Lack of gonadotropin releasing-hormone action on in vivo and in vitro growth hormone release,in rainbow trout (Oncorhynchus mykiss)

In order to understand the mechanisms implicated at the hypothalamo-pituitary level in growth-reproduction interaction in salmonids, the gonadotropin-releasing hormone (GnRH) action on growth hormone (GH) release was studied, in rainbow trout (Oncorhynchus mykiss). In vivo, acute treatment with salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and an sGnRH analogue [(DArg6Pro9)sGnRH] was performed on catheterized fish. The different forms of GnRH have no effect on plasma GH levels of immature and mature fish, but induce a stimulation of gonadotropin (GtH) release in mature fish. In the present work we have adapted and validated a culture system for GH regulation studies. In this system, increasing doses of sGnRH, (DArg6Pro9)sGnRH and cGnRH-II are inactive on GH release (24 hr incubation) in immature or mature fish, but stimulate GtH release in a dose-dependent manner. sGnRH (10⁻⁶ M) has no action on GH release, whatever the incubation time (15 min–24 hr). In a perifusion system, sGnRH also has no action on GH release but stimulates GtH release. The present results obtained using in vivo and in vitro techniques adapted for GH regulation studies, show that GnRH does not function as a growth hormone-releasing factor in rainbow trout as it does in goldfish.     

促性腺激素释放激素及多巴胺对斜带石斑鱼生长激素分泌及其mRNA表达的调控

研究斜带石斑鱼生长激素分泌及其mRNA表达的调控规律对于性别分化的控制、临床药物的选择,以及石斑鱼的增养殖等均具有重要的理论意义和实践意义。本文应用静态孵育系统,采用放射免疫测定法和化学发光液相杂交实验,研究GnRH和DA对斜带石斑鱼GH分泌、GHmRNA合成的调控作用。100nmol／LsGnRH作用斜带石斑鱼脑垂体碎片1也4h,明显促进GH的释放和GHmRNA的合成,并具有时间依存性;10nmol／L～1μmol／LsGnRH作用1h能明显促进斜带石斑鱼脑垂体释放GH,促进GHmRNA的合成,表现出明显的剂量效应。100nmol／L、1μmol／LmGnRH作用1h以一定的剂量依存方式促进GH的释放、促进GHmRNA的合成,但mGnRH的效应比相应剂量的sGnRH的作用弱。APO为DA受体的非选择性激动剂,不同剂量APO对斜带石斑鱼脑垂体碎片的作用结果显示,10nmol／L-1μmol／L APO以剂量依存方式促进斜带石斑鱼脑垂体碎片释放GH、促进GHmRNA的合成：1μmol／LAPO作用12h以上明显促进GH的释放和GHmRNA的合成,并随时间的延长而增加。与sGnRH对斜带石斑鱼GH释放、GHmRNA合成的作用相比,APO的作用较弱。本文研究结果证实GnRH和DA能促进斜带石斑鱼脑垂体GH释放和GHmRNA合成。     

Time- and dose-related effects of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the goldfish pituitary

The goldfish brain contains two molecular forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In a preliminary report, we demonstrated the stimulation of gonadotropin hormone (GtH) subunit and growth hormone (GH) mRNA levels by a single dose of GnRH at a single time point in the goldfish pituitary. Here we extend the work and demonstrate time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH gene expression in vivo and in vitro. The present study demonstrates important differences between the time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH mRNA levels. Using primary cultures of dispersed pituitary cells, the minimal effective dose of cGnRH-II required to stimulate GtH subunit mRNA levels was found to be 10-fold lower than that of sGnRH. In addition, the magnitudes of the increases in GtH subunit and GH mRNA levels stimulated by cGnRH-II were found to be higher than the sGnRH-induced responses. However, no significant difference was observed between sGnRH and cGnRH-II-induced responses in vivo. Time-related studies also revealed significant differences between sGnRH- and cGnRH-II-induced production of GtH subunit and GH mRNA in the goldfish pituitary. In general, the present study provides novel information on time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH mRNA levels and provides a framework for further investigation of GnRH mechanisms of action in the goldfish pituitary.     

The effect of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the pituitary of goldfish, Carassius auratus

The goldfish brain contains at least two forms of gonadotropin-releasing hormone (GnRH): sGnRH and cGnRH-II. In goldfish sGnRH and cGnRH-II are present both in the brain and pituitary, and exert direct effects via specific GnRH receptors stimulating growth hormone (GH) and gonadotropin hormone (GtH) synthesis and secretion. In this study, we investigated the effects of sGnRH and cGnRH-II on GtH subunit (alpha, FSH-beta and LH-beta) and GH mRNA levels in the goldfish pituitary in vivo and in vitro. Injection of goldfish with sGnRH or cGnRH-II (4 microg/fish) stimulated GtH-alpha, FSH-beta and LH-beta mRNA levels after 24 h. For in vitro studies, goldfish pituitary fragments were treated continuously for 12 h with 10(-7) M sGnRH or cGnRH-II. Both sGnRH and cGnRH-II stimulated GtH-alpha, FSH-beta, LH-beta and GH mRNA levels, however, cGnRH-II appeared to have a more pronounced effect. Similar experiments were carried out using cultured dispersed goldfish pituitary cells. In this study, treatments for 12 h with 10(-7) M sGnRH or cGnRH-II also stimulated GtH and GH gene expression. The present results provide a basis for the investigation of the signal transduction pathways that mediate GnRH-induced changes in GtH subunit and GH mRNA levels in the goldfish pituitary.     

Effects of prolactin and growth hormone on plasma levels of lysozyme and ceruloplasmin in rainbow trout

In vivo and in vitro effects of prolactin (PRL) and growth hormone (GH) on plasma levels of lysozyme and ceruloplasmin were examined in the rainbow trout (Oncorhynchus mykiss). Hypophysectomy had no effect on the plasma lysozyme level. Implantation of PRL- or GH-containing cholesterol pellets increased the lysozyme level in a dose-related manner. After hypophysectomy and sham operation, plasma ceruloplasmin was elevated above the level in intact fish, suggesting inflammation caused by the surgery. PRL or GH treatment significantly attenuated the increased level of ceruloplasmin in the operated fish. Expression of lysozyme mRNA was detected in the leucocytes isolated from the peripheral blood by RT-PCR. In vitro administration of PRL or GH showed no effect on the proliferation of isolated leucocytes or on the total protein content; however, lysozyme activity in the medium increased in a dose-related manner. These results suggest that PRL and GH directly stimulate lysozyme production without affecting the proliferation of leucocytes, and the attenuated ceruloplasmin level increased in response to inflammation.     

Effects of gonadotropin-releasing hormone on growth hormone secretion and gene expression in common carp pituitary

Using radioimmuno- and ribonuclease protection assays, we examined the effects of gonadotropin-releasing hormone and its analogs on the growth hormone mRNA level and growth hormone secretion in common carp (Cyprinus carpio) pituitary fragments with static incubation. After a 24 h treatment, sGnRH ([Trp(7),Leu(8)]-LHRH) and sGnRH-A ([D-Arg(6),Pro(9)]-LHRH) (0.1 nM-1 microM) elevated the GH mRNA level and stimulated the GH secretion in a dose-dependent manner, with a higher potency for sGnRH-A. In a time-course experiment, the function of sGnRH and sGnRH-A (10 nM) on GH secretion was observed after 6 h incubation, while no action on the GH mRNA level were noted until 12 h after treatment. Comparing mammalian GnRH, avian GnRH and piscine GnRH, sGnRH and sGnRH-A showed the highest potency in increasing GH mRNA level and GH-release, followed by cGnRH-II ([His(5),Tyr(8)]-LHRH), and finally LHRH and LHRH-A([D-Trp(6), Pro(9)]-LHRH). These findings, taken together, suggest that GnRH not only can influence GH release, but also play a role in the regulation of GH synthesis.     

Permissive Effect of Insulin-like Growth Factor I (IGF-I) on Gonadotropin Releasing-hormone Action on In Vitro Growth Hormone Release,in Rainbow Trout (Oncorhynchus mykiss)

The aim of the work was to study the influence of insulin-like growth factor I (IGF-I) on GnRH-induced GH release by cultured pituitary cells of normally growing rainbow trout (Oncorhynchus mykiss), collected at different stages of gametogenesis. When pituitary cells were pre-incubated with human IGF-I (10⁻⁸ M) for 48 hours they became responsive to sGnRH (10⁻⁸ to 10⁻⁶ M) in the subsequent 24-hour incubation period, depending on the sexual stage, while not IGF-I pre-incubated cells were always non-responsive to GnRH. The permissive effect of IGF-I was detected in immature fish or those at the beginning of the gametogenesis, but not in mature fish. IGF-I inhibition of GH release during the preincubation period varies also with the sexual stage and is greater in immature than in mature fish. The permissive effect of IGF-I seems specific to somatotropes since IGF-I does not modify GnRH action on GtH₂ release. This work suggests that GnRH action on GH release can vary for a particular fish species depending on the physiological status.     

Stimulation of non-specific immune functions in seawater-acclimated rainbow trout, Oncorhynchus mykiss, with reference to the role of growth hormone

The influence of acclimation to seawater (SW) and growth hormone (GH) administration on immune functions was examined in the rainbow trout (Oncorhynchus mykiss). After 3 days acclimation to dilute SW (12 parts per thousand, ppt), an increase in plasma lysozyme activity was observed compared to the fish kept in fresh water (FW). No change was seen in plasma immunoglobulin M (IgM) levels. When they were transferred from dilute SW to full-strength SW (29 ppt) after a single intra-peritoneal injection of ovine or salmon GH, plasma sodium levels of GH-treated fish were significantly lower than those of the control fish injected with Ringer's solution 24 h after the transfer. The plasma level of IgM was not influenced by GH injection in the fish kept in FW nor in those transferred to SW. The administration of GH increased plasma lysozyme activity in the fish in FW, but no further increase was seen after SW transfer. The production of superoxide anions in peripheral blood leucocytes was stimulated by GH in both FW and SW. These results suggest that GH is involved in the stimulation of the non-specific immune functions in SW-acclimated salmonids.     

Changes in brain GnRH mRNA and pituitary GnRH peptide during testicular maturation in barfin flounder

The pleuronectid barfin flounder (Verasper moseri) expresses three forms of gonadotropin-releasing hormone (GnRH) in the brain. To clarify the physiological roles of the respective forms during testicular maturation, changes in brain GnRH mRNA levels and pituitary GnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. Fish hatched in April 2000. The gonadosomatic index remained low until October 2001 and then rapidly increased in January 2002. Fish continued to grow from hatching through testicular maturation. Fish spermiated in March 2002. The amount of seabream GnRH (sbGnRH) mRNA per brain significantly increased in January 2002 and remained at high levels in March 2002. The amounts of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) mRNA per brain did not show significant changes during the experimental periods. Pituitary sbGnRH peptide content significantly increased in March 2002. Pituitary sGnRH peptide and cGnRH-II peptide contents were extremely low compared to sbGnRH peptide levels and showed no significant changes during the experiment. These results indicate that sbGnRH is involved in the testicular maturation of barfin flounder.     

Regulation of gonadotropin beta subunit gene expression by testosterone and gonadotropin-releasing hormones in the goldfish, Carassius auratus

Sex steroids differentially regulate gonadotropin (GTH) beta subunits (FSHbeta and LHbeta) gene expression in the pituitary of goldfish: a strong in vivo inhibitory effect on FSHbeta mRNA production, but a weak stimulatory effect on LHbeta in sexually immature and recrudescent fish. In the present study, to examine a direct effect of testosterone (T) and gonadotropin-releasing hormone (GnRH) on the mRNA levels of FSHbeta and LHbeta subunits in the pituitary, in vitro experiments were performed using dispersed pituitary cells of sexually immature, recrudescent, mature and regressed goldfish. T treatment in vitro did not significantly decrease FSHbeta mRNA levels, but increased that of LHbeta only in the cells of immature fish. Salmon-type GnRH increased FSHbeta mRNA levels in cells of mature fish, but decreased the levels in cells of sexually regressed fish. From these results, it was suggested that: (1) in vivo effect of sex steroids on gene expression of GTH beta subunits is not always exerted on the pituitary; and (2) the different responses of GTH beta subunits by sex steroids between in vivo and in vitro are partly due to a complex pathway through hypothalamic factors, such as GnRH, in the case of in vivo.     

Changes in some endocrinological and non-specific immunological parameters during seawater exposure in the brown trout

The influence of autumnal progressive and direct seawater transfers on ionic parameters, plasma growth hormone (GH) and thyroid hormones (TH) and also on the non–specific immune traits phagocytic activity, lysozyme and non–specific cytotoxicity were examined in 45–55 g brown trout ( Salmo trutta ). In both experiments, the seawater transfer induced the same pattern of endogeneous modifications but they were more pronounced and more lasting after the direct seawater transfer than after the progressive one. In seawater–transferred trout, there was a significant transitory increase of the plasma osmolarity, chloride concentration, GH levels and a transient decrease of the TH. The phagocytic activity of the pronephric leucocytes and the lysozyme concentrations were significantly higher in seawater–transferred trout than in controls. Nevertheless, the non–specific cytotoxicity should not be modified after the seawater exposure. Moreover significant positive correlations were observed between plasma GH and chemiluminescence or lysozyme increases. These data support the hypothesis that GH is involved in the salmonids' non–specific immune potential, especially by stimulating the macrophage functions.     

PKC and ERK are differentially involved in gonadotropin-releasing hormone-induced growth hormone gene expression in the goldfish pituitary

Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus and stimulates the synthesis and secretion of gonadotropin hormones. In addition, GnRH also stimulates the production and secretion of growth hormone (GH) in some fish species and in humans with certain clinical disorders. In the goldfish pituitary, GH secretion and gene expression are regulated by two endogenous forms of GnRH known as salmon GnRH and chicken GnRH-II. It is well established that PKC mediates GnRH-stimulated GH secretion in the goldfish pituitary. In contrast, the signal transduction of GnRH-induced GH gene expression has not been elucidated in any model system. In this study, we demonstrate, for the first time, the presence of novel and atypical PKC isoforms in the pituitary of a fish. Moreover, our results indicate that conventional PKC alpha is present selectively in GH-producing cells. Treatment of primary cultures of dispersed goldfish pituitary cells with PKC activators (phorbol ester or diacylglycerol analog) did not affect basal or GnRH-induced GH mRNA levels, and two different inhibitors of PKC (calphostin C and GF109203X) did not reduce the effects of GnRH on GH gene expression. Together, these results suggest that, in contrast to secretion, conventional and novel PKCs are not involved in GnRH-stimulated increases in GH mRNA levels in the goldfish pituitary. Instead, PD98059 inhibited GnRH-induced GH gene expression, suggesting that the ERK signaling pathway is involved. The results presented here provide novel insights into the functional specificity of GnRH-induced signaling and the regulation of GH gene expression.     

Neurosecretory neurons of the nucleus preopticus (NPO) express salmon GnRH mRNA and show reproduction phase-related variation in the female Indian major carp, Cirrhinus cirrhosus

We studied the expression of sGnRH mRNA in the neurons of the nucleus preopticus (NPO) of the Indian major carp, Cirrhinus cirrhosus, and their correlation with the reproductive status of the fish. Non-radioisotopic in situ hybridization histochemistry protocol employing biotinylated-oligonucleotide probes complementary to salmon GnRH, cichlid GnRH I, catfish GnRH, chicken GnRH II (from cichlid and catfish), and mammalian GnRH, were applied to the sections through the POA of the female Indian major carp Cirrhinus cirrhosus. Incubation with the probe complimentary to salmon GnRH (sGnRH) mRNA from salmon, produced distinct hybridization signal in the cytosol of several neurosecretory neurons of the magnocellular and parvocellular subdivisions of the NPO of the fish collected during February-April (preparatory phase) and May-June (prespawning phase). However, no signal was detected in the NPO of fish collected during July-August (spawning phase). Application of other antisense probes, or sense probe for salmon GnRH mRNA, produced no signal. We suggest that NPO neurons in C. cirrhosus may express sGnRH mRNA, produce GnRH peptide, and play a role in regulation of pituitary-ovary axis.