Salmon GnRH Research Articles

Three distinct types of GnRH receptor characterized in the bullfrog

It has been proposed recently that two types of GnRH receptors (GnRHR) exist in a particular species. Here we present data demonstrating that at least three types of GnRHR are expressed in a single diploid species, the bullfrog. Three different cDNAs, encoding distinct types of bullfrog GnRHR (bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3), were isolated from pituitary and hindbrain of the bullfrog. BfGnRHR-1 mRNA was expressed predominantly in pituitary, whereas bfGnRHR-2 and -3 mRNAs were expressed in brain. The bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3 proteins have an amino acid identity of approximately 30% to approximately 35% with mammalian GnRHRs and approximately 40% to approximately 50% with nonmammalian GnRHRs. Interestingly, bfGnRHR-2 has an 85% amino acid homology with Xenopus GnRHR. Less than 53% amino acid identity was observed among the three bfGnRHRs. All isolated cDNAs encode functional receptors because their transient expression in COS-7 cells resulted in a ligand-dependent increase in inositol phosphate production. Notably, all three receptors exhibited a differential ligand selectivity. For all receptors, cGnRH-II has a higher potency than mGnRH. In addition, salmon GnRH also has a strikingly high potency to stimulate all three receptors. In conclusion, we demonstrated the presence of three GnRHRs in the bullfrog. Their expression in pituitary and brain suggests that bfGnRHRs play an important role in the regulation of reproductive functions in the bullfrog.

Pituitary Levels of Three Forms of GnRH in the Male European Sea Bass (Dicentrarchus labrax, L.) during Sex Differentiation and First Spawning Season

In the present study, levels of three GnRH forms [seabream GnRH (sbGnRH), chicken GnRH-II (cGnRH-II), and salmon GnRH (sGnRH)] were analyzed in the pituitary of male sea bass during sex differentiation and the first spawning season. Plasma levels of gonadotropin (GTH-2), testosterone (T), and 11-ketotestosterone (11-KT) were determined during the same periods. All GnRH forms were present in the pituitary. sbGnRH levels were 9-fold higher than cGnRH-II and 17-fold higher than sGnRH levels. The highest GnRHs levels were detected in November 1995, when fish were 9 months old and when the gonads started to differentiate. Levels of the three forms decreased and remained low during the first spawning season, with the exception of sbGnRH, which showed a significant increase in November 1996. Plasma GTH-2 levels were lowest in November 1995, later increasing 2.5 times during the next months. During the first spawning season, plasma GTH-2 levels peaked in December 1996, 1 month after the peak of sbGnRH. During sex differentiation, plasma T levels were high in November 1995 but decreased over the next months, while levels of 11-KT remained low and unchanged. During the first spawning season, both steroids peaked in January 1997. These results suggest a possible role for all three GnRH forms in achieving gonadal differentiation, while sbGnRH may be the most relevant form in the regulation of the first spawning season in male sea bass. Moreover, GTH-2 and 11-KT may play important roles in gonadal maturation, since plasma GTH-2 and 11-KT levels were high throughout the period of spermiation.

Complementary deoxyribonucleic acid cloning, gene expression, and ligand selectivity of a novel gonadotropin-releasing hormone receptor expressed in the pituitary and midbrain of Xenopus laevis.

We have cloned the full-length complementary DNA (cDNA) for a GnRH receptor from Xenopus laevis pituitary cDNA and determined its gene structure. The cDNA encodes a 368-amino acid protein that has a 46% amino acid identity to the human GnRH receptor. The X laevis GnRH receptor has all of the amino acids identified in the mammalian GnRH receptors as sites of interaction with the GnRH ligand. However, this receptor cDNA shares the same distinguishing structural features of the GnRH receptor that have been characterized from other nonmammalian vertebrates. These include the pair of aspartate residues in the transmembrane domains II and VII compared with the aspartate/asparagine arrangement in mammalian receptors, the amino acid PEY motif in extracellular loop III (SEP in mammals), and the presence of a carboxyl-terminal tail. Previous studies have reported that mammalian GnRH was equipotent to other naturally occurring GnRH subtypes in stimulating LH release from the amphibian pituitary. However, in this study we show that the X. laevis GnRH receptor has ligand selectivity for the naturally occurring GnRHs similar to other nonmammalian GnRH receptors. The order of potency of the GnRHs in stimulating inositol phosphate production in COS-1 cells transiently transfected with the X. laevis GnRH receptor cDNA was chicken GnRH II>salmon GnRH>mammalian GnRH. Transcripts of this GnRH receptor are expressed in the pituitary and midbrain of X. laevis.

Thyroid hormone and estrogen regulate brain region-specific messenger ribonucleic acids encoding three gonadotropin-releasing hormone genes in sexually immature male fish, Oreochromis niloticus.

The present study was undertaken to determine whether T3, estrogen, and 11-ketotestosterone could alter a specific population of GnRH-containing neurons, as indicated by a change in messenger RNA (mRNA) levels in sexually immature male tilapia, Oreochromis niloticus. Two weeks after castration, fish were assigned to four treatment groups. One group served as the control (sesame oil); a single ip injection of (T3; 5 microg/g), estradiol benzoate (EB; 5 microg/g), or 11-ketotestosterone (KT; 5 microg/g) was administered to the remaining three groups. Twenty-four hours after the injection, brains were collected and processed for in situ hybridization histochemistry using 35S-labeled 30-mer antisense oligonucleotide probes complementary to the GnRH-coding region of chicken II, salmon, and seabream GnRH. Computerized image analysis was performed to quantify mRNA concentrations, neuronal numbers, and neuronal size of the terminal nerve-nucleus olfactoretinalis, preoptic, and midbrain GnRH neurons. KT had no effect on any of the above neuronal parameters examined for salmon or seabream GnRH. Neither T3, EB, nor KT was effective to induce changes in midbrain chicken GnRH II mRNA concentrations, neuronal numbers, and neuronal size, indicating that an as yet unknown regulatory mechanism may operate midbrain GnRH neurons. T3 specifically suppressed the concentration of terminal nerve salmon GnRH mRNA, and EB significantly increased preoptic seabream GnRH neuronal numbers. These results are consistent with the hypothesis that thyroid hormone, by suppressing terminal nerve GnRH expression, promotes inhibition of sexual maturation. Furthermore, the failure of KT, a nonaromatizable androgen, to influence preoptic GnRH neurons emphasizes that an estrogenic pathway, at the onset of sexual maturation, is responsible for the recruitment of additional preoptic GnRH neurons that are fundamental to reproduction and behavior.

Differential splicing of three gonadotropin-releasing hormone transcripts in the ovary of seabream (Sparus aurata).

Previous studies demonstrated the presence of high-affinity GnRH binding sites and compounds with GnRH-like activity in the ovary of seabream, Sparus aurata, providing evidence for the role of GnRH as a paracrine/autocrine regulator of ovarian function in this species. In the present study, the expression of three forms of GnRH (salmon, chicken-II, and seabream) genes in this marine teleost species was demonstrated for the first time. Moreover, there is evidence for differential splicing and intronic expression of cGnRH-II and sbGnRH. Treatment of seabream follicle-enclosed oocytes with salmon GnRH stimulated reinitiation of oocyte meiosis, whereas chicken GnRH-II treatment was without effect. Novel information was also provided about organization of cGnRH-II and seabream GnRH transcripts, confirming that GnRH gene organization is maintained through evolution, despite changes in the size and sequence of exons and introns.

Gonadotropin-Releasing Hormone Stimulates Thyroid Activity in a Freshwater Murrel, Channa gachua (Ham.), and Carps, Catla catla (Ham.) and Cirrhinus mrigala (Ham.)

Injections of mammalian GnRH (mGnRH), salmon GnRH (sGnRH), and a homologous murrel, Channa punctatus, GnRH (cGnRH) to a murrel, Channa gachua, and a carp, Catla catla, at a dose of 1 μg/250 g body wt significantly increased plasma thyroxine (T4) levels above control. Piscine GnRHs (sGnRH and cGnRH) had significantly greater stimulatory effects compared with mGnRH. To observe whether this stimulatory effect by GnRHs is direct or indirect, thyroid follicles were isolated from hypobranchial muscles of freshwater murrel, C. gachua and incubated (1×106 follicles/well) in vitro at 30°C for 2 h without hormone and for 3 h with hormones. Addition of these three GnRHs separately at a concentration of 1 μg/well stimulated T4 secretion; sGnRH and cGnRH caused greater secretion of T4 into the medium compared with mGnRH. Specificity of GnRH action in vitro was assessed by using anti-GnRH antibody which significantly (P<0.01) inhibited GnRH-augmented T4 secretion. To gain further insight, 125I uptake by thyroid follicles and formation of [125I]T4 from this radioiodine pool was monitored in the presence or absence of sGnRH. sGnRH greatly augmented 125I uptake by the follicles which resulted a fourfold increase in [125I]T4 formation out of this pool of 125I compared with the control. The results indicate GnRH stimulation of thyroid hormone formation and release in these teleosts suggesting a possible different mode of regulation of thyroid hormone secretion in teleosts.

Primary structure and function of three gonadotropin-releasing hormones, including a novel form, from an ancient teleost, herring.

The evolution of GnRH and the role of multiple forms within the brain are examined. Three forms of GnRH were purified from the brain of Pacific herring (Clupea harengus pallasi) and characterized using Edman degradation and mass spectrometry. Two forms correspond with the known structures of chicken GnRH-II and salmon GnRH that are found in many vertebrate species. The third form, designated herring GnRH (hrGnRH), has a primary structure of pGlu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly-NH2. This novel peptide is a potent stimulator of gonadotropin II and GH release from dispersed fish pituitary cells. The content of hrGnRH in the pituitary was 8-fold that of salmon GnRH and 43-fold that of chicken GnRH-II, which provides supporting evidence that hrGnRH is involved in the release of gonadotropin. Herring is the most phylogenetically ancient animal in which three forms of GnRH have been isolated and sequenced. Our evidence suggests that the existence of three GnRHs in the brain of one species 1) is an ancestral condition for teleosts, 2) has the potential for separate regulation of the distinct GnRHs, and 3) may be an evolutionary advantage for refined control of reproduction in different environments.

Differential expression of three different prepro-GnRH (gonadotrophin-releasing hormone) messengers in the brain of the european sea bass (Dicentrarchus labrax)

The expression sites of three prepro-gonadotrophin-releasing hormones (GnRHs), corresponding to seabream GnRH (sbGnRH: Ser(8)-mGnRH, mammalian GnRH), salmon GnRH (sGnRH: Trp(7)Leu(8)-mGnRH), and chicken GnRH-II (cGnRH-II: His(5)Trp(7)Tyr(8)-mGnRH) forms were studied in the brain of a perciform fish, the European sea bass (Dicentrarchus labrax) by means of in situ hybridization. The riboprobes used in this study correspond to the three GnRH-associated peptide (GAP)-coding regions of the prepro-GnRH cDNAs cloned from the same species (salmon GAP: sGAP; seabream GAP: sbGAP; chicken GAP-II: cIIGAP), which show little oligonucleotide sequence identity (sGAP versus sbGAP: 42%; cIIGAP versus sbGAP: 36%; sGAP versus cIIGAP: 41%). Adjacent paraffin sections (6 mm) throughout the entire brain were treated in parallel with each of the three anti-sense probes and the corresponding sense probes, demonstrating the high specificity of the hybridization signal. The results showed that both sGAP and sbGAP mRNAs had a broader expression in the olfactory bulbs, ventral telencephalon, and preoptic region, whereas cIIGAP mRNA expression was confined to large cells of the nucleus of the medial longitudinal fascicle. In the olfactory bulbs, both the signal intensity and the number of positive cells were higher with the sGAP probe, whereas sbGAP mRNA-expressing cells were more numerous and intensely stained in the preoptic region. Additional isolated sbGAP-positive cells were detected in the ventrolateral hypothalamus. These results demonstrate a clear overlapping of sGAP- and sbGAP-expressing cells in the forebrain of the European sea bass, in contrast to previous reports in other perciforms showing a clear segregation of these two cell populations.

The gonadotropin-releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform

The mammalian gonadotropin-releasing hormone (GnRH-I), which regulates reproduction, was the first isoform of GnRH that was identified in mammals. Recently, we and others have demonstrated the existence of a second isoform of GnRH in the brain of mammals. The presence of a third isoform of GnRH, GnRH-III, in the brain of mammals is reported herein. GnRH-III, extracted from the brain of bovine and human, was purified by high performance liquid chromatography, using two distinct elution programs. In both, GnRH-III was eluted at the same positions as synthetic salmon GnRH, as demonstrated by radioimmunoassay. The luteinizing hormone-releasing activity of purified GnRH-III, using dispersed rat pituitary cells, was found to be similar to that of synthetic salmon GnRH. The total amount of GnRH-III, determined by radioimmunoassay, in the hypothalamus and midbrain of humans and calves is similar to that of GnRH-I. Immunohistochemical studies demonstrated GnRH-III-containing neurons in the hypothalamus and midbrain of human and GnRH-III fibers in the median eminence of rats. The distribution of GnRH-III in the brain suggests that in addition to a putative function as a neurohormone at the hypothalamic–pituitary axis, GnRH-III may have other functions. Our present results suggest that multiple isoforms of GnRH are present in the brain of mammals, and further studies are required in order to elucidate their biological functions.

Two Endogenous Gonadotropin-Releasing Hormones Generate Dissimilar Ca2+ Signals in Identified Goldfish Gonadotropes

Ca2+ signals are involved in the signal transduction of neuroendocrine regulators. In goldfish, two endogenous gonadotropin-releasing hormones, salmon (s)GnRH and chicken (c)GnRH-II, control maturational gonadotropin secretion. Although considerable evidence suggests that sGnRH and cGnRH-II exert their activity on goldfish gonadotropes through a single population of receptors, differences in signal transduction mechanisms between these peptides have been demonstrated. We used ratiometric Fura-2 Ca2+ imaging of single morphologically identified gonadotropes to quantitatively compare the Ca2+ signals evoked by sGnRH and cGnRH-II. The amplitude and the rate of rise of sGnRH- and cGnRH-II-evoked Ca2+ signals increased with concentration. At maximal concentrations, Ca2+ signals generated by cGnRH-II rose significantly faster than those elicited by sGnRH, while other parameters such as the maximum amplitude, average Ca2+ increase, and latency did not differ between the two peptides. Ca2+ signals evoked by sGnRH or cGnRH-II were often spatially restricted to one part of the cell over the duration of the response. We provide a comprehensive account of the spatial and temporal aspects, including calculated kinetics, of GnRH-evoked Ca2+ signals in single identified gonadotropes. This is the first report of quantified differences in Ca2+ signals generated by two endogenous GnRH neuropeptides, which may act through the same receptor population in this cell type.

Induction of apoptosis by salmon GnRH and chicken GnRH-II in the goldfish ovarian follicles

Induction of apoptosis by salmon GnRH and chicken GnRH-II in the goldfish ovarian follicles

Effects of Photoperiod on Salmon GnRH mRNA Levels in Brain of Castrated Underyearling Precocious Male Masu Salmon

Previous studies have suggested that activation of salmon gonadotropin-releasing hormone (sGnRH)-producing neurons is induced by the combined effects of photoperiod and steroid hormones in underyearling males of the masu salmon, Oncorhynchus masou. The present study further assesses the effects of photoperiod and steroid hormones on sGnRH synthetic activity and examines the changes in sGnRH mRNA levels in the brains of castrated underyearling precocious male masu salmon by manipulating the photoperiod for 60 days from August through October. In castrated males in which plasma testosterone levels decreased to low levels, sGnRH mRNA levels in the preoptic area (POA) increased under a short photoperiod (8L-16D), whereas they remained at low levels under a long photoperiod (16L-8D) for a 2-month duration. In sham-operated males, sGnRH mRNA levels in the ventral telencephalon and those in the POA increased in October with testicular maturation even under a long photoperiod with a delay of 1 month compared with the short photoperiod group. These results suggest that preoptic sGnRH-producing neurons receive short photoperiodic signals and that either short photoperiod or steroid hormone secretion is required for the activation of sGnRH synthesis in underyearling precocious male masu salmon.

Effects of sustained administration of testosterone in pre-pubertal sea bass ( Dicentrarchus labrax L)

This study investigated the effects of testosterone (T) on the levels of immunoassayable salmon GnRH (sGnRH) in the brain and the pituitary in relation to blood plasma steroid levels and gonadal activity in juvenile male sea bass ( Dicentrarchus labrax L, <1-year of age). Gonad-intact male fishes were intraperitoneally implanted for 76 days with Silastic pellets containing no steroid (control) or testosterone (T; 100 μg/g body weight). The serum T response, the sGnRH content in whole brain and pituitary and the testicular development at 0, 15, 30 and 76 days following T implantation were assessed. Treatment with T did not significantly affect somatic growth or weight of gonads. However, gonad maturation was histologically more advanced in T-treated fishes compared to control fish. Histological examination showed that 15 days after implantation, 100% of steroid-treated fishes exhibited differentiated gonads, compared to only 50% of the controls. In addition, discrete spermiation was observed in 10% of the steroid-treated fishes 45 days after implantation while spermiation did not occur in the controls. Steroid treatment did not affect plasma oestradiol (E2) levels and all animals exhibited similar basal levels (<0.6 ng E2/ml). Fifteen days after T-implantation, the sGnRH content of brain and pituitary and the plasma levels of T were elevated. Over the following weeks a progressive decrease in all these parameters was observed. A second implant of T provoked a new and significant ( p<0.05) rise of both plasma T and whole brain sGnRH content, indicating that the GnRH system was still responsive to the additional steroid administration. These results provide evidence of a positive feedback of steroids at the brain and/or pituitary level, indicating a possible participation of sex steroids in the acceleration of gonadal differentiation and stimulation of spermatogenesis in pre-pubertal sea bass.

Involvement of γ-Aminobutyric Acid in the Control of GTH-1 and GTH-2 Secretion in Male and Female Rainbow Trout

The potential role of the neurotransmitter γ-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.

In VitroAction of Testosterone in Potentiating Gonadotropin-Releasing Hormone-Stimulated Gonadotropin-II Secretion in Goldfish Pituitary Cells: Involvement of Protein Kinase C, Calcium, and Testosterone Metabolites

Overnight preincubation of goldfish pituitary cell culture with testosterone (T) enhanced the gonadotropin (GTH)-II responses to GTH-releasing hormone (GnRH). In this study, the involvement of GnRH signal transduction components and the requirement for T metabolism in mediating this direct, pituitary cell action of T were examined using cultured pituitary cells from both male and female goldfish. Each sets of related experiments were done in at least two different stages of the gonadal reproductive cycle and similar effects were observed. Overnight treatment with 10 nM T increased GTH-II responses to maximal stimulatory doses (100 nM) of either salmon (s)GnRH or chicken (c)GnRH-II, but not the total cellular GTH-II contents measured prior to and after a 2-h GnRH challenge. T increased the efficacy and sensitivity of the GTH-II response to stimulation by a protein kinase C (PKC) activator, tetradecanoyl phorbol acetate (TPA) without altering the ED50of the dose-response curve. In T-treated cells, addition of a PKC inhibitor attenuated GTH-II responses to 100 nM doses of sGnRH, cGnRH-II, or TPA. T did not affect the GTH-II release stimulated by high concentrations of the Ca2+ionophore ionomycin (100 μM) and the voltage-sensitive Ca2+channel (VSCC) agonist Bay K 8644 (10 μM); similarly, the sensitivity of the GTH-II response to ionomycin and Bay K 8644 was also unaltered. Taken together, these data suggest that T potentiates GnRH-stimulated GTH-II release by enhancing the effectiveness of PKC-dependent pathways, but not by increasing the total Ca2+-sensitive GTH-II pool, the sensitivity of the release response to increases in intracellular Ca2+, or the amount of available GTH-II. However, the VSCC agonist nifedipine reduced sGnRH- and cGnRH-II-elicited GTH-II release in T-treated as well as in non-T-treated cells, suggesting that VSCC dependence is still present in the GnRH-induced response following exposure to T. Since total cGnRH-II binding to pituitary cells was not increased by T, increases in GnRH receptor capacity are unlikely following T treatment. The ability of T to increase GnRH-stimulated GTH-II secretion was not mimicked by 11-ketotestosterone or dihydrotestosterone, but was abolished by coincubation with an aromatase inhibitor. When viewed together, these observations suggest that aromatization of T may be required for the pituitary action of T on GnRH-induced GTH-II release.

Two differing salmon GnRH precursor mRNAs are co-expressed in the brain of sockeye salmon.

The localization of two salmon-type gonadotropin-releasing hormone (sGnRH) precursors, pro-sGnRH-I (short type) and pro-sGnRH-II (long type), was investigated by using in situ hybridization techniques in the brain of the landlocked sockeye salmon, Oncorhynchus nerka. We used 30-mer oligonucleotide probes complementary to pro-sGnRH-I and pro-sGnRH-II cDNA. No significant differences were observed in the localization of sGnRH neurons expressing pro-sGnRH-I and pro-sGnRH-II mRNAs; both were expressed in the olfactory nerve, the olfactory bulbs, the regions between the olfactory bulb and telencephalon, the ventral telencephalon, the preoptic area, and the hypothalamus. Almost all sGnRH neurons examined co-expressed both precursors. The expression of two sGnRH precursors in the same neuron and the wide distribution of such neurons in the brain suggest that there are no functional differences between the two precursors.

Preovulatory Changes in the Levels of Three Gonadotropin-Releasing Hormone- Encoding Messenger Ribonucleic Acids (mRNAs), Gonadotropin I-Subunit mRNAs, Plasma Gonadotropin, and Steroids in the Female Gilthead Seabream, Sparus aurata 1

Gilthead seabream females undergo daily cycles of final oocyte maturation (FOM), ovulation, and spawning throughout their spawning season. FOM consists of lipid droplet and yolk granule coalescence, germinal vesicle (GV) migration, and GV breakdown. Plasma maturational gonadotropin (GtH-II) levels fluctuate throughout the day, reaching a peak at 8 h before spawning, when the GV is at the periphery of the oocyte. The preovulatory GtH-II surge is accompanied by an increase in the plasma levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one and estradiol, while testosterone and 17alpha,20beta,21-trihydroxy-4-pregnen-3-one levels remain unchanged. Concurrent with the preovulatory GtH-II surge, there is an increase in pituitary GtH-II beta subunit mRNA levels followed by an increase in GtH-Ibeta mRNA levels. Gilthead seabream brain contains three different forms of GnRH: salmon (s)GnRH, seabream (sb)GnRH, and chicken (c)GnRH-II. All three GnRH-encoding mRNAs fluctuate throughout the day, reaching highest levels 8 h before spawning, concurrent with the preovulatory GtH-II surge. On the basis of these correlations and of the anatomical organization of the three GnRH systems, it is hypothesized that in the daily-spawning gilthead seabream females, preovulatory GtH-II secretion, and probably synthesis, are induced by a surge of sbGnRH secretion. The involvement of the other two GnRH forms, sGnRH and cGnRH-II, in the control of ovulation and spawning is presumed, on the basis of the elevation of their mRNA levels at the time of the preovulatory GtH-II secretion and spawning.

Activation of Salmon GnRH mRNA Expression Prior to Differentiation of Precocious Males in Masu Salmon

Changes in salmon gonadotropin-releasing hormone (sGnRH) mRNA levels in the brain of underyearling male masu salmon,Oncorhynchus masou,were investigated to clarify sGnRH participation in differentiation of precocious males. Fish were immature with low gonadosomatic index (GSI< 0.100%) on April 16 and May 7. On June 11, fish were differentiated into two groups: future precocious males and immature males. Fish in the former group had high GSI (0.203%) with proliferation of primary spermatocytes in the testis, while the latter had low GSI (0.055%) with primary spermatogonia. sGnRH mRNA levels (the number of neurons expressing sGnRH mRNA and the total number of silver grains per unit area) in the ventral telencephalon (VT) and in the VT plus preoptic area (POA) already increased in May before the differentiation of precocious males. GSI was positively correlated with sGnRH mRNA levels in the VT and in the VT plus POA in April, and with mRNA levels in the POA in May. Activated sGnRH synthesis is concluded to be related to the appearance of precocious males in masu salmon.

Gonadotrophs but not somatotrophs carry gonadotrophin-releasing hormone receptors: receptor localisation, intracellular calcium, and gonadotrophin and GH release.

Gonadotrophs are the primary target cells for GnRH in the pituitary. However, during a limited period of neonatal life in the rat, lactotrophs and somatotrophs respond to GnRH as well. Also, in the adults of a number of teleost fishes (e.g. carp, goldfish, and tilapia but not trout), GnRH is a potent GH secretagogue. In studying hypophysiotrophic actions of the two forms of GnRH present in the African catfish (Clarias gariepinus), chicken GnRH-II ([His5,Trp7,Tyr8]GnRH; cGnRH-II) and catfish GnRH ([His5,Asn8]GnRH; cfGnRH), we have investigated the effects of GnRH on catfish gonadotrophs and somatotrophs. GnRH binding was examined by incubating dispersed pituitary cells attached to coverslips with 125I-labelled [D-Arg6,Trp7,Leu8,Pro9-Net]GnRH (sGnRHa), a salmon GnRH analogue with high affinity for the GnRH receptor. Following fixation and immunohistochemistry using antisera against catfish LH and GH, 125I-labelled sGnRHa was localised autoradiographically and silver grains were quantified on gonadotrophs and somatotrophs. Specific binding of 125I-labelled sGnRHa was restricted to gonadotrophs. Both cfGnRH and cGnRH-II dose-dependently inhibited 125I-labelled sGnRHa binding to gonadotrophs. To substantiate the localisation of functional GnRH receptors, the effects of cfGnRH and cGnRH-II on the cytosolic free calcium concentration ([Ca2+]i) were examined in Fura-2-loaded somatotrophs and gonadotrophs. GnRH-induced increases in [Ca2+]i appeared to be confined to gonadotrophs, in which both endogenous GnRHs caused a single and transient increase in [Ca2+]i. The amplitude of this [Ca2+]i transient depended on the GnRH dose and correlated well with the GnRHs' effect on LH release. In vivo experiments demonstrated that GnRH treatments which markedly elevated plasma LH levels had no effect on plasma GH levels, while a dopamine agonist (apomorphine) significantly elevated plasma GH levels. We conclude that the two endogenous forms of GnRH in the African catfish are not directly involved in the regulation of the release of GH, suggesting that GnRHs cannot be considered as GH secretagogues in teleosts in general.

Distinct expression of GnRH genes in the red seabream brain

This paper reports the molecular cloning of a cDNA encoding the precursor of seabream gonadotropin-releasing hormone (prepro-sbGnRH) and the localization of salmon GnRH (sGnRH) and seabream GnRH (sbGnRH) expressing neurons in the brain of the red seabream (Pagrus major). The cloned prepro-sbGnRH cDNA has a 285 bps open reading frame encoding a 23 amino acid signal peptide, a 10 amino acid sbGnRH, the cleavage site (Gly-Lys-Arg), and a 59 amino acid GnRH-associated peptide. The expression of sGnRH and sbGnRH peptides, and prepro-sGnRH and prepro-sbGnRH mRNA were studied using immunocytochemistry and non-radioactive in situ hybridization, respectively. We found cell bodies that reacted positively with both the sGnRH cRNA probe and anti-sGnRH serum, but not with the sbGnRH cRNA probe or anti-sbGnRH serum in the ganglion of the terminal nerve. Cell bodies that reacted positively with the sbGnRH cRNA probe, anti-sbGnRH serum, and anti-sGnRH serum, but negatively with the sGnRH cRNA probe were found in the preoptic area (POA). Immunocytochemistry showed that a distinct bundle of axons arises in the POA which projected to the pituitary gland. These results suggest that sbGnRH is the most relevant hypophysiotropic form of GnRH.