Goldfish Pituitary Research Articles

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on prolactin and somatolactin release from the goldfish pituitary in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a role in mediating growth hormone and gonadotropin release in the teleost pituitary. In the present study, we examined the immunohistochemical relationship between PACAP nerve fibers and prolactin (PRL)- and somatolactin (SL)-producing cells in the goldfish pituitary. Nerve fibers with PACAP-like immunoreactivity (PACAP-LI) were identified in the neurohypophysis in close proximity to cells containing PRL-LI or SL-LI. Several cells with PRL-LI or SL-LI showed PACAP receptor (PAC 1R)-LI. The cell immunoblot assay method was used to examine the effect of PACAP on PRL and SL release from dispersed goldfish pituitary cells. Treatment with PACAP increased the immunoblot area for PRL- and SL-LI from individual pituitary cells in a dose-dependent manner. The effect of PACAP on the expression of mRNAs for PRL and SL in cultured pituitary cells was also tested. Semiquantitative analysis revealed that the expression of SL mRNA, but not PRL mRNA, was increased significantly by the treatment with PACAP. The effect of PACAP on intracellular calcium mobilization in isolated pituitary cells was also investigated using confocal laser-scanning microscopy. The amplitude of Ca 2+ mobilization in individual cells showing PRL- or SL-LI was increased significantly following exposure of cells to PACAP. These results indicate that PACAP can potentially function as a hypophysiotropic factor mediating PRL and SL release in the goldfish pituitary.

Molecular characterization and sex-related seasonal expression of thyroid receptor subtypes in goldfish

The thyroid hormones, acting through the nuclear thyroid receptors (TRs), play important roles in the growth and development of vertebrates. The present study investigated the molecular structure and season-related expression of the TR isoforms in the male and female goldfish pituitary, brain, liver, gonads, gut, heart, and muscle. Based on sequence alignment with other species, the results demonstrate the presence of: (1) a TRα (TRα-1) consisting of 1496 nucleotides encoding a 466 amino acid protein, (2) a novel splice variant of TRα (TRα-2) containing an out-of-frame deletion of 246 nucleotides in the ligand-binding domain consisting of 1251 nucleotides encoding a 378 amino acid protein, (3) a novel transcript resembling TRα, except for non-homology in the hinge region and a premature stop codon prior to the ligand-binding domain (TRα-truncated; 1418 nucleotides, 206 amino acid protein), and (4) TRβ consisting of 1823 nucleotides encoding a 395 amino acid protein. The findings provide the first demonstration of the presence of a truncated TR isoform in non-mammalian vertebrates. In goldfish, the expression patterns for all TRs subtypes were found to be remarkably similar in both male and female, changing significantly before and during reproductive season. The results provide a frame work for better understanding of the functional significance of novel TR forms and TR subtypes in fish and other vertebrates.

Proteolytic processing and differential distribution of secretogranin-II in goldfish

Secretoneurin (SN) is a 33–34 amino acid neuropeptide derived by endoproteolysis of secretogranin-II (SgII), a chromogranin. A multi-antigenic strategy was used to generate a rabbit polyclonal goldfish SN antiserum that was characterized for Western blot analysis. In the goldfish pituitary two intermediate proteins containing SN and likely processed from the 69.6-kDa SgII precursor were detected. No immunoreactive proteins were observed in the goldfish interrenal, ovary, cerebellum, and telencephalon whereas SgII mRNA was expressed in all these tissues. Immunoreactive levels of the ∼57 kDa product were higher in the pars distalis (PD) than in the neurointermediate lobe (NIL). The abundance of the ∼57 kDa protein indicates that this SgII-product containing the SN sequence is a major stored form in secretory granules of the goldfish pituitary. High expression and processing of SN in the hypothalamus and pituitary suggest important roles for SgII-derived peptides in neuroendocrine tissues.

Molecular Cloning and Tissue Distribution of SF-1-related Orphan Receptors During Sexual Maturation in Female Goldfish

The steroidogenic factor (SF)-1 gene is one of a number of orphan nuclear receptors, which is a key transcriptional regulator in vertebrate reproduction. We have isolated the SF-1 homologue cDNA from the goldfish pituitary and designed primers for SF-1 on the basis of the highly conserved regions of various known SF-1 superfamily genes. SF-1 cDNA contained 1,948 nucleotides including an open reading frame predicted to encode a protein of 503 amino acids. The distribution pattern of SF-1 in a variety of tissues during sexual maturation in female goldfish was also examined by RT-PCR. Significant variations in the relative expression of SF-1 were observed in different tissues in immature and mature female goldfish. SF-1 transcript in pituitary was significantly higher than other tissues tested in immature and mature female goldfish. Lower expression of SF-1 was observed in the liver but was not detected in brain and ovary of the immature female goldfish. Presence of SF-1 was the predominant expression in the pituitary and brain of mature female goldfish. Also, in the mature female goldfish, a weak transcript was detected in liver and ovary. Interestingly, RT-PCR analysis revealed that the expression of SF-1 became higher in the brain and weaker in the liver in maturing female goldfish. Thus, SF-1 may be regulated in goldfish brain and/or liver. Thus is also tissue-specific distribution of SF-1 during sexual maturation in female goldfish.

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.

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-alpha, follicle-stimulating hormone (FSH)-beta and LH-beta mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-alpha-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.

Cloning of Smad2, Smad3, Smad4, and Smad7 from the goldfish pituitary and evidence for their involvement in activin regulation of goldfish FSHβ promoter activity

Follicle-stimulating hormone (FSH), a glycoprotein consisting of an α subunit and a unique β subunit, is essential for gonadal development and function in vertebrates including teleosts. FSH is regulated by a variety of neuroendocrine and endocrine factors, and its biosynthesis is primarily determined by the expression of the β subunit. Although the regulation of FSH biosynthesis has been well documented in mammals, the molecular mechanisms underlying the regulation are poorly understood. Our previous studies demonstrated that activin stimulated goldfish FSHβ expression in the primary pituitary cell culture and enhanced its promoter activity in the mouse gonadotrope cell line LβT-2 cells. However, little is known about the signal transduction pathway involved in the transcriptional activation of this gene by activin. To assess the involvement of intracellular signaling protein Smads in regulating goldfish FSHβ promoter, we first cloned full-length cDNAs for goldfish Smad2, Smad3, Smad4, and Smad7 from the pituitary. All Smads cloned show high sequence conservation with their mammalian counterparts. The spatial expression of these Smads overlapped with that of activin subunits and its receptors in various tissues examined. In addition, we demonstrated that activin induced Smad3 and Smad7 expression, but not Smad2 and Smad4. Co-transfection of Smad2 or Smad3 cDNA into the LβT-2 cells with the reporter construct of goldfish FSHβ promoter significantly enhanced basal and activin-stimulated reporter (SEAP, secreted alkaline phosphatase) expression, while Smad7 completely blocked basal and Smad2/3-stimulated FSHβ activity. Interestingly, the effect of Smad3 was much higher than that of Smad2, suggesting that Smad3 is likely the principal signal transducing molecule involved in activin stimulation of FSHβ expression in the goldfish. This work lays a foundation for further analysis of goldfish FSHβ promoter for the cis-regulatory elements involved in activin signaling.

Seasonal cyclicity of secretogranin-II expression and its modulation by sex steroids and GnRH in the female goldfish pituitary

The secretory vesicle protein secretogranin-II (SgII), a precursor for the bioactive peptide secretoneurin, is expressed at all levels of the goldfish reproductive axis, including the hypothalamus, pituitary and ovaries. These findings led us to hypothesize that SgII is involved in reproduction and is physiologically regulated. We investigated the effects of different sex steroids on pituitary SgII expression throughout the seasonal reproductive cycle of the female goldfish, as well as the effects of GnRH and testosterone on pituitary LHβ subunit, GH, and SgII expression in sexually recrudescent female fish using northern blot analysis. We demonstrated that SgII expression levels vary seasonally, with levels being highest in winter and lowest in spring. Sex steroids did not alter SgII expression at any of the time periods studied. In sexually mature goldfish, injection of a GnRH agonist stimulated the expression of LHβ and SgII specifically in the pars distalis but not the neurointermediate lobe of the pituitary. Although testosterone alone did not alter the expression of either of these genes, it did abolish the stimulatory effects of GnRH on both LHβ and SgII expression. This represents the first study where testosterone is shown to modulate SgII expression in the pituitary.

Effects of cholecystokinin and bombesin on the expression of preprosomatostatin-encoding genes in goldfish forebrain

It was previously demonstrated that both cholecystokinin (CCK) and bombesin (BBS) stimulate growth hormone (GH) secretion in goldfish. Both peptides induce satiety and it was speculated that they integrate satiation and the postprandial increase in GH circulating levels. In the present paper we investigated the effects of CCK and BBS on the forebrain expression of the somatostatin gene family in goldfish to analyze if somatostatin peptides may be part of the effector mechanisms of CCK and BBS. We found that peripherally as well as centrally administered CCK decreases mRNA levels of preprosomatostatin (PSS)-I that encodes for SRIF-14, having no effects on PSS-II and PSS-III, which encode for gSRIF-28 and [Pro 2] SRIF-14, respectively. In addition, a direct action on the pituitary to stimulate GH release, this inhibition of PSS-I expression provides a possible mechanism for CCK to increase postprandial GH levels. On the other hand, BBS inhibits the forebrain expression of PSS-I and PSS-II but does not affect PSS-III regardless of the route of administration. We conclude that this could be the most likely mechanism of action of BBS to increase GH secretion, since there are few BBS-immunoreactive (IR) fibers and BBS binding sites in the anterior pituitary of goldfish.

Follistatin suppresses FSHβ but increases LHβ expression in the goldfish – evidence for an activin-mediated autocrine/paracrine system in fish pituitary

Our previous study demonstrated that recombinant goldfish activin B stimulated goldfish FSHβ but inhibited LHβ expression. Similar to activin B, activin A also exhibited the inverse effects on the expression of the two gonadotropins. The novel dual effects of activins on FSH and LH in the goldfish raise an interesting question as to where the activin comes from in vivo. In the present study, we first demonstrated the expression of activin, its receptors and binding protein follistatin in the goldfish pituitary, leading to a suggestion that an autocrine/paracrine regulatory system involving activin is operative in fish pituitary. To investigate the functionality of the pituitary-derived activin system in the regulation of gonadotropin biosynthesis, we further examined the effects of follistatin, an activin-binding protein, on goldfish FSHβ and LHβ expression. Follistatin not only reversed the effects of exogenous activin on FSHβ and LHβ expression but also had inverse effects on the basal expression of the genes; and its effects were opposite to those of activin. This suggests that the endogenous activin plays roles in controlling the expression of both FSHβ and LHβ genes. It is conceivable that any factors that influence the intrapituitary activin system in vivo will likely affect the biosynthesis of the two gonadotropins in the goldfish.

Nitric oxide produced by a novel nitric oxide synthase isoform is necessary for gonadotropin-releasing hormone-induced growth hormone secretion via a cGMP-dependent mechanism.

The involvement of nitric oxide (NO) in the regulation of goldfish growth hormone (GH) secretion was further characterized using primary cultures of dispersed goldfish pituitary cells. Western blots revealed the presence of an inducible nitric oxide synthase (iNOS)-like protein of approximately 120 kDa in cytosol/plasma membrane extracts. By contrast, brain NOS-immunoreactive proteins of approximately 120-140 kDa were occasionally detected in a cytoskeleton/organelle fraction but were absent from cytosol/plasma membrane extracts. The NO donor sodium nitroprusside (SNP) acutely increased GH secretion but this response was not observed in the presence of either a NO scavenger (PTIO) or a soluble guanylate cyclase inhibitor (ODQ). SNP also significantly increased the levels of cyclic (c)GMP in somatotrope-enriched cell populations. Treatments with 1400W (iNOS inhibitor), PTIO and rutin hydrate (NO scavengers) and ODQ abolished the acute GH-release response to two endogenous gonadotropin-releasing hormones (GnRH). 1400W, rutin hydrate, PTIO and ODQ alone did not significantly alter basal GH secretion. Together, these results establish that an iNOS-like peptide is constitutively present in the pituitary of the goldfish. Furthermore, these data suggest that NO, most likely through the generation of cGMP, is a necessary signal transduction component of GnRH-induced GH secretion.

Quantification of the Effects of Resperine on Gonadotroph Expression in the Pituitary of Goldfish (Carassius auratus)

In many teleosts, the control of gonadotropin II (or luteinizing hormone) secretion is under the dual control of stimulatory and inhibitory neuroendocrine factors. The principal stimulating factor is gonadotropin-releasing hormone and the main inhibitor is dopamine. Inhibiting the activities of dopamine by antidopaminergic drugs potentiates the actions of exogenous gonadotropin-releasing hormone analogs, resulting in a surge release of luteinizing hormone and ovulation and spawning in a number of different species. As the effects of blocking the inhibitory actions of dopamine on gonadotroph cytology have not been studied, goldfish were treated with 2, 4, 6 or 8 injections of reserpine (0.1 mg/kg body weight), at 48 h intervals, and the numbers of gonadotrophic cells studied at 48 h following last injection. After two injections, the number of gonadotrophic cells increased by 189% over controls; after four injections the increase was 234%; after six injections the increase was 259% and after eight injections, 288%. The results suggest that dopamine has an inhibitory influence on the numbers of gonadotrophs.

Estradiol reduces pituitary responsiveness to somatostatin (SRIF-14) and down-regulates the expression of somatostatin sst 2 receptors in female goldfish pituitary

Sex steroid hormones have been shown to regulate somatostatin (SRIF) gene expression in goldfish brain, which in turn influences the regulation of GH secretion. In this study, the influences of sex steroids on pituitary responsiveness to SRIF-14 and the pituitary expression of a type two SRIF receptor (sst 2) were examined. Results from in vitro perifusion of pituitary fragments show that pituitaries from estradiol-primed sexually regressed female fish have significantly lower GH release responsiveness to pulse exposure to SRIF-14 than pituitaries from control or testosterone-treated sexually regressed females. Results from in vitro static culture show that pituitaries from sexually mature female fish have lower GH release responsiveness to SRIF-14 than those from sexually regressed females. In addition, the sst 2 receptor mRNA levels in pituitaries from mature and recrudescent female fish are significantly lower than in sexually regressed female fish. Our results indicate that estradiol acts at the level of the pituitary to regulate GH secretion by influencing the responsiveness to SRIF-14. The underlying mechanism includes, in part, reduction of the expression of sst 2 receptors, presumably leading to the lower number of the receptors available for SRIF binding.

Molecular cloning of estrogen receptor α and expression pattern of estrogen receptor subtypes in male and female goldfish

Estrogen plays an important role in the regulation of gonadotropin production in vertebrates. In this study, we isolated the estrogen receptor (ER) α cDNA from the goldfish pituitary. Primers for ERα were designed based on the similarity of selected regions (C and E domains) of known ER genes. Full-length cDNA sequence for ERα was determined by 3′ and 5′ RACE procedures. ERα cDNA clone was found to contain 2087 nucleotides including an open reading frame that encodes 564 amino acids, with a molecular weight of 62.8 kDa. We also cloned ERβ-1 and ERβ-2 from the published information and investigated the expression pattern of these ER subtypes in a variety of tissues in male and female goldfish by reverse transcriptase-polymerase chain reaction (RT-PCR). Significant variations in the relative expression of ERα, ERβ-1 and ERβ-2 were observed in different tissues in male and female goldfish. Pituitary was found to have the highest expression level of ERα in both male and female goldfish. Significantly, lower levels of ERα expression were observed in the brain, ovary, testis, liver, muscle, heart and intestine. Ovary and testis were found to have higher transcript levels of ERβ-1 with much lower levels in the brain, pituitary, liver, muscle and heart. The ERβ-2 was found to be expressed strongly in the pituitary followed by intestine with lower expression in other tissues. The present study provides molecular characterization of ERα, and information on tissue specific distribution of different ER subtypes in male and female goldfish.

Somatostatin-like receptors in goldfish: cloning of four new receptors

In this study, four somatostatin-like receptor (Sst) cDNAs were identified from goldfish pituitary, using RT-PCR screening and rapid amplification of cDNA ends (RACE) strategies. These include two type-five like Sst (Sst 5B and Sst 5C) and two type-three like Sst receptors (Sst 3A and Sst 3B), designated based on their amino acid sequence similarities to the known mammalian and fish Sst 5 and Sst 3. Both Sst 5C and Sst 3A mRNAs are widely expressed in all brain regions and pituitary; however, Sst 3B expression is restricted to forebrain and Sst 5B expression is mainly detected in pituitary and spinal cord.

Agonist‐Specific and Sexual Stage‐Dependent Inhibition of Gonadotropin‐Releasing Hormone‐Stimulated Gonadotropin and Growth Hormone Release by Ryanodine: Relationship to Sexual Stage‐Dependent Caffeine‐Sensitive Hormone Release

Differential utilization of intracellular Ca2+ stores with specific functional characteristics could be a potential mechanism for coupling various stimuli to specific cellular responses. In the goldfish pituitary, both gonadotropes and somatotropes possess multiple intracellular Ca2+ stores that are differentially coupled to agonist-evoked exocytosis. We investigated the role of ryanodine receptor/Ca2+-release channels (RyR) in basal and gonadotropin-releasing hormone (GnRH)-evoked hormone secretion from cultured gonadotropes and somatotropes using radioimmunoassay for gonadotropin (GTH-II) and growth hormone (GH). As is the case in vivo, the basal and evoked secretion of both hormones varied with seasonal reproductive status. GnRH-stimulated hormone release was three-fold higher in cells from sexually mature animals compared to those in a sexually regressed state. Nanomolar doses of ryanodine evoked significant GTH-II and GH secretion, suggesting that ryanodine-sensitive Ca2+ stores can couple to exocytosis in both cell types. In gonadotropes, 10 microM ryanodine abolished cGnRH-II-evoked GTH-II release in both sexually mature and sexually regressed fish, while sGnRH signalling was mediated by ryanodine-sensitive Ca2+ stores in cells from sexually regressed fish only. Ryanodine-sensitive Ca2+ stores in somatotropes were only involved in cGnRH-II-stimulated GH release during gonadal regression. In contrast, sGnRH-stimulated, but not cGnRH-II-stimulated, GH release was significantly reduced by 1 microM xestospongin C. Although hormone release stimulated by mobilizing caffeine-sensitive Ca2+ pools was also markedly seasonal, it was largely independent of ryanodine-sensitive Ca2+ stores. Ryanodine-sensitive Ca2+ stores in both cell types are not active downstream of ionomycin, BayK 8644, protein kinase C or cyclic adenosine monophosphate signalling pathways, suggesting difference from a classical Ca2+-induced Ca2+ release system. Ours study is the first to suggest that RyR2 may be involved in the seasonal plasticity of pituitary function, which may be related to cyclic changes observed in reproduction and growth.

Identification and characterization of a type five-like somatostatin receptor in goldfish pituitary

In this study, a somatostatin receptor (Sst) cDNA was cloned and sequenced from goldfish pituitary. The cDNA encodes a 390-amino acid type five-like Sst (designated as gfSst 5). The amino acid sequence of the receptor has slightly higher homology to mammalian Sst 5′, compared with other mammalian Sst subtypes and recently identified fish Sst 1, Sst 2 and Sst 3. In CCL39-SRE-Luci cells stably expressing the cloned receptor, agonist radioligand [ 125I]LTT-SRIF 28′, a mammalian SRIF 28 analog, bound to a homogenous population of receptors with high affinity (nM K d). Competition binding studies showed that all three natural goldfish SRIF ligands, SRIF 14, [Pro 2]SRIF 14, and goldfish SRIF 28 (gfSRIF 28), and LTT-SRIF 28 bind the cloned gfSst 5 with high affinity and significantly stimulate [ 35S]GTPγS binding, with SRIF 28 peptides showing higher affinity in receptor binding and potency in [ 35S]GTPγS binding compared with SRIF 14 peptides. The receptor gene is highly and predominately expressed in pituitary; lower levels of the receptor mRNA were also detected in different brain regions by reverse transcriptor-polymerase chain reaction (RT-PCR) followed by Southern blot analysis.

Production of a polyclonal antibody against recombinant goldfish prolactin and demonstration of its usefulness in a non-competitive antigen-capture ELISA

The cDNA encoding the goldfish ( Carassius auratus) prolactin was expressed in Escherichia coli using the pRSETA expression vector. The recombinant goldfish prolactin (gfPRL) produced was a fusion protein containing a hexahistidyl sequence, which facilitated its purification on a Ni 2+ column. The fusion protein was overexpressed in the bacteria as inclusion bodies and was successfully purified under denaturing conditions by one-step affinity chromatography. Repeated immunization of rabbits against the purified recombinant gfPRL allowed the production of a high-titer polyclonal antiserum. The IgG fraction of the antiserum was isolated on an immobilized Protein A–agarose column. The antibody recognized recombinant gfPRL, but not recombinant goldfish growth hormone (gfGH) or goldfish somatolactin (gfSL) on Western analyses. The purified antibody was able to recognize gfPRL, but not gfGH or gfSL, in a non-competitive antigen-capture ELISA. The assay was applied in monitoring the purification of native PRL from goldfish pituitaries.

Molecular characterization of LH-β and FSH-β subunits and their regulation by estrogen in the goldfish pituitary

The gonadal steroids, along with gonadotropin-releasing hormone (GnRH) are involved in the regulation of gonadotropin (GtH) production in vertebrates. Goldfish have an annual reproductive cycle, characterized by seasonal fluctuations in the circulating levels of the reproductive hormones, including 17β-estradiol (E2). The purpose of the present study was to investigate the effect of E2 on basal and GnRH-induced GtH subunit (α, FSH-β and LH-β) gene expression in the goldfish pituitary. Northern analyses were performed to determine changes in steady state mRNA levels. Both in vivo and in vitro treatment with E2 resulted in a stimulation of all three GtH subunit mRNA levels, although a higher concentration was required for the stimulation of the FSH-β subunit mRNA levels. The effect of E2 on GnRH-induced GtH mRNA level was also investigated and demonstrated that E2 influences the GnRH-induced GtH subunit mRNA levels in a seasonally dependent manner. Overall, the present results indicate that E2 stimulates GtH subunit mRNA levels directly at the level of the pituitary in a seasonally dependent manner in goldfish.

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 (α, FSH-β and LH-β) and GH mRNA levels in the goldfish pituitary in vivo and in vitro. Injection of goldfish with sGnRH or cGnRH-II (4 μg/fish) stimulated GtH-α, FSH-β and LH-β 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-α, FSH-β, LH-β 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.