Salmon Gonadotropin-releasing Hormone Research Articles

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.

Extracellular sodium dependence of GnRH-stimulated growth hormone release in goldfish pituitary cells.

In goldfish, gonadotropin-releasing hormone (GnRH) stimulation of growth hormone (GH) release has been shown to involve extracellular Ca2+ entry through voltage-sensitive Ca2+ channels and the activation of protein kinase C (PKC). In this study, the possible involvement of extracellular Na+ in mediating the GH response to GnRH was examined using dispersed pituitary cells. Perifusion with Na(+)-depleted medium reversibly reduced the acute GH response to 5-min pulses of either 10 nM salmon (s)GnRH or 10 nM chicken (c)GnRH-II. Similarly, replacement of normal medium with Na(+)-depleted medium attenuated the long-term GH release response to sGnRH and cGnRH-II under static incubation conditions. These results suggest that GnRH-induced GH release requires the presence of extracellular Na+. Treatment with 5-min pulses of the Na(+)-channel agonist veratridine (10 microM) increased GH release in an extracellular Ca(2+)-dependent manner, presumably due to activation of voltage-sensitive Ca2+ channels resulting from the depolarizing effect of increased Na+ influx. On the other hand, Na+ entry through tetrodotoxin (TTX)-sensitive, voltage-dependent Na+ channels is not involved in GnRH-induced GH release. Application of 250 nM TTX, which abolished the voltage-sensitive Na+ currents in identified goldfish somatotropes, did not affect the acute GH responses to 5-min pulses of sGnRH and cGnRH-II. The possible participation of Na+/H+ antiport in mediating the extracellular Na(+)-dependent GnRH action on GH release was then examined. In static incubation experiments, sGnRH- and cGnRH-II-induced GH secretion were reduced by inhibitors of the Na+/H+ antiport, amiloride and dimethylamiloride (DMA). Likewise, the GH response to the PKC activator, tetradecanoyl phorbol acetate, was attenuated by treatment with Na(+)-depleted medium, amiloride, and DMA. The inhibitory actions of amiloride and DMA were selective as these drugs did not affect the GH response elicited by the Ca2+ ionophore ionomycin and the voltage-sensitive Ca2+ channel agonist, Bay K 8644. Taken together, these results indicate that extracellular Na+ and the Na+/H+ exchanger are involved in the mediation of GnRH-stimulated GH release in goldfish. Furthermore, this dependence on Na+ and Na+/H+ antiport probably occurs distal to the activation of PKC by GnRH.

Primary structure of three forms of gonadotropin-releasing hormone (GnRH) from the pacu brain

Perchlike fish are a vast group of advanced teleosts. The species examined to date have three forms of gonadotropin-releasing hormone (GnRH) within a single species, but the origin of the third GnRH peptide is unknown. In this study, the primary structure of three GnRH peptides is determined from the brain of the pacu, Piaractus mesopotamicus, an example of a teleost that is less advanced than the perchlike fish. The GnRH was purified from pacu brain extracts using high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The three forms identified by chemical sequencing and mass spectrometry are sea bream GnRH (pGlu–His–Trp–Ser–Tyr–Gly–Leu–Ser–Pro–Gly–NH 2, 1113.4 Da); chicken GnRH-II (pGlu–His–Trp–Ser–His–Gly–Trp–Tyr–Pro–Gly–NH 2, 1236.6 Da); and salmon GnRH (pGlu–His–Trp–Ser–Tyr–Gly–Trp–Leu–Pro–Gly–NH 2, 1212.3 Da). In addition, the number of forms of GnRH in the brains of male and female fish was determined separately. The same three forms of GnRH were present in the brains of both sexes as determined by antisera cross-reactivity and elution position from the HPLC column. The results indicate that the pacu brain has the identical forms of GnRH identified in perchlike fish and hence, the origin of three forms occurred earlier in evolution than previously thought.

Effect of d-Lys6 salmon sGnRH alone and in combination with domperidone on the spawning of common carp during the late spawning season

The effect of [d-Lys6] salmon gonadotropin-releasing hormone alone (sGnRH) and in combination with domperidone, a dopamine antagonist, on the spawning of common carp, Cyprinus carpio, was investigated during the late spawning season (when mature fish stop spawning under natural conditions when males and females are put together). Fish were induced to spawn by [d-Lys6]-sGnRH salmon gonadotropin-releasing hormone analogue (sGnRH-X) only when injected in combination with domperidone at doses of 10 μg kg-1 and 20 mg kg-1 body weight respectively. [d-Lys6]-sGnRH-X alone did not induce spawning at a dose as high as 100 μg kg-1 body weight. Fish injected with domperidone alone did not spawn either. These results demonstrate that [d-Lys6]-sGnRH-X in combination with a dopamine antagonist is effective in inducing spawning during the late spawning season.

Presence and activity of compounds with GnRH-like activity in the ovary of seabream Sparus aurata.

The binding and activity of gonadotropin-releasing hormone (GnRH) were characterized in the mature gilthead seabream (Sparus aurata) ovary by use of an analogue of salmon GnRH([D-Arg6,Trp7,Leu8,Pro9-N(Et)]GnRH, sGnRH-A) as labeled ligand. The binding of 125I-sGnRH-A to the seabream ovarian membrane preparation was saturable, displaceable, reversible, and dependent on time, temperature and tissue concentration. Addition of unlabeled s-GnRH-A displaced the radio-ligand in a dose-related manner, indicating the presence of one class of high-affinity binding sites with an equilibrium dissociation constant of 45.5 +/- 6.2 nM. Addition of other GnRH peptides, including salmon GnRH ([Trp7,Leu8]GnRH) and chicken GnRH-II ([His5,Trp7,Tyr8]GnRH), also displaced 125I-sGnRH-A; all these peptides bound with lower affinities than sGnRH-A to the seabream ovarian binding site. In this study, we also demonstrated the presence of compounds with GnRH-like activity in the ovary of seabream. Seabream ovarian extract stimulated pituitary gonadotropin release from the goldfish pituitary and displaced 125I-sGnRH-A binding in the seabream ovary. Furthermore, addition of sGnRH-A to cultured seabream oocytes directly stimulated reinitiation of oocyte meiosis, as indicated by germinal vesicle breakdown. Overall, the present study characterizes GnRH-binding sites in the seabream ovary and supports the hypothesis that GnRH or compounds with GnRH-like activity play an autocrine/paracrine role in the regulation of ovarian function in the seabream ovary.

Neuroendocrine regulation of gonadotrophin II release and gonadal growth in the goldfish, Carassius auratus

The goldfish, a member of the carp family, is a widely used model for reproductive neuroendocrine studies of economically important fish. The two gonadotrophin (GTH) molecules released from the fish anterior pituitary, GTH-I and GTH-II, are structurally similar to tetrapod FSH and LH, respectively. Gonadotrophin II is the best studied, and in goldfish stimulates gonadal growth and steroidogenesis, ovulation and sperm release. Growth hormone also has gonadotrophic actions in fish which enhance gonadal steroidogenesis. The principal stimulatory and inhibitory systems regulating GTH-II release are the gonadotrophin-releasing hormone (GnRH) and dopamine neurones in the preoptic-hypothalamic region. In goldfish there are two native GnRH forms, salmon GnRH and chicken GnRH-I; both stimulate GTH-II release but use different signal transduction pathways. In contrast to mammals, teleost fish do not have a median eminence and the GTH-II cells are thus directly innervated by neurones producing GnRH, dopamine and other stimulatory neurohormones. For most of these factors, the ability to stimulate GTH-II release varies seasonally. The amino acid neurotransmitter, gamma-aminobutyric acid, has the most prominent stimulatory actions which enhance GnRH release and inhibit dopamine turnover in the hypothalamo-pituitary complex. Neuropeptide Y stimulates GTH-II release by a combined direct action on the gonadotroph and also by enhancing GnRH release. Positive and negative sex steroid feedback mechanisms act concurrently to regulate GTH-II release in adults of both sexes. The principal site of positive feedback is the GTH-II cell where testosterone and oestradiol potentiate GnRH-stimulated GTH-II release. Negative feedback by sex steroids involves activation of inhibitory dopamine neurones, thus maintaining tight control over circulating GTH-II concentrations.

Neuroendocrine regulation of gonadotrophin II release and gonadal growth in the goldfish, Carassius auratus.

The goldfish, a member of the carp family, is a widely used model for reproductive neuroendocrine studies of economically important fish. The two gonadotrophin (GTH) molecules released from the fish anterior pituitary, GTH-I and GTH-II, are structurally similar to tetrapod FSH and LH, respectively. Gonadotrophin II is the best studied, and in goldfish stimulates gonadal growth and steroidogenesis, ovulation and sperm release. Growth hormone also has gonadotrophic actions in fish which enhance gonadal steroidogenesis. The principal stimulatory and inhibitory systems regulating GTH-II release are the gonadotrophin-releasing hormone (GnRH) and dopamine neurones in the preoptic-hypothalamic region. In goldfish there are two native GnRH forms, salmon GnRH and chicken GnRH-I; both stimulate GTH-II release but use different signal transduction pathways. In contrast to mammals, teleost fish do not have a median eminence and the GTH-II cells are thus directly innervated by neurones producing GnRH, dopamine and other stimulatory neurohormones. For most of these factors, the ability to stimulate GTH-II release varies seasonally. The amino acid neurotransmitter, gamma-aminobutyric acid, has the most prominent stimulatory actions which enhance GnRH release and inhibit dopamine turnover in the hypothalamo-pituitary complex. Neuropeptide Y stimulates GTH-II release by a combined direct action on the gonadotroph and also by enhancing GnRH release. Positive and negative sex steroid feedback mechanisms act concurrently to regulate GTH-II release in adults of both sexes. The principal site of positive feedback is the GTH-II cell where testosterone and oestradiol potentiate GnRH-stimulated GTH-II release. Negative feedback by sex steroids involves activation of inhibitory dopamine neurones, thus maintaining tight control over circulating GTH-II concentrations.

The maturation of the salmon GnRH system and its regulation by gonadal steroids in masu salmon

Pituitary gonadotropin (GTH) secreting cells and brain gonadotropin-releasing hormone (GnRH) secreting neurons are known to be subjected to feedback control by gonadal steroid in teleosts. In masu salmon, Oncorhynchus masou, salmon GnRH (sGnRH) neurons in the ventral telencephalon (VT) and the preoptic area (POA) are involved in the control of GTH cells because sGnRH synthesis in these areas is activated with gonadal maturation. In this study, we attempted to clarify mechanisms of feedback control of sGnRH neurons by gonadal steroids. We examined the effects of 17α-methyltestosterone (MT) on sGnRH synthesis in yearling and 2-year-old female fish (which were immature during experimentation in May), and the effects of castration on sGnRH synthesis in underyearling precocious male fish in August. sGnRH synthesis in the POA, but not in the VT, was increased by MT administration in 2-year-old females only, indicating higher sensitivity to MT in the preoptic sGnRH neurons. Castration increased sGnRH synthesis in the VT but not in the POA. These results suggest that sGnRH neurons in the VT and those in the POA are differentially regulated by gonadal steroids.

Gonadotropin-releasing hormone and gonadotropin in goldfish and masu salmon

Reproductive activities in vertebrates are regulated by an endocrine system, consisting of the brain-pituitary-gonad axis. In teleosts, gonadotropin-releasing hormone (GnRH) in the brain stimulates gonadotropin (GTH) release in the pituitary gland, but because of lack of the portal vessel, it is not known when and how much GnRH is released for the regulation of GTH release. There are multiple molecular types of GnRH in teleosts and several distinct populations of GnRH neurons in the brain. However, we do not know which types and populations of GnRH neurons regulate reproductive activities. Here we summarize our recent studies on GnRH and GTH in masu salmon Oncorhynchus masou and goldfish Carassius auratus. Immunocytochemistry showed the location and molecular types of GnRH neurons. Salmon (sGnRH) and chicken-II GnRH (cGnRH-II) neuronal fibers were widely distributed in the brain of both masu salmon and goldfish. Only sGnRH fibers were observed in the pituitary of masu salmon, whereas both sGnRH and cGnRH-II fibers were observed in the goldfish pituitary, indicating that species specific GnRH profiles are involved in the regulation of pituitary function in teleosts. A series of experiments in masu salmon and goldfish suggest that among GnRH neuron populations GnRH neurons in the ventral telencephalon and the hypothalamus regulate GTH release, and that GnRH of the terminal nerve origin is not essential to gonadal maturation and ovulation. The biological function of other GnRH neurons remains unkown. Two GTHs appear to be characteristic of teleost; however, regulation of reproduction by these GTHs is a question that remains to be elucidated. In salmonid species, it is proposed that GTH I stimulates early gonadal development, whereas GTH II acts in later stages. When GTH expression was examined in goldfish, both GTH I β and II β mRNA levels in the pituitary gland showed increases in accordance with gonadal development, unlike the sequential expression of GTH subunits in salmonids. The expression of these GTH subunit mRNAs were affected by water temperature, starvation, and steroid hormones in goldfish, but in what manner these two GTHs regulate gonadal development remains to be clarified.

Characterization of molecular variants of GnRH, induction of spermiation and sex reversal using salmon GnRH-A and domperidone in the protogynous diandric fish, Synbranchus marmoratus Bloch, (Teleostei, Synbranchidae)

This paper studies the molecular variants of gonadotropin-releasing hormone (GnRH) present in the brain of the protogynous swamp eel, Synbranchus marmoratus, and the effects of the administration of salmon GnRH analogue (sGnRH-A) and the dopamine receptor antagonist, domperidone (DOM) on final maturation and gamete release in this species. Evidence for the presence of two GnRH variants, sGnRH and cIIGnRH were obtained by reverse phase high-pressure liquid chromatography (RP-HPLC) and radioimmunoassay with different antisera. The effects of treatment with sGnRH-A+DOM were checked by three ways: oocyte and milt release by stripping, histological analysis of the gonadal tissue, and androgen serum levels at different times throughout the experiment. In males, spermiation was induced after three weeks of treatment. In the female group, sGnRH-A+DOM did not induce ovulation at the end of the experiment. Histological analysis of the gonads from the female group showed evidence of sex reversal. All the treated fish had elevated androgen serum levels from the third week, with respect to control fish. In all cases, serum estradiol levels were undetectable. These results suggest that treatment with sGnRH analog and DOM induce sex reversal in female and spermiation in males.

Spontaneous and gonadotropin-releasing hormonem-stimulated cytosolic calcium rises in individual goldfish gonadotrophs

The cytosolic free calcium concentration, [Ca 2+] i, was monitored in single isolated goldfish gonadotrophs with the fluorescent probe Indo-1. It was found that goldfish gonadotrophs exhibit both spontaneous and secretagogue-induced [Ca 2+] i rises. Spontaneous [Ca 2+] i transients showed striking kinetic features and a sensitivity to external Ca 2+ suggesting that they were the consequence of transient Ca 2+ entries. Two kinetically distinct patterns of [Ca 2+] i rises were generated in response to the two native forms of gonadotropin-releasing hormone (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In a part of the gonadotrophs, GnRHs triggered a plateau [Ca 2+] i rise whereas in other responsive cells they induced a series of [Ca 2+] i bursts, each consisting of grouped [Ca 2+] i transients. Both plateau and burst [Ca 2+] i response patterns were due to Ca 2+ entry through plasma membrane Ca 2+ channels, inasmuch as they were suppressed with external Ca 2+ removal. No contribution of Ca 2+ release from thapsigargin-sensitive stores was observed in either response pattern. While in mammalian gonadotrophs GnRH raises [Ca 2+], by mostly acting on internal Ca2+ sequestering stores, our results show that GnRH-stimulated goldfish gonadotrophs rapidly increase Ca 2+ entry to enhance their [Ca 2+] i levels.

A Radioimmunoassay for African Catfish Growth Hormone: Validation and Effects of Substances Modulating the Release of Growth Hormone

A highly sensitive radioimmunoassay has been developed for measuring plasma growth hormone (GH) concentrations in the African catfish (Clarias gariepinus). The lower detection limit of the assay was 0.1 ng/ml and the standard curve had an ED50value of 0.5 ng/ml. The validity of the assay was established and the effects of several neurotransmitters on the release of GH were examined.In vitroexperiments, using a static culture system for dispersed pituitary cells, demonstrated that the GH release in African catfish was affected by growth hormone-releasing hormone and somatostatin. Single intraperitoneal injections with a dopamine agonist, apomorphine, produced significant and dose-dependent increases in plasma GH levels. Unlike carp, goldfish, and tilapia, a super-active analogue of salmon gonadotrophin-releasing hormone did not alter plasma GH levels in African catfish.

Direct influences of temperature on gonadotropin-II release from perifused pituitary fragments of common carp (Cyprinus carpio L.) in vitro

The direct influences of temperature on basal and stimulated gonadotropin-II (GtH-II) release of common carp pituitary fragments in a perifusion system were examined. The pituitaries were taken from sexually recrudescing common carp. The basal GtH-II release of the pituitary fragments was suppressed by low temperature, with a significant decrease in GtH-II release at 10°C compared with those at 20°C and 25°C. The pituitary GtH-II release responses to superactive agonist of salmon gonadotropin-releasing hormone (GnRH) [Arg6, Pro9 NEt]-sGnRH (sGnRH-A), administered in both pulsatile and continuous fashions, were also suppressed by low temperature. With 2 min pulsatile administration, generally there were significantly greater responses to pulsatile sGnRH-A at 20°C and 25°C compared to 10°C and 15°C at the three dosages (1, 10 and 100 nM) tested, although this was not as clear at the low dosage. There were no significant effects of temperature on GtH-II release responses between 20°C and 25°C, and 10°C and 15°C. With continuous administration of 1 or 10 nM sGnRH-A, the initial GtH-II response peaks at the onset of continuous administration, and GtH-II release rates during each hour of continuous sGnRH-A treatment were always greater at the warmer temperature. Continuous exposure of the pituitary fragments at 10°C or 20°C to 10 nM sGnRH-A resulted in an initial peak of GtH-II release followed by desensitization to further sGnRH-A stimulation. This biphasic GtH-II response was not as clear at 1 nM dosage, because a rebound in GtH-II release occurred following the initial desensitization. There was, however, no clear influence of temperature on the GtH-II release pattern in response to continuous sGnRH-A treatment. These results provide evidence for direct influences of temperature on basal GtH-II release and pituitary responsiveness to GnRH in common carp.

Rapid separation of gonadotropin-releasing hormone molecular forms by isocratic high-performance liquid chromatography on an ion-exchange column

The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (clGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.

Cytophysiology of gonadotropin-releasing-hormone neurons in chum salmon (Oncorhynchus keta) forebrain before and after upstream migration.

Cytophysiology of gonadotropin-releasing-hormone neurons in chum salmon (Oncorhynchus keta) was examined before and after upstream migration by an immunocytochemical technique with a specific antiserum to salmon gonadotropin-releasing hormone and an in situ hybridization technique with an oligonucleotide encoding salmon gonadotropin-releasing hormone precursor (pro-salmon gonadotropin- releasing hormone). In the forebrain (olfactory nerve, olfactory bulb, telencephalon, and preoptic area), salmon gonadotropin-releasing hormone-immunoreactive neurons and neurons showing signals for pro-salmon gonadotropin-releasing hormone mRNA were compared between fish from the coastal sea and those from the spawning ground. Neurons in the dorsal region of the olfactory nerve and in the ventral region of the transitional area between olfactory nerve and olfactory bulb showed strong salmon gonadotropin-releasing-hormone immunoreactivity and strong hybridization signals in fish from the coastal sea, but these activities and signals were not observed or were decreased in number in fish from the spawning ground. The neurons in the olfactory bulb, telencephalon, and preoptic area consistently revealed salmon gonadotropin-releasing-hormone immunoreactivity and hybridization signals, and the hybridization signals of salmon gonadotropin-releasing hormone in the telencephalon and the preoptic area were stronger in fish from the spawning ground than in those from the coastal sea. These findings suggest that salmon gonadotropin-releasing-hormone neurons in the olfactory nerve and the transitional area between olfactory nerve and olfactory bulb have different patterns of hormone production than those in the telencephalon and the preoptic area.

Role of Arachidonic Acid and Calmodulin in Mediating Dopamine D1- and GnRH-Stimulated Growth Hormone Release in Goldfish Pituitary Cells

In goldfish, growth hormone (GH) release is stimulated by dopamine via D1 receptors and cAMP-dependent mechanisms and by gonadotropin-releasing hormone (GnRH) through a protein kinase C (PKC) pathway; in addition, both D1 and GnRH actions require extracellular Ca2+. In this study, the involvement of arachidonic acid (AA) and calmodulin (CaM) in mediating the GH responses to D1 and GnRH stimulation was examined using primary cultures of dispersed goldfish pituitary cells. In 2-hr static incubation experiments, the phospholipase A2inhibitor bromophenacylbromide (BPB; 50 μM) decreased the GH responses to the D1 agonist SKF38393 (1 μM), the adenylate cyclase activator forskolin (10 μM), and the cAMP analog 8Br-cAMP (1 mM), but not the responses to salmon (s)GnRH (100 nM), chicken (c)GnRH-II (100 nM), and AA (50 μM). Similarly, the phospholipase A2inhibitor quinacrine (50 μM) and an inhibitor of AA metabolism, nordihydroguaiaretic acid (NDGA; 50 μM), reduced the GH responses to SKF38393, forskolin, and 8Br-cAMP. The response to the dopamine agonist apomorphine (1 μM) was also decreased by NDGA. The GH responses to AA did not add to those of forskolin or SKF38393, but were additive to responses to sGnRH and the PKC activator tetradecanoyl phorbol acetate (TPA; 100 nM). In perifusion experiments, treatment with BPB reduced the acute GH response to 1 μMSKF38393, 10 μMforskolin, or 1 mM8Br-cAMP. Taken together, these results suggest that mobilization and metabolism of AA mediate both acute and prolonged GH responses to D1, but not GnRH. The involvement of AA probably occurs distal to D1-induced cAMP generation. Two-hour static incubation with 10 nMto 10 μMKN62, a CaM-dependent kinase II inhibitor, decreased the GH response to 100 nMsGnRH or cGnRH-II. KN62 (1 μM) similarly decreased the GH response to 1 μMSKF38393, 10 μMforskolin, 1 mM8Br-cAMP, or 100 nMTPA. In perifusion studies, KN62 (1 μM) also reduced the acute GH response to 5-min pulses of 100 nMsGnRH, 100 nMcGnRH-II, or 1 μMSKF38393. These results indicate that CaM mediates the acute, as well as the prolonged, GH responses to GnRH and dopamine. The involvement of CaM likely occurs distal to cAMP and PKC.

Steroid Activation of the Brain–Pituitary Complex Gonadotropic Function in the Triploid Rainbow TroutOncorhynchus mykiss

Testosterone and estradiol were implanted into triploid rainbow trout, and their effects on brain and pituitary salmon gonadotropin-releasing hormone (sGnRH) content and on gonadotropin (GtH2) levels in the pituitary and blood were studied. In the hypothalamic areas, neither steroid altered sGnRH content. In the brain tissues (ventral telencephalon and preoptic areas), testosterone (0.5 mg/kg BW) significantly increased sGnRH contents and larger doses induced a more rapid increase. Estradiol induced a similar increase which occurred later than the response to the same dose of testosterone. Both steroids also stimulated pituitary sGnRH, with estradiol being less potent. Only testosterone induced long-term increases in plasma GtH2 content. Both steroids may act on the synthesis and release of GnRH, with testosterone having a greater influence on the control of gonadotropin secretion, especially at the end of the cycle.

Cloning and Sequencing of the Goldfish Growth Hormone cDNA

This study cloned a cDNA encoding the mature goldfish growth hormone (gGH) using polymerase chain reaction. The nucleotide sequence of the gGH was greater than 90% homologous to that of common carp, silver carp, bighead carp, and grass carp. The amino acid sequence of gGH, determined based on the nucleotide sequence of its cDNA, showed more than 93% homology to that of other mentionedCyprinidspecies, 64% to salmon GH, and 32% to human GH. The cloned gGH was found to lack a cysteine (Cys) residue at position 123 which contrasts with the GH of otherCyprinidspecies studied to date. The goldfish GH contained only four Cys residues that could be aligned with those of other GHs. This is significant from an evolutionary standpoint, suggesting possible divergence among cyprinids. The cloned gGH cDNA was used as a probe in Northern blotting analysis to investigate the effect of salmon gonadotropin-releasing hormone (sGnRH) on goldfish GH mRNA productionin vivo.The results demonstrate for the first time a strong dose-related stimulation of GH synthesis by sGnRH in the goldfish pituitary.

Functional Expression of GnRH Receptors in Xenopus Oocytes Injected with Salmon Brain RNA.

The brain of salmonid fishes contains two types of gonadotropin-releasing hormone (GnRH) peptide, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). Although cDNA encoding the mammalian GnRH (mGnRH) receptor has been cloned and characterized in several mammalian species, structures of cDNAs of other GnRH receptors in non-mammalian vertebrates have not been determined yet. As a step to isolate cDNA clones for the sGnRH and the cGnRH-II receptors from masu salmon (Oncorhynchus masou) brains, we examined functional expression of these receptors electrophysiologically using Xenopus oocyte expression system. Oocytes injected with poly(A)+ RNA of the brain or the pituitary of the masu salmon responded to both sGnRH and cGnRH-II. Oocytes injected with poly(A) + RNA of pituitaries of the masu salmon responded to only sGnRH. The poly(A) + RNA of the brain was further fractionated according to the size. Injection of pooled fractions of poly(A) + RNA of a size range between 5.5 and 6.0 kb induced the oocyte response to sGnRH, but the response to cGnRH-II was not clearly induced. Injection of poly(A) + RNA of about 5.0-5.5 kb in size induced the response to cGnRH-II, but the response to sGnRH was not clearly detected. These results suggest that the sGnRH and cGnRH-II receptors are encoded by different RNA, and that the preference of the agonists of these two receptors are clearly different each other.

Roles of Calcium and Calmodulin in the Mediation of Acute and Sustained GnRH-Stimulated Gonadotropin Secretion from Dispersed Goldfish Pituitary Cells

The possible involvement of extracellular Ca2+([Ca2+]o) in mediating the acute gonadotropin (GtH) response to salmon gonadotropin-releasing hormone (sGnRH) and chicken gonadotropin-releasing hormone-II (cGnRH-II) in goldfish was examined using dispersed pituitary cells in perifusion. Perifusion with Ca2+-deficient medium reduced the GtH responses to 5-min pulses of either GnRH, indicating the participation of [Ca2+]oin acute GnRH action. Using a 10-min GnRH pulse application protocol, the dependence of the acute GtH responses to the two GnRHs on [Ca2+]oentry through voltage-sensitive Ca2+channels (VSCC) was examined using the dihydropyridine VSCC blocker nifedipine and the cation Co2+. Treatment with nifedipine consistently reduced the acute GtH response to either sGnRH or cGnRH-II. Similarly, perifusion with CoCl2reduced the sGnRH-induced GtH release. In contrast to its effects on sGnRH, CoCl2abolished the cGnRH-II-induced GtH release. These results indicate that [Ca2+]oentry through VSCC participates in the acute GtH response to both native GnRHs; however, the cGnRH-II-stimulated acute release is relatively more dependent on [Ca2+]oand VSCC functions than sGnRH-induced secretion. The involvement of calmodulin (CaM) in mediating GnRH action was also examined. Treatment with a CaM antagonist, calmidazolium, or with a Ca2+/CaM-dependent protein kinase II inhibitor, KN62, reduced GtH responses to sGnRH and cGnRH-II in 2-hr static incubation, but not in perifusion studies with dispersed goldfish pituitary cells using 5- or 10-min GnRH pulses. These results suggest that CaM-dependent mechanisms participate in mediating the long-term, but not the acute, GtH response to GnRH. Compared to sGnRH, cGnRH-II-induced GtH release was more sensitive to inhibition by KN62, indicating a higher degree of dependence of cGnRH-II action on CaM. These results extend our understanding of the differential involvement of [Ca2+]oand CaM in mediating the short-term and long-term actions of the two native GnRH peptides on GtH release in goldfish.