NEUROENDOCRINOLOGY
The Highly Conserved Gonadotropin-Releasing
Hormone-2 Form Acts as a Melatonin-Releasing Factor
in the Pineal of a Teleost Fish, the European Sea Bass
Dicentrarchus labrax
Arianna Servili, Christèle Lethimonier, Jean-Jacques Lareyre,
José Fernando López-Olmeda, Francisco Javier Sánchez-Vázquez,
Olivier Kah, and José Antonio Muñoz-Cueto
Departamento de Biología (A.S., J.A.M.-C.), Facultad de Ciencias del Mar y Ambientales, Universidad de
Cádiz, E-11510 Puerto Real, Spain; Neurogenesis and Estrogens (A.S., C.L., O.K.), Unité Mixte de
Recherche Centre National de la Recherche Scientifique 6026, Institut Fédératif de Recherche (IFR) 140;
Institut National de la Santé et de la Recherche Médicale U625, Groupe d’Etude de la Reproduction chez
l’Homme et les Mammifères (GERHM) (C.L.) and Sexualité et Reproduction des Poissons (J.-J.L.), Institut
National de la Recherche Agronomique, Station Commune de Recherches en Ichtyophysiologie,
Biodiversité et Environnement (SCRIBE), IFR140, Université de Rennes 1, Campus de Beaulieu, 35042
Rennes, France; and Departamento de Fisiología (J.F.L.-O., F.J.S.-V.), Facultad de Biología, Universidad de
Murcia, Campus de Espinardo, E-30100 Murcia, Spain
With the exception of modern mammals, most vertebrate species possess two GnRH genes, GnRH-1
and GnRH-2. In addition, in many teleost fish, there is a third gene called GnRH-3. If the main
function of GnRH-1 is unambiguously to stimulate gonadotropin release, the other two GnRH
forms still lack clear functions. This is particularly true for the highly conserved GnRH-2 that encodes
chicken GnRH-II. This GnRH variant is consistently expressed in neurons of the dorsal synencephalon
in most vertebrate groups but still has no clear functions supported by anatomical, pharmacological, and physiological data. In this study performed on a perciform fish, the European sea bass, we
show for the first time that the pineal organ receives GnRH-2-immunoreactive fibers originating
from the synencephalic GnRH-2 neurons. This was shown through a combination of retrograde tracing
and immunohistochemistry, using highly specific antibodies. Supporting the presence of GnRH-2 functional targets, RT-PCR data together with the in situ hybridization studies showed that the sea bass
pineal gland strongly expressed a GnRH receptor (dlGnRHR-II-2b) with clear selectivity for GnRH-2 and,
to a lesser extent, the dlGnRHR-II-1a subtype. Finally, in vitro and in vivo experiments demonstrate
stimulatory effects of GnRH-2 on nocturnal melatonin secretion by the sea bass pineal organ. Altogether, these data provide, for the first time in a vertebrate species, converging evidence supporting
a role of GnRH-2 in the modulation of fish pineal functions. (Endocrinology 151: 2265–2275, 2010)
nRH is a major neuroendocrine initiator of the hormonal cascade controlling reproduction, through its
stimulatory actions on the synthesis and secretion of pituitary gonadotropins. In perciform fishes, as in many
other teleost orders, three different GnRH genes are
present. The type 1 GnRH (GnRH-1) gene is expressed in
ventral forebrain neurons producing a decapeptide whose
G
structure varies from one order to the other. In the sea bass,
this peptide corresponds to sea bream GnRH (sbGnRH)
form, because it was first discovered in sea bream (1–5).
The type 2 GnRH (GnRH-2) gene produces a variant
called chicken GnRH-II (cGnRH-II) that is synthesized by
neurons present at the diencephalic-mesencephalic transition. The type 3 GnRH (GnRH-3) gene encodes another
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2010 by The Endocrine Society
doi: 10.1210/en.2009-1207 Received October 9, 2009. Accepted February 4, 2010.
First Published Online March 9, 2010
Abbreviations: cGnRH-II, Chicken II GnRH; DiI, 1,1⬘-dioctadecyl 3,3,3⬘,3⬘-tetramethylindocarbocyanine perchlorate; GAP, GnRH-associated peptide; GnRHa, GnRH analog; GnRHR, GnRH receptor; IP3, inositol 1,4,5-triphosphate; MD, mid-dark; ML, mid-light; sbGnRH, sea bream GnRH; sGnRH, salmon GnRH.
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variant named salmon GnRH (sGnRH) that is mainly produced in olfactory bulb/telencephalic neurons (4). The
GnRH-1 and GnRH-2 genes have orthologs in all vertebrates, although GnRH-2 seems to be lacking in many
evolved mammalian species. GnRH-1 is consistently
found in neurons that project to the median eminence (tetrapods) or pituitary (fish) and is clearly involved in gonadotropin secretion (6). In teleost fish, GnRH-3 expression overlaps with that of GnRH-1-expressing neurons.
Its functions are largely unknown, although in the sea
bass, there are sGnRH-immunoreactive fibers in the pituitary (4). The functions of the highly conserved GnRH-2
peptide are also a matter of debate, although there is indication that it could be involved in sexual behavior and
possibly the regulation of feeding (7). Currently, there is
no converging morphological, functional, and pharmacological evidence to support a clear function for this highly
conserved GnRH variant in any vertebrate. There is also
very little information as to the nature of GnRH receptors
(GnRHRs) supporting GnRH-2 functions.
GnRH exerts its action through G protein-coupled
GnRHRs (5, 6). In teleost, notably the European sea bass, up
to five GnRHRs have been cloned and characterized (5, 8,
9). These sea bass GnRHRs have been named dlGnRHRII-1a, dlGnRHR-II-1b, dlGnRHR-II-1c, dlGnRHR-II-2a,
and dlGnRHR-II-2b. Apart from dlGnRHR-II-1a that is
strongly expressed in the pituitary (8), there is currently
very little information as to the sites of expression, pharmacology, or functions of these receptors. In contrast to
the mammalian type I receptor, all GnRHRs present in
nonmammalian vertebrates have a long intracellular tail.
In some mammalian species, a type II GnRHR has been
also reported to have an intracellular tail and strong affinity for cGnRH-II (7). From previous studies, it has been
suggested that the disappearance of GnRH-2 in mammals
was accompanied by the loss of the type II receptor gene or
function, at the same time that emerged the type I GnRHR
with very high affinity for GnRH-1 (6).
Reproduction in fish is a rhythmic process entrained by
daily and seasonal changes in environmental factors. The
pineal organ and its main secretion, melatonin, has been
involved in these daily and seasonal reproductive rhythms
in fish, representing both a clock and a calendar (10).
Thus, melatonin actions on the reproductive axis have
been reported at the brain (GnRH, dopaminergic system),
pituitary (FSH/LH), and gonadal (testis and ovary) levels
(see Ref. 10 for a review). However, the modulatory role
of GnRH on pineal function and melatonin secretion remains unexplored in fish.
The GnRH systems have been studied in depth in the
European sea bass, Dicentrarchus labrax (4, 11). In particular, highly specific antibodies have been generated
Endocrinology, May 2010, 151(5):2265–2275
against the precursors of GnRH-1, GnRH-2, and GnRH-3
of this species (4, 12). Such information, together with the
cloning of the five receptors provides outstanding opportunities to correlate the presence of a given peptide to the
expression of a given receptor in GnRH target tissues.
Recently, a tract-tracing study performed in sea bass revealed the presence of pineal projecting cells in the dorsal
synencephalon (Servili, A., and J. A. Muñoz-Cueto, unpublished observation), a region known for the presence
of GnRH-2 cells as stated above (4, 11).
The aim of this paper was to 1) provide evidence on
whether GnRH-2 neurons and the pineal gland appear
anatomically interconnected in sea bass, 2) determine,
both in vivo and in vitro, whether GnRH-2 affects the
nocturnal release of melatonin in this species, 3) establish
using RT-PCR and in situ hybridization whether the pineal expresses GnRHR mRNA, and finally, 4) characterize the pharmacological properties of those GnRHs expressed in the pineal gland of sea bass.
Materials and Methods
Animals
For these studies, we used 1-yr-old (ⱕ250 g) European sea
bass (Dicentrarchus labrax) immature males. Animals were kept
in running seawater under natural light conditions at a temperature and salinity of 19 ⫾ 1 C and 39 ppt, respectively. They were
housed in indoor facilities in the Laboratorio de Cultivos Marinos (University of Cadiz, Puerto Real, Spain) or in the Manuel
Tárrega Institute (San Pedro del Pinatar, Spain). Animals were
fed at libitum once per day and treated in agreement with the
Spanish and European Union regulation concerning the use and
protection of experimental animals.
DiI tract-tracing analysis
Animals (n ⫽ 20) were anesthetized with an overdose of
2-phenoxyethanol and perfused through the heart with fixative
solution. The dorsal cranium was removed for the implantation
of small crystals of 1,1⬘-dioctadecyl 3,3,3⬘,3⬘-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes, Eugene, OR)
into the distal pineal stalk. To avoid accidental contamination,
the whole region was then sealed with warm 3% agar solution and
stored in fresh fixative at 37 C in darkness for 1– 4 months. Only
perfused animals in which brain fixation was optimal were used.
The brains were then dissected out and embedded in 3% agar. Serial
transverse sections of 50 –70 ␮m thick were cut on a Vibratome (St.
Louis, MO), mounted on gelatin-coated slides, and observed and
photographed under a fluorescence Olympus BH-2 photomicroscope. For the precise localization of labeling, we used a sea
bass brain atlas developed in our laboratory (13–15).
Combination of DiI tracing and fluorescence
immunohistochemistry
We performed immunofluorescence staining on selected sections of three sea bass brains processed previously for DiI tracttracing analysis. Tissue sections containing pinealopetal labeled
cells in the dorsal synencephalon were washed for 10 min in
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Coons buffer (pH 7.4) (CBT: 0.01 M barbital, 0.15 M NaCl)
containing 0.2% Triton X-100 and incubated overnight in a
moist chamber at room temperature with an antiserum against
GnRH-2-associated peptide (anti-cIIGAP) at a 1:500 dilution.
This antiserum was generated in guinea pig, and its specificity
was firmly established previously (4, 12). An antibody against
guinea pig IgG coupled to fluorescein isothiocyanate (Jackson
ImmunoResearch Laboratories, West Grove, PA) was used to
reveal immunoreactive GnRH cells and fibers. Slides were coverslipped with Vectashield mounting media (Vector Laboratories, Burlingame, CA), and then observed and photographed under a fluorescence Olympus BH-2 photomicroscope.
Immunohistochemical study of pineal organ
For this study, sagittal brain sections from five sea bass specimens containing the pineal organ were incubated overnight with
antisera against GnRH-1-, GnRH-2-, and GnRH-3- associated
peptides (anti-sbGAP, anti-cIIGAP, and anti-sGAP) at a 1:500
dilution. The specificity of these antisera was tested previously
(4, 12). Subsequently, sections were incubated for 1.5 h at room
temperature with biotin-long space-conjugated goat antiguinea
pig IgG (Jackson ImmunoResearch) diluted 1:1000 in CBT,
rinsed in CBT, and then incubated 1.5 h at room temperature
with peroxidase-conjugated streptavidin complex (Jackson ImmunoResearch) diluted 1:1000 in CBT. Peroxidase activity
was visualized in 0.025% 3,3-diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO) and 0.01%
H2O2. Controls were performed as described previously (4, 12).
Immunohistochemical sections were analyzed and photographed under an Olympus BH-2 photomicroscope.
Analysis of GnRHR expression in pineal organ by
RT-PCR
Rapidly removed pineal organs were obtained from sea bass,
and two pools of five pineals were immediately frozen in liquid
nitrogen. Total RNA was extracted using EUROzol (EuroClone,
Siziano, Italy) according to the manufacturer’s instructions. Total RNA (1 ␮g) was retro-transcribed using a kit that eliminates
genomic DNA contaminations (QuantiTect reverse transcription
kit; QIAGEN Iberia S.L., Madrid, Spain). Specific primer pairs for
different dlGnRHRs (8, 9) (Supplemental Fig. 1 published on
The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) were designed from the sequences available in the GenBank database. RT-PCRs were performed using
the following conditions: denaturation at 95 C for 5 min, followed by 34 cycles of denaturation at 95 C for 45 sec, annealing
at 60 C for 1 min, and elongation steps at 72 C for 1 min before
the final step at 72 C for 7 min more. RT-PCR products were
analyzed by electrophoresis on a 2% agarose gel.
Localization of GnRHR-expressing cells in pineal
organ by in situ hybridization
PCR amplification, subcloning, and sequencing
Total RNA was extracted from adult sea bass brains and
cDNA synthesized as described above. RT-PCR was performed
using specific primers for dlGnRHR-II-1b, dlGnRHR-II-1c, and
dlGnRHR-II-2a sequences available in the GenBank database
(Supplemental Fig. 1). RT-PCR products were purified and
cloned into the pGEM-T Easy Vector (Promega Biotech Ibérica,
Madrid, Spain) for sequencing.
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Riboprobe synthesis
Specific riboprobes were synthesized for the receptors dlGnRHRII-1b, -1c, and -2a using the pGEM-T Easy-based constructs described above. Specific probes for the receptors dlGnRHR-II-1a
(1251 bp) and -II-2b (1143 bp) were synthesized using the plasmids
obtained previously (8) (Lethimonier, C., J.J. Lareyre, O. Kah, unpublished data). The preparation of [␣-35S]dUTP-labeled singlestranded mRNA probes was carried out according to the standard
procedure reported previously (8).
In situ hybridization
Animals (n ⫽ 6) were anesthetized with 2-phenoxyethanol
and perfused with Bouin’s fixative solution. Brains with both
pineal and pituitary glands attached were dissected out and embedded in paraffin. Serial transverse 6-␮m-thick paraffin sections were obtained with a microtome and then mounted in polyL-lysine-coated slides. The protocol for in situ hybridization was
performed as previously described (11) with slight modifications. Serial sections were always hybridized with the correspondent antisense and sense probe pairs of the five GnRHRs cloned
in this species. Pituitary sections hybridized with dlGnRHRII-1a probe were used as positive control of every experiment.
Finally, in situ hybridization sections were analyzed and photographed on an Olympus Provis photomicroscope.
Pharmacological studies of dlGnRHR-II-1a and
dlGnRHR-II-2b subtypes
Inositol 1,4,5-triphosphate (IP3) assay
The cDNAs encoding the full-length open reading frame of
both dlGnRHR-II-1a (accession no. AJ419594) and dlGnRHRII-2b (accession no. AJ606685) receptors were subcloned into
the expression vector pcDNA 3.1-V5-his-TOPO (Life Technologies, Inc. Invitrogen, Carlsbad, CA). For each cDNA, the consensus Kozak sequence (CCATG) was integrated upstream of the
ATG. Eight micrograms of plasmid DNA were transfected into
monolayer cultures of COS-7 cells seeded at 0.7 ⫻ 106 cells/ml
in 100-mm dishes using the Fugene 6 transfection reagent
(Roche, Meylan, France). After 24 h, cells were trypsinized,
seeded into 12-well plates, and grown overnight. Cells were then
labeled with 2 ␮Ci/ml myo[2-3H]inositol (TRK 911; Amersham,
Saclay, France) in 199 medium containing 10% fetal calf serum
(Sigma) and 1% antibiotics (Sigma) and incubated at 37 C for
24 –30 h. Inositol production was assayed as described previously (16). Cells were stimulated with various concentration of
ligands in IP3 buffer [1⫻ Hanks’ balanced salt solution, 20 mM
HEPES, 20 mM LiCl (pH 7.55)] for 1 h at 37 C. The GnRH
ligands used were cGnRH-II (Bachem, Weil Am Rhein, Germany), sGnRH (American Peptides Company Inc., Sunnyvale,
CA) and sbGnRH (Bachem). The reaction was stopped by adding 0.2 ml prechilled 20% perchloric acid and incubated at 4 C
for 30 min. Lysates were then transferred into 1.5-ml tubes,
equilibrated at pH 7.5 with 2 mM EDTA (pH 8) and 500 mM
KOH, and incubated for 1 h at 4 C. Afterward, lysates were
centrifuged for 15 min at 5000 rpm at 4 C. The supernatants were
then loaded onto AG X8 anion exchange resin columns (BioRad, Marnes la Coquette, France) preconditioned with 5 ml 3 M
ammonium formate, 100 mM formic acid solution and then
equilibrated with 10 ml H2O. Resin was washed with 10 ml wash
buffer (60 mM ammonium formate, 5 mM sodium tetraborate)
and eluted with 3 ml 1 M ammonium formate, 100 mM formic
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acid solution. The incorporation of radioactivity in the eluates was
measured in a scintillation counter. Experiments were performed in
triplicate and repeated five times. Data obtained were analyzed with
PRISM software (GraphPad Software, Inc., La Jolla, CA).
cAMP assay
Transient transfections were performed in COS-7 cells maintained under 5% CO2 in DMEM (Sigma) containing 2 mM glutamine (Sigma), 10% fetal bovine serum (Invitrogen), 100 U/ml
penicillin, and 50 ␮g/ml streptomycin (Sigma). Cells were seeded
in six-well dishes at approximately 0.5 ⫻ 106 cells/2 ml. Transient transfections were carried out as described previously (17).
Briefly, 0.5 ␮g expression vector containing the cDNA sequences
of dlGnRHR receptors were transfected in presence of 1 ␮g of the
reporter vector pCREluc (Stratagene, La Jolla, CA), 0.45 ␮g
carrier DNA, and 50 ng pCMV-␤gal used to control transfection
efficiency, using the Fugene 6 transfection reagent (Roche). After
4 h, cells were trypsinized, seeded in 24-well plates, and grown
48 h. Cells were then stimulated with increasing concentrations
of cGnRH-II, sGnRH, and sbGnRH for 6 h at 37 C. Cells were
lysed by 100 ␮l lysis buffer (Promega) and stored at ⫺20 C before
luciferase assay. Luciferase activity was measured in 20 ␮l cellular extract according to the manufacturer’s protocol (Promega), and ␤-galactosidase activity was used to normalize transfection efficiency. The experiment was performed in triplicate,
and the agonist concentrations inducing half-maximal stimulation (EC50) were calculated with PRISM software (GraphPad).
In vitro effects of GnRH treatment on melatonin
secretion in cultured pineal organs
Sea bass pineal organs were rapidly dissected out, treated for
10 min in antibiotic bath with gentamicin, placed individually on
a bed of glass wool in 3-ml culture chambers, and cultured under
a superfusion system as described previously (18). A peristaltic
pump continuously supplied DMEM (Invitrogen) to the chambers (19). All pineal organs were maintained under constant temperature and artificial 12-h light,12-h dark photoperiod cycle
(lights on at 0900 h and lights off at 2100 h, light intensity 500
lux). Different conditions of temperature (20 and 24 C) and
medium flow rates (0.8 and 1 ml/h) were tested. Furthermore,
two distinct GnRH concentrations (10⫺8 and 10⫺12 M) were
used according to a previous study (20).
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phases of each day of culture, by performing melatonin RIA on 3-h
collected medium samples as described previously (18, 21).
In vivo effects of GnRH injections on plasma
melatonin levels
In this experiment, we followed a longitudinal approach in
which every animal was submitted to all the experimental treatments tested. The individual profile of plasma melatonin levels
from each specimen was determined before any manipulation,
after GnRH treatment, and after the vehicle injection. For this
purpose, seven sea bass specimens were individually tagged in
with electronic MUSSIC identification chips (AVID, Norco, CA)
to facilitate their identification, and maintained under controlled
photoperiod (12 h light, 12 h dark) and temperature (19 C)
conditions. On d 0 of the experiment, blood samples were collected from each fish at the middle of the dark phase for melatonin determination by RIA. Melatonin concentrations on these
samples were considered the basal nocturnal plasma melatonin
level of each animal (MD) and represent the negative controls for
injected animals. At the middle of the light period of d 2, GnRH-2
(Sigma-Aldrich) diluted in 0.9% saline solution was injected ip
in anesthetized animals at the concentration of 100 ␮g/kg body
mass, and blood samples were collected again at the following
MD period (MD GnRH). After 7 d resting, fish were ip injected
at the middle of the light phase with 0.9% saline solution with the
same volumes used for the previous GnRH injections. Finally,
blood samples were obtained at the following night (MD vehicle)
and in the middle of the next light phase (ML). All blood samples
were collected and centrifuged at 4 C, 3000 rpm, for 10 min.
Plasma was separated and stored at ⫺80 C until required for
melatonin RIA determination. The individual profile of melatonin secretion was represented for each specimen. To determine
statistically the effects of GnRH injection on plasma melatonin
levels, we have pooled the data and compared the mean values
corresponding to each experimental condition.
Melatonin RIA
Culture medium and plasma melatonin concentrations were
determined using a commercial kit for melatonin-RIA (IBL,
Hamburg, Germany) following the procedure described previously (21).
Experiment 1
In experiment 1, pineal glands were cultured with regular medium in the first night. During the second night of culture, the peristaltic pump was constantly providing culture medium containing
GnRH to the pineal glands for the whole duration of the night (from
2100 – 0900 h). In this first experiment, two distinct GnRH peptides
were tested: GnRH-2 (Sigma-Aldrich) (n ⫽ 4) and GnRH analog
(GnRHa) (Des-Gly10, D-Ala6 LHRH; Sigma-Aldrich) (n ⫽ 7).
Experiment 2
In experiment 2, pineal organs (n ⫽ 2) were submitted to the
same treatments of experiment 1, but the sequence was inverted.
Thus, pineal glands received GnRH-2 administration during the
first night, but they were cultured in absence of GnRH-2 during
the second dark phase.
In both experiments, melatonin concentrations were determined
at the middle of the dark [mid-dark (MD)] and mid-light (ML)
Data analysis
In the in vitro experiments, data are expressed as melatonin
concentration (picograms per milliliter) in the medium collected
during 3 h culture of each pineal organ and also as the mean of
all individual values ⫾ SEM. In the in vivo experiment, data are
expressed as plasma melatonin concentrations (picograms per
milliliter) of each animal and also as the mean of all individual
values ⫾ SEM. Statgraphic Plus 5.1 software was used for data
analysis. Statistical differences between mean values of melatonin levels were determined by repeated-measures ANOVA followed by Student-Newman-Keuls post hoc test with P ⬍ 0.05
considered as statistically significant threshold. When only two
groups were compared (e.g. mean values of melatonin released
by pineal organs incubated with GnRH-2 or GnRHa), statistical
differences were determined by paired t test, with P ⬍ 0.05 considered as statistically significant threshold.
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Results
DiI tract-tracing analysis and combination with
immunofluorescence techniques
The anterograde and retrograde diffusion of DiI labeled
both pinealofugal fibers (Fig. 1A) and pinealopetal neurons (Fig. 1, A, B, C, and E) in the dorsal synencephalon
of sea bass. The pinealofugal terminal field contained
abundant varicose fibers (Fig. 1A) and extended caudally
to the dorsal synencephalon/rostral tegmentum. The labeled cells revealed at this level represented the most caudal population of pinealopetal neurons in sea bass and
exhibited ovoid or rounded perikarya (Fig. 1, B, C, and E).
FIG. 2. Immunohistochemical staining of the pineal organ of sea bass
(sagittal sections). A, Photomicrograph showing cIIGAP (GnRH-2)
immunopositive fibers entering in the pineal organ through the pineal
stalk. The junction of the pineal stalk with the diencephalon is marked
(white arrowheads). B, Magnification of the boxed area in A. Note the
presence of varicose GnRH-2-positive fibers in the pineal stalk, whereas
the telencephalon appears devoid of immunostaining at this level. DIE,
Diencephalon; TEL, telencephalon. Scale bars, 100 ␮m.
Because pinealopetal cells of the dorsal synencephalon showed a similar appearance and position as the
GnRH-2 cells previously observed in this area (4), sections from the DiI neuronal tracing study were stained
using a specific anti-cIIGAP antiserum. The GnRH-2
neuronal population stained at this level consisted of
three to eight large round or ovoid cells per section (Fig.
1, D and F), some of which (one to two per section)
corresponded to DiI-labeled cells (Fig. 1, D and F, white
arrowheads).
FIG. 1. A, B, C, and E, Fluorescence photomicrographs showing the
anterograde and retrograde DiI labeling on transverse sections of the
dorsal synencephalon after the application of the tracer to the pineal
stalk. This synencephalic area contains pinealofugal fibers (A) but also
pinealopetal cells (A, B, C, and E). Note the apical processes of a
pinealopetal labeled cell (A, white arrowhead) reaching the
synencephalic region where the pinealofugal terminal field ends. Also
note the large size of labeled perikarya (B, C, and E) and the absence
of labeling in cell nucleus (B). D and F, GnRH-2 immunohistochemical
staining of sections from the DiI study presented in C and E shows that
pinealopetal cells from this area represent GnRH-2 cells. White
arrowheads mark double-labeled pinealopetal (DiI in red) and
immunopositive GnRH-2 neurons (green). Note that other GnRH-2immunoreactive cells of this area lack DiI labeling (black arrowheads).
Scale bars, 20 ␮m.
Immunohistochemical study of pineal organ
The immunohistochemical study of the pineal organ of
sea bass revealed the presence of GnRH-2-immunopositive fibers coursing in a caudorostral direction through
dorsal surface of the diencephalon and entering in the pineal organ through the pineal stalk (Fig. 2, A and B). No
immunoreactive fibers were found in the sea bass pineal
organ using anti-sGAP and anti-sbGAP antisera or in control sections (Supplemental Fig. 2).
Analysis of GnRHR expression in pineal organ by
RT-PCR
Significant RT-PCR amplification signals in the pineals
were obtained only with the dlGnRHR-II-2b and, to a
lesser extent, with the dlGnRHR-II-1a primer sets (Fig. 3).
No apparent expression was observed for the other three
GnRHRs. The negative control with no cDNA (H2O) run
with the samples in every RT-PCR did not show any
amplification.
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FIG. 3. Electrophoretic profile of amplicons obtained by RT-PCR in 2%
agarose gels. GnRHR expression pattern of sea bass pineal organs. A
strong intense band (249 bp) and a weak one (242 bp), corresponding,
respectively, to the expression of dlGnRHR-II-2b and dlGnRHR-II-1a
genes, are revealed. MW, Molecular weight marker; 2a, dlGnRHR-II-2a;
2b, dlGnRHR-II-2b; 1a, dlGnRHR-II-1a; 1b, dlGnRHR-II-1b; 1c,
dlGnRHR-II-1c; H2O, negative control. Some molecular weight markers
are indicated.
Localization of GnRHR-expressing cells in pineal
organ by in situ hybridization
The in situ hybridization findings were highly consistent with the results obtained by RT-PCR. Indeed, of the
five GnRHRs present in sea bass, only dlGnRHR-II-2b
was intensely expressed in the pineal organ (Fig. 4, A and
B), whereas dlGnRHR-II-1a was also detected but at
lower levels (Fig. 4, D and E). This difference in the intensity of the labeling is the result of a real difference in the
expression levels of the two receptor subtypes. Indeed, in
agreement with previous data (8), the dlGnRHR-II-1a
probe generated a very strong staining in the sea bass pituitary also present on the same sagittal sections (positive
control, Fig. 4F). In contrast, hybridization with the dlGnRHR-II-2b did not result in any signal in the pituitary,
whereas on the same section, the staining was obvious in
the pineal. The expression of dlGnRHR-II-2b was mainly
observed in cells localized in the pineal vesicle (Fig. 4, A
and B), whereas dlGnRHR-II-1a staining was also present
along the pineal stalk (Fig. 4, D and E). No labeling was
observed in the dorsal sac (Fig. 4, D and E). Control sections incubated with sense probes did not exhibit any labeling (Fig. 4C).
Comparative pharmacology of dlGnRHR-II-1a and
dlGnRHR-II-2b
Using two different assays, we showed that both dlGnRHRII-1a and dlGnRHR-II-2b were functional in COS-7 cells
transfected with the corresponding expression vectors. In
both cases, dose-response curves were obtained with each of
the three endogenous ligands tested (Fig. 5). However, the
two receptors showed remarkable differential selectivity.
In the IP3 assay, dlGnRHR-II-2b had a clear preference
for cGnRH-II, which was 5,000 and 71,875 times more
biopotent compared with sGnRH and sbGnRH, respec-
FIG. 4. Dark-field (A and D) and bright-field (B, C, E, and F)
micrographs showing the expression of GnRHR in sea bass pineal
organ and pituitary revealed by in situ hybridization. A and B, A strong
positive staining is detected at the level of the pineal vesicle
corresponding to dlGnRHR-II-2b-expressing cells. Stars show the pineal
lumen, which is not labeled. C, Control sense section showing the
absence of labeling for dlGnRHR-II-2b. D and E, Hybridization signal
revealing dlGnRHR-II-1a-expressing cells along the pineal stalk and
vesicle. Note the presence of melanophores (arrowheads) in this pineal
structure. The dorsal sac appears unlabeled. F, High dlGnRHR-II-1a
expression signal in sea bass pituitary gland used as positive control of
the hybridization procedure. ds, Dorsal sac; ps, pineal stalk; pv, pineal
vesicle. Scale bars, 250 ␮m.
tively (Table 1 and Fig. 5A). In contrast, dlGnRHR-II-1a
showed equal sensitivity to cGnRH-II and sGnRH,
whereas sbGnRH was 105–120 times less efficient than
the two other ligands in this receptor (Table 1 and Fig. 5B).
The same differences in ligand selectivity were also found
in the activation of the cAMP pathway as assessed by the
expression of the CRE-luc reporter gene, although activation of this pathway required higher doses of ligands
(Table 2 and Fig. 5, C and D).
In vitro effects of GnRH administration to cultured
pineal organs on melatonin secretion
After preliminary testing on experimental conditions,
temperature, flow rate of the medium, and doses of the
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Endocrinology, May 2010, 151(5):2265–2275
endo.endojournals.org
FIG. 5. Hormonal responsiveness of the dlGnRHR-II-2b and dlGnRHR-II-1a receptors. A and
B, COS-7 cells were transfected with the dlGnRHR-II-2b and dlGnRHR-II-1a expression vectors
independently. At 48 h after transfection, cells were incubated for 6 h with increasing
concentrations of endogenous sea bass GnRH peptides. Inositol triphosphate production was
monitored as described previously (15). Results are expressed as percentage of maximal
stimulation obtained for cGnRH-II and represent the mean of five independent experiments in
triplicate. C and D, COS-7 cells were cotransfected with the GnRHR expression vectors
together with the cAMP-responsive reporter construct pCRE-Luc and the pCMV-␤gal plasmid
used to normalized transfection efficiency. Cells were incubated for 6 h with increasing doses
of cGnRH-II, sGnRH, or sbGnRH. Hormone-induced cAMP production was indirectly
quantified by measuring the luciferase activity from the reporter vector. Each data point
represents the mean of triplicates of a representative experiment. Note the high selectivity of
the dlGnRHR-II-2b for the cGnRH-II form.
hormonal treatment were set at 20 C, 0.8 ml/min, and
10⫺8 M, respectively. Because no statistical difference in
melatonin release was observed when pineal organs were
incubated with GnRH-2 (n ⫽ 4) or GnRHa (n ⫽ 7) (t test,
P ⬎ 0.05), all pineal organs were combined for analysis.
Incubation with GnRH during the second night increased
nocturnal melatonin secretion (MD GnRH), at least in
nine of 11 pineal organs analyzed (Supplemental Fig. 3A),
TABLE 1. EC50 values of GnRH agonists for IP3
production of COS-7 cells transfected with GnRHR
expression vector
cGnRH-II
( M)
sGnRH
( M)
sbGnRH
(M)
dlGnRHR-II-2b 2.24 ⫻ 10⫺11 1.11 ⫻ 10⫺7 1.61 ⫻ 10⫺6
dlGnRHR-II-1a 1.22 ⫻ 10⫺12 1.06 ⫻ 10⫺12 1.28 ⫻ 10⫺10
IP3 production was measured 6 h after stimulation by GnRHs.
2271
when compared with melatonin released at the first night in absence of
GnRH treatment (MD no GnRH).
Only one pineal of 11 did not exhibit
such change and actually showed higher
concentration of melatonin during the
first night when cultured in the absence
of GnRH (Supplemental Fig. 3A). Results of the second experiment are totally consistent with those obtained in
the first experiment. In this case, pineals
exhibited higher melatonin release
when treated with GnRH-2 (first night)
than in the absence of GnRH-2 (second
night) (Supplemental Fig. 3B).
The mean values of melatonin released at different conditions from the
13 pineal organs analyzed are presented
in Fig. 6A, which shows significantly
higher nocturnal melatonin release in
presence of GnRH (MD GnRH) than in
absence of GnRH treatment (MD no
GnRH). Furthermore, pineals were
clearly rhythmic because they exhibited
lower melatonin secretion during the
day (ML) when compared with the nocturnal values (Fig. 6A).
In vivo effects of GnRH injections
on plasma melatonin levels
The in vivo study also permitted us to
follow the response of individual animals to different conditions in terms
of melatonin production because they
were previously implanted with an electronic tag and could be easily identified.
Increased nocturnal plasma melatonin levels were observed in all animals after GnRH-2 injection (MD GnRH)
in relation to the reference night in the absence of GnRH-2
(MD) (Supplemental Fig. 3C). Vehicle injection (MD vehicle) had no effect and resulted in plasma melatonin levels
similar to those of the reference night (MD) (Supplemental
TABLE 2. EC50 values of GnRH agonists for expression
of CRE-luciferase activity in COS-7 cells transfected with
GnRHR expression vector
dlGnRHR-II-2b
dlGnRHR-II-1a
cGnRH-II
( M)
sGnRH
( M)
sbGnRH
(M)
3.1 ⫻ 10⫺9
6.95 ⫻ 10⫺10
ND
3.02 ⫻ 10⫺10
ND
4.14 ⫻ 10⫺7
Luciferase activity was measured 6 h after stimulation with GnRHs. ND,
Not detected.
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2272
Servili et al.
GnRH2-Pineal Interaction in Sea Bass
FIG. 6. A, Mean values ⫾ SEM of the melatonin released in the
medium from pineals cultured in both in vitro experiments (n ⫽ 13).
ML, Culture medium collected at mid-light period; MD No GnRH,
culture medium collected at night from pineals cultured in the absence
of GnRH treatment; MD GnRH, culture medium collected at night from
pineals exposed to GnRH treatment. B, Mean values ⫾ SEM of the
plasma melatonin levels at the different sampling points and
conditions in the in vivo experiment (n ⫽ 7). MD, Plasma melatonin
concentration determined at midnight in the absence of GnRH
treatment; MD GnRH, plasma melatonin concentration determined at
midnight after GnRH injection; MD VEHICLE, plasma melatonin
concentration determined at midnight after saline solution injection;
ML, plasma melatonin concentration determined at the middle of the
light period. Differences among groups were analyzed by repeatedmeasures ANOVA followed by Student-Newman-Keuls post hoc test.
Different letters indicate statistically significant differences (P ⬍ 0. 05).
Fig. 3C). All animals were rhythmic and exhibited their
lowest plasma melatonin levels during daytime (ML) (Supplemental Fig. 3C). The mean values of plasma melatonin
levels at different conditions are presented in Fig. 6B. Nocturnal plasma melatonin levels were significantly higher in
GnRH-2-injected animals (MD GnRH) than in noninjected control (MD) or in vehicle-injected control (MD
vehicle) animals. Moreover, nocturnal plasma melatonin
levels were significantly higher than diurnal levels (ML)
(Fig. 6B).
Discussion
The present study provides for the first time in a vertebrate
species converging neuroanatomical and physiological evidence showing that GnRH-2 stimulates melatonin release. In addition, these findings were substantiated by the
Endocrinology, May 2010, 151(5):2265–2275
fact that the pineal strongly expressed a receptor with a
high selectivity for GnRH-2 and biopotency upon binding
to this ligand.
First, a tract-tracing study was performed to elucidate
whether the pineal organ interacts directly with the sea
bass GnRH system. We revealed the presence of pinealofugal projections and pinealopetal cells in the dorsal synencephalon of the European sea bass, where GnRH-2 cells
were previously reported (4). In the present study, we also
showed by double staining that GnRH-2 cells were the
source of these synencephalic projections reaching the pineal organ. This is confirmed by the fact that only GnRH-2
fibers were detected in the pineal. In contrast, GnRH-1 or
GnRH-3 fibers, found in the pituitary (4), could not be
observed in the pineal of sea bass, and no GnRH-immunoreactive perikarya were found in this organ. It is also
important to note that GnRH-2 neurons, despite having
extensive projections in the sea bass brain, do not project
to the pituitary (4).
In elasmobranchs, it has been reported that pineal projections end in close topographical proximity to a GnRHimmunoreactive cell population present in this region
(22). Similarly, in the sturgeon, the midbrain tegmentum
containing GnRH-2 cells (23) also receives direct pineal
projections (24). These data, together with the present
results, suggest that the pineal innervation of synencephalic GnRH neurons could be a conserved characteristic
from elasmobranchs to modern teleosts.
The presence of a GnRH innervation in the pineal organ
has been previously reported in other teleosts (25, 26). In
the Indian major carp, pineal GnRH fibers were revealed
using antimammalian LHRH sera, making it difficult to
assess which GnRH form is present in the pineal of this
species (26). In our study, we have used highly specific
antibodies that recognize unambiguously the different
GnRH isoforms (4) and show that GnRH-2, but not
GnRH-1 or GnRH-3, reaches the sea bass pineal organ.
Because GnRH-2 was present in the pineal of sea bass,
we next aimed at investigating whether it could modulate
melatonin release. For this purpose, we performed a set of
experiments in vitro and in vivo. These experiments led to
consistent findings demonstrating that GnRH-2 administration, both in vitro and in vivo, increased the nocturnal
pineal melatonin release and the plasma melatonin levels,
respectively. To our knowledge, the present work constitutes the first evidence of GnRH actions on melatonin
release in fish. In our in vitro and in vivo studies, we followed a longitudinal approach in which every pineal gland
or fish was used as its own control. This approach reduces
the impact of the interindividual variability in basal melatonin levels and responses to treatments. This approach
also permitted identification of those specimens able to
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Endocrinology, May 2010, 151(5):2265–2275
maintain, under the experimental conditions, the rhythmic pattern of melatonin production expected from this
species (18, 27–29). Indeed, it has been reported that some
sea bass specimens under laboratory conditions (27, 28),
as well as some sea bass pineal organs in culture systems
(18), lose their ability to synchronize the melatonin release
to the photoperiodic cycles. Hence, the experimental design used proved very useful to obtain both individual and
group information on the effects of hormonal treatment.
Our results showed that both GnRH-2 and GnRHa administration exerted the same stimulatory effects on pineal
melatonin release. GnRH analogs are known to be highly
potent and to show even higher binding affinities to the
GnRHRs than natural ligands in fish species such as the
winter flounder (30, 31), goldfish (32, 33), catfish (34,
35), stickleback (36), and sea bream (37).
The results obtained in sea bass are consistent with
several findings reported in mammals. Indeed, a stimulatory action of GnRH on pineal melatonin release is observed in pineal glands of female rats (20, 38). The administration of GnRH analogs to women also causes an
increase in nocturnal serum melatonin levels (39). Moreover, GnRH has been reported to influence the protein
synthesis in rat pinealocytes (40). As in sea bass, these
effects in mammals appear to be supported by the presence
of GnRH-immunoreactive elements (41) and GnRHRs
(42) in the pineal gland.
Having shown the presence of GnRH-2 in the pineal
and its effect on nocturnal release of melatonin, the next
step was to identify GnRHRs in the pineal. The sea bass
exhibits five GnRHRs, most of which are still in search of
specific expression sites and functions (5, 8, 9). Only one
of them, dlGnRHR-II-1a, was shown to be strongly expressed in the pituitary (8), where it probably mediates
the response of the gonadotrophs to GnRH stimulation.
This receptor was also shown to be expressed in the
pineal of the sea bass (8), but given the presence of four
other forms (5, 9), we reinvestigated the expression pattern of all GnRHRs in the sea bass pineal organ using
RT-PCR and in situ hybridization. Both techniques provided highly consistent results concerning the GnRHR
types expressed in the pineal. Indeed, we found high expression of dlGnRHR-II-2b and a lower expression of dlGnRHR-II-1a mRNA. The other three receptors were not
detected with either technique. To be able to clarify our
findings, we performed a detailed pharmacological study
of these two GnRHR subtypes.
A number of studies regarding GnRH-mediated signal
transduction have shown that GnRHRs principally activate
phospholipase C via Gq/11 coupling. Phospholipase C in
turn, hydrolyzes phosphatidylinositol 4,5-bisphosphate to
produce IP3 and diacylglycerol. However, recent studies
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2273
based on catfish, bullfrog, and striped bass GnRHRs indicate
that activation of nonmammalian GnRHRs also produces
cAMP via Gs/adenylate cyclase (43– 46), which is the reason
why this pathway was also investigated in this study. The IP3
production in COS-7 cells transfected with dlGnRHR-2b or
dlGnRHR-1a receptors was stimulated by the three sea bass
endogenous ligands: sbGnRH (GnRH-1), cGnRH-II
(GnRH-2), and sGnRH (GnRH-3). This indicates coupling to phospholipase C, but in addition, stimulation of
expression of a CRE-luc reporter gene indicates phosphorylation of cAMP response element-binding protein and
thus implication of adenylate cyclase and protein kinase A.
However, the data of both the IP3 and cAMP assays
showed remarkable differential selectivity of the two receptors for the endogenous ligands. From the EC50 values
(Table 1) calculated as the mean of five independent IP3
assays, it appeared that dlGnRHR-II-2b had a strong preference for cGnRH-II, whereas sGnRH and sbGnRH had
about 5,000 and 70,000 times less biopotency, respectively. In contrast, both cGnRH-II and sGnRH had similar
activities on dlGnRH-R1a, whereas sbGnRH was around
100 times less active than the two other ligands on this
receptor. Using the CRE-luciferase assay, the same selectivity of dlGnRHR-II-2b for cGnRH-II was observed although activation of this pathway required higher doses of
ligand (Table 2). sGnRH had much lower activity on this
pathway, and sbGnRH had none on dlGnRHR-II-2b.
Similarly, CRE-luciferase activity mediated by dlGnRHRII-1a was stimulated equally by cGnRH-II and sGnRH
and less by sbGnRH. Recent data indicate that the presence of the intracellular tail of nonmammalian GnRHRs
is required for activation of the cAMP pathway, because
deletion of the intracellular carboxyl-terminal tail of the
bullfrog GnRHR-1 decreases its ability to induce the
cAMP pathway (47).
Currently, we are able to state that all five sea bass
GnRHRs are functional, and all of them show higher affinity for the ligand GnRH-2 (5, 6) (present work). The
present data clearly showed that receptor dlGnRHR-II-1a
is expressed slightly in the pineal organ but strongly in the
pituitary (present study) (8), representing the only receptor showing some affinity for GnRH-1 and GnRH-3,
which are the hypophysiotropic GnRH forms in sea bass
(4, 5, Lethimonier, C., J.J. Lareyre, O. Kah, unpublished
data). It has been suggested that this GnRHR (dlGnRHRII-1a) is evolving to enhance its affinity for GnRH-1 and
GnRH-3 to better mediate their effects at the pituitary
level (6, 48). On the other hand, we do not know whether
the lack of response of the receptor preferentially expressed in the pineal gland in the presence of GnRH-1 is
due to the absence of receptor binding or an inability of
ligand-receptor complex to transmit the signal to the sec-
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2274
Servili et al.
GnRH2-Pineal Interaction in Sea Bass
ond messenger signaling pathway. Our results indicate
that the dlGnRHR-II-2b subtype, which is the major form
expressed in the pineal gland, is the receptor with the highest biopotency for the non-hypophysiotropic ligand
GnRH-2 (4) (Lethimonier, C., J.J. Lareyre, O. Kah, unpublished data), the only GnRH peptide found in the pineal of sea bass (present study).
In conclusion, our findings provide the first evidence
for direct links between the synencephalic GnRH-2 cells
and the pineal, thus permitting us to assign to GnRH-2 a
functional role in modulating the activity of this photoreceptive organ in sea bass. Moreover, the presence of
pinealofugal terminal fields in the dorsal synencephalon
suggests that both structures appear to be bidirectionally
connected. Interestingly, in all lower vertebrates studied so
far, in which pineal organ exhibits photoreceptor cells,
this synencephalic/tegmental GnRH-2 system appears
highly conserved. On the other hand, several mammalian
species, whose pineal has lost photoreceptor properties,
do not present a GnRH-2 functional system (6, 48). This
fact could represent additional evidence reinforcing the
role of GnRH-2 cells as an intermediary system mediating
the integration/modulation of photoreceptor information
perceived by the pineal organ. In this manner, when the
pineal photoreception is lost and its functions become
dependent of multisynaptic pathways that convey light
information from the retina and involve the suprachiasmatic nucleus, the selective pressure on this neuroendocrine system could be reduced, permitting it to disappear, at least in some species. The evaluation of this
evolutionary consideration will require further research
in fish and other vertebrate groups exhibiting different
circadian organizations.
Acknowledgments
We thank Rosa Vázquez Gómez and all staff from the Planta de
Cultivos Marinos (University of Cádiz) for the maintaining of
animals. We thank Abdeslam El M’Rabet for his help in dissecting sea bass pineal organs.
Address all correspondence and requests for reprints to: José
A. Muñoz-Cueto, Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, E-11510
Puerto Real, Spain. E-mail: [email protected]; or Olivier
Kah, Neurogenesis and Estrogens, Unité Mixte de Recherche
Centre National de la Recherche Scientifique 6026, Institut Fédératif de Recherche 140, Campus de Beaulieu, 35042 Rennes
cedex, France. E-mail: [email protected].
The research leading to these results has received funding
from the Spanish Ministry of Science and Innovation (MICINN)
(AGL2001-07984-C02-02/ACU) to J.A.M.-C., a European
project (PUBERTIMING) to O.K. and the European Commu-
Endocrinology, May 2010, 151(5):2265–2275
nity’s Seventh Framework Program (FP7/2007-2013) under
Grant Agreement 222719 LIFECYCLE. A.S. was supported by
a predoctoral fellow from the Junta de Andalucía.
Disclosure Summary: The authors have nothing to disclose.
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