Download Neurobiological mechanisms of puberty in higher primates

Human Reproduction Update, Vol.10, No.1 pp. 67±77, 2004
DOI: 10.1093/humupd/dmh001
Neurobiological mechanisms of puberty in higher primates
Tony M.Plant1 and Mandi L.Barker-Gibb
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
1
To whom correspondence should be addressed. E-mail: [email protected]
Puberty in humans is comprised of two developmental processes; namely, gonadarche and adrenarche. Of the two,
gonadarche is fundamentally the most important, and this review examines the neurobiological mechanisms that
®rst prevent, and later trigger, progression into this critically important phase of human development when the
ability to ®rst reproduce is established. The review draws extensively upon results obtained by studies of the rhesus
monkey (Macaca mulatta), a representative higher primate which, like man, exhibits a postnatal pattern in activity
of the hypothalamic±pituitary±gonadal axis that is characterized by a prolonged period of relative quiescence from
late infancy until the initiation of the pubertal process. The proximate cause of the prepubertal quiescence in this
neuroendocrine axis is the arrest or restraint of the pulsatile mode of hypothalamic GnRH release by a neurobiological brake that holds in check release of this decapeptide, without seeming to down-regulate the transcriptional
activity of the gene encoding GnRH (GnRH-I). Thus, if neurogenomes control the onset of gonadarche, they must
reside upstream from that of the GnRH neuron. The genetic and physiological factors (with a particular emphasis
on leptin) that time the application and duration of the prepubertal brake on GnRH release are also considered.
Key words: g-aminobutyric acid/gonadarche/GnRH/leptin/neuropeptide Y
Introduction
Puberty is the phase of development during which the potential to
reproduce is ®rst realized. In our own species, puberty is typically
initiated at the end of the ®rst, or the beginning of the second,
decade of life, and its onset is recognized by maturation of the
genitalia, development of secondary sexual characteristics, acceleration in growth, changes in affect and, in the female, by the
occurrence of menstruation. Paediatric endocrinologists generally
consider that human puberty is the result of two independent
physiological processes, namely gonadarche and adrenarche
(Witchel and Plant, 2003). Gonadarche refers to the activation of
the ovary and testis at the end of the prepubertal phase of
development and leads to the dramatic increase in gonadal steroid
production and the completion of gametogenesis. The unfolding of
gonadarche is manifest by thelarche and menarche in girls, and by
testicular enlargement and virilization in boys. Adrenarche, on the
other hand, refers to the maturation of the adrenal cortex that leads
to increased secretion of adrenal androgens, namely dehydroepiandrosterone, dehydroepiandrosterone sulphate and androstenedione, and is manifest by the appearance of sexual hair, a process
termed pubarche. It is interesting to note that adrenarche appears to
be peculiar to man and the Great Apes (see Plant, 1994), and the
absence of adrenarche in humans does not appear to prevent
fertility nor markedly to in¯uence the timing and tempo of
gonadarche (Sklar et al., 1980; Grumbach, 2002). Thus, from a
biological perspective it would seem reasonable to argue that
adrenarche should be viewed as a corollary of the pubertal process
rather than as an integral and fundamental component of this
important developmental event. From a clinical perspective,
however, the impact of pubertal disorders of adrenal androgen
secretion on development of the neuroendocrine axis governing
the ovary should not be overlooked. This is particularly relevant to
the emerging relationship between hyperandrogenism and polycystic ovarian syndrome (IbaÁnÄez et al., 2000). With the foregoing
argument in mind, the present review examines our current
understanding of the physiological, genomic and genetic mechanisms that underlie gonadarche and that regulate the timing of
this process in higher primates.
Role of hypothalamic GnRH
That an intermittent hypophysiotrophic gonadotrophin-releasing
stimulus is obligatory for operation of the pituitary±gonadal axis in
the adult was established by the classic work of Knobil and his
colleagues in the 1970s (Belchetz et al., 1978). This group was
able to restore gonadotrophin secretion in hypothalamic-lesioned
monkeys with an intravenous infusion of synthetic GnRH
(mammalian GnRH, GnRH-I) only when the decapeptide was
administered in an intermittent mode. Spontaneous pulsatile
release of GnRH by the brain is generated by a diffusely
distributed hypothalamic network of several hundred neurons
that express the GnRH-I gene (Silverman et al., 1994). Although it
is now generally recognized that the mechanism responsible for
Human Reproduction Update Vol. 10 No. 1 ã European Society of Human Reproduction and Embryology 2004; all rights reserved
67
T.M.Plant and M.L.Barker-Gibb
Figure 1. Sex differences in the duration and intensity of the neurobiological
brake imposed upon pulsatile GnRH release during prepubertal development
in the rhesus monkey, as re¯ected by the time-courses of circulating
concentrations of LH (top panel) and FSH (lower panel) in animals
ovariectomized (closed data points) and orchidectomized (stippled area) at 1
week of age. Note that the prepubertal brake on the secretion of
gonadotrophin is most prolonged and marked in the male. This sex
difference is further exaggerated when night-time gonadotrophin
concentrations are examined (not shown but may be found in Plant, 1985
and Pohl et al., 1995). Vertical bars above data points indicate SEM.
Reproduced with permission from Plant (1994), ã Lippincott Williams &
Wilkins 1994.
pulsatility in this neuronal network, the so-called GnRH pulse
generator, is intrinsic to the GnRH neurons themselves (Wetsel
et al., 1992; Terasawa et al., 1999, 2002), the ability of rat
retrochiasmatic hypothalamic explants (where GnRH axons have
been severed from their cell bodies) to sustain pulsatile GnRH
release (Prunelle et al., 1997) is dif®cult to explain.
The case may be that a pulsatile GnRH drive is critical for
sustained gonadotrophin secretion and if compromised, for
example in severe undernutrition and during strenuous exercise
training, individuals become hypogonadotrophic (Cameron, 1996;
Warren and Stiehl, 1999). This, in turn, leads to a hypogonadal
state that in women presents as secondary amenorrhoea and
anovulation. Thus, the pituitary±gonadal axis may be viewed as a
slave to the hypothalamic GnRH pulse generator, and this analogy
should be borne in mind as the role of GnRH in initiating
gonadarche is considered.
Parenthetically, it should be noted that a second GnRH (GnRHII) is also found in the primate hypothalamus (Chen et al., 1998;
Latimer et al., 2001), and the gene encoding the GnRH-II receptor
is expressed by the anterior pituitary (Millar et al., 2001; Neill
et al., 2001). Although intravenous administration of a bolus of
GnRH-II to the rhesus macaque is able to elicit an LH discharge
(Densmore and Urbanski, 2003), the role of this peptide in
regulating the pituitary±gonadal axis in general, and the initiation
of gonadarche in particular, is unknown.
68
Although direct measurement of temporal changes in GnRH
concentrations in portal blood during puberty have not been
reported for any species of primate, the concept that an increased
release in pulsatile GnRH is responsible for gonadarche is well
founded. A marked increase in hypothalamic release of this
decapeptide at the time of puberty in the female rhesus monkey has
been described using the technique of push±pull perfusion
(Watanabe and Terasawa, 1989), and the notion that neither
pituitary nor gonad are limiting to the onset of gonadarche was
graphically underlined by Knobil and his colleagues, who, in the
late 1970s, treated premenarcheal female rhesus monkeys with
uninterrupted intermittent intravenous infusions of GnRH and
elicited premature ovulatory menstrual cycles (Wildt et al., 1980).
Cognate observations of the male rhesus monkey have subsequently been made (Plant, 1988; Plant et al., 1989a), and the
condition of GnRH-dependent precocious gonadarche in man
(Witchel and Plant, 2003) further supports the pivotal role of
increased hypothalamic GnRH release in triggering the pubertal
activation of the pituitary±gonadal axis.
Interestingly, the pubertal increase in pulsatile GnRH release
does not re¯ect the acquisition by the primate hypothalamus of the
capacity to generate such an intermittent mode of secretion.
Rather, it represents the re-expression of this capacity, a potential
that has been held in a state of check since infancy (Plant, 2001).
The ability of the network of GnRH neurons to generate a pulsatile
discharge of the decapeptide originates during early fetal development (Jaffe, 1989) at the time when these neurons, which are
born in the olfactory bulb, are completing their migration to the
mediobasal hypothalamus, where they reside in the brain of the
adult primate (Ronnekleiv and Resko, 1990). The cell and
molecular biology underlying the guidance systems utilized by
migrating GnRH neurons is currently an area of intense investigation (Hardelin, 2001; Tobet et al., 2001; Allen et al., 2002;
Wray, 2002).
While some authors have argued that gonadarche may be
viewed as a continuum of a developmental process that starts
during early fetal development (Delemarre-van de Waal, 2002;
Grumbach, 2002), the value of this concept to higher primates is
not readily apparent. This is because the mechanism that times
gonadarche in these species is one that regulates the delay or
prevention of this developmental process (Pescovitz, 2000),
which, in the case of man, is capable of manifesting itself many
years before the normal age of puberty. Thus, while the
hypothalamus of the infant primate generates a robust mode of
pulsatile GnRH release, this `adult' mode of activity is soon
restrained, albeit not abolished, leading to a relatively hypogonadotrophic state, which guarantees the quiescence of the ovary and
testis during childhood. Therefore, the timing of gonadarche in
primates is governed by the duration of the interval during which
the activity of a `mature' hypothalamic GnRH neuronal network to
generate a robust pulsatile pattern of activity is restrained.
A non-gonadal control system delays gonadarche
The classic description by Conte et al. (1975) nearly 30 years ago
of the developmental pattern of circulating gonadotrophin concentrations from birth to 20 years of age in patients with gonadal
dysgenesis, demonstrated that the restraint imposed on pulsatile
GnRH release during childhood (2±10 years of age) was applied in
Mechanisms of primate puberty
the absence of the gonad. Studies of agonadal monkeys have
substantiated the early clinical data (Plant, 1985, 1994; Pohl et al.,
1995) and have also demonstrated that the restraint exerted on
hypothalamic GnRH release prior to puberty is noticeably more
intense in the male than in the female (Figure 1). This important
sex difference, which is presumably determined by exposure to
testicular testosterone secretion in utero, is probably the reason
why idiopathic precocious gonadarche is seen most frequently in
girls (Witchel and Plant, 2003), and why delayed gonadarche due
to constitutive delay in growth and maturation (CD) is observed
more frequently in boys (Sedlmeyer and Palmert, 2002). The
hypogonadotrophic state resulting from the non-gonadal prepubertal brake on pulsatile GnRH release is ampli®ed by ovarian and
testicular feedback, and particularly by ovarian estradiol inhibition
(Conte et al., 1975; Pohl et al., 1995), but this ®nding does not
distract from the conclusion that the pivotal component of
quiescence in the reproductive axis of the child is extragonadal.
The nature of the neurobiological brake delaying
gonadarche
Paradigms
Before discussing progress on this front, it is important to
comment brie¯y on the experimental paradigms that have been
employed to examine this issue. Generally, studies of mechanisms
of puberty have been conducted in the gonadally intact situation.
Intuitively, this approach would seem reasonable, but on closer
inspection it becomes clear that such a paradigm is not always the
most appropriate, particularly when the neurobiological basis of
the onset of puberty is being examined. This is because the
resurgence of GnRH pulsatility that activates gonadarche results
concomitantly in a dramatic and progressive increase in circulating
concentrations of sex steroids, which are capable of remodelling
both molecular and structural components of the hypothalamus
and other regions of the central nervous system (Witkin et al.,
1991; Witkin, 1996; Plant, 2001). Thus, in the intact situation,
secondary and tertiary actions of increasing steroid hormone levels
in the brain will, at best, confound the interpretation of any
primary developmental changes in microstructure and/or gene
expression observed in association with the resurgence of GnRH
pulsatility. Thus, studies conducted in this laboratory to probe the
molecular biology of the brake on pulsatile GnRH release have
employed an agonadal paradigm.
GnRH
The ®rst gene that we examined in the context of the brake was,
naturally, GnRH-I. Surprisingly, in the agonadal male, hypothalamic GnRH-I mRNA levels show only a modest increase in
association with the pubertal resurgence in GnRH pulsatility (El
Majdoubi et al., 2000a). Moreover, in intact animals the pubertal
increase in GnRH-I gene expression is greatly dampened (Vician
et al., 1991; Ma et al., 1994), presumably as a result of imposing
gonadal steroid feedback (El Majdoubi et al., 1998, 2000b;
Krajewski et al., 2003), and is not associated with a corresponding
change in the hypothalamic content of GnRH (Fraser et al., 1989;
El Majdoubi et al., 2000a). The modest increase in hypothalamic
GnRH-I expression together with the constitutively expressed
peptide content may be contrasted with the molecular state of the
gonadotroph at corresponding stages of development. This pituitary cell, as might be expected, is profoundly down-regulated
prior to the resurgence of pulsatile GnRH release (S. J. Winters and
T.M.Plant, unpublished observation), while the GnRH neuron at
this stage of development is essentially indistinguishable from that
in the adult hypothalamus.
Not only is the GnRH-I gene and its peptide expressed at high
levels prior to the pubertal resurgence of pulsatile release, but a
pattern of peptide secretion similar to that of the pubertal state may
be elicited with remarkable ease from the hypothalamus of the
juvenile by applying a repetitive intermittent chemical stimulation
with the glutamate receptor agonist, N-methyl-D-aspartate (Gay
and Plant, 1988). Taking these ®ndings together, it may be
concluded that transcriptional regulation of GnRH-I probably does
not play a critical role in the onset of gonadarche. On the contrary,
the molecular machinery for generating an adult pattern of GnRH
release is extant in the prepubertal situation, and thus it would
seem that genomics of the GnRH neuron do not play a pivotal role
in timing the pubertal resurgence of pulsatile GnRH release.
Rather, the signal controlling the timing of gonadarche originates
upstream from the GnRH neuronal network. Before discussing
upstream signals to the network of GnRH-I neurons, it is
interesting to note that the spontaneous resurgence in pulsatile
GnRH release in the agonadal situation occurs explosively, with
the pattern of release increasing from an essentially apulsatile
mode to a near-adult circhoral mode over an epoch of 40 days
(Suter et al., 1998).
Upstream signals
For the last few years we have been pursuing the hypothesis that a
major component of the upstream brake on pulsatile GnRH release
is provided by hypothalamic neurons expressing neuropeptide Y
(NPY) (Plant and Shahab, 2002). This hypothesis is based on the
following ®ndings. First, central administration of NPY arrests
GnRH pulsatility in agonadal postpubertal male and female
monkeys (Pau et al., 1995; Kaynard et al., 1990; Shahab et al.,
2003). Second, ontogenetic changes in NPY gene expression and
peptide content in the mediobasal hypothalamus of the agonadal
male monkey are inversely related to that of GnRH release at the
three major stages of postnatal development; namely, infancy±
juvenile development±puberty (El Majdoubi et al., 2000a).
Moreover, this pattern in the ontogeny in NPY gene expression
is correlated with a structural remodelling of the interactions
between NPY axonal varicosities and GnRH perikarya in the
hypothalamus of the male rhesus monkey (Plant, 2001; Plant and
Shahab, 2002). In the mediobasal hypothalamus of the juvenile,
the number of appositions between NPY varicosities and GnRH
perikarya are greater than that in the pubertal animal. Although the
issue of whether the NPY varicosities are truly synaptic has not
been de®nitively established, ultrastructural analysis has con®rmed that there is a decrease in overall synaptic input to the
GnRH perikarya at this critical developmental transition (Perera
and Plant, 1997).
To date, ®ve NPY receptors have been cloned (Michel et al.,
1998), and all are G-protein-coupled receptors that are linked to
inhibitory pathways, which, upon activation, lead to membrane
hyperpolarization (Sun et al., 1998). The hypothalamic NPY
receptors that underlie the ability of NPY to arrest pulsatile GnRH
release remain to be fully elucidated. In the postpubertal situation,
69
T.M.Plant and M.L.Barker-Gibb
the inhibition of hypothalamic GnRH release by centrally
administered NPY is mediated, at least in part, by the NPY Y1
receptor, but the pharmacological data supporting a role of this
NPY receptor in mediating the prepubertal brake on pulsatile
GnRH release are tenuous (Shahab et al., 2003). In another
approach to test the hypothesis that increased hypothalamic NPY
signalling is a major component of the brake that is exerted on
pulsatile GnRH release prior to puberty, we and others have looked
for loss of function mutations in the NPY Y1 receptor in children
with GnRH-dependent precocious gonadarche (Barker-Gibb et al.,
2002a; Freitas et al., 2003). In a total of 35 patients, a heterozygous
substitution of T for G at nucleotide 1330 of exon 3 of the gene
encoding the Y1 receptor was found in one girl with precocity.
Although this mutation results in the substitution of lysine by
threonine at position 374 in the carboxy terminus of the receptor,
the mother of this subject was found to have the same
heterozygous mutation, yet had apparently normal pubertal
development (Freitas et al., 2003).
Before leaving the role of NPY in the control of GnRH
secretion, it should be noted that NPY also exerts a stimulatory
action on GnRH release in the monkey (Woeller et al., 1991; Gore
et al., 1993; Pau et al., 1995). For a discussion of this conundrum,
the reader is referred to an earlier review by this laboratory (Plant,
2001).
A considerable body of evidence obtained primarily for studies
of the female is available to support the idea that g-aminobutyric
acid (GABA) is also a major component of the neurobiological
brake that is imposed on GnRH release prior to puberty (Terasawa
and Fernandez, 2001). Using hypothalamic perfusion, release of
GABA in the median eminence of the female rhesus monkey
declined as pulsatile GnRH release increased at the onset of
gonadarche (Mitsushima et al., 1994). Moreover, acute inactivation of the GABAA receptor or reduction in GABA tone in this
region of the hypothalamus in the prepubertal female monkey with
bicuculline or antisense oligodeoxynucleotide for the mRNA
encoding the GABA-synthesizing enzyme, glutamic acid decarboxylase 67 (GAD 67), respectively elicited an immediate
discharge of GnRH (Mitsushima et al., 1994, 1996). Furthermore,
chronic repetitive administration of bicuculline into the base of the
third cerebroventricle at ~15 months of age in pregonadarcheal
monkeys leads to premature menarche and precocious ®rst
ovulation (Keen et al., 1999). Although changes in hypothalamic
expression of GAD 65 and 67 have not been reported during
puberty in the female rhesus monkey, in the male of this species
they appear unremarkable at this stage of development (Urbanski
et al., 1998; El Majdoubi et al., 2000a).
Additional support for a role of GABA comes from clinical
observations. Bourguignon et al. (1997) reported that administration of loreclezole, a GABA agonist, to an 11 month old girl with
epilepsy and precocious gonadarche decreased LH secretion and
led to a regression in pubertal development. Pubertal delay has
also been observed in epileptic boys treated with valproic acid, a
drug with GABAergic activity (El-Khayat et al., 2003). It should
be noted, however, that the pharmacology of valproic acid is
complex, and others have reported that this drug and other
antiepileptic agents do not invariable delay sexual maturation
(RaÈttyaÈ et al., 1999).
GABA receptors, including the GABAA receptor, are coupled to
Cl± channels, and it is generally recognized that GABA activation
70
of these channels in neurons in the adult brain leads to an inward
Cl± current that hyperpolarizes the membrane (Kaila, 1994).
Indeed, it is generally recognized that GABA is the major
inhibitory neurotransmitter in the hypothalamus (Decavel and van
den Pol, 1990). Thus, until recently it had been assumed that the
site of action of GABA was directly on the GnRH neuron of the
prepubertal monkey. In this regard, in the rodent brain, GABA
synapses have been reported on GnRH neurons (Leranth et al.,
1985), which express GABAA receptor subunits (Jung et al., 1998;
Pape et al., 2001).
In contrast to the inhibitory action of GABA in the adult brain,
the action of GABA on many neuronal systems in the developing
embryonic brain is excitatory (Cherubini et al., 1991). The switch
in the electrophysiological action of GABA on developing
neurons, which, in the rat, generally occurs around postnatal
days 7±14 (Cherubini et al., 1991), appears to be related to an
increased expression of a potassium chloride co-transporter, KCC2
(Rivera et al., 1999; Ganguly et al., 2001). Under physiological
conditions, KCC2 extrudes K+ and Cl± and thereby maintains
intracellular Cl± at concentrations lower than those predicted by
the Nernst equilibrium. Thus, GABAA receptor activation in
neurons expressing KCC2 will lead to an inward Cl± current and
hyperpolarization. Whether GnRH neurons exhibit this developmental switch in expression of KCC2, and therefore respond to
GABA with inhibition in early development and with excitation
later, is currently a controversial issue. Two studies, both utilizing
transgenic mice expressing either b-galactosidase or green ¯uorescent protein under the control of the GnRH-I promoter, in order
to visualize GnRH neurons in acute hypothalamic slices, have
addressed this issue (DeFazio et al., 2002; Han et al., 2002). Both
studies monitored electrophysiological activity using the gramicidin perforated patch technique, which enables membrane
responses of a neuron to be recorded without changing the
intracellular Cl± concentration and, therefore, the resting potential
of the cell. In one study, mmol/l concentrations of GABA were
added to the medium bathing the slice, and in the other, GABA at
mmol/l concentrations was applied as a very brief `puff' to the
immediate vicinity of a GnRH-I neuron within the slice being
studied. In the former study, the classic switch in GABA action
was noted, while in the latter, GABA was shown to be excitatory at
all stages of development. This issue is further confounded by the
®nding that expression of KCC2 in GnRH-I neurons in the
hypothalamus of the adult mouse is heterogeneous (Leupen et al.,
2003), indicating that subpopulations of GnRH neurons may
respond differently to direct GABA inputs.
While the switch in GABA action in the central nervous system
(CNS) of rodents occurs postnatally (Cherubini et al., 1991), in
primates it is to be expected that this developmental event would
occur prenatally. Thus, if GnRH-I neurons in higher primates
manifest this developmental switch, the action of increased GABA
release during prepubertal development to restrain pulsatile GnRH
release could be accounted for by a direct action of the
neurotransmitter on GnRH-I neurons. Conversely, if GnRH-I
neurons in the postnatal primate brain remain `embryonic' and are
excited by GABA, then an indirect action of GABA on inhibitory
afferents of the GnRH-I network, such as NPY, would have to be
invoked.
In addition to NPY and GABA, glial-derived growth factors,
such as transforming growth factor a (TGF-a) have been
Mechanisms of primate puberty
implicated in the control of the pubertal resurgence of pulsatile
GnRH-I secretion. The action of TGF-a on GnRH neurons is
stimulatory and indirect, involving autocrine/paracrine release of
prostaglandin E2 from neighbouring astrocytes (Ojeda et al.,
2003). Release of GnRH is then effected by the prostaglandin.
Although most studies of the glial regulation of GnRH-I release at
the time of puberty have been conducted in the rat (Ojeda et al.,
2003), TGF-a gene expression in the hypothalamus has been
shown to be dramatically increased in the pubertal female rhesus
monkey (Ma et al., 1994) and marginally elevated in the agonadal
male monkey at a comparable stage of development (El Majdoubi
et al., 2000a). Additional circumstantial evidence for this view is
provided by the ®nding that harmatomas removed from patients
with GnRH-dependent precocious gonadarche have, on occasion,
been found to be rich in astrocytes containing TGF-a, but devoid
of neurons expressing GnRH-I (Jung and Ojeda, 2002).
Although attempts have been made to develop integrated
models of the neurobiological brake that is exerted upon pulsatile
GnRH-I release from the primate hypothalamus prior to puberty,
they have not been entirely satisfactory (Plant, 2001; Terasawa and
Fernandez, 2001) for the following reason. The idea that
hypothalamic NPY neurons comprise an essential component of
the brake has been developed from studies of the male rhesus
monkey, while the GABA hypothesis has been evolved from
studies of the female rhesus monkey. Caution is needed in
extrapolating ®ndings in the male to the female and vice versa,
since the duration and intensity of the neurobiological brake
exerted on GnRH release during prepubertal development is
greater in the male than in the female (Figure 1; Pohl et al., 1995).
Whether this sex difference is purely quantitative or represents
fundamental differences in the neurobiology is unknown. Another
issue that remains to be clari®ed is whether the neurobiological
mechanism underlying the resurgence of pulsatile GnRH release at
the time of gonadarche is a simple reversal of that responsible in
late infancy for inhibition of the same network (El Majdoubi et al.,
2000c).
Genomics and the neurobiological brake
The proximal step in mediating the function of a genome is the
appropriate temporal and cell speci®c expression of a gene or
subset of genes. Thus, if physiological genomics underlie
gonadarche, the ®rst condition that must be met is that a
programmed change or changes in gene expression in speci®c
neuronal (and/or glial cells) phenotypes precede the resurgence in
pulsatile GnRH release. The fact that a gene, for example NPY, is
expressed or repressed in a particular set of neurons upstream to
the GnRH neurons, however, is in itself insuf®cient to argue that
neurogenomics are playing a fundamental role in regulating the
timing of gonadarche. Information in the genome may be
expressed as a result of an intrinsic event originating within a
subset of neurons or the niche, in which the group of neurons
reside. Such control by the genome is most clearly operational
during early embryogenesis where gene expression unfolds in a
programmed manner leading to particular cell lineages, to
organogenesis and to differentiated function, as, for example,
has been demonstrated for the anterior pituitary (Dasen and
Rosenfeld, 2001). In the context of the timing of gonadarche, the
extent to which intrinsic developmental control of gene expression
occurs upstream from the GnRH neuron is unknown.
Alternatively, a non-hypothalamic (perhaps even a non-CNS)
cue might activate signalling pathways leading to expression of a
gene, protein synthesis and changes in the activity of upstream
neurons and/or glial components that would result in the downstream resurgence of pulsatile GnRH release. While such a control
system is conceptually appealing (see below), since it would
provide a mechanism for coordinating attainment of adult stature
with the onset of sexual maturation, it should not be viewed as a
truly genomic mechanism.
Mechanisms timing the delay to gonadarche
The timing of the duration of the neurobiological brake on
pulsatile GnRH release during juvenile development is presumably governed by a CNS `clock' or by a control system, termed a
`somatometer', that tracks some aspect of somatic growth and
development (Plant et al., 1989b). In the case of the somatometer
hypothesis, the attainment of a particular proportion of fat has long
be suggested to be a cue for the onset of gonadarche in girls (Frisch
and Revelle, 1970; Frisch et al., 1973), and interest in this notion
was resurrected with the discovery of leptin, a circulating protein
hormone, that is produced by expression of the ob gene in
adipocytes, and that plays a major role in the regulation of appetite
and food intake (Friedman and Halaas, 1998; Schwartz et al.,
2000).
The idea that leptin is necessary, in a permissive sense, for
gonadarche to unfold at an appropriate time is supported by several
clinical observations. First, primary hypogonadotrophic hypogonadism at 23±34 years of age has been reported in association with
morbid obesity in one male and two female subjects with a
missense mutation in the leptin gene (Strobel et al., 1998; Ozata
et al., 1999). Second, hypogonadotrophism and failure to undergo
pubertal development at age 13.5, 19 and 19 years of age
respectively has been described in three sisters homozygous for
mutations in the leptin receptor gene (Clement et al., 1998). Third,
primary amenorrhoea at age 15, 17 and 17 years respectively was
reported in three patients with congenital generalized lipodystrophy associated with Seip±Berardinelli syndrome (Oral et al.,
2002). Although one of the female subjects with leptin de®ciency
subsequently entered puberty spontaneously (Ozata et al., 1999),
the foregoing clinical observations suggest that leptin signalling is
required for normal gonadarche. On the other hand, normal
pubertal development was reported in two female subjects with
severe lipodystrophy (Andreelli et al., 2000), and in one girl with
congenital contractual arachnodactyly (Galler et al., 2001), all of
whom were markedly hypoleptinaemic. Interestingly, although the
lipodystrophic subjects with normal pubertal development had
measurable concentrations of circulating immunoactive leptin
(0.7±1.6 ng/ml), they were no greater than those in the subjects
with Seip±Berardinelli syndrome (0.8±1.0 ng/ml), who failed to
enter puberty. The most parsimonious explanation for these
inconsistent ®ndings is that immunoactive leptin concentrations in
the blood may not accurately re¯ect the bioavailability of the
circulating hormone.
Additional support for the notion that leptin is necessary for
gonadarche to occur is provided by studies of the effects of
hormone replacement in a small number of leptin-de®cient
subjects of pubertal or postpubertal age but exhibiting absent or
71
T.M.Plant and M.L.Barker-Gibb
Figure 2. Temporal relationship between circulating concentrations of leptin
(mean 6 SEM) and the nocturnal elevation in plasma testosterone (T) at the
time of initiation of gonadarche in the male rhesus monkey. Hormone levels
were aligned to the week during which a sustained nocturnal elevation in
plasma T was observed (week 0) when the monkeys (n = 6) were 27±31
months of age. Reproduced with permission from Plant and Durrant (1997),
ã The Endocrine Society 1997.
incomplete gonadarche. In a 9 year old girl with a frameshift
mutation in the leptin gene and a pubertal bone age (12.5 years), a
pulsatile pattern of LH secretion, indicative of a pubertal mode of
GnRH pulse generator activity, was observed after 12 months of
replacement with recombinant leptin to correct her morbid obesity
(Farooqi et al., 1999). The bone age of this subject may be
signi®cant because it is generally recognized that, in normal
children, bone age is a better predictor of gonadarche than
chronological age (Plant et al., 1989b). Continued leptin replacement to this girl was associated with menarche at 12.1 years of age
(bone age of 13.4 years), and 1.5 years later Tanner stage 3 was
noted (Farooqi et al., 2002). Leptin replacement was also reported
to facilitate menstruation in the women with primary or secondary
amenorrhoea in association with congenital general lipodystrophy
(Oral et al., 2002).
It seems reasonable to conclude from the foregoing discussion
that a permissive action of leptin is required for gonadarche. The
question now becomes whether leptin also serves as the signal that
times the pubertal resurgence of pulsatile GnRH release and is
therefore responsible for timing the termination of the delay of
gonadarche. For this notion to be valid, two conditions must be
satis®ed: (i) an elevation in plasma leptin must precede the
pubertal resurgence of pulsatile GnRH, and (ii) a premature
elevation of circulating leptin would elicit precocious GnRH
release. With regard to the ®rst condition, cross-sectional and
longitudinal studies of both boys and girls indicate in general that
circulating leptin concentrations increase during pubertal development (Blum et al., 1997; Carlsson et al., 1997; Clayton et al.,
1997; Garcia-Mayor et al., 1997; Ahmed et al., 1999; AnkarbergLindgren et al., 2001). Parenthetically, the pubertal increases in
circulating leptin concentrations in man are inversely related to
72
changes in levels of the soluble leptin receptor (Kratzsch et al.,
2002; Mann et al., 2003), indicating that the rise in bioavailable
leptin at this stage of development may be more robust than that of
total immunoactive leptin. The binning of data with respect to
Tanner stage, and the paucity of detailed sequential information on
both leptin levels and LH pulsatility during transition from Tanner
stage 1 to stage 2, however, does not allow the question of whether
circulating leptin increases immediately prior to the initiation of
gonadarche to be addressed. One exception to this situation is
provided by the longitudinal study of eight boys by Mantzoros
et al. (1997), who reported that elevations in circulating leptin
concentrations occurred prior to nocturnal elevation in testosterone secretion, a robust marker of the onset of gonadarche in boys
(Boyar et al., 1974; Judd et al., 1974). As previously noted (Mann
and Plant, 2002), however, the leptin levels reported in the latter
study were extremely variable, and the seemingly tantalizing
increments of circulating levels of leptin were actually rather
trivial in many of the subjects.
Studies of the relationship between circulating leptin levels and
the onset of gonadarche in other species of higher primate have
been restricted to the rhesus monkey, and only the male has been
examined. In the male monkey, as in boys, an early marker of the
initiation of gonadarche is a nocturnal elevation of testosterone
secretion (Plant, 1985). In contrast to boys, however, the
longitudinal relationship between the onset of nocturnal testosterone secretion and plasma leptin levels is unremarkable (Figure 2;
Plant and Durrant, 1997). Results from cross-sectional studies of
the monkey (Urbanski and Pau, 1998; Mann et al., 2000) are also
consistent with the view that gonadarche in the male is triggered in
the absence of a rise in circulating leptin.
With regard to the second condition that needs to be met if leptin
is to be considered as a candidate for the trigger of gonadarche,
data are available for the monkey. In this species, a continuous
intravenous infusion of recombinant human leptin to agonadal
juvenile males, equivalent in age to 4±5 year old boys, did not
trigger precocious activation of GnRH pulse generator activity
(Figure 3; Barker-Gibb et al., 2002b). Also of interest in this
regard is the report that moment-to-moment patterns of LH
secretion, and FSH and testosterone concentrations, remained
prepubertal in a 5 year old leptin-de®cient boy during 12 months of
replacement treatment with recombinant human leptin.
Replacement was calculated to achieve circulating leptin levels
that were not >150% of those in normal children of comparable
age and gender (Farooqi et al., 2002).
Two other disorders of pubertal development in man that
provide potential paradigms relevant to the role of leptin in timing
gonadarche merit comment. First, circulating leptin concentrations
were unchanged at the time delayed gonadarche was initiated in
boys with CD (Gill et al., 1999). Second, while circulating leptin
levels have been reported in both boys and girls with GnRHdependent precocious gonadarche (Palmert et al., 1998; Heger
et al., 1999), understandably measurements of the hormone prior
to manifestations of the pathophysiological condition were not
reported.
Taking the foregoing considerations together, the role of leptin
in primate gonadarche may be summarized as follows. A threshold
concentration of plasma leptin, which appears to be quite low, is
needed for this developmental process to unfold (Mann and Plant,
2002): a notion that is consistent with the ®nding that, in
Mechanisms of primate puberty
Figure 3. Continuous intravenous infusion of recombinant (r) human (h)
leptin (5 mg/kg body weight for 16 days), which resulted in a 5-fold
elevation in circulating leptin concentrations (top panel, closed data points),
failed to produce a premature resurgence in pulsatile GnRH release (as
re¯ected by circulating LH concentrations; lower panel, closed data points)
in an agonadal juvenile male rhesus monkey ~18 months of age. The
spontaneous pubertal resurgence in pulsatile GnRH release is observed at
~26 months of age (Suter et al., 1998). Open data points show leptin and LH
concentrations during vehicle infusion to the same animal. The absence of
LH pulsatility cannot be accounted for by pituitary insensitivity since the
administration of a `physiological' bolus of GnRH (300 ng, intravenous) on
day 8 and 16 (arrow) produced a robust discharge of LH. It should also be
noted that growth hormone secretion was ampli®ed during the leptin infusion
(not shown) indicating that the heterologous leptin was bioactive in the
monkey. Broken vertical line indicates start of leptin and vehicle infusions
on day 0. Reprinted with permission from Barker-Gibb et al. 2002b, ã The
Endocrine Society 2002.
underweight postpubertal women, regular menstruation was
associated with leptin levels in the range of 1.9 ng/ml (KoÈpp
et al., 1997). Although this action of the hormone may be viewed
as obligatory for gonadarche to unfold, leptin does not provide the
signal that times the resurgence of pulsatile GnRH release and,
therefore, the cue that terminates the prepubertal state. Rather,
leptin allows for the expression of this critical hypothalamic event,
which is triggered by a mechanism that remains elusive. This
permissive action of leptin on hypothalamic GnRH release at
puberty is also necessary for maintenance of gonadal function
throughout postpubertal development. Leptin also appears to
subserve a similar role in rodents (Cheung et al., 1997, 2001).
Genetics and gonadarche
The recognition that genetics play a role in the timing of
gonadarche has been recently highlighted (Palmert and Boepple,
2001). In general, studies that have described age of menarche in
monozygotic and dizygotic twins report a greater concordancy in
the timing of this developmental index in monozygotic twins than
in dizygotic twins (Gedda and Brenci, 1975; Fischbein 1977a,b;
Sharma, 1983; Treloar and Martin, 1990; Kaprio et al., 1995).
Other indicators of the progression of gonadarche, such as peak
height velocity and plasma LH concentrations have also been
reported to exhibit greater concordancy in monozygotic twins
(Wasada et al., 1978; Sharma, 1983). The impact of genetics in the
timing of gonadarche is further indicated by the ®ndings of a high
degree of heritability in the age of menarche (Gedda and Brenci,
1975; Fischbein 1977a). Age of menarche also varies with racial/
ethnic group, although when evaluating epidemiological data it
should be noted that study designs have not been uniform, most
have been retrospective and few have evaluated different ethnic
populations within the same geographical location and socioeconomic niche. Notwithstanding, studies of different ethnic
groups living in the USA have revealed that black girls have a
signi®cantly earlier age of menarche than their white counterparts
(Herman-Giddens et al., 1997; Biro et al., 2001; Sun et al., 2002;
Chumlea et al., 2003). Similarly, girls in London of AfroCaribbean and Indo-Pakistani parentage have earlier age of
menarche than white girls of British parentage (Ulijaszek et al.,
1991). It should be noted that when a genetic in¯uence is
established it remains unclear whether the genotype is directly
determining the timing of the application and duration of the
neurobiological brake on prepubertal GnRH release (and therefore
dictating the delay of gonadarche), or simply governing permissive
factors that are required for the resurgence of pulsatile GnRH
release to be manifest once the brake has been withdrawn.
Mutations of several genes involved in differentiation and
development of the hypothalamic±pituitary±gonadal axis have
been reported (Kalantaridou and Chrousos, 2002), and, not
surprisingly, such mutations have profound effects on pubertal
development. For example, mutations of the KAL-1 gene, which
encodes anosmin, an extracellular glycoprotein that appears to be
involved in the migration of GnRH neurons from the olfactory
bulb to the hypothalamus during fetal development (Hardelin,
2001), may lead to hypogonadotrophic hypogonadism and,
therefore, to the absence of gonadarche. Such mutations, however,
although providing great insight into the early development of the
reproductive axis, contribute little to our understanding of the
control system that times the pubertal resurgence of pulsatile
GnRH release. Quantal progress on this front is to be anticipated
with the identi®cation, should they exist, of speci®c `puberty
genes', i.e. those that directly dictate, during prepubertal development, the timing of the application and removal of the
neurobiological brake restraining the activity of the hypothalamic
GnRH pulse generator. To date, however, attempts to identify
`puberty genes' by screening for mutations in patients with GnRHdependent precocious gonadarche have not been realized (BarkerGibb et al., 2002a; Freitas et al., 2003).
Although similar analyses of patients with delayed gonadarche
due to CD have not been reported to date, genotyping of
individuals with this `normal' variant of pubertal development
may be rewarding. Indeed, a recent study of a relatively large
number of subjects with CD indicated a genetic predisposition, as
re¯ected by the ®nding that parents of children with CD recall a
later age of menarche and the adolescent growth spurt than those
of a control group of children (Sedlmeyer and Palmert, 2002;
Sedlmeyer et al., 2002).
73
T.M.Plant and M.L.Barker-Gibb
Conclusions
The fundamental process underlying puberty in higher primates
including man is gonadarche, which is triggered by a resurgence in
pulsatile GnRH release from the hypothalamus. The potential for
robust pulsatile GnRH secretion is extant well before the initiation
of puberty, and the mechanism that times gonadarche should
therefore be viewed as one that governs the delay, rather than the
onset, of this critical stage in the ontogeny of reproductive
function. The developmental delay is initiated by a neurobiological brake imposed during infancy on the neuronal network
responsible for generating a pulsatile mode of GnRH release.
Withdrawal of the brake several years later allows gonadarche to
unfold. Interestingly, the pubertal resurgence of pulsatile GnRH
release appears to be largely emancipated from transcriptional
control of GnRH-I, the gene encoding GnRH. Instead, the brake is
comprised of upstream neuronal and perhaps glial signals that
regulate release of GnRH synthesized by molecular machinery
that, postnatally, does not appear to be subjected to marked
developmental regulation. The nature of the upstream cues
remains incompletely understood, but NPY and GABA have
emerged as prime candidates from studies of the male and female
rhesus monkey respectively. Complete resolution of this issue is
likely to be confounded by the sex differences in the duration and
intensity of the neurobiological brake, particularly if they are
re¯ected in the underlying neurobiology. The pubertal resurgence
of GnRH release is also associated with structural remodelling in
the hypothalamus. As of yet, speci®c `puberty genes', i.e. those
that time the application of the neurobiological brake, have not
been identi®ed, but their existence is suggested because of the high
degree of heritability in the age of gonadarche. Whether these
genes are expressed in the central nervous system or in somatic
systems is also an open question. Lastly, for a resurgence in
pulsatile GnRH release to be manifest following removal of the
neurobiological brake at the end of the prepubertal phase of
development, several permissive factors must be in place. Leptin is
one of these.
Acknowledgements
Work conducted in the author's laboratory was supported by NIH
grants HD 13254 and HD 08610.
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