 1994) et de LH (Vijayan et McCann, 1978). Certains travaux
ont montré que l’effet direct du GABA sur des neurones foetaux à GnRH immortalisés de la
lignée GT-1 (Hales et al., 1994; Martinez de la Escalera et al., 1994), c’est-à-dire considérés
comme immatures (Wetsel, 1995), ou des neurones à GnRH issus de placode olfactive
(Kusano et al., 1995), était stimulant via les récepteurs de type GABAA. Des effets stimulants
directs du GABA, sur les neurones à GnRH, ont également été rapportés chez la souris
(DeFazio et al., 2002). Contrairement au récepteur GABAB dont le rôle est principalement
inhibiteur (Masotto et al., 1989; Martinez de la Escalera et al., 1994; Moore et al., 2002), le
récepteur GABAA paraît impliqué à la fois dans des effets inhibiteurs et stimulants (Martinez
de la Escalera et al., 1994). Néanmoins, la majorité des études in vivo ont rapporté un rôle
inhibiteur du GABA qui pourrait être modulé de façon indirecte par des circuits neuronaux
formant des synapses avec les neurones à GnRH. Ainsi, le GABA ou son agoniste, le
muscimol, injecté chez des rats au niveau de l’aire préoptique ou du 3ème ventricule, cause une
inhibition de la sécrétion de LH (Akema et al., 1990; Herbison et al., 1991). D’autres ont
montré ce même effet aussi bien sur la sécrétion pulsatile que sur le pic préovulatoire de LH
(Donoso et Banzan, 1984; Adler et Crowley, 1986; Herbison et Dyer, 1991). Plus récemment,
l’équipe de Funabashi (2002) a confirmé son effet inhibiteur sur la sécrétion pulsatile de
GnRH in vitro. Ces effets inhibiteurs durant le développement ont également été constatés
chez la guenon immature qui présente une puberté précoce suite à l’infusion de bicuculline ou
d’un oligonucléotide anti-sens pour la glutamate décarboxylase, enzyme impliquée dans la
synthèse du GABA, au niveau de l’éminence médiane (Mitsushima et al., 1994; Keen et al.,
1999; Terasawa et al., 1999). Notre laboratoire a également mis en évidence un effet
inhibiteur du GABA chez des rats âgés de 5 ou 15 jours sur la sécrétion pulsatile de GnRH in
vitro, à partir d’explants hypothalamiques, lorsque l’on utilisait de la bicuculline ou des
oligonucléotides anti-sens. Cet effet disparaissait avec l’âge et l’apparition de la puberté, et
requérait l’activation du récepteur GABAA puisque le blocage de ce dernier par la bicuculline
ou par un oligonucléotide anti-sens induisait une augmentation de la fréquence de sécrétion
pulsatile de GnRH in vitro (Bourguignon et al., 1997). Ces résultats ont été confirmés tout
récemment par l’équipe de Terasawa qui a montré que la bicuculline stimule fortement la
sécrétion de kisspeptine, qui augmente la sécrétion de GnRH (Terasawa et al., 2010). Les
effets opposés du GABA pourraient aussi s’expliquer par les différents sous-types de
récepteurs impliqués. Ainsi, l’effet direct du GABA, modulé par le récepteur de type GABAA,
22
Introduction
est stimulant pour certains neurones du SNC (Smith et al., 1995). Contrairement à ces effets,
ceux qui le sont par le récepteur GABAB, sur la sécrétion de GnRH, semblent inhibiteurs de
façon univoque (Akema et al., 1990; Akema et Kimura, 1993; Lagrange et al., 1995). Par
ailleurs, certaines études ont montré une diminution du contenu en GABA avant la puberté au
niveau de l’aire préoptique chez le rat et de l’éminence médiane chez le singe, ce qui suggère
que les taux élevés de GABA chez l’animal immature pourraient contribuer à limiter la
sécrétion de GnRH avant le début de la puberté (Goroll et al., 1993; Mitsushima et al., 1994).
- Les effets stimulants de la noradrénaline sur la sécrétion de LH ont été rapportés chez le
singe (Bhattacharya et al., 1972) et chez le rat (Kalra et al., 1972). Des expériences in vivo
chez le singe ont également montré un effet stimulant de la noradrénaline sur la sécrétion de
GnRH (Terasawa et al., 1988) via une activation du récepteur α1-adrénergique (Kaufman et
al., 1985; Gore et Terasawa, 1991; Terasawa et al., 1998). L’hypothèse selon laquelle la
noradrénaline serait impliquée dans la puberté et le cycle oestral fut émise par Weiner et
Ganong (1971). Elle repose sur l’observation de la diminution du contenu hypothalamique en
noradrénaline et du retard de l’OV après le traitement de rats femelles par un agent bloquant
la synthèse des monoamines. Bien que les neurones à GnRH expriment les récepteurs αadrénergiques (Hosny et Jennes, 1998), les études d’Ojeda et son équipe (1982) tendent plutôt
à démontrer que la noradrénaline agit via la sécrétion d’autres facteurs tels que la PGE2, qui
est également un facteur stimulant la libération de la GnRH (Ojeda et al., 1979; Gearing et
Terasawa, 1991). Ces effets ont également été observés dans notre modèle d’explants
hypothalamiques (Parent et al., 2005). Chez le primate, comme chez le rongeur, la
noradrénaline serait donc impliquée dans le contrôle de la sécrétion de GnRH au cours du
développement, mais elle ne semble pas jouer un rôle majeur dans l’initiation de la puberté.
- La neurokinine B (NKB): ce peptide appartenant à la famille des tachykinines est, à l’instar
d’autres cotransmetteurs, exprimé par certains neurones à kisspeptine (Rometo et Rance,
2008). De très récentes études chez l’Homme ont montré que la NKB et son récepteur NK3R,
couplé aux protéines G et induisant l’activation de la phospholipase C, pouvaient être
impliqués dans le déclenchement de la puberté. En effet, des mutations au niveau des gènes
TAC3 et TACR3, codant respectivement pour la NKB et le NK3R, provoquent un
hypogonadisme hypogonadotrope idiopathique normosmique (Topaloglu et al., 2001). La
NKB pourrait donc jouer un rôle majeur de concert avec la kisspeptine et d’autres
neurotransmetteurs, dont le mécanisme d’action n’a pas encore été élucidé, dans l’activation
du générateur de pulsatilité de la GnRH permettant la sécrétion du peptide hypothalamique au
niveau de l’éminence médiane (Topaloglu, 2010).
23
Introduction
- La kisspeptine, originellement identifiée comme suppresseur des métastases, joue un rôle
crucial dans la gouvernance de l’établissement de la puberté et la fonction reproductrice chez
l’adulte (Seminara et al., 2003; Seminara et Kaiser, 2005; de Roux, 2006; Kauffman et al.,
2007). Des mutations inactivant le GPR54 induisent un hypogonadisme hypogonadotrope
chez l’humain. Les souris «null», atteintes de cette même pathologie, ne présentent pas de
maturation des organes reproducteurs, menant à une infertilité. Ce nouveau rôle du système
kisspeptine/GPR54 a mené à des études, chez le singe Rhésus, qui ont permis d’identifier les
ARNm du récepteur et de son ligand dans l’hypothalamus médio-basal, colocalisés avec les
neurones à GnRH, démontrant ainsi une activité dépendante de la sécrétion des
gonadotrophines FSH et LH par la kisspeptine (Plant, 2006). Ces études ont également montré
un taux croissant des transcrits de GPR54 et kiss-1 au moment de la puberté. L’ensemble de
ces données indique ainsi un rôle direct de la kisspeptine dans la stimulation des neurones à
GnRH durant la puberté (Kuohung et Kaiser, 2006). L’activation de GPR54, par
administration de kisspeptine, est suffisante pour activer précocement l’axe gonadotrope chez
les rongeurs et singes immatures. La kisspeptine hypothalamique fonctionnerait donc comme
un intégrateur essentiel pour les influx périphériques, incluant les stéroïdes gonadiques et les
signaux nutritionnels, en contrôlant la sécrétion de GnRH et des gonadotrophines (TenaSempere, 2006). Par ailleurs, une administration centrale ou intraveineuse (périphérique)
aigüe de kisspeptine à des hommes sains augmente de façon marquante le niveau de LH
plasmatique et significativement celui de la FSH plasmatique et de la testostérone. Ainsi, la
signalisation kisspeptine/GPR54 a été suggérée pour contrôler la cyclicité ovarienne à travers
la régulation de deux modes de sécrétion de la GnRH: d’une part, une population de neurones
à kisspeptine, localisée dans le noyau arqué, pourrait être impliquée dans la génération de
«pulses» de GnRH et la modulation de l’action du rétrocontrôle négatif des oestrogènes sur la
sécrétion de GnRH; d’autre part, une autre population de neurones à kisspeptine, localisée
dans le noyau périventriculaire antéro-ventral, déclencherait la sécrétion de GnRH (Maeda et
al., 2005). Le mécanisme précis par lequel la kisspeptine régule ces deux modes de sécrétion
de GnRH reste encore à déterminer.
- L’ocytocine fait partie des neuromodulateurs impliqués dans la maturation de l’axe
hypothalamo-hypophyso-gonadique. L’ARNm codant pour l’ocytocine est détecté dans
l’hypothalamus de rat à partir du 16ème ou 17ème jour de vie foetale (Almazan et al., 1989;
Laurent et al., 1989) alors que sa forme mature n’apparaît que 4 à 5 jours plus tard, au
moment de la naissance. Le taux d’ARNm codant pour l’ocytocine continue ensuite à
augmenter pendant les premières semaines de vie post-natale. Les récepteurs de l’ocytocine
24
Introduction
apparaissent au niveau du cerveau au cours de la fin de vie foetale et du début de la vie postnatale (Barberis et Tribollet, 1996), et le système ne connaît son organisation définitive que
quelques semaines après le sevrage, un certain nombre de récepteurs apparaissant au moment
de la puberté. Le système ocytocinergique se met donc en place assez tardivement au cours de
la vie foetale et continue d’évoluer au cours du développement. Un effet stimulant de
l’ocytocine sur la libération de GnRH a été rapporté chez le rat mâle adulte ainsi que chez la
femelle lors de l’après-midi du proestrus (Rettori et al., 1997; Selvage et Johnston, 2001).
Notre laboratoire a également montré que la sécrétion pulsatile de GnRH in vitro était
stimulée en présence d’ocytocine et de PGE2 chez des rats nouveaux-nés (Parent et al., 2005).
En outre, l’inhibition de l’ocytocine, par un antagoniste de son récepteur, diminue
significativement l’amplitude du pic préovulatoire de LH chez la femme (Evans et al., 2003),
ceci plaidant pour un rôle physiologique de l’ocytocine endogène. Ces observations ont été
confirmées par notre équipe qui a montré que l’administration d’un antagoniste de l’ocytocine
chez des rats femelles prépubères augmentait l’intervalle de pulsatilité (IP) de GnRH in vitro,
suggérant que l’ocytocine endogène restait effective dans le contrôle facilitateur de la
fréquence pulsatile de GnRH in vitro. En outre, l’ocytocine causait une réduction de l’IP de
GnRH in vitro de façon dose-dépendante, prévenue en présence d’un inhibiteur de la synthèse
de la PGE2 tandis que l’utilisation d’un antagoniste de l’ocytocine augmentait l’IP de GnRH.
Par ailleurs, la sécrétion de GnRH in vitro, évoquée par le glutamate, n’était pas influencée
par l’ocytocine, suggérant l’implication d’un autre site de stimulation au sein de
l’hypothalamus. L’ocytocine pourrait ainsi faire partie d’un système de sauvegarde pour
maintenir la sécrétion de GnRH dans certaines conditions défavorables (Parent et al., 2008).
Cependant, il faut noter que les souris «knock-out» pour le gène de l’ocytocine présentent une
fertilité normale (Kimura et al., 1999), ce qui souligne probablement le rôle redondant de
l’ocytocine au sein du système de régulation de la sécrétion de GnRH.
- Les neurones sécrétant le neuropeptide Y (NPY) sont largement distribués dans
l’hypothalamus (MacDonald et al., 1988) et sont principalement impliqués dans les
mécanismes régulant la balance énergétique et l’appétit (Kalra et al., 1999). Il a été démontré
que le NPY avait un effet stimulant sur la sécrétion de LH au moment du pic préovulatoire
(Brann et al., 1991; Kalra et al., 1992). Cependant, l’effet de ce neuropeptide sur la sécrétion
de GnRH et son implication dans l’installation de la puberté restent discutés. En effet, on lui
attribue des effets stimulants ou inhibiteurs sur la sécrétion de GnRH selon l’espèce étudiée,
l’environnement stéroïdien, la voie ou la durée d’administration (Kalra et Crowley, 1984;
Khorram et al., 1987; Kalra et al., 1992; Minami et Sarkar, 1992; Besecke et Levine, 1994;
25
Introduction
Pau et al., 1995; Pierroz et al., 1995). Pour l’équipe de Plant, qui a étudié son rôle chez le
singe, ce peptide est responsable d’un effet inhibiteur majeur chez l’animal prépubère et cette
inhibition diminue avec l’apparition de la puberté (Majdoubi et al., 2000). Pour d’autres, il est
aussi impliqué dans le déclenchement de la puberté (Sutton et al., 1988; Minami et al., 1990)
alors que certains observent un effet inhibiteur du NPY sur la maturation sexuelle dans des
conditions d’insuffisance nutritionnelle (Gruaz et al., 1993; Pierroz et al., 1995). Les travaux
menés au sein du laboratoire ont montré que le NPY stimule la fréquence de la sécrétion
pulsatile de GnRH in vitro via des médiateurs différents de ceux mis en jeu par la leptine
(Lebrethon et al., 2000). Toutes ces données indiquent donc que le NPY peut jouer un rôle
dans l’apparition de la puberté, mais qu’il doit être intégré avec l’ensemble des
neuromédiateurs impliqués à la fois dans la régulation de l’appétit et la balance énergétique
d’une part, et la reproduction d’autre part. Une altération de la balance énergétique peut
entraîner des perturbations de la fonction de reproduction (Badger et al., 1985; Cagampang et
al., 1990) ainsi qu’un retard de puberté (Kennedy et Mitra, 1963; Foster et Olster, 1985) par
inhibition de la sécrétion de LH.
- Au cours de ces dernières décennies, il a été démontré que les cellules gliales pouvaient
intervenir dans la fonction neuronale via la production de signaux tels que l’augmentation des
taux de calcium intracellulaire suite à une stimulation au glutamate (Cornell-Bell et al., 1990),
la sécrétion directe de glutamate (Parpura et al., 1994) ou celle de substances gliales
spécifiques telles que les facteurs de croissance qui peuvent réguler la sécrétion de GnRH et
qui agissent via des récepteurs de type tyrosine kinase (bFGF et TGFα) ou sérine-thréonine
kinase (TGFβ et PGE2):
- le bFGF stimule la prolifération de neurones à GnRH immortalisés (Voigt et al.,
1996) et maintient leur survie (Tsai et al., 1995). A l’inverse, il peut stimuler indirectement
les cellules gliales (Gallo et al., 2000).
- le TGFα agit de façon indirecte sur la sécrétion de GnRH (Ojeda et al., 1990) via la
libération de PGE2 (Ma et al., 1997). Il joue également un rôle dans le déclenchement de la
puberté puisque la surexpression de son gène au niveau de l’hypothalamus murin provoque
l’initiation précoce du cycle oestral (Ma et al., 1994; Rage et al., 1997). Par ailleurs, le TGFα
pourrait aussi être impliqué dans l’émergence de la puberté chez l’Homme puisque les
hamartomes (malformations hypothalamiques responsables de l’apparition de puberté
précoce) expriment fortement le TGFα, par l’intermédiaire des cellules gliales (Jung et al.,
1999).
26
Introduction
- le TGFβ, produit par les cellules astrocytaires, stimule la sécrétion de GnRH in vitro
(Gonzalez-Manchon et al., 1991; Melcangi et al., 1995; Calogero et al., 1998). Ses récepteurs,
situés sur le péricaryon (Ojeda et al., 1990), sont exprimés par des neurones à GnRH
immortalisés (Buchanan et al., 2000) ainsi que par des neurones à GnRH de rat (Prevot et al.,
2000; Bouret et al., 2004). Les effets in vivo du TGFβ sur les neurones à GnRH se limitent
uniquement à une action stimulante sur les transcrits de GnRH et non sur la sécrétion de cette
dernière (Bouret et al., 2004).
- L’inhibition de la PEP et de l’EP 24.15 augmentent la sécrétion des gonadotrophines chez le
rat (Lasdun et Orlowski, 1990; Wu et al., 1997). Des études in vitro menées au sein du
laboratoire ont montré que ces deux endopeptidases inhibent la sécrétion de GnRH via la
GnRH1-5 qui bloque l’activation des récepteurs au NMDA (Bourguignon et al., 1994).
Contrairement à l’EP 24.15, dont l’expression ne varie pas au cours du développement
(Pierotti et al., 1991), une variation de l’activité de la PEP au cours du développement est
observée, avec un maximun d’activité durant les deux premières semaines de vie post-natale
(Kato et al., 1980; Yamanaka et al., 1999). La réduction de la production de la GnRH1-5
pourrait donc permettre une augmentation de l’activation des récepteurs au NMDA et, à ce
titre, participer à l’accélération développementale de la fréquence de sécrétion pulsatile de
GnRH observée in vitro au moment de la puberté (Yamanaka et al., 1999).
- Dès les années ‘70, il est apparu que la PGE2 était libérée par l’éminence médiane sous
l’effet de la noradrénaline et de la dopamine, et stimulait la libération de GnRH (Ojeda et al.,
1979). L’injection systémique ou intracérébrale d’un inhibiteur de la synthèse de ces
prostaglandines induit une diminution de la sécrétion de LH et suggère ainsi un rôle
physiologique de ces hormones (Ojeda et al., 1999). Plus récemment, il a été observé que la
PGE2, sécrétée par les cellules gliales, module l’effet facilitateur du TGFα et des neurégulines
sur la sécrétion de GnRH. En outre, les systèmes glutamatergiques et la PGE2 sont interdépendants puisque le glutamate stimule la libération de PGE2 qui, à son tour, induit une
libération de glutamate astrocytaire (Bezzi et al., 1998). La PGE2, synthétisée au niveau
central et dans l’utérus, a été décrite comme un stimulateur potentiel de la sécrétion de GnRH
(MacDonald et Wilkinson, 1990). De nombreuses études lui suggèrent un rôle dans
l’éminence médiane où elle est sécrétée par les astrocytes en réponse au TGFα (MeijsRoelofs et al., 1991). Par ailleurs, la PGE2 semble agir davantage sur les terminaisons
nerveuses des neurones à GnRH que sur le générateur de pulsatilité lui-même, ce qui pourrait
expliquer l’absence d’augmentation de la fréquence pulsatile de GnRH en présence de PGE2
27
Introduction
dans notre modèle d’incubation hypothalamique. Enfin, les effets combinés de la PGE2 et de
l’ocytocine suggèrent que la modulation de la fréquence pulsatile de GnRH requiert différents
facteurs (Parent et al., 2005).
- Le NO est un neurotransmetteur gazeux dont le rôle, dans le contrôle de la formation de la
paroi vasculaire, a été découvert à la fin des années ‘70 (Gruetter et al., 1979). Il est produit à
partir de l’arginine grâce à la NO synthase (NOS) qui existe sous différentes formes et peut
être détectée au niveau neuronal et endothélial (Forstermann et al., 1991). Des expériences in
vitro ont montré que le NO était capable de stimuler la sécrétion de GnRH à partir d’explants
d’éminence médiane (Rettori et al., 1993). Ce médiateur a aussi été impliqué dans la survenue
du pic préovulatoire de GnRH chez le rat (Prevot et al., 1999). La proximité anatomique de
cellules immunopositives pour la NOS neuronale et les neurones à GnRH plaident en faveur
d’une implication du NO dans la régulation de l’activité des corps cellulaires du neurone à
GnRH (Herbison et al., 1996). Par ailleurs, il a été démontré que la sécrétion pulsatile du NO
varie au cours du cycle oestral avec, le jour du proestrus, une augmentation de l’amplitude qui
s’accompagne d’une augmentation de la libération de GnRH (Knauf et al., 2001; Clasadonte
et al., 2008).
2. Facteurs périphériques
- La leptine: découverte il y a une quinzaine d’années, il s’agit d’une protéine de 167 acides
aminés sécrétée essentiellement par le tissu adipeux (Zhang et al., 1994). Son inactivation se
traduit par une obésité, une hyperphagie et un hypogonadisme avec tous les troubles qui s’en
suivent. Chez l’Homme comme chez les rongeurs, la concentration plasmatique et le taux de
transcrits de leptine sont étroitement liés avec le degré de la masse adipeuse (Maffei et al.,
1995; Considine et al., 1996). De par sa forte concentration au niveau du plexus choroïde (Fei
et al., 1997), le récepteur à la leptine, qui présente des homologies avec les membres de la
famille des récepteurs aux cytokines de classe I (Tartaglia, 1997), pourrait participer au
transport de la leptine dans le SNC. Bien que des récepteurs à la leptine aient été localisés sur
des neurones à GnRH immortalisés (Magni et al., 1999), des études immunocytochimiques
chez le rat (Hakansson et al., 1998) et le singe (Finn et al., 1998) n’ont pas mis en évidence ce
type de récepteur sur les neurones à GnRH, suggérant que des neurotransmetteurs
intermédiaires interviennent dans l’action de la protéine (Bouret et al., 2004). La leptine
exerce une partie de ces effets dans la régulation de l’homéostasie énergétique en agissant sur
les neurones orexigéniques (Cheung et al., 1997; Mercer et al., 1997; Kask et al., 1998). En
outre, il a également été montré que la leptine joue un rôle important dans la maturation et la
28
Introduction
fonction de l’axe reproducteur (Magni et al., 2000). En effet, l’administration intrapéritonéale
de leptine recombinante humaine à des souris homozygotes ob/ob (dont le gène codant pour la
leptine a été muté) permet d’assurer la maturation de l’axe hypothalamo-hypophysogonadique (Chehab et al., 1996), s’accompagnant, quel que soit le sexe, d’une augmentation
des concentrations plasmatiques de FSH et LH (Barash et al., 1996) et d’une cyclicité oestrale
normale chez les femelles (Ahima et al., 1996). Par ailleurs, des injections de leptine à des
souris prépubères nourries ad libitum provoquent une avancée de l’âge de la puberté (Chehab
et al., 1997). Enfin, une augmentation des taux plasmatiques de la leptine a été observée avant
le début de la puberté chez le singe (Suter et al., 2000).
- En 1999, Kojima et son équipe ont isolé la ghréline, une hormone peptidique orexigène de
28 acides aminés sécrétée par l’estomac, qui joue un rôle important dans l’initiation et la
régulation de la prise alimentaire (Horvath et al., 2001; Olszewski et al., 2003). La
concentration plasmatique de ghréline chez l’Homme augmente de façon très significative
avant chaque repas et diminue rapidement ensuite (Cummings et al., 2001). Chez le rongeur,
l’administration chronique périphérique de ghréline provoque une augmentation du poids
corporel en réduisant le métabolisme lipidique (Tschop et al., 2000). En dehors du tractus
gastro-intestinal, la ghréline est exprimée au niveau d’un type de neurones adjacents au 3ème
ventricule, entre les noyaux arqué, paraventriculaire, dorso-médian et ventro-médian
hypothalamiques (Cowley et al., 2003). Ces neurones envoient des afférences dans des
circuits hypothalamiques importants pour la régulation de la prise alimentaire. Ainsi, la
ghréline agit sur des neurones déjà sensibles à la leptine (Traebert et al., 2002). Plusieurs
études expérimentales ont permis de démontrer que les effets orexigènes de la ghréline
augmentent l’expression hypothalamique des gènes codant pour le NPY chez le rat (Kamegai
et al., 2001) et bloquent les effets anorexigènes de la leptine (Shintani et al., 2001). Il a
également été suggéré que la ghréline peut jouer un rôle physiologique dans la fonction de
reproduction via son action sur la sécrétion pulsatile de LH. Un certain nombre de données
établissent, en effet, une action inhibitrice de l’hormone sur la sécrétion pulsatile de LH chez
des rates ovariectomisées et traitées par l’E2 (Furuta et al., 2001), ou non ovariectomisées
(Fernandez-Fernandez et al., 2005). En outre, l’administration périphérique et chronique de
ghréline chez le singe ovariectomisé diminue la sécrétion pulsatile de LH (Vulliémoz et al.,
2004). Nous avons également démontré, au sein de notre laboratoire, qu’une administration
intrapéritonéale de ghréline chez des rats âgés de 15 et 50 jours augmente significativement
l’IP de GnRH, à partir des explants hypothalamiques incubés in vitro. Lorsque les explants
hypothalamiques sont directement incubés en présence de ghréline, une réduction de l’IP a
29
Introduction
lieu à 15 jours et cet effet est complètement inhibé en présence d’un antagoniste du récepteur
au NPY. Ceci suggère que les effets opposés, in vivo et in vitro, de la ghréline requièrent la
présence du récepteur au NPY, et qu’elle agit avant et après la maturité sexuelle (Lebrethon et
al., 2007). L’ensemble des données reprises ci-dessus indique donc un rôle de la ghréline dans
la régulation de l’axe hypophyso-gonadique, et plus particulièrement en cas de balance
énergétique négative.
- L’«insuline growth factor 1», facteur de croissance ressemblant à l’insuline de type 1 (IGF1), impliqué dans la régulation de la sécrétion de GnRH, stimule, via des récepteurs de type
tyrosine kinase, la libération de GnRH à partir d’éminences médianes issues d’explants
hypothalamiques in vitro et entraîne une puberté précoce quand il est administré à des rats
immatures (Hiney et al., 1991; Hiney et al., 1996). Ses effets pourraient s’exercer directement
au niveau des neurones à GnRH depuis qu’il a été rapporté l’expression de trois types de
récepteurs à l’insuline sur des neurones à GnRH immortalisés (Oison et al., 1995). Par contre,
d’autres études ont montré que l’IGF-1 n’est pas nécessaire à la maturation sexuelle du rat
femelle et qu’un excès d’IGF-1 circulant n’a pas d’effet sur l’installation de la puberté (Gruaz
et al., 1997). D’autre part, des travaux réalisés au sein de notre laboratoire ont mis en
évidence un effet inhibiteur de l’IGF-1 sur la sécrétion pulsatile de GnRH in vitro chez des
rats âgés de 25 et 50 jours. En effet, nous avons montré que l’IGF-1 pouvait également
diminuer la sécrétion de GnRH via un produit de dégradation du peptide, l’IGF-1(1-3), qui
inhibe la sécrétion de GnRH induite par une stimulation des récepteurs au NMDA
(Bourguignon et al., 1993). Par conséquent, les effets régulateurs de l’IGF-1 sur la sécrétion
de GnRH pourraient être directs ou indirects.
Les facteurs périphériques gonadiques (stéroïdes et inhibine) ne seront pas discutés dans cette
partie de l’introduction.
30
Introduction
II. Mise en situation du problème
II. MISE EN SITUATION DU PROBLEME
Résumé: Dans ce second chapitre, nous expliquerons en quoi la différenciation sexuelle et la
programmation du système reproducteur sont influencées par les stéroïdes sexuels et les
perturbateurs endocriniens (PEs). Au travers d’exemples dramatiques tels que les cancers et des
troubles de la reproduction causés par le diéthylstilbestrol, nous démontrerons que la perturbation
endocrinienne concerne l’Homme et la faune. L’analyse est complexe en raison des différents facteurs
(nature, demi-vie, dose-réponse, …) qui interviennent dans le processus perturbateur. La nature et les
effets généraux du dichlorodiphényltrichloroéthane (DDT), que nous avons précisément étudié, seront
davantage décrits. Nous expliquerons également la raison pour laquelle les modifications de timing
pubertaire dans la population générale mais aussi les pubertés précoces anormalement fréquentes
chez les enfants migrants, ont soulevé la question de l’effet des PEs. En outre, nous résumerons les
études mettant en relation les PEs et le timing pubertaire chez l’Homme ainsi que celles qui
concernent plus particulièrement le DDT. Enfin, nous détaillerons l’étude clinique menée en Belgique
chez des enfants de l’adoption internationale qui est, précisément, à l’origine de notre travail.
A. Différenciation sexuelle, programmation foetale et perturbation
endocrinienne
Le concept classique de la différenciation sexuelle suggère que le cerveau mâle se
développe sous l’influence des sécrétions testiculaires (testostérone aromatisée en oestradiol)
tandis que, chez la femelle, aucune stimulation hormonale n’est requise dans ce processus
(Feder et Whalen, 1965; Grady et al., 1965; Plapinger et McEwen, 1973; Lamprecht et al.,
1976; Baum, 1979; Booth, 1979; Dörner, 1981; Jacobson et Gorski, 1981). De par sa
localisation au sein de l’hypothalamus (Wagner et Morrell, 1997; Roselli et al., 1998;
Naftolin et al., 2001), l’aromatase, nécessaire à la synthèse de l’oestradiol à partir de la
testostérone, est directement impliquée dans la différenciation sexuelle du cerveau du rat
mâle, chez lequel une augmentation de son activité enzymatique est observée durant les
premiers jours post-natals (Tsuruo et al., 1994; Roselli et al., 1998; Amateau et al., 2004).
Plusieurs études réalisées sur des souris ArKO, dont le gène codant pour l’aromatase est
déficient, ont cependant permis d’émettre l’hypothèse d’un possible rôle de l’oestradiol dans
le développement neuronal contrôlant le comportement sexuel chez la femelle (Bakker et al.,
2002 et 2003). Par ailleurs, Bakker et ses collègues ont montré qu’une exposition prénatale
aux oestrogènes masculinise et déféminise le cerveau, l’AFP protégeant le cerveau femelle
des effets des stéroïdes sexuels (2006). Il a également été observé qu’une exposition du foetus
31
Mise en situation du problème
ovin en présence de testostérone provoque une altération du timing pubertaire et de la
cyclicité oestrale par la perturbation neuroendocrinienne du rétrocontrôle positif de
l’oestradiol (Unsworth et al., 2005), ainsi qu’une diminution du nombre de neurones à GnRH
présentant un marquage positif pour la protéine c-Fos en réponse à l’E2 (Wood et al., 1996).
Au cours du développement des rongeurs, les stéroïdes jouent un rôle permissif dans
l’initiation de la puberté puisque de très anciennes expériences montrèrent déjà que des
extraits placentaires induisent un avancement de la puberté (Frank et al., 1925) et que
l’administration d’E2 chez des rats femelles immatures provoque l’apparition précoce du
cycle oestral (Ramirez et Sawyer, 1965). Il y a plus de 25 ans, Ojeda et son équipe ont montré
que l’hypothalamus immature est nettement plus sensible à des facteurs stimulant la sécrétion
de GnRH, et ce de façon oestrogéno-dépendante (Ojeda et al., 1986), démontrant ainsi que les
oestrogènes jouent un rôle précoce dans l’accélération de la maturation hypothalamique
conduisant à la puberté. Plusieurs arguments cliniques indiquent une entrée en puberté plus
rapide chez les filles que chez les garçons, le déterminisme de cette différence étant précoce
puisqu’elle persiste, chez le singe, après castration néonatale (Plant, 1994). En outre, chez les
ovins, l’administration prénatale de testostérone chez la femelle entraîne un retard du
déclenchement pubertaire, ce qui suggère l’importante influence de la vie intra-utérine à cet
égard (Kosut et al., 1997).
La majorité des études relatant les effets des perturbateurs endocriniens (PEs) ont été
réalisées suite à leur exposition durant la gestation ou chez le nouveau-né, ces deux périodes
étant critiques pour la détermination des effets des stéroïdes sexuels sur l’homéostasie de la
reproduction (McCarthy et al., 2009). Les évènements neuroendocriniens affectés par les PEs
in vivo incluent l’établissement de la puberté centrale femelle, notamment gonadotrophinesdépendante (Gore, 2008) et l’ovulation déclenchée par le pic des gonadotrophines
(Svabieasfahani et al., 2006; Steinberg et al., 2008), ainsi que le comportement sexuel chez
les mâles (Viglietti-Panzica et al., 2005) et les femelles (Patisaul et al., 2001; Funabashi et al.,
2003; Rubin et al., 2006; Steinberg et al., 2007), ce qui démontre que ces processus seraient
donc régulés par les stéroïdes sexuels et potentiellement perturbés par les PEs de façon
différente chez les deux genres.
B. Historique de la découverte des PEs et questions actuelles
La notion de PE est apparue à la fin du 20ème siècle pour désigner toute molécule
exogène capable d’interférer avec la synthèse, la sécrétion, le transport, la liaison, l’action ou
l’élimination des hormones naturelles, qui contrôlent l’homéostasie, la reproduction, le
32
Mise en situation du problème
développement et le comportement (Kavlock et al., 1996). Selon l’Organisation Mondiale de
la Santé (O.M.S.), les PEs sont des substances chimiques, biologiques ou physiques d’origine
naturelle ou artificielle étrangères à l’organisme qui peuvent interférer, à très faible dose (du
même ordre de grandeur que les concentrations physiologiques des hormones), avec le
fonctionnement du système endocrinien et induire (en mimant ou inhibant l’action des
hormones endogènes, ou en modulant leur transport et/ou la synthèse hormonale) ainsi des
effets délétères sur cet organisme ou sur ses descendants.
L’un des premiers PEs identifié est le diéthylstilbestrol (DES), qui a défrayé la
chronique il y a plusieurs décennies, après avoir été prescrit massivement et à hautes doses à
des femmes enceintes pour traiter des fausses couches entre 1938 et 1971 aux Etats-Unis. Les
traitements par cette molécule oestrogénique, non stéroïdienne et synthétique, ont provoqué
des perturbations endocriniennes chez les mères, mais ont également causé, chez leurs filles,
des troubles de la reproduction et, dès l’âge de 30 ou 40 ans, des cancers du vagin et du col de
l’utérus (Herbst et al., 1971; Herbst et Anderson, 1990). Le DES a également été utilisé
comme anabolisant en élevage, provoquant des désordres hormonaux chez des enfants ayant
consommé des résidus de ce composé présents dans des denrées animales à des quantités
anormalement élevées. Bien que le DES n'ait plus été prescrit depuis plus de 30 ans, ses effets
continuent à être observés: en effet, les femmes qui y ont été exposées par l’intermédiaire de
leur mère affichent aujourd’hui des anomalies du tractus reproducteur et un taux d'infertilité
accru (Merino, 1991; Schrager et Potier, 2004), sans évoquer des effets trans-générationnels
qui peuvent aussi impliquer la descendance. Le DES, de part sa nature oestrogénique, est
encore régulièrement utilisé dans de nombreuses études expérimentales parce que considéré
comme référence afin de comparer et étudier les effets d'autres substances potentiellement
oestrogéniques.
Dans de nombreux pays industrialisés, des évidences d’effets anti-androgéniques et
oestrogéniques respectivement sont davantage observées: d’une part la diminution de la
fertilité masculine (altérations morphologiques et baisse de la production de spermatozoïdes)
et l’augmentation de la fréquence de cancers des testicules et de la prostate (Toppari et al.,
1996; Skakkebaek et al., 2001), d’autre part la fréquence accrue de cancers du sein (Snedeker,
2001) et le déclenchement pubertaire féminin de plus en plus précoce (Proos et al., 1991;
Bourguignon et al., 1992; Bridges et al., 1994; Herman-Giddens et al., 1997; Virdis et al.,
1998; Lee et al., 2001; Aksglaede et al., 2009; Roelants et al., 2009). Des études
épidémiologiques d’abord, des expériences en laboratoire ensuite, ont montré que l'exposition
33
Mise en situation du problème
à des molécules hormono-mimétiques est, au moins en partie, responsable de ces
phénomènes.
Par ailleurs, des études rapportaient également des effets des PEs à des doses
d’exposition environnementales sur la faune sauvage. Au début des années ’60 déjà, la baisse
de fertilité des visons d’Amérique du Nord, constatée par les éleveurs de la région des Grands
Lacs, fut attribuée aux polluants bio-accumulés par les poissons et ingérés sous forme de
farines
animales.
En
1962,
Carson
mis
en
évidence
la
toxicité
du
dichlorodiphényltrichloroéthane (DDT), un puissant insecticide abondamment utilisé à cette
époque, sur l’appareil reproducteur des oiseaux. Par la suite, d’autres études, notamment la
découverte de l'altération des fonctions reproductrices des alligators sauvages de Floride,
relanceront les travaux de recherche sur ce thème au début des années ‘90. Il s’ensuivra alors
de nombreuses publications soulignant le danger de certaines substances chimiques sur la
reproduction et le développement, aussi bien chez l’Homme que sur la faune sauvage (Carlsen
et al., 1992; Colborn et Clément, 1992).
A travers la méthylation de l’ADN et l’acétylation des protéines histones, l’épigénome
contrôle systématiquement l’expression des gènes durant le développement, à la fois in utero
et durant la vie post-natale. De par sa nature labile, l’épigénome est très sensible aux
perturbations environnementales durant la vie prénatale, ce qui peut conduire à de nombreux
états pathologiques tels que des dysfonctionnements métaboliques, des désordres de la
balance énergétique, des fonctions thyroïdiennes et reproductrices ainsi que des risques accrus
de cancers. En effet, des conséquences néfastes trans-générationnelles, suite à l’exposition à
des PEs, ont été constatées aussi bien dans la faune sauvage que sur des animaux de
laboratoire ainsi que chez l’Homme, ce qui suggère la façon dont la transmission de ces
modifications épigénétiques peuvent être sujettes à la sélection des espèces (Crews et
McLachlan, 2006): l’exposition à ces substances hormono-mimétiques peut profondément
altérer la physiologie des organismes et, ultérieurement, avoir un impact nuisible significatif
sur des populations entières, ce phénomène non génomique pouvant prendre des dizaines
d’années avant que l’effet ne se manifeste sur les caractères phénotypiques (Gore, 2008;
Patisaul et Adewale, 2009; Bernal et Jirtle, 2010; Walker et Gore, 2011).
Parmi les nombreux paramètres qui peuvent influencer la réponse aux PEs, la nature
de ces derniers occupe une place de choix: naturelle ou synthétique, oestrogénique ou antiandrogénique, végétale, insecticides, fongicides ou herbicides, organochlorés ou biphényls
polychlorés (PCBs)/-bromés (PBBs), dioxines, dérivés phénoliques, phtalates, métaux lourds,
… La demi-vie des PEs varie selon leur structure chimique et le milieu dans lequel ils se
34
Mise en situation du problème
trouvent. Une tendance à l’augmentation de leur demi-vie a été observée au fil des décennies:
Carlson et ses collègues (2010) viennent de démontrer, après avoir analysé des données issues
d’un intervalle de 34 ans, que la plupart des contaminants des Grands Lacs américains
présentent des changements significatifs d’un point de vue de leur nature et de leur propriété
physico-chimiques. Pour exemple, les demi-vies des PCBs étaient de 3-6 ans jusqu’à la moitié
des années ’80, et des récentes données montrent qu’elles se situent actuellement aux
alentours de 15-30 ans. Des changements dans la structure de l’alimentation, de la dynamique
des pêches et des climats pourraient être à l’origine de ces modifications. Par ailleurs, le type
de milieu (oxydant ou réducteur, aqueux ou huileux, …) dans lequel sont véhiculés les PEs
joue également un rôle important dans le maintien ou non de leur structure et propriété
chimiques (Ying et al., 2003; Ying et Kookana, 2005).
Plusieurs études rapportent que l’exposition, au cours du développement, à des
substances oestrogéniques à de très faibles doses résulte en une courbe dose-réponse non
monotone, en forme de U. Pour exemple, chez des souris mâles exposées in utero à l’E2, au
DES ou au bisphénol A (BPA), le poids de la prostate augmente fortement à de très faibles
doses, et de façon moins marquée en présence de doses plus élevées (Nagel et al., 1997; vom
Saal et al., 1997). En revanche, d’autres études significativement plus robustes ne recouvrent
pas cette tendance non monotone (Ashby et al., 1999; Cagen et al., 1999a et 1999b; Chapin et
al., 1999; Kwon et al., 2000; Tyl et al., 2002). Lorsque, néanmoins, ce genre de courbe doseréponse en forme de U est obtenu, faut-il encore pouvoir déterminer si les effets observés à
faibles doses ont des conséquences néfastes à long terme. En effet, Putz et ses collègues
(2001a et 2001b) ont rapporté une évolution non monotone transitoire des poids de la prostate,
des testicules et de l’épididyme chez des rats nouveau-nés exposés à des faibles doses d’E2,
l’effet étant attribuable à l’accélération de l’établissement de la puberté. Il est donc également
primordial de connaître les éventuelles prédispositions à une pathologie, ce qui est cependant
très difficile lorsque l’on utilise des modèles d’animaux.
Dans son environnement quotidien, l’Homme et plus généralement l’ensemble du
règne animal est probablement exposé à une grande variété de PEs, d’origine et de nature
différente ou non, agissant en mixtures (Kolpin et al., 2002). Bien que plusieurs études in vivo
et in vitro indiquent généralement que les effets de ces mixtures soient additifs (Gray et al.,
2001; Payne et al., 2001; Payne et al., 2002; Silva et al., 2002; Hotchkiss et al., 2004; Brian et
al., 2005; Gray et al., 2006; Howdeshell et al., 2007; Rider et al., 2008), il semble qu’ils le
sont davantage, à savoir synergiques, puisque des composés mélangés à des concentrations
inactives si testés seuls deviennent actifs lorsqu’ils sont mélangés sous forme de mixtures
35
Mise en situation du problème
(Kortenkamp, 2008). Dans les études où une seule classe de PEs a été testée, on observe que
ces derniers agissent soit comme des agonistes des oestrogènes, soit comme des antagonistes
des androgènes, le rapport oestrogène/androgène étant le déterminant de l’effet final (Rivas et
al., 2002). Le type d’administration des substances, la durée et l’âge à leur exposition sont
d’autres facteurs qui influencent aussi les effets des PEs.
C. Exemple du DDT
1. Structure et propriétés
Le DDT est un insecticide organochloré qui, à température et pression normales, se
présente sous forme de solide incolore hydrophobe. Synthétisé la première fois par Othmar
Zeidler en 1874, le DDT fut produit massivement au début de la seconde guerre mondiale par
Paul Hermann Müller et utilisé avec beaucoup de succès dans la lutte contre les moustiques
transmettant le paludisme et le typhus ainsi que d'autres insectes vecteurs de maladies ou
affectant les récoltes agricoles. Il faudra attendre les publications de Carson, rapportant que le
DDT était cancérigène et empêchait la reproduction des oiseaux en amincissant la coquille de
leurs œufs (1962), pour que l’utilisation de ce pesticide soit interdite dans les pays
industrialisés au début des années ‘70. Entre-temps, après avoir constaté le déclin des
balbuzards et autres grands oiseaux aux alentours de la rivière de Carman (1967), Cooley
supposa un lien avec l'utilisation du DDT et lança alors une grande campagne nationale à
travers les Etats-Unis contre son utilisation. Au cours des années ‘70 et ‘80, le DDT a été
remplacé par des produits moins persistants et plus chers, mais de nos jours, il reste toujours
très utilisé dans les pays tropicaux et/ou en voie de développement où l’O.M.S. le
recommande en l’absence d’alternative pour lutter contre la mortalité due à la malaria. La
contamination par le DDT continue via la consommation de nourriture (Key et Reeves, 1994;
Kelce et al., 1995; Clark et al., 1998; Partsch et Sippel, 2001; Parent et al., 2003).
Outre le principal isomère p,p’-DDT (Fig. 4) qui représente approximativement 80 %
de la répartition de l’insecticide dans l’environnement, il existe également l’isomère o,p’DDT (Fig. 4) où l'un des atomes de chlore est déplacé autour du cycle benzénique en position
ortho. Le DDT se comporte comme un agoniste des oestrogènes et/ou un antagoniste des
androgènes par l’intermédiaire du sous-produit dichlorodiphénylchloroéthylène (DDE), son
principal produit de décomposition. La demi-vie du DDT, qui se fixe et s’accumule dans de
nombreux sols via les eaux de ruissellement et de percolation, est évaluée entre 2 et 15 ans: il
s’agit donc d’un polluant organique persistant dont les lents processus de dégradation incluent
36
Mise en situation du problème
la volatilisation et la biodégradation aérobie et anaérobie. Par contre, rapidement
apidement dégradé par
une exposition à la lumière (dont l’effet est inhibé par les substances humiques) et une
augmentation du pH (Quan et al.,
al., 2005), le DDT est transformé en DDE, dont l’isomère p,p’p,p’
DDE (Fig. 4) est biologiquement
ment plus actif (demi-vie
(demi
de 5-77 ans) que son congénère o,p’o,p’
DDE (Fig. 4).. Cependant, lorsque le DDT est répandu de façon intensive et chronique sur le
sol, la concentration du DDE peut persister de façon inchangée pendant plus de 20 ans
(Thomas et al., 2008).
Figure 4: Représentations semi-développées
développées des principaux isomères du DDT et du DDE.
Ce puissant pesticide tue les moustiques et autres insectes cibles en ouvrant les canaux
sodiques de leurs neurones,, ce qui les détruit instantanément, conduisant à des spasmes, puis à
la mort. Bien que l’o,p’-DDT
DDT ne soit pas l’isomère prédominant dans les préparations de DDT
D
utilisées comme insecticide, nous l’avons privilégié pour nos recherches in vivo pour diverses
raisons: celui-ci
ci a été le plus utilisé dans les travaux antérieurs (Gellert et al.,
al. 1974; Gladen et
al., 2000; Diel et al.,, 2002; Tomiyama et al., 2003; Denham et al.,, 2005; Ouyang et al., 2005)
et nous permet ainsi des comparaisons. En outre, il est plus oestrogénique et moins toxique
que le p,p’-DDT (Heinrichs et al.,
al. 1971; Wrenn et al., 1971; Gellert et al.,, 1972; Faber et al.,
1991). Enfin, dans notre expérience
rience préalable in vitro, l’o,p’-DDT
DDT s’est avéré plus rapidement
actif que le p,p’-DDT, dès 1-22 h d’incubation
d’incubat
et à des doses plus faibles.
37
Mise en situation du problème
2. Effets chez l’Homme
Avant les années 2000, peu d’études prouvaient que le DDT était toxique et pouvait
avoir un effet néfaste chez les humains. Des études ont conclu que les taux tissulaires de DDT
et DDE étaient plus élevés chez des malades atteints de cancer que pour ceux mourant
d’autres maladies (Dacre et Jennings, 1970; Wasserman et al., 1976) mais selon d’autres
auteurs, aucune relation de la sorte n’a pu être établie (Maier-Bode, 1960; Robinson et al.,
1965; Hoffman et al., 1967). En 1993, Wolf a montré une corrélation significative entre la
concentration des métabolites du DDT dans le sang et les risques de développer le cancer du
sein chez la femme. Dans une cohorte de pêcheurs du Michigan, l'exposition au DDT de
mères ingérant du poisson a été mesurée et l’âge à la puberté a été déterminé chez 151 filles:
l’exposition in utero à des taux élevés de DDE est associée avec un âge à la ménarche avancé
(Vasiliu et al., 2004). Chez des enfants de Caroline du Nord (316 filles et 278 garçons),
aucune association significative entre une exposition au DDE et l’âge à la puberté n'a été
trouvée. De même, aucun effet n’a été observé chez des garçons à la suite d’une exposition in
utero au DDE (Gladen et al., 2000). En 2002 et 2003, 1196 paires mères-enfants ont
systématiquement été recrutées dans 25 maternités à travers la Flandre: le taux de p,p’-DDE
dans le sang de cordon ombilical ou plasmatique était mesurable dans presque tous les
échantillons prélevés (Koppen et al., 2009). En Chine, une corrélation significative entre
l’ingestion quotidienne et la concentration de DDT et ses métabolites dans le lait maternel de
deux populations de Pékin et Shenyang a été déterminée: une tendance temporelle démontre
une diminution du taux sérique de DDT parallèlement à une augmentation du rapport
DDE/DDT (Tao et al., 2008). Récemment, Eskenazi et ses collègues (2009) ont rédigé une
synthèse de 494 études publiées de 2003 à 2008 se focalisant sur les effets du DDT chez
l’Homme, et ont rapporté que les restrictions d’utilisation du pesticide au cours des dernières
décennies sont observables du point de vue de la diminution d’exposition humaine au DDT.
Cependant, les concentrations sanguines de DDT et DDE étaient élevées chez les individus
issus de pays où le DDT reste encore largement utilisé ou a seulement récemment été interdit.
En outre, ils ont également constaté que l’exposition au DDT et/ou au DDE est associée à des
effets néfastes tels que le cancer du sein, un risque accru de diabète, la diminution de la
qualité du sperme et l’affectation du neuro-développement chez les enfants.
3. Effets d’insecticides chez les rongeurs
Peu d’études expérimentales ont investigué les effets du DDT sur le système
reproducteur des rongeurs. Mussi et son équipe (2005) ont observé une augmentation des taux
38
Mise en situation du problème
des ERs dans le cerveau de souris mâles adultes qui ont été exposées à l’insecticide. D’autres
ont montré que lorsque des souris en gestation (11-17èmes jours) sont nourries avec de l’o,p’DDT ou du méthoxychlor (MXC), les progénitures présentent une distance ano-génitale
diminuée à la naissance (Palanza et al., 2001). Le MXC a été développé pour remplacer le
DDT tout en gardant un spectre d’action similaire. Comparé au DDT, il est plus facilement
excrété et s’accumule moins dans les sols (Kapoor et al., 1997). Par l’intermédiaire de son
produit métabolite, l’hydroxyphényltrichloroéthane, il stimule l’activité oestrogénique en se
liant aux ERs intracellulaires (Bulger et al., 1978a et 1978b). A la fin des années ’80, Gray Jr
et ses collègues (1988 et 1989) ont rapporté que le MXC, administré durant le sevrage, cause
un avancement de l’âge à l’OV et au premier oestrus. Une décennie plus tard, il a été montré
qu’une exposition durant 3 jours post-natals (JPN 21-23) au MXC cause aussi un avancement
de l’âge à l’OV ainsi qu’une diminution du nombre de cycle oestral régulier (Laws et al.,
2000). L’équipe de Gray avait également observé que la longueur des cycles oestraux est
augmentée jusqu’à ce qu’un oestrus permanent soit observé, après une administration de
MXC (Gray Jr et al., 1988; Gray Jr et al., 1989). Ingéré oralement ou injecté en sous-cutané
(JPN 21-23), le MXC provoque une augmentation du poids utérin (Odum et al., 1997; Laws
et al., 2000). A noter également que le MXC réduit le taux des transcrits de GnRH
indépendamment des ERs, mais stimule la sécrétion de GnRH modulée par les ERs sur la
lignée GT1-7 de neurones à GnRH immortalisés (Roy et al., 1999; Gore, 2002).
Largement utilisé dans les pays développés, le lindane (hexachlorocyclohexane) est
très persistant dans l’environnement et s’accumule tout au long de la chaîne alimentaire. Un
traitement en présence de ce pesticide est suivi de périodes d’oestrus ou de dioestrus
permanents. Lorsqu’il est administré aux JPN 21-110 ou 125, l’âge à l’OV est retardé (Cooper
et al., 1989). Dans la même étude, il a également été observé que le lindane réduit le volume
de l’hypophyse, induit un taux élevé de FSH et faible de LH, et cause une diminution du poids
utérin ainsi qu’une perturbation du cycle ovarien.
D. Relations entre exposition pré/post-natale aux PEs et timing pubertaire
chez l’Homme
Tandis que la majorité des PEs étudiés chez les filles induisent une programmation
normale ou précoce (Tableau 2), les dioxines et les phytoestrogènes causent un retard du
développement des seins (Den Hond et al., 2002; Leijs et al., 2008; Wolff et al., 2008).
Cependant, les auteurs de ces études ne constatèrent pas que l’âge à la ménarche était affecté
39
Mise en situation du problème
par ces deux types de substances (Den Hond et al., 2002; Leijs et al., 2008), constat qui est
confirmé dans d’autres études (Strom et al., 2001; Warner et al., 2004). En revanche, la
distinction entre une programmation normale de la thélarche et une ménarche précoce a été
rapportée suite à une exposition aux PBBs, soulignant l’importance d’étudier des signes
impliquant probablement plusieurs mécanismes à différentes périodes du processus pubertaire
(Blanck et al., 2000). A l’exception d’une étude (Denham et al., 2005) mentionnant une
ménarche précoce après exposition aux PCBs, ces derniers ont été majoritairement associés à
une période normale de développement des seins et d’apparition des règles (Gladen et al.,
2000; Den Hond et al., 2002; Vasiliu et al., 2004; Yang et al., 2005; Wolff et al., 2008). Une
thélarche (Krstevska-Konstantinova et al., 2001) et une ménarche (Vasiliu et al., 2004;
Ouyang et al., 2005) précoces ont été rapportées à la suite d’une exposition au DDT et/ou au
DDE tandis que d’autres auteurs ont rapporté une période pubertaire normale (Gladen et al.,
2000; Denham et al., 2005; Wolff et al., 2008). Chez les guenons, Golub et ses collègues
(2003) ont observé une croissance retardée des tétons et une courte phase folliculaire après
l’exposition au MXC. Prises ensembles, toutes les études précitées ne permettent pas de
démontrer significativement des effets différents selon la période d’exposition pré- ou postnatale aux PEs.
Quelques études (Tableau 3) suggèrent qu’il n’y a pas d’effet du DDT ou du DDE, ni
des dioxines (Gladen et al., 2000; Den Hond et al., 2002) chez les garçons. Quant à
l’exposition aux PCBs, elle est associée à une programmation normale (Gladen et al., 2000;
Mol et al., 2002) ou retardée (Den Hond et al., 2002; Guo et al., 2004) du développement
pubertaire mâle. Il est à noter qu’aucune situation de puberté avancée n’est rapportée chez les
garçons, à l’inverse des filles.
Des données plus abondantes ont été obtenues chez les filles (Tableau 2) que chez les
garçons (Tableau 3), ce qui pourrait impliquer des erreurs méthodologiques puisqu’un signe
précis de maturation est fourni par l’âge à la ménarche chez les filles qui expérimentent plus
sérieusement l’établissement de la puberté avec le développement des seins, à l’opposé d’une
augmentation moins perceptible du volume testiculaire chez les garçons (Parent et al., 2003).
40
Mise en situation du problème
Tableau 2: Variations de la période pubertaire chez la jeune fille
en fonction d’une exposition pré- et/ou post-natale aux PEs.
Période
pubertaire
Exposition
DDE
(+ DDT)
Précoce
Prénatale
Post-natale
Ménarche
(Vasiliu
et al., 2004)
Ménarche
(Ouyang
et al., 2005)
B2 (KrstevskaKonstantinova et al., 2001)
Normale
Prénatale
Post-natale
Ménarche (Denham
et al., 2005)
et
B2 (Wolff
et al., 2008)
Ménarche et B3
(Gladen et al., 2000)
Retardée
Prénatale
Singe
(Golub et
al., 2003)
MXC
PBBs
Post-natale
Ménarche
(Blanck et al., 2000)
PCBs
Ménarche
(Denham
et al., 2005)
Dioxines
Phtalates
B2
(Blanck et al., 2000)
Ménarche
Ménarche et B2
(Vasiliu
(Den Hond
et al., 2004;
et al., 2002);
Yang
B2 (Wolff
et al., 2005)
et al., 2008)
Ménarche et B3
(Gladen et al., 2000)
Ménarche
(Leijs
Ménarche
et al., 2008;
(Den Hond
Warner
et al., 2002)
et al., 2004)
B2
(Leijs et
al., 2008)
B2
(Den
Hond et
al., 2002)
B2
(Colon
et al., 2000)
B2
(Wolff et
al., 2008)
DDE: dichlorodiphénylchloroéthylène; DDT: dichlorodiphényltrichloroéthane; MXC: méthoxychlor; PBBs:
biphényls polybromés; PCBs: biphényls polychlorés; B2-B3: stades 2 et 3 de Tanner du développement des
seins.
Phytoestrogènes
Ménarche
(Strom et al., 2001)
41
Mise en situation du problème
Tableau 3: Variations de la période pubertaire chez le jeune garçon
en fonction d’une exposition pré- et/ou post-natale aux PEs.
Période
pubertaire
Exposition
DDE
(+ DDT)
Précoce
Prénatale
Post-natale
Normale
Prénatale
Post-natale
Retardée
Prénatale
Post-natale
G3-G5 et VPT
(Gladen et al., 2000)
G3-G5 et VPT
Longueur
(Gladen et al., 2000)
P et G
du pénis
(Den
Hond
G
et
VT
PCBs
(Guo
et al., 2002)
(Mol
et al., 2004)
et al., 2002)
P et G
(Den Hond
Dioxines
et al., 2002)
DDE: dichlorodiphénylchloroéthylène; DDT: dichlorodiphényltrichloroéthane; PCBs: biphényls polychlorés; G:
stade de Tanner du développement génital; P: stade de Tanner du développement de pilosité pubienne; VPT:
vélocité du pic de taille; VT: volume testiculaire.
Dans le cadre d’une étude clinique menée par le Docteur Marina KrstevskaKonstantinova (2001) au sein de notre unité de recherche, l’hypothèse selon laquelle la
migration d’un pays étranger sous-développé vers la Belgique (pays occidental industrialisé)
pourrait résulter en un changement d’exposition aux PEs, et par conséquent causer une
précocité sexuelle, fut émise. Le sérum de 145 enfants migrants (135 filles et 10 garçons), pris
en charge pour une puberté précoce et traités par des agonistes de la GnRH en Belgique, fut
collecté pour la détection de huit pesticides organochlorés. Les analyses réalisées par le
Professeur Corinne Charlier révélèrent la présence de p,p’-DDE, un métabolite et marqueur
d’exposition au p,p’-DDT (insecticide encore largement utilisé dans les pays en voie de
développement), à une concentration sérique 10 fois plus élevée que le seuil de détection chez
18 % (26 enfants: 15 adoptés et 11 non adoptés) des patients migrants, tandis que le taux de ce
résidu était en dessous de cette limite chez 13 des 15 patients natifs Belges (KrstevskaKonstantinova et al., 2001). Par ailleurs, d’une part, le taux de p,p’-DDE chez ces enfants
migrants était d’autant plus élevé qu’ils avaient séjourné longtemps dans leur pays d’origine
et, d’autre part, ce taux diminuait de façon d’autant plus significative que le temps écoulé
depuis leur arrivée en Belgique était important (Parent et al., 2003). Ces observations nous
amenèrent à notre modèle expérimental d’incubation statique d’explants hypothalamiques de
jeunes rats femelles pour étudier l’influence des PEs dans l’apparition de la puberté centrale et
sur le système de reproduction: les travaux réalisés par le Docteur Valérie Matagne ont
montré que l’E2 entraîne une accélération de la fréquence de sécrétion pulsatile de GnRH
uniquement chez les rates immatures de 5 et 15 jours (2004), via un mécanisme inhérent à la
différenciation sexuelle périnatale du cerveau. Il était ainsi prouvé que l’E2 stimule
42
Mise en situation du problème
rapidement une augmentation de la fréquence pulsatile de GnRH chez le rat femelle
immature,
ure, contribuant ainsi à l’apparition d’une précocité sexuelle.
Comme illustré dans la Fig. 5,
5, l’hypothèse selon laquelle la maturation
hypothalamique pourrait être stimulée tandis que les gonadotrophines hypophysaires seraient
inhibées via un effet de rétrocontrôle
contrôle négatif durant l’exposition à un signal environnemental
oestrogénique, a été émise. Ces évènements préviendraient toute manifestation de maturation
centrale et seule la puberté périphérique
pér
pourrait être alors observée. La migration causerait
un retrait des PEs et l’inhibition hypophysaire conséquente disparaîtrait, permettant la
maturation hypothalamique pour entraîner la cascade hypophyso-ovarienne
hypophyso ovarienne et induire la
puberté centrale.
Figure 5: Représentation schématique du mécanisme potentiel de précocité sexuelle après une exposition
transitoire à l’insecticide oestrogénique DDT chez des jeunes filles migrantes issues de l’adoption internationale.
Durant l'exposition au DDT, la maturation neuroendocrinienne
neuroendocrinienne est déclenchée mais non traduite par une
stimulation ovarienne,
ne, à cause du rétrocontrôle
rétrocontrôle négatif (inhibiteur) actionné par les gonadotrophines
hypophysaires. Après migration dans un environnement
enviro
exempt de DDT, le rétrocontrôle
contrôle inhibiteur disparaît
rapidement et la maturation centrale est exprimée, menant à la puberté centrale.
43
Mise en situation du problème
E. Implication des facteurs environnementaux, autres que les PEs, dans la
puberté précoce
Rappelons d’abord que les critères internationaux retenus pour définir une puberté
précoce chez les jeunes filles sont l’apparition des seins (thélarche) avant l’âge de 8 ans et/ou
des règles (ménarche) avant celui de 10 ans. Il y a une trentaine d’années, Frisch et McArthur
ont suggéré qu’une masse graisseuse critique doit être atteinte pour permettre l’apparition des
premières règles (1974). Par la suite, chez des adolescentes anorexiques (Pugliese et al., 1983)
ainsi que chez des gymnastes soumises à un entraînement intensif avec des contraintes
diététiques (Theintz, 1994), la fonction hypothalamo-hypophyso-ovarienne était inhibée,
renforçant l’hypothèse selon laquelle le régime alimentaire influence le timing pubertaire. En
ce qui concerne une éventuelle influence saisonnière, quelques études ont démontré que la
ménarche débute plus fréquemment en hiver qu’en été chez les filles normales (Bojlen et
Bentzon, 1974; Albright et al., 1990; Cohen, 1993), suggérant un effet inhibiteur de
l’exposition à la lumière. Pourtant, d’autres auteurs ont indiqué que, dans les zones arctiques,
les mois sombres d’hiver sont associés à une fonction hypophyso-gonadique réduite et un
faible taux de conception (Rojansky et al., 1992). En outre, il est également probable que la
privation psycho-sociale puisse aussi jouer un rôle dans ces conditions particulières (Dominé
et al., 2006).
Plusieurs études ont mis en lumière une incidence élevée de pubertés précoces parmi
des enfants migrants, dont ceux issus d’une adoption internationale, dans différents pays
d’Europe occidentale, parmi lesquels la Suède, le Danemark, les Pays-Bas, l’Italie, la France
et la Belgique (Proos et al., 1991). Le risque que ces enfants développent une puberté précoce
a été estimé 15 à 20 fois supérieur par rapport à leurs congénères autochtones au Danemark
(Teilmann et al., 2006; Aksglaede et al., 2009) et même jusqu’à 80 fois en Belgique (Roelants
et al., 2009). Il y a une dizaine d’années déjà, deux études américaines de grande ampleur ont
également fourni la preuve d’un établissement précoce de la puberté (Herman-Giddens et al.,
1997; Lee et al., 2001). Des signes précurseurs tels que l’âge à la thélarche sont davantage
affectés que celui à la ménarche. Les évènements pubertaires initiaux ont tendance à
apparaître plus précocement tandis que les signes finaux semblent être observés à des âges
plus élevés (Papadimitriou et al., 2008; Roelants et al., 2009). Puisque ces changements
pubertaires sont concomitants avec l’épidémie d’obésité observée aux Etats-Unis, l’hypothèse
d’une implication pathophysiologique de la masse graisseuse, probablement via l’action de la
leptine, due à des évolutions sanitaire et nutritionnelle/alimentaire amplifiées par le processus
44
Mise en situation du problème
d’industrialisation, a été émise (Herman-Giddens et al., 1997; Lee et al., 2001; Himes, 2006).
Cependant, les récents changements pubertaires observés au Danemark ne sont pas liés à des
variations du tissu adipeux (Aksglaede et al., 2009), laissant penser que d’autres facteurs tels
les PEs pourraient être impliqués (Teilmann et al., 2002). L’étude clinique menée par
Krstevska-Konstantinova (2001), décrite ci-avant, démontre que le DDT, par le biais de son
métabolite DDE qui présente une très longue demi-vie, peut être un candidat à l’origine de ce
phénomène, mais de nombreux autres PEs peuvent également être impliqués et induire une
précocité pubertaire périphérique.
45
Mise en situation du problème
III. Objectifs du travail
III. OBJECTIFS DU TRAVAIL
Comme décrit précédemment dans l’introduction de ce travail, l’hypothalamus est la
région du cerveau qui, après intégration des messages centraux et périphériques, active le
réseau des neurones à GnRH, dont la sécrétion stimulera le système hypophyso-gonadique.
Cette stimulation mènera au déclenchement de la puberté et à l’installation des fonctions de
reproduction. Ce travail se focalise essentiellement sur les effets oestrogéniques des PEs,
particulièrement ceux du DDT, sur la sécrétion de GnRH, et plus généralement sur l’axe
hypothalamo-hypophysaire et ses conséquences sur la maturation sexuelle chez le rat femelle
immature.
Rappelons brièvement le contexte et la justification de notre travail: des recherches
cliniques portant sur la puberté précoce chez les enfants, essentiellement des filles, de
l’adoption internationale en Belgique avaient mis en évidence, dans le sérum de ces
migrantes, un résidu de l’insecticide DDT, à savoir le DDE (Krstevska-Konstantinova et al.,
2001; Parent et al., 2003) dont les propriétés anti-androgéniques sont établies, reflétant ainsi
indirectement une exposition antérieure au DDT, lui-même oestrogénique. L’élucidation des
effets oestrogéniques versus anti-androgéniques résiderait donc dans la balance additionnelle
des deux composés, mais également dans les concentrations respectives de l’un et l’autre en
fonction du temps après l’exposition (Rasier et al., 2008). Par ailleurs, des travaux réalisés au
sein du laboratoire avaient démontré les effets stimulants de l’E2 sur la sécrétion pulsatile de
GnRH chez le rat femelle immature et précisé certains mécanismes de récepteurs impliqués
dans ces effets (Matagne et al., 2004). L’ensemble de ces données nous a naturellement
conduit à rechercher si, et par quel(s) mécanisme(s), le DDT pouvait influencer la sécrétion de
GnRH ainsi que la maturation de la fonction hypophyso-ovarienne chez le rat femelle.
Les chapitres I et II de ce mémoire résument l’analyse de la littérature et l’hypothèse
de travail sur lesquelles nos recherches se sont construites, et qui ont fait l’objet de deux
publications de revue (Rasier et al., 2006; Bourguignon et al., 2010). Nous avons d’abord
comparé les processus de maturation hypothalamo-hypophyso-gonadique chez l’humain et les
rongeurs, qui sont notre modèle expérimental. Nous avons ensuite distingué les mécanismes
de puberté centrale (à savoir dépendante physiologiquement d’une activation de la sécrétion
de GnRH et des gonadotrophines) et périphérique (non physiologique et indépendante de cette
activation hypothalamo-hypophysaire). En effet, les PEs pourraient agir selon ces deux
modalités, stimulant à la fois les maturations périphérique et hypothalamique. Enfin, nous
46
Objectifs du travail
avons revu les effets des principaux types de PEs sur l’axe hypothalamo-hypophysogonadique, la maturation sexuelle et le tractus reproducteur.
Dans la mesure où les observations cliniques de puberté précoce ont été effectuées
chez des sujets exposés précédemment au PE qu’est le DDT et ensuite soustraits à cette
exposition du fait de leur migration, nous avons postulé que l’insecticide pouvait, par un effet
oestrogénique, stimuler la maturation hypothalamique sans que cet effet ne se traduise au
niveau hypophysaire tant que l’individu restait exposé au PE. Il existe une grande sensibilité
hypophysaire au rétrocontrôle inhibiteur chez l’individu immature et cela pourrait ainsi
expliquer que l’âge pubertaire ne soit pas avancé, voire même qu’il soit retardé chez les sujets
qui restent exposés à ce PE. Remarquons toutefois qu’une telle hypothèse n’a pas été vérifiée
jusqu’ici car nous ne disposons pas de données sur la période pubertaire en rapport avec
l’exposition aux PEs dans les pays d’origine des enfants migrants. Suite à la migration dans
un environnement moins ou non exposant à ce PE, le rétrocontrôle inhibiteur peut diminuer,
laissant alors la maturation avancée des centres neuroendocriniens s’exprimer par une puberté
centrale. L’objectif de notre travail est donc de modéliser, chez le rat femelle, les effets
précoces d’une exposition transitoire au DDT afin de vérifier l’hypothèse que nous venons
d’énoncer.
Dans la première partie (chapitre V) qui rapporte nos travaux personnels, nous avons
étudié, en comparaison avec l’E2, les effets d’une administration de différentes doses de
DDT, transitoire ou prolongée par voie sous-cutanée, chez le rat femelle immature. Dans ces
conditions, l’effet sur la maturation sexuelle in vivo, la sécrétion de LH en réponse à
l’injection de GnRH ainsi que les concentrations sériques des différents isomères et dérivés du
DDT ont été mesurés. L’effet sur la sécrétion hypothalamo-hypophysaire a également été
étudié ex vivo dans les mêmes conditions.
Dans la deuxième partie (chapitre VI) dans laquelle la suite de nos résultats est
rapportée, nous nous sommes attachés à préciser in vitro le(s) mécanisme(s) impliqué(s) dans
les effets neuroendocriniens obtenus précédemment in vivo. Nous avons notamment étudié la
façon dont le DDT et ses dérivés peuvent mimer les effets rapides de l’E2 sur la réponse
sécrétoire de GnRH. Nous y avons également investigué in vitro les types de récepteurs et
le(s) mécanisme(s) d’action impliqué(s) dans les effets des PEs sur la sécrétion de GnRH tant
dans sa forme spontanément pulsatile que sur la réponse déclenchée par le glutamate.
La discussion générale (chapitre VII) intègre nos différentes observations quant à
l’influence des PEs sur la sécrétion de GnRH (fréquence et amplitude). Parmi les aspects qui
donnent matière à discussion, nous envisagerons si les neurones à GnRH sont la cible directe
47
Objectifs du travail
des PEs ou si d’autres neurones, voire les cellules gliales, sont impliqués. La période critique
et les autres paramètres d’exposition seront également discutés. Nous tentons de conclure
quant à l’hypothèse de travail formulée initialement et nous dégagerons la contribution de nos
travaux pour la progression de la compréhension des mécanismes d’action des PEs dans le
déclenchement de la sécrétion de GnRH. Pour terminer, nous évoquerons quelques pistes qui
restent ouvertes pour des recherches ultérieures.
48
Objectifs du travail
IV. Méthodologie
IV. METHODOLOGIE
A. Incubation d’explants hypothalamiques
Après décapitation, les hypothalami rétrochiasmatiques de rats femelles âgés de 1 à 50
jours ont rapidement été prélevés. Pour ce faire, deux incisions sagittales ont d’abord été
effectuées au niveau des sillons hypothalamiques latéraux. Ensuite, une première incision
transversale antérieure a été réalisée en arrière de la limite caudale du chiasma optique, et une
seconde au niveau de la limite antérieure des corps mamillaires. Finalement, une section
frontale oblique, d’avant en arrière et de haut en bas, a été effectuée à 2-3 mm de profondeur,
au niveau de la commissure antérieure. Ces explants ne contiennent, en principe, que des
fibres nerveuses de neurones à GnRH et leurs terminaisons, ces dernières étant localisées dans
l’éminence médiane, ainsi que des cellules gliales. L’aire préoptique n’étant pas prélevée, les
péricaryons de neurones à GnRH ne sont, en principe, pas présents dans ces explants (Fig. 6).
En effet, une étude menée au sein de notre laboratoire a montré, par immuno-histochimie, que
l’explant rétrochiasmatique ne contenait aucun corps cellulaire identifiable de neurone à
GnRH. En outre, une transcription inverse suivie d’une réaction de polymérisation en chaîne
semi-quantitative a démontré que le rapport des ARNm codant pour la GnRH, présents dans
l’explant rétrochiasmatique et l’explant total comprenant l’aire préoptique, était de 1:600
(Purnelle et al., 1997). Un argument supplémentaire repose sur l’étude des récepteurs NMDA:
les neurones à GnRH issus des lignées immortalisées GT1-1 et GT1-7 expriment le récepteur
NMDA (Urbanski et al., 1994; Mahesh et al., 1999), ce qui donne à penser que celui-ci est
encodé dans les péricaryons de ces neurones. Or, des oligonucléotides anti-sens ciblant les
ARNm de la sous-unité 2A des récepteurs NMDA (NR-2A) n’affectaient la sécrétion de
GnRH, en réponse au NMDA, que s’ils étaient incubés avec l’explant total uniquement,
suggérant que ces récepteurs étaient encodés dans les péricaryons présents dans l’aire
préoptique mais absents dans l’explant rétrochiasmatique (Bourguignon et al., 1997). On peut,
dès lors, penser que l’explant étudié ne contient que très peu, voire aucun péricaryon de
neurone à GnRH. Par ailleurs, des études plus récentes ont montré que les stéroïdes sexuels
modulent, de façon directe et localisation-dépendante, l’expression de kiss-1 dans le cerveau
antérieur (Gottsch et al., 2006): ils inhibent son expression dans le noyau arqué, suggérant que
les neurones à kisspeptine servent d’éléments intermédiaires pour réguler le rétrocontrôle
négatif de la sécrétion de GnRH; en revanche, dans le noyau antéro-ventral, les stéroïdes
sexuels stimulent l’expression de kiss-1, ce qui laisse à penser que ces neurones pourraient
49
Méthodologie
jouer un rôle dans le rétrocontrôle positif (pic préovulatoire de LH chez la femelle et
comportement sexuel chez le mâle) dans cette région du cerveau (Smith et al., 2006; Roa et
Tena-Sempere, 2007) non prélevée dans les expériences que nous avons réalisées. On
mentionnera que nous avons étudié les effets du DDT in vitro mais également ex vivo et que
dans ce deuxième cas de figure, toutes les structures intra- et extra-hypothalamiques
potentiellement impliquées ont été exposées au DDT. Le mécanisme d’action qui induit la
sécrétion de GnRH observée dans notre modèle d’incubation d’explants hypothalamiques
rétrochiasmatiques sera discuté dans le chapitre VII de ce travail.
NHA
ERC
CA
NVM
NA
CM
APOM
OVLT
EM
CO
NSC
Figure 6: Représentation schématique d’une coupe sagittale du cerveau de rat, incluse la région
hypothalamique prélevée (encadré vert). APOM: aire préoptique médiane; CA: commissure antérieure; CM:
corps mamillaire; CO: chiasma optique; EM: éminence médiane; ERC: explant rétrochiasmatique; NA: noyau
arqué; NHA: noyau hypothalamique antérieur; NSC: noyau suprachiasmatique; NVM: noyau ventro-médian;
OVLT: organum vasculosum laminae terminalis.
Une fois prélevés, les explants ont été incubés individuellement à l’aide d’un système
statique dans une atmosphère de 95 % en O2 et 5 % en CO2 durant une période de 4 à 6 h.
Quinze explants au maximum, dans chaque expérience, étaient incubés dans 500 µl de milieu
«minimum essential medium» enrichi en glucose, en magnésium et en glycine pour atteindre
des concentrations respectives de 2,5.10-2 M, 10-3 M et 10-8 M. Des travaux antérieurs réalisés
au sein du laboratoire ont montré que ces concentrations constituaient les conditions
optimales pour observer une sécrétion pulsatile de GnRH in vitro. A des concentrations plus
50
Méthodologie
élevées, le magnésium agit comme antagoniste non compétitif des récepteurs NMDA et
inhibe la sécrétion de GnRH, aussi bien que l’absence de glycine. En effet, cette dernière est
requise comme agoniste d’un site non allostérique de celui du glutamate sur ces mêmes
récepteurs.
Comme déjà mentionné dans l’introduction de ce travail, un rétrocontrôle inhibiteur de
la sécrétion de GnRH a été observé dans notre modèle expérimental. Il est modulé par la
GnRH1-5, produit de dégradation de la GnRH par la prolyl-endopeptidase hypothalamique
(Yamanaka et al., 1999). C’est la raison pour laquelle le milieu a également été enrichi en
bacitracine (2.10-5 M), un inhibiteur de l’enzyme précitée. Le milieu a ensuite été prélevé et
renouvelé toutes les 7,5 min, et les échantillons ont été congelés à -20°C jusqu’à ce que la
concentration de GnRH soit mesurée dans chaque fraction à l’aide d’un dosage radioimmunologique ultra-sensible (Fig. 7).
95 % d’O2
+
5 % de CO2
MEM
+ glucose (25 mM)
+ Mg2+ (1 mM)
+ glycine (10 nM)
+ bacitracine (20 mM)
Explant
hypothalamique
H2O à 37°C
Milieu renouvelé
toutes les 7,5 min
Stockage à -20°C
jusqu’à dosage
radio-immunologique
des échantillons
Figure 7: Représentation schématique de l’incubation des explants hypothalamiques et du prélèvement du
milieu contenant la GnRH.
Ce modèle d’incubation d’explants hypothalamiques permet d’étudier deux paramètres
de la sécrétion de GnRH in vitro. Le premier est la sécrétion pulsatile spontanée qui permet de
quantifier le niveau basal de sécrétion de GnRH (bien qu’il soit régulièrement inférieur au
seuil de détection, à savoir 5 pg/fraction de 7,5 min) ainsi que la fréquence et l’amplitude des
à-coups sécrétoires de GnRH (Bourguignon et Franchimont, 1984). Ces différentes
composantes sécrétoires peuvent être étudiées en conditions contrôles et en présence d’agents
divers (oestrogéniques, antagonistes ou inhibiteurs dans le cadre de ce travail) maintenus dans
le milieu d’incubation pendant la durée totale de l’expérience (Fig. 8). Dans des études
réalisées antérieurement au sein du laboratoire, il s’est avéré que la fréquence était le plus
51
Méthodologie
souvent affectée par l’âge de l’animal, entre la naissance et 25 jours (Matagne et al., 2004).
Ainsi, ce paramètre est le plus important dans nos travaux et se décline en terme d’IP. En
revanche, le niveau de sécrétion basale et l’amplitude des à-coups sécrétoires varient
rarement, à l’exception des explants étudiés à différentes phases du cycle oestral: l’amplitude
est augmentée l’après-midi du proestrus (Parent et al., 2000). Le second paramètre, la réponse
sécrétoire induite, est mesurable grâce à la capacité qu’a l’éminence médiane de répondre à
des sécrétagogues (Fig. 8), à savoir le glutamate pour ce travail (Bourguignon et al., 1995;
Purnelle et al., 1997). Cet acide aminé, utilisé à une concentration de 10-2 M, a été ajouté au
milieu d’incubation pendant 7,5 min toutes les 37,5 min. Nos travaux antérieurs ont montré
que la concentration de glutamate requise était très élevée, comme dans les autres études
menées avec des explants où les concentrations nécessaires sont supérieures à celles utilisées
en culture monocellulaire de neurones à GnRH (Mahachoklertwattana et al., 1994; Spergel et
al., 1995). Nos conditions posent donc la question des effets potentiellement toxiques de cet
acide aminé excitateur: la concentration utilisée est considérée comme non toxique
puisqu’une réponse sécrétoire de GnRH similaire peut être obtenue et maintenue inchangée
durant une période de 4 à 6 h d’incubation (Bourguignon et al., 1989b; Matagne et al., 2005).
Afin d’éviter le biais de la libération initiale de substances diverses dans le milieu, y compris
la GnRH, secondairement à la dissection de l’explant et le biais du rétrocontrôle inhibiteur qui
peut en résulter, les explants sont incubés en MEM seul pendant 30 min avant la première
stimulation par le glutamate.
Figure 8: Représentation de profils de sécrétion pulsatile spontanée (gauche) et de réponse sécrétoire de
GnRH induite par du glutamate (droite) à partir d’incubation d’explants hypothalamiques de rats âgés de 15
jours.
52
Méthodologie
B. Dosage de la GnRH
Le dosage radio-immunologique est une méthode précise d’analyse compétitive basée
sur la réaction de deux antigènes (Ag) identiques, l’un étant marqué par un radio-isotope, avec
leur anti-sérum (Berson et Yalow, 1968). Précisément, le dosage de la GnRH (Dluzen et
Ramirez, 1981) repose sur la compétition entre la GnRH marquée (GnRH* ou traceur) à l’125I
par la méthode de Greenwood et la GnRH non marquée (GnRH° ou hormone froide) pour
l’anticorps (Ac) hautement spécifique CR11-B81 (dilution finale 1:80000) généreusement
fourni par le Dr V.D. Ramirez (Urbana, IL, U.S.A.). La réaction peut être représentée comme
suit:
GnRH* + GnRH° + Ac
GnRH*Ac + GnRH°Ac
Lorsque la GnRH* et l’Ac sont ajoutés en quantité connue et constante, la GnRH° détermine
la quantité de GnRH*Ac. Une courbe standard représentant la relation entre le pourcentage de
GnRH*Ac en fonction de la concentration en GnRH° ajoutée dans le milieu est établie. Pour
ce dosage, la gamme de concentrations s’étend de 0,5 à 125 pg par 100 µl de milieu.
La séparation de la GnRH* et du complexe GnRH*Ac est réalisée par immunoprécipitation. Le principe consiste à précipiter le complexe Ag-Ac par l’ajout, au milieu, d’Ac
anti-γ-globulines dirigés contre les γ-globulines de l’animal utilisé pour produire l’Ac
primaire. L’Ac polyclonal, pour sa part, a été produit chez le lapin et sa précipitation a été
obtenue par un Ac anti-γ-globuline de lapin préparé chez le mouton (Ac secondaire).
Brièvement, le dosage de la GnRH se déroule en cinq étapes:
- pré-incubation pendant 16-18 h à 4°C de 100 µl de milieu en présence de l’Ac primaire;
- incubation pendant 16-18 h à 4°C en présence de la GnRH* et du sérum (dilution 1:100) de
lapin normal;
- précipitation du complexe Ag-Ac par immuno-précipitation (polyéthylène glycol 6000 + Ac
secondaire, dilution 1:200) pendant 20 min à température ambiante;
- centrifugation (3300 tours/min) des échantillons pendant 20 min à 4°C;
- décantation des échantillons et comptage du précipité au moyen d’un compteur γ.
Deux mesures ont été réalisées à partir de chaque échantillon. Les coefficients de variation
intra- et inter-«assay» sont respectivement de 14 % et 18 % (Bourguignon et al., 1989a;
Bourguignon et al., 1989b). Le seuil de détection est de 5 pg/fraction de 500 µl prélevée
toutes les 7,5 min.
53
Méthodologie
C. Dosage de la LH
Les concentrations sériques de LH ont été mesurées en conditions basales et après
stimulation à la GnRH. L’intervalle de 30 min a été choisi sur base d’études rapportées par
Ramaley (1982) et Tena-Sempere et al. (2004), qui indiquaient un pic de sécrétion de LH
après cet intervalle.
Après une période de 2 h d’incubation à température ordinaire pour permettre la
coagulation, le sang collecté est centrifugé durant 5 min à 2000 g. Le sérum est alors récupéré
et la concentration de LH mesurée en double dans un volume de 100 µl selon une méthode
utilisant un double Ac et des réactifs généreusement fournis par l’Institut National de la Santé
(N.I.H., U.S.A). La LH1-8 de rat a été marquée à l’125I par la méthode de la chloramine-T. Les
coefficients de variation intra- et inter-«assay» étaient respectivement de 7 % et 9 %, et le
seuil de détection de 5 ng/ml de sérum.
D. Dosage des isomères du DDT et ses métabolites
Cette partie expérimentale du travail a été réalisée en collaboration avec le Laboratoire
de Toxicologie Clinique et Environnementale du Professeur Corinne Charlier au Centre
Hospitalier Universitaire de Liège. L’identification et la quantification des isomères o,p’-DDT
et p,p’-DDT ainsi que de leurs molécules dérivées (o,p’-DDE et p,p’-DDE) sériques ont été
réalisées au moyen d’un chromatographe en phase gazeuse couplé à un spectromètre de
masse. La préparation des échantillons a nécessité une extraction liquide-liquide suivie d’une
extraction en phase solide. L’éluat était évaporé jusqu’à sec, reconstitué avec de l’hexane et
injecté dans le chromatographe. Les échantillons standards de référence des quatre isomères
ont été fournis par la firme Dr Ehrenstorfer (Augsburg, Allemagne) et l’aldrine a été utilisée
comme standard interne. La limite de quantification valait 10 fois la déviation standard des
résultats de l’échantillon blanc, à savoir 0,5 ppb pour tous les isomères. Les coefficients de
variation étaient respectivement de 7,1 %, 7,7 %, 6,1 % et 8,2 % pour l’o,p’-DDT, le p,p’DDT, l’o,p’-DDE et le p,p’-DDE. La limite de détection était de 1 ng/ml de sérum (Charlier et
Plomteux, 2002). Etant donné la distribution logarithme-normale des valeurs mesurées, les
changements de concentrations sériques d’o,p’-DDT ont été comparés après une
transformation logarithmique, puis la moyenne géométrique a été calculée et les données ont
été présentées selon une échelle logarithmique.
54
Méthodologie
E. Détermination des phases du cycle oestral
Les animaux étaient examinés quotidiennement pour constater la perforation de la
membrane vaginale et déterminer ainsi l’âge à l’OV. Une fois cet évènement observé, des
frottis vaginaux étaient réalisés l’après-midi de chaque jour jusqu’au JPN 60. En collaboration
avec le Département d’Anatomie et de Pathologie du Professeur Jacques Boniver au Centre
Hospitalier Universitaire de Liège, les coupes sur lesquelles avaient été étalés ces frottis
vaginaux, étaient colorées au moyen de la méthode Papanicolaou afin de déterminer la
périodicité du cycle oestral. L’âge au premier oestrus était déterminé lorsque les frottis
vaginaux présentaient des cellules cornées suivant une phase pro-oestrale, cette dernière étant
caractérisée à la fois par la présence de cellules stratifiées et cornées (Ojeda et Urbanski,
1994).
F. Justification du choix de l’o,p’-DDT
Lorsque les explants hypothalamiques étaient incubés in vitro en conditions contrôles,
l’IP de GnRH ne variait pas avec le temps (Fig. 9). Durant une incubation continue de 4 h en
présence d’E2 à 10-7 M, l’IP de GnRH était significativement réduit après 1–2 h et davantage
après 3–4 h. Cet effet dépendait directement de la concentration d’E2 et de son temps
d’incubation: à la concentration de 10-8 M d’E2, l’IP restait inchangé après 1–2 h et diminuait
significativement après 3–4 h; à 10-9 M, l’E2 entraînait une réduction significative après 5–6 h
seulement. Les deux isomères actifs du DDT induisaient également une réduction de l’IP de
GnRH, directement dépendante de la concentration et du temps d’incubation, qui était
significative après 3–4 h à 10-5 M. A une concentration de 10-4 M, les deux isomères
montraient un effet davantage précoce et qui était également plus important après 3–4 h. Testé
à 10-6 M, le p,p’-DDT n’avait pas d’effet durant une incubation de 6 h tandis que l’o,p’-DDT
causait un effet déjà significatif, mais seulement après 5–6 h. Ce résultat significatif obtenu
avec l’isomère o,p’-DDT a justifié son utilisation dans toutes les expériences réalisées en
présence de l’insecticide. Aucun effet n’était observé avec le p,p’-DDE à des concentrations
similaires (Fig. 9). Les données disponibles dans la littérature avaient souvent été obtenues
avec l’o,p’-DDT et nous donnaient un argument supplémentaire pour l’utilisation de cet
isomère afin de pouvoir intégrer nos observations avec celles effectuées antérieurement.
55
Méthodologie
70
1-2 h d’incubation
3-4 h d’incubation
IP de GnRH (min)
65
5-6 h d’incubation
60
a
b
55
a
b
50
a
b
a
b
a
a
b
a
a
a
b
a
b
45
40
10-9
CONTRÔLE
10-8 10-7
E2
10-6 10-5 10-4
o,p’-DDT
a
b
10-6 10-5 10-4
p,p’-DDT
10-6 10-5 10-4 M
p,p’-DDE
Figure 9: Effets de l’E2 (n=8-13), des isomères du DDT (n=4-6) et du p,p’-DDE (n=5-9) in vitro sur la
fréquence de sécrétion pulsatile de GnRH (n=19 pour les contrôles) à partir d’explants hypothalamiques obtenus
chez des rats femelles âgés de 15 jours. Les données sont calculées en fonction du temps (2 ou 3 périodes de 2 h
consécutives) d’incubation in vitro. a: p<0,05 versus contrôle; b: p<0,05 versus données obtenues durant les 2
premières heures d’incubation.
G. Statistiques
La survenue d’à-coups sécrétoires significatifs de GnRH a été mesurée au moyen du
logiciel PULSAR (Merriam et Wachter, 1982; Bourguignon et al., 1987). L’IP de GnRH a été
calculé comme étant le temps entre deux à-coups sécrétoires successifs de GnRH durant une
période d’incubation de 2 h. Selon que les distributions étaient normales ou pas, les périodes
d’incubation ont été respectivement comparées selon le test apparié t de Student ou le test
apparié de Wilcoxon. Une différence d’effet était considérée comme significative lorsque
p<0,05. Dans les cas où tous les explants d’une même condition présentaient une valeur
similaire, la déviation standard était nulle et n’était donc pas représentée.
La réponse sécrétoire (pg/fraction de 7,5 min) a été calculée comme la différence entre
la quantité de GnRH sécrétée au moment de l’à-coup et celle obtenue immédiatement
auparavant. Si cette valeur se trouvait sous la limite de détection, elle était assimilée à celle-ci
pour le calcul. Les données ont été rassemblées et analysées selon le test «one-way» ANOVA
si la distribution était normale, suivi d’un post-test à comparaison multiple de Newman-Keuls.
Si les données n’étaient pas distribuées de façon normale, le test de Kruskal-Wallis était
utilisé, suivi d’un post-test à multiples comparaisons de Dunn (effet significatif lorsque
p<0,05 pour les deux distributions). Afin de comparer les résultats obtenus dans toutes les
56
Méthodologie
expériences, les valeurs absolues ont été transformées en pourcentages de la réponse
sécrétoire de GnRH observée dans le contrôle et considérée comme 100 %. Un niveau de
signification identique a été obtenu avec les deux formes de données, à savoir les valeurs
absolues et les pourcentages.
57
Méthodologie
V. Maturation avancée de la sécrétion
de GnRH et précocité sexuelle
après exposition de rats femelles
immatures à l’E2 ou au DDT
V. MATURATION AVANCEE DE LA SECRETION DE GnRH
ET PRECOCITE SEXUELLE APRES EXPOSITION
DE RATS FEMELLES IMMATURES A L’E2 OU AU DDT
A. Objectifs de l’étude
Dans notre première étude expérimentale, nous avons étudié les effets d’une
administration sous-cutanée, néonatale (JPN 6-10 et 6-15) ou prolongée (JPN 6-40), chez des
rats femelles, de deux doses (10 et 100 mg/kg.jour) d’o,p’-DDT. Les paramètres suivis étaient
la fonction hypothalamo-hypophysaire évaluée au travers de la sécrétion de GnRH étudiée ex
vivo et des taux sériques de LH, en conditions basales et 30 min après une injection souscutanée de 1 µg/kg de GnRH (JPN 15). En outre, nous avons évalué la maturation sexuelle
(jours de l’OV et du premier oestrus ainsi que déroulement du cycle oestral jusqu’au JPN 60).
A titre de comparaison, nous avons étudié les effets de l’E2 (0,01, 0,1 et 1 mg/kg.jour), utilisé
comme substance de référence (Fig. 11). Le but du travail était de mimer, respectivement, les
situations dans lesquelles se trouvent les enfants adoptés qui migrent dans notre pays après la
période néonatale, et qui sont donc soustraits à une exposition permanente au DDT, et celle
des enfants qui ne migrent pas et restent ainsi exposés au DDT jusqu’à leur période
pubertaire. Nous avons voulu déterminer si les doses d’o,p’-DDT administrées in vivo en
injection sous-cutanée aux JPN 6-10 étaient du même ordre que les données de la littérature et
comment elles se situaient par rapport aux concentrations utilisées in vitro. Dès lors, après une
période d’administration sous-cutanée (JPN 6-10) d’o,p’-DDT, les concentrations sériques des
isomères du DDT et de ses métabolites ont été mesurées aux JPN 15 et 22 ainsi qu’aux jours
de l’OV et du premier oestrus (Fig. 11).
58
Puberté après exposition précoce au DDT
E2 0,01, 0,1 ou 1 mg/kg.jour; o,p’-DDT 10 ou 100 mg/kg.jour
E2 0,01 mg/kg.jour; o,p’-DDT 10 mg/kg.jour
E2 0,01 mg/kg.jour;
o,p’-DDT 10 mg/kg.jour
Sécrétion pulsatile
de GnRH in vitro et ex vivo
-Taux sérique basal de LH et en réponse
(après délai de 30 min) à 1 µ g/kg de GnRH
-Taux sérique des isomères du DDT et du DDE
IP de GnRH (min)
90
Examens quotidiens pour l’OV et frottis vaginaux
journaliers (pour le 1er oestrus et le cycle oestral)
jusqu’au JPN 60
70
1er
oestrus
OV
50
Contrôle
30
Naissance
5
10
15
20
25
30
40
50
Age (jours)
Figure 11: Schéma expérimental d’étude in vivo des effets d’un traitement à l’E2 ou à l’o,p’-DDT par des
injections sous-cutanées quotidiennes pendant 5, 10 ou 35 jours chez des rats femelles à partir de l’âge de 6
jours. La procédure est présentée selon la réduction développementale de l’IP de GnRH observée in vitro en
fonction de l’âge (n=6). Les âges moyens à l’OV et au premier oestrus sont également représentés.
B. Résultats
Pour les détails des données, le lecteur pourra se référer aux articles originaux en annexes IV
& VI de ce travail.
Comme montré à la Fig. 12A, après 5 jours (JPN 6-10) de traitement avec 0,01, 0,1 et
1 mg/kg d’E2, l’âge à l’OV était significativement avancé. Cet évènement avait lieu
également plus précocement après traitement avec 10 et 100 mg/kg d’o,p’-DDT. Le premier
oestrus apparaissait significativement plus tôt après 0,01 et 0,1 mg/kg d’E2 ainsi qu’après 10
mg/kg d’o,p’-DDT. Après 1 mg/kg d’E2 ou 100 mg/kg d’o,p’-DDT, l’âge au premier oestrus
ne changeait pas. Lorsque l’intervalle de temps entre l’OV et le premier oestrus était mesuré,
une augmentation dose-dépendante significative était observée avec 0,1 et 1 mg/kg d’E2 ou
100 mg/kg d’o,p’-DDT (Fig. 12B). Par ailleurs, une période prolongée d’administration souscutanée quotidienne de 0,01 mg/kg d’E2 entre les JPN 6 et 40 induisait l’OV à un âge
significativement avancé tandis que l’âge au premier oestrus d’un cycle normal était
59
Puberté après exposition précoce au DDT
significativement retardé. En effet, cet évènement était observé après un oestrus permanent
maintenu jusqu’à quelques jours après la fin du traitement (Fig. 12A). Quelle que soit la
condition, aucune différence dans la longueur du cycle oestral n’était observée jusqu’au JPN
60 (Tableau 4).
0,01
Véhiculant E2
0,1
1 mg/kg.jour
o,p’-DDT
10
Période de traitement
Période de traitement
JPN 6-10
A
JPN 6-40
JPN 6-10
*
50
B
Age (jours) à la puberté
*
*
*
30
20
10
40
1er oestrus
OV
*
OV
1er oestrus
*
30
20
10
OV
Intervalle (jours) entre l’OV et le 1er oestrus
15
40
100 mg/kg.jour
*
JPN 6-40
30
10
20
5
10
0
0
15
*
*
10
5
0
1er oestrus
Figure 12: Effets de l’E2 ou de l’o,p’-DDT (n=10) administrés par injections sous-cutanées chez des rats
femelles aux JPN 6-10 ou 6-40 sur l’âge à l’OV et au premier oestrus (A), ainsi que sur l’intervalle de temps
entre l’OV et le premier oestrus (B). *: p<0,05 versus véhiculant (huile de sésame).
Tableau 4: Moyenne ± déviation standard (n=10) de la longueur du cycle oestral
(intervalle de temps entre deux oestrus consécutifs) observée jusqu’au JPN 60.
Période d’exposition
JPN 6-10
JPN 6-40
Traitement
Dose
(mg/kg.jour)
Longueur du cycle oestral
(jours)
Véhiculant
-
4,7 ± 1,4
E2
0,01
0,1
1
5,2 ± 1,6
5,5 ± 1,8
4,6 ± 1,5
o,p’-DDT
10
100
5,3 ± 2,0
5,5 ± 1,4
Véhiculant
E2
0,01
4,8 ± 1,2
4,9 ± 1,8
60
Puberté après exposition précoce au DDT
La Fig. 13 montre qu’après des injections sous-cutanées quotidiennes durant 5 jours
(JPN 6-10) avec 0,01, 0,1 et 1 mg/kg d’E2, l’IP de GnRH étudié ex vivo au JPN 15 était
significativement réduit selon un effet dose-réponse. Un tel effet était également observé
après un traitement avec 10 et 100 mg/kg d’o,p’-DDT, mais était significatif seulement avec
100 mg/kg. Lorsque 0,01 mg/kg d’E2 ou 10 mg/kg d’o,p’-DDT étaient administrés pendant
une plus longue période, à savoir 10 jours (JPN 6–15), l’IP de GnRH mesuré ex vivo était
aussi significativement réduit au JPN 15. La plus faible dose d’o,p’-DDT qui n’avait pas
d’effet significatif après 5 jours d’exposition devenait donc significativement efficace après
10 jours.
0,01
Véhiculant E2
0,1
1 mg/kg.jour
o,p’-DDT
Période de traitement
JPN 6-10
IP de GnRH (min) au JPN 15
*
*
10
100 mg/kg.jour
JPN 6-15
*
65
60
55
50
45
40
Figure 13: Effets de l’E2 ou de l’o,p’-DDT injecté en sous-cutané chez des rats femelles durant les JPN 6-10
(n=10) ou JPN 6-15 (n=5) sur l’IP de GnRH mesuré ex vivo à 15 jours. *: p<0,05 versus véhiculant (huile de
sésame).
Le taux sérique de LH au JPN 15 était significativement diminué après 0,01, 0,1 et 1
mg/kg d’E2 entre les JPN 6 et 10 ainsi qu’après 10 et 100 mg/kg d’o,p’-DDT (Fig. 14).
Cependant, le niveau sérique de LH diminuait aussi avec l’âge (aux JPN 15 et 22, à l’OV et
au premier oestrus) après une administration sous-cutanée d’huile de sésame (Fig. 15). Dès
lors, la diminution des taux de base observée pouvait traduire une composante de
rétrocontrôle inhibiteur aussi bien que développementale. Lorsque la réponse de la LH à la
GnRH était étudiée après avoir injecté le véhiculant, une réduction progressive de la réponse
de LH était observée au cours de la maturation sexuelle, à l’inverse de ce qui est observé chez
l’humain en période prépubertaire. Après administration de 10 mg/kg d’o,p’-DDT entre les
JPN 6 et 10, la réponse de LH à la GnRH n’était pas affectée au JPN 15 et montrait une
réduction, non significative, au JPN 22. En revanche, elle diminuait significativement plus
61
Puberté après exposition précoce au DDT
rapidement que chez les témoins les jours suivants, à savoir à l’âge de l’OV et du premier
oestrus (Fig. 15).
0,01
Véhiculant E2
0,1
1 mg/kg.jour
o,p’-DDT
10
Période de traitement
JPN 6-10
LH sérique (ng/ml) au JPN 15
*
100 mg/kg.jour
JPN 6-15
*
*
75
50
25
0
Figure 14: Effets de l’E2 ou de l’o,p’-DDT injecté en sous-cutané chez des rats femelles durant les JPN 6-10
(n=10) ou JPN 6-15 (n=5) sur les concentrations sériques de LH mesurées ex vivo à 15 jours. *: p<0,05 versus
véhiculant (huile de sésame).
après huile de sésame,
500
après o,p’-DDT (10 mg/kg.jour),
Taux sérique de LH (ng/ml)
30 min après stimulation à la GnRH
450
JPN 6-10
au jour de l’OV
400
au jour du 1er oestrus
350
300
250
200
150
100
*
*
50
0
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Age (jours post-nataux)
Figure 15: Effets de l’huile de sésame ou de l’o,p’-DDT injecté en sous-cutané chez des rats femelles durant
les JPN 6-10 (n=7) sur les concentrations sériques de LH mesurées ex vivo 30 min après une injection souscutanée de GnRH à 4 âges différents (JPN 15 et 22, à l’OV et au premier oestrus). *: p<0,05 versus véhiculant
(huile de sésame).
62
Puberté après exposition précoce au DDT
Durant une administration sous-cutanée quotidienne de 10 mg/kg d’o,p’-DDT
prolongée (période JPN 6–40), les animaux ont développé un statut sanitaire altéré avec une
activité ainsi qu’une alimentation réduites. Au JPN 40, leur poids était significativement
réduit (Tableau 5). Dans de telles conditions, il n’était pas possible de distinguer les effets
directs du PE sur la maturation sexuelle et la reproduction des effets indirects dus à un trouble
du statut nutritionnel connu pour affecter la maturation sexuelle (Lebrethon et al., 2007).
Tableau 5: Moyenne ± déviation standard (n=10) du poids corporel (g) mesuré au JPN 40
après une exposition prolongée (JPN 6-40). *: p<0,05 versus véhiculant (huile de sésame).
Véhiculant
E2 0,01 mg/kg.jour
o,p’-DDT 10 mg/kg.jour
149,0 ± 9,9
130,0 ± 11,5*
111,3 ± 8,3*
Comme déjà mentionné précédemment, afin d’évaluer si les doses d’o,p’-DDT
administrées in vivo aux JPN 6-10 étaient du même ordre que les concentrations utilisées in
vitro, les concentrations sériques des isomères du DDT et de ses métabolites ont été mesurées
après administration in vivo d’o,p’-DDT: les rats femelles ont reçu une injection sous-cutanée
quotidienne de 10 mg/kg d’o,p’DDT lors des JPN 6-10. La première mesure du taux sérique
de l’isomère était prise 5 jours après la fin du traitement. Sept jours plus tard, le niveau chutait
drastiquement. L’OV et le premier oestrus avait lieu tôt, respectivement aux JPN 23 et 27,
confirmant nos données présentées ci-avant. Le taux sérique d’o,p’-DDT était également très
bas à l’OV et davantage encore au premier oestrus (Fig. 16). L’isomère p,p’-DDT ainsi que
les dérivés o,p’-DDE et p,p’-DDE n’étaient pas détectables dans le sérum des rats traités au
moment de l’étude et aucun des composés étudiés n’était détectable dans le sérum des rats
contrôles.
63
Puberté après exposition précoce au DDT
Taux d’o,p’-DDT
sérique (ng/ml)
3000
o,p’-DDT
10 mg/kg
1000
10-6 M
OV
300
100
1er oestrus
30
10
3
≤1
5
10
15
20
25
30
Age (jours)
Figure 16: Moyenne géométrique ± déviation standard (n=6) des taux sériques d’o,p’-DDT à 4 âges différents
après l’administration sous-cutanée d’o,p’-DDT pendant 5 jours (JPN 6-10): aux JPN 15 et 22, aux âges à l’OV
et au premier oestrus. La concentration sérique moyenne d’o,p’-DDT au JPN 15 correspond à 10-6 M.
Contrairement aux données recueillies chez le rat, à savoir la présence unique de
l’isomère o,p’-DDT dans le sérum, chez l’humain, Parent et al. (2003) n’ont détecté aucun
isomère du DDT dans le sérum des patients ayant été exposés précédemment à l’insecticide.
A l’inverse, le taux sérique de p,p’-DDE, un dérivé possible du DDT, était mesurable.
64
Puberté après exposition précoce au DDT
Etrangers adoptés
300,0
o,p’-DDT 10 mg/kg.jour
JPN 6-10
Etrangers non adoptés
Belges natifs, origine organique
Belges natifs,
r = -0,41
origine idiopathique p = 0,02
30,0
Taux sérique d’o,p‘-DDT (ng/ml)
Taux sérique de p,p‘-DDE (ng/ml)
100,0
10,0
3,0
1,0
0,3
≤0,1
2
4
6
8
10 12
Temps depuis l’immigration (années)
2,5
5 7,5 10 12,5 15 17,5
Temps après l’exposition au DDT (jours)
Figure 17: Comparaison des taux sériques de p,p’-DDE (gauche) et d’o,p’-DDT (droite) au cours du temps, à
la suite d’une exposition au DDT chez l’humain (gauche) et à l’o,p’-DDT chez le rat (droite). Les patients avec
précocité pubertaire sont soit natifs Belges avec une puberté précoce centrale, d’origine organique ou
idiopathique, soit des patients migrants étrangers, adoptés ou non. Les données des patients étrangers sont
représentées en fonction du temps écoulé depuis l’immigration.
C. Discussion
La présente étude a été réalisée afin de vérifier expérimentalement l’influence d’une
exposition précoce et transitoire au DDT, un insecticide banni dans les pays industrialisés
depuis une trentaine d’années mais encore largement répandu dans les pays en voie de
développement. Notre hypothèse de travail est que cette exposition précoce et transitoire au
DDT peut influencer la maturation hypothalamo-hypophysaire chez le rat femelle et, qu’en
outre, un mécanisme central orchestre cette précocité sexuelle, telle qu’observée chez des
jeunes filles migrantes adoptées dans nos pays occidentaux.
L’incubation des explants hypothalamiques en présence d’E2 ou des isomères du DDT
cause une augmentation concentration- et temps-dépendante de la fréquence de sécrétion
pulsatile de GnRH. Pour obtenir des effets similaires, le rapport effectif mesuré dans nos
conditions expérimentales entre l’E2 et le DDT est de 1/1000, ce qui est cohérent avec celui
rapporté par Diel et al. en 2002. Comme également constaté dans d’autres conditions par
Bulger et al. (1978), le p,p’-DDE n’a pas non plus d’effet, suggérant que cet isomère n’est pas
doué de propriétés oestrogéniques. Bien que des concentrations supra-physiologiques d’E2
65
Puberté après exposition précoce au DDT
soient requises pour déceler un effet dans les premières heures d’incubation, de plus faibles
concentrations sont toutefois effectives après plusieurs heures d’incubation. Un tel délai peut
s’expliquer par une lente diffusion des réactifs au sein de l’explant. Cette hypothèse rejoint un
constat fait pour d’autres substances puisque l’utilisation de plus grandes concentrations en
acides aminés excitateurs sont nécessaires dans les modèles d’explants, comparés aux
systèmes de cultures neuronales (Matagne et al., 2003). Une autre explication résiderait dans
le fait que les effets de l’E2 résulteraient de mécanismes génomiques et, par conséquent, que
son délai d’action soit plus lent. Ainsi, les effets de l’E2 sur la sécrétion de GnRH pourraient
impliquer, à la fois, une composante rapide et probablement non génomique, telle qu’illustrée
par Matagne et al. en 2005 dans notre modèle d’étude, et une composante lente et génomique.
Dans ce dernier cas, les cellules-cibles seraient autres que des neurones à GnRH puisque les
corps cellulaires de ces neurones sont absents des explants rétrochiasmatiques que nous avons
prélevés (Purnelle et al., 1998). En outre, Herbison (1998) et, plus récemment, Herbison et
Pape (2001), ont également rapporté que l’E2 exerce in vivo des effets complexes sur la
fonction des neurones à GnRH, suggérant des effets à long-terme ou génomiques via des
liaisons au ERα et/ou β.
Après l’administration d’E2 à des rat femelles immatures, une inhibition de la
sécrétion de LH est observée (Caligaris et al., 1972; Andrews et Ojeda, 1977). Cet évènement
susceptible de résulter d’une action directe sur l’hypophyse ne permet pas de conclure quant à
des effets indirects éventuels au niveau hypothalamique. En outre, une réduction
développementale des concentrations de LH sérique a lieu entre le JPN 15 et le jour de l’OV,
à la fois en sécrétion basale et en réponse à la GnRH (Ojeda et al., 1977; Ramaley, 1982), de
telle sorte que les taux de LH réduits peuvent résulter soit d’un effet de rétrocontrôle négatif,
soit d’une maturation accélérée, ou les deux à la fois. Par conséquent, la maturation
hypothalamo-hypophysaire a été étudiée à la fois en mesurant le taux de sécrétion de GnRH
durant l’incubation des explants hypothalamiques et la réponse de LH à la GnRH après
l’administration du stéroïde ou de l’insecticide in vivo. L’exposition de rats femelles infantiles
à l’E2 ou à l’o,p’-DDT pendant 5 ou 10 jours a été suivie par une augmentation dosedépendante de la fréquence de sécrétion pulsatile de la GnRH et une diminution du taux
sérique de LH au JPN 15, suggérant une stimulation de la maturation hypothalamique et une
inhibition du rétrocontrôle négatif ou une maturation précoce de la sécrétion hypophysaire.
Un effet sur le rétrocontrôle négatif au niveau hypothalamique et/ou hypophysaire est
supporté par le fait que l’élévation, après castration, des taux sériques de LH, est réduite après
un traitement à l’o,p’-DDT chez des rats matures ou néonatals (Gellert et al., 1972; Gellert et
66
Puberté après exposition précoce au DDT
al., 1974). Cependant, Faber et al. (1991) n’ont observé aucun changement dans la sécrétion
basale ou en réponse à la GnRH (50 ng/kg) de LH, plusieurs semaines après un traitement à
l’o,p’-DDT (0,5 mg/jour; 40 mg/kg.jour lorsque la dose est calculée en fonction des mesures
des poids des ratons utilisés dans nos expériences) entre les JPN 1 et 10. Ces résultats
divergents des nôtres pourraient s’expliquer par le fait que Faber et son équipe ont exposé les
ratons à l’insecticide dès la naissance, les ont castrés au JPN 21 et/ou que la stimulation à la
GnRH (dont la dose est 20 fois inférieure) a eu lieu plus tard (JPN 42). Aux JPN 15 et 22, la
réponse inchangée de la LH en réponse à la GnRH suggère soit l’absence d’effets, soit que la
résultante des effets combinés, inhibiteurs et stimulateurs, est nulle à ces âges.
L’OV pourrait résulter soit d’un effet périphérique de l’E2 ou de l’o,p’-DDT, soit
d’une activité hypophyso-ovarienne, ou les deux à la fois. Une composante centrale ayant lieu
le jour de l’OV est suggérée par la brusque réduction de la réponse de LH à la GnRH. Quant
au premier oestrus précoce, il confirme probablement l’activité de l’axe hypothalamohypophyso-ovarien. Un tel mécanisme pathophysiologique serait cohérent avec une
implication du DDT dans la précocité sexuelle apparaissant chez des enfants issus de
l’adoption internationale après leur migration (Krstevska-Konstantinova et al., 2001; Parent et
al., 2003; Rasier et al., 2006). Dans une récente étude danoise traitant du cas de tels enfants, il
a été montré que des changements développementaux des taux d’hormones hypophysoovariennes sont observables avant l’établissement de la puberté et supportent un mécanisme
hypothalamo-hypophysaire de la puberté précoce (Teilmann et al., 2006). Depuis que
Krstevska-Konstantinova et ses collaborateurs ont rapporté en 2001 qu’il existe une relation
entre la précocité sexuelle et la détection du p,p’-DDE dans le sérum des filles migrantes, il a
été démontré qu’une ménarche précoce a lieu après une exposition prénatale (Vasiliu et al.,
2004) ou post-natale (Ouyang et al., 2005) au DDE. Cependant, d’autres auteurs n’ont
observé aucun changement à l’âge de la ménarche après une exposition prénatale ou postnatale au DDE (Gladen et al., 2000; Denham et al., 2005) et le traitement post-natal de
guenons avec du MXC résulte en un développement des mamelons et une ménarche retardée
(Golub et al., 2003). Chez les rongeurs, de telles observations ont également été réalisées
puisque des pesticides tels que le MXC et le lindane provoquent, respectivement, une
précocité et un retard de l’âge à l’OV. Cependant, ces insecticides causent des perturbations
de la cyclicité oestrale (Cooper et al., 1989; Gray Jr et al., 1989; Laws et al., 2000). En outre,
un taux réduit de LH sérique a été rapporté après une administration de lindane (Cooper et al.,
1989).
67
Puberté après exposition précoce au DDT
Bien que nous n’ayons pas étudié en détail le cycle oestral et la reproduction, relevons
que l’intervalle entre l’OV et le premier oestrus est d’autant plus long que les animaux ont été
exposés à des doses plus élevées d’o,p’-DDT. Un élément critique à ce sujet est la
vulnérabilité, chez le rongeur, du mécanisme hypothalamique qui préside au contrôle de
l’ovulation. Celui-ci est perturbé par toute exposition périnatale à des stéroïdes sexuels ou
agents apparentés, et nous aurait empêchés d’observer les effets éventuels sur l’apparition du
cycle oestral. C’est pourquoi nous avons démarré le traitement par l’o,p’-DDT au JPN 6.
Nous pensons que cet âge est une limite et que l’observation, a priori paradoxale, que 10
mg/kg d’o,p’-DDT causent un premier oestrus précoce qui n’est plus observé après 100
mg/kg, résulte d’une possible interférence de la dose plus élevée avec le mécanisme de la
différenciation sexuelle de l’hypothalamus. Citons les données chez l’humain qui rapportent
une chute de 32 % de la probabilité de grossesse chez des femmes dont la mère présentait, 28
à 31 ans auparavant, des taux sériques de p,p’-DDT aux alentours de 10 µg/L au moment de
l’accouchement (Cohn et al., 2003). Un léger effet anorexigène de l’E2 est également
observé, ce qui est cohérent avec les études rapportées précédemment par Ramirez et Sawyer
(1965), et Ramirez (1981). Quant à l’o,p’-DDT, il cause une forte diminution du poids
corporel, suggérant un possible effet toxique de l’insecticide (Tomiyama et al., 2003).
Testant des doses d’o,p’-DDT 10 fois plus élevées et administrées oralement pendant
7 jours, Mussi et al. (2005) ont trouvé une concentration plasmique de 7,20 µg/L, qui est
environ 20 fois plus élevée que le niveau que nous avons observé 5 jours après la fin du
traitement dans nos conditions expérimentales. Aucun dérivé du DDT, en particulier l’o,p’DDE, n’a pu être détecté dans nos conditions après le traitement à l’o,p’-DDT, suggérant que,
chez le rat, peu de transformations du DDT en DDE ont lieu dans ces conditions d’exposition.
Cependant, Tomiyama et al. (2003) ont trouvé du DDE plasmique à des concentrations 10
fois plus basses que celles du DDT, à peine 2 jours après le début de l’exposition au DDT.
Ces résultats contradictoires pourraient s’expliquer par des différences dans le taux de
dégradation selon la nature de l’isomère du DDT, la dose et l’âge à l’administration puisque
l’équipe japonaise a injecté du p,p’-DDT entre les JPN 36 et 42 tandis que nous avons
administré 10 fois moins d’o,p’-DDT aux JPN 6-10.
En conclusion de cette première partie expérimentale, des preuves que le DDT pourrait
influencer la maturation hypothalamo-hypophysaire des femelles infantiles sont fournies par
une accélération développementale précoce de la sécrétion de GnRH in vitro et ex vivo et une
réduction précoce de la réponse de LH à la GnRH in vivo. Il est également démontré que ces
substances déclenchent une puberté précoce in vivo lorsque les individus immatures sont
68
Puberté après exposition précoce au DDT
exposés transitoirement au DDT. Dans les mêmes conditions de quantités administrées, les
effets à long terme n’ont pu être étudiés en raison d’effets toxiques.
69
Puberté après exposition précoce au DDT
VI. Mécanismes d’interaction des PEs
sur la sécrétion de GnRH induite
par le glutamate
VI. MECANISMES D’INTERACTION DES PEs
SUR LA SECRETION DE GnRH
INDUITE PAR LE GLUTAMATE
A. Objectifs de l’étude
Dans cette seconde étude expérimentale, nous nous sommes attachés à préciser le(s)
mécanisme(s) in vitro impliqué(s) dans les effets neuroendocriniens obtenus précédemment in
vivo. Nous avons particulièrement étudié la façon avec laquelle le DDT et ses dérivés peuvent
mimer les effets rapides de l’E2 sur la réponse sécrétoire de GnRH induite par le glutamate,
un sécrétagogue préalablement testé dans notre laboratoire (Bourguignon et al., 1989a). Nous
avons ensuite déterminé les types de récepteurs impliqués dans les effets des PEs sur la
sécrétion de GnRH tant dans sa forme spontanément pulsatile que sur la réponse induite par le
glutamate. Les effets du MXC, un insecticide développé et commercialisé afin de remplacer le
DDT banni depuis le début des années ’70 dans les pays occidentaux, et le BPA, largement
utilisé dans la fabrication de nombreux produits tels que les boîtes de conserve, les canettes de
soda, les tétines de biberons ou encore les résines dentaires, ont également été comparés avec
ceux du DDT.
B. Résultats
Pour les détails des données, le lecteur pourra se référer aux articles originaux en annexes IV
& VI de ce travail.
Des stimulations répétées en présence de glutamate seul, pendant 7,5 min toutes les
37,5 min, déclenchent une sécrétion de GnRH reproductible et maintenue pendant 4 h. Une
co-incubation avec des concentrations croissantes d’E2 induit une augmentation
concentration-dépendante de la réponse sécrétoire de GnRH. Ces effets sont significatifs à
partir de 10-8 M d’E2 et augmentent davantage en présence, respectivement, de 10-7 M et 10-6
M du stéroïde. Une augmentation du même type a lieu en présence de 10-5 M et 10-4 M d’o,p’DDT. Une stimulation finale, avec uniquement du glutamate, induit une réponse similaire à
celle obtenue initialement (Fig. 18).
70
PEs et réponse sécrétoire de GnRH
Réponse sécrétoire de GnRH induite par le glutamate
(pg/fraction de 7,5 min)
35
Glu 10-2 M
A
25
15
≤5
10-6M
E2 10-10 à 10-6 M
35
-7
Glu 10-2 M
10-10M
10-8M
10 M
B
10-9M
25
15
≤5
o,p’-DDT 10-8 à 10-4 M
35
Glu 10-2 M
10-8M
10-7M
10-6M
10-4M
C
10-5M
25
15
≤5
60
120
180
240
300
Temps (min)
Figure 18: Profils représentatifs de la sécrétion de GnRH induite par le glutamate à partir d’explants
hypothalamiques obtenus chez des rats femelles âgés de 15 jours et incubés dans du «minimum essentiel
medium» pendant 4 h. Le glutamate est ajouté (toutes les 37,5 min, pendant 7,5 min) seul (A) et avec des
concentrations croissantes d’E2 (B) ou d’o,p’-DDT (C), elles-mêmes incubées pendant 7,5 min avant la
stimulation au glutamate et durant celle-ci. Le seuil de détection est de 5 pg/7,5 min.
L’isomère p,p’-DDT est aussi effectif qu’o,p’-DDT: à 10-5 M et 10-4M, il augmente
significativement la sécrétion de GnRH. Par contre, le p,p’-DDE, un dérivé stable du DDT, et
le MXC, un insecticide dérivé du DDT, n’ont aucun effet. Quant au BPA, son effet n’est
significatif qu’à 10-4M, et lorsque le 4-nonylphénol est utilisé à la même concentration, il ne
montre aucun effet (Fig. 19).
71
PEs et réponse sécrétoire de GnRH
Réponse sécrétoire de GnRH induite par le glutamate
(en % par rapport au contrôle)
260
A
E2
220
*
*
180
*
140
100
60
260
220
180
140
B
o,p’-DDT
p,p’-DDT
p,p’-DDE
MXC
*
*
* *
100
60
260
220
*
BPA
4-NP
C
180
140
100
60
10-10
10-9
10-7
10-8
10-6
10-5
10-4
Concentration (M)
Figure 19: Effets de concentrations croissantes d’E2 (n=4) (A) et de différents PEs (4-NP: 4-nonylphénol;
n=4-7) (B & C) sur la réponse sécrétoire de GnRH induite par le glutamate, à partir d’explants hypothalamiques
de rats femelles âgés de 15 jours. La réponse sécrétoire de GnRH (moyenne ± déviation standard) est calculée
comme étant la différence entre les taux de GnRH dans la fraction, immédiatement collectée avant et durant
l’exposition au glutamate (pg/fraction de 7,5 min). Ces différences sont ensuite converties en pourcentages du
contrôle (glutamate seul). *: p<0,05 versus contrôle.
La réduction significative de l’IP de GnRH obtenue en présence d’E2 à 10-7 M ou
d’o,p’-DDT à 10-4 M après 3–4 h d’incubation in vitro est complètement levée lorsque les
sous-types AMPA/kaïnate du récepteur au
glutamate sont saturés par le 6,7-
dinitroquinoxaline-2,3-dione (DNQX). Les effets du stéroïde et de l’insecticide sont
également totalement inhibés en présence de l’antagoniste des ERs, l’ICI 182.780. Par
ailleurs, lorsque l’α-naphtoflavone est utilisé pour antagoniser le récepteur aryl hydrocarbone
(AhR) aux dioxines, la diminution significative de l’IP de GnRH, causée par l’o,p’-DDT,
n’est pas constatée alors que les effets de l’E2 sont atténués, mais restent cependant
significatifs (Fig. 20).
72
PEs et réponse sécrétoire de GnRH
Figure 20: Effets du DNQX (n=3-4) (B), de l’ICI 182.780 (n=3-4) (C) et de l’α-naphtoflavone (n=3-4) (D) sur
l’IP de GnRH durant l’incubation d’explants hypothalamiques chez des rats femelles âgés de 15 jours en
présence de 10-7 M d’E2 (n=9) ou 10-4 M d’o,p’-DDT (n=9) (A) in vitro. Un profil représentatif de la sécrétion
pulsatile de GnRH est montré dans chaque condition et la moyenne (± déviation standard) de l’IP de GnRH
observé durant les 3-4 h d’incubation sont donnés. *: p<0,05 traitement versus contrôle (n=9).
La sécrétion de GnRH induite par le glutamate est fortement réduite par le DNQX.
Cette réduction fait suite à la suppression de la contribution du sous-type kaïnate, puisque que
l’antagoniste de la sous-unité AMPA, le SYM 2206, n’affecte pas significativement la
réponse sécrétoire de GnRH. En présence de DNQX, l’E2 et l’o,p’-DDT augmentent
significativement la sécrétion de GnRH induite par le glutamate, bien que l’amplification de
la réponse sécrétoire par l’E2 soit moindre que dans les conditions contrôles. En présence du
SYM 2206, l’amplification de l’effet, par l’E2 et l’o,p’-DDT, est significative mais cependant
moins marquée que dans les conditions contrôles, indiquant une contribution du sous-type
AMPA. Lorsque l’ICI 182.780 est utilisé, l’amplification, par l’E2 et l’o,p’-DDT, de la
sécrétion de GnRH induite par le glutamate est complètement inhibée. En outre, l’αnaphtoflavone, utilisé dans les mêmes conditions, inhibe la réponse sécrétoire de GnRH
causée seulement par l’o,p’-DDT, alors que l’effet amplificateur de l’E2 reste inchangé (Fig.
21).
73
PEs et réponse sécrétoire de GnRH
Réponse sécrétoire de GnRH induite par le glutamate
(% du contrôle)
170
a
150
A
a
b, c
b
130
b, c
b, d
110
90
70
50
30
170
o,p’-DDT
E2
10-4M
(Contrôle)10-7M
a
150
DNQX DNQX
DNQX
10-6M
+ E2 + o,p’-DDT
(contrôle)
SYM
10-6M
(contrôle)
SYM
SYM
+ E2 + o,p’-DDT
b
B
a
130
c
110
d
d
90
70
50
30
o,p’-DDT
E2
(Contrôle) 10-7M 10-4M
ICI
10-7M
(contrôle)
ICI
ICI
+ E2 + o,p’-DDT
α -naphto α -naphtoα -naphto
10-7M
+ E2 + o,p’-DDT
(contrôle)
Figure 21: Effets d’antagonistes (n=5) des sous-types AMPA/kaïnate (DNQX) et AMPA (SYM 2206) du
récepteur au glutamate (A), des ERs (ICI 182.780) et du AhR (α-naphthoflavone) (B) sur l’amplification de la
sécrétion de GnRH, induite par le glutamate et causée par l’E2 ou l’o,p’-DDT. La réponse sécrétoire de GnRH
(moyenne ± déviation standard) est calculée comme étant la différence entre les taux de GnRH dans la fraction
immédiatement collectée avant et durant l’exposition au glutamate (pg/fraction de 7,5 min). Ces différences sont
ensuite converties en pourcentages du contrôle (glutamate seul ou glutamate avec l’antagoniste). Des explants
hypothalamiques de rats femelles âgés de 15 jours ont été utilisés. p<0,05 versus glutamate seul (a); versus
glutamate + antagoniste (b); versus glutamate + E2 (c); versus glutamate + o,p’-DDT (d).
La staurosporine et le chlorure de chélérythrine, respectivement des inhibiteurs des
protéines kinases A (PKA) et C (PKC), ainsi que le PD98059, inhibiteur des kinases
«mutagen activating proteins» (MAPK), inhibent complètement l’amplification, par l’E2 et
l’o,p’-DDT, de la sécrétion de GnRH induite par le glutamate (Tableau 6).
74
PEs et réponse sécrétoire de GnRH
Tableau 6: Effets d’inhibiteurs des kinases (n=5) sur la réponse sécrétoire de GnRH induite par le glutamate
(% du contrôle). *: p<0,05 versus glutamate.
Inhibiteur
Staurosporine
Kinase inhibée
Protéines
kinases
A&C
Glutamate
100,0
E2 10-7 M
150,4 ± 7,6*
o,p’-DDT 10-4 M
142,0 ± 4,7*
90,6 ± 6,4
95,5 ± 7,6
96,8 ± 4,4
93,7 ± 6,5
Chlorure de
chélérythrine
Protéine
kinase
C
93,8 ± 6,4
90,6 ± 6,4
PD 98059
Kinases
MAP
(ERK1/2)
101,6 ± 7,9
100,0 ± 9,1
96,8 ± 4,4
Lorsque l’E2 à 10-8 M est appliqué de façon intermittente durant des périodes de 15
min, répétées toutes les 37,5 min, l’amplification de la sécrétion de GnRH induite par le
glutamate ne varie pas au cours d’une incubation de 4 h. Après arrêt de l’exposition à l’E2,
une réponse similaire à celle obtenue initialement est observée. Lorsque l’E2 est appliqué de
façon continue, l’amplification de la réponse sécrétoire de GnRH est identique à celle
observée lorsque l’E2 est utilisé par intermittence. Cependant, après 3,5 h d’incubation, une
augmentation supplémentaire de la sécrétion de GnRH induite par le glutamate est causée par
l’E2, cette dernière étant significative et persistant même après avoir stoppé l’incubation en
présence d’E2. Un tel effet est également observé avec 10-5 M d’o,p’-DDT, mais
l’augmentation de la réponse sécrétoire est moindre et ne persiste pas après un arrêt de la
stimulation par l’insecticide (Fig. 22).
75
PEs et réponse sécrétoire de GnRH
contrôle
E2 10-8M: pdt 15 min, toutes les 37,5 min
E2 10-8M: incubation continue
o,p’-DDT 10-5M: incubation continue
Réponse sécrétoire de GnRH induite par le glutamate
(% du contrôle)
160
b
b
140
a
a a
a
a
120
a a
a
a
a
a
a
a
a
a
a
a
100
80
60
37,5
75
112,5
150
187,5
225
262,5
300
Temps (min)
Figure 22: Réponses sécrétoires de GnRH (n=4) induites par le glutamate (appliqué pendant 7,5 min, toutes
les 37,5 min) durant une incubation de 5 h. Les explants hypothalamiques de rats femelles âgés de 15 jours ont
été incubés de façon intermittente (pendant 15 min, toutes les 37,5 min) ou de façon continue en présence d’E2
ou d’o,p’-DDT. La stimulation par le stéroïde ou l’insecticide a débuté 60 min après le début de l’expérience et a
été stoppée après 262,5 min. a: p<0,05 versus réponse sécrétoire initiale; b: p<0,05 versus réponse sécrétoire
amplifiée par l’E2 ou l’o,p’-DDT.
C. Discussion
Dans cette seconde étude expérimentale, nous fournissons la preuve que plusieurs PEs
peuvent directement stimuler la sécrétion de GnRH induite par le glutamate. De tels effets ont
lieu rapidement, à savoir endéans 15 min, et augmentent davantage après plusieurs heures
d’exposition. Nous nous sommes particulièrement focalisés sur l’o,p’-DDT, dont nous
montrons, dans la première partie expérimentale de ce travail (chapitre V), qu’une exposition
précoce à cet isomère résulte en une accélération de la sécrétion pulsatile de GnRH et une
précocité sexuelle (Rasier et al., 2007). Nous démontrons également que le mécanisme
d’action de l’o,p’-DDT implique plusieurs récepteurs (AMPA, ERs et AhR) et kinases
intracellulaires (A, C et MAPK).
Afin de comparer l’amplification, par l’E2 ou l’o,p’-DDT, de la sécrétion de GnRH
induite par le glutamate, les données ont été calculées en pourcentages de la réponse
76
PEs et réponse sécrétoire de GnRH
sécrétoire obtenue en présence de l’antagoniste seul. Les concentrations de PEs utilisées in
vitro dans cette étude sont cohérentes avec les taux sériques mesurés après une administration
in vivo. Comparée avec d’autres modèles in vitro impliquant des neurones à GnRH,
l’incubation statique d’explants hypothalamiques maintient les neurones à GnRH dans leur
environnement neurono-glial original, ce qui peut contribuer à conserver la capacité de
sécrétion pulsatile de GnRH si elle n’est pas l’expression d’une propriété intrinsèque des
neurones à GnRH (Matagne et al., 2004; Rasier et al., 2007). C’est sans doute le cas dans nos
conditions où les péricaryons des neurones à GnRH ne sont pas inclus dans l’explant
(Purnelle et al., 1997). Il faut tout de même souligner que les concentrations requises pour des
réactifs comme le glutamate sont plus élevées que celles utilisées dans les cultures cellulaires
(Donoso et al., 1990; Ojeda et Urbanski, 1994; Kuehl-Kovarik et al., 2002; Matagne et al.,
2005; Rubin et al., 2006). Cette différence est toujours inexpliquée (diffusion des réactifs,
dégradation, …) et soulève même le point d’éventuelles erreurs de mesure des données in vivo
ainsi que de possibles effets excitotoxiques modulés via le sous-type NMDA du récepteur au
glutamate. Cependant, la capacité des explants à répondre à des stimulations répétées de
glutamate est maintenue pendant plusieurs heures, ce qui suggère que l’intégrité fonctionnelle
de l’appareil neurono-glial persiste et permet de maintenir le processus sécrétoire. Les
concentrations d’E2 et d’o,p’-DDT, utilisées in vitro pour les études mécanistiques, sont
cohérentes avec celles utilisées par d’autres (Urbanski et al., 1996; Clark et al., 1998; Diel et
al., 2002). Elles ont été choisies sur base de mesures préliminaires de l’amplification de la
sécrétion de GnRH induite par le glutamate en fonction des concentrations du stéroïde et de
l’insecticide. La concentration effective maximale des diférents PEs testés n’a pas été
déterminée, mais un effet concentration-dépendant peut être observé.
Comme observé avec l’E2, les deux isomères du DDT augmentent rapidement la
sécrétion de GnRH induite par le glutamate, cet effet étant directement dépendant de la
concentration. Le rapport E2/PE des concentrations effectives observé avec les isomères du
DDT et le BPA est cohérent avec d’autres études in vitro (Clark et al., 1998; Desaulniers et
al., 2005; Rasier et al., 2006). Quant aux autres PEs testés, ils ne montrent aucun effet aux
concentrations étudiées. Le manque d’effet du DDE s’explique par sa nature essentiellement
anti-androgénique, le DDE étant beaucoup moins oestrogénique que le DDT. Une récente
étude rapporte que l’effet oestrogénique du MXC serait modulé par ses métabolites mono-OH
MXC et bis-OH MXC après la métabolisation du cytochrome P450 (Miller et al., 2006). Ceci
pourrait expliquer son absence d’effet dans nos conditions expérimentales.
77
PEs et réponse sécrétoire de GnRH
Des études antérieures réalisées au sein de notre laboratoire ont montré que les effets
de l’E2 sur la sécrétion pulsatile de GnRH apparaissent en quelques heures (Matagne et al.,
2004) et que la sécrétion de GnRH induite par le glutamate a lieu en quelques minutes
(Matagne et al., 2005). Même si le BPA s’est avéré capable de stimuler la sécrétion de GnRH,
nous nous sommes focalisés sur le DDT, du fait de sa possible implication dans la précocité
sexuelle des enfants migrants (Krstevska-Konstantinova et al., 2001; Parent et al., 2003) et la
preuve expérimentale des effets hypothalamo-hypophysaires chez le rat (Heinrichs et al.,
1971; Gellert et al., 1972; Rasier et al., 2007). Les deux principaux mécanismes impliqués
dans la perturbation endocrinienne sont des effets agonistes au niveau des ERs ou
oestrogéniques, comme montré pour le DDT, et des effets antagonistes au niveau des
récepteurs androgéniques ou anti-androgéniques, comme montré pour le DDE (Kelce et al.,
1995; Clark et al., 1998). Dans nos conditions expérimentales, il a été montré qu’à la fois les
oestrogènes et les androgènes sont capables d’amplifier la sécrétion de GnRH, les effets des
androgènes étant aromatase-dépendants et modulés ultimement via les oestrogènes (Matagne
et al., 2004).
Remarque: les effets reportés dans ce travail tombent uniquement dans la catégorie oestrogénique.
Des rats femelles âgés de 15 jours ont été choisis puisqu’il a été montré au sein du
laboratoire que la sécrétion de GnRH in vitro est affectée de façon maximale par l’E2 à cet
âge (Matagne et al., 2004 et 2005). L’accélération de la sécrétion pulsatile de GnRH, causée
par l’E2, est inhibée par l’ICI 182.780 ainsi que par le DNQX, confirmant nos précédentes
observations (Matagne et al., 2004). L’implication des sous-types AMPA/kaïnate du récepteur
au glutamate dans la stimulation de l’E2, sur la fréquence pulsatile de GnRH, est supportée
par la co-localisation hypothalamique de ces récepteurs avec les ERs (Diano et al., 1998).
Etant donné que les effets de l’o,p’-DDT sont également inhibés par l’ICI 182.780 et le
DNQX, ce PE pourrait engager les mêmes récepteurs que l’E2. Cependant, la voie
d’activation des effets de l’o,p’-DDT serait partiellement différente: en effet, nos données
indiquent le possible rôle du AhR dans la modulation des effets de l’o,p’-DDT sur la sécrétion
de GnRH. Le AhR est un récepteur ubiquitaire se liant aux ligands endogènes ainsi qu’aux
xénobitiques telle que la dioxine (Harper et al., 2006). L’o,p’-DDT pourrait donc être un
ligand du AhR, puisque ses seuls effets sur la sécrétion de GnRH sont antagonisés par l’αnaphthoflavone. L’hypothèse envisagée serait que le complexe o,p’-DDT-AhR s’insère dans
la machinerie de signalisation de l’E2 et soit transcriptionnellement fonctionnel dans la région
régulatoire des gènes répondant à l’activation des ERs (Ohtake et al., 2003).
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PEs et réponse sécrétoire de GnRH
Après avoir montré que l’o,p’-DDT stimule la fréquence de sécrétion pulsatile de
GnRH (Rasier et al., 2007), nous avons mené une approche similaire pour étudier la sécrétion
de GnRH induite par le glutamate. Le rôle primordial de l’implication simultanée des soustype NMDA et kaïnate dans la réponse sécrétoire de GnRH a été démontré dans des études
précédentes (Bourguignon et al., 1989b; Matagne et al., 2004 et 2005; Parent et al., 2005). En
revanche, un antagoniste sélectif du récepteur AMPA n’affecte pas la sécrétion de GnRH
induite par le glutamate, indiquant qu’il n’y a aucune implication du sous-type AMPA dans de
telles conditions. Cependant, ces récepteurs apparaissent jouer certains rôles dans les effets
amplifiés par l’E2 et l’o,p’-DDT, confirmant les travaux qui rapportent la stimulation de
l’expression du sous-type AMPA par l’E2 dans l’hypothalamus de rat (Diano et al., 1997). A
notre connaissance, nous rapportons pour la première fois l’implication du sous-type AMPA
dans des effets rapides des PEs. Quelques études récentes ont toutefois déjà montré que les
récepteurs AMPA modulent l’effet plastique, induit par l’E2, dans différentes populations de
neurones (Tsurugizawa et al., 2005; Todd et al., 2007). Plus précisément, les récepteurs
AMPA ont été mis en évidence dans l’implication des changements morphologiques
neuronaux induits par l’E2 dans le noyau ventro-médial de l’hypothalamus, exclusivement
chez des rats femelles (Todd et al., 2007).
Non seulement le temps d’incubation, mais aussi la continuité de l’exposition du
stéroïde ou du PE sont des critères cruciaux puisque la composante lente de la réponse
sécrétoire n’apparaît pas si l’incubation est discontinue. Par contre, nous n’avons pas de
preuve de l’imprégnation de la réponse causée par des stimulations répétées de glutamate. En
outre, des effets rapides et non génomiques in vitro de l’E2 peuvent avoir lieu en quelques
secondes à quelques minutes dans des conditions variées pour influencer des évènements
cellulaires tels que des courants induits par le kaïnate dans des neurones de l’hippocampe
(Improta-Brears et al., 1999) et des cascades de seconds messagers dans ce même organe (Gu
et al., 1999) ou des neurones hypothalamiques (Lagrange et al., 1999; Abraham et al., 2004).
Les effets amplificateurs de l’E2 et de l’o,p’-DDT, sur la sécrétion de GnRH induite
par le glutamate, sont inhibés par les inhibiteurs des PKA, PKC et MAPK (Zhang et al.,
2010). Ces observations confirment donc nos précédents résultats et fournissent une preuve
supplémentaire d’une composante rapide et intracellulaire des effets des PEs.
En résumé, cette seconde étude expérimentale montre que les PEs, en particulier
l’o,p’-DDT, peuvent moduler la sécrétion de GnRH in vitro à partir de l’hypothalamus
femelle immature via à la fois des effets rapides et lents, avec l’implication des récepteurs
AMPA, des ERs et du AhR.
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PEs et réponse sécrétoire de GnRH
VII. Discussion générale et perspectives
VII. DISCUSSION GENERALE ET PERSPECTIVES
Résumé: Dans cette discussion générale, nous redéfinirons brièvement la problématique qui nous a
motivés à réaliser ce travail de recherche. Nous intégrerons ensuite les résultats que nous avons
observés dans les connaissances actuelles de l’influence des PEs sur le timing pubertaire et la
maturation sexuelle après une exposition néonatale. Finalement, nous explorerons quelques pistes de
recherche pour l’avenir.
Changements séculaires du timing pubertaire et facteurs envisagés
Les deux ou trois dernières décennies ont été marquées par des changements de l’âge
au début de la puberté qui apparaissent assez complexes. En effet, les premiers enfants à
entrer en puberté sont de plus en plus jeunes, en particulier les filles pour le début du
développement des seins ainsi que montré aux Etats-Unis, au Danemark et en Belgique
(Herman-Giddens et al., 1997; Lee et al., 2001; Aksglaede et al., 2009; Roelants et al., 2009).
Une étude belge a montré que les garçons connaissaient semblable phénomène pour le début
du développement testiculaire (Roelants et al., 2009). Toutefois, d’autres manifestations de la
puberté comme les premières règles chez la fille surviennent à un âge stable et la fin de la
puberté (cycles réguliers ou ovulatoires chez la fille et volume testiculaire adulte chez le
garçon) est atteinte de plus en plus tard par un certain nombre de sujets. Dans notre travail,
nous nous sommes focalisés sur le début de plus en plus précoce de la puberté chez la fille et
les facteurs qui pourraient y contribuer.
Les facteurs familiaux sous-tendus par les facteurs génétiques sont prépondérants dans
le déterminisme du timing pubertaire, mais ceux-ci ne peuvent être de nature à expliquer les
changements observés. En outre, des mécanismes tels que des polymorphismes ne peuvent
pas être étudiés dans la mesure où les gènes qui contrôlent les variations physiologiques de la
puberté restent peu connus. Un gène candidat est le variant LIN28B d’un gène qui encode un
régulateur du processing des µRNAs (Ong et al., 2009). Toutefois, ce variant ne peut
expliquer que 2 % de l’ensemble des variations physiologiques de timing qui s’étalent sur 4 à
5 années.
Ni l’appartenance ethnique, ni les différences d’origine géographique, ne peuvent, à
elles seules, expliquer la tendance à un avancement de l’âge au début de la puberté observé
durant les dernières décennies. Une telle conclusion a été tirée sur base de l’analyse des
origines des enfants de l’adoption internationale qui développent une puberté précoce
80
Discussion générale et perspectives
(Krstevska-Konstantinova et al., 2001). Les facteurs nutritionnels, en particulier l’excédent
d’apport énergétique, et l’obésité qui en découle ont été invoqués comme de possibles
responsables, notamment via la sécrétion accrue de leptine qui y est associée (Frisch et
Revelle, 1970 et 1971; Frisch et al., 1973). Ainsi, chez une fillette âgée de 9 ans, déficiente en
leptine et traitée par cette hormone, la sécrétion pulsatile nocturne de LH (GnRH) apparaît
(Farooqi et al., 1999). Chez l’humain comme chez le rongeur, la leptine est donc un pré-requis au
fonctionnement normal de l’axe hypophyso-gonadique (Ahima et al., 1996; Chehab et al., 1996;
Köpp et al., 1997; Montague et al., 1997; Strobel et al., 1998; Farooqi et al., 1999; Magni et al.,
2000; Welt et al., 2004). Dès lors, certains pensent que la leptine pourrait causer un
déclenchement de la puberté avancée aux Etats-Unis puisque l’avance du début de la puberté
dans ce pays coïncide avec ce que d’aucuns considère comme une épidémie d’obésité. On
remarquera toutefois que des données similaires rapportées récemment au Danemark
concernant l’avance de l’âge du début du développement des seins ne sont pas associées à une
modification séculaire de la corpulence des enfants qui est stable dans ce pays. Ceci suggère
l’implication d’autres facteurs. Un enjeu lié au rôle possible des facteurs nutritionnels est
celui de la période de la vie à laquelle ces effets s’exerceraient. Dans le mécanisme évoqué cidessus, l’hypothèse porte sur un effet de la nutrition durant la période qui précède
immédiatement le début de la puberté. On peut, par ailleurs, se demander s’il n’existe pas,
plus tôt dans la vie, des périodes ou fenêtres critiques au cours desquelles le timing d’un
processus comme le déclenchement de la puberté serait programmé et éventuellement
influencé par des facteurs comme la nutrition. A ce sujet, même si nous avons développé
l’hypothèse de l’action des PEs chez les enfants de l’adoption internationale, nous avons
également souligné que ces mêmes enfants sont plus susceptibles que d’autres d’avoir connu
divers stress anté- et post-natals, parmi lesquels des restrictions tant sur le plan nutritionnel
qu’affectif (Dominé et al., 2006).
Programmation intra-utérine du timing pubertaire
La question de la fenêtre critique de la période intra-utérine (que nous n’avons pas
investiguée dans nos travaux expérimentaux) a déjà été évoquée dans le chapitre II,
notamment sur base des PEs, en particulier le DES. Il est utile, dans cette discussion générale,
de retourner à l’hypothèse originale de Barker et Osmond (1986) qui portait sur des aspects en
relation avec la nutrition. Barker, un épidémiologiste anglais, s’est intéressé aux facteurs qui
pouvaient expliquer les variations de mortalité d’une région à l’autre du Royaume-Uni, à la
suite d’accidents coronariens. Il a observé que les variations régionales du taux de mortalité
81
Discussion générale et perspectives
par infarctus du myocarde étaient en relation directe et étroite avec la mortalité néonatale dans
ces mêmes régions, 60 années plus tôt. Partant du constat connu que la mortalité néonatale est
inversement liée au poids de naissance, il a montré que le risque de décès par accidents
coronariens était d’autant plus élevé que le poids de naissance était faible. Il en a émis
l’hypothèse que les conditions nutritionnelles durant la vie prénatale et post-natale précoce
pouvaient déterminer, en quelque sorte programmer, des perturbations ultérieures favorisant
les accidents coronariens. En l’occurrence, les travaux ultérieurs ont montré que le retard de
croissance intra-utérin (RCIU) était associé à un risque accru de syndrome métabolique
pouvant expliquer l’athérosclérose et les accidents coronariens qui en découlent (Ibanez et al.,
2009). Dans une perspective causale, différents facteurs sont à envisager en amont du RCIU.
Parmi ceux-ci, les restrictions nutritionnelles durant la vie intra-utérine mais aussi les PEs
dont certains se sont avérés responsables d’un RCIU à court terme (Ibanez et al., 1998; Ibanez
et al., 2007), mais aussi d’un syndrome métabolique à long terme (Hugo et al., 2008; BenJonathan et al., 2009). Des mécanismes et concepts communs (Fig. 23) pourraient ainsi soustendre les observations de Barker et les effets du DES détaillés au chapitre II. La période
intra-utérine et les premiers mois ou années de la vie constituent des périodes critiques durant
lesquelles des évènements ultérieurs sont programmés et susceptibles d’être influencés par
divers facteurs en raison de la plasticité des systèmes de régulation. Des modifications de
l’environnement foetal qui touchent à la nutrition, au métabolisme ou aux hormones peuvent
interférer avec la programmation physiologique et les mécanismes sont complexes. Par
exemple, dans le cas de la régulation du métabolisme énergétique, une réduction de la
production d’adiponectine est observée ainsi qu’une réduction de la sensibilité à la leptine qui
joue un rôle organisationnel sur la mise en place de l’homéostasie du métabolisme
énergétique, tôt dans la vie (Hugo et al., 2008; Ben-Jonathan et al., 2009). Des modifications
épigénétiques qui portent notamment sur la méthylation et l’acétylation de l’ADN
apparaissent comme centrales dans ces processus (Crews et McLachlan, 2006). Ces
modifications induites par les facteurs environnementaux modifient l’expression de l’ADN,
de telle sorte que l’acquis module l’inné. En outre, certains travaux récents suggèrent que les
modifications épigénétiques seraient transmissibles aux générations suivantes si l’ADN des
gamètes est concerné (Gore , 2008; Patisaul et Adewale, 2009; Bernal et Jirtle, 2010; Walker
et Gore, 2011). Après une latence variable de quelques mois à plusieurs dizaines d’années,
diverses manifestations surviennent et constituent en quelque sorte un «lifelong syndrome».
Ainsi, un RCIU est prédictif de diverses perturbations hormonales et métaboliques tout au
82
Discussion générale et perspectives
long de la vie (Tableau 7), parmi lesquelles une adrénarche prématurée, une insulinorésistance et un syndrome métabolique (Ibanez et al., 1998; Ibanez et al., 2007).
Evènement
environnemental:
nutritionnel,
métabolique,
endocrinien, …
DE- ou REPROGRAMMATION
Manifestations
diverses:
Périodes
critiques
intra-utérine
et post-natale
précoce:
SYNDROMES
PLASTICITE
Latence variable
Barker et Osmond, 1986
Herbst et al., 1971
?
Mère
DES
RCIU
Syndrome
métabolique
Systèmes
endocrinien,
reproducteur,
métabolisme,
SNC
Décès
Coronaropathie
Filles adultes
Cancers vagin
Figure 23: Origines développementales des perturbations du contrôle physiologique de l’homéostasie (balance
énergétique et reproduction) selon les hypothèses de Barker et Herbst. DES: diéthylstilbestrol; RCIU: retard de
croissance intra-utérin; SNC: système nerveux central.
Dans le cas du DES qui a évidemment été banni mais dont les effets sont toujours
étudiés dans la descendance des femmes exposées au traitement, les constats des
conséquences de l’exposition foetale ont été élargi à diverses anomalies du système
reproducteur chez la femme et également chez l’homme (Herbst et Anderson, 1990; Merino,
1991; Schrager et Potter, 2004). A côté de ces aspects repro-toxiques, les données récentes
chez l’animal indiquent que le DES peut avoir des effets neuro-toxiques, notamment sur
l’hippocampe (SanMartin et al., 1999; Sato et al., 2002; Ogiue-Ikeda et al., 2008) et des effets
métabo-toxiques qui apparaissent à l’âge adulte (Zangar et al., 1992; Piersma et al., 2002), à
la suite d’une exposition précoce (Tableau 7). Un autre exemple où les données animales et
humaines sont convergentes, est le syndrome de dysgénésie testiculaire proposé par
Skaekebbaek et son équipe (2001): cette entité rassemble des anomalies génitales à la
naissance chez le garçon (hypospadias et cryptorchidie), une oligospermie et des cancers de la
lignée germinale dans le testicule (Tableau 7). Chez la fille, des signes de puberté précoce
peuvent survenir. Parmi les PEs susceptibles d’expliquer ces manifestations, retenons les
phtalates, agents associés à diverses matières plastiques, sans exclure d’autres substances.
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Discussion générale et perspectives
EVENEMENTS INTRA-UTERINS OU
POST-NATALS PRECOCES
Tableau 7: Manifestations possibles durant la vie entière suite à des évènements périnataux.
MANIFESTATIONS ULTERIEURES POSSIBLES
Nouveau-né
Enfant
Adolescent
Adulte
RCIU
Adrénarche
HyperandroHTA, obésité
Insufprématurée
génie ovarienne
viscérale,
fisance
hypertriglycérinutridémie et diabète de
tionnelle
type II
Cancers vaginaux
MalformaPuberté avancée
Cancer du sein
tions utérines
Exposition Hypospadias
Oligospermie et
au DES
et
cancer de la prostate
cryptorchidie
Altérations de
Syndrome
neurogenèse
métabolique
de
l’hippocampe
Développement
précoce
Exposition
des seins
aux
phtalates
Hypospadias
Oligospermie et
et
cancer testiculaire
cryptorchidie
Syndrome de dysgénésie testiculaire
DES: diéthylstilbestrol; HTA: hypertension artérielle; RCIU: retard de croissance intra-utérin.
Quelle est l’évidence que le timing pubertaire fait l’objet d’une programmation intrautérine? Dans les modèles animaux comme le mouton qui se caractérise par un timing plus
précoce du début de la puberté chez le mâle que la femelle, l’implication d’une
programmation intra-utérine a été montrée. En effet, l’exposition foetale aux androgènes
masculinise le timing pubertaire chez les femelles et, dans la même mesure, les organes
génitaux externes (Kosut et al., 1997).
Rôle des PEs dans les variations du timing pubertaire
Ainsi que nous l’avons discuté dans le chapitre II, pratiquement toutes les formes de
puberté précoce centrale touchent davantage la fille que le garçon. Les raisons de ce
dimorphisme sexuel restent non élucidées. Il est remarquable que le modèle murin que nous
avons exploité se caractérise par un semblable dimorphisme puisque la sécrétion pulsatile de
GnRH à partir d’explants hypothalamiques d’animaux prépubères est accélérée par des
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Discussion générale et perspectives
concentrations d’E2 plus faibles chez la femelle que chez le mâle (Matagne et al., 2004).
Dans d’autres espèces (oiseaux et poissons notamment), un dimorphisme sexuel est bien
démontré notamment sur base de l’expression préférentielle de l’aromatase dans le cerveau du
mâle (Balthazart, 1991; Vizziano-Cantonnet et al., 2010). Le lien entre ce dimorphisme et le
comportement sexuel est bien établi, et une relation possible avec la maturation sexuelle n’a
pas été invoquée à notre connaissance. Par ailleurs, l’aromatase localisée dans le cerveau est
impliquée dans des troubles de la différenciation sexuelle chez des poissons traités par l’o,p’DDT (Kuhl et al., 2005).
Bien que la raison de la tendance à un début plus avancé de la maturation pubertaire
des fillettes ne soit pas encore évidente, il apparaît que l’exposition chronique à des agents
toxiques environnementaux, qui affectent principalement le système reproducteur, pourrait y
contribuer (Potashnik et al., 1984; Rogan et al., 1999; Mocarelli et al., 2000; Hama et al.,
2001; Sahamoto et al., 2001). En Belgique, un taux de p,p’-DDE anormalement élevé a été
mesuré dans le sérum d’enfants immigrants en raison de l’adoption internationale pour la
plupart et qui avaient développé une puberté précoce après leur arrivée en Belgique. Cette
dernière pourrait être associée à une exposition au DDT dans leur pays d’origine et, ensuite à
une soustraction de l’environnement exposé, en raison de la migration en Belgique
(Krstevska-Konstantinova et al., 2001; Parent et al., 2003). Le mécanisme permettant
d’expliquer l’apparition d’une puberté précoce, dont la fréquence est 80 fois plus élevée chez
ces jeunes filles que chez les autochtones, serait le suivant (Fig. 5): le DDT, doué de
propriétés oestrogéno-mimétiques, entraîne à la fois un effet de promotion de la maturation
hypothalamique (que nous avons étayé par nos travaux) mais également un rétrocontrôle
hypohysaire négatif (inhibiteur). Ce dernier est particulièrement efficace chez le sujet
prépubère qui y est très sensible (Grumbach, 1998). Il masque la maturation hypothalamique
qui ne peut s’exprimer si les gonadotrophines sont freinées lorsque les enfants restent exposés
à la substance dans leur pays d’origine. La migration en Belgique entraîne une diminution ou
une disparition de l’exposition et une levée du rétrocontrôle inhibiteur hypophysaire. Ceci
permet à la maturation hypothalamique déjà enclenchée de s’exprimer sous la forme d’une
puberté précoce centrale qui est, en fait, secondaire à une origine périphérique (KrstevskaKonstantinova et al., 2001; Parent et al., 2003; Rasier et al., 2006). Il a été montré que les
cohortes d’enfants de l’adoption internationale se caractérisent par un âge au début de la
puberté globalement avancé (Parent et al., 2003). Le problème concerne donc l’ensemble de
ces enfants migrants et non un sous-groupe seulement. Cela dit, l’ensemble de ces enfants a
aussi été exposé au DDT et la relation de cause à effet entre cette exposition et l’avance du
85
Discussion générale et perspectives
timing pubertaire est très difficile à démontrer. Nous avons imaginé de rechercher, via un
questionnaire auto-administré, les enfants adoptés avec puberté différée et de pouvoir mesurer
chez ceux-ci les taux de p,p’-DDE en postulant qu’ils pouvaient être plus bas à conditions
égales d’âge à l’immigration et de délai depuis l’immigration. Ce projet s’est avéré
impraticable.
On peut s’interroger sur le rôle du p,p’-DDE mis en évidence dans le sérum des
fillettes souffrant de puberté précoce. Dans la publication initiale rapportant les observations
cliniques (Krstevska-Konstantinova et al., 2001), le DDE a été considéré comme un reflet de
l’exposition antérieure au DDT dont il est dérivé. Il n’est toutefois pas exclu que le DDE ait
contribué en tant que tel à la puberté précoce. Un concept mécanistique de la perturbation
endocrinienne avancé récemment repose davantage sur le rapport entre effets oestrogéniques
et androgéniques que sur un effet mimant ou antagonisant exclusivement les stéroïdes de l’un
ou l’autre type. Une illustration clinique est apportée par la survenue de développement
mammaire précoce suite à une exposition aux phtalates qui sont essentiellement doués
d’activité anti-androgénique (Colon et al., 2000). Une autre illustration est celle d’un jeune
garçon âgé de 4 ans atteint d’un syndrome de Peutz-Jeghers et qui, dans ce cadre, présente
une gynécomastie bilatérale secondaire à la présence d’adénomes testiculaires qui sécrètent
l’aromatase (Coen et al., 1991). En l’absence de toute élévation des sécrétions androgéniques,
cette situation qui altère le rapport androgènes/oestrogènes suffit pour causer la gynécomastie.
Chez le rat, semblable conclusion a été tirée sur base de l’exposition conjointe à un agent antiandrogénique et au DES, puissant oestrogène de synthèse (Rivas et al., 2002). Ainsi, les
manifestations oestrogéniques causées par le DDT pourraient avoir été potentiées par les
effets anti-androgéniques du DDE chez les fillettes exposées à l’insecticide.
Choix des conditions expérimentales (dérivés étudiés et périodes d’exposition)
Puisque nos observations cliniques ont été faites essentiellement chez des filles
(Krstevska-Konstantinova et al., 2001) et que l’E2 influence préférentiellement la sécrétion de
GnRH au niveau de l’hypothalamus des rats femelles (Matagne et al., 2004), ce modèle
expérimental a été utilisé pour réaliser notre travail, dont les deux objectifs principaux étaient
d’une part évaluer les effets des PEs sur le déclenchement de la puberté in vivo et d’autre part
comprendre le(s) mécanisme(s) d’action impliqué(s) dans ces effets in vitro, chez le rat
femelle.
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Discussion générale et perspectives
Parmi les deux isomères du DDT, dont les effets sur la fréquence pulsatile de GnRH in
vitro sont similaires, l’o,p’-DDT a été préféré dans nos investigations in vivo pour son
oestrogénicité plus marquée et sa toxicité plus faible (Heinrichs et al., 1971; Gellert et al.,
1972). En outre, il s’agit de l’isomère utilisé dans de nombreuses études antérieures portant
sur le contrôle neuroendocrinien de la reproduction (Wrenn et al., 1971; Gellert et al., 1974;
Faber et al., 1991). Les doses utilisées pour les études in vivo ont été déterminées selon nos
données in vitro et sont comparables à celles utilisées dans d’autres études chez les rongeurs
(Desaulniers et al., 2005; Mussi et al., 2005). Dans nos travaux, il n’a pas été possible de tirer
de conclusions sur base des concentrations des isomères du DDT et de ses dérivés dans le
sérum. Les conditions d’exposition au DDT chez le rat et les jeunes filles migrantes sont, par
ailleurs, difficiles à comparer sur base des seules informations disponibles chez les patientes,
à savoir les concentrations sériques de p,p’-DDE mesurées plusieurs années plus tard. En
outre, il est à noter que l’extrapolation des doses et des durées d’exposition aux substances
chimiques chez les animaux de laboratoire ne représente pas toujours les conditions que les
humains rencontrent dans leur habitat durant leur vie. Certaines études expérimentales, chez
le rat nouveau-né soumis à une exposition précoce au DDT, ont rapporté des concentrations
sériques environ 20 fois plus élevées que celles mesurées chez les jeunes filles migrantes
(Heinrichs et al., 1971; Tomiyama et al., 2003; Mussi et al., 2005). Dans nos conditions,
aucun dérivé du DDT, en particulier l’o,p’-DDE, n’a été détecté après un traitement avec
l’o,p’-DDT, suggérant une faible transformation du DDT en DDE chez le rat. Cependant,
Tomiyama et son équipe (2003) ont détecté du DDE plasmatique jusqu’à des concentrations
10 fois plus basses que celles du DDT, à peine 2 jours après le début d’exposition à
l’insecticide. Par ailleurs, lorsque les PEs sont utilisés en mixtures, leurs effets requièrent des
concentrations plus faibles qu’attendu (Rajapakse et al., 2004). En outre, des effets
apparaissent en présence d’une mixture de substances à des concentrations qui n’entraînent
pas d’effets lorsque ces substances sont utilisées individuellement (Christiansen et al., 2009).
Des poissons exposés à des mixtures composées de métabolites du DDT ainsi que de dérivés
des PBBs et des PCBs présentent un puberté précoce, une augmentation du rapport
mâles/femelles et des différences de poids corporel, supportées par des modulations de la
régulation des gènes impliqués dans la signalisation endocrinienne et la croissance (Lyche et
al., 2010). Il est plus que probable que les patientes migrantes ont été exposées à une mixture
de perturbateurs dans leurs pays d’origine et à d’autres mixtures après leur migration en
Belgique. Etudier l’effet de mixtures n’était pas possible dans nos conditions car nous
ignorons les substances en cause et leurs concentrations. Les protocoles expérimentaux s’en
87
Discussion générale et perspectives
seraient trouvés alourdis de manière inacceptable et des conclusions n’auraient pas pu être
tirées concernant le DDT.
La fenêtre d’âge des JPN 6-10 coïncide avec la période périnatale chez les enfants. En
ce qui concerne la maturation du SNC, le rat nouveau-né est en effet à un stade de maturation
moins avancé que l’humain. Une période de 5 jours de traitement est relativement courte pour
l’espérance de vie mais significative si l’on se réfère à la courte période de 3 semaines entre la
naissance et la maturation sexuelle chez le rat. Quant à l’utilisation de rats femelles âgés de 15
jours pour l’étude de l’IP de GnRH, elle est basée sur nos résultats antérieurs qui montrent
que la sécrétion de GnRH in vitro est affectée de façon maximale par l’E2 à cet âge (Matagne
et al., 2004). Elle est aussi affectée à 5 jours, mais un tel âge se situe dans la fenêtre critique
de la différenciation sexuelle du cerveau, pouvant causer des interférences possibles avec la
programmation de la cyclicité oestrale (Heinrichs et al., 1971; Gellert et al., 1972). Un
traitement durant la seconde semaine de vie post-natale est donc un compromis pour affecter
potentiellement la maturation sexuelle en limitant les interférences autant que faire se peut
avec le mécanisme d’ovulation sexuellement différencié. Cependant, de telles perturbations
ont probablement encore lieu à ces âges de JPN 6-10 car des concentrations d’E2 et de DDT
élevées n’induisent plus un premier oestrus précoce contrairement aux faibles doses; par
contre, elles entraînent un délai prolongé entre l’OV et le premier oestrus. Si le concept de
fenêtre critique de programmation est étendu à d’autres aspects que l’ovulation, notamment au
contrôle de la balance énergétique, l’exposition au DDT se situe certainement dans la fenêtre
critique de programmation. En effet, le développement d’un syndrome d’obésité et d’insulinorésistance après malnutrition intra-utérine est empêché par un traitement à la leptine entre les
JPN 3 et 13 chez le rat (Vickers et al., 2005).
Diverses études ont clarifié le possible rôle de la nature des PEs et des paramètres
d’exposition (âge, dose et durée) dans les effets sur la maturation sexuelle. Chez les rongeurs,
des pesticides tels que le MXC (oestrogénique/anti-androgénique) et le lindane (faiblement
oestrogénique/anti-androgénique) causent, respectivement, une précocité et un retard de l’âge
à l’OV, et tous deux entraînent des perturbations de la cyclicité oestrale (Cooper et al., 1989;
Gray Jr et al., 1989; Laws et al., 2000). En outre, des taux sériques réduits de LH sont
rapportés après une administration de lindane (Cooper et al., 1989).
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Discussion générale et perspectives
Mécanisme central versus périphérique de la puberté précoce expérimentale
Après l’administration d’E2 à des rongeurs immatures, plusieurs études ont rapporté
une inhibition de la sécrétion de LH (Caligaris et al., 1972; Andrews et Ojeda, 1977) et une
réduction développementale des concentrations sériques de LH entre le JPN 15 et l’OV, à la
fois en condition basale et en réponse à la GnRH (Ojeda et al., 1977; Ramaley, 1982). Dans
nos conditions d’exposition à l’o,p’-DDT pendant 5 ou 10 jours, nous observons
simultanément au JPN 15 une augmentation de la fréquence de la sécrétion pulsatile de GnRH
in vitro et une diminution des taux de LH sériques in vivo, ce qui suggère la coexistence d’une
stimulation de la maturation hypothalamique et d’une inhibition hypophysaire par un
rétrocontrôle négatif. Toutefois, dans le contexte murin où une diminution des taux de LH
survient physiologiquement à cette période, l’observation d’une telle diminution pouvait aussi
résulter d’une maturation précoce de la sécrétion hypophysaire. Un rétrocontrôle inhibiteur au
niveau hypothalamique et/ou hypophysaire est supporté par la réduction des niveaux sériques
de LH après traitement de rats matures ou nouveau-nés avec de l’o,p’-DDT (Gellert et al.,
1972; Gellert et al., 1974). Cependant, aucun changement dans la sécrétion basale de LH et en
réponse à la GnRH n’est observé dans d’autres études 6 semaines après un traitement à l’o,p’DDT entre les JPN 1 et 10 (Faber et al., 1991). Le taux de LH inchangé en réponse à la GnRH
aux JPN 15 et 22, dans notre étude expérimentale, pourrait s’expliquer par la coexistence
d’effets inhibiteurs et stimulateurs qui se neutralisent à ces âges (Rasier et al., 2007).
A côté des effets des PEs sur le système hypothalamo-hypophysaire, des effets directs
sont possibles au niveau des tissus-cibles des stéroïdes sexuels. Ainsi, une OV précoce peut
résulter soit d’un effet périphérique du DDT sur l’épithélium vaginal, soit d’une activité
hypophyso-ovarienne précoce, soit des deux à la fois. Dans nos expériences in vivo, un effet
central au moment de l’OV est suggéré par la brusque réduction de la LH en réponse à la
GnRH observée chez les animaux traités. Par ailleurs, un possible premier oestrus précoce
indique une activité prématurée de l’axe hypothalamo-hypophyso-ovarien. Un tel mécanisme
physiopathologique qui implique un effet promoteur de la maturation au niveau
neuroendocrinien est cohérent avec l’implication du DDT dans la précocité sexuelle
apparaissant après migration chez les enfants issus de l’adoption internationale (KrstevskaKonstantinova et al., 2001; Parent et al., 2003; Rasier et al., 2006). Dans une étude danoise, il
a été montré que les changements développementaux des taux hormonaux hypophysoovariens étaient observables avant l’établissement de la puberté et supportaient un mécanisme
hypothalamo-hypophysaire de puberté précoce (Teilmann et al., 2006). Un objectif de nos
89
Discussion générale et perspectives
expérimentations était d’étudier les effets d’une exposition prolongée au DDT de manière à
reconstituer la situation des enfants exposés et non migrants. Une OV précoce suivie par un
oestrus permanent est observée après l’administration d’E2 pendant les JPN 6-40. Une fois le
traitement arrêté, le premier oestrus apparaît à un âge fortement retardé, ce qui indique à la
fois un effet stimulateur périphérique du stéroïde et une inhibition centrale durant
l’exposition. Un léger effet anorexigène de l’E2 est aussi observé, ce qui confirme les
observations précédemment rapportées par Ramirez et Sawyer (1965) et par Ramirez (1981),
tandis que l’o,p’-DDT cause une diminution dramatique du poids corporel, suggérant un
possible effet toxique de l’insecticide (Tomiyama et al., 2003).
Mécanisme hypothalamique de l’effet du DDT sur la sécrétion de GnRH
En contraste avec d’autres modèles in vitro impliquant uniquement les neurones à
GnRH notamment en tant que lignée cellulaire immortalisée, l’incubation statique d’explants
hypothalamiques permet d’étudier la sécrétion pulsatile de GnRH in vitro comme le résultat
de la fonction du neurone à GnRH préservé dans son environnement neurono-glial original
qui régule et maintient cette sécrétion pulsatile de GnRH (Matagne et al., 2004; Rasier et al.,
2007). Notons que les concentrations requises de certains réactifs tels que le glutamate sont
plus élevées que celles utilisées dans les cultures cellulaires (Donoso et al., 1990; Ojeda et
Urbanski, 1994; Kuehl-Kovarik et al., 2002; Matagne et al., 2005; Rubin et al., 2006). Cette
différence qui relève probablement d’aspects méthodologiques (diffusion du réactif,
dégradation, …), peut justifier un questionnement sur la validité de nos données in vitro.
Cependant, la capacité des explants à répondre à des stimulations répétées de glutamate est
maintenue pendant plusieurs heures et reste parfaitement régulière. Ceci suggère l’intégrité
fonctionnelle de l’appareil neurono-glial impliqué dans le processus sécrétoire. Les
concentrations des substances utilisées in vitro pour les études mécanistiques sont, par
ailleurs, cohérentes avec celles utilisées par d’autres (Urbanski et al., 1996; Clark et al., 1998;
Diel et al., 2002) et ont été choisies après une étude concentration-réponse sur l’amplification
de la sécrétion de GnRH induite par le glutamate. De manière similaire à l’E2, les deux
isomères du DDT augmentent rapidement la sécrétion de GnRH induite par le glutamate de
façon concentration-dépendante. Le ratio E2:PE en terme d’activité biologique qui est
observé avec les isomères du DDT et le BPA est cohérent avec d’autres études (Clark et al.,
1998; Desaulniers et al., 2005; Rasier et al., 2006). Le p,p’-DDE, connu pour être doué d’une
activité anti-androgénique et faiblement oestrogénique (Sohoni et Sumpter, 1998), n’est pas
90
Discussion générale et perspectives
actif en tant que tel dans notre système malgré les concentrations élevées utilisées in vitro.
Soulignons toutefois que les conclusions tirées in vitro ne peuvent être simplement
transposées aux conditions in vivo.
Des plus faibles concentrations d’E2 que celles requises pour un effet endéans 1 à 2
heures deviennent effectives après plusieurs heures d’incubation, ce qui peut impliquer la
diffusion progressive des réactifs dans l’explant, une hypothèse cohérente avec le fait que des
plus grandes concentrations de substances comme celles observées pour les acides aminés
sont nécessaires dans les modèles d’explants contrairement à d’autres systèmes de cultures
neuronales (Matagne et al., 2003). Par ailleurs, la mise en jeu de mécanismes génomiques
dont la traduction et l’expression des effets de l’E2 est une autre explication possible.
D’éventuels mécanismes génomiques sont, en effet, impliqués dans les actions de l’E2 via les
ERs α et/ou β (Herbison, 1998; Herbison et Pape, 2001), à côté des effets rapides ou non
génomiques illustrés par le Docteur Valérie Matagne au sein de notre laboratoire (Matagne et
al., 2005). Dans le cas d’effets génomiques, la cible de l’E2 serait sans doute différente des
neurones à GnRH puisque les corps cellulaires de GnRH sont absents des explants
rétrochiasmatiques que nous avons étudiés (Purnelle et al., 1998). Quant aux effets rapides de
l’E2, ils peuvent avoir lieu dans les secondes ou les minutes qui suivent l’application d’un
stimulus. Parmi ceux-ci, mentionnons les courants induits par le kaïnate dans les neurones
hippocampaux (Improta-Brears et al., 1999) et les cascades de seconds messagers dans
l’hippocampe (Gu et al., 1999) ou les neurones hypothalamiques (Lagrange et al., 1999;
Abraham et al., 2004) ainsi que le récepteur 30 couplé aux protéines G dans les neurones à
GnRH (Noel et al., 2009). Nous fournissons aussi la preuve que l’o,p’-DDT peut directement
stimuler la sécrétion de GnRH induite par le glutamate endéans 15 minutes, avec une
augmentation de cette réponse sécrétoire après quelques heures d’exposition (Rasier et al.,
2007). Par ailleurs, les effets du DDT sont également observés sur la sécrétion pulsatile après
1 à 2 heures d’incubation. L’ensemble des observations suggère que ces effets impliquent à la
fois des mécanismes non génomiques (rapides ou à court terme) et des mécanismes
génomiques (lents ou à long terme) comme mentionnés pour l’E2 (Matagne et al., 2004;
Matagne et al., 2005) dans les mêmes conditions.
Quelques études ont montré que le sous-type AMPA des récepteurs au glutamate
module les effets plastiques induits par l’E2 dans différentes populations de neurones
(Tsurugizawa et al., 2005; Todd et al., 2007) et qu’ils sont impliqués dans les changements
morphologiques induits par l’E2 dans le noyau ventro-médian de l’hypothalamus,
spécifiquement chez les rats femelles (Todd et al., 2007). Ce récepteur apparaît également
91
Discussion générale et perspectives
jouer un rôle non négligeable dans les effets de l’E2 à la fois sur la sécrétion pulsatile de
GnRH (Rasier et al., 2007) et sa sécrétion induite par le glutamate. L’o,p’-DDT apparaît
impliquer les mêmes récepteurs que l’E2 et, par conséquent, le sous-type AMPA des
récepteurs au glutamate est une classe supplémentaire de récepteur impliqué dans les effets du
DDT, ce qui constitue la première preuve de son implication dans les effets rapides des PEs.
Cependant, la voie des récepteurs impliqués dans les effets de l’o,p’-DDT est
partiellement différente de celle d’E2, avec une participation du AhR, comme indiqué par la
réduction préférentielle des effets de l’o,p’-DDT par son antagoniste. L’investigation des
effets de l’insecticide via cette voie est justifiée puisque Ohtake et ses collègues (2003) ont
rapporté que les dioxines peuvent mimer l’effet des oestrogènes via un mécanisme qui
implique l’activation des ERs par le complexe AhR - translocateur nucléaire. Nos données
indiquent aussi le rôle possible du AhR dans la modulation des effets de l’o,p’-DDT sur la
sécrétion de GnRH. Il est donc concevable que le complexe o,p’-DDT-AhR agisse en réaction
croisée
avec
la
machinerie
de
signalisation
de
l’E2
et
forme
un
complexe
transcriptionnellement actif dans la région régulatrice des gènes répondant au ERα (Ohtake et
al., 2003). En dehors de cette étape initiale, la voie semble similaire à l’E2 avec, comme déjà
mentionné, l’implication du sous-type AMPA des récepteurs au glutamate dans les effets
rapides, et les ERs à la fois dans les effets rapides et lents (Rasier et al., 2007). En outre, les
effets amplificateurs de l’o,p’-DDT sur la sécrétion de GnRH induite par le glutamate sont
prévenus par les inhibiteurs de la PKA et C, et des MAPK, ce qui fournit une preuve
supplémentaire d’une composante rapide intracellulaire des effets des PEs.
En résumé, nous avons mis en évidence un effet stimulant des isomères
oestrogéniques du DDT sur la sécrétion pulsatile de GnRH ainsi que sur la sécrétion de
GnRH induite par le glutamate chez le rat femelle immature. Le DDT entraîne un effet
stimulant similaire à celui de l’E2, mais à des concentrations 100 à 1000 fois plus élevées
que le stéroïde. Cette différence de concentration est cohérente avec les observations
faites dans d’autres modèles in vivo et in vitro (Smeets et al., 1999; Legler et al., 2002).
Par nos travaux, nous fournissons une preuve que le DDT peut influencer la maturation
hypothalamo-hypophysaire femelle infantile via une accélération développementale
précoce de la sécrétion de GnRH démontrée in vitro et une réduction précoce de la LH
en réponse à la GnRH démontrée in vivo. Nous montrons aussi que cet insecticide cause
une puberté précoce in vivo quand des individus immatures y sont exposés durant une
période limitée. Les PEs, en particulier l’o,p’-DDT, peuvent moduler la sécrétion de
92
Discussion générale et perspectives
GnRH in vitro dans l’hypothalamus femelle immature à travers à la fois des effets
rapides et lents avec l’implication des ERs, du AhR et du sous-type AMPA des
récepteurs au glutamate, ainsi que via les kinases intracellulaires A, C et MAPK.
Perspectives
La poursuite de travaux sur les effets de l’exposition anté- et périnatale à différents
PEs est indispensable pour une compréhension réelle des relations exposition-désordre chez
les animaux femelles immatures et, par extension, chez les jeunes filles. Parmi les alternatives
devant lesquelles se trouvent les investigateurs, la suivante nous paraît cruciale: soit étudier
isolément les effets d’une exposition précoce limitée à une substance bien caractérisée comme
PE en vue d’approfondir les aspects mécanistiques (ce que nous avons réalisé dans ce travail);
soit étudier les effets d’une exposition prolongée à une mixture de substances qui nous
rapproche des conditions réelles dans le règne animal et chez l’humain. En effet, ce n’est pas
l’exposition individuelle à une seule substance mais plutôt l’exposition cumulative à de
nombreux agents chimiques qui détermine les effets observés (Soto et al., 1994; Soto et al.,
1997). Le choix posé dans le laboratoire est le premier avec l’objectif d’étendre les aspects de
l’homéostasie qui sont évalués au-delà de l’axe reproducteur et d’évaluer aussi l’impact sur le
contrôle de la balance énergétique (Bourguignon et al., 2010). L’option prise est d’étudier le
DES, un PE banni mais dont les effets sur différents systèmes sont bien caractérisés.
Notamment, une exposition néonatale au DES pendant 5 jours entraîne une obésité avec
insulino-résistance à l’âge adulte (Newbold et al., 2008). Les données les plus récentes du
laboratoire (non encore publiées) indiquent que ce PE cause une diminution précoce de
sensibilité de la sécrétion de GnRH à la leptine. On notera avec intérêt que dans ce cas, la
puberté du rat femelle apparaît fortement retardée.
La puberté est un phénomène dont l’expression des gènes oestrogéno-sensibles
responsables de son déclenchement et la régulation par les déterminants moléculaires sont
complexes (Sisk et Foster, 2004) et encore largement méconnus. Les perspectives que nous
dessinons au terme de ce travail sont aussi orientées vers la biologie moléculaire et la
recherche de biomarqueurs d’exposition aux PEs, notamment le gène oct-2 (Ojeda et al.,
1999) et le facteur de transcription TTF-1/ebp/Nkx2.1 (Kugler et al., 2001). Il serait possible
que l’o,p’-DDT, après liaison au ER, stimule la transcription de ces gènes et, par là-même,
accélère l’apparition de la puberté. A cette fin, il serait intéressant de comparer l’expression
des gènes cibles en présence de doses faibles versus fortes de PEs. L’interaction de l’o,p’-
93
Discussion générale et perspectives
DDT avec le système GPR54/kisspeptine (de Roux et al., 2003; Seminara et al., 2003), lequel
est un acteur important dans la régulation de la sécrétion de GnRH, pourra également être
investigué. L’implication de ce système dans la perturbation neuroendocrinienne a d’ailleurs
été démontrée récemment (Tena-Sempere, 2010; Servili et al., 2011). Enfin, d’autres études
devront également permettre de définir de façon plus détaillée la contribution relative des
formes α et β du ER ainsi que les sous-types AMPA et kaïnate des récepteurs au glutamate.
Les effets des PEs sur la puberté ne représentent, en réalité, que la partie visible de
l’iceberg et beaucoup d’autres effets potentiels méritent d’être investigués plus avant. Les
modèles étudiés peuvent comporter les neurones à GnRH isolés dans les lignées cellulaires
immortalisées aussi bien que l’animal in toto soumis à des stress précoces comme la
malnutrition ou l’inflammation chronique chez la rate gestante. En outre, les implications
pour d’autres structures du SNC, en particulier le cortex cérébral et l’hippocampe, méritent
d’être étudiées (Parent et al., 2011).
D'autres substances toxiques déclenchent des cascades de signalisation qui ont pour
effet de modifier la structure biochimique du récepteur hormonal. Pour exemple, la
phosphorylation (catalysée par une protéine kinase) d'un récepteur hormonal modifie les
propriétés biochimiques de ce dernier, y compris ses interactions avec d’autres molécules, ses
propriétés de liaison et ses fonctions. Or, certaines hormones nécessitent des composés ou des
complexes accessoires pour fonctionner normalement, et la perturbation de la libération de ces
complexes cellulaires nécessaires à l'activité hormonale est un autre mécanisme d'action
possible des PEs.
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Discussion générale et perspectives
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Annexes
Estradiol Stimulation of Pulsatile Gonadotropin-Releasing Hormone Secretion in
Vitro : Correlation with Perinatal Exposure to Sex Steroids and Induction of
Sexual Precocity in Vivo
V. Matagne, G. Rasier, M.-C. Lebrethon, A. Gérard and J.-P. Bourguignon
Endocrinology 2004 145:2775-2783 originally published online Feb 26, 2004; , doi: 10.1210/en.2003-1259
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Endocrinology 145(6):2775–2783
Copyright © 2004 by The Endocrine Society
doi: 10.1210/en.2003-1259
Estradiol Stimulation of Pulsatile GonadotropinReleasing Hormone Secretion in Vitro: Correlation with
Perinatal Exposure to Sex Steroids and Induction of
Sexual Precocity in Vivo
V. MATAGNE, G. RASIER, M.-C. LEBRETHON, A. GÉRARD,
AND
J.-P. BOURGUIGNON
Developmental Neuroendocrinology Unit, Center for Cellular and Molecular Neurobiology, University of Liège, Centre
Hospitalier Universitaire, Sart-Tilman, B-4000 Liège, Belgium
Our aim was to study the effect of estradiol (E2) on pulsatile
GnRH secretion in vitro in relation to sex and development.
When hypothalamic explants obtained from 5- and 15-d-old
female rats were exposed to E2 (10ⴚ7 M), a reduction of GnRH
interpulse interval (IPI) occurred but not at 25 and 50 d of age.
This effect was prevented by the estrogen receptor antagonist
ICI 182.780 and the AMPA/kainate receptor antagonist DNQX
but not by the AMPA and N-methyl-D-aspartate receptor antagonists SYM 2206 and MK-801. E2 did not affect GnRH IPI in
hypothalamic explants obtained from male rats. Therefore,
the possible relation between the female-specific effects of E2
in vitro and perinatal sexual differentiation was investigated.
When using explants obtained from female rats masculinized
through testosterone injection on postnatal d 1, E2 was no
I
N CONDITIONS SUCH AS congenital adrenal hyperplasia (1, 2), sex-steroid-secreting tumors (3), or gonadotropin-independent sexual precocity (4, 5), the secondary
occurrence of central precocious puberty has led to the hypothesis that sex steroids could promote hypothalamic maturation. In addition, the different forms of central precocious
puberty are remarkably more frequent in the female than in
the male (6), suggesting that the female hypothalamus could
be more sensitive to triggering signals such as sex steroids.
Hypothalamic mechanisms, however, are difficult to study
in humans because they can only be assessed indirectly
through pituitary gonadotropin secretions that are directly
inhibited by a potent negative feedback, particularly in the
immature individual (7). In the rat, evidence has been provided that sexual maturation could be advanced after estrogen administration before puberty (8). Since we have set up
a rat hypothalamic explant paradigm in which developmental changes in frequency of pulsatile GnRH secretion can be
observed (9), we first aimed at studying the developmental
and sex-related effects of sex steroids in the hypothalamus in
vitro.
Abbreviations: AMPA, ␣-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; ATD, 1,4,6-androstatrien-3,17-dione; DNQX, 6,7-dinitroquinoxaline-2,3-dione; E2, estradiol; ER, estrogen receptor; GABA, ␥-aminobutyric acid; IPI, interpulse interval; NMDA, N-methyl-d-aspartate; T,
testosterone; V.O., vaginal opening.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
longer effective in vitro at 5 and 15 d. In addition, with explants obtained from male rats demasculinized through perinatal aromatase inhibitor treatment, E2 became capable of
decreasing GnRH IPI in vitro at 15 d. To study the possible
pathophysiological significance of early hypothalamic E2 effects, female rats received a single E2 injection on postnatal
d 10. This resulted in reduced GnRH IPI in vitro on d 15 as well
as advancement in age at vaginal opening and first estrus. In
conclusion, E2 decreases the GnRH IPI in the immature
female hypothalamus in vitro through a mechanism that
depends on perinatal brain sexual differentiation and that
could be involved in some forms of female precocious puberty.
(Endocrinology 145: 2775–2783, 2004)
In previous studies, we found that the developmental
changes in frequency of pulsatile GnRH secretion in vitro
were associated with an increase in stimulatory glutamatergic inputs (10 –12), an increase in activity of glutaminase (an
enzyme involved in glutamate biosynthesis) (13), and a reduction in prolyl endopeptidase activity that resulted in a
decreased inhibitory GnRH autofeedback mediated by the
GnRH1–5 degradation product through interaction at the Nmethyl-d-aspartate (NMDA) subtype of glutamate receptors
(12, 14). Moreover, in vivo studies in rats and monkeys have
shown the involvement of glutamate receptors in the experimental induction of precocious puberty using NMDA receptor agonists and delayed puberty using NMDA receptor
antagonists (15–18). Our second aim was to study the possible involvement of glutamate receptors in estradiol (E2)
effects on GnRH secretion in vitro.
The impact of E2 on adult reproductive function depends
on brain sexual differentiation during the perinatal period
(19, 20). In females, neonatal injection of testosterone (T)
disrupts estrous cyclicity in the adult and induces male sexual behavior (19). In males orchidectomized at birth, an LH
surge (20) can be induced by E2 treatment in adulthood, and
the male brain can also be demasculinized using an aromatase inhibitor within the first hours after birth (21, 22). Our
third aim was to determine whether perinatal sexual differentiation could influence the effects of E2 on pulsatile GnRH
secretion in vitro. In addition, we aimed to study whether the
effects of E2 on GnRH pulse frequency in vitro could be
observed after E2 treatment of immature female rats in vivo
2775
2776
Endocrinology, June 2004, 145(6):2775–2783
and whether this effect could possibly be associated with
subsequent precocious puberty.
Materials and Methods
Animals and reagents
Pregnant rats and 5-, 15-, 25-, and 50-d-old male and female Wistar
rats were purchased from the University of Liège and housed under
standardized conditions (22 C, lights on from 0630 to 1830 h, food and
water ad libitum). Each litter contained nine pups, and weaning occurred
at the age of 3 wk. The day of birth was considered as postnatal d 1. All
experiments were carried out with the approval of the Belgian Ministry
of Agriculture and the Ethical Committee at the University of Liege.
The incubation medium MEM (phenol red-free MEM) was purchased
from Life Technologies, Inc. (Invitrogen Corp., Merelbeke, Belgium).
Sesame oil was purchased from VWR international (Leuven, Belgium).
The aromatase inhibitor ATD (1,4,6-androstatrien-3,17-dione) was purchased from Steraloids Inc. (Newport, RI). The NMDA receptor antagonist MK-801 was obtained from Merck Sharp & Dohme research laboratory (Rahway, NJ). The AMPA/kainate receptor antagonist DNQX
(6,7-Dinitroquinoxaline-2,3-dione), the AMPA receptor antagonist SYM
2206
(4-aminophenyl-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7methylenedioxyphtalazine) and the estrogen receptor (ER) antagonist
ICI 182.780 were purchased from Tocris (Fisher Bioblock Scientific,
Illkirch, France). 17␤-E2 and T were purchased from Sigma-Aldrich
(Bornem, Belgium); and R76713, racemic Vorozole, an aromatase inhibitor, was obtained from Janssen Pharmaceutica (Beerse, Belgium). All
steroids were dissolved initially in absolute ethanol and subsequently in
incubation medium to achieve a final ethanol concentration of 0.01%.
R76713 was initially diluted in 20% polyethyleneglycol (3.35 mg/ml)
and subsequently in the incubation medium to achieve a 10⫺5 m solution
containing 0.02% polyethyleneglycol. All the other drugs were diluted
directly in the incubation medium.
Hypothalamic explant incubation and hormone assays
The animals were rapidly decapitated between 1000 and 1100 h, and
trunk blood was collected. After decapitation, the retrochiasmatic hypothalamus was rapidly dissected and transferred into a static incubator
as described in detail previously (23, 24). The medium consisted of
phenol red free MEM supplemented with glucose, magnesium, glycine,
and bacitracin (25 mm, 1 mm, 10 nm, 20 ␮m, respectively). The incubation
FIG. 1. Schematic illustration of the in
vivo experiments, with treatment and
investigations represented on an age
scale.
Matagne et al. • Developmental Effect of E2 on GnRH Secretion
medium was collected and renewed every 7.5 min and kept frozen until
assayed. Each experiment included 12–16 hypothalamic explants, which
were incubated individually, each in a separate chamber. In each experiment, at least two explants were incubated in MEM alone and used
as controls. GnRH was measured in duplicate using a RIA method with
intra- and interassay coefficients of 14 and 18%, respectively (24, 25). The
CR11-B81 anti-GnRH antiserum (final dilution, 1:80,000) was kindly
provided by Dr. V. D. Ramirez (Urbana, IL) (26). The data below the limit
of detection (5 pg/7.5 min) were assigned that value. After 4 h clotting
at room temperature, trunk blood was centrifuged (10 min at 1500 ⫻ g).
Serum was collected and stored at ⫺20 C until assayed. The LH assay
material was kindly provided by the National Institute of Diabetes and
Digestive and Kidney Diseases program. The samples were measured
in duplicate.
Study protocols
In vitro experiments. The effect of E2 on pulsatile GnRH secretion was
studied using explants obtained from female and male rats at 5, 15, 25,
and 50 d of age. The steroid was used for the whole 4-h experimental
period. The explants were incubated with 10⫺9–10⫺7 m E2 in initial
experiments. Subsequently, explants obtained from 15-d-old male and
female rats were used and incubated using 10⫺7 m E2. To study the
implication of ionotropic glutamate receptor subtypes, the antagonists
MK-801 (3.10⫺6 m), DNQX (10⫺6 m), or SYM2206 (10⫺6 m) were used
together with E2 or alone as control. The dosage of the antagonists was
selected based on previous experiments (23, 24). The effect of T was
studied by incubation with T (10⫺7 m). The aromatase involvement in
the T effect was studied through coincubation of T with the aromatase
inhibitor R76713 (10⫺5 m), which was also used alone as control. The
involvement of ER was studied using the ER antagonist ICI 182.780 (10⫺7
m) in the incubation medium together with E2 or alone as control.
In vivo experiments. In Fig. 1, a schematic summary of the in vivo experiments is shown on an age scale.
Protocol A. Masculinization of female rats. On postnatal d 1, female pups
received either a single sc injection of 1.25 mg T in 100 ␮l sesame oil (27)
or vehicle only, for control purposes. The animals were killed either on
postnatal d 5 or on d 15, and pulsatile GnRH secretion was studied in
vitro in the presence of 10⫺7 m E2 or in MEM alone.
Protocols B and C. Demasculinization of male rats. In protocol B, pregnant
rats were injected daily sc with the aromatase inhibitor ATD (5 mg in
Matagne et al. • Developmental Effect of E2 on GnRH Secretion
100 ␮l sesame oil) from d 17–21 of gestation. In the control group,
pregnant rats were injected with vehicle. In the offspring, the male pups
were injected sc with 3 mg ATD in 50 ␮l sesame oil on postnatal d 1, 6,
and 11 (28). The control rats were injected with the vehicle only. The
animals were killed either on postnatal d 5 or 15, and pulsatile GnRH
secretion was studied in vitro with or without the addition of 10⫺7 m E2.
In protocol C, ATD was administered as silicone implants (length, 5 mm;
inner diameter, 1.6 mm; outer diameter, 2.4 mm; Degania Silicone Silclear tubing, Degania Bet, Israel). Within 5 h after birth, the pups were
anesthetized through ice-cooling and surgically implanted in the neck
(29). The animals were killed on d 15 to obtain the hypothalamic explants
and to study pulsatile GnRH secretion with or without 10⫺7 m E2.
Protocol D. Single E2 administration in immature female rats. Ten-day-old
female rats received a single sc injection of E2 (10 mg/kg given in 100
␮l sesame oil) (30, 31), which is comparable to the dose used by others
(32). This particular age was selected to be outside of the critical period
for sexual differentiation of the brain (31), and a single injection was
given to avoid the persisting negative feedback effect that could result
from repeated or prolonged administration. To increase the likelihood
of an effect after a single administration, we used a massive dose that
was greater than in the previous experiments showing induction of
precocious puberty (8). There were three groups of 14 rats injected either
with E2 or with sesame oil or saline. On d 15, five rats from each group
were killed to study pulsatile GnRH secretion in vitro in the absence of
any steroid in the incubation medium. Serum LH was also measured
using trunk blood. In the remaining animals, sexual maturation was
subsequently evaluated by daily examination for imperforation of the
vaginal membrane (vaginal opening, V.O.). Thereafter, vaginal smears
were taken every day in the afternoon until postnatal d 60. Slides of
vaginal smears were colored following the papanicolaou method and
examined to detect the occurrence of estrus cycle. The age at the first
estrus was considered when vaginal smears contained primary leukocytes after the first proestrus phase, which was characterized by cornified cells (33).
Statistical analysis
The mean LH levels (ng/ml) were calculated after log transform and
expressed as geometric mean.
GnRH secretory pulses were detected using the PULSAR program for
PC (basic version by S. Rosberg) (34). The cut-off criteria for peak detection were determined empirically and were G1 ⫽ 2.5 and G2 ⫽ 2 (35).
Peak splitting parameter was set at 2.7, and the intraassay coefficient was
used as B coefficient (35). Data concerning the interpulse interval (IPI)
from different experiments were pooled and expressed as mean ⫾ sd.
The mean (⫾sd) amplitude of GnRH secretory pulses was calculated
(pg/7.5 min fraction), and the data from different experiments were
analyzed separately by t test to avoid biases due to interassay variations
FIG. 2. Representative profiles of pulsatile GnRH secretion from six individual hypothalamic explants obtained at
15 d in female (left and middle panels)
and male (right panels) rats and incubated in control conditions or together
with different concentrations of E2
(mean ⫾ SD). GnRH IPI is given together with the number of explants
studied in each condition (n). *, P ⬍ 0.05
(controls vs. E2 10⫺8 M, t ⫽ 6.127, df ⫽
27; controls vs. E2 10⫺7 M, t ⫽ 12.4, df ⫽
44). The limit of detection was 5 pg/7.5
min.
Endocrinology, June 2004, 145(6):2775–2783 2777
between experiments. In several instances, all the explants in a group
showed a similar GnRH IPI. Then, sd was zero and could not be represented in the figures. Similarly, when V.O. occurred at the same age
in all the rats in a control group, the sd was zero and could not be
represented. Significant effect of treatment was determined by t test
(control vs. E2 incubation). The effect of antagonist on control and treated
groups was determined by ANOVA test (one-way ANOVA) followed
by Newman-Keuls post test when the threshold for significance of difference (P ⬍ 0.05) was reached.
Results
As shown in Fig. 2, incubation of individual hypothalamic
explants obtained in 15-d-old female rats with different E2
concentrations (10⫺9–10⫺7 m) resulted in a significant doserelated decrease of GnRH IPI. E2 effectiveness occurred in
the nanomolar range as observed in other studies (36, 37). In
subsequent experiments, we used the highest concentration
because it was the most efficient in our paradigm. In explants
from 15-d-old male rats, no significant decrease of GnRH IPI
was caused by 10⫺7 m of E2 (Fig. 2). With explants from
5-d-old female rats, E2 (10⫺7 m) caused a significant decrease
in GnRH IPI, whereas no effect of E2 on pulsatile GnRH
secretion could be observed at 25 or 50 d in the female or at
any age studied in the male (Table 1). GnRH pulse amplitude
(mean ⫾ sd, pg/7.5 min) was not affected by E2 at 5 d or 25 d
in both sexes. In 15-d-old female rats, discrepant data were
obtained because GnRH pulse amplitude was slightly, but
significantly, decreased in one experiment (mean amplitude ⫾ sd; controls, 9.9 ⫾ 0.2 pg/7.5 min; E2, 7.4 ⫾ 0.4 pg/7.5
min; t ⫽ 5.657; df ⫽ 16), whereas a significant increase in
amplitude was seen in a second experiment (controls, 10.3 ⫾
0.4 pg/7.5 min; E2, 15.6 ⫾ 2 pg/7.5 min; t ⫽ 7.7; df ⫽ 19). In
a third experiment, amplitude was found to be higher and
more variable with no significant changes.
As previously reported (23, 35), the NMDA receptor antagonist MK-801 resulted in a significant increase of GnRH
IPI (59 ⫾ 3 vs. 68 ⫾ 4 min, control vs. MK-801, Fig. 3) when
hypothalamic explants of 15-d-old rats were used. In contrast, the AMPA/kainate receptor antagonist (DNQX) or the
specific AMPA receptor antagonist (SYM 2206) did not affect
the GnRH IPI in similar conditions (control, 59 ⫾ 3 min;
2778
Endocrinology, June 2004, 145(6):2775–2783
Matagne et al. • Developmental Effect of E2 on GnRH Secretion
DNQX, 60 ⫾ 4 min; SYM2206, 62 ⫾ 1 min). When similar
experiments were performed in the presence of 10⫺7 m of E2
(Fig. 3), the reduction of GnRH IPI caused by E2 was not
observed anymore in the presence of DNQX (59 ⫾ 2 min).
Using SYM 2206, the E2 effect was still observed (62 ⫾ 1 min
vs. 48 ⫾ 2 min, SYM 2206 vs. E2/SYM 2206). Likewise, the E2
effect was still observed in the presence of MK-801 (68 ⫾ 4
vs. 58 ⫾ 1 min, MK-801 vs. E2/MK-801). With hypothalamic
explants obtained from female rats and incubated in the
presence of the ER antagonist ICI 182.780, the reduction of
the GnRH IPI caused by E2 was absent (Table 2). T was as
effective as E2 in decreasing GnRH IPI in female explants,
and this effect was significantly prevented by an aromatase
inhibitor (R76713, 10⫺5 m) that had no effect when used
alone. When explants obtained from male rats were incubated either with E2 or with T, no change in GnRH IPI was
observed (Table 2).
Because E2 could affect the GnRH IPI in the female only,
we studied the involvement of steroid imprinting in the
perinatal period. As shown in Fig. 4, after female rats were
androgenized through a sc injection of T on postnatal d 1, the
hypothalamic explants studied on d 5 and 15 did not show
any significant change in GnRH IPI when incubated with E2
(84 ⫾ 2 vs. 90 ⫾ 0 min at 5 d, 58 ⫾ 1 vs. 61 ⫾ 2 min at 15 d,
TABLE 1. Effect of E2 (10⫺7 M) on the IPI of pulsatile GnRH
secretion in vitro using male and female rat (n) hypothalamic
explants
Females
Males
Age
(d)
Control
Estradiol
Control
Estradiol
5
15
25
50
85 ⫾ 15 (15)
59 ⫾ 3 (24)
35 ⫾ 3 (6)
34 ⫾ 0 (3)
75 ⫾ 7a (14)
44 ⫾ 6a (22)
32 ⫾ 2 (7)
34 ⫾ 3 (3)
83 ⫾ 15 (11)
60 ⫾ 1 (11)
41 ⫾ 2 (6)
34 ⫾ 3 (3)
87 ⫾ 2 (11)
56 ⫾ 4 (11)
45 ⫾ 10 (8)
35 ⫾ 2 (3)
Data are mean ⫾ SD (min, a P ⬍ 0.05, E2 vs. control, 5-d-old female
rats: t ⫽ 2.16, df ⫽ 27; 15-d-old female rats: t ⫽ 12.4, df ⫽ 44).
FIG. 3. Effect of antagonists of NMDA
(MK-801), AMPA/kainate (DNQX), and
AMPA receptors (SYM 2206) on GnRH
IPI (mean ⫾ SD, min) using (n) hypothalamic explants obtained from 15-dold female rats and incubated in control
conditions or with E2. NS, Nonsignificant; *, P ⬍ 0.05 (MK-801, F ⫽ 79.27,
df ⫽ 3; DNQX, F ⫽ 68.23, df ⫽ 3;
SYM2206, F ⫽ 67.69, df ⫽ 3.
E2 vs. control). With control explants from female rats injected neonatally with the vehicle, E2 could significantly
decrease the GnRH IPI in vitro at both ages (75 ⫾ 0 vs. 86 ⫾
10 min at 5 d, 45 ⫾ 6 vs. 56 ⫾ 7 min at 15 d, E2 vs. control).
When male rats were demasculinized through perinatal administration of the aromatase inhibitor ATD, E2 caused a
significant decrease in GnRH IPI at 15 d (48 ⫾ 4 min vs. 60 ⫾
0 min, E2 vs. control). A nonsignificant effect was observed
at 5 d (83 ⫾ 5 vs. 86 ⫾ 6 min, E2 vs. control). Using explants
from male rats treated perinatally with vehicle, E2 had no
effect on the GnRH IPI. To decrease the stress due to manipulation, we implanted male pups with a capsule containing ATD within 5 h after birth. In this case, the results obtained were similar to those obtained in ATD-injected pups
(Table 3), which indicates that neonatal inhibition of aromatase activity is sufficient for E2 effect to occur.
After a single massive injection of E2 on d 10 in vivo, the
GnRH IPI studied in vitro on d 15 was found to be significantly reduced (Fig. 5). On the same day, serum LH was
decreased in the E2-treated animals. In the E2-injected female
rats, the age at V.O. (27 ⫾ 1 d) and at first estrus (33 ⫾ 1 d)
was significantly advanced in comparison with controls injected with vehicle (V.O., 31 ⫾ 1 d; first estrus, 37 ⫾ 2 d) or
saline (V.O., 33 ⫾ 0 d; first estrus, 38 ⫾ 1 d). Age at V.O. was
also advanced after vehicle injection when compared with
saline, but age at first estrus was not significantly different
between these two groups.
Discussion
The developmental increase in GnRH pulse frequency is
an important event for the pubertal changes in gonadotropin
secretion and has been observed in vivo in the female (38) and
the male rat (39) as well as in the female monkey (40) and in
vitro, using our rat explant paradigm (9). The critical age
period for such changes, however, was earlier in vitro (9) than
Matagne et al. • Developmental Effect of E2 on GnRH Secretion
Endocrinology, June 2004, 145(6):2775–2783 2779
in vivo (38, 39). Isolation of the hypothalamus could suppress
extrahypothalamic inhibiting inputs and account for earlier
manifestation of the accelerated GnRH pulse generator. In
this study, the developmental acceleration of pulsatile GnRH
secretion in vitro was found to be stimulated by E2 specifically in the immature female hypothalamus and through a
TABLE 2. Effect of E2 and testosterone and ER antagonist (ICI
182.781) and aromatase inhibitor (R76713) on the IPI of pulsatile
GnRH secretion in vitro using (n) 15-d-old rat hypothalamic
explants
Steroid antagonist
None
ICI 182.780 (10⫺7
R76713 (10⫺5 M)
None
M)
Sex
Control
F
F
F
M
59 ⫾ 3 (24)
61 ⫾ 1 (4)
59 ⫾ 6 (4)
60 ⫾ 1 (11)
E2 (10⫺7
M)
44 ⫾ 6a (22)
57 ⫾ 1 (5)
57 ⫾ 2 (11)
T (10⫺7
M)
46 ⫾ 4a (6)
56 ⫾ 6 (4)
51 ⫾ 4 (4)
Data are mean ⫾ SD (min, a P ⬍ 0.05, control vs. E2: t ⫽ 12.4, df ⫽
44; control vs. T: t ⫽ 11.36, df ⫽ 28)
FIG. 4. E2 effect in vitro on the GnRH IPI (mean ⫾ SD)
obtained using (n) hypothalamic explants from female
rats androgenized neonatally through sc injection of
1.25 mg T on postnatal d 1 (upper panel) and from male
rats treated perinatally using 5 mg of the inhibitor of
aromatase ATD injected daily sc to the dams on d
17–21 of gestation and 3 mg to the pups on postnatal
d 1, 6, and 11 (lower panels). *, P ⬍ 0.05, control vs. E2
(5-d-old female rats, vehicle, t ⫽ 2.379, df ⫽ 8; 15-d-old
female rats, vehicle, t ⫽ 2.484, df ⫽ 6; 15-d-old male
rats, ATD, t ⫽ 11;54, df ⫽ 6).
mechanism dependent on perinatal brain sexual differentiation. In addition, E2 administration to immature female rats
in vivo resulted in both stimulation of pulsatile GnRH secretion in the hypothalamus and subsequent precocious puberty. The physiological significance of our data in the normal immature rat was uncertain due to normally limited or
absent exposure to sex steroids in such young animals. However, ultrasensitive E2 assay showed much higher plasma
levels in prepubertal girls than in boys, which was proposed
to be responsible for the faster maturation in girls than in
boys (41). Considering the abnormal early exposure to sex
steroids, our observations could provide a pathophysiological basis for central precocious puberty occurring as a complication of peripheral sexual precocity.
Using the rat model, we explored the possible pathophysiological significance of accelerated hypothalamic maturation induced by abnormal early exposure to sex steroids, a
putative mechanism of central precocious puberty occurring
2780
Endocrinology, June 2004, 145(6):2775–2783
Matagne et al. • Developmental Effect of E2 on GnRH Secretion
secondarily to peripheral precocity (1– 4). In the female rat,
chronic treatment with low doses of E2 between postnatal d
5 and 30 resulted in early peripheral signs of puberty as
shown by early V.O. (42). Because administration of high
doses of E2 before d 10 caused brain masculinization with
loss of estrus cyclicity (19, 31), we elected to use a single E2
administration on d 10 to avoid interfering with this sexually
differentiated process, so that the timing of first estrus cyclicity, a presumably centrally driven pubertal event, could
be studied. The E2 administration in vivo resulted in a reduction of serum LH 5 d after E2 injection, which is in
agreement with pituitary negative feedback effects that characterize immature female rats (42, 43). A decrease in the age
at V.O., as well as at first estrus, was also observed. These
results are in agreement with other findings (32) and point
out that the hypothalamic-pituitary-gonadal axis is sensitive
to E2 maturational effect in vivo both peripherally (V.O.) and
centrally (first estrus). In addition, the study of pulsatile
GnRH secretion in vitro provided evidence of concomitant
advance in the developmental acceleration of GnRH pulse
frequency that could account for subsequent occurrence of
central precocious puberty. This pattern was similar to several clinical conditions such as congenital adrenal hyperplasia (1, 2), sex steroid secreting tumors (3), and gonadotropinindependent sexual precocity (4, 5), where central precocious
puberty occurred after treatment suppressing the peripheral
excess of sex steroids. So, these conditions account for withdrawal of sex steroids after a period of exposure (6) such as
obtained here after an acute massive E2 administration.
E2 effects on GnRH neurons or GnRH secretion in the
hypothalamus were shown in several conditions using different paradigms. Using hypothalamic explants from adult
female monkeys, an increase in GnRH release was observed
in vitro 12 and 48 h after E2 treatment in vivo, whereas LH
secretion was reduced after 12 h and increased after 48 h (44).
Such a discrepancy between E2 effects on GnRH secretion in
TABLE 3. Effect of perinatal inhibition of aromatase activity in
15-d-old male rats; ATD was delivered through silastic implants
with or without (control) aromatase inhibitor
Implants
Empty
ATD
Control
59 ⫾ 1 (3)
60 ⫾ 0 (5)
E2 (10⫺7
M)
58 ⫾ 1 (3)
48.9 ⫾ 2a (6)
Data are mean ⫾ SD (min, a P ⬍ 0.05, E2 vs. control, ATD implants:
t ⫽ 10.73, df ⫽ 3). N (n) explants were used in each condition.
vitro and gonadotropin secretion in vivo were consistent with
our in vivo data showing reduced serum LH levels and increased frequency of pulsatile GnRH secretion 5 d after a
single E2 administration. This suggested a possible coexistence of negative feedback effects at the pituitary level and
stimulating effects in the hypothalamus. Because E2 commonly resulted in inhibition of pulsatile LH secretion, particularly in immature rodents (45, 46), a concomitant stimulating hypothalamic effect could be difficult to demonstrate
through the study of LH secretion. Noteworthy were some
possible inhibitory effects of E2 on the GnRH pulse generator
that could also occur as shown through electrophysiological
recording in the hypothalamus (47). A stimulating effect of
E2 on GnRH output in vivo was described in female rats (48)
and ewes (49). In these experiments, the mass of GnRH
release increased in possible relation to the preovulatory
GnRH and/or LH surge. However, these results were obtained in adult ovariectomized animals (44, 48, 49), a condition different from the present study in intact immature
rats. It is possible that stimulating effects of E2 observed in
vitro persist in the adult female rat but could not be observed
in the conditions of the present study. We showed earlier that
the amplitude of GnRH secretion by hypothalamic explants
obtained around 1600 h in cycling female rats was increased
in the afternoon of proestrus, presumably in relation with the
sex steroid induced preovulatory surge (50). For consistency
and practical reasons, all the experiments reported here were
carried out starting at 1000 h and in the absence of the
preoptic area. Further experiments that start at 1600 h with
explants obtained from adult female rats and including the
preoptic area are warranted to study the possible effects of
E2 on either GnRH pulse frequency and/or amplitude in
vitro. In this study, E2 increased GnRH pulse frequency
within minutes after starting incubation in vitro. Such a rapid
effect suggests a mechanism different from the preovulatory
surge and also deserves further studies. A rapid effect of E2
on LH secretion was reported in the ovariectomized female
guinea pig that was attributed to direct E2 effects on the
pituitary responsiveness rather than on the GnRH output
(51). Our data raises the question as to whether E2 acts
directly on GnRH neurons or on other target structures in the
hypothalamus to increase GnRH pulse frequency. Recently,
GnRH neurons were shown to express ER␣ protein (52) and
ER␤ protein and mRNA (53). Using hypothalamic slices from
GnRH-GFP transgenic mice, E2 was able to decrease the
FIG. 5. Effect of a single sc injection of E2 (10 mg/kg, postnatal d 10) in female rats on GnRH IPI in vitro (n ⫽ 4 in each group, F ⫽ 21.50, df ⫽
2). Measurement of serum LH (n ⫽ 5 in each group, F ⫽ 7.094, df ⫽ 2) as well as age at V.O. (n ⫽ 10 in each group, F ⫽ 133.2, df ⫽ 2) and
age at first estrus (n ⫽ 10 in each group, F ⫽ 31.85, df ⫽ 2). The control groups were injected sc either physiological saline (saline) or vehicle
(sesame oil). Data are mean ⫾ SD. *, P ⬍ 0.05.
Matagne et al. • Developmental Effect of E2 on GnRH Secretion
GnRH neuron firing rate (54) and increase the number of
quiescent GnRH neurons (55), whereas membrane excitability was increased as well (56). Although these observations
support a direct involvement of GnRH neurons in hypothalamic effects of E2, we used retrochiasmatic explants where
GnRH cell bodies were virtually absent (57). Therefore, an
indirect E2 effect on GnRH secretion through interneurons or
glial cells could occur. Such cells have been shown to play an
important role in the control of GnRH secretion throughout
development (58). In addition, an E2 effect could occur in the
terminal part of GnRH neurons because, using median eminence explants, an increased output of basal GnRH release
was observed within 5–10 min of incubation with E2 (36).
Since the onset of puberty involves an activation of hypothalamic glutamatergic inputs to GnRH neurons (59), we
evaluated glutamate receptor involvement in the E2-induced
decrease in GnRH IPI. As expected from the earlier demonstration implicating NMDA receptor activation in pulsatile
GnRH/LH secretion (16, 23), the NMDA receptor antagonist
MK-801 resulted in increased GnRH IPI in vitro. However,
MK-801 did not affect E2 stimulation of an increased GnRH
pulse frequency, suggesting that NMDA receptors were not
primarily involved in the mechanism of E2 effects. Previous
studies showed that non-NMDA receptors are involved in
adult female reproductive life, because nearly half of activated GnRH neurons expressed kainate receptor subunits in
the afternoon of proestrus (60). In addition, the AMPA/
kainate receptor antagonist DNQX can block the E2-induced
LH surge in OVX rats (61). In the present study, E2 stimulation of GnRH pulse frequency was shown to involve specifically the kainate receptor subtype. As reported previously
(24), DNQX alone had no effect on pulsatile GnRH secretion
in vitro. Based on the evidence of an E2-kainate receptor
interaction involved in the control of GnRH secretion, the
absence of kainate receptor antagonist effect on the timing of
puberty could be explained by the silent role of such receptors in the physiological absence of sex steroids in the immature animal. The implication of kainate receptor in E2
effect was further supported by hypothalamic colocalization
of kainate receptor with ER (62). The preferential involvement of kainate receptors in E2 effect on GnRH secretion in
vitro was consistent with the potentiating effect of E2 on
kainate-evoked currents in hippocampal neurons (63) or in
oxytocinergic neurons (37). The developmental disappearance of E2 effect in the female rat hypothalamus could also
be linked to the developmental increase in activity of glutamatergic inputs and/or decrease of GABAergic inputs
shown in the primate (64) and the rodent (10, 11, 35, 65). E2
was reported either to increase ␥-aminobutyric acid (GABA)
turnover in relation with negative feedback effects (66) or to
decrease GABA levels just before the E2-induced LH surge
(67). In addition, kainate down-regulates GABA release in
mediobasal hypothalamic explants from ovariectomized female rats treated with E2 but not in untreated animals (68).
It could be hypothesized that E2 effects were involving the
GABAergic pathway because they were seen only in a period
of high activity of that inhibitory amino acid. Such indications of involvement of the GABAergic pathway in E2 effects
with developmental changes point to the need of further
studies using our in vitro paradigm.
Endocrinology, June 2004, 145(6):2775–2783 2781
An important finding was that the E2 stimulatory effects
on GnRH pulse frequency were only seen using hypothalamic explants obtained from female rats but not using explants obtained from male rats. Such a sex-related effect was
not simply dependent on E2 because T was as effective as E2
in the female, provided that T could be aromatized into E2.
Further confirmation of the key role of E2 came from experiments showing the inhibitory effect of ICI 182.780 on E2
stimulation of GnRH pulse frequency. Such an involvement
of ER was not inconsistent with the rapid, possibly nongenomic effect of E2, because ICI 182.780 was shown to
prevent some rapid E2 effects such as those involving NO
(36). The sexual dimorphism of E2 effects could be related
with the sexually divergent effects of kainate on LH secretion
that was found to be inhibited in female rats and stimulated
in males (69). In addition, the occurrence of E2 effects in the
immature female rat could be related to the sexually dimorphic expression of ER␤ that was reported in the rat hypothalamus before 21 d of age (70). These developmental variations in ER␤ expression are consistent with our observation
that this age period is characterized by a particular sensitivity
to E2 for increase in GnRH pulse frequency.
The dimorphic effects of E2 could depend also on sexual
brain differentiation. If so, we postulated that the E2 effect
should occur in hypothalamic explants from demasculinized
male rats, whereas this effect should be absent using hypothalamic explants obtained from masculinized females. It
was well established that neonatal exposure to T or E2 induced masculinization of female rats, resulting in disappearance of female sexual behavior, absence of the E2-induced
LH surge (30), and the occurrence of a delayed anovulatory
syndrome in adult life (19, 31). In the male, blockade of
aromatase activity can induce appearance of female sexual
behavior (21, 28). Therefore, we used these two strategies to
investigate the possible early programming of the E2 effect
on GnRH pulse frequency that we had confirmed was occurring. Such findings raised the issue of pre- or perinatal
programming of sexually dimorphic events that can be different among species. For instance, in the ovine species, the
timing of puberty is sexually dimorphic and is clearly affected by fetal exposure to T (71).
In summary, evidence is provided in this paper that E2 can
rapidly stimulate an increase in GnRH pulse frequency in the
immature female hypothalamus. It is suggested that such a
mechanism can contribute to some form of sexual precocity,
a hypothesis which deserves further studies.
Acknowledgments
We thank Prof. J. Boniver for assistance of his team in papanicolaou
staining of the vaginal smears. We also thank Dr. J. Bakker for her help
and advice concerning the in vivo demasculinization experiments. We
are grateful to Prof. J. Balthazart for helpful criticisms and constant
support.
Received September 19, 2003. Accepted February 19, 2004.
Address all correspondence and requests for reprints to: Jean-Pierre
Bourguignon, M.D., Ph.D., Division of Pediatric and Adolescent Medicine, CHU Sart-Tilman, B-4000 Liège, Belgium. E-mail: jpbourguignon@
ulg.ac.be.
This work was supported by grants from the French Community of
Belgium (ARC 99/04 –241), the Fonds de la Recherche Scientifique Médicale (3.4515.01), the Faculty of Medicine at the University of Liège, the
2782
Endocrinology, June 2004, 145(6):2775–2783
Belgian Study Group for Pediatric Endocrinology, and the European
Commission supporting the EDEN project.
Matagne et al. • Developmental Effect of E2 on GnRH Secretion
24.
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Genetic Control of Puberty
HORMONE
RESEARCH
Horm Res 2005;64(suppl 2):41–47
DOI: 10.1159/000087753
© Free Author
Early Onset of Puberty: Tracking
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Genetic and Environmentalsonal
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Key Words
Gonadotropin releasing hormone Endocrine
disrupting chemicals Hypothalamic hamartoma Secular trend Dichlorodiphenyltrichloroethane (DDT)
Abstract
Under physiological conditions, factors affecting the genetic control of hypothalamic functions are predominant in determining the individual variations in timing
of pubertal onset. In pathological conditions, however,
these variations can involve different genetic susceptibility and the interaction of environmental factors. The
high incidence of precocious puberty in foreign children
migrating to Belgium and the detection in their plasma
of a long-lasting 1,1,1-trichloro-2,2-bis(4-chlorophenyl)
ethane (DDT) residue suggest the potential role of environmental endocrine disrupting chemicals in the early
onset of puberty. This hypothesis was confirmed by experimental data showing that temporary exposure of immature female rats to DDT in vivo results in early onset
of puberty. We compared the gene expression profile of
hypothalamic hamartoma associated or not with precocious puberty in order to identify gene networks responsible for both hamartoma-dependent sexual precocity
and the onset of normal human puberty. In conclusion,
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pathological variations in the timing of puberty may provide unique information about the interactions of either
environmental conditions or genetic susceptibility with
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hypothalamic
mechanism
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sexual maturation, as shown by examples of precocious
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Introduction
Puberty results from the awakening of a complex neuroendocrine machinery initiated by an unknown primary
mechanism. Sexual maturation is the culmination of a
complex sequence of events that leads to activation of the
gonadotropic axis. The hypothalamus integrates multiple
peripheral and central signals leading to the resurgence of
pulsatile gonadotropin releasing hormone (GnRH) secretion which controls the secretion of luteinizing hormone
and follicle stimulating hormone.
Abnormally precocious sexual development has been
defined as the occurrence of Tanner stage B2 before 8
years in girls and Tanner stage G2 before 9 years in boys.
However, two recent studies in the United States [1, 2],
reviewed by Lee et al. [3], highlighted an unexpected ad-
Dr. Jean-Pierre Bourguignon
Division of Paediatric and Adolescent Medicine
Centre Hospitalier Universitaire Sart-Tilman
BE–4000 Liège (Belgium)
Tel. +32 43 667247, Fax +32 43 667246, E-Mail [email protected]
vance in physiological age at the onset of breast development, thus raising the question of a possible advancement
in the timing of puberty. Recently updated Belgian data
indicate that menarcheal age has remained stable for the
past 20 years at around 13 years of age but that the third
centile for stage B2 in girls and 4 ml testicular volume in
boys has decreased to 7.7 and 7.6 years, respectively [4].
In addition, early pubertal development and an increased
incidence of sexual precocity have been noticed in children migrating to a number of western European countries [5]. These differences in timing of puberty seem to
result from interactions of environmental factors irrespective of genetic susceptibility, since children migrating
from several continents and belonging to several ethnic
groups are involved [5].
Hypothalamic hamartoma is a condition frequently
associated with severely precocious puberty possibly resulting from overexpression of some central genes independently of environmental factors.
We have chosen two conditions associated with precocious puberty, i.e. hypothalamic hamartoma and migration after exposure to endocrine disrupting chemicals, in
order to explore some of the possible environmental and
genetic factors likely involved in early sexual maturation.
Environmental Factors Possibly Involved in
Early Onset of Puberty
Environmental effects on the mechanism of pubertal
onset may start during intrauterine life. Some studies
have reported an earlier menarcheal age in girls with low
birth weight or in utero growth retardation [6, 7]. In the
general population, however, some authors found no significant correlation between birth weight and menarcheal age [8], while others reported that thin newborns entered puberty earlier [9, 10].
Nutrition is likely to play a key role in the timing of
puberty and could explain, at least partially, the downward secular trend in the timing of puberty [5]. This is
suggested by the direct relationship between body
weight and age at onset of puberty [11, 12]. Also, girls
with early menarcheal age are more likely to be obese
[8]. However, the relation between fatness and menarcheal age could be either causal or consequential and
may involve genetic participation. Leptin, insulin-like
growth factor-I and glucose have been shown to be involved in the control of GnRH secretion, but their role
in the timing of puberty remains controversial [5].
42
Horm Res 2005;64(suppl 2):41–47
Leptin, however, appears to be a permissive factor important in physiological conditions and critical in some
disorders, with an effect independent of body weight in
hypothalamic amenorrhea [13]. The effects of fat mass
may also interact with the effect of some endocrine disrupters that have a high affinity for lipids and are stored
in fat tissue, thus creating conditions for persistence of
systemic effects.
Precocious Puberty in Migrating Children as a Model
for Studying Environmental Factors Involved in the
Onset of Puberty
Following an initial report from Sweden [14], sexual
precocity has been described in children migrating from
developing countries, primarily through international
adoption [5]. In some of these studies, cohorts of foreign
adopted children were evaluated: not only was the absolute frequency of sexual precocity increased but advanced puberty was also a general feature (fig. 1) instead
of precocity being a feature of a particular subset of cohorts. The differences in country of origin were unlikely
to bias the findings, because in these studies the average
menarcheal age was advanced in comparison with data
from the foster countries as well as from the countries
of origin [5]. In addition to the cohort studies of foreign
adopted children, additional evidence of early pubertal
timing was provided by the observation of sexual precocity in individual foreign adopted patients described
as an entity in Italy [15] and in France [16] or in comparison with the whole group of patients seen for central
precocious puberty in Copenhagen [17] and in Belgium
[18]. In this latter country, foreign migrating children
represented 28% of patients seen with central precocious
puberty, accounting for an 80-fold increased risk of sexual precocity in comparison with native Belgian children [18]. The migrating children came from different
ethnic and national backgrounds and were migrating
either for international adoption or together with their
original family and without any history of deprivation.
It has been hypothesized that moving to Belgium could
result in a change in exposure to endocrine disrupting
chemicals when a child moves from the home country
to the foster country, thus causing the occurrence of sexual precocity.
Endocrine disrupting chemicals are widespread environmental substances that have been introduced by man
and may influence the endocrine system in a harmful
manner [19, 20]. They may play a role in disorders of
human sex differentiation and in alterations to the reproductive organs and functioning. Screening for eight
Parent /Rasier /Gerard /Heger /Roth /
Mastronardi /Jung /Ojeda /Bourguignon
Fig. 1. Average (mean or median) age at
menarche in foreign adopted children compared with average age at menarche in the
foster countries and the countries of origin.
NCHS = National Center for Health Statistics.
organochlorine pesticides in the serum of foreign migrating children with precocious puberty revealed the presence of 1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene
(p,p-DDE), a persistent derivative of the pesticide DDT,
while p,p-DDE levels were undetectable in the serum of
native Belgian patients. DDT, although banned in the US
and western Europe, is still in use in developing countries
and behaves as an oestrogen agonist or androgen antagonist [21, 22]. A pathophysiological mechanism of precocious puberty in these children has been proposed [5, 18]
and is schematically depicted in figure 2: DDT, like oestradiol [23, 24], is able to promote hypothalamic maturation, exerting an inhibitory effect at the pituitary level
that is most effective in prepubertal individuals. This prevents the gonadal manifestation of hypothalamic effects
and signs of sexual maturation. Migration may interrupt
exposure to endocrine disrupters and precocious puberty
might then result from withdrawal of their negative feedback effect and/or from accelerated hypothalamic maturation. A stimulatory effect of sex steroids at the hypothalamic level is suggested by our experimental data. Oestra-
diol increased the frequency of pulsatile GnRH secretion
from hypothalamic explants of 15-day-old female rats.
This stimulatory effect was preferentially seen in immature female rats and was observed neither in older females
nor in males [23]. Such sexual dimorphism, which is dependent on perinatal brain sexual differentiation, is interesting to note since precocious puberty affects girls
more often than it does boys for reasons which are still
unclear. Moreover, when oestradiol was administered in
vivo between days 5 and 10, pulsatile GnRH secretion
was accelerated, which is typical of the process of hypothalamic maturation, and precocious puberty was observed with early vaginal opening and first oestrus [23,
24]. Similarly, DDT accelerated pulsatile GnRH secretion in immature female rats and induced precocious puberty when administered in vivo [24]. DDT effects, like
oestradiol effects [24, 25], are mediated by oestrogen receptors and involve the kainate subtype of glutamate receptors. Moreover, DDT also acts through the orphan
dioxin receptor [24].
Precocious Puberty: Genetic and
Environmental Factors
Horm Res 2005;64(suppl 2):41–47
43
Fig. 2. Pathophysiological relevance of endocrine disrupting chemicals in precocious puberty observed in migrat-
ing children. LH = Luteinizing hormone; FSH = follicle stimulating hormone.
Genetic Factors Possibly Involved in Early
Onset of Puberty
Evidence for genetic regulation of puberty is provided
by studies showing a correlation between the ages at
which a mother and her daughter attain puberty [5, 26],
as well as twin correlation studies indicating that 70–80%
of the variance in pubertal timing can be explained by
genetic factors [5, 26]. Population studies showing that
age at puberty varies among ethnic groups also suggest a
genetic control of puberty. The genetic control of the variance in pubertal timing is likely to be a complex polygenic trait [26] and those genes still remain to be discovered. Some candidate genes, such as those controlling sex
steroid biosynthesis, action and metabolism, have been
found to exhibit polymorphisms associated with possible
variations in pubertal timing in a given population. In
Japan, early menarche was linked to the A2 polymorphism of the CYP17 gene controlling androgen biosynthesis and thereby possibly accounting for increased serum oestradiol levels [27]. However, in American girls,
the CYP17 alleles were not associated with early breast
44
Horm Res 2005;64(suppl 2):41–47
development. Instead, this event was strongly associated
with the A4 allele of CYP3, an enzyme involved in testosterone catabolism [28]. More recently, evidence has
been presented showing that familial central precocious
puberty is determined by an autosomal dominant mode
of transmission with incomplete penetrance affecting
mostly girls [29].
Krewson et al. [30] assessed a panel of chromosome
substitution strains in mice and reported that the timing
of vaginal opening differed between two inbred strains.
Their findings showed that chromosome 6 and 13 harbour quantitative trait loci regulating pubertal timing in
mice. This lays the foundations to map these loci and establish the identities of the genes responsible. Moreover,
this new tool could lead to the discovery of new genes involved in sexual maturation in humans.
The central activation of puberty requires the participation of neuronal and astroglial networks regulated
by upstream transcriptional factors. Some of these upstream genes have recently been identified. Oct-2, a
POU domain gene originally described in cells of the immune system, transactivates transforming growth factor
Parent /Rasier /Gerard /Heger /Roth /
Mastronardi /Jung /Ojeda /Bourguignon
(TGF)- promoter in glial cells and thus activates a gene
involved in facilitating the onset of puberty. Oct-2 mRNA
transcripts increase during juvenile development before
puberty takes place and brain lesions inducing puberty
result in the rapid and selective increase in Oct-2 transcript in TGF--containing astrocytes surrounding the
lesion [31].
A role for the GPR54/KiSS-1 system recently emerged
when two independent reports showed that mutations
and deletions of the GPR54 gene were found in patients
suffering from idiopathic hypogonadotropic hypogonadism [32, 33], and this condition has been reproduced in
mice carrying a disrupted GPR54 locus [33]. In addition,
recent studies have shown that GPR54 gene expression
increases in the mouse hypothalamus at puberty [34], and
that the expression of both the GPR54 gene and the gene
encoding the KiSS precursor increase in the non-human
primate hypothalamus at the time of puberty [35]. Furthermore, chronic central administration of KiSS-1 has
now been shown to precociously activate the gonadotropic axis [34, 35].
One of our laboratories has compared gene expression
profiles between the cortex and the hypothalamus and
identified changes that are specific to the neuroendocrine
brain throughout puberty in the non-human primate.
These results support the concept that the onset of puberty requires concerted changes in gene transcription
and intracellular signalling cascade but also activation of
cell-cell communication pathways that may be critical for
neuron-neuron and glial-neuron information transfer.
Two genes showed a robust increase throughout puberty:
TTF-1 and a novel gene called chromosome 14 open reading frame 4 (C14ORF4). TTF-1 is a homeodomain gene
of the Nkx family that has been implicated in the control
of female reproductive capacity [36]. C14ORF4, now
named EAP1 (enhanced at puberty-1), appears to be a
transcriptional regulator of genes involved in the transsynaptic control of GnRH secretion [37]. Although the
involvement of these genes in precocious puberty has not
yet been demonstrated, they represent interesting candidates since they are part of the gene network leading to
sexual maturation.
Hypothalamic Hamartoma as a Model for Studying
Genetic Factors Involved in the Onset of Puberty
Hamartomas are rare congenital non-neoplastic lesions containing mature brain tissue in a heterotopic location. They are almost exclusively located at the base of
the hypothalamus. In most cases, they contain neurons
and astroglial cells of normal aspect in addition to epen-
Precocious Puberty: Genetic and
Environmental Factors
dymoglial-like cells. Astrocytes seem to be increased.
Neuronal processes reaching adjacent structures are frequently observed [38]. Most hypothalamic hamartomas
are symptomatic and are associated with precocious puberty [39] and/or gelastic seizures. Sexual precocity caused
by a hypothalamic hamartoma occurs at a much earlier
age than idiopathic precocious puberty of central origin
[40]. The mechanism by which hypothalamic hamartomas cause precocious puberty is unknown and they represent a model of choice to identify the genes involved in
the early onset of puberty and, subsequently, those involved in the physiological onset of puberty.
Several mechanisms have been proposed to explain
precocious puberty in hypothalamic hamartomas, including mechanical pressure, autonomous GnRH secretion, transsynaptic activation via myelinated fibres connecting the hamartoma to the hypothalamus, and secretion of glial products able to stimulate the patient’s GnRH
neuronal network [41].
Some hypothalamic hamartomas do not induce sexual
precocity despite a hypothalamic location similar to that
of hamartomas associated with precocious puberty. This
observation argues against the idea of mechanical factors
underlying hypothalamic hamartoma-induced precocious puberty. Moreover, this activation of puberty occurs only if the lesion affects areas of the hypothalamus
near to the GnRH neuronal network.
The detection of GnRH neurons in some hypothalamic hamartomas [42] led to the hypothesis that these neurons represented neurosecretory cells able to function independently, activate endogenous GnRH secretion and
induce premature sexual maturation [42, 43]. However,
Jung et al. [44] reported two cases of hypothalamic hamartoma associated with precocious puberty in which no
GnRH neurons were found. Instead of containing GnRH
neurons, the hamartomas displayed astrocytes expressing
TGF- and its erbB1 receptor. TGF- is known to mediate the facilitatory effect of astroglial cells on GnRH secretion [45]. Moreover, cells genetically engineered to
produce TGF- were found to induce sexual maturation
in female rats when grafted near GnRH nerve terminals
or GnRH cell bodies [46]. Hypothalamic hamartomas
have been found to produce several neuropeptides in addition to GnRH and TGF- [41].
Because hypothalamic hamartomas might express the
same transcriptional and signalling networks required for
the initiation of normal puberty, we compared the gene
expression profile of a hamartoma associated with precocious puberty with three hamartomas not accompanied
by advanced sexual maturation [47]. The hamartomas
Horm Res 2005;64(suppl 2):41–47
45
were surgically removed due to intractable seizure activity. Total RNA was extracted, amplified, labelled and hybridized to a microarray chip containing 18,400 genes.
Hierarchical cluster analysis identified a subset of genes
whose expression was increased at least twofold in the
hamartoma with precocious puberty compared with each
of the other three hamartomas without precocious puberty. These genes encode proteins involved in three key
cellular processes: transcriptional regulation, cell-cell signalling and cell adhesiveness. Thus, they may be part of
biological complex relevant to the ability to induce puberty. It is interesting to note that some of these genes
were first described as tumour-related genes. Similarly,
microarrays of hypothalamic gene expression in the monkey identified a subset of tumour-related genes increased
at onset of puberty [48].
Conclusion
Pathological variations in the timing of puberty may
provide unique information about the interactions of either environmental conditions or genetic susceptibility
with the hypothalamic mechanism regulating the onset of
sexual maturation. Normal individual variability in the
timing of puberty involves familial, ethnic and gender
patterns and is likely to depend on genetic control of the
expression of signals or signal receptors in the hypothalamus. This physiological process is likely to be less influ-
enced by environmental factors. These environmental
factors may play a prominent role in situations such as
the secular advancement in onset of breast development
seen in some industrialized countries or the increased incidence of precocious puberty in children migrating to
such countries. Another condition associated with precocious puberty is hypothalamic hamartoma containing the
key transcriptional and signalling networks required to
initiate the pubertal process. Hypothalamic hamartomas
provide us with valuable hints towards the identification
of gene networks responsible for both hamartoma-dependent sexual precocity and the onset of normal human
puberty.
We have mentioned the potential role of DDT in precocious puberty in migrating children, but there are several other endocrine disrupters still to be studied. Similarly, the study of hypothalamic hamartoma gene profile
expression has allowed us to identify some of the potential
factors involved in precocious puberty amongst several
other possible candidates.
Acknowledgments
Anne-Simone Parent is a scientific research worker for the
Fonds National de la Recherche Scientifique, Belgium. This work
was supported by the Fonds de la Recherche Scientifique Médicale
(grant 3.4515.01), the Belgian Study Group for Paediatric Endocrinology, and the European Commission (contract no. QLRT-200100269).
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Precocious Puberty: Genetic and
Environmental Factors
Horm Res 2005;64(suppl 2):41–47
47
Molecular and Cellular Endocrinology 254–255 (2006) 187–201
Female sexual maturation and reproduction after prepubertal
exposure to estrogens and endocrine disrupting chemicals:
A review of rodent and human data
G. Rasier a , J. Toppari b , A.-S. Parent a , J.-P. Bourguignon a,∗
a
Developmental Neuroendocrinology Unit, Center for Cellular and Molecular Neurobiology, University of Liège,
University Hospital Center, B36, +1, B-4000 Liège (Sart-Tilman), Belgium
b Departments of Physiology and Pediatrics, University of Turku, FI-20520 Turku, Finland
Abstract
Natural hormones and some synthetic chemicals spread into our surrounding environment share the capacity to interact with hormone action and
metabolism. Exposure to such compounds can cause a variety of developmental and reproductive detrimental abnormalities in wildlife species and,
potentially, in human. Many experimental and epidemiological data have reported that exposure of the developing fetus or neonate to environmentally
relevant concentrations of some among these endocrine disrupters induces morphological, biochemical and/or physiological disorders in brain
and reproductive organs, by interfering with the hormone actions. The impact of such exposures on the hypothalamic–pituitary–gonadal axis and
subsequent sexual maturation is the subject of the present review. We will highlight epidemiological human studies and the effects of early exposure
during gestational, perinatal or postnatal life in female rodents.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Female puberty; Endocrine disrupters
1. Introduction
Since the 1990s, the plausibility that some environmental
and industrial chemicals could cause a number of disorders in
hormonally regulated biological systems and, ultimately, affect
harmfully the human, has preoccupied biologists, toxicologists
and epidemiologists. In addition, the time of exposure during
development is a clue since evidence has accumulated that disturbances of the (very) early life environment have an impact on
the child and, further, adult health. Those adverse substances,
which are widespread in our surrounding environment, are called
“endocrine disrupting chemicals (EDCs)” or “endocrine disrupters”. They interfere with the endocrine system and thus
may affect the development and/or reproduction in many animal species (Colborn et al., 1993; Marshall, 1993; Toppari et al.,
1996). They consist of two groups: natural substances such as
∗ Corresponding author at: Division of Pediatric and Adolescent Medicine,
University Hospital Center, B-4000 Liège (Sart-Tilman), Belgium.
Tel.: +32 4 366 72 47; fax: +32 4 366 72 46.
E-mail address: [email protected] (J.-P. Bourguignon).
0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2006.04.002
animal hormones and phytoestrogens, and man-made industrial
chemicals, including pesticides, phenol derivatives, phthalates,
dioxins, . . ..
Many EDCs have an estrogenic activity. Mechanistic experiments indicate that these chemicals interact with the estrogen
receptors (ERs) and stimulate or inhibit some sensitive cell types
in a number of different ways (Ferguson et al., 2000; Gutendorf
and Westendorf, 2001; Holmes et al., 2004). There are also many
compounds which exhibit other effects, including antiestrogenic, androgenic or antiandrogenic. The compounds may act
on the receptor level or influence the synthesis and metabolism
of hormones, receptors and transcription factors. They can affect
the hypothalamic–pituitary–gonadal (HPG) axis, with consequences on sexual development and functioning (Clark et al.,
1998; Gray, 1998; Toppari and Skakkebaek, 1998; Takeyoshi et
al., 2002; Toppari, 2002).
In this review, we will briefly describe the processes of sexual
differentiation and maturation in the rodent. The mechanisms of
central versus peripheral puberty will be opposed and the reasons
for using the immature female rodent to study EDC effects will
be discussed in the light of our previous clinical observations.
We will then review the data obtained in rodents and humans
with different chemicals.
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Fig. 1. Schematic representation of the main pubertal events in the female. Comparison between laboratory rat (A) and human (B) based on a similar lifespan
time scale.
2. Comparative sexual differentiation and maturation
in female rodents and humans
The reproductive system of female rodents (primarily rats
and mice) shares a number of characteristics with the human.
Consequently, these animals have been extensively used in biological research and are routinely employed in laboratory studies
designed to elucidate the mammalian mechanisms of reproductive development and functions. The murine female puberty
is a transitional process which encompasses vaginal opening
(VO) and first ovulation (Fig. 1A). VO is the initial sign of the
estrogenic rise that accompanies the onset of puberty and first
ovulation followed by estrous cyclicity as the initial sign of a central drive of ovarian activity (Ramirez and Sawyer, 1965). The
corresponding events in the human are the onset of breast development (stage B2) and menarche, respectively. In the rat, the
mean age at VO varies among the strains and with raising conditions. It usually takes place around 35 days. The mean interval
between VO and first estrus is 4.2 ± 1.1 days. In the laboratory
rat, sexual maturation starts very early in life, when compared
with the human female on a time scale determined by the lifespan (Fig. 1A and B) and the individual variations in timing of
pubertal events are comparatively much less than observed in the
human. The occurrence of the first ovulatory episode is an ovarian response to a massive secretory surge of the gonadotrophins
(follicle-stimulating hormone and luteinizing hormone, FSH and
LH). In the rodent, programming of the central mechanism of
ovulation takes place between the last week of prenatal life and
the first week of postnatal life (Naftolin, 1994). Early exposure
to sex steroids causes disturbances in that process (Matagne et
al., 2004) whereas, in the human female, there is no convincing
evidence of a similar mechanism (Grumbach and Styne, 2003).
3. Central versus peripheral puberty
The mechanism of onset of female puberty has been reviewed
recently by Ojeda and Terasawa (2002). There are several
endpoints that involve two different pathophysiologic mechanisms. In central puberty, estrogens are produced following
ovarian stimulation by the HP system (Fig. 2A). In peripheral
puberty, endogenous estrogens are produced in a gonadotrophinindependent manner (Fig. 2B) or they come from exogenous
administrations. The study of sex steroid sensitive tissues such
as uterus, vagina and breasts or anogenital distance (AGD)
for estrogenic effects (e.g. VO) may provide an insight into
central as well as peripheral puberty. The study of pituitary
gonadotrophins and hypothalamic gonadotrophin-releasing hormone (GnRH) provides information on the central components
and the possible effects of steroids and EDCs at those levels. In
a previous study involving some among us, central sexual precocity was reported to occur very frequently in foreign migrating girls, as a possible consequence of early and temporary
exposure to dichlorodiphenyltrichloroethane (DDT) (KrstevskaKonstantinova et al., 2001; Parent et al., 2003). As illustrated
schematically in Fig. 2C and D, it was hypothesized that, during exposure to an estrogenic EDC, hypothalamic maturation
could be stimulated while pituitary gonadotrophins are inhibited
through a negative feedback effect. This prevents any manifestation of central maturation and only peripheral puberty can thus
be observed (Fig. 2C). Migration causes withdrawal from the
EDC and the consequent pituitary inhibition disappears, allowing the hypothalamic maturation to turn on the pituitary–ovarian
cascade of pubertal events, i.e. central puberty (Fig. 2D).
4. Prepubertal exposure to EDCs: female study
endpoints in rodents
In consistency with the above observations, we were interested in delineating the central effects of sex steroid and EDC on
the HP mechanism of puberty. In the rat, GnRH can be detected
in the hypothalamus by the gestation day (GD) 12 and the levels of this decapeptide gradually increase until a few days after
the parturition (Aubert et al., 1985). A steep rise takes place
through the second suckling week, followed by a further increase
that continues until puberty (Chiappa and Fink, 1977). Moore
and Wray (2000) indicated that GnRH neurons in the olfactory
placode can already exhibit neuroendocrine secretory properties. Indeed, embryonic GnRH neurons from nasal explants
were capable of synthesizing, secreting and rapidly replenishing stores of GnRH peptide, and Wetsel et al. (1991) showed
that the immature GnRH-secreting GT1-7 cell line secreted
pro-GnRH. The postnatal ontogeny of frequency of pulsatile
GnRH secretion in rat hypothalamic explants revealed a developmental acceleration before 21 days of age (Bourguignon and
Franchimont, 1984) while others found such an increase to occur
later in vivo (Sisk et al., 2001; Harris and Levine, 2003). Data
from Ford and Ebling (2000) showed that GnRH transcript level
increased markedly between birth and postnatal day (PND) 12
and that endogenous glutamatergic signal played a role as potential regulator during development, allowing maintenance of a
sufficient level in adulthood (Gore et al., 1999). These data indicate that GnRH synthesis is not a rate-limiting factor for the
onset of puberty. Such a concept is in agreement with our observation that an adult pattern of pulsatile GnRH secretion from
G. Rasier et al. / Molecular and Cellular Endocrinology 254–255 (2006) 187–201
189
Fig. 2. Schematic illustration of the hypothalamic–pituitary–gonadal (HPG) axis function in different conditions: stimulation in physiological or precocious central
puberty (A), inhibition in peripheral puberty due to steroids of extra-gonadal origin (B), hypothalamic stimulation and PG inhibition in the presence of an estrogenic
EDC (C) and HPG stimulation after withdrawal from the EDC (D).
neonatal hypothalamic explants can be obtained during intermittent stimulation by glutamate (Parent et al., 2005). Indeed,
an increase in glutamatergic input to GnRH neurons plays a role
in the elevated GnRH release and gene expression that occurs
at the initiation of puberty (Gore et al., 1996). Then, the responsiveness of the GnRH neurons to neurotransmitter stimulations
becomes enhanced, as a potent inhibitory influence of estradiol (E2) declines (Docke et al., 1981; Ojeda et al., 1986). The
effects of steroids or some EDCs on such endpoints will be discussed below. In our laboratory, the developmental increase in
frequency of pulsatile GnRH secretion in vitro has been shown
to be particularly sensitive to sex steroids administered in vitro
or in vivo around the age of 10–15 days (Matagne et al., 2004).
Thus, the immature female rat provides a condition with high
sensitivity of the HP unit to the inhibitory and stimulatory effects
of estrogens.
In the human, preliminary observations indicate that onset
of puberty can be vulnerable to environmental effects on the
neuroendocrine processes, culminating in the emergence of disorders in mature reproductive functions (Parent et al., 2003).
The pubertal alterations can be either an advancement or a
delay of maturation, maybe depending on the chemical and
the conditions of the insult (age at exposure, doses, mixture
effects, gender). In experimental conditions, the sensitivity to
these effects can vary among species. Here, we will focus on
the influence of several types of EDCs on the HPG axis after
prepubertal (gestational, perinatal and/or postnatal) exposure of
the female rodent in vivo. More specifically, we will address the
impact on sexual maturation and the reproductive tract after such
exposures, with emphasis on the comparative effects of low or
high doses, prolonged or acute exposure and subcutaneous (s.c.)
or oral administration. EDC impact using in vitro hypothalamic
or pituitary explant and GnRH neuronal cell line models will
also be discussed.
5. Effects of different EDCs on sexual maturation and
reproductive functions
5.1. E2, a reference natural estrogen
E2 is the main natural estrogenic hormone in the female. It
is principally secreted by the ovaries and is known to exert a
vast number of endocrine and metabolic functions in the mammals, notably on the HP unit during development and on the
estrous cyclic activity. It was shown that the administration of
ovarian steroids (E2 and progesterone) in ovariectomized rats
not only influenced the release of hypothalamic GnRH but also
the processing of GnRH precursor forms (Drouva et al., 1986).
These effects involve a negative (inhibitory) feedback control
of GnRH and the pituitary gonadotrophins which is very potent
in the immature individual (Grumbach and Styne, 2003). In the
mature female, a so-called positive (stimulating) feedback is
also exerted by E2 and leads to the preovulatory GnRH and
gonadotrophin (FSH and LH) surge (Levine, 1997; Terasawa,
2001).
E2 has been usually administered postnatally starting either
at birth or by PND 5–12, i.e. at the end of the critical period of
sex differentiation or during the third week of life (Table 1). In all
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Table 1
Data on effects of estradiol on several hypothalamic–pituitary–ovarian outcomes
Age at treatment
Dose
(per kg day)
VO
PND 1–10
10 ␮g
Advanced
PND 1–21
PND 5-VO
PND 10
2 mg
0.5 ␮g
10 mg
Advanced
Advanced
Advanced
PND 12-VO
PND 21–23
10 ␮g
Advanced
Advanced
PND 23–26
PND 25–42
PND 26-VO
5 ␮g
0.3 ␮g
0.5 ␮g
Advanced
Advanced
Advanced
1st estrus
Cyclicity
HP axis
Irregular then
persistent estrus
Advanced
Advanced
Irregular then
persistent estrus
Ref.
Kato et al. (2003)
LH
GnRH pulse frequency
FSH/LH response to GnRH
Advanced
Uterus
weight
Marty et al. (1999)
Ramirez and Sawyer (1965)
Matagne et al. (2004)
Nass et al. (1984)
Ashby et al. (1997a,b),
Odum et al. (1997)
Korach et al. (1978)
Edgren et al. (1966)
Ramirez and Sawyer (1965)
PND: postnatal day; VO: vaginal opening; HP: hypothalamic–pituitary.
instances, VO was advanced as a possible result of either direct
peripheral effects or early HPG maturation or both. The age at
first estrus was usually not mentioned except in two studies. In
the pioneering study of Ramirez and Sawyer (1965), injections
of E2-benzoate (E2B, 0.5 ␮g/kg day) starting from PND 5 or 26
until VO in rats caused an advancement in age at both VO and
first estrus. When administered after PND 20, E2 also advanced
the age at VO (Edgren et al., 1966; Ashby et al., 1997a,b; Odum
et al., 1997). Early VO and first estrus were also observed in our
laboratory after a single massive dose of E2 (10 mg/kg day) given
on PND 10 (Matagne et al., 2004). Cycling disturbances were
found after treatment during the first 10 postnatal days (Kato et
al., 2003) as a possible result of disturbed sex differentiation of
central nervous system. However, cycling disorders were seen
as well after treatment between PND 12 and VO, suggesting
another additional mechanism. Evidence of pituitary inhibition
has been obtained after E2 treatment starting on PND 12 since
gonadotrophin response to GnRH was diminished by 5 months
of age (Nass et al., 1984). Studies using immortalized GnRHsecreting cell line GT1-7 revealed that E2 down-regulated GnRH
transcript levels, indicating also a hypothalamic inhibition (Roy
et al., 1999). However, E2 was found to stimulate GnRH secretion by these GT1-7 cells, an effect mediated by transforming
growth factor (TGF)␤1 derived from astrocytes (Zwain et al.,
2002). Recently, a stimulation of GnRH pulse frequency was
observed using hypothalamic explants of immature female rats
either incubated with E2 in vitro or after E2 administration
in vivo (Matagne et al., 2004). Scarce data on pituitary function were obtained. After E2 treatment between PND 5 till VO,
increased plasma LH was seen on PND 30 (Ramirez and Sawyer,
1965). Obviously, the interpretation of those data is difficult
due to differences in age at treatment, dose and age at study.
Korach et al. (1978) showed that uterus weight was doubled
after injection of 5 ␮g/kg day of E2 between PND 23 and 26.
Other studies reported also an uterine weight increase after later
treatment (Ramirez and Sawyer, 1965; Gould et al., 1998). In
a single study of rats fed early (from birth to weaning) with
E2 (2–4 mg/kg day), decreased uterine weights were observed
(Marty et al., 1999). However, as discussed below, early treatment with other estrogenic compounds resulted in uterine weight
increase. These data point to a prominent and presumably direct
stimulatory peripheral effect in the case of late treatment and,
possibly, after early treatment as well.
Structural changes can also occur in the ovaries and peripheral
estrogen sensitive organs. After 10 ␮g/kg day of E2 was administered between PND 1 and 10, polycystic ovaries were observed
on PND 80 (Kato et al., 2003). In 2001, Ikeda et al. showed that a
neonatal exposure to E2B (1 mg/kg day, PND 1–5) caused disorders in ovarian development and differentiation before PND 21.
Several studies in mice exposed during the same period reported
that E2 affected the mammary gland development and structure
in the immature and adult animals (Mori et al., 1976; Warner,
1976; Tomooka and Bern, 1982; Bern et al., 1983; DiPaolo and
Jones, 2000). An in utero exposure of late fetal rats (GD 16–20)
to E2B (50 ␮g/kg day) caused a smaller AGD in the offspring at
birth (Levy et al., 1995).
5.2. Two synthetic estrogens: diethylstilbestrol (DES) and
ethynylestradiol (EE2)
The clinical concept of EDCs has arised from the longterm adverse effects of DES. This synthetic nonsteroidal estrogenic chemical has been administered in high doses to pregnant
women as an antiabortive medication to prevent miscarriage
and other pregnancy complications between 1938 and 1971
in the United States (U.S.). By that year, the U.S. Food and
Drug Administration issued a warning about its use after having found a relationship between an in utero exposure to this
synthetic estrogen and the development of clear cell adenocarcinoma of the vagina and cervix in young women whose mothers
had taken this substance while they were pregnant (Herbst and
Anderson, 1990). Although DES has not been given for more
than about 30 years, its effects continue to be seen. Indeed,
women who were exposed to it through their mother display
structural reproductive tractus anomalies and an increased infertility rate (Merino, 1991; Schrager and Potter, 2004). DES has
been considered as a paradigmatic compound for estrogenic
EDCs and is often used as reference to study the effects of other
putative estrogenic substances. It does not bind ␣-fetoprotein,
an estrogen-binding plasma protein, and can thus bind ERs in
G. Rasier et al. / Molecular and Cellular Endocrinology 254–255 (2006) 187–201
191
Table 2
Data on effects of diethylstilbestrol on several hypothalamic–pituitary–ovarian outcomes
Age at treatment
GD 1–21
GD 1–PND 21
GD 9–16
GD 11-PND 20
PND 1–5
Dose (␮g/kg day)
5
6.5
VO
Cyclicity
HP axis
Advanced
Irregular
Irregular
FSH/LH
Uterus weight
Levy et al. (1995)
Kubo et al. (2003)
Newbold et al. (1983)
Newbold et al. (1998)
Haney et al. (1984), Newbold et al. (1983)
0.01
2.5
100
15
0.002
PND 12-VO
PND 21–23
PND 20–22
0.2
PND 21–40
5
PND 23–26
7
Ref.
Irregular
Irregular
then
Advanced
Irregular
Irregular then persistent
estrus or diestrus
Advanced
Kwon et al. (2000), Mori et al. (1976)
Huseby and Thurlow (1982), McLachlan et
al. (1982), Tomooka and Bern (1982),
Newbold et al. (1998), Branham et al. (1988)
Nass et al. (1984)
Sharpe et al. (1995), Ashby et al. (1997a,b),
Cagen et al. (1999), Nagao et al. (2000),
Atanassova et al. (2000)
Kim et al. (2002)
Irregular then persistent
estrus
Irregular then persistent
estrus
Korach et al. (1978)
GD: gestational day; PND: postnatal day; VO: vaginal opening; HP: hypothalamic–pituitary.
the fetal tissues much more actively than endogenous estrogens
(McLachlan and Newbold, 1987).
As expected from the clinical observations made using DES,
experimental administration of this compound often started in
prenatal life though postnatal treatment was considered as well
(Table 2). Irrespective of the period of treatment, VO was found
to be advanced by DES when studied (Nass et al., 1984; Kim et
al., 2002; Kubo et al., 2003). Disturbances in estrous cyclicity
were commonly observed when DES was administered during
either gestational or postnatal period or both. The animals displayed irregular cycles followed by persistent estrus or diestrus,
causing reduced fertility (0.01 ␮g/kg day) or complete sterility
(100 ␮g/kg day), depending on DES dose. Exposure during fetal
life and suckling period (6.5 ␮g/kg day) caused a decrease in
FSH and LH secretion at 3 months of age, indicating a negative feedback effect on the HP axis (Kubo et al., 2003). The
uterotrophic response to DES was evidenced in many studies,
uterine weight being doubled even after a short period of postnatal exposure (PND 20–22 or 23–26, 0.2–7 ␮g/kg day). Uterine
abnormalities were also observed when 0.01–100 ␮g/kg day of
DES were given prenatally (McLachlan et al., 1982; Newbold et
al., 1983, 1984) and after a PND 1–5 exposure to 0.01 ␮g/kg day,
resulting in permanent disorders of the reproductive ability
(Branham et al., 1988; Halling and Forsberg, 1993).
Some investigations focused on DES effects on the ovarian
histoarchitecture. Anovulatory ovaries were observed (Döhler et
al., 1984; Tarttelin and Gorski, 1988; Vancutsem and Roessler,
1997). Haney et al. (1984) reported that mice exposed in utero
to 100 ␮g/kg day displayed an enlargement of the ovarian interstitial compartments at 3 months of age. Others showed that
administration of DES in mice between PND 1 and 5 affected
the growth of mammary glands (Mori et al., 1976; Warner, 1976;
Huseby and Thurlow, 1982; Tomooka and Bern, 1982; Bern et
al., 1983; DiPaolo and Jones, 2000). When pregnant mice were
fed with DES from GD 11 to GD 17, the AGD of the offspring
at birth was decreased (Palanza et al., 2001).
EE2 is actually the most frequently used estrogenic component of the anovulatory contraceptive pills in women. This
synthetic estrogenic compound is eliminated out of the body
through the urines (Bolt et al., 1973; Bolt, 1979), thus contaminating the environment via the waste water. The experimental
EE2 effects were mainly tested postnatally. In a study where
age at VO was assessed, an advancement was observed after
100 ␮g/kg day, PND 21–35 (Laws et al., 2000). It was demonstrated (Odum et al., 1997) that an exposure through oral gavage
was less effective than s.c. injection (2–400 ␮g/kg day, PND
21–23). Laws et al. (2000) reported also that the number of
regular cycles was reduced, pointing to peripheral and central
effects. Investigations of the uterotrophic response showed an
increase in uterus weight after 0.3–100 ␮g/kg day, during PND
21–23 (Freyberger et al., 2001; Laws et al., 2000). Similar results
were observed when EE2 was administered in early life, PND
1–5 (Branham et al., 1988).
5.3. Phytoestrogens
Plants produce estrogen-like substances, called phytoestrogens. They are nonsteroidal EDCs naturally carried from food.
There is a growing body of literature showing that these estrogens are capable of binding to ERs and exerting estrogenic
responses in mammals (Martin et al., 1978; Nelson et al.,
1984; Hopert et al., 1998). They include mainly the isoflavones
(e.g. genistein found in soy derivatives) and the coumestanes
(principally coumestrol present in several vegetables). Transresveratrol (RVT), a stilbene derivative, is a phytoalexin found
in grapes, red wine, peanuts and other fruits. Little information is known about its estrogenic potential (Gehm et al.,
1997).
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Table 3
Data on effects of phytoestrogens on several hypothalamic–pituitary–ovarian outcomes
Age at treatment
Dose
(mg/kg day)
EDCs
VO
Cyclicity
GD 1–PND 21
GD 16–20
1.5
5
25
0.1
0.5
18
20
RVT
Genistein
Genistein
Coumestrol
Genistein
RVT
Coumestrol
Genistein
Coumestrol
Delayed
Delayed
Delayed
Irregular
PND 1–5
3 days
PND 21–23
PND 21–27
PND 22–60
Uterus
weight
then
Prolonged estrus irregular
Advanced
Advanced
Irregular
Irregular
Ref.
Kubo et al. (2003)
Levy et al. (1995)
Gallo et al. (1999)
Medlock et al. (1995)
Jefferson et al. (2005)
Freyberger et al. (2001)
Baker et al. (1999)
Gallo et al. (1999)
Whitten and Naftolin (1992)
GD: gestational day; PND: postnatal day; EDCs: endocrine disrupting chemicals; RVT: trans-resveratrol; VO: vaginal opening.
Phytoestrogens were studied in rodents during all the periods of life till adulthood (Table 3). Discrepant effects were
obtained when VO was studied depending on species and time
of exposure. Administration of genistein (5 mg/kg day) during
fetal life (GD 16–20) delayed the age at VO (Levy et al., 1995)
while when given between PND 21 and 27, VO was advanced
(Gallo et al., 1999). VO advancement was also obtained
after coumestrol administration during PND 22–60 (Whitten
and Naftolin, 1992). Early exposure to RVT (1500 ␮g/kg day)
during fetal and suckling periods caused a delay in age at
VO (Kubo et al., 2003). Irregular cycles, i.e. increase or
decrease in cycle duration, were reported in several studies
(Whitten and Naftolin, 1992; Gallo et al., 1999; Freyberger et
al., 2001; Jefferson et al., 2005). Coumestrol was also found to
inhibit the GnRH transcript expression in GT1-7 cells (Bowe et
al., 2003). McGarvey et al. (2001) showed that coumestrol profoundly inhibited the pulsatile LH secretion with concomitant
reduction of the frequency of hypothalamic multiunit electrical
activity volleys. In addition, the GnRH-induced pituitary LH
release in vitro in rat was completely suppressed. Genistein,
however, had no effects on pulsatile LH secretion. As observed
for VO, contradictory results were also obtained concerning the
effects on uterus. Uterine weight was increased by 25 mg/kg day
of genistein given GD 16–20 (Levy et al., 1995; Gallo et al.,
1999) and 20–80 mg/kg day of coumestrol given PND 21–23
(Baker et al., 1999) whereas Medlock et al. (1995) reported
an initial increase and subsequent decrease in uterine weight
after 100 ␮g/kg day of coumestrol given PND 1–5. A decrease
in uterine weight was caused by RVT (18, 58 or 575 mg/kg day)
given on 3 consecutive days (Freyberger et al., 2001). Gallo et
al. (1999) described disturbances in the distribution of ovarian
follicular size after genistein. Moreover, Jefferson and Newbold
(2000) reported multi-oocyte follicles in the ovaries when mice
were exposed to environmentally relevant dose (50 mg/kg day,
s.c.) of genistein. Levy et al. (1995) demonstrated that pregnant
rats injected with 5 mg/kg day of genistein during GD 16–20 had
their offspring with a smaller AGD at birth.
Studies of nutritional effects on the timing of human puberty
have focused on questions around obesity, fat and protein contents of the food, and there is very little information on possible
effects of phytoestrogens (Berkey et al., 2000).
5.4. DDT and other insecticides
Many organochlorine pesticides are extremely persistent and
tend to bioaccumulate. The insecticide DDT can behave as an
estrogen agonist and/or an androgen antagonist. It has been
banned in the U.S. and Western European countries since the
late 1960s but is still used extensively in developing countries
where contamination continues via the consumption of foods
(Key and Reeves, 1994; Kelce et al., 1995; Clark et al., 1998;
Partsch and Sippel, 2001; Parent et al., 2003). Two DDT isomers
exist, p,p - and o,p -DDT, the first one representing approximately 80% of DDT spread in the environment. Methoxychlor
(MXC) was developed to replace DDT and to have a similar
spectrum of intended effects while being more readily excreted.
It has a much reduced tendency to accumulate in the nature compared to DDT (Kapoor et al., 1970). It stimulates the estrogenic
activity presumably through the ability of some of its metabolite
hydroxyphenyltrichloroethane (HPTE) to bind to intracellular
ERs (Bulger et al., 1978a,b). The effects of the broad spectrum insecticide lindane, still relatively widespread in developed
nations as well as in the third world, will also be discussed in
this review. It can affect the endocrine system through its isomer
hexachlorocyclohexane that is long persistent in the environment
and tends to bioaccumulate along food chains.
Data from Gray et al. (1988, 1989) reported that MXC
(25–200 mg/kg day) given from weaning onwards resulted in
advancement in age at VO and first estrus. Laws et al.
(2000) showed that a 3-day exposure (PND 21–23) to MXC
(50 mg/kg day) also caused advancement in age at VO (Table 4).
Lindane (5–40 mg/kg day) given PND 21–110 or 125 was found
to delay the age at VO (Cooper et al., 1989). This observation
suggests that, during prolonged exposure to a compound with
evidence of few or no peripheral estrogenic effects, the central
inhibitory effects can be prominent. Consistent with this interpretation, Cooper et al. (1989) observed that 20–40 mg/kg day of
lindane caused a reduced size of pituitary gland associated with
high FSH and low LH concentrations, along with a decrease
in uterine weight (Table 4) and a disruption of the ovarian
cyclicity. Further about a putative central effect of pesticides,
Gray et al. (1988, 1989) observed that, following administration of 200 mg/kg day of MXC, the length of estrous cycles
G. Rasier et al. / Molecular and Cellular Endocrinology 254–255 (2006) 187–201
193
Table 4
Data on effects of pesticides on several hypothalamic–pituitary–ovarian outcomes
Age at treatment
Dose
(mg/kg day)
EDCs
PND 21–23
PND 21–35
25 days
PND 21–110
50
MXC
VO
Cyclicity
HP axis
Uterus
weight
Laws et al. (2000)
Cooper et al. (1989)
Advanced
5
10
Lindane
Lindane
20
Lindane
Delayed
Ref.
Irregular
Irregular then
persistent estrus or
diestrus
Pituitary weight
FSH /LH
PND: postnatal day; EDCs: endocrine disrupting chemicals; MXC: methoxychlor; VO: vaginal opening; HP: hypothalamic–pituitary.
was increased until a persistent estrus was seen. A decrease in
the number of regular (4–5 days) estrous cycles was also noted
(Laws et al., 2000). Treatment with 10 mg/kg day of lindane
(Cooper et al., 1989) was followed by periods of persistent estrus
or diestrus (Table 4). Using the GnRH-secreting GT1-7 cell line,
MXC was found to down-regulate the GnRH transcript levels
through its metabolite HPTE (Roy et al., 1999; Gore, 2002).
MXC given orally or s.c. (50 or 500 mg/kg day, PND 21–23)
accounted for an increase in uterine weight (Odum et al., 1997;
Laws et al., 2000). When pregnant mice (GD 11–17) were fed
with o,p -DDT or MXC, a decrease of the AGD was observed
in the offspring at birth (Palanza et al., 2001).
Since the persistent DDT derivative p,p -DDE was found
in the serum of foreign migrating children developing sexual
precocity in Belgium (Krstevska-Konstantinova et al., 2001;
Parent et al., 2003), we have been interested in delineating the
possible effects of DDT on the HP axis. Preliminary observations (Rasier et al., 2005) indicate that o,p -DDT could have
stimulatory effects on GnRH secretion similar to those of E2
with involvement of ERs and the kainate subtype of glutamate
receptor as well as the orphan dioxin receptor following a mechanism described by Ohtake et al. (2003). Additional mechanisms
could exist since DDT was reported recently to stimulate brain
aromatase (Kuhl et al., 2005). We obtained preliminary data
showing that exposure of immature female rats for 5 days to o,p DDT as well as E2 in 1000:1 concentration ratio could result in
early developmental acceleration of GnRH secretion and subsequent sexual precocity (Rasier et al., 2005). This is consistent
with the above-mentioned model of central precocity occurring
as a consequence of exposure to and withdrawal from an EDC.
High levels of p,p -DDE was found in 26 immigrant girls
with precocious puberty as compared to 15 Belgian patients
with the same diagnosis (Krstevska-Konstantinova et al., 2001).
Only two Belgian girls had detectable serum DDE concentration, whereas the mean concentration in immigrant girls was
10 times higher than the detection limit. Three of the immigrant children with high DDE levels had been born in Belgium,
suggesting trans-placental and lactational exposure, because the
half-life of DDE is very long. The DDE levels correlated positively with the age of immigration and negatively with the time
since immigration, suggesting that the source of contamination
was in the home country (Parent et al., 2003).
In the Michigan anglers cohort, exposure to DDT of fisheating mothers and their controls was measured, and the tim-
ing of puberty in 151 daughters was studied (Vasiliu et al.,
2004). In utero exposure to high levels of DDE was associated with an advanced age at menarche. In the North Carolina
Infant Feeding study of 316 girls and 278 boys, no significant
exposure–outcome associations between DDE and pubertal timing were found (Gladen et al., 2000).
Pubertal delay was associated to a high endosulfan exposure
in a recent Indian study where 117 boys from a highly contaminated area were compared to 90 matched control boys from
uncontaminated area (Saiyed et al., 2003). Antisteroidogenic
properties of endosulfan could contribute to this effect.
5.5. Bisphenol A (BPA) and phenol derivatives
BPA is a most common EDC in our environment. It is used
in the manufacture of polycarbonate plastics and epoxy resins,
from which a variety of products are made, including reusable
milk and food storage containers, baby formula bottles, interior
lacquer-coating of food cans or dental sealants, indicating that
humans are exposed widely to this chemical (Krishnan et al.,
1993). Indeed, BPA has been detected in the human umbilical
cord blood, pointing to a possible fetal contamination (Sakurai
and Mori, 2000; Brock et al., 2001). This compound has been
found to compete with E2 for binding to ERs (Krishnan et al.,
1993; Hiroi et al., 1999). However, its affinity for those receptors
has been shown to be much weaker than that of E2 (Kwon et al.,
2000).
As shown in Table 5, when administered (s.c.) postnatally
starting either from birth or later in life, BPA (600–800 mg/
kg day, PND 21–23; 1 or 4 mg/kg day, PND 1–10) was found
to advance the age at VO (Ashby and Tinwell, 1998; Kato et al.,
2003). It was also shown that a fetal exposure (GD 11–17) to a
low dose (2.4 ␮g/kg day) reduced the time interval between VO
and first estrus (Howdeshell et al., 1999). Early and prolonged
administration (GD 6–PND 21; 1.2 mg/kg day) also accounted
for disruption in estrous cyclicity (Rubin et al., 2001) while a
single administration (100 mg/kg day, 25 days, orally) resulted
in a decrease of the number of regular cycles (Laws et al., 2000)
with subsequent persistent estrus (Ashby and Tinwell, 1998;
Kato et al., 2003). Rubin et al. (2001) showed that serum LH
levels decreased in adulthood, suggesting the involvement of a
negative feedback effect. Concerning the reproductive tract, an
uterotrophic response was observed after a 3-day exposure (PND
21–23) to 37.5–150 mg/kg day (Ashby and Tinwell, 1998; Stein-
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G. Rasier et al. / Molecular and Cellular Endocrinology 254–255 (2006) 187–201
Table 5
Data on effects of bisphenol A on several hypothalamic–pituitary–ovarian outcomes
Age at treatment
Dose
(per kg day)
GD 6–PND 21
GD 11–17
1.2 mg
2.4 ␮g
PND 1–10
1 mg
4 mg
PND 21–23
Cyclicity
HP axis
Irregular
LH
Number of days
between VO and 1st
estrus
Advanced
Uterus
weight
Ref.
Rubin et al. (2001)
Howdeshell et al. (1999)
Kato et al. (2003)
Irregular then persistent estrus
37.5 mg
200 mg
600 mg
PND 23–25
25 days
VO
Advanced
Irregular then persistent estrus
100 mg
Steinmetz et al. (1998)
Laws et al. (2000), Ashby and
Odum (2004)
Ashby and Tinwell (1998)
Markey et al. (2001)
Laws et al. (2000), Markey et al.
(2001)
Irregular
GD: gestational day; PND: postnatal day; VO: vaginal opening; HP: hypothalamic–pituitary.
metz et al., 1998), 200 mg/kg day, orally (Laws et al., 2000) or
200–800 mg/kg day (Ashby and Odum, 2004). Taken together,
those data indicate stimulatory peripheral effects and inhibition
of the central mechanism of ovulatory cycles. Kato et al. (2003)
observed polycystic ovaries in adulthood. Markey et al. (2001)
showed that when mice were exposed in utero (GD 9–20) to
low, presumably environmentally relevant doses of BPA (25 or
250 ␮g/kg day, s.c.), changes in histoarchitecture of mammary
glands occurred. Very recently, Munoz-de-Toro et al. (2005)
reported same disorders in adult mice when 25 or 250 ng/kg day
of BPA were administered from GD 9 till PND 4.
A number of alkylphenol compounds in the environment
have been also reported to affect estrogenic activity in the
endocrine system. Octylphenol, notably used in the manufacture of plastics, has been detected in food and water consumed
by animals and people. When 100–200 mg/kg day were given
orally from PND 21 until VO, an advancement in the age at
VO was observed (Gray and Ostby, 1998). A 10 mg/kg day
(s.c.) was found to increase uterine weight in prepubertal rats
(Bicknell et al., 1995). Similar results were reported by Laws et
al. (2000) with 200 mg/kg day (PND 21–35, orally), along with
disruption in estrous cyclicity after 20–40 mg/kg day s.c. (Blake
and Ashiru, 1997) and disorders in pituitary hormone levels
after 80 mg/kg day s.c. (Blake and Bookfor, 1997). Nonylphenol, which is widely used in the production of many surfactants
and plastics, has been reported to advance the age at VO after
50–100 mg/kg day, PND 21–35 (Laws et al., 2000). When given
between PND 21 and 40 at the same doses, advancement in age
at VO and irregular estrous cycles following an increase length
of diestrus were also seen (Kim et al., 2002). An uterotrophic
response was observed (Odum et al., 1997; Laws et al., 2000)
after treatment with 100–200 mg/kg day s.c. during PND 20–22
(Kim et al., 2002). Thus, alkylphenols accounted to effects very
comparable to those of BPA.
To our knowledge there are no human studies on the association of alkylphenol or BPA exposure to timing of puberty.
Assessment of exposure is difficult as compared to persistent
compounds such as PCBs and DDE that can be measured years
after the exposure.
5.6. Polychlorinated biphenyls (PCBs)
There is paucity of information describing the effects of exposure to PCBs. They are lipophilic industrial chemicals often
detected in human breast milk, serum or tissues. They can have
estrogenic and antiandrogenic activities. The effects of two PCB
mixtures, Aroclor 1221 (A1221) and A1254, were tested on
the hypothalamic neuronal GT1-7 cells by Gore et al. (2002).
A1221 increased GnRH transcript and peptide levels. A1254
had different effects, inhibiting GnRH transcription at high concentrations and elevating transcript levels at low concentrations.
Moreover, it did not alter GnRH peptide levels. When pregnant female rats were exposed (GD 1–21) via food containing
40 mg/kg day of a reconstituted PCB mixture (composed according to the congener-pattern in human breast milk), Hany et al.
(1999) showed that the breastfed pups exhibited elevated uterine
weights on PND 21.
There are already several epidemiological studies that have
assessed exposure to PCBs in relation to the timing of puberty.
In a Belgian study comparing children (120 girls and 80 boys)
from rural and urban areas, PCB congeners 138, 153 and 180
were measured in serum, and the pubertal stage was assessed by
trained physicians (Staessen et al., 2001; Den Hond et al., 2002).
No association of PCB levels to pubertal development in girls
was observed, whereas in boys, a significant delay of puberty was
found in urban areas and in association with high PCB levels.
In the North Carolina Infant Feeding Study, no association of
PCB exposure to the self-reported timing of puberty (including
age at menarche) among 316 girls and 278 boys were found,
although there was a nonsignificant tendency to early maturation
in the girls in the highest prenatal exposure group (Gladen et al.,
2000). Two studies from the Great Lake area, Michigan in U.S.,
found no correlation of PCB exposure to self-reported timing
of puberty in 327 (Blanck et al., 2000) or 151 girls (Vasiliu
G. Rasier et al. / Molecular and Cellular Endocrinology 254–255 (2006) 187–201
et al., 2004). Similar results were found in a boy cohort (196
boys) from Faroe Islands, i.e. no association of PCB exposure
to the timing of puberty (Mol et al., 2002). In Yucheng, 55 boys
who were accidentally exposed to high PCB and polychlorinated
dibenzofuran (PCDF) levels were reported to have shorter penile
length than the control boys at the same age, suggesting pubertal
delay (Guo et al., 2004). In summary, epidemiological studies
have not revealed any association of PCB exposure with the
timing of puberty in girls, whereas in boys, there are two studies
suggesting a link to delayed puberty and two studies showing
no effect.
5.7. Polybrominated biphenyl (PBB)
Animal feed was contaminated with polybrominated
biphenyl in a farm in Michigan in the 1970s causing a high
exposure of thousands of people via milk and other products
from contaminated cows. Perinatal exposure of children was
estimated by measuring PBB in serum of mothers some years
after exposure. The girls that had been exposed to high PBB
levels through lactation had an earlier age at menarche and earlier pubic hair development than those who were less exposed
through breastfeeding, whereas no differences were found in
breast development. The girls reported their pubertal development themselves, and this may have caused more inaccuracy
and variation in timing of breast development than other pubertal landmarks (Blanck et al., 2000).
5.8. Phthalates
Phthalates are plasticizers and thus can contaminate environment and humans by consumption of food or drinks contained
in plastic package or bottles. Ashby et al. (1997a,b) reported
that when 182.6 ␮g/kg day of butyl benzyl phthalate (BBP) was
administered orally to rats from conception until weaning, VO
was advanced in the offspring. Similarly, pups whose mother
was exposed from the first day of gestation until birth to dibutyl
phthalate DBP (12 or 50 mg/kg day, orally) had age at VO and
first estrus advanced (Salazar et al., 2004). Nagao et al. (2000)
conducted a study in pregnant female rats using a 500 mg/kg day
oral dose of BBP. The AGD was increased in the offspring at
birth.
Puerto Rican epidemy of thelarche has prompted studies
on several putative endocrine disrupters, including phthalates
(Colon et al., 2000). In the case–control study of 41 girls with
thelarche and 35 controls, two-thirds of the cases had measurable
phthalate levels in serum, whereas only 14% of the controls had
detectable phthalates. However, the phthalate profile in serum
raised a concern about possible technical errors (or contamination), because diethyl hexyl phthalate concentrations were high
as compared to other phthalates (McKee, 2004).
5.9. Dioxins
Immature female rats were given single oral doses (0.3–
60 ␮g/kg) of the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on PND 22 and equine chorionic
195
gonadotrophin (eCG) 24 h later to induce follicular development. A dose-dependently increase in ovarian weight and a
decrease in number of animals ovulating were observed. Peak
serum levels of FSH and LH were decreased by TCDD. In
hypophysectomized immature rats treated with eCG and exogenous LH, number of rats ovulating was reduced by TCDD (10
and 60 ␮g/kg), suggesting that TCDD alters reproductive functions via effects on the HP axis as well as through direct effects
on the ovary (Li et al., 1995). Single doses (0.03–30 ␮g/kg) of
TCDD were also administered orally by gastric intubation to 22day-old rats. Peaks of FSH and LH were observed immediately
after treatment and 1 day later. This dose-dependent elevation
indicated that TCDD induces release of gonadotrophins. Such a
stimulation was at least partially due to a direct pituitary effect
since TCDD caused a release of LH in vitro from pituitary halves
and primary pituitary cell cultures (Li et al., 1997). Recently, it
was also demonstrated that 8 or 32 ␮g/kg of TCDD administered
to immature female rats induced a premature increase in FSH
and LH in vivo, this effect being facilitated by administration of
a long-acting E2 (Petroff et al., 2003).
Retrospective analyses of the age at menarche in girls exposed
to TCDD in Seveso 1976 found no association (Warner et al.,
2004). However, it remained open whether the timing of exposure was appropriate in terms of pubertal effects. In the Yucheng
rice oil accident, children were exposed to PCDF that were
formed through heat-degradation of PCB contaminants in food
preparation (Guo et al., 2004). As described earlier, the exposed
boys had smaller penises as compared to control subjects at the
same age. In the Belgian study of children from rural and two
urban areas, dioxin exposure was estimated with the Calux assay
that measures the total dioxin-like activity in serum (Staessen
et al., 2001; Den Hond et al., 2002). Higher dioxin-like activities were measured in children from the urban suburbs than
in those from the rural area. There was no correlation of the
activity with the age at menarche or pubic hair development, but
breast development to the adult stage was inversely correlated
to dioxin-like activity, i.e. high exposure was associated with a
developmental delay (Den Hond et al., 2002). In boys, there was
no correlation of dioxin-like activity with landmarks of pubertal
development, although testes of boys in the polluted urban areas
were significantly smaller than those in the rural area (Den Hond
et al., 2002). Dioxins act through the aryl hydrocarbon receptor
(AhR) and have antiestrogenic effects in vitro (Wormke et al.,
2003) which might contribute to the slow breast development
in highly exposed girls. Estrogenic effects, however, have been
reported as well through interaction of the dioxin–AhR–nuclear
translocator complex with ERs (Ohtake et al., 2003).
5.10. Lead
High lead levels in blood were reported to be associated to a
delayed age at menarche and delayed pubic hair development in
two studies that were based on the National Health and Nutrition Examination Survey in U.S. (NHANES III) (Selevan et al.,
2003; Wu et al., 2003). Breast development was also found to
be delayed in the study including 2186 girls (Selevan et al.,
2003).
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G. Rasier et al. / Molecular and Cellular Endocrinology 254–255 (2006) 187–201
5.11. Geographic trends
High incidence of central precocious puberty was recently
reported in a small region in Northwest Tuscany, Viareggio, but
the cause for the difference to the neighbouring areas remains
open (Massart et al., 2005). However, the regional differences
may give clues to further exploration of potential causal agents.
Viareggio area has many small navy yards and greenhouses that
may be sources of pollutants contributing to the effect.
6. Concluding remarks
During past decades, the birth rate has declined in a number
of industrialized countries (Davis et al., 1998). The reason of
this reduction is not really clear, but it has been suggested that
chronic exposures to toxic environmental agents, that predominantly affect the reproductive system, could lead to this lower
rate (Potashnik et al., 1984; Rogan et al., 1999; Mocarelli et al.,
2000; Hama et al., 2001; Sakamoto et al., 2001). The time interval between exposure to such substances can be very long and
early prenatal and postnatal life is probably a critical window for
exposure. For instance, a 32% fall in probability of pregnancy
was found in daughters of mothers with 10 ␮g/l of p,p -DDT in
blood drawn at the time of delivery, 30 years earlier (Cohn et
al., 2003).
Indeed, we are continuously exposed to a number of natural and artificial chemical substances which bioaccumulate in
the environment. These substances, mostly showing estrogenic
activity, potentially interfere with the endocrine system, altering
the hormone actions through the ER (␣ and/or ␤) pathway (synthesis and/or receptor binding). While EDC effects on female
reproduction have been a matter of concern for several decades,
female pubertal development has emerged more recently as
a possible endpoint. Current knowledge of exposure–outcome
relationships in pubertal timing is limited to few epidemiological studies. In many of those studies, timing of pubertal stages
and menarche were self-reported and the assessment of exposure indirect, both of which cause considerable variation to data.
The size of the study populations varies also largely. High levels of DDE in serum of immigrant girls with central precocious
puberty was found (Krstevska-Konstantinova et al., 2001) and
a high perinatal exposure to DDE levels was also associated to
early menarche in the so-called Michigan angler cohort (Vasiliu
et al., 2004), whereas in the North Carolina Infant Feeding Study,
no association was found between DDE exposure and pubertal
timing (Gladen et al., 2000). A high perinatal exposure to PBB
was associated to early menarche and pubic hair development,
but not to timing of breast development (Blanck et al., 2000).
Dioxins and PCBs do not seem to affect pubertal development
in girls, but one study reported an association of PCBs to a
delay in pubertal development in boys (Den Hond et al., 2002).
Endosulfan exposure was also associated with slower pubertal
development in boys (Saiyed et al., 2003) and lead exposure
with delayed pubertal timing in girls (Selevan et al., 2003; Wu
et al., 2003).
The animal data tend to substantiate effects of early exposure to EDCs on HPG axis with alterations of sexual maturation
and reproductive functions in female rodents. It has always been
controversial to extrapolate from data that are obtained in laboratory animals in which the dose and time of chemical exposure
do not always represent conditions that humans face in their
habitat during a life time. However, we cannot ignore the increasing evidence coming from these studies when human populations are exposed to the same chemicals during developmental
stages.
When all instances of EDC exposure are taken together, both
peripheral and central effects were observed. In most cases,
advancement in the age at VO was reported though some EDCs
such as genistein, RVT and lindane accounted for some delay.
The advancement in age at VO suggests a peripheral effect occurring directly on vaginal epithelium, the acceleration being a sign
of a premature estrogenic activity and the delay, an antiestrogenic effect. Uterine weight increase, which is known to be a
marker of estrogenicity, was also commonly observed when
studied after exposure to estrogenic EDCs. Since genistein as
well as RVT can act as mixed agonists/antagonists at the ERs
(Miodini et al., 1999; Bowers et al., 2000), a prominent antagonistic pathway is suggested by the delay. Little is known about
the mechanism of action of HPTE, the active isomer of lindane,
Cooper et al. (1989) suggesting an antiestrogenic effect.
After an exposure to E2, the age at first estrus was advanced as
noted for VO. This effect suggests a central stimulatory involvement, resulting from acceleration in hypothalamic maturation.
Such an hypothesis was supported by the increase in GnRH
pulse frequency seen after in vivo and in vitro E2 administration (Matagne et al., 2004). Irregular cyclicity followed by
persistent estrus or diestrus, associated with subfertility or complete sterility was shown in most studies with all types of EDCs,
indicating an additional disturbing impact on the hypothalamic
functions. The interpretation of such findings is complex since
it can involve disorders of the perinatally programmed central
mechanism of ovulation that is highly sensitive to interactions
at ERs and inhibitory effects disordering hypothalamic function
later in life. The latter component is consistent with the direct
action of coumestrol centrally to reduce the frequency of the
hypothalamic GnRH pulse generator (McGarvey et al., 2001).
In addition, Bowe et al. (2003) showed that ER␤ was involved
in the suppression of GnRH transcript expression by coumestrol
in GnRH-secreting neuronal GT1-7 cells. Direct central stimulatory effects are consistent with some findings using the GT1-7
cell line, where E2 and A1221 directly stimulated GnRH gene
expression, through interaction with specific receptors expressed
in these cells. As effects of A1221 on GnRH gene expression
were not blocked by ICI 182,780, it indicated that some but not
all of its effects were mediated by the classical ER (␣ and/or
␤) pathway (Gore et al., 2002). TCDD appeared to stimulate
premature gonadotrophin release in the gonadotrophin-primed
immature rat by interacting with an E2-sensitive neural signal
(Petroff et al., 2003). Nass et al. (1984) concluded that exposure
to exogenous estrogen prior to puberty precipitates the decline in
estrous cyclicity associated with the loss of gonadotrophin surge
response presumably due to an alteration in hypothalamic GnRH
release. Again evidence of both stimulatory and inhibitory central effects is provided by these observations.
G. Rasier et al. / Molecular and Cellular Endocrinology 254–255 (2006) 187–201
Besides hypothalamic effects, EDC could have direct action
at the pituitary gland level. It was shown that coumestrol inhibited pituitary LH pulses by reducing responsiveness to GnRH
via an ER-mediated process (McGarvey et al., 2001).
When EDC effects are considered, the exposure issue including age, dose and mixture effects is critical. The observations
reviewed here deal with in vivo data which can be different
from in vitro models commonly used to compare biopotency of
EDCs. Except for DES which is known to be as or even more
potent than E2, the dose-related potency ratio between E2 and
other EDCs usually ranges between 1:1000 and 1:10,000. However, because differences in doses are usually associated with
differences in age at treatment, duration and route of administration, comparisons are difficult. In utero and/or early postnatal administration causes generally more severe disturbances
involving central effects whereas late postnatal exposure mainly
cause an uterotrophic response and other peripheral disorders.
Most authors reported that s.c. injections were more effective or
required a lower dose to induce an effect than an oral gavage. Differences in absorption, metabolic activation and/or elimination
of tested chemicals can occur following the exposure subcutaneously or orally (Klaassen and Eaton, 1991).
A thorough assessment of effects of exposure to EDCs before,
around and/or after birth is indispensable for a real understanding
of the exposure–disorder relationships in the immature female
animals and, by extension, in the young girls. We have also
to remind that it is not the individual exposure to a single
xenoestrogen but rather the cumulative exposure to multiple
xenoestrogens that determines the intensity of the effects (Soto
et al., 1994, 1997). Finally, disorders of female sexual development can be a relatively early manifestation of EDC effects that
should be put in perspective as warning towards more severe
and later consequences such as fertility disorders and neoplasia
of estrogen-sensitive tissues.
Acknowledgements
This work has been supported by grants from the “Fonds
de la Recherche Scientifique Médicale” (grant 3.4515.01), the
Belgian Study Group for Paediatric Endocrinology and the European Commission (contract no. QLRT-2001-00269).
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BIOLOGY OF REPRODUCTION 77, 734–742 (2007)
Published online before print 27 June 2007.
DOI 10.1095/biolreprod.106.059303
Early Maturation of Gonadotropin-Releasing Hormone Secretion and Sexual Precocity
after Exposure of Infant Female Rats to Estradiol or Dichlorodiphenyltrichloroethane1
Grégory Rasier, Anne-Simone Parent, Arlette Gérard, Marie-Christine Lebrethon, and
Jean-Pierre Bourguignon2
Developmental Neuroendocrinology Unit, Centre for Cellular and Molecular Neurobiology, University of Liège,
University Hospital, B-4000 Liège (Sart-Tilman), Belgium
ABSTRACT
INTRODUCTION
An increase in the frequency of pulsatile gonadotropinreleasing hormone (GnRH) secretion in vitro and a reduction
in LH response to GnRH in vivo characterize hypothalamicpituitary maturation before puberty in the female rat. In girls
migrating for international adoption, sexual precocity is frequent
and could implicate former exposure to the insecticide
dichlorodiphenyltrichloroethane (DDT), since a long-lasting
DDT derivative has been detected in the serum of such children.
We aimed at studying the effects of early transient exposure to
estradiol (E2) or DDT in vitro and in vivo in the infantile female
rat. Using a static incubation system of hypothalamic explants
from 15-day-old female rats, a concentration- and timedependent reduction in GnRH interpulse interval (IPI) was seen
during incubation with E2 and DDT isomers. These effects were
prevented by antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4 propionic acid (AMPA)/kainate receptors and
estrogen receptor. Also, o,p 0 -DDT effects were prevented by
an antagonist of the aryl hydrocarbon orphan dioxin receptor
(AHR). After subcutaneous injections of E2 or o,p 0 -DDT between
Postnatal Days (PNDs) 6 and 10, a decreased GnRH IPI was
observed on PND 15 as an ex vivo effect. After DDT
administration, serum LH levels in response to GnRH were not
different from controls on PND 15, whereas they tended to be
lower on PND 22. Subsequently, early vaginal opening (VO) and
first estrus were observed together with a premature age-related
decrease in LH response to GnRH. After prolonged exposure to
E2 between PNDs 6 and 40, VO occurred at an earlier age, but
first estrus was delayed. We conclude that a transient exposure
to E2 or o,p 0 -DDT in early postnatal life is followed by early
maturation of pulsatile GnRH secretion and, subsequently, early
developmental reduction of LH response to GnRH that are
possible mechanisms of the subsequent sexual precocity. The
early maturation of pulsatile GnRH secretion could involve
effects mediated through estrogen receptor and/or AHR as well
as AMPA/kainate subtype of glutamate receptors.
During the past decade, precocious puberty (PP) was
reported to occur frequently in foreign girls after migration for
adoption in different countries of Western Europe. Among the
possible pathophysiologic mechanisms, we have hypothesized
that migration could result in withdrawal from exposure to
estrogenic endocrine-disrupting chemicals (EDCs) in the home
country and cause sexual precocity to occur following early
hypothalamic maturation caused by the EDCs [1, 2]. Those
environmental substances are known to interact with the
reproductive system in a harmful manner [3, 4]. The
involvement of EDCs in sexual precocity in migrating girls
was suggested based on the detection of p,p 0 -dichlorodiphenyldichloroethene (p,p 0 -DDE) in the serum of those patients
[1]. With a half-life of several decades, p,p 0 -DDE is a
persistent derivative of the insecticide dichlorodiphenyltrichloroethane (DDT), which has been banned in the United
States of America and Western European countries since the
late 1960s [5, 6] but is still used extensively in developing
countries [2]. This EDC is known to act as an estrogen receptor
(ER) agonist and/or androgen receptor antagonist, both in vitro
and in vivo [7, 8]. In the above situation, because p,p 0 -DDE
levels were related directly to the length of stay in the country
of origin and inversely to time since immigration [2], it was
thought to be a marker of previous exposure to DDT during
early life.
In immature animals, direct effects on peripheral tissues,
such as the vaginal epithelium, were reported after exposure to
estradiol (E2) or estrogenic EDCs and were consistent with
peripheral sexual precocity [7–9]. In 1971, Heinrichs et al.
reported that administration of 1 mg o,p 0 -DDT in neonatal
female rats on Postnatal Days (PNDs) 2–4 resulted in earlier
vaginal opening (VO) and first estrus and delayed anovulatory
syndrome [10]. They hypothesized that exposure to DDT in
early life could cause premature hypothalamic-pituitary
maturation and disturb the hypothalamic control of ovulation
through unknown mechanisms. Since the o,p 0 -isomer of DDT
had estrogenic uterotrophic properties [11], the question arose
as to whether central PP could coexist or follow peripheral PP
after exposure to DDT [2, 9]. A hypothalamic effect of
estrogenic substances in the female individual was supported
by our previous observation that E2 preferentially stimulated
gonadotropin-releasing hormone (GnRH) pulse frequency in
the immature female rat hypothalamus in vitro through a
mechanism dependent on the perinatal sexual differentiation.
In addition, a single massive dose of E2 given on PND 10
caused early maturation of pulsatile GnRH secretion, with
early VO and first estrus subsequently [12]. The aim of the
present study was to investigate, both in vitro and in vivo, the
effects of an early transient exposure of the immature female
rat hypothalamus and pituitary gland to DDT in comparison
with E2 and the mechanisms involved in such effects. Two
environment, estradiol, gonadotropin-releasing hormone,
hypothalamus, puberty
1
Supported by the European Commission (EDEN project, contract
QLRT-2001-00269), the Léon Frédéricq Foundation, the Belgian Study
Group for Pediatric Endocrinology, and grants 3.4515.01 and
3.4573.05 from the National Belgian Fund for Scientific Research.
2
Correspondence: Jean-Pierre Bourguignon, Division of Pediatric
Endocrinology and Adolescent Medicine, University Hospital, B-4000
Liège (Sart-Tilman), Belgium. FAX: 32 4 366 72 46;
e-mail: [email protected]
Received: 5 December 2006.
First decision: 6 January 2007.
Accepted: 26 June 2007.
Ó 2007 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
734
735
PUBERTY AFTER EARLY EXPOSURE TO DDT
FIG. 1. Experimental design to study in
vivo the effects of E2 or o,p 0 -DDT treatment
started in 6-day-old female rats as daily s.c.
injections and maintained for 5, 10, or 35
days. The procedure is represented in
relation to age with reference to the
developmental reduction in GnRH IPI
observed in vitro (n ¼ 6). The average ages
at VO and first estrus are also represented.
particular endpoints were chosen based on developmental
characteristics: the frequency of pulsatile GnRH secretion by
hypothalamic explants in vitro that shows a prepubertal
acceleration between PNDs 10 and 25, as illustrated in Figure
1 [13, 14], and the LH secretory response to a synthetic GnRH
administration in vivo that shows a prepubertal reduction until
PND 36 [15, 16].
GnRH Stimulation Test and LH Assay
Infantile female Wistar rats were purchased from the University of Liège.
They were housed in standardized conditions with lactating dams (228C, lights
on from 0630 to 1830 h, food and water ad libitum). Each litter contained 5–10
pups. The day of birth was considered PND 1. The weaning occurred on PND
21. All experiments were carried out with the approval of the Belgian Ministry
of Agriculture and the Ethical Committee at the University of Liège.
At PNDs 15 and 22, and at VO and first estrus, serum LH levels were
measured in basal conditions (s.c. injection of vehicle or o,p 0 -DDT) and 30 min
after stimulation through s.c. injection of 1 lg/kg GnRH. These conditions
were used after testing different time points (15, 30, and 45 min) following
GnRH at different ages (15 and 20 days and at VO) and were consistent with
the conditions reported by others [16, 20].
After 2 h of clotting at room temperature, trunk blood collected from the
killed animals was centrifuged (5 min at 2000 3 g). The serum was collected
and stored at 208C until assayed. Serum LH levels were determined in
duplicate in a volume of 100 ll using a double-antibody method and
radioimmunoassay kits kindly supplied by the National Institutes of Health (Dr.
A.F. Parlow, National Institute of Diabetes and Digestive & Kidney Diseases,
National Hormone and Peptide Program, Torrance, CA). Rat LH-I-8 was
labeled with 125I by the chloramine-T method. The hormone concentrations
were expressed using the reference preparation rLH-RP-3 as a standard. The
intraassay and interassay coefficients of variation were 7% and 9%,
respectively. The sensitivity limit of the assay was 5 ng/ml.
Hypothalamic Explant Incubation and GnRH Assay
Reagents
The developmental variations in GnRH pulse frequency in vitro were
studied using hypothalamic explants obtained in female rats on PNDs 1, 5, 10,
15, 20, 25, 30, and 50. For the in vitro study of hypothalamic explants, 15-dayold animals were used. After decapitation, the hypothalamus was rapidly
dissected. The limits to obtain the retrochiasmatic hypothalamus were the
caudal margin of the optic chiasm, the caudal margin of the mammillary bodies,
and the lateral hypothalamic sulci [14]. Each explant was transferred into an
individual chamber in a static incubator, as described in detail previously [12,
14]. Each chamber contained 500 ll minimum essential medium (MEM)
supplemented with glucose, magnesium, glycine, and bacitracin to achieve final
concentrations of 25 3 103, 103, 108, and 2 3 105 M, respectively. The
explants were incubated in an atmosphere of 95% O2/5% CO2 for a total period
varying between 4 and 6 h. The incubation medium was collected and renewed
every 7.5 min and was kept frozen until assayed.
The GnRH release in the incubation medium of hypothalamic explants was
measured in duplicate using a radioimmunoassay method with intraassay and
interassay coefficients of variation of 14% and 18%, respectively [17, 18]. The
highly specific CR11-B81 anti-GnRH antiserum (final dilution 1:80 000) was
kindly provided by Dr. V. D. Ramirez (Urbana, IL) [19]. The data below the
limit of detection (5 pg/7.5-min fraction) were assigned that value.
The MEM was purchased from Life Technologies Invitrogen Corp.
(Merelbeke, Belgium). E2 (17b-estradiol or 3,17b-dihydroxy-1,3,5(10)-estratriene); the two DDT isomers, o,p 0 -DDT (2,4 0 -DDT) and p,p 0 -DDT (4,4’DDT); and p,p 0 -DDE (4,4 0 -DDE) were purchased from Sigma-Aldrich
(Bornem, Belgium). P,p 0 -DDT represents approximately 80% of the insecticide
still commonly used in developing countries. O,p 0 -DDT is an equally active
isomer of the insecticide that accounts for 15%–20% of technical grade DDT.
In endocrine studies, o,p 0 -DDT has been particularly studied due to its
prominent estrogenic property and relatively less toxic activity. P,p 0 -DDE is a
long-lasting derivative of p,p 0 -DDT, with a half-life of several years. The aamino-3-hydroxy-5-methylisoxazole-4 propionic acid (AMPA)/kainate subtype
of glutamate receptor antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione) and
the ER antagonist ICI 182 780 (7a,17b-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-1,3,5(10)-estratriene-3,17b-diol) were purchased from Tocris Fisher
Bioblock Scientific (Illkirch, France), whereas the aryl hydrocarbon orphan
dioxin receptor (AHR) antagonist a-naphtoflavone (7,8-benzoflavone) was
purchased from Sigma-Aldrich. In all experiments, the steroid and insecticides
were dissolved initially in absolute ethanol (Labonord, Templenars, Belgium)
and, subsequently, in the incubation medium or sesame oil (Calbiochem VWR
International, Leuven, Belgium) for in vitro or in vivo studies, respectively, to
MATERIALS AND METHODS
Animals
736
RASIER ET AL.
serum LH levels. When the treatment period was PNDs 6–40, there were 10
rats followed in each condition to study VO and estrous cyclicity.
Statistical Analysis
FIG. 2. Effects of E2, DDT isomers, and p,p 0 -DDE in vitro on the
frequency of pulsatile GnRH secretion from hypothalamic explants
obtained in 15-day-old female rats. The data are calculated in relation
to time (two or three consecutive 2-h periods) of incubation in vitro. a: P ,
0.05 versus control; b: P , 0.05 versus data obtained during the initial 2-h
period.
achieve a final ethanol concentration of 0.01% or 1%. The antagonists were
directly diluted in the incubation medium.
Study Protocols
In vitro experiments. The in vitro effects of E2 or DDT on the frequency of
pulsatile GnRH secretion were studied. The compounds (steroid, DDT isomers,
and antagonists) were used for a whole 4- or 6-h experimental incubation
period. A concentration-response study was carried out with explants incubated
with 109 to 107 M of E2 and 106 to 104 M of o,p 0 -DDT, p,p 0 -DDT, or p,p 0 DDE. The effects of antagonists were studied in the presence of maximal
effective concentrations of E2 and o,p 0 -DDT. Those antagonists were chosen
because they were shown to prevent E2 effects on GnRH secretion in our
hypothalamic explant conditions [12, 21], and it was our hypothesis that DDT
effects, if any, could be mediated through mechanisms similar to E2 effects.
The antagonist DNQX (106 M) was used to study the involvement of the
AMPA/kainate subtype of glutamate receptors. The implication of ER was
studied using the antagonist ICI 182 780 (107 M). To investigate the
implication of AHR, the antagonist a-naphtoflavone (107 M) was used. The
concentration of these three antagonists was selected based on previous data
from our laboratory and other studies [12, 17, 21–23]. It was shown previously
that when used alone, the AMPA/kainate subtype of glutamate receptor and ER
antagonists did not affect pulsatile GnRH secretion [12], and the absence of
effects of the different antagonists when used alone was double checked in this
study.
In vivo experiments. The procedures are schematically summarized in
Figure 1. The animals received a daily s.c. administration of steroid or
insecticide for 5, 10, or 35 days (E2: 0.01, 0.1, or 1 mg/kg/day for PNDs 6–10
and 0.01 mg/kg/day for PNDs 6–15 and PND 6–40; o,p 0 -DDT: 10 or 100 mg/
kg/day for PNDs 6–10 and 10 mg/kg/day for PNDs 6–15). The dose of E2 and
o,p 0 -DDT was adjusted for increasing body weight of rats. The chemicals
dissolved in absolute ethanol were diluted in 50 ll sesame oil for s.c. injection,
as described in other studies [24, 25]. When the treatment period was PNDs 6–
10, 20 rats were studied in each treated group in comparison with 20 controls
injected with vehicle alone. On PND 15, 10 rats from each group were killed to
study the pulsatile GnRH secretion in vitro and serum LH levels. In each group,
the 10 remaining animals were then examined daily for imperforation of the
vaginal membrane to determine age at VO. Thereafter, vaginal smears were
taken every day in the afternoon until PND 60. Slides of vaginal smears were
colored using the Papanicolaou method to detect the occurrence of estrous
cyclicity and to follow cycling. The age at first estrus was considered when
vaginal smears contained primary cornified cells after the first proestrous phase,
which is characterized by both stratified and cornified cells [26]. In subsequent
experiments to study LH response to GnRH on PNDs 15 and 22, and at the
time of VO and first estrus, seven animals were killed in each group at each
age. When the treatment period was PNDs 6–15, there were five rats in each
group that were killed on PND 15 to study the GnRH pulse frequency and
When pulsatile GnRH secretion was studied, the peaks were detected using
the PULSAR program for PC [27]. The cutoff criteria for peak detection were
determined empirically and were G1 ¼ 2.5 and G2 ¼ 2.0. Peak splitting
parameter was set at 2.7, and intraassay coefficient of variation was used as B
coefficient [28]. The GnRH interpulse interval (IPI) was calculated as the time
interval between two consecutive peaks. The IPI was calculated during different
time periods of incubation (1–2 h and 3–4 h or 5–6 h). Depending on the
normal or nonnormal distribution of IPI data in the different study periods,
comparisons were made using the paired Student t-test with P , 0.05 as the
threshold for significance (GraphPad Prism software for PC) or the Wilcoxon
matched pairs test, respectively. In several instances, all the explants in a group
showed a similar IPI value. In this case, SD was null and could not be
represented.
When comparisons were made between steroid and/or insecticide effects on
LH levels and age at VO or first estrus in different conditions, raw data were
pooled and analyzed by the one-way ANOVA test when normally distributed,
followed by a multiple-comparison Newman-Keuls post-hoc test when the
threshold for significance of differences (P , 0.05) was reached. When data
were not normally distributed, the Kruskal-Wallis test was used, followed by a
multiple-comparison Dunn post-hoc test. For the experiment run PNDs 6–40,
an unpaired t-test was employed. All results are expressed as mean 6 SD.
RESULTS
In Vitro Treatments
In control conditions of hypothalamic explant incubation in
vitro (Fig. 1), the GnRH IPI showed a reduction between PNDs
10 and 25, confirming our data in the male [13]. When
hypothalamic explants obtained at 15 days were incubated in
control conditions (Fig. 2), the GnRH IPI did not change with
time (1–2 h: 60.4 6 1.7 min; 3–4 h: 59.6 6 1.8 min; and 5–6
h: 60.0 6 0.0 min). During a 4-h continuous incubation with
107 M of E2, the GnRH IPI was reduced significantly after 1–
2 h (49.0 6 3.9 min) and further after 3–4 h (45.4 6 1.7 min).
This effect was dependent on E2 concentration and incubation
time: with 108 M E2, the GnRH IPI was unchanged after 1–2
h and decreased significantly after 3–4 h; with 109 M E2, a
significant reduction occurred after 5–6 h only (52.5 6 0.0
min). The two active isomers of DDT also caused a
concentration- and time-dependent reduction in GnRH IPI that
was significant after 3–4 h using 105 M of both isomers (o,p 0 DDT: 50.4 6 5.7 min; p,p 0 -DDT: 52.5 6 0.0 min). At a 104
M concentration, both isomers resulted in an earlier effect that
was also greater after 3–4 h (o,p 0 -DDT: 45.0 6 0.0 min; p,p 0 DDT: 52.5 6 0.0 min). When used at 106 M, p,p 0 -DDT had
no effect during a 6-h incubation, and o,p 0 -DDT showed a
significant effect only after 5–6 h of incubation (52.5 6 0.0
min). No effect could be obtained using p,p 0 -DDE at similar
concentrations.
The significant reduction of GnRH IPI caused by 107 M E2
or 104 M o,p 0 -DDT after 3–4 h of incubation in vitro (Fig. 3A)
was totally prevented when the AMPA/kainate subtype of
glutamate receptors was antagonized by coincubation with
DNQX (Fig. 3B). Likewise, the effects of E2 and o,p 0 -DDT
were totally prevented in the presence of the ER antagonist ICI
182 780 (Fig. 3C). When a-naphtoflavone was used to
antagonize the AHR, the significant decrease in GnRH IPI
caused by o,p 0 -DDT was not observed any more, whereas the
E2 effect was attenuated but remained significant (Fig. 3D).
In Vivo Treatments
As shown in Figure 4A, after 5 days of treatment with 0.01,
0.1, and 1 mg/kg E2 (PNDs 6–10), the age at VO (controls:
PUBERTY AFTER EARLY EXPOSURE TO DDT
737
FIG. 3. Effects of 106 M DNQX, an antagonist of the AMPA/kainate subtype of glutamate receptors (B), 107 M of ICI 182 780, an ER antagonist (C), and
107 M a-naphtoflavone, an AHR antagonist (D) on the GnRH IPI during incubation of hypothalamic explants obtained in 15-day-old female rats in the
presence of 107 M E2 or 104 M o,p 0 -DDT (A) in vitro. A representative profile of pulsatile GnRH secretion is shown in each condition, and the mean (6
SD) IPI observed during 3–4 h of incubation are given. *P , 0.05 treatment versus control conditions.
FIG. 4. Effects of E2 or o,p 0 -DDT injected
s.c. in female rats for PNDs 6–10 or PNDs
6–40 on the ages at VO and first estrus (A)
and the interval between VO and first estrus
(B). *P , 0.05 versus vehicle (sesame oil).
738
RASIER ET AL.
TABLE 1. Mean 6 SD (n ¼ 10) of estrous cycle length (interval between
two consecutive estrus) observed until PND 60.
Treatment
PND 6–10
Vehicle
E2
o,p’-DDT
PND 6–40
Vehicle
E2
Dose (mg/kg)
—
0.01
0.10
1.00
10.00
100.00
—
0.01
Estrous cycle length (days)
4.7
5.2
5.5
4.6
5.3
5.5
6
6
6
6
6
6
1.4
1.6
1.8
1.5
2.0
1.4
4.8 6 1.2
4.9 6 1.8
30.1 6 0.6 days) was significantly earlier (23.0 6 0.7, 22.7 6
1.3, and 24.6 6 0.5 days, respectively). The VO was also
earlier after 10 and 100 mg/kg o,p 0 -DDT (22.6 6 0.5 and 24.6
6 0.7 days, respectively). The first estrus was observed on
PND 31.9 6 0.7 in controls and occurred significantly earlier
after 0.01 and 0.1 mg/kg of E2 (27.2 6 0.7 and 27.5 6 1.1
days), as well as after 10 mg/kg o,p 0 -DDT (26.4 6 1.5 days).
After 1 mg/kg E2 or 100 mg/kg o,p 0 -DDT, the age at first estrus
did not change. When the time interval between VO and first
estrus (Fig. 4B) was calculated (controls: 1.8 6 0.9 days), a
significant dose-dependent increase was observed after 0.1 and
1 mg/kg E2 or 100 mg/kg o,p 0 -DDT. No differences in estrus
cycle length were observed until PND 60 (Table 1).
As shown in Figure 5A, after five daily s.c. injections of
0.01, 0.1, and 1 mg/kg E2 (PNDs 6–10), the GnRH IPI studied
ex vivo on PND 15 (controls: 60.0 6 0.0 min) was
significantly reduced (54.4 6 3.3, 52.5 6 0.0, and 47.0 6
3.4 min, respectively). A reduction of GnRH IPI was also seen
after treatment with 10 and 100 mg/kg/day o,p 0 -DDT, which
was only significant with 100 mg/kg (55.0 6 3.6 min). When
0.01 mg/kg E2 or 10 mg/kg o,p 0 -DDT was administered for a
FIG. 5. Effects of E2 or o,p 0 -DDT injected
s.c. in female rats for PNDs 6–10 or PNDs
6–15 on the GnRH IPI (A) and serum LH
concentrations (B) studied ex vivo at 15
days of age. *P , 0.05 versus vehicle
(sesame oil).
FIG. 6. Effects of treatment of female rats with o,p 0 -DDT (10 mg/kg/day)
or sesame oil for PNDs 6–10 on serum LH levels studied 30 min after
subcutaneous injection of 1 lg/kg GnRH at four age points: PND 15, PND
22, the day of VO, and the day of first estrus. The data are mean 6 SD of
LH levels and age in seven rats studied in each group. *P , 0.05 versus
vehicle (sesame oil).
longer period of 10 days (PNDs 6–15), the GnRH IPI ex vivo
was significantly reduced on PND 15 as well.
As shown in Figure 5B, the serum LH level on PND 15
(controls: 41.1 6 19.9 ng/ml) was significantly decreased after
0.01, 0.1, and 1 mg/kg E2 between PNDs 6 and 10 (18.9 6
12.7, 23.7 6 13.8, and 9.7 6 3.0 ng/ml, respectively) and after
10 and 100 mg/kg o,p 0 -DDT (22.7 6 18.5 and 15.7 6 6.0 ng/
ml, respectively). However, the serum LH level also decreased
with age after sesame oil (control) administration (Fig. 6): 48.2
6 46.7 ng/ml on PND 15, 25.8 6 19.3 on PND 22, 16.5 6
13.7 on PND 30.6 6 1.5 (at VO), and 10.5 6 7.3 on PND 35.0
PUBERTY AFTER EARLY EXPOSURE TO DDT
6 1.2 (at first estrus). When the LH response to GnRH was
studied after vehicle or 10 mg/kg o,p 0 -DDT given for PNDs 6–
10, the LH response was not affected on PND 15 and showed
some but not significant reduction on PND 22, whereas it
dropped significantly in the next days, at the time of early VO
and first estrus.
An extended period of daily s.c. administration of 0.01 mg/
kg E2 for PNDs 6–40 (Fig. 4A) caused a significantly earlier
age at VO (22.1 6 1.0 vs. 31.8 6 1.0 days in controls),
whereas the age at first estrus was significantly delayed (47.1
6 0.8 vs. 35.4 6 1.7 days in controls), and the interval
between VO and first estrus was markedly increased (20.6 6
1.2 vs. 3.6 6 2.2 days in controls). The first estrus was
observed after a permanent estrus lasting until a few days after
the end of treatment. Then, no differences in estrous cycle
length were observed until PND 60 (Table 1). After daily s.c.
administration of 10 mg/kg o,p 0 -DDT for PNDs 6–40, the
animals showed altered health status with reduced activity and
feeding. On PND 40, their weight was significantly decreased
to 111.3 6 8.3 g (130.0 6 11.5 g after E2 and 149.0 6 9.9 g in
controls). In such conditions, it was no longer possible to
separate direct o,p 0 -DDT effects on sexual maturation and
reproduction from indirect effects through disordered nutritional status.
DISCUSSION
The hypothalamic explant paradigm used in this study
provided an opportunity to observe pulsatile GnRH secretion in
vitro as the result of GnRH neuron function in its original
surrounding neuronoglial apparatus, which regulates GnRH
secretion [29]. Moreover, the developmental changes in
frequency of pulsatile GnRH secretion retained in this model
[13] made possible the study of interaction with maturational
processes, although the critical age period for the developmental increase in GnRH pulse frequency was earlier in vitro
[14] than in vivo [30, 31]. Such a difference could involve
suppression of inhibitory extrahypothalamic inputs when
explants are deafferented from the rest of the brain. However,
the critical period of the second and third postnatal weeks in
our conditions is consistent with neuroendocrine maturation
preceding the peripheral changes of sexual maturation (VO,
testicular growth). Since we incubated retrochiasmatic hypothalami containing prominently axons and terminals of GnRHsecreting neurons [32], we tend to interpret our observations as
the effects of presynaptic regulation by the surrounding
neurons and glial cells. Other in vitro paradigms involving
GnRH neurons, such as GnRH-secreting neuronal cell lines
[33–35], primary cultures of hypothalamic neurons [36–38], or
hypothalamic slices from transgenic mice carrying reporter
genes linked to the GnRH promoter [39] could enable one to
study directly the regulatory mechanisms at the GnRH neuron
level.
The present study was designed to test experimentally the
hypothesis that early and transient exposure to pesticide, as
seen in internationally adopted children, could influence
hypothalamic-pituitary maturation and account for some
central mechanism in the sexual precocity occurring frequently
in such conditions. Since p,p 0 -DDE, which was detected in the
serum of those children, was thought to result from previous
exposure to DDT, this EDC was used for in vivo treatment.
After we observed that o,p 0 -DDT and p,p 0 -DDT were almost
equally effective on GnRH pulse frequency in vitro, the o,p 0 DDT isomer was preferred for in vivo investigations based on
its greater estrogenicity and lower toxicity than p,p 0 -DDT. In
addition, o,p 0 -DDT was the isomer used in former studies on
739
the neuroendocrine control of reproduction [10, 11, 40–42].
The doses that we used for in vivo studies and the E2:o,p 0 -DDT
concentration ratio were based on our in vitro data reported in
the present study and were comparable with those used in other
studies in rodents [43, 44]. The 10 and 100 mg/kg doses were
in the same range as the amounts given by others either
neonatally [10] or around the time of weaning [11]. After early
postnatal administration, lower doses appeared to have no
effects on age at VO and gonadotropin secretion [41]. It was
not possible in this study to measure the concentrations of DDT
isomers and derivatives in serum and tissues. The quantitative
relevance of our conditions of exposure to DDT when
compared to the exposure of migrating girls was difficult to
assess, since the only available information was p,p 0 -DDE
concentrations measured in serum several years later. After
early DDT treatment in neonatal rats for 3 days, p,p 0 -DDE
measured in adipose tissue 4–5 mo later was not different from
controls, suggesting clearance of the insecticide and its residues
after that long period [10]. After 7 days of daily oral intake of
p,p 0 -DDT in a daily dose equivalent to 106 mg/kg in 5-wk-old
rats, serum p,p 0 -DDE levels attained a mean level of 0.66 mg/l
[45]. Such a serum concentration was about 20 times higher
than the serum concentrations found in migrating children [1].
We, however, observed significant neuroendocrine effects
using a 10-times-lower DDT dose that would presumably
result in lower serum DDT levels. Further comparison would
require measurement of the different DDT isomers and residues
at different time points after stopping DDT treatment in the
animals. The interpretation of doses and exposure is even more
complex, since migrating children are likely to be exposed to
various EDCs both in the original and foster countries. It has
been reported that when chemicals are used in mixtures,
combination effect could require lower concentrations than
expected based on simply additive effects [46]. Moreover,
when animals undergo low-dose exposure, the dose-effect
relationship is not linear with comparatively greater effects of
low versus high doses. Although these aspects have not been
investigated in the present study, they could be important for
the pathophysiologic relevance of our findings.
The age window of PNDs 6–10 could be consistent with
early postnatal period in human infants. A 5-day period of
treatment was relatively short for the lifespan but significant
with respect to the short time period between birth and sexual
maturation in the rat. The use of female rats aged 15 days to
study GnRH IPI was based on our previous data showing that
GnRH secretion in vitro was maximally affected by E2 in such
conditions [12]. With hypothalamic explants from younger
females (5 days), GnRH secretion was also found to be
responsive to E2, but such age falls in the critical window of the
brain sexual differentiation, causing possible interferences with
programming of estrus cyclicity. This conclusion was drawn by
Heinrichs et al., who found early VO and delayed persistent
estrus to occur after DDT treatment for PNDs 2–4 [10].
Discrepant observations were made when o,p 0 -DDT treatment
was started at 3 wk of age for several weeks: Gellert et al.
found VO to occur earlier [11], whereas Wrenn et al. found no
change in age at VO that was hastened only when treatment
was given before the age of 3 wk [40]. In the present study,
treatment during the second week of postnatal life was a
compromise to possibly affect sexual maturation without
interfering with the sexually differentiated mechanism of
ovulation. However, such interferences probably did occur
using the highest E2 and DDT concentrations, since they did
not result in early first estrus as opposed to lower doses.
Alternatively, toxic effects could have occurred, although the
nonsignificantly affected growth in those short treatment
740
RASIER ET AL.
conditions did not support such an hypothesis. Three periods
were chosen for in vivo exposure based on the attempt to
mimic the either temporary or persisting exposure of children
either migrating from or staying in developing countries. In the
case of temporary exposure, two different lengths were studied
(until PND 10 or 15) in order to investigate whether duration
could influence the effect. This was confirmed since, after
exposure for 10 instead of 5 days, the lowest dose of DDT
became significantly effective in reducing the GnRH IPI, and
the lowest dose of E2 became more effective.
As many other EDCs, the DDT isomers were shown to
exhibit both estrogenic and antiandrogenic properties, whereas
p,p 0 -DDE retained only antiandrogenic activity [7, 8]. Because
the clinical observation was made in girls [1], and E2 was
found to preferentially influence GnRH secretion in the female
rat hypothalamus [12], female rats were used in the present
study. In our experimental conditions, it was shown previously
that both E2 and testosterone could accelerate GnRH pulse
frequency, the effect of androgens being aromatase dependent
and ultimately mediated through estrogens [12]. Therefore, we
hypothesized that the estrogenic activity of DDT was also
involved in the pathogenesis of sexual precocity [1, 2, 9]. This
hypothesis was consistent with our previous observation of
sexual precocity after a single massive administration of E2 on
PND 10 [12], although the use of lower doses for a longer time
period needed to be investigated. For all these reasons, E2 was
used as a positive control in this work to provide a comparison
basis with DDT effects in vitro and in vivo.
Direct incubation of hypothalamic explants with E2 or DDT
isomers resulted in a concentration- and time-dependent
increase in GnRH pulse frequency. The 1:1000 potency ratio
of E2:DDT found in our conditions was consistent with other
studies [47]. P,p 0 -DDE had no effect, which is in agreement
with the absence of estrogenlike effects reported in other
conditions [48]. Although supraphysiologic concentrations of
E 2 were required for an effect within 1–2 h, lower
concentrations became effective after few hours of incubation.
Such a delay could be explained by the slow diffusion of
reagents into the explant, a hypothesis consistent with the
observation that greater concentrations of compounds like
excitatory amino acids are needed in explant paradigms
compared with neuronal culture systems [49]. Another
explanation could be the latency before the possibly genomic
mechanisms involved in E2 effects became effective. Then E2
effects on GnRH secretion could have a rapid, presumably
nongenomic component, as illustrated by Matagne et al. [21] in
our paradigm, together with a slow genomic component. In this
case, the target would be other cells than GnRH neurons, since
GnRH cell bodies are absent from the studied retrochiasmatic
explants [32]. Herbison [50] and, more recently, Herbison and
Pape [51] have also reported that E2 exerts complex effects on
the GnRH neuronal function, including long-term or genomic
effects through binding to ERa and/or ERb subtypes. In our
system, the acceleration of pulsatile GnRH secretion caused by
E2 was prevented by ICI 182 780, an a/b ER antagonist, as well
as by DNQX, an antagonist of the AMPA/kainate subtype of
glutamate receptors, confirming our previous observations
[12]. The involvement of AMPA/kainate subtypes of glutamate
receptors in E2 stimulation of GnRH pulse frequency was
further supported by the hypothalamic colocalization of those
receptors together with ER [52]. Since the effects of o,p 0 -DDT
were also prevented by ICI 182 780 and DNQX, this EDC
appeared to involve the same receptors as E2. However, the
receptor pathway involved in o,p 0 -DDT effects could be partly
different, with a participation of the orphan dioxin AHR, as
indicated by the preferential reduction of o,p 0 -DDT effects by
the antagonist a-naphtoflavone. The investigation of the
insecticide effect through this pathway was justified, since
Ohtake et al. [23] reported a few years ago that dioxins can
mimic the effect of estrogens via a mechanism that involves the
activation of ER by the transcriptionally active AHR-aryl
hydrocarbon nuclear translocator complex. Further studies
could delineate the relative contribution of the a- and bisoforms of ER and the AMPA and kainate subtypes of
glutamate receptors.
After E2 administration in immature rodents, an inhibition
of LH secretion was commonly observed [53, 54] and impeded
the use of variations in LH secretion to investigate indirectly
hypothalamic effects. In addition, there is physiologically a
developmental reduction in serum LH concentrations between
PND 15 and VO, both basally and in response to GnRH [15,
16], so that decreased LH levels could result from either
negative feedback effects or accelerated maturation or both.
Therefore, hypothalamic-pituitary maturation was assessed
through evaluation of GnRH secretion during explant incubation and study of LH response to GnRH after steroid or EDC
administration in vivo. We elected to study GnRH secretion at
15 days using explants from female rats based on our previous
studies showing that the frequency of pulsatile GnRH secretion
in vitro was maximally stimulated by E2 in such conditions and
through a mechanism dependent on perinatal brain sexual
differentiation [12]. The exposure of infantile female rats to E2
or o,p 0 -DDT for 5 or 10 days was followed by a dosedependent increase in GnRH pulse frequency and a decrease in
serum LH levels on PND 15, suggesting a stimulation of
hypothalamic maturation and a negative feedback inhibition or
an early maturation of the pituitary secretion. A negative
feedback component at the hypothalamic and/or pituitary level
was supported by the reduction in the postcastration rise of
serum LH levels that was reported after treatment of mature or
neonatal rats with o,p 0 -DDT [11, 41]. However, no change in
LH secretion basally and in response to GnRH was found in
another study 6 wk after o,p 0 -DDT treatment between PNDs 1
and 10 [42]. The unchanged LH response to GnRH on PNDs
15 and 22 suggests that the result of the combined inhibitory
and stimulatory effects at those stages is a steady state. Then,
early VO could result from either a peripheral effect of E2 or
DDT or early pituitary-ovarian activity or both. A central
component at the time of VO is suggested by the brisk
reduction in LH response to GnRH that was observed in the
treated animals. Subsequently, early first estrus possibly
confirmed premature activity of the hypothalamic-pituitaryovarian axis. Such a pathophysiologic mechanism could be
consistent with an involvement of DDT in the sexual precocity
occurring after migration in internationally adopted children [1,
2, 9]. In a recent Danish study of such children, it was shown
that developmental changes in pituitary-ovarian hormone levels
were observable before onset of puberty and supported a
hypothalamic-pituitary mechanism of early puberty [55]. Since
our report of an association between sexual precocity and the
detection of p,p 0 -DDE in the serum of migrating girls [1], early
menarche was found to occur after prenatal or postnatal
exposure to DDE and/or DDT [56, 57]. However, others did
not observe changes in menarcheal age after prenatal or
postnatal exposure to DDE [58, 59], and postnatal treatment of
female monkeys with methoxychlor resulted in delayed nipple
development and menarche [60]. Further studies are warranted
to clarify the possible role of EDC nature and parameters of
exposure (age, dose, duration) in those discrepant effects. In
rodents, discrepant observations were made as well, since
pesticides such as methoxychlor and lindane resulted, respectively, in precocity and delay in the age at VO, but both
PUBERTY AFTER EARLY EXPOSURE TO DDT
insecticides caused disturbances of estrous cyclicity [61–63],
and decreased serum LH levels were reported after lindane
administration [61]. The GnRH neuron itself could be targeted
by EDCs, since the phytoestrogen coumestrol caused inhibitory
effects on GnRH transcript expression in GT1–7 GnRHsecreting neuronal cells through the b subtype of ER [64].
When E2 was injected for PND 6–40, early VO followed by
permanent estrus was observed. After E2 treatment was
stopped, first estrus appeared at a markedly delayed age. This
could indicate a peripheral stimulatory effect of the steroid
together with a central inhibition during exposure. A slight
anorexigenic effect of E2 was observed, consistent with studies
previously reported by Ramirez and Sawyer [65] and by
Ramirez [66], whereas o,p 0 -DDT caused a dramatic decrease in
body weight, suggesting a possible toxic effect of the
insecticide [45].
In summary, evidence that DDT could influence the
infantile female hypothalamic pituitary maturation was provided through early developmental acceleration of the GnRH
secretion in vitro and early reduction of LH response to GnRH
in vivo. It was also demonstrated that this chemical caused a
precocious onset of puberty in vivo when immature individuals
were transiently exposed to DDT. Further studies will aim to
delineate its mechanism of action and address whether the
GnRH neurons or other cell structures are primary targets.
13.
14.
15.
16.
17.
18.
19.
20.
ACKNOWLEDGMENT
21.
We thank Pr. J. Boniver (Department of Anatomy and Pathology) for the
assistance of his lab in Papanicolaou staining.
22.
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BIOLOGY OF REPRODUCTION 77, 734–742 (2007)
Published online before print 27 June 2007.
DOI 10.1095/biolreprod.106.059303
Early Maturation of Gonadotropin-Releasing Hormone Secretion and Sexual Precocity
after Exposure of Infant Female Rats to Estradiol or Dichlorodiphenyltrichloroethane1
Grégory Rasier, Anne-Simone Parent, Arlette Gérard, Marie-Christine Lebrethon, and
Jean-Pierre Bourguignon2
Developmental Neuroendocrinology Unit, Centre for Cellular and Molecular Neurobiology, University of Liège,
University Hospital, B-4000 Liège (Sart-Tilman), Belgium
ABSTRACT
INTRODUCTION
An increase in the frequency of pulsatile gonadotropinreleasing hormone (GnRH) secretion in vitro and a reduction
in LH response to GnRH in vivo characterize hypothalamicpituitary maturation before puberty in the female rat. In girls
migrating for international adoption, sexual precocity is frequent
and could implicate former exposure to the insecticide
dichlorodiphenyltrichloroethane (DDT), since a long-lasting
DDT derivative has been detected in the serum of such children.
We aimed at studying the effects of early transient exposure to
estradiol (E2) or DDT in vitro and in vivo in the infantile female
rat. Using a static incubation system of hypothalamic explants
from 15-day-old female rats, a concentration- and timedependent reduction in GnRH interpulse interval (IPI) was seen
during incubation with E2 and DDT isomers. These effects were
prevented by antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4 propionic acid (AMPA)/kainate receptors and
estrogen receptor. Also, o,p 0 -DDT effects were prevented by
an antagonist of the aryl hydrocarbon orphan dioxin receptor
(AHR). After subcutaneous injections of E2 or o,p 0 -DDT between
Postnatal Days (PNDs) 6 and 10, a decreased GnRH IPI was
observed on PND 15 as an ex vivo effect. After DDT
administration, serum LH levels in response to GnRH were not
different from controls on PND 15, whereas they tended to be
lower on PND 22. Subsequently, early vaginal opening (VO) and
first estrus were observed together with a premature age-related
decrease in LH response to GnRH. After prolonged exposure to
E2 between PNDs 6 and 40, VO occurred at an earlier age, but
first estrus was delayed. We conclude that a transient exposure
to E2 or o,p 0 -DDT in early postnatal life is followed by early
maturation of pulsatile GnRH secretion and, subsequently, early
developmental reduction of LH response to GnRH that are
possible mechanisms of the subsequent sexual precocity. The
early maturation of pulsatile GnRH secretion could involve
effects mediated through estrogen receptor and/or AHR as well
as AMPA/kainate subtype of glutamate receptors.
During the past decade, precocious puberty (PP) was
reported to occur frequently in foreign girls after migration for
adoption in different countries of Western Europe. Among the
possible pathophysiologic mechanisms, we have hypothesized
that migration could result in withdrawal from exposure to
estrogenic endocrine-disrupting chemicals (EDCs) in the home
country and cause sexual precocity to occur following early
hypothalamic maturation caused by the EDCs [1, 2]. Those
environmental substances are known to interact with the
reproductive system in a harmful manner [3, 4]. The
involvement of EDCs in sexual precocity in migrating girls
was suggested based on the detection of p,p 0 -dichlorodiphenyldichloroethene (p,p 0 -DDE) in the serum of those patients
[1]. With a half-life of several decades, p,p 0 -DDE is a
persistent derivative of the insecticide dichlorodiphenyltrichloroethane (DDT), which has been banned in the United
States of America and Western European countries since the
late 1960s [5, 6] but is still used extensively in developing
countries [2]. This EDC is known to act as an estrogen receptor
(ER) agonist and/or androgen receptor antagonist, both in vitro
and in vivo [7, 8]. In the above situation, because p,p 0 -DDE
levels were related directly to the length of stay in the country
of origin and inversely to time since immigration [2], it was
thought to be a marker of previous exposure to DDT during
early life.
In immature animals, direct effects on peripheral tissues,
such as the vaginal epithelium, were reported after exposure to
estradiol (E2) or estrogenic EDCs and were consistent with
peripheral sexual precocity [7–9]. In 1971, Heinrichs et al.
reported that administration of 1 mg o,p 0 -DDT in neonatal
female rats on Postnatal Days (PNDs) 2–4 resulted in earlier
vaginal opening (VO) and first estrus and delayed anovulatory
syndrome [10]. They hypothesized that exposure to DDT in
early life could cause premature hypothalamic-pituitary
maturation and disturb the hypothalamic control of ovulation
through unknown mechanisms. Since the o,p 0 -isomer of DDT
had estrogenic uterotrophic properties [11], the question arose
as to whether central PP could coexist or follow peripheral PP
after exposure to DDT [2, 9]. A hypothalamic effect of
estrogenic substances in the female individual was supported
by our previous observation that E2 preferentially stimulated
gonadotropin-releasing hormone (GnRH) pulse frequency in
the immature female rat hypothalamus in vitro through a
mechanism dependent on the perinatal sexual differentiation.
In addition, a single massive dose of E2 given on PND 10
caused early maturation of pulsatile GnRH secretion, with
early VO and first estrus subsequently [12]. The aim of the
present study was to investigate, both in vitro and in vivo, the
effects of an early transient exposure of the immature female
rat hypothalamus and pituitary gland to DDT in comparison
with E2 and the mechanisms involved in such effects. Two
environment, estradiol, gonadotropin-releasing hormone,
hypothalamus, puberty
1
Supported by the European Commission (EDEN project, contract
QLRT-2001-00269), the Léon Frédéricq Foundation, the Belgian Study
Group for Pediatric Endocrinology, and grants 3.4515.01 and
3.4573.05 from the National Belgian Fund for Scientific Research.
2
Correspondence: Jean-Pierre Bourguignon, Division of Pediatric
Endocrinology and Adolescent Medicine, University Hospital, B-4000
Liège (Sart-Tilman), Belgium. FAX: 32 4 366 72 46;
e-mail: [email protected]
Received: 5 December 2006.
First decision: 6 January 2007.
Accepted: 26 June 2007.
Ó 2007 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
734
735
PUBERTY AFTER EARLY EXPOSURE TO DDT
FIG. 1. Experimental design to study in
vivo the effects of E2 or o,p 0 -DDT treatment
started in 6-day-old female rats as daily s.c.
injections and maintained for 5, 10, or 35
days. The procedure is represented in
relation to age with reference to the
developmental reduction in GnRH IPI
observed in vitro (n ¼ 6). The average ages
at VO and first estrus are also represented.
particular endpoints were chosen based on developmental
characteristics: the frequency of pulsatile GnRH secretion by
hypothalamic explants in vitro that shows a prepubertal
acceleration between PNDs 10 and 25, as illustrated in Figure
1 [13, 14], and the LH secretory response to a synthetic GnRH
administration in vivo that shows a prepubertal reduction until
PND 36 [15, 16].
GnRH Stimulation Test and LH Assay
Infantile female Wistar rats were purchased from the University of Liège.
They were housed in standardized conditions with lactating dams (228C, lights
on from 0630 to 1830 h, food and water ad libitum). Each litter contained 5–10
pups. The day of birth was considered PND 1. The weaning occurred on PND
21. All experiments were carried out with the approval of the Belgian Ministry
of Agriculture and the Ethical Committee at the University of Liège.
At PNDs 15 and 22, and at VO and first estrus, serum LH levels were
measured in basal conditions (s.c. injection of vehicle or o,p 0 -DDT) and 30 min
after stimulation through s.c. injection of 1 lg/kg GnRH. These conditions
were used after testing different time points (15, 30, and 45 min) following
GnRH at different ages (15 and 20 days and at VO) and were consistent with
the conditions reported by others [16, 20].
After 2 h of clotting at room temperature, trunk blood collected from the
killed animals was centrifuged (5 min at 2000 3 g). The serum was collected
and stored at 208C until assayed. Serum LH levels were determined in
duplicate in a volume of 100 ll using a double-antibody method and
radioimmunoassay kits kindly supplied by the National Institutes of Health (Dr.
A.F. Parlow, National Institute of Diabetes and Digestive & Kidney Diseases,
National Hormone and Peptide Program, Torrance, CA). Rat LH-I-8 was
labeled with 125I by the chloramine-T method. The hormone concentrations
were expressed using the reference preparation rLH-RP-3 as a standard. The
intraassay and interassay coefficients of variation were 7% and 9%,
respectively. The sensitivity limit of the assay was 5 ng/ml.
Hypothalamic Explant Incubation and GnRH Assay
Reagents
The developmental variations in GnRH pulse frequency in vitro were
studied using hypothalamic explants obtained in female rats on PNDs 1, 5, 10,
15, 20, 25, 30, and 50. For the in vitro study of hypothalamic explants, 15-dayold animals were used. After decapitation, the hypothalamus was rapidly
dissected. The limits to obtain the retrochiasmatic hypothalamus were the
caudal margin of the optic chiasm, the caudal margin of the mammillary bodies,
and the lateral hypothalamic sulci [14]. Each explant was transferred into an
individual chamber in a static incubator, as described in detail previously [12,
14]. Each chamber contained 500 ll minimum essential medium (MEM)
supplemented with glucose, magnesium, glycine, and bacitracin to achieve final
concentrations of 25 3 103, 103, 108, and 2 3 105 M, respectively. The
explants were incubated in an atmosphere of 95% O2/5% CO2 for a total period
varying between 4 and 6 h. The incubation medium was collected and renewed
every 7.5 min and was kept frozen until assayed.
The GnRH release in the incubation medium of hypothalamic explants was
measured in duplicate using a radioimmunoassay method with intraassay and
interassay coefficients of variation of 14% and 18%, respectively [17, 18]. The
highly specific CR11-B81 anti-GnRH antiserum (final dilution 1:80 000) was
kindly provided by Dr. V. D. Ramirez (Urbana, IL) [19]. The data below the
limit of detection (5 pg/7.5-min fraction) were assigned that value.
The MEM was purchased from Life Technologies Invitrogen Corp.
(Merelbeke, Belgium). E2 (17b-estradiol or 3,17b-dihydroxy-1,3,5(10)-estratriene); the two DDT isomers, o,p 0 -DDT (2,4 0 -DDT) and p,p 0 -DDT (4,4’DDT); and p,p 0 -DDE (4,4 0 -DDE) were purchased from Sigma-Aldrich
(Bornem, Belgium). P,p 0 -DDT represents approximately 80% of the insecticide
still commonly used in developing countries. O,p 0 -DDT is an equally active
isomer of the insecticide that accounts for 15%–20% of technical grade DDT.
In endocrine studies, o,p 0 -DDT has been particularly studied due to its
prominent estrogenic property and relatively less toxic activity. P,p 0 -DDE is a
long-lasting derivative of p,p 0 -DDT, with a half-life of several years. The aamino-3-hydroxy-5-methylisoxazole-4 propionic acid (AMPA)/kainate subtype
of glutamate receptor antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione) and
the ER antagonist ICI 182 780 (7a,17b-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-1,3,5(10)-estratriene-3,17b-diol) were purchased from Tocris Fisher
Bioblock Scientific (Illkirch, France), whereas the aryl hydrocarbon orphan
dioxin receptor (AHR) antagonist a-naphtoflavone (7,8-benzoflavone) was
purchased from Sigma-Aldrich. In all experiments, the steroid and insecticides
were dissolved initially in absolute ethanol (Labonord, Templenars, Belgium)
and, subsequently, in the incubation medium or sesame oil (Calbiochem VWR
International, Leuven, Belgium) for in vitro or in vivo studies, respectively, to
MATERIALS AND METHODS
Animals
736
RASIER ET AL.
serum LH levels. When the treatment period was PNDs 6–40, there were 10
rats followed in each condition to study VO and estrous cyclicity.
Statistical Analysis
FIG. 2. Effects of E2, DDT isomers, and p,p 0 -DDE in vitro on the
frequency of pulsatile GnRH secretion from hypothalamic explants
obtained in 15-day-old female rats. The data are calculated in relation
to time (two or three consecutive 2-h periods) of incubation in vitro. a: P ,
0.05 versus control; b: P , 0.05 versus data obtained during the initial 2-h
period.
achieve a final ethanol concentration of 0.01% or 1%. The antagonists were
directly diluted in the incubation medium.
Study Protocols
In vitro experiments. The in vitro effects of E2 or DDT on the frequency of
pulsatile GnRH secretion were studied. The compounds (steroid, DDT isomers,
and antagonists) were used for a whole 4- or 6-h experimental incubation
period. A concentration-response study was carried out with explants incubated
with 109 to 107 M of E2 and 106 to 104 M of o,p 0 -DDT, p,p 0 -DDT, or p,p 0 DDE. The effects of antagonists were studied in the presence of maximal
effective concentrations of E2 and o,p 0 -DDT. Those antagonists were chosen
because they were shown to prevent E2 effects on GnRH secretion in our
hypothalamic explant conditions [12, 21], and it was our hypothesis that DDT
effects, if any, could be mediated through mechanisms similar to E2 effects.
The antagonist DNQX (106 M) was used to study the involvement of the
AMPA/kainate subtype of glutamate receptors. The implication of ER was
studied using the antagonist ICI 182 780 (107 M). To investigate the
implication of AHR, the antagonist a-naphtoflavone (107 M) was used. The
concentration of these three antagonists was selected based on previous data
from our laboratory and other studies [12, 17, 21–23]. It was shown previously
that when used alone, the AMPA/kainate subtype of glutamate receptor and ER
antagonists did not affect pulsatile GnRH secretion [12], and the absence of
effects of the different antagonists when used alone was double checked in this
study.
In vivo experiments. The procedures are schematically summarized in
Figure 1. The animals received a daily s.c. administration of steroid or
insecticide for 5, 10, or 35 days (E2: 0.01, 0.1, or 1 mg/kg/day for PNDs 6–10
and 0.01 mg/kg/day for PNDs 6–15 and PND 6–40; o,p 0 -DDT: 10 or 100 mg/
kg/day for PNDs 6–10 and 10 mg/kg/day for PNDs 6–15). The dose of E2 and
o,p 0 -DDT was adjusted for increasing body weight of rats. The chemicals
dissolved in absolute ethanol were diluted in 50 ll sesame oil for s.c. injection,
as described in other studies [24, 25]. When the treatment period was PNDs 6–
10, 20 rats were studied in each treated group in comparison with 20 controls
injected with vehicle alone. On PND 15, 10 rats from each group were killed to
study the pulsatile GnRH secretion in vitro and serum LH levels. In each group,
the 10 remaining animals were then examined daily for imperforation of the
vaginal membrane to determine age at VO. Thereafter, vaginal smears were
taken every day in the afternoon until PND 60. Slides of vaginal smears were
colored using the Papanicolaou method to detect the occurrence of estrous
cyclicity and to follow cycling. The age at first estrus was considered when
vaginal smears contained primary cornified cells after the first proestrous phase,
which is characterized by both stratified and cornified cells [26]. In subsequent
experiments to study LH response to GnRH on PNDs 15 and 22, and at the
time of VO and first estrus, seven animals were killed in each group at each
age. When the treatment period was PNDs 6–15, there were five rats in each
group that were killed on PND 15 to study the GnRH pulse frequency and
When pulsatile GnRH secretion was studied, the peaks were detected using
the PULSAR program for PC [27]. The cutoff criteria for peak detection were
determined empirically and were G1 ¼ 2.5 and G2 ¼ 2.0. Peak splitting
parameter was set at 2.7, and intraassay coefficient of variation was used as B
coefficient [28]. The GnRH interpulse interval (IPI) was calculated as the time
interval between two consecutive peaks. The IPI was calculated during different
time periods of incubation (1–2 h and 3–4 h or 5–6 h). Depending on the
normal or nonnormal distribution of IPI data in the different study periods,
comparisons were made using the paired Student t-test with P , 0.05 as the
threshold for significance (GraphPad Prism software for PC) or the Wilcoxon
matched pairs test, respectively. In several instances, all the explants in a group
showed a similar IPI value. In this case, SD was null and could not be
represented.
When comparisons were made between steroid and/or insecticide effects on
LH levels and age at VO or first estrus in different conditions, raw data were
pooled and analyzed by the one-way ANOVA test when normally distributed,
followed by a multiple-comparison Newman-Keuls post-hoc test when the
threshold for significance of differences (P , 0.05) was reached. When data
were not normally distributed, the Kruskal-Wallis test was used, followed by a
multiple-comparison Dunn post-hoc test. For the experiment run PNDs 6–40,
an unpaired t-test was employed. All results are expressed as mean 6 SD.
RESULTS
In Vitro Treatments
In control conditions of hypothalamic explant incubation in
vitro (Fig. 1), the GnRH IPI showed a reduction between PNDs
10 and 25, confirming our data in the male [13]. When
hypothalamic explants obtained at 15 days were incubated in
control conditions (Fig. 2), the GnRH IPI did not change with
time (1–2 h: 60.4 6 1.7 min; 3–4 h: 59.6 6 1.8 min; and 5–6
h: 60.0 6 0.0 min). During a 4-h continuous incubation with
107 M of E2, the GnRH IPI was reduced significantly after 1–
2 h (49.0 6 3.9 min) and further after 3–4 h (45.4 6 1.7 min).
This effect was dependent on E2 concentration and incubation
time: with 108 M E2, the GnRH IPI was unchanged after 1–2
h and decreased significantly after 3–4 h; with 109 M E2, a
significant reduction occurred after 5–6 h only (52.5 6 0.0
min). The two active isomers of DDT also caused a
concentration- and time-dependent reduction in GnRH IPI that
was significant after 3–4 h using 105 M of both isomers (o,p 0 DDT: 50.4 6 5.7 min; p,p 0 -DDT: 52.5 6 0.0 min). At a 104
M concentration, both isomers resulted in an earlier effect that
was also greater after 3–4 h (o,p 0 -DDT: 45.0 6 0.0 min; p,p 0 DDT: 52.5 6 0.0 min). When used at 106 M, p,p 0 -DDT had
no effect during a 6-h incubation, and o,p 0 -DDT showed a
significant effect only after 5–6 h of incubation (52.5 6 0.0
min). No effect could be obtained using p,p 0 -DDE at similar
concentrations.
The significant reduction of GnRH IPI caused by 107 M E2
or 104 M o,p 0 -DDT after 3–4 h of incubation in vitro (Fig. 3A)
was totally prevented when the AMPA/kainate subtype of
glutamate receptors was antagonized by coincubation with
DNQX (Fig. 3B). Likewise, the effects of E2 and o,p 0 -DDT
were totally prevented in the presence of the ER antagonist ICI
182 780 (Fig. 3C). When a-naphtoflavone was used to
antagonize the AHR, the significant decrease in GnRH IPI
caused by o,p 0 -DDT was not observed any more, whereas the
E2 effect was attenuated but remained significant (Fig. 3D).
In Vivo Treatments
As shown in Figure 4A, after 5 days of treatment with 0.01,
0.1, and 1 mg/kg E2 (PNDs 6–10), the age at VO (controls:
PUBERTY AFTER EARLY EXPOSURE TO DDT
737
FIG. 3. Effects of 106 M DNQX, an antagonist of the AMPA/kainate subtype of glutamate receptors (B), 107 M of ICI 182 780, an ER antagonist (C), and
107 M a-naphtoflavone, an AHR antagonist (D) on the GnRH IPI during incubation of hypothalamic explants obtained in 15-day-old female rats in the
presence of 107 M E2 or 104 M o,p 0 -DDT (A) in vitro. A representative profile of pulsatile GnRH secretion is shown in each condition, and the mean (6
SD) IPI observed during 3–4 h of incubation are given. *P , 0.05 treatment versus control conditions.
FIG. 4. Effects of E2 or o,p 0 -DDT injected
s.c. in female rats for PNDs 6–10 or PNDs
6–40 on the ages at VO and first estrus (A)
and the interval between VO and first estrus
(B). *P , 0.05 versus vehicle (sesame oil).
738
RASIER ET AL.
TABLE 1. Mean 6 SD (n ¼ 10) of estrous cycle length (interval between
two consecutive estrus) observed until PND 60.
Treatment
PND 6–10
Vehicle
E2
o,p’-DDT
PND 6–40
Vehicle
E2
Dose (mg/kg)
—
0.01
0.10
1.00
10.00
100.00
—
0.01
Estrous cycle length (days)
4.7
5.2
5.5
4.6
5.3
5.5
6
6
6
6
6
6
1.4
1.6
1.8
1.5
2.0
1.4
4.8 6 1.2
4.9 6 1.8
30.1 6 0.6 days) was significantly earlier (23.0 6 0.7, 22.7 6
1.3, and 24.6 6 0.5 days, respectively). The VO was also
earlier after 10 and 100 mg/kg o,p 0 -DDT (22.6 6 0.5 and 24.6
6 0.7 days, respectively). The first estrus was observed on
PND 31.9 6 0.7 in controls and occurred significantly earlier
after 0.01 and 0.1 mg/kg of E2 (27.2 6 0.7 and 27.5 6 1.1
days), as well as after 10 mg/kg o,p 0 -DDT (26.4 6 1.5 days).
After 1 mg/kg E2 or 100 mg/kg o,p 0 -DDT, the age at first estrus
did not change. When the time interval between VO and first
estrus (Fig. 4B) was calculated (controls: 1.8 6 0.9 days), a
significant dose-dependent increase was observed after 0.1 and
1 mg/kg E2 or 100 mg/kg o,p 0 -DDT. No differences in estrus
cycle length were observed until PND 60 (Table 1).
As shown in Figure 5A, after five daily s.c. injections of
0.01, 0.1, and 1 mg/kg E2 (PNDs 6–10), the GnRH IPI studied
ex vivo on PND 15 (controls: 60.0 6 0.0 min) was
significantly reduced (54.4 6 3.3, 52.5 6 0.0, and 47.0 6
3.4 min, respectively). A reduction of GnRH IPI was also seen
after treatment with 10 and 100 mg/kg/day o,p 0 -DDT, which
was only significant with 100 mg/kg (55.0 6 3.6 min). When
0.01 mg/kg E2 or 10 mg/kg o,p 0 -DDT was administered for a
FIG. 5. Effects of E2 or o,p 0 -DDT injected
s.c. in female rats for PNDs 6–10 or PNDs
6–15 on the GnRH IPI (A) and serum LH
concentrations (B) studied ex vivo at 15
days of age. *P , 0.05 versus vehicle
(sesame oil).
FIG. 6. Effects of treatment of female rats with o,p 0 -DDT (10 mg/kg/day)
or sesame oil for PNDs 6–10 on serum LH levels studied 30 min after
subcutaneous injection of 1 lg/kg GnRH at four age points: PND 15, PND
22, the day of VO, and the day of first estrus. The data are mean 6 SD of
LH levels and age in seven rats studied in each group. *P , 0.05 versus
vehicle (sesame oil).
longer period of 10 days (PNDs 6–15), the GnRH IPI ex vivo
was significantly reduced on PND 15 as well.
As shown in Figure 5B, the serum LH level on PND 15
(controls: 41.1 6 19.9 ng/ml) was significantly decreased after
0.01, 0.1, and 1 mg/kg E2 between PNDs 6 and 10 (18.9 6
12.7, 23.7 6 13.8, and 9.7 6 3.0 ng/ml, respectively) and after
10 and 100 mg/kg o,p 0 -DDT (22.7 6 18.5 and 15.7 6 6.0 ng/
ml, respectively). However, the serum LH level also decreased
with age after sesame oil (control) administration (Fig. 6): 48.2
6 46.7 ng/ml on PND 15, 25.8 6 19.3 on PND 22, 16.5 6
13.7 on PND 30.6 6 1.5 (at VO), and 10.5 6 7.3 on PND 35.0
PUBERTY AFTER EARLY EXPOSURE TO DDT
6 1.2 (at first estrus). When the LH response to GnRH was
studied after vehicle or 10 mg/kg o,p 0 -DDT given for PNDs 6–
10, the LH response was not affected on PND 15 and showed
some but not significant reduction on PND 22, whereas it
dropped significantly in the next days, at the time of early VO
and first estrus.
An extended period of daily s.c. administration of 0.01 mg/
kg E2 for PNDs 6–40 (Fig. 4A) caused a significantly earlier
age at VO (22.1 6 1.0 vs. 31.8 6 1.0 days in controls),
whereas the age at first estrus was significantly delayed (47.1
6 0.8 vs. 35.4 6 1.7 days in controls), and the interval
between VO and first estrus was markedly increased (20.6 6
1.2 vs. 3.6 6 2.2 days in controls). The first estrus was
observed after a permanent estrus lasting until a few days after
the end of treatment. Then, no differences in estrous cycle
length were observed until PND 60 (Table 1). After daily s.c.
administration of 10 mg/kg o,p 0 -DDT for PNDs 6–40, the
animals showed altered health status with reduced activity and
feeding. On PND 40, their weight was significantly decreased
to 111.3 6 8.3 g (130.0 6 11.5 g after E2 and 149.0 6 9.9 g in
controls). In such conditions, it was no longer possible to
separate direct o,p 0 -DDT effects on sexual maturation and
reproduction from indirect effects through disordered nutritional status.
DISCUSSION
The hypothalamic explant paradigm used in this study
provided an opportunity to observe pulsatile GnRH secretion in
vitro as the result of GnRH neuron function in its original
surrounding neuronoglial apparatus, which regulates GnRH
secretion [29]. Moreover, the developmental changes in
frequency of pulsatile GnRH secretion retained in this model
[13] made possible the study of interaction with maturational
processes, although the critical age period for the developmental increase in GnRH pulse frequency was earlier in vitro
[14] than in vivo [30, 31]. Such a difference could involve
suppression of inhibitory extrahypothalamic inputs when
explants are deafferented from the rest of the brain. However,
the critical period of the second and third postnatal weeks in
our conditions is consistent with neuroendocrine maturation
preceding the peripheral changes of sexual maturation (VO,
testicular growth). Since we incubated retrochiasmatic hypothalami containing prominently axons and terminals of GnRHsecreting neurons [32], we tend to interpret our observations as
the effects of presynaptic regulation by the surrounding
neurons and glial cells. Other in vitro paradigms involving
GnRH neurons, such as GnRH-secreting neuronal cell lines
[33–35], primary cultures of hypothalamic neurons [36–38], or
hypothalamic slices from transgenic mice carrying reporter
genes linked to the GnRH promoter [39] could enable one to
study directly the regulatory mechanisms at the GnRH neuron
level.
The present study was designed to test experimentally the
hypothesis that early and transient exposure to pesticide, as
seen in internationally adopted children, could influence
hypothalamic-pituitary maturation and account for some
central mechanism in the sexual precocity occurring frequently
in such conditions. Since p,p 0 -DDE, which was detected in the
serum of those children, was thought to result from previous
exposure to DDT, this EDC was used for in vivo treatment.
After we observed that o,p 0 -DDT and p,p 0 -DDT were almost
equally effective on GnRH pulse frequency in vitro, the o,p 0 DDT isomer was preferred for in vivo investigations based on
its greater estrogenicity and lower toxicity than p,p 0 -DDT. In
addition, o,p 0 -DDT was the isomer used in former studies on
739
the neuroendocrine control of reproduction [10, 11, 40–42].
The doses that we used for in vivo studies and the E2:o,p 0 -DDT
concentration ratio were based on our in vitro data reported in
the present study and were comparable with those used in other
studies in rodents [43, 44]. The 10 and 100 mg/kg doses were
in the same range as the amounts given by others either
neonatally [10] or around the time of weaning [11]. After early
postnatal administration, lower doses appeared to have no
effects on age at VO and gonadotropin secretion [41]. It was
not possible in this study to measure the concentrations of DDT
isomers and derivatives in serum and tissues. The quantitative
relevance of our conditions of exposure to DDT when
compared to the exposure of migrating girls was difficult to
assess, since the only available information was p,p 0 -DDE
concentrations measured in serum several years later. After
early DDT treatment in neonatal rats for 3 days, p,p 0 -DDE
measured in adipose tissue 4–5 mo later was not different from
controls, suggesting clearance of the insecticide and its residues
after that long period [10]. After 7 days of daily oral intake of
p,p 0 -DDT in a daily dose equivalent to 106 mg/kg in 5-wk-old
rats, serum p,p 0 -DDE levels attained a mean level of 0.66 mg/l
[45]. Such a serum concentration was about 20 times higher
than the serum concentrations found in migrating children [1].
We, however, observed significant neuroendocrine effects
using a 10-times-lower DDT dose that would presumably
result in lower serum DDT levels. Further comparison would
require measurement of the different DDT isomers and residues
at different time points after stopping DDT treatment in the
animals. The interpretation of doses and exposure is even more
complex, since migrating children are likely to be exposed to
various EDCs both in the original and foster countries. It has
been reported that when chemicals are used in mixtures,
combination effect could require lower concentrations than
expected based on simply additive effects [46]. Moreover,
when animals undergo low-dose exposure, the dose-effect
relationship is not linear with comparatively greater effects of
low versus high doses. Although these aspects have not been
investigated in the present study, they could be important for
the pathophysiologic relevance of our findings.
The age window of PNDs 6–10 could be consistent with
early postnatal period in human infants. A 5-day period of
treatment was relatively short for the lifespan but significant
with respect to the short time period between birth and sexual
maturation in the rat. The use of female rats aged 15 days to
study GnRH IPI was based on our previous data showing that
GnRH secretion in vitro was maximally affected by E2 in such
conditions [12]. With hypothalamic explants from younger
females (5 days), GnRH secretion was also found to be
responsive to E2, but such age falls in the critical window of the
brain sexual differentiation, causing possible interferences with
programming of estrus cyclicity. This conclusion was drawn by
Heinrichs et al., who found early VO and delayed persistent
estrus to occur after DDT treatment for PNDs 2–4 [10].
Discrepant observations were made when o,p 0 -DDT treatment
was started at 3 wk of age for several weeks: Gellert et al.
found VO to occur earlier [11], whereas Wrenn et al. found no
change in age at VO that was hastened only when treatment
was given before the age of 3 wk [40]. In the present study,
treatment during the second week of postnatal life was a
compromise to possibly affect sexual maturation without
interfering with the sexually differentiated mechanism of
ovulation. However, such interferences probably did occur
using the highest E2 and DDT concentrations, since they did
not result in early first estrus as opposed to lower doses.
Alternatively, toxic effects could have occurred, although the
nonsignificantly affected growth in those short treatment
740
RASIER ET AL.
conditions did not support such an hypothesis. Three periods
were chosen for in vivo exposure based on the attempt to
mimic the either temporary or persisting exposure of children
either migrating from or staying in developing countries. In the
case of temporary exposure, two different lengths were studied
(until PND 10 or 15) in order to investigate whether duration
could influence the effect. This was confirmed since, after
exposure for 10 instead of 5 days, the lowest dose of DDT
became significantly effective in reducing the GnRH IPI, and
the lowest dose of E2 became more effective.
As many other EDCs, the DDT isomers were shown to
exhibit both estrogenic and antiandrogenic properties, whereas
p,p 0 -DDE retained only antiandrogenic activity [7, 8]. Because
the clinical observation was made in girls [1], and E2 was
found to preferentially influence GnRH secretion in the female
rat hypothalamus [12], female rats were used in the present
study. In our experimental conditions, it was shown previously
that both E2 and testosterone could accelerate GnRH pulse
frequency, the effect of androgens being aromatase dependent
and ultimately mediated through estrogens [12]. Therefore, we
hypothesized that the estrogenic activity of DDT was also
involved in the pathogenesis of sexual precocity [1, 2, 9]. This
hypothesis was consistent with our previous observation of
sexual precocity after a single massive administration of E2 on
PND 10 [12], although the use of lower doses for a longer time
period needed to be investigated. For all these reasons, E2 was
used as a positive control in this work to provide a comparison
basis with DDT effects in vitro and in vivo.
Direct incubation of hypothalamic explants with E2 or DDT
isomers resulted in a concentration- and time-dependent
increase in GnRH pulse frequency. The 1:1000 potency ratio
of E2:DDT found in our conditions was consistent with other
studies [47]. P,p 0 -DDE had no effect, which is in agreement
with the absence of estrogenlike effects reported in other
conditions [48]. Although supraphysiologic concentrations of
E 2 were required for an effect within 1–2 h, lower
concentrations became effective after few hours of incubation.
Such a delay could be explained by the slow diffusion of
reagents into the explant, a hypothesis consistent with the
observation that greater concentrations of compounds like
excitatory amino acids are needed in explant paradigms
compared with neuronal culture systems [49]. Another
explanation could be the latency before the possibly genomic
mechanisms involved in E2 effects became effective. Then E2
effects on GnRH secretion could have a rapid, presumably
nongenomic component, as illustrated by Matagne et al. [21] in
our paradigm, together with a slow genomic component. In this
case, the target would be other cells than GnRH neurons, since
GnRH cell bodies are absent from the studied retrochiasmatic
explants [32]. Herbison [50] and, more recently, Herbison and
Pape [51] have also reported that E2 exerts complex effects on
the GnRH neuronal function, including long-term or genomic
effects through binding to ERa and/or ERb subtypes. In our
system, the acceleration of pulsatile GnRH secretion caused by
E2 was prevented by ICI 182 780, an a/b ER antagonist, as well
as by DNQX, an antagonist of the AMPA/kainate subtype of
glutamate receptors, confirming our previous observations
[12]. The involvement of AMPA/kainate subtypes of glutamate
receptors in E2 stimulation of GnRH pulse frequency was
further supported by the hypothalamic colocalization of those
receptors together with ER [52]. Since the effects of o,p 0 -DDT
were also prevented by ICI 182 780 and DNQX, this EDC
appeared to involve the same receptors as E2. However, the
receptor pathway involved in o,p 0 -DDT effects could be partly
different, with a participation of the orphan dioxin AHR, as
indicated by the preferential reduction of o,p 0 -DDT effects by
the antagonist a-naphtoflavone. The investigation of the
insecticide effect through this pathway was justified, since
Ohtake et al. [23] reported a few years ago that dioxins can
mimic the effect of estrogens via a mechanism that involves the
activation of ER by the transcriptionally active AHR-aryl
hydrocarbon nuclear translocator complex. Further studies
could delineate the relative contribution of the a- and bisoforms of ER and the AMPA and kainate subtypes of
glutamate receptors.
After E2 administration in immature rodents, an inhibition
of LH secretion was commonly observed [53, 54] and impeded
the use of variations in LH secretion to investigate indirectly
hypothalamic effects. In addition, there is physiologically a
developmental reduction in serum LH concentrations between
PND 15 and VO, both basally and in response to GnRH [15,
16], so that decreased LH levels could result from either
negative feedback effects or accelerated maturation or both.
Therefore, hypothalamic-pituitary maturation was assessed
through evaluation of GnRH secretion during explant incubation and study of LH response to GnRH after steroid or EDC
administration in vivo. We elected to study GnRH secretion at
15 days using explants from female rats based on our previous
studies showing that the frequency of pulsatile GnRH secretion
in vitro was maximally stimulated by E2 in such conditions and
through a mechanism dependent on perinatal brain sexual
differentiation [12]. The exposure of infantile female rats to E2
or o,p 0 -DDT for 5 or 10 days was followed by a dosedependent increase in GnRH pulse frequency and a decrease in
serum LH levels on PND 15, suggesting a stimulation of
hypothalamic maturation and a negative feedback inhibition or
an early maturation of the pituitary secretion. A negative
feedback component at the hypothalamic and/or pituitary level
was supported by the reduction in the postcastration rise of
serum LH levels that was reported after treatment of mature or
neonatal rats with o,p 0 -DDT [11, 41]. However, no change in
LH secretion basally and in response to GnRH was found in
another study 6 wk after o,p 0 -DDT treatment between PNDs 1
and 10 [42]. The unchanged LH response to GnRH on PNDs
15 and 22 suggests that the result of the combined inhibitory
and stimulatory effects at those stages is a steady state. Then,
early VO could result from either a peripheral effect of E2 or
DDT or early pituitary-ovarian activity or both. A central
component at the time of VO is suggested by the brisk
reduction in LH response to GnRH that was observed in the
treated animals. Subsequently, early first estrus possibly
confirmed premature activity of the hypothalamic-pituitaryovarian axis. Such a pathophysiologic mechanism could be
consistent with an involvement of DDT in the sexual precocity
occurring after migration in internationally adopted children [1,
2, 9]. In a recent Danish study of such children, it was shown
that developmental changes in pituitary-ovarian hormone levels
were observable before onset of puberty and supported a
hypothalamic-pituitary mechanism of early puberty [55]. Since
our report of an association between sexual precocity and the
detection of p,p 0 -DDE in the serum of migrating girls [1], early
menarche was found to occur after prenatal or postnatal
exposure to DDE and/or DDT [56, 57]. However, others did
not observe changes in menarcheal age after prenatal or
postnatal exposure to DDE [58, 59], and postnatal treatment of
female monkeys with methoxychlor resulted in delayed nipple
development and menarche [60]. Further studies are warranted
to clarify the possible role of EDC nature and parameters of
exposure (age, dose, duration) in those discrepant effects. In
rodents, discrepant observations were made as well, since
pesticides such as methoxychlor and lindane resulted, respectively, in precocity and delay in the age at VO, but both
PUBERTY AFTER EARLY EXPOSURE TO DDT
insecticides caused disturbances of estrous cyclicity [61–63],
and decreased serum LH levels were reported after lindane
administration [61]. The GnRH neuron itself could be targeted
by EDCs, since the phytoestrogen coumestrol caused inhibitory
effects on GnRH transcript expression in GT1–7 GnRHsecreting neuronal cells through the b subtype of ER [64].
When E2 was injected for PND 6–40, early VO followed by
permanent estrus was observed. After E2 treatment was
stopped, first estrus appeared at a markedly delayed age. This
could indicate a peripheral stimulatory effect of the steroid
together with a central inhibition during exposure. A slight
anorexigenic effect of E2 was observed, consistent with studies
previously reported by Ramirez and Sawyer [65] and by
Ramirez [66], whereas o,p 0 -DDT caused a dramatic decrease in
body weight, suggesting a possible toxic effect of the
insecticide [45].
In summary, evidence that DDT could influence the
infantile female hypothalamic pituitary maturation was provided through early developmental acceleration of the GnRH
secretion in vitro and early reduction of LH response to GnRH
in vivo. It was also demonstrated that this chemical caused a
precocious onset of puberty in vivo when immature individuals
were transiently exposed to DDT. Further studies will aim to
delineate its mechanism of action and address whether the
GnRH neurons or other cell structures are primary targets.
13.
14.
15.
16.
17.
18.
19.
20.
ACKNOWLEDGMENT
21.
We thank Pr. J. Boniver (Department of Anatomy and Pathology) for the
assistance of his lab in Papanicolaou staining.
22.
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Oxytocin Facilitates Female Sexual Maturation through a Glia-to-Neuron
Signaling Pathway
Anne-Simone Parent, Grégory Rasier, Valérie Matagne, Alejandro Lomniczi, Marie-Christine Lebrethon, Arlette
Gérard, Sergio R. Ojeda and Jean-Pierre Bourguignon
Endocrinology 2008 149:1358-1365 originally published online Nov 26, 2007; , doi: 10.1210/en.2007-1054
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Printed in U.S.A.
Endocrinology 149(3):1358 –1365
Copyright © 2008 by The Endocrine Society
doi: 10.1210/en.2007-1054
Oxytocin Facilitates Female Sexual Maturation through
a Glia-to-Neuron Signaling Pathway
Anne-Simone Parent, Grégory Rasier, Valérie Matagne, Alejandro Lomniczi, Marie-Christine Lebrethon,
Arlette Gérard, Sergio R. Ojeda, and Jean-Pierre Bourguignon
Developmental Neuroendocrinology Unit (A.-S.P., G.R., M.-C.L., A.G., J.-P.B.), University of Liège, 4000 Liège, Belgium; and
Division of Neuroscience (V.M., A.L., S.R.O.), Oregon National Primate Research Center, Beaverton, Oregon 97006
It has been earlier proposed that oxytocin could play a facilitatory role in the preovulatory LH surge in both rats and
humans. We here provide evidence that oxytocin also facilitates sexual maturation in female rats. The administration of
an oxytocin antagonist for 6 d to immature female rats decreased GnRH pulse frequency ex vivo and delayed the age at
vaginal opening and first estrus. The in vitro reduction in
GnRH pulse frequency required chronic blockade of oxytocin
receptors, because it was not acutely observed after a single
injection of the antagonist. Hypothalamic explants exposed to
the antagonist in vitro showed a reduced GnRH pulse frequency and failed to respond to oxytocin with GnRH release.
O
XYTOCIN PLAYS a crucial role in reproduction. The
peptide plays a pivotal role in parturition and lactation in many species (1) and acts centrally to influence maternal and mating behavior in rodents (2– 4). In addition to
this involvement in reproductive behavior, oxytocin has
been shown to stimulate GnRH secretion from medial basal
hypothalamic explants of adult male rats (5) and of cycling
female rats on the afternoon of proestrus (6). Using hypothalamic explants from male rats, one of our laboratories
recently showed that neonatal pulsatile GnRH secretion in
vitro is facilitated by oxytocin and that this stimulatory effect
is mimicked by prostaglandin E2 (PGE2) (7).
Sexual maturation involves an acceleration of pulsatile
GnRH secretion (8 –10). This activation is elicited by neuronal
as well as astroglial factors produced by cells functionally
connected to GnRH neurons (11). The neuronal networks
involved in the transsynaptic regulation of GnRH secretion
mainly comprise neurons that use excitatory and inhibitory
amino acids for neurotransmission in addition to the newly
discovered kisspeptin-GPR54 signaling system (12, 13).
However, additional neuronal systems that either stimulate
or inhibit GnRH secretion have been described, including
noradrenergic, dopaminergic, and opiatergic neurons (14).
More recently, oxytocin neurons have been involved in the
facilitatory control of GnRH secretion (5–7). The recent findings that oxytocin stimulates GnRH secretion in prepubertal
male rats (7) and that administration of an oxytocin antag-
First Published Online November 26, 2007
Abbreviations: AACOCF3, Arachidonylfluoromethyl ketone; OTR,
oxytocin receptor; PGE2, prostaglandin E2; PLA2, phospholipase A2.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
Prostaglandin E2 (PGE2) mimicked the stimulatory effect of
oxytocin on GnRH pulse frequency, and inhibition of PG synthesis blocked the effect of oxytocin, suggesting that oxytocin
accelerates pulsatile GnRH release via PGE2. The source of
PGE2 appears to be astrocytes, because oxytocin stimulates
PGE2 release from cultured hypothalamic astrocytes. Moreover, astrocytes express oxytocin receptors, whereas GnRH
neurons do not. These results suggest that oxytocin facilitates
female sexual development and that this effect is mediated by
a mechanism involving glial production of PGE2. (Endocrinology 149: 1358 –1365, 2008)
onist blunted the preovulatory LH peak in women (15)
prompted us to study the role of oxytocin in female puberty.
Thus, we aimed at studying in vivo the possible delaying
effects of an oxytocin antagonist on female sexual maturation
and used an explant paradigm to define in vitro the mechanism underlying this effect. In particular, we aimed at determining whether PGE2 mediates the facilitatory effect of
oxytocin on pulsatile GnRH secretion, a pathway suggested
by the ability of oxytocin to stimulate PGE2 release from the
rat hypothalamus (5), and the effectiveness of PGE2 to stimulate GnRH release (16) via PGE2 receptors expressed in
GnRH neurons (17).
Materials and Methods
Animals
Female Wistar rats used for in vivo studies and in vitro experiments
to measure pulsatile GnRH release were housed in temperature- and
light-controlled conditions and were given ad libitum access to water and
standard rat pellets. The prepubertal animals were housed with their
mothers until weaning at 3 wk of age. Except on d 1 when rats were used
irrespective of gender, only female rats were used. The day of birth was
considered as postnatal d 1. Two-day-old female rats of the Sprague
Dawley strain purchased from Charles River Laboratories (Wilmington,
MA) were used for RNA extraction and preparation of astrocyte cultures. For comparative purposes, RNA was also extracted from the
hypothalamus of 2-d-old female mice (FVB/NTAC strain; Taconic, Hudson, NY). The use of rats and mice was approved by the University of
Liege and the Oregon National Primate Research Center Animal Care
and Use Committees in accordance with the National Institutes of Health
guidelines for the use of animals in research.
Incubation of hypothalamic explants and GnRH RIA
The animals were decapitated between 1000 and 1100 h, and tissue
fragments that included the preoptic region and the medial basal hypothalamus were rapidly dissected and transferred into a static incubator. In each experiment, 12–15 explants were studied individually for
1358
Parent et al. • Oxytocin and Female Sexual Maturation
4 – 6 h through collection and renewal of the incubation medium (0.5 ml)
every 7.5 min. This procedure has been described in detail in previous
publications (7, 10, 18). The incubation medium was phenol red-free
MEM (Life Technologies, Inc., Invitrogen Corp., Merelbeke, Belgium)
supplemented with glycine (10 nm), magnesium (1 mm), and glucose (25
mm). The incubation medium was supplemented with 20 ␮m bacitracin,
an inhibitor of GnRH degradation by endopeptidases. The medium
samples were frozen until the GnRH RIA was performed. GnRH was
measured in duplicate samples using a highly sensitive RIA (18, 19) and
an antiserum (20) generously provided by Dr. Y. F. Chen and V. D.
Ramirez (Urbana, IL). The intra- and interassay coefficients of variation
were 14 and 18%, respectively (18, 19). Values below the limit of detection of the assay (5 pg/7.5 min) were assigned that value.
Endocrinology, March 2008, 149(3):1358 –1365
1359
designed using the Primer Select software (DNASTAR Inc., Madison, WI)
and were as follows: mouse OTR (XM_144956.6) sense 5⬘-TTCTACGGGCCCGACCTGCTGTGT-3⬘ and antisense 5⬘-CTGTGCGGATTTTGGCCTTGGAGA-3⬘, rat OTR (NM_012871.2) sense 5⬘-TTCTATGGGCCCGACCTGCTGTGT-3⬘ and antisense 5⬘-CCGTGCGGATTTTGGCCTTGGAGA-3⬘,
mouse cyclophilin (NM_008907) sense 5⬘-GGCAAATGCTGGACCAAACACAA-3⬘ and antisense 5⬘-GGTAAAATGCCCGCAAGTCAAAAG-3⬘,
and rat cyclophilin (M19533) sense 5⬘-CTTTGCAGACGCCGCTGTCTCTTTTCGCCG-3⬘ and antisense 5⬘-GCATTTGCCATGGACAAGATGCCAGGA-3⬘. PCR products were resolved on a 2% agarose gels and
visualized by ethidium bromide staining. Both rat and mouse OTR
primers amplify a 505-bp PCR product.
Combined immunohistochemistry-in situ hybridization
Cell culture
Astrocytes were isolated from the hypothalamus of 1- to 2-d-old rats
and cultured as described previously (21, 22). After a growth period of
8 –10 d in 75-cm3 culture flasks containing DMEM-F12 medium supplemented with 10% calf serum, the astrocytes were isolated from contaminant cells by overnight shaking at 250 rpm and were replated on
10-cm3 dishes for RT-PCR or 12-well plates for PGE2 release experiments. After reaching 90% confluence, the medium was replaced with
a serum-free, astrocyte-defined medium consisting of DMEM devoid of
phenol red, supplemented with 2 mm l-glutamine, 15 mm HEPES, 5
␮g/ml insulin, and 100 ␮m putrescine. The cells were used 2 d later for
RT-PCR or PGE2 release experiments. For RT-PCR, the cells were snapfrozen on dry ice before extraction of total RNA. To assess the effect of
oxytocin on PGE2 release, the cells were incubated for 16 h with oxytocin
acetate (10⫺8 m; Sigma Chemical Co., St. Louis, MO). TGF␣ (100 ng/ml;
PreproTech Inc., Rocky Hill, NJ) was used as a positive control. After
stimulation, the medium was collected and stored at ⫺85 C before PGE2
assay.
The immortalized GnRH-producing cells GT1-7 (kindly provided by
Dr. R. Weiner, University of California, San Francisco, CA) were cultured in 10-cm dishes in DMEM containing 10% fetal calf serum. After
reaching 70 – 80% confluence, the cells were washed with PBS and frozen
on dry ice before RNA extraction.
Measurement of PGE2 release
PGE2 released from astrocytes in response to TGF␣ or oxytocin was
measured by RIA as described previously (23).
RNA extraction and RT-PCR
Total RNA from rat and mouse hypothalami was prepared by the acid
phenol-extraction method. Tissues were homogenized (100 mg/ml) in
Tri Reagent (Molecular Research Center, Cincinnati, OH), and the aqueous and organic phases were separated by the addition of 0.1 vol bromochloropropane (Sigma) followed by centrifugation at 4 C. One volume of isopropanol was added to the aqueous phase, and RNA was
precipitated by overnight incubation at ⫺20 C. Samples were centrifugated at 14,500 ⫻ g for 15 min at 4 C. The pellets were washed in 70%
ethanol and then air dried for 5 min. The RNA pellets were then resuspended in diethylpyrocarbonate-treated H2O, and the suspension
was incubated with DNA-free DNase I (two units per reaction) from
Ambion (Austin, TX) for 30 min at 37 C. RNA concentrations were
determined spectrophotometrically, and RNA integrity was verified on
denaturing agarose gels.
RT2-PCR was used to detect oxytocin receptor (OTR) mRNA in cultured
hypothalamic astrocytes, GT1-7 cells, and hypothalami obtained from 2-dold female rats and mice. Five hundred nanograms of total RNA were
reverse transcribed using Omniscript RT Kit (QIAGEN, Valencia, CA)
according to the manufacturer’s instructions. PCR was performed in a
volume of 25 ␮l containing 1 ␮l RT product, 2.5 ␮l 10⫻ buffer (HotStar Taq
Polymerase Kit; QIAGEN), 1 ␮l 10 mm dNTPs (HotStar Taq Polymerase Kit;
QIAGEN), 0.15 ␮l HotSar Taq Polymerase (QIAGEN), and 0.5 ␮l OTR
primers (50 ␮m) or 0.5 ␮l of a set of primers (50 ␮m) that amplify cyclophilin
mRNA, a constitutively expressed mRNA. After an initial incubation at 95
C for 15 min, the samples were amplified for 35 cycles consisting of 30 sec
at 94 C (denaturing), 30 sec at 64 C (annealing), and 1 min at 72 C (extension)
and then incubated 10 min at 72 C (final extension). The primers were
To determine whether OTR mRNA is expressed in GnRH neurons of
the rat hypothalamus, we used a combined immunohistochemistry-in
situ hybridization procedure described earlier in detail (17, 24). GnRH
neurons were stained with a monoclonal antibody to GnRH (25) diluted
1:3000, and the reaction was developed to a brown color with 3,3⬘diaminobenzidine. After completing the GnRH immunohistochemical
procedure, the sections were mounted on glass slides and dried overnight under vacuum before hybridization with a [35S]UTP-labeled rat
(r)OTR cRNA probe described below. After an overnight hybridization
at 55 C, the slides were washed and processed for cRNA detection. After
a final dehydration step in graded alcohols, the slides were dipped in
NTB-2 emulsion and were exposed to the emulsion for 3 wk at 4 C. At
this time, the slides were developed, counterstained with 0.1% methyl
green, quickly dehydrated, dried, and coverslipped for microscopic
examination. All reagents used for the immunohistochemical procedure
were prepared in diethylpyrocarbonate-treated water.
The OTR cRNA probe used was prepared by in vitro transcription of
a cDNA template cloned into the pGEM-T vector (Promega, Madison,
WI) and that derived from the 505-bp PCR product described above.
Study protocols
Effect of an oxytocin antagonist on sexual maturation. From d 15–20 of age,
16 immature female rats received a daily ip injection of 200 ␮g/kg of an
oxytocin antagonist, des-Gly-NH2d(CH2)2[d-Tyr2, Thr4]vasotocin (10⫺7
m), generously provided by Dr. Maurice Manning, Medical University
of Ohio, Toledo, OH. This antagonist is selective for the OTR with respect
to vasopressin receptors, in particular the closely related vasopressin
receptor V1a (26). Such selectivity allowed us to consider the effect of the
antagonist as specifically due to functional disruption of the intended
target.
The antagonist was diluted in saline. The control group consisted of
16 immature female rats receiving an ip injection of the saline vehicle
from d 15–20 at 0800 h. On d 20, 2 h after the last injection, eight of the
oxytocin antagonist-treated rats and eight of the control rats were killed,
and the hypothalamus was dissected and incubated for 4 h either in
regular medium or in the presence of oxytocin (10⫺8 m). The other half
of the control and oxytocin antagonist-treated groups were inspected
daily from d 20 on for vaginal opening. Subsequently, vaginal lavages
were obtained daily for 6 wk to determine the age at first estrus (27), and
the cells were visualized after staining using the Papanicolaou method.
The second experiment followed the same protocol, except that the
rats were injected daily between d 10 and 20 of age to determine whether
an earlier neutralization of oxytocin actions was more effective to delay
sexual maturation.
To determine whether the effect of the oxytocin antagonist administration in vivo on GnRH pulse frequency in vitro resulted from an acute
effect of the last dose injected or a chronic effect of the 6-d treatment, a
group of 16 20-d-old female rats received only one injection of oxytocin
antagonist or saline at 0800 h and were killed 2 h later. The hypothalami
were dissected and incubated as above.
Dose dependency of oxytocin stimulatory effect on pulsatile GnRH secretion in
vitro. Because the above in vitro paradigm allowed us earlier to detect an
increase in frequency of pulsatile GnRH secretion between 10 and 25 d
of age in male rats (8), the present studies were performed using explants
of an intermediate age (15 d). Pulsatile GnRH secretion was evaluated
after exposing the explants to 10⫺10, 10⫺9, 10⫺8, or 10⫺7 m oxytocin for
1360
Endocrinology, March 2008, 149(3):1358 –1365
Parent et al. • Oxytocin and Female Sexual Maturation
4 h (four explants per concentration) in comparison with eight explants
incubated under control conditions.
Effect of oxytocin on GnRH pulse frequency in vitro. Pulsatile GnRH secretion was studied for 4 h, starting at 1000 h, using explants obtained from
1-, 5-, 15-, and 50-d-old female rats. The 50-d-old rats were used irrespective of the phase of the estrous cycle. In a previous study, we showed
that changes in pulsatile GnRH secretion related to the phase of the
estrous cycle were detected only when the experiments were started
around 1600 h (28). The explants were incubated with saline vehicle,
oxytocin alone, or a combination of oxytocin and the oxytocin antagonist
(n ⫽ 5 for each condition), as outlined above.
Oxytocin-PGE2 interactions in vitro. Pulsatile GnRH secretion was studied
for 4 h using explants obtained from 1- and 15-d-old female rats. Pulsatile
GnRH secretion was studied under control conditions, in the presence
of oxytocin (10⫺8 m) or PGE2 (10⫺6 m) or in the presence of arachidonylfluoromethyl ketone (AACOCF3, 25 ␮m; Biomol Research Laboratories, Plymouth Meeting, PA), a blocker of phospholipase A2 (PLA2)
that results in inhibition of PGE2 synthesis. This inhibitor was used alone
or together with oxytocin (10⫺8 m).
Statistical analysis
Pulses of GnRH secretion were identified using the Pulsar program,
as described previously (29). The individual interpulse interval as well
as the mean ⫾ sem interpulse interval was calculated. In several instances, all the interpulse intervals were equal, thus accounting for an
sem equal to zero.
The effect of the different agents on GnRH pulse amplitude and
frequency was analyzed by one-way ANOVA followed by the StudentNewman-Keuls test. The effect of the oxytocin antagonist on the age at
vaginal opening and first estrus was analyzed by unpaired t test. The
threshold for significant difference was P ⬍ 0.05.
Results
Effect of an oxytocin antagonist on female sexual
maturation
In rats treated with an oxytocin antagonist for 6 d (d 15–20),
vaginal opening was delayed compared with rats injected
with the vehicle (35 ⫾ 0 vs. 33 ⫾ 0 d, P ⬍ 0.0001; Fig. 1A). The
age at first estrus, which defines the age of first ovulation,
was not affected (Fig. 1B). When treatment with the oxytocin
antagonist started earlier (d 10 instead of 15), both vaginal
opening and the age at first estrus were significantly delayed
(P ⬍ 0.0001; Fig. 1, C and D).
In vitro study of GnRH pulse frequency under control
conditions using hypothalamic explants from female rats at
5, 10, 15, 20, 25, and 30 d of age showed a decrease of GnRH
interpulse interval between d 10 and 20 (Fig. 1E). When the
hypothalamic explants of the rats treated in vivo with the
oxytocin antagonist were studied in vitro on d 20, the GnRH
interpulse interval was significantly increased with respect
to explants obtained from vehicle-treated animals (51 ⫾ 3 vs.
44 ⫾ 3 min, respectively, P ⬍ 0.001; Figs. 1E and 2, A and B).
This increase resulted from the chronic administration of the
FIG. 1. Effect of in vivo administration of an oxytocin antagonist on
the onset of puberty in female rats and on pulsatile hypothalamic
GnRH release. A, Age at vaginal opening in vehicle-treated rats and
rats treated with an oxytocin antagonist from postnatal d 15–20. B,
Age at first estrus of the same animals. Bars are means, and vertical
lines are SEM. Each group represents the mean of seven to 10 animals.
C, Age at vaginal opening in female rats treated with the oxytocin
antagonist or vehicle from postnatal d 10 –20. D, Age at first estrus
of the same animals. Bars are means, and vertical lines are SEM. Each
group represents the mean of seven to 10 animals. E, GnRH interpulse interval during female rat development using hypothalamic
explants from 5-, 10-, 15-, 20-, 25-, and 30-d-old rats as well as hypothalamic explants of 20-d-old rats incubated in vitro after a 6-d
treatment (d 15–20) with an oxytocin antagonist. Bars represent the
mean ⫾ SEM of four to five explants for each age. *, P ⬍ 0.001 vs.
vehicle-treated group; &&, P ⬍ 0.001 vs. preceding age; &, P ⬍ 0.05
vs. preceding age.
Parent et al. • Oxytocin and Female Sexual Maturation
FIG. 2. Representative profiles of GnRH secretion from hypothalamic explants of 20-d-old female rats. The rats received a daily
injection of vehicle (A and D) or 200 ␮g/kg of an oxytocin antagonist
(B and E) from d 15–20 or only one injection of oxytocin antagonist on
d 20 (C). The explants were incubated with 10⫺8 M oxytocin (D and E)
or without the hormone (A–C). Each group consists of five explants.
antagonist because the GnRH interpulse interval was not
affected by a single in vivo administration of the antagonist
on d 20 (45 ⫾ 0 min; Fig. 2C). Exposure of the explants from
rats injected with the vehicle to oxytocin in vitro resulted in
a significant reduction in GnRH interpulse interval (40 ⫾ 4
vs. 44 ⫾ 3 min, P ⬍ 0.01; Fig. 2, A and D). This was consistent
with our previous data showing a facilitatory effect of oxytocin on pulsatile GnRH secretion from male hypothalami
(7). Administration of the oxytocin antagonist for 5 d in vivo
did not affect this in vitro effect of oxytocin (48 ⫾ 4 vs. 51 ⫾
3 min, P ⬍ 0.05; Fig. 2, B and E), likely due to displacement
of decreasing antagonist concentrations by an excess of oxytocin from common binding sites.
Dose dependency and ontogeny of oxytocin and oxytocin
antagonist effect on pulsatile GnRH secretion in vitro
Incubation of hypothalamic explants from 15-d-old female
rats with oxytocin resulted in a dose-dependent reduction in
GnRH interpulse interval, which was significant at concentrations of 10⫺9 to 10⫺7 m (Fig. 3). When explants from 1-, 5-,
and 15-d-old rats were examined, the mean GnRH interpulse
interval was significantly (P ⬍ 0.001) reduced by incubation
with 10⫺8 m oxytocin (Fig. 4). Conversely, with the exception
of d 5, GnRH pulse frequency was significantly decreased by
addition of the oxytocin antagonist to the incubation medium, in the absence of exogenous oxytocin. This effect is
consistent with previous results obtained using explants
from male rats (7). Control hypothalami showed a GnRH
interpulse interval that decreased gradually throughout sexual maturation, reaching minimal values at 50 d of age (Fig.
4). At this time, neither oxytocin nor the antagonist was any
longer effective in altering pulsatile GnRH release. When
explants from 5-d-old rats were incubated simultaneously
with oxytocin and its antagonist, the effect of oxytocin on
GnRH interpulse interval was totally inhibited (Fig. 4).
Oxytocin-PGE2 interactions in vitro
Because oxytocin has been shown to induce PGE2 release
from medial basal hypothalamic explants (5), we hypothe-
Endocrinology, March 2008, 149(3):1358 –1365
1361
FIG. 3. Effects of increasing oxytocin concentrations on the GnRH
interpulse interval (mean ⫾ SEM) of hypothalamic explants from 15d-old female rats (n ⫽ 4 explants per group). NS, Not significantly
different from vehicle-treated group.
sized that PGE2 might mediate the stimulatory effect of oxytocin on pulsatile GnRH secretion. As shown in Fig. 5, both
oxytocin and PGE2 were able to significantly decrease GnRH
interpulse interval at the two ages studied (1 and 15 d).
AACOCF3, an inhibitor of PLA2, was more effective in blocking the effect of oxytocin on d 1 than on d 15. When used
alone AACOCF3 had no effect.
Study of OTR expression in GnRH neurons
The actions of oxytocin are mediated by activation of G
protein-coupled receptors (30). To determine whether GnRH
neurons express the OTR gene, we examined the preoptic
region of two immature 28-d-old female rats. GnRH neurons
were identified by immunohistochemistry and OTR mRNA
by in situ hybridization. In agreement with earlier findings
(31), OTR transcripts were expressed at low abundance in
cells scattered throughout the suprachiasmatic region. However, they were not detected in GnRH neurons. Instead,
FIG. 4. Effect of an oxytocin antagonist on GnRH interpulse interval
and on the increase in GnRH pulse frequency elicited by oxytocin
during postnatal development of the female rat hypothalamus. Hypothalamic explants from 1-, 5-, 15-, and 50-d-old rats were used. Bars
represent means (n ⫽ 4), and vertical lines are SEM. A group of hypothalamic explants from 5-d-old rats was incubated in presence of
both oxytocin and oxytocin antagonist. *, P ⬍ 0.001 vs. group incubated with vehicle alone; §, P ⬍ 0.001 vs. group treated with oxytocin
alone.
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Endocrinology, March 2008, 149(3):1358 –1365
Parent et al. • Oxytocin and Female Sexual Maturation
FIG. 5. Effects of PGE2 on GnRH pulse frequency and of an inhibitor
of PLA2 synthesis (AACOCF3) on the increase in GnRH pulse frequency elicited by oxytocin on hypothalamic explants from 1- and
15-d-old female rats (n ⫽ 4 per group). Bars are means, and vertical
lines represent SEM. *, P ⬍ 0.001 vs. control group incubated with
vehicle; §, P ⬍ 0.001 vs. group treated with oxytocin.
hybridization signals were seen in cells adjacent (Fig. 6, A
and B) or near (Fig. 6C) these neurons.
Study of OTR mRNA in hypothalamic astrocytes and
GT1-7 cells
Detection of OTR mRNA by RT-PCR showed that the
transcripts are abundant in cultured rat hypothalamic astrocytes and in the rat medial basal hypothalamus (Fig. 7A). In
contrast, they were absent in immortalized mouse GnRH
neurons but clearly evident in cultured mouse hypothalamic
astrocytes and mouse medial basal hypothalamus (Fig. 7B),
suggesting that oxytocin might act on astrocytes to stimulate
GnRH release indirectly, instead of acting on GnRH neurons.
PGE2 release by oxytocin from hypothalamic astrocytes
TGF␣ stimulates GnRH release via a glial intermediacy
that involves activation of astrocytic erbB1 receptors and the
FIG. 7. OTR mRNA can be detected by RT-PCR in hypothalamic
astrocytes but not in immortalized GnRH-producing cells. A, OTR
mRNA is present in cultured rat hypothalamic astrocytes (Astr-1 and
Astr-2) and rat medial basal hypothalamus (MBH). B, Absence of OTR
mRNA in GT1-7 cells (GT), in contrast with its presence in both
cultured mouse hypothalamic astrocytes (Astr) and mouse MBH. C,
Stimulation of PGE2 release from rat hypothalamic astrocyte cultures
by oxytocin (10⫺8 M) or TGF␣ (100 ng/ml). Bars are means, and
vertical lines are SEM. Numbers in parentheses are number of wells per
group. **, P ⬍ 0.01; *, P ⬍ 0.05 vs. basal release. Cyclo, Cyclophilin
mRNA; MM, molecular markers; ⫺RT, no RT reaction.
subsequent release of PGE2 (32). We hypothesized that oxytocin action on GnRH neurons could be similarly mediated
by astrocytic PGE2. We measured PGE2 release from rat
hypothalamic astrocytes in primary culture after 16 h of
exposure to oxytocin (10⫺8 m) or TGF␣ (100 ng/ml). As
shown in Fig. 7C, oxytocin induced a significant (P ⬍ 0.01)
increase in PGE2 release, which was, however, lower than
that elicited by TGF␣.
Discussion
FIG. 6. Absence of OTR mRNA in GnRH neurons of the immature
(28-d-old) female rat preoptic region, as assessed by combined immunohistochemistry (GnRH)/in situ hybridization (OTR) using a
[35S]UTP-labeled OTR cRNA. A and B, Examples of OTR mRNApositive cells (arrows) adjacent to GnRH neurons (brown staining)
lacking detectable OTR mRNA transcripts. C, OTR mRNA-positive
cells (arrows) in the vicinity of GnRH neurons lacking OTR mRNA.
Bars, 20 ␮m.
In this paper, we provide evidence that oxytocin facilitates
sexual maturation in the female rat. We also show that oxytocin accelerates GnRH pulse frequency in vitro from hypothalamic explants obtained from female rats of different prepubertal ages and that this effect is mostly lost in adulthood.
Using medial basal hypothalamic explants obtained from
adult male rats, acceleration of GnRH pulsatile release by
oxytocin concentrations as low as 10⫺10 m was previously
Parent et al. • Oxytocin and Female Sexual Maturation
reported (5). The present study, using hypothalamic explants
from female rats, is in agreement with those earlier findings.
That endogenous oxytocin is physiologically involved in
the control of female puberty is suggested by the delayed
sexual maturation resulting from blocking oxytocin actions
during the infantile period of postnatal development. Of
note, the oxytocin antagonist did not affect pulsatile GnRH
secretion from 5-d-old explants, in contrast to the inhibitory
effects observed earlier on d 1 and later on d 15. This ineffectiveness at 5 d may be related to a low availability of
endogenous oxytocin at this age, instead of a reduced OTR
response, because oxytocin was as effective on d 5 as on d 1
or 15 to increase GnRH pulse frequency. It is possible that
after the effect of oxytocin of maternal origin dissipates
shortly after birth, a maturational process is required for
endogenous oxytocin to be released at physiologically relevant amounts in the offspring. Earlier studies have shown
that significant amounts of mature oxytocin are detected in
the hypothalamus only after the second week of postnatal life
(33, 34). In agreement with this hypothesis, a study reported
the absence of effect of the oxytocin antagonist on age at
vaginal opening and first estrus when administered between
1 and 7 d of age. In contrast, they observed a delayed vaginal
opening and first estrus after exposure to oxytocin during the
same period (35). Our in vitro data suggest that the stimulatory effect of oxytocin on GnRH secretion decreases with
age. This could be due to an age-related increase in oxytocin
clearance or a reduced activity of its receptor. No study has
specifically reported the ontogeny of oxytocin expression in
the rat hypothalamus during pubertal maturation. However,
Chibbar et al. (36) have shown that oxytocin mRNA levels
increase in rats after puberty, after sex steroids stimulation.
The regulation of the OTR appears to be complex as well.
Although the receptor is expressed prenatally in the rat brain
(37), the same study described a progressive decrease in
receptor expression in certain regions, such as the hippocampus and the parietal cortex during postnatal development
(37). Autoradiography experiments have shown the appearance of OTRs in the rat ventromedial hypothalamus at the
time of puberty, probably under the stimulation of estrogens
(38). To our knowledge, expression of the OTR in the hypothalamus during pubertal development has never been studied. The age-related decrease in oxytocin effectiveness in our
model might be related to a decrease in expression of its
receptor, as suggested by the aforementioned studies. Alternatively, a change in receptor affinity for oxytocin may
also occur as the animal matures. However, only a limited
subset of oxytocin neurons project to targets located within
the hypothalamus (39). Thus, changes of oxytocin expression
affecting the overall population of oxytocin neurons might
make it difficult to identify changes occurring only in those
subsets of oxytocin neurons innervating the hypothalamus.
Beside its role in puberty, oxytocin has been suggested to
play a role in the preovulatory LH surge. Using explants
obtained at different phases of the estrous cycle, it has been
shown that oxytocin can stimulate GnRH release in the afternoon of proestrus only (6). In our study, neither oxytocin
nor the antagonist was any longer effective in significantly
altering the frequency of pulsatile GnRH secretion in adult
female rats. However, these data were obtained using ex-
Endocrinology, March 2008, 149(3):1358 –1365
1363
plants studied in the morning, whereas we have shown earlier that the amplitude of GnRH secretion was increased in
the afternoon of proestrus (28). Data concerning oxytocin
effects on the human menstrual cycle are controversial. In
contrast to the data showing an effect of oxytocin and an
oxytocin antagonist on the endogenous LH surge (15, 40), a
recent study showed that neither oxytocin nor its antagonist
had any effect on basal and GnRH-induced gonadotropin
secretion in the late follicular phase of the normal menstrual
cycle (41).
A major finding of the present study is that blockade of
endogenous oxytocin actions by in vivo administration of a
specific antagonist delays the initiation of female puberty.
The onset of puberty is characterized by an increase in GnRH
pulse generator activity (7). The main hypothalamic neurotransmitters/neuromodulators involved in this activation
have been extensively characterized (14). They include excitatory and inhibitory amino acids such as ␥-aminobutyric
acid and glutamate (11) and the newly described neuropeptide kisspeptin (12, 13). Our results suggest the existence of
an independent oxytocin-mediated pathway contributing to
the central regulation of the pubertal process. When endogenous oxytocin actions are blocked transiently (single injection of the antagonist), the timing of puberty is not affected.
A delay is observed only when the antagonist is given for 6 d
at the end of the infantile period, and becomes more evident
when the treatment is initiated at an even earlier age. The age
at which the antagonist is effective and the need for a sustained blockade for the antagonist to act effectively suggests
that oxytocin regulates the pubertal process by modifying
hardwiring events that take place during infantile development. The nature of these events has not been elucidated, but
it may be related to the ability of oxytocin to cause morphological changes in glial cells and neurons (42). Other studies
have shown that both neuronal remodeling induced by oxytocin (43) and permanent sex-related changes in glial morphology that occur during early postnatal development are
mediated by PGE2 (44). Thus, our in vivo and in vitro data
suggest the convergence of different regulatory mechanisms.
The stimulatory effect of oxytocin on PGE2 release from
astrocytes in primary culture as well as the stimulatory effect
of oxytocin on GnRH secretion from hypothalamic explants
in vitro suggest a rapid stimulatory effect of oxytocin. Oxytocin stimulation leads to a release of PGE2 that is able to
directly stimulate GnRH release (17). The necessity of repeated in vivo injections suggests a long-term effect potentially involving morphological changes, as discussed above.
The oxytocin antagonist was administered by ip injection,
leading to the question of its transfer across the blood-brain
barrier. The common understanding is that oxytocin antagonists cross the blood-brain barrier in small amounts (45– 47).
However, oxytocin antagonists have been shown to induce
behavioral changes when administered peripherally, implying a rate of brain transfer sufficient to induce central effects
(48 –50). Because these behavioral effects were observed
shortly after a single peripheral injection of the oxytocin
antagonist (50), it would appear unlikely that the antagonist
failed to cross the blood-brain barrier in our experiments. In
the present study, the concordant reduction in GnRH pulse
frequency observed both ex vivo after systemic administra-
1364 Endocrinology, March 2008, 149(3):1358 –1365
tion of the antagonist and in vitro demonstrates that the
antagonist is acting centrally after peripheral administration.
However, a peripheral effect of the injected oxytocin antagonist cannot be excluded. Oxytocin and its receptor are both
expressed in rodent, human. and nonhuman primate ovaries
(51–53), and the local production of oxytocin seems to play
a role in the regulation of ovarian function during the estrous
cycle (reviewed in Ref. 54). To our knowledge, no effect of
oxytocin on ovarian maturation has been yet shown.
Oxytocin neurons are located in the supraoptic and the
paraventricular nuclei, and their axons terminate in several
areas including the median eminence and the rostral hypothalamus (39). Our results suggest that oxytocin does not
stimulate pulsatile GnRH release by acting directly on GnRH
neurons. Instead, the stimulatory effect of oxytocin on GnRH
secretion appears to be mediated by PGE2 released from
astrocytes. These findings are in keeping with earlier reports
showing the involvement of PGE2 in mediating oxytocin
actions in other cellular systems (55). PGE2 is a potent GnRH
secretagogue (16), which upon release from glial cells, binds
to specific receptors located on GnRH neurons to elicit GnRH
release (17) (reviewed in Ref. 56) However, a very recent
study using double-label immunofluorescence reported the
expression of OTR in 10% of the GnRH neurons in female rat
hypothalamus (57), suggesting a possible direct action of
oxytocin on GnRH neurons. Our results did not reveal the
presence of OTR mRNA in GnRH neurons. Our combined
immunohistochemistry/in situ hybridization procedure
might not be sensitive enough to detect low levels of transcripts in such a small fraction of neurons.
Cytosolic PLA2 is a major enzyme involved in prostaglandin production by generating arachidonic acid, the precursor
of the prostaglandins, from membrane glycerophospholipids. Blocking PLA2 with AACOCF3 prevented the effect of
oxytocin on GnRH pulse frequency, implicating prostaglandins in this process. That the prostaglandin involved is PGE2
of glial origin was evidenced by the presence of OTRs in
astrocytes but not GnRH neurons and the ability of hypothalamic astrocytes to release PGE2 in response to oxytocin.
Because the effect of oxytocin was more evident after 16 h,
it is possible that the effect of oxytocin on PGE2 release may
involve an increase of cyclooxygenase 2 expression, because
it is observed in supraoptic neurons and astrocytes (43).
Although the stimulatory effect of PGE2 on GnRH release
appears to be more prominent in the median eminence (16),
the main terminal field for GnRH axons, PGE2 is also able to
stimulate GnRH neurons when directly applied to the preoptic region (58). That this is an important site of action in
mediating oxytocin-induced GnRH release is suggested by
the previous observation that during the preovulatory GnRH
release, a stimulatory effect of oxytocin requires the presence
of the preoptic area in addition to the median eminence (6).
Spontaneous mutations in the oxytocin gene have not been
reported in either rodents or humans. With the exception of
an inability to eject milk, no reproductive defects have been
reported to occur in oxytocin knockout mice (59). Because it
frequently occurs when describing knockout animals, a detailed analysis of potential defects in pubertal maturation
resulting from oxytocin deficiency has not been undertaken,
but is necessary. Given the complexity of the neuroendocrine
Parent et al. • Oxytocin and Female Sexual Maturation
mechanisms involved in controlling puberty, the failure of
unconditional gene targeting to verify findings made via
other means can be best explained as due to early compensatory mechanisms set in motion in response to the gene
deletion.
In summary, our results show that oxytocin can act on the
immature female hypothalamus to accelerate pulsatile
GnRH release and to advance the onset of female puberty.
Our results also show that this effect requires the intermediacy of OTR-containing astroglial cells that respond to oxytocin with PGE2 release.
Acknowledgments
We thank Ms. Maria Costa for expert technical assistance.
Received July 31, 2007. Accepted November 13, 2007.
Address all correspondence and requests for reprints to: Jean-Pierre
Bourguignon, Developmental Neuroendocrinology Unit, CHU Sart Tilman, 4000 Liège, Belgium. E-mail: [email protected].
This work was supported by National Institutes of Health Grants
HD25123 and U54 HD18185 through cooperative agreement as part of
the Specialized Cooperative Center’s Program in Reproduction and
Infertility Research, National Institute of Child Health and Human Development, and RR00163 for the operation of the Oregon National Primate Research Center (S.R.O.). A.-S.P. is a fellow of the Belgian “Fonds
National de la Recherche Scientifique” (FNRS). This work was supported by grants from the Fonds de la Recherche Scientifique Médicale,
Grants 3.4515.01 and 3.4573.05 (J.P.B.).
Disclosure Statement: The authors have nothing to disclose.
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endocrine community.
TOXICOLOGICAL SCIENCES 102(1), 33–41 (2008)
doi:10.1093/toxsci/kfm285
Advance Access publication November 20, 2007
Mechanisms of Interaction of Endocrine-Disrupting Chemicals with
Glutamate-Evoked Secretion of Gonadotropin-Releasing Hormone
Grégory Rasier,* Anne-Simone Parent,* Arlette Gérard,* Raphaël Denooz,† Marie-Christine Lebrethon,* Corinne Charlier,† and
Jean-Pierre Bourguignon*,1
*Developmental Neuroendocrinology Unit, Centre for Cellular and Molecular Neurobiology; and †Clinical, Forensic and Environmental Toxicology
Laboratory, University of Liège, University Hospital, B-4000 Liège (Sart-Tilman), Belgium
Received August 17, 2007; accepted October 15, 2007
In previous studies, we detected a dichlorodiphenyltrichloroethane (DDT) derivative in the serum of children with sexual
precocity after migration from developing countries. Recently, we
reported that DDT stimulated pulsatile gonadotropin-releasing
hormone (GnRH) secretion and sexual maturation in the female
rat. The aim of this study was to delineate the mechanisms of
interaction of endocrine-disrupting chemicals including DDT with
GnRH secretion evoked by glutamate in vitro. Using hypothalamic explants obtained from 15-day-old female rats, estradiol
(E2) and DDT caused a concentration-related increase in
glutamate-evoked GnRH release while p,p#-dichlorodiphenyldichloroethene and methoxychlor had no effect. The effective DDT
concentrations in vitro were consistent with the serum concentrations measured in vivo 5 days after exposure of immature rats
to 10 mg/kg/day of o,p#-DDT. Bisphenol A induced some
stimulatory effect, whereas no change was observed with
4-nonylphenol. The o,p#-DDT effects in vitro were prevented
partially by a selective antagonist of the a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate
receptors. A complete prevention of o,p#-DDT effects was caused
by an estrogen receptor (ER) antagonist as well as an aryl
hydrocarbon receptor (AHR) antagonist and inhibitors of protein
kinases A and C and mitogen-activated kinases. While an
intermittent incubation with E2 caused no change in amplification
of the glutamate-evoked GnRH release for 4 h, continuous
incubation with E2 or o,p#-DDT caused an increase of this
amplification after 3.5 h of incubation. In summary, DDT
amplifies the glutamate-evoked GnRH secretion in vitro through
rapid and slow effects involving ER, AHR, and AMPA receptor
mediation.
Key Words: gonadotropin-releasing hormone; endocrinedisrupting chemicals; glutamate receptors; estrogen receptors;
aryl hydrocarbon receptor.
1
To whom correspondence should be addressed at Division of Paediatric
Endocrinology and Adolescent Medicine, University Hospital, B-4000 Liège
(Sart-Tilman), Belgium. Fax: þ32-4-366-72-46. E-mail: jpbourguignon@
ulg.ac.be.
The persisting secular shift in timing of puberty toward early
onset in United States (Herman-Giddens et al., 1997; Lee et al.,
2001) raised the issue of the possible role of environmental
factors including endocrine-disrupting chemicals (EDCs)
(Buck-Louis et al., in press). In other countries such as
Belgium and Denmark, a 20–80 times increased risk of sexual
precocity in internationally adopted children (KrstevskaKonstantinova et al., 2001; Teilmann et al., 2006) also led to
the question of possible EDC involvement (Parent et al., 2003).
Based on the measurement of p,p#-dichlorodiphenylchloroethene (DDE) levels in the serum of those children, it was
hypothesized that migration could account for early puberty
after transient exposure to the estrogenic insecticide dichlorodiphenyltrichloroethane (DDT) in the home country (KrstevskaKonstantinova et al., 2001; Parent et al., 2003). That
hypothesis was consistent with the early report of precocious
puberty in female rats treated neonatally with o,p#-DDT by
Heinrichs et al. (1971) who showed inhibitory effects on
pituitary gonadotropin secretion and suggested a facilitatory
hypothalamic mechanism (Gellert et al., 1972).
Very recently, we administered o,p#-DDT for 5 or 10 days in
infantile female rats, and we found pulsatile gonadotropinreleasing hormone (GnRH) secretion in vitro to accelerate
prematurely and precocious puberty to occur subsequently
(Rasier et al., 2007). In that study, the effects on GnRH
secretion became significant after several hours, and both the
estrogen receptors (ERs) and the orphan dioxin receptor (aryl
hydrocarbon receptor [AHR]) appeared to be involved in DDT
effects on pulsatile GnRH secretion. In previous studies from
our laboratory, estradiol (E2) was shown to cause a rapid
(within 7.5–15 min) amplification of the GnRH release evoked
by glutamate, a main excitatory neurotransmitter in the brain.
This effect involved the a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA)-kainate receptor subtypes,
the a-subunit of ERs, and different intracellular kinases
(Matagne et al., 2005). Beside those rapid effects, initially
inactive concentrations of E2 (Matagne et al., 2005) and DDT
(Rasier et al., 2007) were shown to have effects appearing with
time, indicating slow and possibly genomic effects. This was
Ó The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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34
RASIER ET AL.
the reason why the glutamate-evoked secretion of GnRH was
studied over several hours in the present study.
Our aims were as follows: (1) to study whether DDT and
derivatives could have rapid effects similar to E2 on the
glutamate-evoked release of GnRH in vitro; (2) to compare
DDT effects with those of other EDCs such as methoxychlor
(MXC) and bisphenol A (BPA). MXC is another pesticide that
has been developed to replace DDT and to have a similar
spectrum of intended effects while being more readily
eliminated from the body and less persisting in nature. MXC
estrogenic activity is presumably related to the ability of its
metabolite hydroxyphenyltrichloroethane to bind to intracellular ERs (Miller et al., 2006). BPA is a common EDC used in
the manufacture of a variety of products including reusable
milk and food storage containers, baby formula bottles, interior
lacquer coating of food cans, or dental sealants. This
compound has been found to compete with E2 for binding to
ERs though its affinity for those receptors is much less than
that of E2 (Kwon et al., 2000). (3) To study whether DDT and
derivatives could have effects on the glutamate-evoked release
of GnRH over a period of several hours and the receptor
mechanisms involved in such effects.
MATERIALS AND METHODS
Animals
Fifteen-day-old female Wistar rats were purchased from the University of
Liège. They were housed in standardized conditions with lactating dams (22°C,
lights on from 06:30 to 18:30 h, food and water ad libitum) till the time of
sacrifice. Each litter contained 5–10 pups. The day of birth was considered as
postnatal day (PND) 1. All experiments were carried out with the approval of
the Belgian Ministry of Agriculture and the Ethical Committee at the University
of Liège.
Incubation of Hypothalamic Explants
After decapitation, the hypothalamus was rapidly dissected. The limits to
obtain the retrochiasmatic hypothalamus were the caudal margin of the optic
chiasm, the caudal margin of the mammillary bodies, and the lateral
hypothalamic sulci. Each explant was transferred into an individual incubation
chamber, as described in detail previously (Bourguignon et al., 1989a; Matagne
et al., 2004), which contained 500 ll of phenol red–free minimum essential
medium (MEM) supplemented with glucose, magnesium, glycine, and
bacitracin to achieve final concentrations of 25 3 103, 103, 108, and 2 3
105M, respectively. The explants were incubated in an atmosphere of 95%
O2–5% CO2. The incubation medium was collected and renewed every 7.5 min
and kept frozen until assayed. Since some ‘‘traumatic’’ peptide release was
observed for the first 7.5–15 min of incubation, the explants were incubated for
30 min before the first glutamate stimulation.
Assays
The GnRH release in the incubation medium was measured in duplicate
using a radioimmunoassay method with intra- and interassay coefficients of
variation of 14 and 18%, respectively (Bourguignon et al., 1989a,b). The
highly specific CR11-B81 anti-GnRH antiserum (final dilution 1:80,000) was
kindly provided by Dr V. D. Ramirez (Urbana, IL) (Dluzen and Ramirez,
1981). The data below the limit of detection (5 pg/7.5-min fraction) were
assigned that value.
The identification and quantification of p,p#-DDT, o,p#-DDT, p,p#-DDE,
and o,p#-DDE in serum were performed using a gas chromatographic analyzer
coupled to an ion-trap mass spectrometer (MS) detector (PolarisQ, Thermo
Scientific, Waltham, MA). Sample preparation included a liquid-liquid extraction (petroleum ether:diethylether, 98:2) followed by a solid-phase extraction
(Bond Elut Certify, Varian, Harbor City, CA). The eluate was evaporated to
dryness, reconstituted with n-hexane, and then injected into the gas chromatograph. The column was a HP-5 Trace (30 m 3 0.25 mm internal diameter) from
Agilent (Waldbronn, Germany). For detection, the MS was operated in the
electronic impact mode (70 eV). Multiple reaction monitoring was used for
identification and quantification. All solvents were of the highest purity
available and pesticide-grade quality (Baker, Phillipsburg, NJ). Reference
standards of all pesticides were obtained from Dr Ehrenstorfer (Augsburg,
Germany). Aldrine was used as internal standard. The calibration curve was
constructed from 0.5 to 20 ppb, and samples dilutions were applied to this
concentration range. The limits of quantification were defined as 10 times the
SD of the results of a blank sample. These limits were 0.5 ppb for p,p#-DDT,
o,p#-DDT, p,p#-DDE, and o,p#-DDE, and the coefficients of variation were 7.7,
7.1, 8.2, and 6.1%, respectively. Based on the amount of serum used, the limit
of detection was 1 ng/ml serum (Charlier and Plomteux, 2002).
Reagents
The phenol red–free MEM (supplemented with Earle’s and L-glutamine)
was purchased from Life Technologies Invitrogen Corporation (Merelbeke,
Belgium). E2 (17b-estradiol or 3,17b-dihydroxy-1,3,5(10)-estratriene), the two
DDT isomers, o,p#-DDT (2,4#-DDT) and p,p#-DDT (4,4#-DDT), p,p#-DDE
(4,4#-DDE), MXC (1,1,1-trichloro-2,2-bis-(4-methoxyphenyl)ethane), BPA
(2,2-bis-(4-hydroxyphenyl)-propane), 4-nonylphenol (4-NP), bacitracin, the
AHR antagonist a-naphthoflavone (7,8-benzoflavone), the protein kinase (PK)
A and C inhibitor staurosporine, and the PKC inhibitor chelerythrine chloride
(1,2-dimethoxy-N-methyl(1,3)benzodioxolo(5,6-c)phenanthridinium chloride)
were purchased from Sigma-Aldrich (Bornem, Belgium). The AMPA/kainate
subtype of glutamate receptor antagonist DNQX (6,7-dinitroquinoxaline-2,3dione), the AMPA receptor selective antagonist SYM 2206 (4-aminophenyl1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7-methylenedioxyphtalazine) and
the ER antagonist ICI 182,780 (7a,17b-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-1,3,5(10)-estratriene-3,17b-diol) were purchased from Tocris Fisher
Bioblock Scientific (Illkirch, France). The mitogen-activated protein kinase
(MAPK, extracellular signal-regulated kinase 1/2) inhibitor PD98059 (2#amino-3#-methoxyflavone) was purchased from Calbiochem (VWR International, Leuven, Belgium). In all experiments, steroid or EDCs were dissolved
initially in absolute ethanol (Labonord, Templenars, Belgium) and subsequently in the incubation medium to achieve a final ethanol concentration of
0.01%. All the other drugs were directly diluted in the incubation medium.
Experimental Protocols
In vivo experiments. Female rats received a daily sc administration of
o,p#-DDT (10 mg/kg/day) or vehicle (sesame oil) for 5 days (PND 6–10). The
insecticide was first dissolved in absolute ethanol and then diluted in 50 ll of
sesame oil for sc injection with 24 rats studied in the treated group in
comparison with 24 controls. On PND 15 and 22, on the day of vaginal opening
(VO) and on the day of first estrus, six rats from each group were sacrificed to
study the serum levels of the DDT isomers. A daily examination for
imperforation of the vaginal membrane was achieved to determine age at
VO. Thereafter, vaginal smears were taken every day in the afternoon until first
estrus. Slides of vaginal smears were colored using the Papanicolaou method to
detect the occurrence of estrous cyclicity (Papanicolaou and Traut, 1941). The
age at first estrus was considered when vaginal smears contained primary
cornified cells after the first proestrous phase which is characterized by both
stratified and cornified cells (Ojeda and Urbanski, 1994). The in vivo conditions
for these experiments were similar to a previous study (Rasier et al., 2007).
In vitro experiments. The effects of E2 or EDCs were studied on the
glutamate-evoked GnRH release from hypothalamic explants obtained in
15-day-old female rats. Glutamate was incubated with the explant for a 7.5-min
ENDOCRINE DISRUPTERS AND GnRH SECRETION
fraction, at a submaximal concentration (102M) which was shown previously
to be required to evoke GnRH secretion in vitro (Matagne et al., 2005). Such
a glutamate challenge was repeated every 37.5 min for a period of 4–5 h. The
102M concentration of glutamate was presumably not resulting in any toxic
effect since a similar GnRH secretory response could be obtained throughout
a 4-h experiment (Bourguignon et al., 1989b; Matagne et al., 2005). Each
experiment on the effects of E2 or EDCs started and finished with a control
glutamate stimulation in the absence of any other compound. The glutamateevoked release of GnRH was compared in control conditions and during
coincubation with E2 or EDCs added in the incubation medium. These
substances were used at increasing concentrations (E2: 1010 to 106M; p,p#DDT and o,p#-DDT: 108 to 104M; p,p#-DDE, MXC, BPA, and 4-NP: 109
to 104M) for two 7.5-min fractions, before and during incubation with
glutamate. In some experiments, the effects of E2 or EDCs were studied in the
presence of antagonists or inhibitors that were used for three consecutive
7.5-min fractions so that they were firstly incubated alone, secondly with the
steroid or EDCs, and thirdly with the two substances and glutamate. DNQX
and SYM 2206 (106M) were used to study the involvement of the AMPA/
kainate subtype of glutamate receptors on the amplification of the glutamateevoked GnRH release caused by E2 (107M) or o,p#-DDT (104M). The
implication of ERs was studied using the antagonist ICI 182,780 (107M). To
investigate the implication of AHR, the antagonist a-naphthoflavone (107M)
was used. The concentration of these four antagonists was selected based on
previous data from this laboratory and another study (Ojeda and Urbanski,
1994; Rasier et al., 2007). To investigate intracellular pathways and kinase
involvement in E2 or o,p#-DDT effects, the PKA and C inhibitor staurosporine
(107M), the PKC inhibitor chelerythrine chloride (105M), and the MAPK
inhibitor PD98059 (5 3 105M) were used.
Statistical Analysis
A minimum of four explants were used for each condition in an experiment.
The secretory response (pg/7.5-min fraction) was calculated as the difference
between the levels in the fractions collected immediately prior to and during
exposure to the glutamate. Raw data were pooled and analyzed by a one-way
analysis of variance test followed by a Newman-Keuls posttest when the
threshold for significance of difference ( p < 0.05) was reached (GraphPad
Prism software for PC). In order to compare the results obtained among
experiments in which the control data were different due to the use of receptor
antagonists in some experiments, the absolute levels (determined by
radioimmunoassay as pg/7.5-min fraction) were transformed as percentages
of the glutamate-evoked release observed in control conditions and regarded as
100%. The statistical analysis was performed using the absolute as well as
transformed data, and identical significance levels were obtained in both
conditions. The time-related changes in serum concentrations of o,p#-DDT
were compared after log transformation of the data because they were
lognormally distributed. Geometric means were calculated, and data were
represented on a log scale. All other results are expressed as mean ± SD.
RESULTS
Serum Levels of DDT Isomers and Derivatives After In Vivo
Treatment
We showed earlier that an early exposure to o,p#-DDT
between PND 5 and 10 accelerated pulsatile GnRH secretion
and induced a precocious puberty in female rats (Rasier et al.,
2007). In order to determine if the concentrations of o,p#-DDT
achieved in vivo were consistent with the concentrations used
in vitro in the present study, we measured DDT isomers and
metabolites serum concentrations after in vivo administration of
o,p#-DDT. Five days after a 5-day treatment with o,p#-DDT,
35
FIG. 1. Geometric mean (± SD) serum levels of o,p#-DDT at four time
points after administration of o,p#-DDT for 5 days (PND 6–10): PND 15,
PND 22, on the day of VO, and on the day of first estrus. The average serum
concentration on PND 15 is equivalent to 106M (n ¼ 6).
the mean serum level of this isomer was 361 ng/ml. Seven days
later, the level dropped to 23 ng/ml. VO and first estrus
occurred early on PND 23 and 27, respectively, confirming
our previous findings (Rasier et al., 2007). The mean serum
o,p#-DDT level was 22 ng/ml at VO and 4 ng/ml at first
estrus (Fig. 1). The p,p#-DDT isomer and the p,p#-DDE and
o,p#-DDE derivatives were undetectable in the serum of the
treated rats at any time of the study, and none of the studied
compounds were detectable in the serum of control rats.
Concentration-Response Effects of E2 or EDCs on
Glutamate-Evoked GnRH Release
In Figure 2 are illustrated three representative concentrationresponse profiles of the glutamate-evoked GnRH release from
retrochiasmatic hypothalamic explants obtained at 15 days in
female rats. Repeated challenges with glutamate alone for
7.5 min every 37.5 min resulted in a reproducible release of
GnRH which was maintained for 4 h (Fig. 2A). Coincubation
with increasing concentrations of E2 caused a concentrationrelated increase in glutamate-evoked GnRH release (Fig. 2B).
As shown in Figure 3A, this effect became significant using
108M of E2 (128.0 ± 4.9% of control) and further increased to
162.6 ± 15.1% and 195.0 ± 6.4% using 107M and 106M of
E2, respectively. A concentration-response increase in glutamateevoked GnRH release was also caused by o,p#-DDT (Figs. 2C
and 3B), 105 and 104M being required for a significant
effect (130.8 ± 4.5% and 173.7 ± 7.6%, respectively). After
repeated glutamate stimulation in the presence of increasing
concentrations of E2 or o,p#-DDT, a final stimulatory challenge
with glutamate alone could evoke a release similar to that
obtained initially (Figs. 2B and C). The p,p#-DDT isomer was
as effective as o,p#-DDT since the 105 and 104M
concentrations accounted for a significant increase in GnRH
release (135.7 ± 8.8% and 185.3 ± 18.1%, respectively).
36
RASIER ET AL.
FIG. 2. Representative profiles of glutamate-evoked GnRH secretion from
hypothalamic explants obtained at 15 days in female rats and incubated in
MEM for 4 h. Glutamate was added repeatedly (every 37.5 min) for 7.5 min
alone (A) and together with increasing concentrations of E2 (B) or o,p#-DDT
(C) which were also incubated for an additional 7.5 min before glutamate
stimulation. The limit of detection was 5 pg/7.5 min.
In contrast, p,p#-DDE, a stable DDT derivative, and MXC,
a DDT-related EDC, had no effect (Fig. 3B). BPA was
significantly stimulatory when used at 104M (201.7 ± 74.2%),
whereas the 105M concentration did not result in any effect
(94.2 ± 22.0%). When used at similar concentrations, 4-NP did
not show any effect (Fig. 3C).
Receptors and Signaling Involved in E2 or EDC Effects on
Glutamate-Evoked GnRH Release
When DNQX, an antagonist of the AMPA/kainate subtypes
of glutamate receptors, was used, the glutamate-evoked release
of GnRH was markedly reduced versus control (5.8 ± 0.4% vs.
11.2 ± 1.3 pg/7.5 min). This was presumably due to
suppression of the kainate receptor contribution to the
glutamate-evoked release since the selective AMPA antagonist
SYM 2206 did not significantly reduce the glutamate-evoked
release of GnRH (10.8 ± 0.8 vs. 11.4 ± 1.1 pg/7.5 min). To
allow comparison of the amplification by 107M of E2 or
104M of o,p#-DDT of the glutamate-evoked GnRH secretion,
the data were calculated as percentages of the secretory
response obtained in the presence of the antagonist alone. In
the presence of DNQX, both E2 and o,p#-DDT could still
significantly increase the glutamate-evoked secretion of GnRH
though the amplification of the response by E2 was less than
that in control conditions. In the presence of the specific
AMPA antagonist SYM 2206, both the E2 and o,p#-DDT
amplification of the glutamate-evoked GnRH release were
significant but less marked than in control conditions,
indicating a contribution of AMPA receptors (Fig. 4A). When
the ER antagonist ICI 182,780 was used (Fig. 4B), the E2 and
o,p#-DDT amplification of the glutamate-evoked GnRH
secretion were completely prevented. When the AHR antagonist a-naphthoflavone was used, only the increase in GnRH
secretory response caused by o,p#-DDT was prevented,
whereas the E2 potentiating effect was unchanged (Fig. 4B).
Inhibitors of PKA and C (staurosporine and chelerythrine
chloride) as well as MAPK (PD98059) all prevented
completely the amplification by E2 and o,p#-DDT of the
glutamate-evoked GnRH release (Table 1).
When 108M of E2 was applied intermittently for 15-min
episodes that were repeated every 37.5 min, the GnRH release
evoked by glutamate showed a similar increase during a 4-h
study period (Fig. 5). After stopping exposure to E2 at the end
of the experiment, the explants recovered a response similar to
that obtained initially. When E2 was applied continuously, the
glutamate-evoked GnRH release showed a significant increase
similar to that observed when E2 was used intermittently. After
3.5 h of incubation, however, a further increase in glutamateevoked GnRH secretion (148.2 ± 2.1%) was caused by E2 and
some significant increase persisted at the end of the experiment
(114.9 ± 1.4%), after stopping incubation with E2. A similar
time-dependent effect was observed using 105M of o,p#DDT, except that the increase in glutamate-evoked secretion
was less marked and did not persist after being back to control
conditions at the end of the experiment.
DISCUSSION
In the present study, we provide evidence that several EDCs
can directly stimulate the GnRH secretion evoked by
glutamate. Such effects occur rapidly, within 15 min and
further increase after several hours of exposure. Emphasis was
put on o,p#-DDT since we showed recently that early exposure
to this chemical resulted in acceleration of pulsatile GnRH
secretion and sexual precocity (Rasier et al., 2007). The
concentrations used in vitro for our study were shown to be
consistent with serum levels achieved after in vivo administration. We found that the mechanism of o,p#-DDT effects
involves several receptors (ERs, AHR, and AMPA) and
intracellular kinases (A, C, and MAPK).
ENDOCRINE DISRUPTERS AND GnRH SECRETION
37
FIG. 3. Concentration-response effect of E2 (A) and several EDCs (B and C) on glutamate-evoked secretory response of GnRH using hypothalamic explants
from 15-day-old female rats. The GnRH secretory response (mean ± SD) was calculated as the difference between the levels in the fraction collected immediately
prior to and during exposure to the glutamate (pg/7.5-min fraction). Those differences were then converted in percentage of controls (glutamate alone). *p < 0.05
versus control.
When compared with other in vitro paradigms involving
GnRH neurons, the static incubation of hypothalamic explants
maintains GnRH neurons in their original surrounding
neurono-glial environment and such explants retain the
capacity of pulsatile secretion of GnRH (Matagne et al.,
2004; Rasier et al., 2007). Though these two characteristics
may account for some functional relevance of our observations
in vitro, the required concentrations of some reagents such as
glutamate are higher than using cell cultures (Donoso et al.,
1990; Kuehl-Kovarik et al., 2002; Matagne et al., 2005; Ojeda
and Urbanski, 1994; Rubin et al., 2006). This discrepancy
which is still unexplained (reagent diffusion, degradation, . . .)
raises the issue of possible irrelevance of the in vitro data as
well as concerns regarding possible excitotoxic effects
mediated via the N-methyl-D-aspartic acid (NMDA) receptor
subtype. However, the capacity of the explants to respond to
repeated glutamate stimulation is sustained for several hours,
suggesting the functional integrity of the neurono-glial
apparatus involved in the secretory process. The concentrations
of E2 and o,p#-DDT used in vitro for the mechanistic studies
are consistent with those used by others (Clark et al., 1998;
Diel et al., 2002; Urbanski et al., 1996). They have been
selected after a concentration-response study on amplification
of the glutamate-evoked secretion of GnRH. For each EDC, the
maximal effective concentration has not been determined but
a concentration-response effect can be observed. Similar to E2,
the two DDT isomers increased rapidly the GnRH secretion
evoked by glutamate in a concentration-dependent manner.
The E2:EDC ratios of biocapacity observed with DDT isomers
and BPA were consistent with other in vitro studies (Clark
et al., 1998; Desaulniers et al., 2005; Rasier et al., 2006). The
other tested EDCs did not show any effect at the studied
concentrations. The lack of effect of DDE can be explained by
its prominent anti-androgenic nature. In fact, DDE is
considered to be much less estrogenic than DDT. The absence
of effect of MXC could be explained by a recent study showing
that its estrogenic effects might be mediated by its metabolites,
mono-OH MXC and bis-OH MXC (1,1,1-trichloro-2,2-bis(4hydroxyphenyl)ethane), after cytochrome P450 metabolization
(Miller et al., 2006). It is important that the serum o,p#-DDT
concentrations measured 5 days after a treatment in vivo with
the EDC are consistent with the effective concentrations
in vitro. Using a 10 times higher dose of p,p#-DDT in vivo given
orally for 7 days, others found a plateau plasma concentration
of 7.20 lg/l, that is about 20 times higher than the level we
have observed 5 days after termination of treatment in our
conditions (Mussi et al., 2005). No DDT derivatives, in
particular o,p#-DDE, could be detected in our conditions after
o,p#-DDT treatment, suggesting that, in the rat, few transformation of DDT into DDE had occurred. Tomiyama et al. (2003),
38
RASIER ET AL.
FIG. 4. Effects of antagonists of AMPA/kainate (DNQX) and AMPA (SYM 2206) subtypes of glutamate receptors (A), ERs (ICI 182,780), and AHR (anaphthoflavone) (B) on the increase in glutamate-evoked GnRH secretion caused by E2 or o,p#-DDT. The GnRH secretory response (mean ± SD) was calculated
as the difference between the levels in the fraction collected immediately prior to and during exposure to the glutamate (pg/7.5-min fraction). Those differences
were then converted in percentage of control (glutamate alone or glutamate with antagonist). Hypothalamic explants from 15-day-old female rats were used. p <
0.05 versus glutamate alone (a), versus glutamate þ antagonist (b), versus glutamate þ E2 (c), versus glutamate þ o,p#-DDT (d).
however, found plasma DDE to attain concentrations 10 times
lower to those of DDT as soon as 2 days after starting exposure
to DDT. Some explanation for this discrepancy could relate to
differences in degradation rate depending on the nature of DDT
isomer, the treatment dose, and the age at administration since
they administered p,p#-DDT between PND 36 and 42 while we
gave 10 times less of o,p#-DDT on PND 6–10.
In previous studies from our laboratory, the effects of E2 on
pulsatile GnRH secretion occurring within hours (Matagne
et al., 2004) were found to parallel the effects on glutamate-
evoked GnRH release occurring within minutes (Matagne
et al., 2005). Therefore, after showing that o,p#-DDT
stimulated frequency of pulsatile GnRH secretion (Rasier
et al., 2007), we followed a similar approach toward
a mechanistic study based on glutamate-evoked secretion.
Along the same line, 15-day-old female rats were used since
GnRH secretion in vitro was maximally affected by E2 at that
age (Matagne et al., 2004, 2005). While BPA was found to be
also capable of stimulating the glutamate-evoked GnRH
secretion, we put emphasis on DDT because of the possible
TABLE 1
Effect of Kinase Inhibitors on the Glutamate-Evoked GnRH Release (% of Controls)
Inhibitor
Inhibited kinase
Glutamate
þE2 107M
—
Staurosporine
Chelerythrine chloride
PD98059
—
PKA and PKC
PKC
MAPKs (ERK1/2)
100.0
90.6 ± 6.4
93.8 ± 6.4
101.6 ± 7.9
150.4
95.5
90.6
100.0
Note. ERK1/2, extracellular signal-regulated kinase; n ¼ 5; *p < 0.05 versus glutamate.
±
±
±
±
7.6*
7.6
6.4
9.1
þo,p#-DDT 104M
142.0
96.8
93.7
96.8
±
±
±
±
4.7*
4.4
6.5
4.4
ENDOCRINE DISRUPTERS AND GnRH SECRETION
FIG. 5. Secretory response of GnRH to 102M of glutamate used for
7.5 min every 37.5 min for 5 h. Starting after 60 min and ending after 262.5 min,
the explants are incubated intermittently (for 15 min, every 37.5 min) or
continuously with E2 or o,p#-DDT. a: p < 0.05 versus initial glutamate
secretory response; b: p < 0.05 versus previous E2-potentiated or o,p#-DDT–
potentiated glutamate secretory response.
involvement of the insecticide in sexual precocity of migrating
children (Krstevska-Konstantinova et al., 2001; Parent et al.,
2003) and the experimental evidence of hypothalamic-pituitary
effects in the rat (Gellert et al., 1972; Heinrichs et al., 1971).
Overall, the effects of DDT appear to be comparable to those of
E2, and ERs play a key role in mediating such effects. The two
main mechanisms involved in endocrine disruption are
agonistic effects at ERs or estrogenic as shown for DDT, and
antagonistic effects at androgen receptors or anti-androgenic as
shown for DDE (Clark et al., 1998; Kelce et al., 1995). In our
experimental conditions, it was shown previously that both
estrogens and androgens were capable of potentiating the
GnRH secretion, the effects of androgens being aromatase
dependent and ultimately mediated through estrogens
(Matagne et al., 2004). Thus, the effects reported here fall
within the estrogenic category.
The AMPA receptors are an additional class of receptors
involved in DDT effects. The prominent role of NMDA and
kainate receptors in glutamate-mediated pulsatile GnRH
secretion and the involvement of both NMDA and kainate
receptors in the glutamate-evoked release of GnRH have been
demonstrated in earlier studies (Bourguignon et al., 1989b;
Matagne et al., 2004, 2005; Parent et al., 2005). By contrast,
a selective AMPA antagonist does not affect, neither pulsatile
GnRH secretion (Rasier et al., 2007) nor the glutamate-evoked
release as shown here, indicating no involvement of the AMPA
receptor subtype in such conditions. These receptors, however,
appear to play some role in the effects of E2 and o,p#-DDT on
both the pulsatile secretion of GnRH (Rasier et al., 2007) and
its release evoked by glutamate. Since those two effects occur,
39
respectively, after 1–2 h and 7.5–15 min, the former were
thought to possibly involve genomic mechanisms and the latter
nongenomic. In consistency with a time-dependent involvement of two mechanisms, E2 and o,p#-DDT cause rapidly an
increase in glutamate-evoked GnRH release and a further
increase occurring after few hours as a slow effect. Not only
time but also continuity of the exposure to the steroid is critical
since the slow component of the increased responsiveness to
glutamate does not appear when exposure is discontinuous. In
contrast, we had no evidence of priming of the response caused
by the repeated glutamate stimulation. Herbison (1998), and
Herbison and Pape (2001) have reported that E2 exerts
complex effects on GnRH neuronal function including longterm or genomic effects through binding to ERa- and/or bsubtypes. Furthermore, in vitro rapid nongenomic effects of E2
can occur within seconds to minutes in various conditions to
influence cellular events such as kainate-induced currents in
hippocampal neurons (Improta-Brears et al., 1999) and second
messenger cascades in hippocampal (Gu et al., 1999) or
hypothalamic neurons (Abraham et al., 2004; Lagrange et al.,
1999). Regarding a possible genomic effect, E2 has been
shown to stimulate AMPA glutamate receptor expression in the
rat hypothalamus (Diano et al., 1997). This could be one of the
mechanisms explaining AMPA involvement in E2 or EDC
effects on GnRH secretion. As far as we know, this is the first
report showing the involvement of the AMPA subtype of
glutamate receptors in rapid EDC effects. A few recent studies
showed that AMPA receptors mediate plastic effects induced
by E2 in different populations of neurons (Todd et al., 2007;
Tsurugizawa et al., 2005). Particularly, AMPA receptors have
been shown to be involved in the neuronal morphological
changes induced by E2 in the ventromedial nucleus of the
hypothalamus in female rats, specifically (Todd et al., 2007).
Both E2 and o,p#-DDT potentiating effects on the glutamateevoked GnRH release could be prevented by PKA and C and
MAPK inhibitors. These observations confirm our earlier
report for E2 and provide further evidence of a rapid
intracellular component of EDC effects.
Our data also indicate the possible role of AHR in mediating
o,p#-DDT effects on GnRH secretion. As reviewed recently,
the AHR is an ubiquitous receptor system binding endogenous
ligand as well as xenobiotics such as dioxins (Harper et al.,
2006). In this study, it appears that o,p#-DDT could be a ligand
of AHR since its effects on GnRH release are antagonized by
a-naphthoflavone. Only the glutamate-evoked GnRH secretion
amplified by o,p#-DDT is prevented by a-naphthoflavone,
suggesting that DDT can stimulate GnRH secretion through
a mechanism partially different from that of E2. Beyond the
presumably initial step in o,p#-DDT effects, the pathway seems
to be similar to E2 with involvement of the AMPA receptors in
rapid effects and also ERs in rapid and slow effects (Rasier
et al., 2007). To integrate these observations, it is conceivable
that the o,p#-DDT–AHR complex results in a cross talk
with the E2 signaling machinery and forms a functional
40
RASIER ET AL.
transcriptional complex in the regulatory region of ERaresponsive genes (Ohtake et al., 2003).
Recently, kisspeptin (kiss-1), a tumor suppressing peptide,
and its receptor GPR54 have been identified as major actors of
the regulation of GnRH secretion (de Roux et al., 2003;
Seminara and Kaiser, 2005). Kiss-1 and GPR54 are expressed
in the medial basal hypothalamus, and kiss-1 has a direct action
on GnRH neurons which express GPR54. Recent studies have
shown that kisspeptin neurons in the anteroventral periventricular nucleus express a-subtype of ERs and that E2 might
exert a positive feedback on GnRH secretion during LH surge
by stimulating kiss-1 expression (Adachi et al., 2007).
Knowing that crucial role of kiss-1 in regulation of GnRH
secretion and its modulation by E2, we could hypothesize that
kiss-1 could be a potential target for estrogenic EDCs.
In summary, this study shows that EDCs, in particular
o,p#-DDT, can modulate the GnRH secretion in vitro in the
immature female hypothalamus through both rapid and slow
effects with the involvement of estrogen, dioxin, and AMPA
receptor pathways.
FUNDING
European Commission (EDEN project, QLRT-2001-00269);
Faculty of Medicine at the University of Liège (Léon Frédéricq
Foundation); Belgian Study Group for Paediatric Endocrinology.
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Molecular and Cellular Endocrinology 324 (2010) 110–120
Contents lists available at ScienceDirect
Molecular and Cellular Endocrinology
journal homepage: www.elsevier.com/locate/mce
Review
Neuroendocrine disruption of pubertal timing and interactions between
homeostasis of reproduction and energy balance
Jean-Pierre Bourguignon ∗ , Grégory Rasier, Marie-Christine Lebrethon,
Arlette Gérard, Elise Naveau, Anne-Simone Parent
Developmental Neuroendocrinology Unit, GIGA Neurosciences, University of Liège and Department of Pediatrics, CHU de Liège, Belgium
a r t i c l e
i n f o
Article history:
Received 18 December 2009
Received in revised form 23 February 2010
Accepted 23 February 2010
Keywords:
Hypothalamus
Puberty
Gonadotropin releasing hormone
Endocrine disrupters
Energy balance
Nutrition
a b s t r a c t
The involvement of environmental factors such as endocrine disrupting chemicals (EDCs) in the timing
of onset of puberty is suggested by recent changes in age at onset of puberty and pattern of distribution
that are variable among countries, as well as new forms of sexual precocity after migration. However, the
evidence of association between early or late pubertal timing and exposure to EDCs is weak in humans,
possibly due to heterogeneity of effects likely involving mixtures and incapacity to assess fetal or neonatal
exposure retrospectively. The neuroendocrine system which is crucial for physiological onset of puberty
is targeted by EDCs. These compounds also act directly in the gonads and peripheral sex-steroid sensitive
tissues. Feedbacks add to the complexity of regulation so that changes in pubertal timing caused by EDCs
can involve both central and peripheral mechanisms. In experimental conditions, several neuroendocrine
endpoints are affected by EDCs though only few studies including from our laboratory aimed at EDC
involvement in the pathophysiology of early sexual maturation. Recent observations support the concept
that EDC cause disturbed energy balance and account for the obesity epidemic. Several aspects are linking
this system and the reproductive axis: coexisting neuroendocrine and peripheral effects, dependency on
fetal/neonatal programming and the many factors cross-linking the two systems, for instance leptin,
adiponectin, Agouti Related Peptide (AgRP). This opens perspectives for future research and, hopefully,
measures preventing the disturbances of homeostasis caused by EDCs.
© 2010 Published by Elsevier Ireland Ltd.
Contents
1.
2.
3.
4.
5.
6.
7.
Introduction: rationale for endocrine disruption of pubertal timing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sexually dimorphic evidence of endocrine disruption of pubertal timing in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Direct and indirect EDC effects on neuroendocrine maturation at puberty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experimental models of EDC neuroendocrine effects on the reproductive axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
In vitro studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
In vivo studies and critical periods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A rodent model of EDC neuroendocrine effects on sexual maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Endocrine disruption of puberty and reproduction in relation to homeostasis of energy balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Experimental data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Human data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction: rationale for endocrine disruption of
pubertal timing
∗ Corresponding author at: Department of Pediatrics, CHU ND des Bruyères, Rue
de Gaillarmont, 600, B4032 CHENEE, Belgium. Tel.: +32 4 367 92 75/96 87;
fax: +32 4 367 94 29.
E-mail address: [email protected] (J.-P. Bourguignon).
0303-7207/$ – see front matter © 2010 Published by Elsevier Ireland Ltd.
doi:10.1016/j.mce.2010.02.033
Puberty is the life period when pituitary–gonadal maturation
leads to a series of physical changes and ultimately, achievement
of reproductive capacity. A central event in the onset of puberty is
Author's personal copy
J.-P. Bourguignon et al. / Molecular and Cellular Endocrinology 324 (2010) 110–120
an increase in frequency and amplitude of Gonadotropin Releasing Hormone (GnRH) secretion in the hypothalamus. This event
is controlled by redundant inhibitory or excitatory mechanisms
that respectively disappear or appear at the onset of puberty
(Bourguignon, 2004). It is generally agreed that variations in pubertal timing within a physiological 5-year period are predominantly
determined by genetic factors while environmental factors play
a comparatively minor role (Parent et al., 2003). A robust landmark of environmental effects on pubertal timing arose through
the secular advance in menarcheal age. Because this observation
was made between the mid-19th and the mid-20th centuries both
in USA and Western Europe and more recently in developing countries (Parent et al., 2003), the likely explanation was thought to
be improvement in health and nutritional status with industrialization. Accordingly, the end or slowdown of this process seen
between 1960 and 2000 was expected. Around the year 2000 however, two large American studies provided evidence of earlier onset
of puberty (Herman-Giddens et al., 1997; Lee et al., 2001). Very
recently relatively similar findings were obtained in Denmark and
Belgium (Aksglaede et al., 2009; Roelants et al., 2009). As opposed
to the previous changes, the recent observations were heterogeneous: they could differ among countries; initial signs such as onset
of breast development were more affected than subsequent signs
such as menarcheal age (Herman-Giddens et al., 1997; Lee et al.,
2001; Aksglaede et al., 2009; Roelants et al., 2009); age distribution
showed skewing towards earlier ages for initial signs and towards
later ages for final signs (Roelants et al., 2009; Papadimitriou et al.,
2008). Because those changes in pubertal timing were concomitant with the epidemic of obesity in USA, the pathophysiological
involvement of fat mass, possibly through leptin (Herman-Giddens
et al., 1997; Lee et al., 2001; Himes, 2006) was hypothesized in that
country. However, the recent changes in pubertal timing in Denmark were not associated with changes in adiposity (Aksglaede et
al., 2009). Thus other factors including endocrine disrupting chemicals (EDCs) could be involved (Teilmann et al., 2002).
Additional evidence of environmental effects on pubertal timing
in humans came from studies in children migrating for international adoption. As cohorts, they appeared to mature earlier than
children in the foster countries and in the countries of origin (Proos
et al., 1991; Parent et al., 2003). Also, sexual precocity requiring
GnRH agonist therapy was much more common in those migrat-
111
ing children than in others (Krstevska-Konstantinova et al., 2001;
Teilmann et al., 2006). Based on increased serum levels of DDE, a
derivative of the estrogenic insecticide DDT found among migrating children, we hypothesized that early exposure to this EDC
and subsequent withdrawal due to migration could account for a
neuroendocrine pathogenetic mechanism of secondary central precocious puberty (Krstevska-Konstantinova et al., 2001; Parent et al.,
2003). Many other EDCs however could possibly be involved and
could result in peripheral precocity as well (see below). The bias
accounting for DDT study came from the very long half-life of its
derivative DDE. Moreover, it was likely that other factors including
recovery from earlier nutritional as well as psychosocial deprivation could play some role in this particular condition (Dominé et
al., 2006).
2. Sexually dimorphic evidence of endocrine disruption of
pubertal timing in humans
It is challenging to link exposure to particular EDCs and health
issues such as disorders of pubertal timing for several reasons
(Buck Louis et al., 2008; Diamanti-Kandarakis et al., 2009). Humans
as well as animals are likely exposed to a variety of EDCs acting
as mixtures with time-related changes in compounds and doses.
Mixtures result in more than additive effects since compounds
mixed at concentrations that were inactive when used as single EDCs were shown to become active when used as mixtures
(Kortenkamp, 2008). The study of mixtures, however, is complex
and laborious. So far neuroendocrine studies have been performed
with single classes of EDCs only. When exposure is assessed at the
time of pubertal disorders, there has been a very long period since
fetal/perinatal life, the most critical time for EDC effects. Though
such factors could affect the relevance of the studied relationship
between EDCs and pubertal disorders, we will review the available information that is mainly based on the study of single EDCs.
Prenatal and postnatal exposure will be separated whenever possible as well as effects in males and females. Sexual dimorphism
is indeed a critical issue when EDC effects are considered. As a
whole, EDCs appear to work either as estrogen agonists or as androgen antagonists with the ratio Estrogen/Androgen actions as an
ultimate determinant of EDC effects (Rivas et al., 2002). Consistent with this concept is the observation that premature breast
Table 1
Variations in pubertal timing in relation with pre- and/or post-natal exposure of female humans to endocrine disrupters.
Pubertal timing
Early
Normal
Exposure
Prenatal
Postnatal
DDE (+DDT)
Menarche (Vasiliu
et al., 2004)
Menarche (Ouyang
et al., 2005)
B2 (Krstevska-Konstantinova et al., 2001)
Prenatal
Delayed
Postnatal
Prenatal
Menarche (Denham et
al., 2005)
B2 (Wolff et al., 2008)
Menarche and B3 (Gladen et al., 2000)
Methoxychlor
PBBs
PCBs
Monkey (Golub et
al., 2003)
Menarche (Blanck et al., 2000)
Menarche
(Denham et al.,
2005)
Dioxins
Phthalates
Phytoestrogens soy
formula
Postnatal
B2 (Blanck et al., 2000)
Menarche (Vasiliu
B2/menarche (Den
et al., 2004; Yang et
Hond et al., 2002); B2
al., 2005)
(Wolff et al., 2008)
Menarche and B3 (Gladen et al., 2000)
Menarche (Leijs et
al., 2008; Warner
et al., 2004)
Menarche (Den Hond
et al., 2002)
B2 (Leijs et al.,
2008)
B2 (Den Hond et
al., 2002)
B2 (Colon et al.,
2000)
Menarche (Strom et al.,
2001)
B2 (Wolff et al.,
2008)
DDE: dichlorodiphenyldichloroethene; DDT: dichlorodiphenyltrichloroethane; PBB: polybrominated biphenyl; PCB: polychlorinated biphenyl; and B2, B3: Tanner’s stages 2
and 3 of breast development.
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Table 2
Variations in pubertal timing in relation with pre- and/or post-natal exposure of male humans to endocrine disrupters.
Pubertal timing
Exposure
Early
Prenatal
Normal
Postnatal
Delayed
Prenatal
Postnatal
DDE (+DDT)
G3-5, PHV (Gladen et al., 2000)
PCBs
G3-5, PHV (Gladen et al., 2000)
G, TV (Mol et al., 2002)
Dioxin
Prenatal
Postnatal
Penile length (Guo et
al., 2004)
P and G (Den Hond
et al., 2002)
P and G (Den Hond
et al., 2002)
DDE: dichlorodiphenyldichloroethene; DDT: dichlorodiphenyltrichloroethane; PCB: polychlorinated biphenyl; G: Tanner’s stage of genital development; P: Tanner’s stage
of pubic hair development; PHV: peak height velocity; and TV: testicular volume.
development can occur after exposure to phthalates that are considered to act primarily as androgen antagonists (Colon et al.,
2000). Further, an estrogenic compound such as DDT can generate the anti-androgenic sub-product DDE that can also indirectly
reflect previous exposure to DDT, further complicating the elucidation of estrogenic versus anti-androgenic effects (Rasier et al.,
2008).
Virtually, any clinical evidence of pubertal development (except
testicular growth) can result from either centrally driven maturation involving the hypothalamic–pituitary system or direct
peripheral interaction in the tissues targeted by sex steroids or both
mechanisms. Here, the clinical manifestations will be reviewed
irrespective of the underlying mechanism. In a subsequent section, the neuroendocrine mechanisms will be delineated. More
data were obtained in girls (Table 1) than in boys (Table 2). This
could involve methodological biases since a precise timer of maturation is provided by menarcheal age in girls who also experience
more obvious onset of puberty with the development of breasts
as opposed to the less perceptible increase in testicular volume in
boys (Parent et al., 2003).
While the majority of EDCs studied in girls accounted for normal
or early timing (Table 1), dioxins and phytoestrogens were associated with delayed timing of breast development (Den Hond et al.,
2002; Leijs et al., 2008; Wolff et al., 2008). Menarcheal age however did not seem to be affected by dioxins or phytoestrogens in
the same studies (Den Hond et al., 2002; Leijs et al., 2008) and in
others (Strom et al., 2001; Warner et al., 2004). Likewise, dissociation between normal timing of breast development and early
menarche was reported in relation to exposure to PBBs (Blanck et
al., 2000). These findings emphasize the importance of studying
different pubertal signs possibly involving different mechanisms
at several times throughout the pubertal process. Except one study
reporting early menarche in relation to exposure to PCBs (Denham
et al., 2005), this EDC was found to be associated with normal timing
of both breast development and menarche (Gladen et al., 2000; Den
Hond et al., 2002; Vasiliu et al., 2004; Yang et al., 2005; Wolff et al.,
2008). Early breast development (Krstevska-Konstantinova et al.,
2001) and early menarche (Vasiliu et al., 2004; Ouyang et al., 2005)
were reported in relation to exposure to DDT and or DDE whereas
normal pubertal timing was found by others (Gladen et al., 2000;
Denham et al., 2005; Wolff et al., 2008). In the female monkey,
delayed nipple growth and short follicular phase were seen after
exposure to methoxychlor (Golub et al., 2003). Overall, those studies did not enable to show different effects depending on prenatal
or postnatal period of exposure to the EDCs.
In boys, the few studies available (Table 2) suggest no effects of
DDT, DDE and dioxins (Gladen et al., 2000; Den Hond et al., 2002).
Exposure to PCBs was found to be associated with either normal
(Gladen et al., 2000; Mol et al., 2002) or delayed timing (Den Hond
et al., 2002; Guo et al., 2004) of male pubertal development.
3. Direct and indirect EDC effects on neuroendocrine
maturation at puberty
Puberty involves a cascade of physiological events resulting from CNS and neuroendocrine maturation (Table 3).
Because sex steroids can act at the different levels of the
hypothalamic–pituitary–gonadal system, the imbalance between
estrogen and androgen effects caused by EDCs (Rivas et al., 2002)
potentially affects all those levels. Gender dimorphism is another
important aspect both peripherally and centrally.
Changes in gonads and peripheral tissues responsive to sex
steroids were the first endpoints studied to show EDC effects.
Direct disruption of the peripheral reproductive system involves
predominantly effects mimicking estrogens in the female while
counteracted androgen effects are involved peripherally in the male
(Diamanti-Kandarakis et al., 2009). Since the reproductive system
is regulated by feedback loops between gonads and peripheral tissues on the one hand and pituitary gland, hypothalamus and CNS
on the other hand, direct peripheral effects of EDCs can secondar-
Table 3
Some possible mechanisms of EDC maturational effects on the hypothalamic–pituitary–gonadal system in female and male individuals.
Level possibly targeted by EDCs
Developmental effects of increased Estrogen/Androgen balance
Female
Mechanisms
Male
CNS: suprahypothalamic afferences
Hypothalamus: GnRH neurons and
surrounding neurono-glial system
Structural changes?
Facilitation (or inhibition) of pulsatile GnRH secretion
Female more sensitive than the male?
Alteration of sexually dimorphic control of ovulation
Primary central/neuroendocrine
or
Secondary to altered feedback effects of
gonadal hormones
Pituitary gland: gonadotrophic cells
Early pubertal stimulation
or
Increased prepubertal inhibition (negative feedback)
Response to neuroendocr. effects
or
Peripheral feedback
Gonads: sex steroid production/effects
and gametogenesis
Peripheral tissues: sex steroid effects
Alteration of folliculogenesis
Alteration of spermatogenesis
Early/increased stimulation of
estrogen sensitive tissues
(breast, uterus)
Delayed/reduced stimulation
of androgen sensitive tissues
(penis, prostate)
Primary peripheral
or
Secondary to altered neuroendocrine control
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113
Table 4
In vitro evidence of neuroendocrine disruption of the reproductive system.
EDC
Expos.
Sex
Endpoint
PCBs
GT1–7 neurons
–
GT1–7 neurons
–
Aroclor1221: increased GnRH mRNAs and peptide release; neurite outgrowth.
Aroclor 1254: biphasic effects and neurotoxicity (Gore et al., 2002)
Early elevation and late reduction of GnRH release; apoptotic and neurotoxic
effects (Dickerson et al., 2009)
MXC
GT1–7 neurons
–
Coumestrol
DDT
GT1–7 neurons
Rat hyp. explants
–
F
Dioxin
Adult rat
F
ER independent reduction of GnRH mRNAs; ER mediated stimulation of GnRH
release (Gore, 2002)
ER␤ mediated inhibition of GnRH mRNA expression (Bowe et al., 2003)
ER and AhR receptor mediated stimulation of pulse frequency of GnRH
secretion and glutamate-evoked release (Rasier et al., 2007, 2008)
TCDD in vitro: no effect on pulsatile GnRH release (Trewin et al., 2007)
MXC: methoxychlor; PCBs: polychlorinated biphenyls; ER: estrogen receptor; AhR: arylhydrocarbon receptor; DDT: dichlorodiphenyltrichloroethane; and TCDD: 2,3,7,8tetrachlorodibenzo-p-dioxin.
ily influence central mechanisms through changes in endogenous
peripheral hormones. Consequently, changes in neuroendocrine
and pituitary function could result indirectly from altered peripheral feedback.
Changes in hypothalamic and pituitary function could also
result directly from EDC neuroendocrine effects. Centrally, the
neuroendocrine control of reproduction through the preovulatory
gonadotropin surge and its alteration following exposure to sex
steroids during fetal or perinatal life has been known for several
decades as a specific female feature (Gorski, 1968). In such conditions, changes in gonadal and peripheral tissue function could be
determined by EDC neuroendocrine effects. Due to obvious limits in
assessment of neuroendocrine function in the clinical setting, the
use of experimental models is required to tackle neuroendocrine
effects of EDC.
4. Experimental models of EDC neuroendocrine effects on
the reproductive axis
In Tables 4 and 5 are summarized some findings providing
experimental evidence of EDC neuroendocrine effects. The available data are quite heterogeneous as far as the studied EDCs, the
experimental models and the endpoints. Since, so far, only few
studies have addressed the issue of neuroendocrine disruption of
sexual maturation, we have extended our review of the mecha-
Table 5
In vivo evidence of neuroendocrine disruption of the reproductive system.
EDC
Expos.
Sex
Endpoint
DES
Quail embryo
M
MXC
Fetal ewe
F
Reduced vasotocin in medial preoptic nucleus and bed nucleus of stria
terminalis; suppression of male copulatory behavior (Viglietti-Panzica et al.,
2005)
Delayed LH surge (Savabieasfahani et al., 2006) – secondary to altered
folliculogenesis?
BPA
Fetal ewe
F
Fetal rat
Fetal/neonatal mouse
M
F
Neonatal rat
Neonatal rat
Adult OVX rat
M
M/F
F
Neonat. mouse
Neonat. fish
M
M/F
Neonat. rat
F
Fetal rat
F
Fetal rat
F
Adult fish
M
DDT
PCBs
Dioxin
Fish larvae
Fetal rat
M
Isoflavone
Adult rat
F
Coumestrol
Adult OVX rat
F
Reduced magnitude of LH surge (Savabieasfahani et al., 2006) – secondary to
altered folliculogenesis?
Increased ER␤ mRNA expression in POA (Ramos et al., 2003)
Decline in tyrosine hydroxylase neurons in POA and suppression of sex-related
rearing behavior (Rubin et al., 2006)
Increased anxiety behavior and body weight (Patisaul and Bateman, 2008)
Reduced hypothalamic Kiss-1 expression (Navarro et al., 2009)
Increased progesterone receptor expression in POA and VMN; reduced sexual
receptivity (Funabashi et al., 2003)
Increased brain estrogen receptor (Mussi et al., 2005)
Increased activity and expression of brain aromatase and male to female sex
reversal (Kuhl et al., 2005)
Increased frequency of pulsatile GnRH secretion, sexual precocity, disturbed
estrus cyclicity (Rasier et al., 2007)
Aroclor 1221: increased GnRH mRNAs in POA-anterior hypothalamus and
unchanged timing of vaginal opening; Aroclor 1254: no effects (Gore, 2008)
Aroclor 1221: altered female mating behavior (Steinberg et al., 2007) and
reduced proestrus LH surge (Steinberg et al., 2008)
Aroclor 1254: reduced tryptophan hydroxylase activity and GnRH content in
POA (Khan and Thomas, 2001)
TCDD: prevention of brain aromatase upregulation by estradiol (Cheshenko et
al., 2007)
TCDD: increased content and reduced release of GnRH by hypothalamic
explants (Clements et al., 2009)
Increased ER␤ receptors in PVN; reduced upregulation of oxytocin receptors
via ER␣ in VMN; reduced sexual receptivity (Patisaul et al., 2001)
Reduced multiunit electrical activity in hypothalamus and inhibition of LH
pulse amplitude and frequency (McGarvey et al., 2001)
MXC: methoxychlor; BPA: bisphenol A; DES: diethylstilbestrol; PCBs: polychlorinated biphenyls; PVN: paraventricular nucleus; VMN: ventromedial nucleus; POA: preoptic
area; OVX: ovariectomized; ER: estrogen receptor; DDT: dichlorodiphenyltrichloroethane; and TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin.
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nisms of EDC effects to the overall neuroendocrine control of the
reproductive system.
4.1. In vitro studies
In vitro models allowed direct evaluation of EDC effects on
neurono-glial function (Table 4). A debated question is as to
whether sex steroids and EDCs that have the estrogen receptor
as common target influence the GnRH neuron directly or indirectly. In a previous review, we concluded that GnRH neurons could
be directly involved in responding to estrogens (Matagne et al.,
2003). Maturational characteristics could influence estrogen effects
on GnRH neurons. GT1 cells, a model for GnRH neurons that are
responsive to estrogens and EDCs, have been obtained from immortalization at an embryonic stage and are somehow developmentally
arrested. Direct early responsiveness of GnRH neurons to estrogens
at early developmental stage is supported by a recent study showing that neurogenesis in cultured olfactory placode was directly
stimulated by estradiol (Agça et al., 2008). Later in adulthood, estrogen effects on GnRH neurons may become indirect since studies
with cell specific deletion of the different ER subtypes indicated that
estradiol positive feedback at the time of the preovulatory surge
primarily involved other estrogen responsive cells than GnRH neurons (Herbison, 2008). Species differences could also occur since it
was shown recently that orchidectomy caused increased amplitude
of pulsatile GnRH release from monkey but not from rat hypothalamic explants (Woller et al., 2010). Using cultured immortalized
GnRH neurons, variable changes in GnRH transcripts and peptide
release were obtained depending on the EDC and the concentrations used, the response being possibly non-linear and biphasic
(Gore et al., 2002; Gore, 2002; Bowe et al., 2003; Dickerson et al.,
2009). PCBs also accounted for neurotoxic effects with increased
expression of cleaved caspase-9 (Gore et al., 2002; Dickerson et
al., 2009). We used hypothalamic explants to study the effects
of DDT, an estrogenic EDC, in relation to sexual maturation. Our
paradigm included axons and terminals of the final effector, i.e. the
GnRH neuron (Purnelle et al., 1997) as well as the afferent neuronoglial apparatus possibly involved in EDC effects. The explants also
retained some developmental characteristics since they released
GnRH in a pulsatile manner with a frequency increasing from birth
to onset of puberty (Bourguignon et al., 1992). We used hypothalamic explants of immature female rats aged 15 days because they
were found earlier to be responsive to estradiol through an increase
in frequency of pulsatile GnRH secretion (Matagne et al., 2004). In
such conditions, DDT resulted in effects similar to estradiol (Rasier
et al., 2008). However, Trewin et al. (2007) could not observe any
change in pulsatile GnRH secretion caused by dioxin using hypothalamic explants from cycling adult female rats. The age difference
could account for such discrepant observations since we did not
observe at 25 and 50 days the stimulatory estradiol effects seen at
5 and 15 days (Matagne et al., 2004). Though such in vitro models provided an opportunity to study directly the mechanisms of
neuroendocrine EDC effects, the conditions were not comparable
to those in vivo both in terms of GnRH neuron function/regulation
and environmental exposure to the studied EDCs. The explants or
the immortalized neurons were deafferented from physiological
neurono-glial inputs. Concentrations higher than those toxicologically relevant in humans were required likely due to low diffusion
in the explants (Matagne et al., 2003). This reinforced the value of
in vivo models.
4.2. In vivo studies and critical periods
Suggestive evidence of EDC neuroendocrine effects was
obtained in vivo by studying physiological processes known to
involve hypothalamic or CNS regulation (Table 5). Except phytoe-
strogen effects that were studied in adult animals, the vast majority
of studies were performed after exposure during fetal and/or early
postnatal life. This is consistent with the concept of critical periods
in early life that appeared to determine the effects of sex steroids
(McCarthy et al., 2009) as well as nutrition (Gluckman and Hanson,
2004) on the homeostasis of reproduction and energy balance. The
neuroendocrine events affected by EDCs in vivo include central,
i.e. gonadotropin-dependent onset of puberty (Rasier et al., 2007;
Gore, 2008), ovulation that is dependent on stimulation by the
gonadotropin surge (Savabieasfahani et al., 2006; Steinberg et al.,
2008) and sexual behavior in males (Viglietti-Panzica et al., 2005)
and females (Patisaul et al., 2001; Funabashi et al., 2003; Rubin et
al., 2006; Steinberg et al., 2007). Not unexpectedly, those three sexually dimorphic processes are likely regulated by sex steroids and
possibly disrupted by EDCs differently in males and females in different species. Prenatal exposure of the ovine fetus to testosterone
caused alteration of pubertal timing and estrus cyclicity through
neuroendocrine alteration of estradiol positive feedback (Unsworth
et al., 2005), including marked reduction of FOS-positive GnRH
neurons in response to estradiol (Wood et al., 1996). In similar
conditions, fetal lamb exposure to the EDCs methoxychlor or BPA
accounted for delayed or severely reduced LH surge, respectively,
without change in pubertal timing (Savabieasfahani et al., 2006). As
emphasized by the authors, the exposed animals also had ovarian
anomalies, growth retardation and metabolic disorders that could
contribute to disrupted reproductive function. A single prenatal
administration of PCB mixture on gestational day 16 caused postnatal growth retardation (Gore, 2008) and disturbed sexual behavior,
prominently in females (Wang et al., 2002; Steinberg et al., 2008).
Behavioral evidence of transgenerational effects came from female
preference of male with no history of exposure, three generations after the progenitors were exposed to vinclozolin (Crews et
al., 2007). These findings might suggest epigenetic changes in the
neuroendocrine components of sexual behavior regulation. Direct
evidence of epigenetic mechanisms in the hypothalamus in relation
with sexual differentiation further provide rationale for studies on
EDC effects on epigenetics of the neuroendocrine system (Murray
et al., 2009).
Other endpoints in experimental studies on neuroendocrine
effects include expression or transcripts of sex steroid receptors
(Patisaul et al., 2001; Funabashi et al., 2003; Ramos et al., 2003;
Mussi et al., 2005) and enzymes involved in sex steroid metabolism
or dependent on sex steroid effects (Khan and Thomas, 2001; Kuhl
et al., 2005; Rubin et al., 2006). In this respect, aromatase deserves
special attention due to its involvement in the control of sexually
differentiated neuroendocrine functions including sexual behavior
(Balthazart et al., 2006). In the zebra fish, dioxins were shown to
alter estrogen upregulation of aromatase (Cheshenko et al., 2007).
Several authors including our group have studied direct or indirect appraisal of GnRH synthesis and secretion (Khan and Thomas,
2001; McGarvey et al., 2001; Rasier et al., 2007; Gore, 2008) While
we found that in vivo early postnatal exposure of female rat to DDT,
an estrogenic EDC, resulted in premature developmental increase
in GnRH pulse frequency in vitro (Rasier et al., 2007), Clements
et al. (2009) reported recently that, in male rats, GnRH release in
vitro was reduced after fetal exposure to dioxin, another estrogenic EDC. This stresses again the diversity of conditions including
gender, compound and period of exposure that could account for
discrepant data. Finally, neuropeptides dependent on sex steroid
effects (Patisaul et al., 2001; Viglietti-Panzica et al., 2005) including the recent demonstration of reduced Kiss-1 expression by BPA
(Navarro et al., 2009) and non-sexual aspects of behavior (Patisaul
and Bateman, 2008). In summary, several neuroendocrine studies
on EDC effects were performed but the conclusions drawn from
such studies remain rather limited due to the many variables that
may influence the response to EDCs, e.g. dose, age at exposure,
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115
Fig. 1. Schematic illustration of the mechanisms of estradiol and o,p -DDT effects on GnRH secretion. Three pathways are identified with both rapid, presumably non-genomic
effects and slow, presumably genomic effects: (1) binding the classical estrogen receptor (ER) and causing genomic effects after activation through the estrogen responsive
element (ERE); (2) binding some ER and interacting with the AMPA subtype of glutamate receptor with subsequent involvement of intracellular kinases; and (3) binding the
aryl hydrocarbon (AHR) orphan dioxin receptor, and cross talking with the E2 signalling machinery to form a functional transcriptional complex in the regulatory region of
ER-responsive genes (Ohtake et al., 2003).
duration of exposure, route of administration, gender and mixtures.
Comparison between neuroendocrine effects and studies on effects
in other parts of the CNS may help in elucidation of alterations
caused by EDCs during CNS development (Naveau et al., 2010).
5. A rodent model of EDC neuroendocrine effects on sexual
maturation
Using hypothalamic explants of immature female rats aged 15
days, we showed that o,p -DDT directly stimulated GnRH pulse frequency in vitro. The mechanism (Fig. 1) involved both the ER and
the aryl hydrocarbon receptor (AHR) as well as the AMPA subtype of
glutamate receptors (Rasier et al., 2008). The DDT effects were not
only dose-dependent but also time-dependent with both rapid and
slow effects since the glutamate-evoked release of GnRH increased
as soon as after 7.5 min of incubation and further after 4 h of incubation (Rasier et al., 2008). These in vitro findings provided the
rationale for an in vivo study of DDT effects on pubertal timing
through a central/hypothalamic mechanism. The aim was to expose
female rats early and transiently to DDT in order to model the neuroendocrine effects of the pesticide that could account for sexual
precocity in girls migrating for international adoption (KrstevskaKonstantinova et al., 2001; Parent et al., 2003). Because fetal or
early postnatal exposure to testosterone or estradiol would masculinize the CNS and alter the mechanism of estrus cycling, the
animals were exposed to DDT on postnatal days 6–10. We reported
in earlier studies that exposure to estradiol within this age window resulted in sexually differentiated effects on GnRH secretion
both in vitro and in vivo and subsequent female sexual precocity
(Matagne et al., 2004). In similar age conditions, in vivo exposure
to DDT followed by ex vivo study of GnRH release by hypothalamic explants resulted in premature acceleration of pulsatile GnRH
secretion (Rasier et al., 2007). Both vaginal opening and first estrus
occurred earlier after exposure to estradiol or DDT though the time
interval between the two events was increased. Our findings could
involve central as well as peripheral mechanisms of sexual precocity. In order to obtain further evidence of neuroendocrine effects of
DDT in vivo, the response of pituitary LH to a bolus administration
of synthetic GnRH was studied. LH response was shown to reflect
previous stimulation by endogenous GnRH and, in man, increased
LH response was regarded as the evidence of neuroendocrine maturation leading to the so-called central puberty (Carel et al., 2009). A
different situation occurred in the female rat since neuroendocrine
maturation was associated with a developmental reduction of LH
response, a confounding observation with LH reduction due to negative feedback such as seen in peripheral precocity. Nevertheless,
we observed a premature developmental reduction in LH response
after exposure to DDT that possibly resulted from neuroendocrine
effects (Rasier et al., 2007). Based on the rodent model, our interpretation of sexual precocity after migration is summarized in Fig. 2
(Parent et al., 2003; Rasier et al., 2006, 2007). During exposure to
DDT, estrogenic effects can account for both peripheral and central
(neuroendocrine) stimulation. However, due to concomitant negative feedback inhibition at the pituitary level, the central effects
are not translated into gonadotropin stimulation of the ovaries until
the pituitary inhibition disappears following migration in a DDTfree environment. In a study of internationally adopted girls aged
5–8 years before they eventually showed clinical evidence of sexual precocity, Teilmann et al. (2007) reported that serum FSH and
estradiol levels were already elevated in several girls, confirming
early pituitary–ovarian activity after migration. The above mechanism is comparable to that operating in other conditions with
peripheral precocious puberty (e.g. congenital adrenal hyperplasia, adrenal or gonadal tumours) followed by secondary central
precocious puberty after the peripheral disorders is cured by medical or surgical treatment (Parent et al., 2003). Consistent with this
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Fig. 2. Schematic representation of the putative mechanism of sexual precocity after transient exposure to the estrogenic insecticide DDT in girls migrating for international
adoption. During exposure to DDT, neuroendocrine maturation is promoted but not translated into ovarian stimulation due to negative feedback inhibition of the pituitary
gonadotropes. After migration in a DDT-free environment, the negative feedback does no longer operate and allows central maturation to lead to gonadotropin-dependent
puberty (modified from Rasier et al., 2006).
concept, central precocity should not be manifested in conditions
of persisting exposure to DDT. We attempted to demonstrate that
continued administration of DDT to the female rats was not associated with evidence of precocious central maturation but we failed
due to toxic effects including malnourishment and growth failure
(Rasier et al., 2007).
We previously reviewed the effects of EDCs on sexual maturation in laboratory rodents (Rasier et al., 2006). Peripheral effects
can be biphasic, low doses causing early puberty and high doses
delayed puberty such as shown using triphenyltin in the female rat
(Grote et al., 2006) and phthalates in the male (Ge et al., 2007). In the
few studies where hypothalamic pituitary function was assessed,
LH secretion was reduced together with early vaginal opening after
DES (Kubo et al., 2003) or irregular cycling after BPA (Rubin et al.,
2006). Still, the possibly coexisting central and peripheral effects
remain a matter of confusion. As an example, BPA administration
to neonatal female rats for 4 days resulted in early vaginal opening
and acyclicity but no change in sexual receptivity and FOS induction in GnRH neurons after steroid priming (Adewale et al., 2009).
This suggested predominant peripheral effects in those conditions
and highlighted the importance of simultaneous study of central
and peripheral effects.
6. Endocrine disruption of puberty and reproduction in
relation to homeostasis of energy balance
The neuroendocrine system in the hypothalamus constitutes a
single place for different aspects of homeostasis including reproduction and energy balance. It is also involved, during fetal and
neonatal life, in programming mechanisms accounting for the
“developmental origin of health and diseases” (Gluckman and
Hanson, 2004). Therefore, it appeared interesting to integrate some
of the many connections between homeostasis of reproduction and
homeostasis of energy balance, during both prenatal/neonatal and
postnatal life (Fig. 3).
6.1. Experimental data
Fetal malnourishment as well as fetal exposure to endocrine
disrupters such as DES and BPA were shown to possibly result
in low birth weight, early puberty, ovulatory disorders, obesity
in adulthood and metabolic syndrome (Gluckman and Hanson,
2004; Newbold et al., 2008, 2009; Sloboda et al., 2009; Heindel
and vom Saal, 2009). The consequences of fetal malnourishment
on adult adiposity excess and metabolic syndrome could be prevented by neonatal or early postnatal leptin treatment indicating
the critical role of this peripheral peptide in fetal/neonatal programming (Vickers et al., 2005). Leptin has appeared to be not only
an anorexigenic hormone produced by the mature adipocyte but
also a structural organizer of the hypothalamic circuitry controlling
energy balance during a critical period including fetal life and the
first 3 weeks of postnatal life in rodents (Bouret et al., 2004; Bouret
and Simerly, 2007). Bouret (2010) hypothesized that anomalies in
early leptin organizational effects in relation with nutritional disturbances during that critical window could predispose to later
obesity and metabolic disorders. Leptin could also be involved after
fetal/neonatal exposure to sex steroids since neonatal androgenization resulted in marked reduction of leptin mRNA levels in the
pituitary gland of female rats on days 14 and 22 (Morash et al.,
2001). Further involvement of leptin in relation to sex steroids and
EDCs was suggested by the reduced serum leptin levels observed
neonatally in association with reduced anogenital distance after
fetal exposure to phthalates (Boberg et al., 2008). During postnatal life, leptin is also obviously an important link between energy
balance and reproduction. As shown in Fig. 4, facilitatory effects on
GnRH secretion were observed after a single leptin administration
in 15-day-old rats but not at 50 days, further supporting the concept of early critical window before the age of 3 weeks in rodents
(Parent et al., 2003; Lebrethon et al., 2007). Together with leptin as
peripheral messenger, a common hypothalamic mediator of early
EDC effects on energy balance and reproduction could be the Agouti
Related Peptide (AgRP), an endogenous orexigenic antagonist at
melanocortin receptors. In our laboratory, AgRP treatment in vivo
or in vitro (Fig. 4) was found to cause deceleration of frequency
of pulsatile GnRH secretion from male rat hypothalamic explants
(Lebrethon et al., 2007). This effect was similarly seen in immature
and pubertal animals suggesting a role in the neuroendocrine control of the reproductive axis but no involvement in the mechanism
of puberty. Exposure of fetal and neonatal mice to BPA was shown to
result in hypomethylation of a metastable epiallele locus upstream
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J.-P. Bourguignon et al. / Molecular and Cellular Endocrinology 324 (2010) 110–120
117
Fig. 3. Schematic representation of the interactions between homeostasis of reproduction and energy balance as well as hypothalamic effectors and peripheral effectors in
the neuroendocrine mechanisms involved in effects on pubertal timing.
Fig. 4. Upper panel: representative profiles of GnRH secretion by hypothalamic explants from 15-day-old male rats incubated in the presence of leptin, ghrelin or Agoutirelated peptide (AgRP). In each condition, the mean ± SD interpulse interval is given (number of explants studied). Lower panel: mean interpulse interval observed in vitro
using explants from 15- or 50-day-old male rats injected with leptin, ghrelin or AgRP in vivo. *p < 0.05 versus controls (modified from Lebrethon et al., 2007).
Author's personal copy
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J.-P. Bourguignon et al. / Molecular and Cellular Endocrinology 324 (2010) 110–120
to the agouti gene that was associated with increased ectopic agouti
gene expression (Dolinoy et al., 2007) known to result in yellow coat
color and adult obesity (Dolinoy, 2008). Fertility however was not
impaired in those BPA exposure conditions and adult weight was
not studied.
EDCs affect adipocytes directly at different periods of life
through induction of cell differentiation and adipogenesis (Grün et
al., 2006) as well as lipid accumulation in differentiated adipocytes
(Wada et al., 2007). Gestational and lactational exposure to BPA
resulted in increased adipogenesis at weaning in female rats (Somm
et al., 2009). This is in contrast to the reduced adiposity and
increased insulin sensitivity in mice fed from conception to adulthood with a phytoestrogen rich diet (Cederroth et al., 2008). EDC
effects on adipocytes could be increased due to storage in the adipose tissue of several EDCs that are lipophilic. Adiponectin, a factor
protecting against obesity and insulin resistance, could play a role
as well. Using mature abdominal adipocytes in vitro, BPA reduced
the secretion of several adipokines including adiponectin (Hugo et
al., 2008; Ben-Jonathan et al., 2009). Through such an effect, EDC
could facilitate GnRH secretion since immortalized GnRH neurons
were shown to express adiponectin receptors and GnRH release
was reduced in the presence of adiponectin (Wen et al., 2008). The
fetal balance between estrogenic and androgenic steroids that is
disturbed by EDCs can also be directly affected by sex steroids such
as treatment of pregnant ewe with testosterone. In these conditions, pubertal timing in female lambs was shifted from the female
to the male pattern as a function of the degree of virilization of
external genitalia (Kosut et al., 1997). Postnatal events may worsen
the consequences of disturbed programming: postnatal weight
excess due to overfeeding amplified the reproductive disturbances
following prenatal androgenization of the female lamb (Steckler et
al., 2009).
ture pubarche, hyperinsulinism, ovarian hyperandrogenism and
polycystic ovary syndrome (PCOS). These findings can be correlated with several experimental effects of manipulation of nutrition
or exposure to sex steroids or EDCs in the fetus as detailed
above. Of particular interest is the context of this discussion is
the observation that IUGR is associated with increased visceral
adiposity and reduced serum levels of adiponectin in childhood
(Ibáñez et al., 2009). The latter effect parallels BPA-induced reduction of adiponectin production by adipocytes (Hugo et al., 2008;
Ben-Jonathan et al., 2009). In addition, breast-fed IUGR newborns
show lower adiposity and higher serum leptin levels than controls
(Ibáñez et al., 2010), an interesting finding in the face of prevention
of insulin resistance and obesity by postnatal leptin administration
in the rat born IUGR (Vickers et al., 2005).
7. Perspectives
Understandably, the main focus of studies on EDCs has been
the reproductive system for several decades with fertility and
hormone-dependent cancers as the most critical issues. More
recently, genital malformations and disorders of pubertal timing
have emerged as earlier manifestations of a spectrum of disturbances all likely to result from EDC effects during the fetal and
neonatal critical age windows. While the interactions between
nutrition and reproduction have been known for decades, it is
only recently that EDCs have appeared to alter the homeostasis
of both reproduction and energy balance. These two systems share
neuroendocrine control in relation with feedback from peripheral
tissues through circulating factors. Further studies should scrutinize the common mechanisms and factors possibly linking EDCs
with disturbances of those two systems.
Acknowledgments
6.2. Human data
In the clinical setting, the interaction between nutrition and
reproduction has been viewed for almost 40 years as an issue for
adolescent and adult females in the perspective of energy availability determining reproductive system function. Such a concept that
was proposed by Frisch and Revelle (1970) became further substantiated when leptin was discovered as a key messenger from adipose
tissue to the hypothalamus and controlling the reproductive system via stimulation of GnRH secretion (rev in Parent et al., 2003).
As already stated in the introduction, such a mechanism substantiated a putative link between obesity epidemics and earlier onset
of puberty. In the field of endocrine disruption, fetal and neonatal
determination of reproductive disorders in adulthood arose four
decades ago following the observations of genital cancer in the
female offspring of mothers treated with DES during pregnancy
(Herbst et al., 1971). Similarly consistent with the early life origins
of diseases, it was suggested recently that obesity and metabolic
syndrome could involve primary peripheral mechanisms through
early impact of nutrition on increased adipogenic and lipogenic
capacity of adipocytes (Muhlhausler and Smith, 2009). Such a concept is consistent with the recent finding that fast weight gain in
infancy between birth and 9 months predicts increased adiposity at
10 years and early menarcheal age (Ong et al., 2009). The hypothesis was also raised that EDC effects on adipose tissue and energy
balance could provide the basis of a toxicological mechanism in
the obesity epidemic (Baillie-Hamilton, 2002; Heindel, 2003). All
together, those findings indicate that both the human reproductive system and energy balance system share a possible fetal/early
postnatal determinism of adult disorders under the influence of
early nutritional conditions and/or early exposure to EDCs.
Ibáñez et al. (1998, 2007) reported that intrauterine growth
retardation (IUGR) was associated with increased risk of prema-
This work was supported by grants from the European Commission (EDEN project, contract QLRT-2001-00269), the Fonds
National de la Recherche Scientifique (FRS-FNRS 3.4.573.05F and
3.4.567.09F), the Faculty of Medicine at the University of Liège (Léon
Frédéricq Foundation) and the Belgian Study Group for Paediatric
Endocrinology.
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