Perspectives on fish gonadotropins and their receptors.

Teleosts lack a hypophyseal portal system and hence neurohormones are carried by nerve fibers from the preoptic region to the pituitary. The various cell types in the teleost pituitary are organized in discrete domains. Fish possess two gonadotropins (GtH) similar to FSH and LH in other vertebrates; they are heterodimeric hormones that consist of a common alpha subunit non-covalently associated with a hormone-specific beta subunit. In recent years the availability of molecular cloning techniques allowed the isolation of the genes coding for the GtH subunits in 56 fish species representing at least 14 teleost orders. Advanced molecular engineering provides the technology to produce recombinant GtHs from isolated cDNAs. Various expression systems have been used for the production of recombinant proteins. Recombinant fish GtHs were produced for carp, seabream, channel and African catfish, goldfish, eel, tilapia, zebrafish, Manchurian trout and Orange-spotted grouper. The hypothalamus in fishes exerts its regulation on the release of the GtHs via several neurohormones such as GnRH, dopamine, GABA, PACAP, IGF-I, norepinephrine, NPY, kisspeptin, leptin and ghrelin. In addition, gonadal steroids and peptides exert their effects on the gonadotropins either directly or via the hypothalamus. All these are discussed in detail in this review. In mammals, the biological activities of FSH and LH are directed to different gonadal target cells through the cell-specific expression of the FSH receptor (FSHR) and LH receptor (LHR), respectively, and the interaction between each gonadotropin-receptor couple is highly selective. In contrast, the bioactivity of fish gonadotropins seems to be less specific as a result of promiscuous hormone-receptor interactions, while FSHR expression in Leydig cells explains the strong steroidogenic activity of FSH in certain fish species.

Comparative gene expression of gonadotropins (FSH and LH) and peptide levels of gonadotropin-releasing hormones (GnRHs) in the pituitary of wild and cultured Senegalese sole (Solea senegalensis) broodstocks.

The Senegalese sole (Solea senegalensis) is a valuable flatfish for aquaculture, but it presents important reproductive problems in captivity. Spawning is achieved by wild-caught breeders but cultured broodstocks fail to spawn spontaneously and, when they do, eggs are unfertilized. To gain knowledge on the physiological basis underlying this reproductive dysfunction, this study aimed at analyzing comparative hormone levels between wild and cultured broodstocks at the spawning season. The Senegalese sole gonadotropin (GTH) subunits, FSHbeta, LHbeta and GPalpha, were cloned and qualitative (in situ hybridization) and quantitative (real-time PCR) assays developed to analyze pituitary GTH gene expression. In females, FSHbeta and GPalpha mRNA levels were higher in wild than in cultured broodstocks, whereas in males all three subunits were highest in cultured. By ELISA, three GnRH forms were detected in the pituitary, displaying a relative abundance of GnRH2>GnRH1>GnRH3. All GnRHs were slightly more abundant in wild than cultured females, whereas no differences were observed in males. Plasma levels of vitellogenin and sex steroids were also analyzed. Results showed endocrine differences between wild and cultured broodstocks at the spawning period, which could be related to the endocrine failure of the reproductive axis in cultured breeders.

Modulation of pituitary dopamine D1 or D2 receptors and secretion of follicle stimulating hormone and luteinizing hormone during the annual reproductive cycle of female rainbow trout.

The two gonadotrophins follicle stimulating hormone (FSH) and luteinizing hormone (LH) have distinct temporal expression and release profiles in fish, but little is known regarding their neuroendocrine control, especially for FSH. The present experiments were performed on previtellogenic, mature and preovulatory female trout. The catecholamine synthesis inhibitor, alpha-methyl-p-tyrosine, increased plasma LH and FSH concentrations of mature fish. The dopamine agonist apomorphine decreased and the dopamine antagonist domperidone increased plasma LH concentration of preovulatory fish and delayed ovulation, but did not modify plasma FSH concentration. The dopamine D2 agonist bromocryptine inhibited LH release in cultured gonadotrophs from mature and preovulatory fish, but not from previtellogenic fish. Bromocryptine also significantly inhibited basal and salmon gonadotrophin releasing-hormone (sGnRH)-induced FSH release from cultured gonadotrophs of mature fish, but not of preovulatory fish, and increased FSH release from gonadotrophs of previtellogenic fish. The dopamine D1 agonist SKF 38393 had no observed effect on the release of FSH and LH, at any reproductive stage studied. The D1 agonist SKF 38393, the D2 agonist bromocriptine and sGnRH had no observed effects on cell contents of FSH and LH. Taken together, these data suggest that, at the level of the pituitary, dopamine inhibits LH release as vitellogenesis proceeds, via activation of dopamine D2 receptors. We demonstrate for the first time in fish a control of FSH release (a dopamine control), especially in mature fish which have low circulating concentrations of FSH.

Distribution of glutamic acid decarboxylase mRNA in the forebrain of the rainbow trout as studied by in situ hybridization.

By using degenerate primers designed from glutamate decarboxylase (GAD) sequences of mammals, Xenopus and Drosophila, a 270-bp cDNA fragment was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) from cerebellum total RNA of rainbow trout. This partial cDNA shows 90% identity with mammalian GAD 65 and presents the Asn-Pro-His-Lys (NPHK) sequence corresponding to the pyridoxal-binding region of porcine DOPA decarboxylase or mammalian GAD. The distribution of GAD 65 mRNA-expressing neurons in the forebrain of the trout was studied by in situ hybridization using either digoxigenin- or 35S-labeled probes. The results demonstrate that gamma-amino butyric acid (GABA) neurons are widely distributed throughout the forebrain, with a high density in the periventricular regions. In this study, we report their precise distribution in the telencephalon and diencephalon. GAD mRNA-expressing cells were particularly abundant in the preoptic region and the mediobasal hypothalamus, two major neuroendocrine and estrogen-sensitive regions in fish. The presence of GAD mRNA-expressing neurons was observed in visually related structures such as the suprachiasmatic nucleus, the pretectal region, and the thalamus. Immunohistochemistry with antibodies directed against mouse GAD failed to demonstrate the presence of immunoreactive cell bodies, but showed a very high concentration of GAD-immunoreactive fibers in many brain regions, notably in the preoptic area, hypothalamus, and neurohypophyseal digitations of the pituitary, in particular in the proximal pars distalis. These results indicate that GABA neurons are ideally placed to modulate neuroendocrine activities at the hypothalamic and pituitary levels and to participate in the processing of sensorial information.

Involvement of gamma-aminobutyric acid in the control of GTH-1 and GTH-2 secretion in male and female rainbow trout.

The potential role of the neurotransmitter gamma-aminobutyric acid (GABA) in the control of the secretion of the two pituitary fish gonadotropins (GTH-1 and GTH-2) was investigated in male and female rainbow trout (Oncorhynchus mykiss). The presence of glutamate decarboxylase-positive fibers in the neurohypophyseal digitations adjacent to the gonadotropic cells was demonstrated by means of double immunohistochemistry, providing a morphofunctional support for potential GABA-gonadotropin interactions in both sexes. In spermiating males, in vivo treatment with GABA did not affect basal gonadotropin release, but stimulated GTH-1 release when coadministered with a gonadotropin-releasing hormone analogue (GnRHa), and potentiated GnRHa-stimulated GTH-2 release. In vitro, using dispersed pituitary cells, GABA stimulated basal GTH-1 and GTH-2 secretion, in a dose-dependent manner, and potentiated salmon GnRH effect on both hormones. In mature females, GABA induced in vivo a strong elevation of plasma GTH-2 levels after 2- 6 h of injection, but had no effect in vitro. GABA treatment in vivo was also stimulatory in recrudescent females, slightly increasing plasma GTH-2 levels in both saline- and GnRHa-treated fish (GnRHa alone has no effect at this stage). Immature fish were unresponsive to GABA/GnRHa treatments but, after steroid implantation [testosterone (T) or estradiol] for 13 days, injection of GABA stimulated GTH-2 release in vivo (also GTH-1 slightly in T-implanted fish). In conclusion, GABA has an overall stimulatory action on GTH-1 and GTH-2 secretion in rainbow trout, which depends on the sex and the reproductive stage of the fish. The stimulatory action of GABA might be exerted, at least in part, directly onto the gonadotropes, as it stimulates basal and GnRH-induced GTH-1 and GTH-2 secretion from dispersed pituitary cells.

Release of pituitary gonadotrophins GtH I and GtH II in the rainbow trout (Oncorhynchus mykiss): modulation by estradiol and catecholamines.

The present study focused on the role of catecholaminergic neurons and estrogens on the release of gonadotropins I and II in immature and early vitellogenic female rainbow trout. The ovariectomy-induced increase of GtH I blood levels (from about 10 to 15 ng/ml) was prevented in vitellogenic fish by E2 supplementation. E2 implantation of immature fish decreased blood GtH I levels (from about 6 to 1 ng/ml). Blood levels of GtH II were low (about 0.5 ng/ml) and not altered by ovariectomy and E2 treatment. These data demonstrate that estrogens exert a negative feedback on the release of GtH I in trout. A treatment with alpha-methyl-p-tyrosine (MPT), an inhibitor of catecholamine synthesis, increased blood GtH II levels of sham-operated vitellogenic fish and ovariectomized fish implanted with E2, but had no effects in ovariectomized fish. MPT did not modify blood GtH I levels in any experimental group. A treatment of E2-implanted immature or vitellogenic fish with the dopamine antagonist pimozide also increased blood GtH II levels, but did not significantly change blood GtH I levels. These data demonstrate that release of GtH II, but not of GtH I, depends on an E2-activated DA inhibitory tone.

Purification of gonadotropin II from a teleost fish, the hybrid striped bass, and development of a specific enzyme-linked immunosorbent assay.

A highly purified gonadotropin II (GtH II), referred to as striped bass GtH II (stbGtH II), and its alpha and beta subunits were prepared from pituitaries of sexually mature hybrid striped bass. Pituitary glycoproteins were extracted with ethanol and intact stbGtH II purified by gel-filtration chromatography on Sephadex G-100, ion-exchange chromatography (IEC) on DE-52, and fast-performance liquid chromatography (FPLC) on Superdex 75. The presence of GtHs during the purification procedure was monitored by characteristic elution on reversed-phase high-performance liquid chromatography (rpHPLC) and in vitro steroidogenic activity. The stbGtH II alpha and beta subunits were purified from the pituitary ethanol extract by gel-filtration, IEC, and rpHPLC, and their identities assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), rpHPLC, and N-terminal amino acid sequencing. Molecular weights of intact stbGtH II and its alpha and beta subunits, determined by SDS-PAGE, were 34.5, 14.8, and 20.4 kDa, respectively. The stbGtH II beta subunit was used to produce specific antibodies, and a competitive enzyme-linked immunosorbent assay was developed using intact stbGtH II for the standard curve. The sensitivity of the assay was 156 pg/ml (15.6 pg/well) and the intra- and interassay coefficients of variation (at 50% binding) were 7.7% (n = 16) and 8.7% (n = 10), respectively. Physiological validation of the assay was performed by measuring changes of plasma GtH II levels in mature striped bass females, after injection of GnRHa ([d-Ala6,Pro9-NEt]-mGnRH, 100 microg/kg BW). A maximum surge of GtH II in plasma was observed at 12 hr postinjection (22.5 +/- 3. 01 ng/ml), whereas GtH II levels in control fish (around 4 ng/ml) remained unchanged. Displacement curves obtained with serial dilutions of plasma and pituitaries from a number of perciform species were parallel to the standard curve, indicating that this assay can be used for GtH II measurements in a variety of fish species.

Identification of vitellogenin receptors in the ovary of a teleost fish, the Mediterranean sea bass (Dicentrarchus labrax).

The characterization of vitellogenin (VTG) receptors in ovarian membranes from vitellogenic female sea bass (Dicentrarchus labrax) is described. Incubation of membrane proteins with radiolabeled VTG (125I-VTG) after SDS-electrophoresis showed specific binding of 125I-VTG to a protein band of 100 kDa. Filter binding assays showed that binding of 125I-VTG to membrane receptors was saturable with increasing amounts of 125I-VTG. Scatchard analysis of the saturation data revealed a single class of binding sites with an apparent KD of 1.04 x 10(-8) M. The specificity of the VTG receptors was tested in competition assays; binding of 125I-VTG to ovarian membranes was completely abolished with an excess of purified sea bass VTG (cold VTG, VTG degree) or plasma from estradiol (E2)-treated fish, while the addition of control male plasma (without VTG) caused negligible effect.