Download Identification of immunoreactive FSH and LH cells in the cichlid fish

Anat Embryol (2006)
DOI 10.1007/s00429-006-0086-0
O R I GI N A L A R T IC L E
Matı́as Pandolfi Æ Fabiana L. Lo Nostro
Akio Shimizu Æ Andrea G. Pozzi Æ Fernando J. Meijide
Graciela Rey Vazquez Æ M. Cristina Maggese
Identification of immunoreactive FSH and LH cells in the cichlid fish
Cichlasoma dimerus during the ontogeny and sexual differentiation
Accepted: 24 February 2006
Springer-Verlag 2006
Abstract Follicle-stimulating hormone (FSH) and
luteinizing hormone (LH) expressing cells were detected
in pituitary, brain and ovary of the Perciform cichlid fish
Cichlasoma dimerus. This detection was carried out by
immunohistochemistry (IHC) and Western blot techniques using antisera of the Cyprinodontiform Fundulus
heteroclitus raised against the conservative region of the
teleost bFSH and the bLH subunits. The estimated
molecular weights were 24 kDa for LH and 19 and
15 kDa for FSH. In the adult pituitary, both cell types
were distributed along mid and ventral zones of the
proximal pars distalis (PPD, mid-immunoreactive cells),
and along the ventral and dorsal external border of the
pars intermedia (PI, high-immunoreactive cells). Double
IHC showed that FSH and LH are mainly expressed in
different pituitary cells. FSH cells were detected in the
pituitary around day 21 after hatching (ah) (prior to sex
differentiation), while LH cells were detected by day
60 ah (during the sexual differentiation period). A correlation between gonadal sex differentiation and FSH
was demonstrated in a 15 days organ culture system.
FSH and LH neurons were localized in the nucleus
lateralis tuberis and their fibers project through the
ventral hypothalamus, preoptic area and neurohypophysis. FSH neurons differentiated on day 21 ah, while LH
neurons appeared on day 15 ah. In the ovary, the
immunoreactivity for both FSH and LH was restricted
to the cytoplasm of previtellogenic and early vitellogenic
oocytes.
Keywords Teleostei Æ Cichlidae Æ Gonadotropins Æ
Sexual differentiation Æ Ontogeny
Introduction
M. Pandolfi (&) Æ F. L. Lo Nostro Æ F. J. Meijide
G. R. Vazquez Æ M. C. Maggese
Laboratorio de Embriologı́a Animal, Departamento de Biodiversidad y Biologı́a Experimental, Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires, Ciudad Universitaria,
Pabellón 2, Buenos Aires C1428EHA, Argentina
E-mail: pandolfi@bg.fcen.uba.ar
Tel.: +54-11-45763348
Fax: +54-11-45763384
M. Pandolfi Æ F. L. Lo Nostro Æ A. G. Pozzi Æ M. C. Maggese
Consejo Nacional de Investigaciones y Tecnicas (CONICET),
Departamento de Biodiversidad y Biologı́a Experimental, Facultad
de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Ciudad Universitaria, Pabellón 2, Buenos Aires C1428EHA,
Argentina
A. Shimizu
National Research Institute of Fisheries Science,
Fisheries Research Agency, Kanazawa,
Yokohama 236-8648, Japan
A. G. Pozzi
Laboratorio de Biologı́a del Desarrollo,
Departamento de Biodiversidad y Biologı́a Experimental,
Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Universitaria,
Pabellón 2, Buenos Aires C1428EHA, Argentina
Follicle-stimulating hormone (FSH) and luteinizing
hormone (LH) are glycoproteins that are synthesized in
the vertebrate pituitary, and their principal role is the
control of gametogenesis and gonadal steroidogenesis in
adults. In several tetrapod and teleost species, two different types of gonadotropins (GtHs) were isolated and
it was demonstrated that each GtH contains two different subunits named a and b (Yoshiura et al. 1999;
Yaron et al. 2001). In a given vertebrate species, the
aFSH and aLH subunits are identical while b subunits
are different, which gives the specific biological function
of each GtH. In salmonid species, characterized for
spawning only once a year, the different roles of FSH
and LH were studied in detail (Suzuki et al. 1988; Tyler
et al. 1991). The functions of FSH as well as its variation
along the reproductive cycle are not very clear in salmonid species examined so far. In general, FSH is considered as a vitellogenic hormone, while LH is related
with gonadal maturation and steroidogenesis. Several
studies conducted during the development of teleost
fishes, demonstrated different plasma levels and different
expression patterns of FSH and LH in the pituitary
(Dufour et al. 2000). Taken together, these results suggest specific functions of FSH and LH in the onset of
puberty and control of gametogenesis.
Traditionally it was thought that LH and FSH were
exclusively synthesized in vertebrate pituitary cells.
However, immunoreactivity to these hormones was detected in rodent and human brains (Croxatto et al. 1964;
Emanuelle et al. 1981; Hostetter et al. 1981). Likewise, in
the salmon Oncorhynchus nerka, it was reported that
some neurons from preoptic nuclei expressed FSH and
LH-like glycoproteins (Parhar et al. 1995). In the tilapia
Oreochromis niloticus, FSH and LH were cloned and
sequenced, and the presence of their respective mRNAs
in the brain was confirmed by RT-PCR (Parhar et al.
2003). Although the precise functions of the brain-derived FSH and LH are still unknown, the distribution of
their ir-axons along the brain and neurohypophysis
suggest a probable neuromodulator function in brain
nuclei related with the olfactory system and the control
of pituitary endocrine cells. On the other hand, cell
bodies that express FSH and LH belong to preoptic
areas strongly associated with reproductive behavior
(Demski and Sloan 1985, Foran and Bass 1999).
A few studies were focused on the ontogeny and
functions of GtHs during early development: salmon
Salmo gairdneri (Van der Hurk 1982), carp Cyprinus
carpio (Van Winkoop et al. 1987), rainbow trout O.
mykiss (Saga et al. 1993; Feist and Schreck 1996),
platyfish Xiphophorus maculates (Magliulo Cepriano
et al. 1994), pejerrey Odontesthes bonariensis (Miranda
et al. 2001) and gilthead seabream Sparus aurata (Garcia
Ayala et al. 2003). These studies revealed a marked
species-specific variation in the chronological appearance of LH and FSH cells during the ontogeny.
Particularly, the identification of FSH cells using
heterologous antisera was very difficult to perform due
to cross-reactions or low specificity between FSH molecules from different species (Vissio et al. 1996; Pandolfi
et al. 2001a). Antisera raised against the mummichog
Fundulus heteroclitus FSH and LH were successfully
used to recognize GtH cells in several species of teleost
fishes, because they recognized conserved regions of
FSH and LH from teleosts (Shimizu et al. 2003a, b).
One of the aims of this study was to identify GTH
cells in adult pituitary and brain, and during the early
development of the South American cichlid fish Cichlasoma dimerus. This freshwater species adapts easily to
captivity and spawns with high frequency during
8 months of the year, providing an appropriate model
for developmental studies.
In terms of gonadal development, this species was
classified as a differentiated gonochorist, in which
ovarian differentiation (day 42) preceded testicular differentiation (day 72), revealed by the onset of meiosis
(Meijide et al. 2005). A correlation between the innervation of the pituitary gland by GnRHI fibers and the
onset of the sexual differentiation process was already
reported for this species suggesting that GTHs may be
involved in this process (Pandolfi et al. 2002). The sec-
ond aim of this study was to evaluate the effect of FSH
on the gonadal differentiation in vitro.
Materials and methods
Animals and tissue processing
Thirty C. dimerus adult fish (12 males and 18 females,
11 ± 2 cm and 45 ± 8 g) were collected from Esteros
del Riachuelo, Corrientes, Argentina (2725¢S 5815¢W)
and kept in large aquaria at 27 ± 1C and 12:12 h
photoperiod. Aquaria were well aerated and provided
with external filtration. Larvae were obtained from
natural spawnings of adult specimens. Adult animals
were sacrificed during the reproductive season (Spring–
Summer). Pituitaries, brains and gonads were fixed in
Bouin’s solution for 24 h. Samples were then dehydrated
and embedded in paraplast (Fisherbrand, Fisher, WA,
USA). For immunohistochemistry (IHC) techniques,
pituitaries were sagittally sectioned at 7 lm while brains
and gonads were transversally sectioned at 10 lm. Some
other sections, randomly selected, were stained with
hematoxilin–eosin, Masson tricromic and periodic-acid
schiff (PAS) techniques to further characterize the
morphology of gonadotrops.
For developmental IHC analysis, daily samples of
larvae were obtained from hatching to 15 days after
hatching (ah), and every 3 days from day 15 to 90 ah. At
each stage, ten animals were used.
Antisera
Antisera used for this study were raised in rabbit against
bFSH and bLH of the cyprinodontiform F. heteroclitus
(mummichog) (Fh). The generation and characterization
of these antisera were previously described (Shimizu and
Yamashita 2002). These antisera were specifically raised
against conservative sites of teleost GtHs. For this study,
anti-Fh (50-60) bFSH and anti-Fh (91-106) bLH were
used.
Epitope unmasking
In order to enhance antigen immunoreactivity, sections
were treated for epitope unmasking after deparaffination
and rehydratation. Sections were dipped in an epitope
unmasking solution (Target Unmasking Fluid, TUF,
Sanbio BV) for 10 min at 90C, cooled at room temperature (RT) and finally washed in distilled water.
Single immunohistochemistry
After the epitope unmasking treatment, sections were
rewashed in phosphate buffered saline (PBS, pH 7.4) and
incubated for 15 min in a 0.75% gelatine solution and
30 min in PBS containing 5% non-fat dry milk at RT
(blocking solution). Next, they were incubated with the
primary antisera anti-bFSH (1:1,000) or anti-bLH
(1:2,000) for 18 h at RT. The sections were then washed
in PBS and incubated for 45 min at RT with a biotynilated anti-rabbit IgG (1:600) (Vector LAB, Burlingame,
CA). Amplification of the signal was achieved by incubating the sections with a peroxidase-conjugated streptavidin (Dako, Carpinteria, CA) diluted 1:300 for 1 h at
RT. After three washes in PBS, peroxidase activity was
visualized with 0.1% 3,3¢-diaminobenzidine (DAB) in
TRIS buffer (pH 7.6) and 0.03% H2O2. Sections were
lightly counterstained with hematoxylin, mounted,
examined with a NIKON microphot FX microscope
and digitally photographed (Nikon, Coolpix 4500).
To confirm the specificity of the immunostaining,
control sections were incubated with the primary antisera (in their working dilution) preabsorbed with an
excess of its respective antigen (100 ng/ll). To avoid
false positives caused by the IHC itself, replacement of
primary antisera with PBS and omission of secondary
antisera were also performed.
bFSH and bLH double immunohistochemistry
After the epitope unmasking treatment, some sections
were washed in distilled water and then in PBS for
20 min. After that, they were preincubated in blocking
solution (5% non-fat dry milk) for 1 h and finally with
the anti-Fh (91-106) bLH diluted 1:2,000 for 18 h at RT.
Sections were then washed in PBS and incubated for 1 h
at RT with an anti-rabbit IgG coupled to peroxidase
(1:200) (Dako, Carpinteria, CA). Sections were washed
10 min in PBS and developed with DAB. As a result,
bLH expressing cells showed a dark brown color.
As both primary antisera were raised in rabbit, it was
necessary to perform a blocking protocol against free
epitopes in order to avoid interactions between the first
and second antigen detection systems. After developing
bLH cells with DAB, sections were washed again in
distilled water and then deep in a 10 mM glicine-HCl
(pH 2) solution for 1 h at 40C and after that, in a 8 M
urea solution for 1 h at 40C. Sections were then washed
in distilled water and PBS and preincubated again in
blocking solution for 1 h at RT, and finally with the
anti-Fh (50-60) bFSH for 18 h at RT. After a 10 min
wash in PBS, sections were incubated with a 1:200
dilution of anti-rabbit IgG coupled to alkaline phosphatase (Dako, Carpinteria, CA) for 1 h at RT. Sections
were washed for 10 min and then developed using an
alkaline phosphatase kit (BCIP/NBT, Vector Blue,
Dako). As a result of this technique, bFSH expressing
cells displayed a dark blue color.
Western blot analysis
To test the specificity of F. heteroclitus bFSH and bLH
antisera in C. dimerus, an analysis on 15% sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE) followed by Western blot was performed.
Five adult males and five adult females were anesthetized with 0.1% benzocaine and killed by decapitation. Ten pituitaries and two ovaries were homogenized
in 1 ml of Tris–HCl buffer 50 mM pH 7.4 with 10 ll of
protease inhibitor cocktail (Sigma, Saint Louis, MO).
Samples were processed for SDS-PAGE followed by
Western blot as was previously described for this species
(Pandolfi et al. 2005). The primary antisera dilutions
were 1:2,000 for anti-Fh (50-60) bFSH and 1:4,000 for
anti-Fh (91-106) bLH. Control lanes of pituitary homogenates were performed by replacing the primary
antisera with Tris buffer and by incubating pituitary
homogenates with the preadsorbed primary antibodies
in their working dilution with and excess of LH or FSH
(Fig. 2e, f).
Treatment of undifferentiated gonads using recombinant
human FSH (rhFSH) in vitro
Eighty 30-day-old larvae were anesthetized in a 0.1%
benzocaine solution, quickly washed in 70% ethanol and
transferred to sterilized capsules containing physiological solution. The undifferentiated gonads, together with
the kidney and some portions of the dorsal body wall,
were dissected under stereoscopic microscope and
transferred to multi-well culture plates containing 300 ll
of culture media (L15 in a 60% V/V dilution, supplemented with 10% fetal bovine serum, 10 mM Hepes,
100 IU/ml of penicillin and 100 lg/ml of streptomycin).
Gonads were maintained in culture for 15 days at 25C
and the media was completely changed every 2 days.
Two independent experiments with N = 40 each were
performed. The viability of the gonads (measured by the
cytoarchitecture of fixed gonads after 15 days in culture)
was always above 80% in every treatment. The following protocol was used:
Days 1–3 (30–33 days ah): The 40 gonads only with
culture media.
Days 4–11 (34–41 ah): 10 gonads only with culture
media (control), and 30 gonads with the three different
concentrations of rhFSH (10 gonads/concentration: 0.5,
1 and 2 lg/ml).
Days 12–15 (42–45 ah): Again, the 40 gonads only
with culture media.
The rhFSH concentrations were chosen based on
some published works related to the effect of this hormone on the onset of meiosis (Maekawa et al. 1995; Ito
and Ave 1999; Yazawa et al. 2002).
After 15 days in culture media, gonads were fixed in
Bouin’s solution and embedded in paraffin, then sectioned and stained with hematoxilin–eosin. The onset of
meiosis in control and treated group was observed. Data
analysis was performed with non-parametric statistics
using a frequency analysis with a v2 test and William’s
correction for adjusting the analysis to our sample size
(N).
Results
Localization of bFSH and bLH-ir cells in the adult
pituitary
The pituitary of C. dimerus has two clearly different
zones: the adenohypohysis (ADH) and the neurohypohysis (NH), and, as in all teleosts, the NH sends deep
branches to the ADH, especially into the pars intermedia (PI). Gonadotrops are PAS + basophilic cells
localized mainly in the proximal pars distalis (PPD) and
the external border of the PI.
bLH expressing cells
A sagittal pituitary section inmunostained with anti-Fh
(91-106) bLH is shown in Fig. 1a. A moderated LH-ir
cell population is located in central, ventral and
Fig. 1 Single label
immunohistochemistry (IHC).
a Sagittal section of an adult
pituitary immunostained with
anti-Fh (91-106) bLH. b Details
of LH-ir cells in central zones of
the PPD. Typical vacuolated
cytoplasm can be observed in
the inset. c Detail of high LH-ir
cells in the external border of
the PI. d Sagittal section of an
adult pituitary immunostained
with anti-Fh (50-60) bFSH.
e FSH-ir cells (arrowheads) in
central zones of the PPD.
f FSH-ir cells in the external
border of the PI where some
FSH ir-fibers could be observed
in the NH. Scale bars: 20 lm.
HYP hypothalamus, IR
infundibular recess, PI pars
intermedia, PPD proximal pars
distalis, RPD rostral pars
distalis
marginal zones of the PPD (Fig. 1b). There is also a high
ir-population in the external border of the PI (Fig. 1c).
These cells are arranged in cords or small groups along
the PPD. They have conspicuous processes directed towards the NH branches (Fig. 2c), and they show a
vacuolated cytoplasm (Fig. 1b, inset).
bFSH expressing cells
A sagittal pituitary section inmunostained with anti-Fh
(50-60) bFSH is observed in Fig. 1d. A population of
FSH cells showing a moderate immunoreactivity is detected in central and ventral zones of the PPD (Fig. 1e).
There is also a FSH high ir-population in the external
border of the PI (Fig. 1f). In general, FSH-ir cells are
scarce and smaller when compared with LH cells, and
localized in more anterior positions of the PPD. These
endocrine cells are arranged in cords of two or three cells
A
D
B
E
C
F
or isolated. They are spherical or poliedrical in shape,
and they show a vacuolated cytoplasm.
A double IHC for FSH and LH in an adult pituitary
section can be observed in Fig. 2a, b. LH cells show a
brown color and FSH cells a blue one (Fig. 2c, d). Both
FSH and LH cells present the same distribution in midsagittal and lateral sections. No apparent differences in
cell number and immunoreactivity could be detected
between males and females.
Western blot analysis showed that C. dimerus bLH
has a MW of 24 kDa in pituitary. For bFSH two specific
ir-bands were obtained: 19 and 15 kDa (Fig. 2e, f).
ir fibers in the NH (Fig. 1f). These ir-fibers can also be
observed in the infundibular stalk, preoptic area and
ventral hypothalamus. The neuronal cells bodies that
send these projections are located in the preoptic area
(nucleus parvocellular and magnocellular) and in the
hypothalamic nucleus lateralis tuberis (Fig. 3b). Although both GtHs are expressed in the same brain areas,
this expression does not occur in the same neurons.
Treatment of sections with preabsorbed antisera abolished fibers and cell bodies immunostaining.
Ontogeny of FSH and LH endocrine cells and neurons
Localization of bFSH-ir and bLH neurons
and their projections in adults
bFSH expressing cells
Adult pituitaries show not only ir-endocrine cells but
also a lot of LH-ir fibers (Fig. 3a) and some scarce FSHFig. 2 bFSH and bLH double
IHC. Double IHC in sagittal
sections of an adult male (a),
and an adult female (b),
showing LH cells in brown and
FSH cells in blue. c, d Details of
FSH (blue) and LH (brown)
cells in the PPD, where the LH
cell processes directed towards
the NH branches can be
observed (arrows in c). Scale
bars: 20 lm. e Western blot
analysis of pituitary
homogenates showing a single
ir-band for LH (24 kDa) and
two ir-bands for FSH (15 and
19 kDa). f Western blot
analysis of pituitary
homogenates incubated with
the preadsorbed antibodies: 1
anti-LH preadsorbed with
FSH, 2 anti-LH preadsorbed
with LH, 3 anti-FSH
preadsorbed with LH, 4 antiFSH preadsorbed with FSH.
HYP hypothalamus, IR
infundibular recess, PI pars
intermedia, PPD proximal pars
distalis, RPD rostral pars
distalis
At day 21 ah, the first pituitary FSH-ir cells are detected
in the anterior ADH (Fig. 3c). Close to the onset of the
sexual differentiation period (day 42 ah) the number of
A
B
C
D
E
F
Fig. 3 Single label IHC. a LHir fibers in the NH (arrows)
contacting the PPD and PI.
b LH-ir cell bodies and fibers in
the hypothalamic NLT.
c Sagittal section at the
pituitary level of 21-day-old
larvae in which FSH cells could
be detected in ventral zones of
the PPD (arrowheads).
d Coronal section of 21-day-old
larvae in which a small
population of FSH neurons in
the NLT was detectable.
e Sagittal section at the
pituitary level of 15-day-old
larvae in which a strong
innervation of the PI by LH-ir
fibers (arrowheads) could be
detected. Details of
hypothalamic fibers in the inset.
f Sagittal section at the pituitary
level of 60-day-old larvae in
which the expression of LH
pituitary cells starts. Scale bars:
20 lm. HYP hypothalamus, ir
immunoreactive, IR
infundibular recess, NH
neurohypophysis, NLT nucleus
lateralis tuberis, PI pars
intermedia, PPD proximal pars
distalis, RPD rostral pars
distalis
A
B
C
D
E
F
FSH-ir cells increases and they can be observed along
the PPD and the external border of the PI. At least at
day 90, when this ontogenetic study finished, the number
of FSH-ir cells is still increasing. Preoptic and hypothalamic FSH-ir neurons are synchronically differentiated with FSH-ir pituitary cells at day 21 ah (Fig. 3d).
bLH expressing cells
At day 15 ah, some neuronal bodies in the hypothalamus sending their projections through the PI are
observed (Fig. 3e). The pituitary ir-cells are detected
later than the respective neurons. The differentiation
of this population occurred at day 60 ah, after the
sexual differentiation period was started. The first ircells are detected in central zones of the PPD (Fig. 3f).
Data of this ontogenetic study is summarized in
Table 1.
Expression of FSH and LH in gonads
Along the ontogenetic study immunoreactivity for FSH
and LH was also detected in ovaries. This was evidenced
by the presence of FSH and LH ir-material in the
cytoplasm of the oocytes of this species. Ovaries sampled
during winter season and during the pre and postspawning periods of summer season were treated for
GtHs IHC. In all cases, the FSH-ir and LH material was
Table 1 Ontogenetic expression of bFSH and bLH pituitary cells
and neurons in C. dimerus
Day
after
hatching
Body
length
(mm)
bFSH
pituitary
cells
bFSH
neurons
15
21
60
6 ± 0.4
8 ± 0.6
17 ± 1.5
+
+
+
+
bLH
pituitary
cells
+
bLH
neurons
+
+
+
Fig. 4 Single label IHC.
Transversal sections of adult
female ovary. a Previtellogenic
oocytes with a FSH-ir
cytoplasm. b Ovarian control
section treated with the
preabsorbed anti-FSH.
c Previtellogenic oocytes with a
LH-ir cytoplasm. d Ovarian
control section treated with the
preabsorbed anti-LH. Controls
were counterstained with
hematoxylin. Scale bars: 20 lm.
e Western blot analysis of
ovarian homogenates showing a
single ir-band for LH (24 kDa)
and 2 ir-bands for FSH (15 and
19 kDa)
A
B
C
D
E
localized within the cytoplasm of previtellogenic and
early vitellogenic oocytes (Fig. 4a, c). Ovarian control
sections treated with the preabsorbed anti-FSH or antiLH are shown (Fig. 4b, d). Western blot analysis of
ovarian homogenates revealed the presence of the same
ir-bands detected in the pituitary (Fig. 4e). On the other
hand, no immunoreactivity was detected in the testis.
Table 2 Percentage of viability, and of undifferentiated and differentiated gonads in both experiments
Viability (%) Undifferentiated Differentiated
gonads (%)
gonads (%)
Control
0.5 lg/ml rhFSH
1 lg/ml rhFSH
2 lg/ml rhFSH
85
90
90
85
100
28
11
12
0
72
89
88
Significant differences were observed in the percentage of differentiated gonads among the three treatments when compared to the
control. N = 20 for each treatment, P < 0.05
Effect of rhFSH on undifferentiated gonads in vitro
The undifferentiated gonads belonging to 30-day-old
larvae could be maintained in culture conditions for
15 days, and most of the gonads treated with rhFSH
underwent sexual differentiation (Table 2). We named
‘‘undifferentiated gonads’’ (UG) those containing only
undifferentiated germ cells (Fig. 5a, b), and ‘‘differentiated gonads’’ (DG) those in which early meiotic cells
(oocytes) were detected (Fig. 5c, d). During normal gonadal development in C. dimerus, ovarian differentiation
starts around day 42, while the onset of testicular differentiation occurs 30 days later (Meijide et al. 2005).
Then, DGs at day 45 under culture conditions were
considered as ovaries under the process of sex differentiation, because testicular differentiation does not occur
until later in development. It is important to notice that
none of the control gonads (placed 15 days in culture
media without rhFSH) underwent sexual differentiation
while most of the treated gonads did (Table 2). No sig-
Fig. 5 Culture of
undifferentiated gonads.
a Transversal section of an
explant treated for 15 days in
control culture conditions (day
45 of larval development), in
which an undifferentiated
gonad was observed (arrow).
b Details of undifferentiated
gonad with germ cells
surrounded by somatic cells.
c Transversal section of an
explant treated for 15 days in
culture conditions containing
rhFSH (day 45 of larval
development), in which a
differentiated gonad was
observed. d Detail of a
differentiated gonad with
oogonia and oocytes. In all
cases, sections were stained with
hematoxilin–eosin. Scale bars:
20 lm. G gonad, Gc germ cell,
K kidney, M muscle, MS
mesentery, N notocord, Nt
neural tube, Og oogonia, Oo
pachytene oocytes, Sc somatic
cell
A
B
C
D
nificant differences were observed between the two
experiments. There were no significant differences between the three rhFSH concentrations used in this study.
Significant differences were observed between control
and treatments (P < 0.05).
Discussion
The synthesis of FSH and LH in different cellular types
was already reported in several teleost species: mummichog (Calman et al. 2001), mediterranean yellowtail
(Garcia Ayala et al. 1998), red seabream (Kagawa et al.
1998), pejerrey (Miranda et al. 2001) and salmon (Nozaki et al. 1990). However, in other species, the use of
heterologous antisera failed to detect two types of
gonadotrops (Vissio et al. 1996; Saga et al. 1999; SeguraNoguera et al. 2000; Pandolfi et al. 2001a). The results of
our immunohistochemical analysis revealed that in adult
C. dimerus, FSH and LH are mainly produced in different cell populations of the adenohypophysis. The
precise location of the different GtH cells was based on
two previous anatomical and embryological studies on
C. dimerus pituitary gland (Pandolfi et al. 2001a, b). In
this species, FSH expressing cells are mainly located in
central and ventral zones of the PPD and, in a minor
proportion, in the marginal border of the PI. LH
expressing cells show a similar pattern but with a higher
cell density in the PPD when compared to FSH. The
immunoreactivity differences observed between PPD
cells (moderated) and PI cells (high) for both GTHs
could reflect a differential regulation of their production
according to the period chosen for this study (reproductive season).
The antisera used for this study were obtained from
the mummichog F. heteroclitus (Cyprinodontidae; Cyprinodontiforms, Acantopterygii). The synthetic peptides
used as antigens, carried the conserved sequences of
teleost FSH and LH (Shimizu and Yamashita 2002;
Shimizu et al. 2003a, b). These antisera were successful
on detecting gonadotrops of several acantopterygian
species (Shimizu et al. 2003a, b). For the anti-Fh (50-60)
bFSH, the only way of obtaining positive reactions is
pre-treating sections at 90C for 10 min in the
unmasking solution. It is probable that the FSH molecule experience a partial epitope masking due to the
histological procedures performed (fixation, dehydration, etc.).
The specificity of our GTHs detection system was
reinforced by the preabsortion controls and by the
Western blot of pituitary and ovary homogenates. C.
dimerus bLH showed a MW of 24 kDa, 3 kDa higher
than that of the mummichog bLH (21 kDa). This difference in MW may reflect a major glycosilation degree
of C. dimerus bLH. For bFSH, two specific ir-bands
were obtained (C. dimerus: 19 and 15 kDa; mummichog:
18 kDa). It is probable that the 15 kDa bFSH band is a
degradation product of the 19 kDa one, or a different
isoform recognized by the same antisera.
The vacuolated cytoplasm of FSH and LH cells in
adults probably corresponds to the typical development
of the RER previously described by electron microscopy
in several teleost species (Quesada et al. 1988; Garcı́a
Ayala et al. 1998). This vacuolization of gonadotrops
seems to correlate mainly with age/sexual status, because
they were not observed in larvae or juveniles.
During the study of the adult pituitary, the presence of
FSH-ir and LH-ir fibers was detected in the NH and
infundibular stalk. Serial brain sections showed that the
cells bodies, which project those fibers, were located in
the POA (parvocelullar and magnocelluar preoptic nuclei) and in the hypothalamus (nucleus lateralis tuberis).
In the control sections, the immunostaining was completely abolished. In salmon and tilapia, FSH and LHlike immunoreactivity was found in the preoptic nucleus.
Also in the tilapia, the mRNA brain levels for these
GTHs were quantified and they were lower than the ones
quantified for pituitary (Parhar et al. 2003). Immunoreactivity for LH was also reported in arcuate nucleus
neurons, pars tuberalis cells and hypothalamic axons
from rodents (Gross and Page 1979; Hostetter et al.
1981). It was then hypothetized that the brain derived
LH was originated from the pituitary LH that reached
the brain from the circulatory system or by selective
absorption (tanocytosis) from the brain–spinal fluid
(Pacold et al. 1978). Experimental evidences showed that
after hypophysectomy, it is possible to measure pituitary
hormones in brain (Emanuel et al. 1981; Hojvat el al.
1982). The precise role of brain-derived FSH and LH in
C. dimerus is not very clear, but, the abundance of iraxons in the ventral forebrain and in the NH suggests the
existence of a neuromodulatory function together with a
possible function in the control of pituitary cells activity.
In rodents, it was shown that an intracerebral injection of
LH modulated not only the synthesis and release of
pituitary LH (Emanuele et al. 1981) but also the electric
activity of some hypothalamic neurons (Kawakami and
Sakuma 1974; Teresawa et al. 1969). Moreover, in the
tilapia it was clearly demonstrated the FSH and LH
expressing neurons also express AVT, and this neurohypohysial hormone was synthesized in preoptic neurons, which are strongly related with the control of some
reproductive behaviors (Demski and Sloan 1985; Foran
and Bass 1999; Parhar et al. 2003). Interestingly, FSH
and LH neurons are expressed in POA regions that also
express GnRH1 and GnRH3 in this species (Pandolfi
et al. 2005). Although this occurs in morphological different neurons, it is possible that some synapsis between
GnRH and FSH and/or LH neurons take place. Future
studies will be focused in the relationship between GnRH
and GTHs at the brain level.
Another fact that reinforces the specificity of our
detection system is the difference in the timing of
appearance of LH and FSH both in brain and pituitary:
LH neurons (day 15), FSH pituitary cells and neurons
(day 21), LH pituitary cells (day 60). In this species, FSH
cells appeared prior to the onset of sexual differentiation, while LH cells were differentiated several days after
this process started. Then, we decided to study only the
effect of FSH on sex differentiation, because at the day
the experiment started (day 30) and ended (day 45) there
was no expression of pituitary LH. This variation in the
timing of expression of FSH and LH was also observed
in several teleost species (Mal et al. 1989; Nozaki et al.
1990; Saga et al. 1993; Magliulo-Cepriano et al. 1994;
Miranda et al. 2001). The results of this studies suggested that in salmonid species and paltyfish, as in C.
dimerus, the differentiation of FSH cells preceded the
differentiation of LH cells. Only in pejerrey, it was
shown a first appearance of LH cells. It was described
that GtHs are important for the regulation of the
hypothalamus–pituitary–gonad axis (Schreibman et al.
1982). In salmonids, FSH, but not LH, was involved in
the gonadal initial grow and development (Saga et al.
1993). When C. dimerus undifferentiated gonads were
left alone in culture media, the onset of gonadal differentiation did not occur as observed in vivo, suggesting
an important role of the brain–pituitary–axis during sex
differentiation in this species. We postulated that one of
the factors involved in this process is FSH because its
expressing cells were already differentiated at the larval
period chosen for this study and because it was demonstrated to be involved in the onset of meiosis (Ito and
Abe 1999; Yazawa et al. 2002). When treated with
rhFSH, 80% of the gonads were differentiated to presumptive ovaries, based on the morphological description available for this species (Meijide et al. 2005). The
results of this experiment reinforce our previous
hypothesis about a correlation between the innervation
of pituitary cells by GnRH1 fibers and the release of
FSH that will be acting in any of the important steps of
the sexual differentiation process (Pandolfi et al. 2002).
Another extracerebral region in which FSH and LH
were found is the ovary. The presence of FSH and LHlike material was detected in previtellogenic and vitellogenic oocytes cytoplasm. The specificity of this immunostaining was confirmed by preabsorption tests and
western blot. A recent study in another Perciform, the
seabream Sparus aurata, showed a novel expression of
FSH and LH in the ovary by isolating their corresponding genes (Wong and Zohar 2004). It is probable
that these ovarian-derived GtHs act as autocrine and/or
paracrine factors involved in intraovarian communication between the oocytes and the follicular cells.
Acknowledgments The authors would like to thank Dr. Frank van
den Berg (University of Amsterdam) for his aid in the epitope
unmasking protocol, and Dr. P. Vissio (Universidad de Buenos
Aires) for her constant and disinterested advice. This work was
supported by grants from UBACyT X-118 and PICT E-14271.
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