Amino Acid Neurotransmitters and Dopamine in Brain and Pituitary of the Goldfish: Involvement in the Regulation of Gonadotropin Secretion

An isocratic high-performance liquid chromatographic technique was developed to measure levels of gamma-aminobutyric acid (GABA), glutamate, and taurine in the brain and pituitary of goldfish. Accuracy of this procedure for quantification of these compounds was established by evaluating anesthetic and postmortem effects and by selectively manipulating GABA concentrations by intraperitoneal administration of the glutamic acid decarboxylase (GAD) inhibitor 3-mercaptopropionic acid or the GABA transaminase inhibitor gamma-vinyl GABA. The technique provided a simple, rapid, and reliable method for evaluating the concentrations of these amino acids without the use of complex gradient chromatographic systems. To investigate the relationship between neurotransmitter amino acids and the control of pituitary secretion of gonadotropin, the effects of injection of taurine, GABA, or monosodium glutamate on GABA, glutamate, taurine, and, in some instances, monoamine concentrations in the brain and pituitary were evaluated and related to serum gonadotropin levels. Injection of taurine caused an elevation in serum gonadotropin concentrations. In addition, injection of the taurine precursor hypotaurine but not the taurine catabolite isethionic acid elevated serum gonadotropin levels. Intracerebroventricular injection of either GABA or taurine also elevated serum gonadotropin concentrations. Pretreatment of recrudescent fish with alpha-methyl-p-tyrosine reduced pituitary dopamine concentrations and also potentiated the serum gonadotropin response to taurine. Injection of monosodium glutamate caused an increase of glutamate content in the pituitary at 24 h; this was followed by a decrease at 72 h after administration. Pituitary GABA, taurine, and dopamine concentrations underwent a transient depletion after monosodium glutamate administration, and this was associated with an elevation of serum gonadotropin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of dopamine and norepinephrine on in vitro spontaneous and gonadotropin-releasing hormone-induced gonadotropin release by dispersed cells or fragments of the goldfish pituitary

Enzymatically dispersed goldfish pituitary cells or freshly prepared goldfish pituitary fragments continue to secrete gonadotropin spontaneously in a column perifusion system. After the establishment of basal secretion rates, treatment of dispersed pituitary cells with 5 and 500 nM dopamine, or pituitary fragments with 50 and 100 nM dopamine, decreased the amount of gonadotropin released into the perifusate. Perifusion with 500 nM dopamine also abolished the gonadotropin-release response to a 10 nM solution of a luteinizing hormone-releasing hormone analogue in both perifusion systems. Perifusion of pituitary dispersed cells or fragment preparations obtained from sexually regressed goldfish with 50 nM norepinephrine consistently increased the amount of gonadotropin released into the perifusate. These results provide in vitro evidence for direct dopamine inhibition of spontaneous gonadotropin release, blockade by dopamine of gonadotropin-releasing hormone actions, and norepinephrine stimulation of gonadotropin secretion in goldfish.     

注射促黄体素释放激素类似物和地欧酮诱导大鳍■和长吻■排卵的研究

对于性成熟的大鳍Ｍｙｓｔｕｓｍａｃｒｏｐｔｅｒｕｓ（Ｂｌｅｅｋｅｒ）野生鱼,单独注射多巴胺的抑制剂地欧酮（ＤＯＭ）不能影响血清促性腺激素（ＧＴＨ）水平,也不能诱导排卵;单独注射类似物ＬＨＲＨ－Ａ,虽能使血清ＧＴＨ水平显著升高,但仅产生较低的排卵率;而当ＤＯＭ与ＬＨＲＨ－Ａ结合注射却显著增强ＬＨＲＨ－Ａ促进血清ＧＴＨ水平升高的作用,并诱导出较高的排卵率。对性成熟的长吻ＬｅｉｏｃａｓｉｓｌｏｎｇｉｒｏｓｔｒｉｓＧｕｎｔｈｅｒ野生鱼,使用ＬＨＲＨ－Ａ＋ＤＯＭ作２次注射诱导排卵的效果也与注射ＬＨＲＨ－Ａ加脑垂体这一传统诱导排卵方法相似。Ｌｉｎｐｅ方法（ＬＨＲＨ－Ａ＋ＤＯＭ,作１次或２次注射）避免了采集、保存脑垂体不便给生产带来的麻烦,在科鱼类的人工繁殖中,具有较高的推广价值。     

诱导大鳍鱯和长吻鮠排卵过程中血清GTH水平的变化

繁殖期从嘉陵江收集性成熟的大鳍■ 和长吻 野生亲鱼,用Ｌｉｎｐｅ方法（即ＬＨＲＨ－Ａ加多巴胺Ｄ２受体拮抗剂地欧酮）或传统的ＬＨＲＨ—Ａ加脑垂体的方法进行催产,定时取血样,用放射免疫方法测定催产过程中血清ＧＴＨ水平的变化,进一步证实鲇形目鱼类ＧＴＨ的分泌受到下丘脑分泌的促性腺激素释放激素ＧｎＲＨ和多巴胺的双重调节;排卵和产卵也是以血清ＧＴＨ的急剧升高为先导的,而最终能否排卵还有赖于血清ＧＴＨ峰是否超过“排卵阈值”。尽管催产后的大鳍 和长吻 雄鱼血清ＧＴＨ水平也有一个高峰出现,但血清ＧＴＨ水平升高幅度都大大低于雌鱼,这种现象在硬骨鱼类可能具有普遍性。     

多马胺能药物对鲇鱼促性腺激素（GtH）分泌活动的影响

以珠江流域鲇鱼（silurus asotus)为实验材料,研究了多巴胺（DA）能药物（DA及其D－2型受体拮抗物 ,DOM）对鲇鱼促性腺激素（GtH)释放的影响,结果表明,在性腺发育的各个时期,单独注射DOM（5ug/g)均不能显著提高鲇鱼血液基础GtH水平,当DOM与LHRH－A联合注射时能显著增强LHRH－A刺激GtH释放的作用;DA只能抑制GnRH诱导的GtH释放,对基础GtH释放无抑制作用,这种生殖内分泌调节方式与鲇形目的革胡子鲇（Clarias gariepinus)和大鳍Hu（Mystus macropterus)相似,而与鲤形目的鲁科（Cyrpindiae)鱼类不同。     

The role of domperidone in the regulation of prolactin release in rats

Effects of domperidone, a dopamine antagonist, on prolactin release in female rats were studied. Oral administration of domperidone for 14 days caused a significant increase in serum prolactin levels in mature female rats. The routes by which domperidone exerted its effects on prolactin release were studied by a in vitro incubation system using rat pituitary tissues. Pituitary halves were incubated with (1) domperidone, (2) dopamine, (3) dopamine plus domperidone, (4) hypothalamic extracts from rats which had been treated with control meal (control hypothalamic extract), (5) control hypothalamic extract plus domperidone, and with (6) hypothalamic extract from rats which had been treated with domperidone for 14 days (domperidone-treated hypothalamic extract). Pituitary halves, when incubated alone, released a significant amount of prolactin into the incubation medium after 24 hours incubation, which was completely inhibited by dopamine or control hypothalamic extract. The addition of domperidone could not reverse the inhibitory effect of dopamine or control hypothalamic extract. On the other hand, domperidone-treated hypothalamic extract showed no inhibitory effects on prolactin release. These results indicated that domperidone could increase serum prolactin levels in female rats by acting primarily at the hypothalamus.     

Recent trends in research on induced spawning of fish in aquaculture

Since 1973, the treatment of sexually mature fish with brain hormones (i. e. neurohormones) in order to induce spawning activity has gradually been replacing the hypophysation, although the latter is still widely used. Some brain-hormone analogues have a hightened spawning-inducing effect. Since the discovery that dopamine inhibits gonadotropic hormone release, dopamine antagonists—pimozide or domperidone—are injected before or together with brain-hormone analogues. This double treatment, i. e. the suppression of dopamine inhibition followed by neurohormone stimulation, has become a current technique in aquaculture. The discovery of the pulsatile release of gonadotropic hormone from the pituitary hints at the possibility of using techniques in which exogenous hormones are injected in pulses. In the past few years, induction of spawning through the control of photoperiod and/or temperature has become increasingly important.     

Testosterone and estradiol potentiate the serum gonadotropin response to gonadotropin-releasing hormone in goldfish

The effects of gonadal steroids on gonadosomatic index (GSI; gonad wt/total body wt x 100), pituitary gonadotropin (GTH) content, and serum GTH response to [D-Ala6,Pro9-Net]-luteinizing hormone-releasing hormone (LHRH-A) were investigated throughout the seasonal reproductive cycle of the goldfish. Gonad-intact female fish were implanted i.p. for 5 days with silastic pellets containing no steroid (blank), testosterone (T; 100 micrograms/g), or estradiol (E2; 100 micrograms/g). The serum GTH response at 6 h following i.p. injection of saline or 0.1 microgram/g LHRH-A was assessed. In blank-implanted, saline-injected animals, seasonal variations in GSI, pituitary GTH content, and serum GTH levels were evident; maximal and minimal levels were noted in the spring and summer months, respectively. In blank-implanted fish, LHRH-A effectively stimulated GTH release in females undergoing gonadal recrudescence (late autumn and winter) and in sexually mature (spring) females, but not in sexually regressed (summer and early autumn) females. Implantation of T or E2 raised serum steroid levels to those found during ovulation in goldfish. Steroid treatments did not affect unstimulated serum GTH levels at any time of the year. Testosterone effectively potentiated the serum GTH response to LHRH-A during the entire reproductive cycle, whereas the positive effects of E2 were evident in sexually regressed and post-spawning females only. Both T and E2 potentiated the GTH response to LHRH-A in male fish. To examine the involvement of T aromatization in mediating its actions on induced GTH secretion, male and female fish were implanted with T or the nonaromatizable androgens 5 alpha-dihydroxytestosterone (DHT; 100 micrograms/g) and 11-keto-testosterone (11-KT; 250 micrograms/animal). Testosterone potentiated the GTH response to LHRH-A in both males and females whereas DHT and 11-KT were without effect. Furthermore, the positive action of T on induced GTH secretion was blocked by 2-day pretreatment with the aromatase inhibitor 1,4,6-androstatrien-3,17-dione (100 or 300 micrograms/g). Multiple i.p. injections of hCG (0.2 microgram/g every 3 days for 39 days), probably through stimulation of endogenous T secretion, resulted in potentiation of the GTH response to LHRH-A in mature male goldfish. These results clearly demonstrate that T, through aromatization to E2, can increase pituitary responsiveness to exogenous LHRH-A in gonad-intact male and female goldfish.     

Presynaptic inhibition of excitatory input from the substantia nigra to caudate nucleus neurons by a substituted quinolinone derivative, 7-[3-(4-(2,3-dimethylphenyl)piperazinyl)propoxy]-2(1H)-quinolinone (OPC-4392)

The effects of a newly synthesized quinolinone derivative, 7-[3-(4-(2,3-dimethylphenyl)piperazinyl) propoxy]-2(1H)-quinolinone (OPC-4392) on neuronal activities of the caudate nucleus (CN) were investigated in cats anesthetized with alpha-chloralose using a microiontophoretic method. In the CN neurons of which spikes elicited by stimulation of the pars compacta of substantia nigra (SN) were suppressed by iontophoretically applied domperidone, a dopamine D-2 receptor antagonist, application of OPC-4392 (100-200 nA) inhibited the spike generation induced by SN stimulation. Conversely, the CN neurons insensitive to domperidone were unaffected by OPC-4392. Iontophoretic application of CPC-4392 up to 200 nA did not affect glutamate-induced firing of the CN neurons, of which the firing was blocked by dopamine less than 100 nA. In addition, OPC-4392 did not inhibit firing induced by bromocriptine, a dopamine D-2 agonist; while domperidone suppressed the bromocriptine-induced firing without affecting the glutamate-induced firing. These results suggest that OPC-4392 acts on the dopaminergic nerve terminals and inhibits excitatory transmission from the SN to the CN.     

Dopamine D-2 receptor-mediated excitation of caudate nucleus neurons from the substantia nigra

Microiontophoretic studies using cats anesthetized with alpha-chloralose were performed to elucidate whether the excitatory response of caudate nucleus (CN) neurons upon stimulation of the pars compacta of the substantia nigra (SN) is mediated by the dopamine D-1 or D-2 receptor. There were rare convergent inputs from the SN and motor cortex (MC) in the CN neurons. Iontophoretic application of haloperidol and domperidone (dopamine D-2 receptor antagonist) produced dose-dependent inhibition of spikes elicited by SN stimulation in 25 of 42 and 50 of 82 CN neurons, respectively, however, no alterations of spikes elicited by MC stimulation occurred in any 11 neurons tested. Iontophoretically applied SCH 23390 (D-1 antagonist) did not inhibit the SN-induced spikes in any CN neurons, of which spikes were inhibited by domperidone. These results suggest that the SN-induced spikes are mediated by dopamine, which acts on postsynaptic D-2 receptors.     

Excitation by dopamine D-2 receptor agonists, bromocriptine and LY 171555, in caudate nucleus neurons activated by nigral stimulation

Electrophysiological studies using cats anesthetized with alpha-chloralose were carried out to determine whether or not the dopamine D-2 receptor mediates the excitation of the caudate nucleus (CN) neurons activated by stimulation of the substantia nigra (SN). Microiontophoretic application of domperidone (D-2 antagonist) produced a significant inhibition of spikes elicited by SN stimulation in 20 of 27 CN neurons. When bromocriptine and LY 171555 (D-2 agonists) were iontophoretically applied to the CN neurons in which the SN-induced spikes were inhibited by domperidone, an increase in spontaneous firing rate was observed in 18 of 20 neurons and all of 10 neurons tested, respectively. However, no alterations of firing occurred with bromocriptine or LY 171555 in any 7 neurons in which the SN-induced spikes were not affected by domperidone. The increase in firing rate by the D-2 agonists was apparently antagonized during simultaneous application of domperidone and haloperidol, but not affected during application of SCH 23390 (D-1 antagonist). These results strongly suggest that the spike generation of the CN neurons upon SN stimulation is mediated by the dopamine D-2 receptor.     

Coexistence of inhibitory dopamine D-1 and excitatory D-2 receptors on the same caudate nucleus neurons

Microiontophoretic studies using cats anesthetized with alpha-chloralose were performed to determine whether or not dopamine D-1 and D-2 receptors co-exist in the same caudate nucleus (CN) neurons that receive inputs from the substantia nigra (SN), and in which spikes elicited by SN stimulation were blocked by domperidone, a selective D-2 antagonist. Iontophoretic application of dopamine produced a dose-dependent inhibition of spontaneous firing in 2 of 4 spontaneously active CN neurons and an increase in firing in the remaining 2 neurons. However, dopamine inhibited the glutamate-induced firing in 31 of 32 CN neurons that were not spontaneously active. Similar inhibition with iontophoretically applied SKF 38393, a selective D-1 agonist, was observed in 33 of 34 spontaneously inactive neurons tested. When the effects of dopamine, SKF 38393 and bromocriptine (D-2 agonist) were examined on the same CN neurons, the inhibitory effects of both dopamine and SKF 38393 were seen in 14 of 15 neurons, and both an inhibition by SKF 38393 and an excitation by bromocriptine were observed in 15 of 17 neurons. The inhibitory effects of dopamine and SKF 38393 were antagonized by haloperidol and SCH 23390 (D-1 antagonist) without being affected by domperidone. Furthermore, the dopamine-induced inhibition was converted to an excitation during simultaneous application of SCH 23390 in 6 of 10 CN neurons, and this excitation was antagonized by domperidone. These results strongly suggest that the inhibitory D-1 and excitatory D-2 receptors co-exist on the same CN neurons receiving inputs from the SN.     

Direct effects of triiodothyronine on production of anterior pituitary hormones and gonadal steroids in goldfish

The present study investigated the effects of triiodothyronine (T3) on pituitary gonadotropin (GTH) subunits, thyroid stimulating hormone (TSH) β subunit, and growth hormone (GH) mRNA levels, as well as gonadal steroid secretion during different stages of reproduction in goldfish. Goldfish pituitary cells cultured with T3 exhibited lower tshβ mRNA levels in all reproductive stages and lower luteinising hormone β (lhβ) mRNA levels in early recrudescence, whereas gh and fshβ mRNA levels were not altered. T3 injections significantly reduced circulating oestrogen (OE2) concentrations in early and mid recrudescent male goldfish, but were without effect on the circulating level of OE2 in female fish. T3 injections also reduced circulating levels of testosterone in both male and female goldfish during the mid stage of gonadal recrudescence. In vitro culture of goldfish ovarian follicles at the late stage of gonadal recrudescence, in the presence of T3, resulted in reduced OE2 secretion; no consistent effect of T3 on testosterone secretion was observed in cultured goldfish ovarian follicles and testis. These findings support the hypothesis that T3 impairs reproduction by inhibiting production of gonadal steroids and pituitary luteinising hormone production in goldfish. Mol. Reprod. Dev. 79: 592–602, 2012. © 2012 Wiley Periodicals, Inc.     

Evidence for alternative splicing of a dopamine D2 receptor in a teleost

Previous attempts at identifying an alternatively spliced dopamine (DA) D2 receptor in teleosts have proven unsuccessful. We provide evidence of a splicing event of a goldfish D2 (gfD2b1) receptor in the neuroendocrine brain of adult goldfish that produces a spliced short isoform (gfD2b1S). We also identify an additional novel D2b paralog (gfD2b2) that does not appear to be alternatively spliced in adult fish during the reproductive cycle. Relatively high mRNA levels of gfD2b1S were observed in the neuroendocrine brain and pituitary of sexually immature fish compared with sexually regressing fish. Real-time RT-PCR revealed that intraperitoneal injection of either SCH 23390 or sulpiride-D1- or D2-specific antagonists, respectively-decreased mRNA levels of gfD2b1S by 3.9-fold without affecting the unspliced isoforms. We suggest that the expression of the spliced D2 receptor modulates the inhibitory tone of DA throughout the reproductive cycle. The deduced amino acid sequence of gfD2b1S lacks 29 amino acids in the same region as the short isoform of mammalian D2. We propose that the gfD2b1S splice variant is the teleost ortholog of mammalian D2S. The hypothesis that D2 receptor splicing is a relatively recent innovation in higher tetrapods is not supported by our results.     

Hyperactivity induced by stimulation of separate dopamine D-1 and D-2 receptors in rats with bilateral 6-OHDA lesions

The effects of DA agonists and antagonists with different dopamine (DA) D-1 and D-2 receptor selectivity have been studied in rats with bilateral 6-OHDA lesions. The D-1 agonist SK & F 38393, the D-2 agonist pergolide and the mixed agonist apomorphine all induced marked hyperactivity in lesioned rats in doses which were without stimulant effect in sham-operated animals. The hyperactivity induced by SK & F 38393 was blocked by the DA D-1 antagonist SCH 23390, but unaffected by the D-2 antagonists spiroperidol or clebopride. Pergolide-induced hyperactivity showed the reverse selectivity. The mixed D-1/D-2 antagonists, cis(Z)-flupentixol and cis(Z)-clopenthixol, however blocked the effect of both agonists. Apomorphine-induced hyperactivity was neither blocked by selective D-1 nor D-2 antagonists, but was dose-dependently inhibited by cis(Z)-flupentixol and cis(Z)-clopenthixol. Potent blockade was also obtained by combined treatment with SCH 23390 and spiroperidol, indicating the need of blocking both D-1 and D-2 receptors simultaneously. The results indicate that D-1 and D-2 receptor function can be independently manipulated in denervated rats and they confirm similar results obtained in rats with unilateral 6-OHDA lesions using circling behaviour.     

Effects of brain lesions on gonadotrop ultrastructure and serum gonadotropin levels in goldfish

Summary Brain lesions that destroyed the anterior preoptic region or the pituitary stalk in sexually mature (= completed ovarian recrudescence) goldfish caused a significant increase in serum gonadotropin levels for at least 11 days postoperatively. These results confirmed previous findings indicating the presence of a gonadotropin release-inhibitory factor. Electron-microscopic investigation revealed that the gonadotrops were depleted of the small secretory granules, had marked dilations of the cisternae of the endoplasmic reticulum and extensive development of the Golgi apparatus. This indicated both secretion and synthesis, and correlated with the prolonged increase in serum gonadotropin resulting from the lesions.Supported by grants from Natural Sciences and Engineering Research Council of Canada to W.S. Hoar and R.E. Peter     

Levels of specifically bound [3H]ketanserin compared with levels of serotonin (5HT) in the brain regions of juvenile and sexually recrudescing female rainbow trout, Oncorhynchus mykiss

This study compared the distribution of specifically bound [3H]ketanserin (Bsp) with serotonin (5HT) in brain regions of juvenile and sexually recrudescing female trout. Amounts of Bsp varied widely among brain regions and consistently differed between juvenile and sexually recrudescing females. Levels of Bsp were significantly greater in the hypothalamus than the olfactory lobe, which were at least threefold greater than in all other tissues examined (Kruskal-Wallis test, p < 0.05). Bsp densities in the hypothalamus, preoptic area, and optic lobe were significantly greater in juveniles compared with corresponding tissues from sexually recrudescing females (Mann-Whitney U test, p < 0.05); in contrast, Bsp in olfactory lobe and spinal cord did not differ significantly between the two classes of fish. 5HT concentration was determined by high performance liquid chromatography - electrochemical detection (HPLC-EC) analysis. Biogenic amine standards eluted in a stereotypic pattern, with peaks consistently separable in time. 5HT concentration was significantly greater in hypothalamus than in olfactory lobe and undetectable in the pituitary (Kruskal-Wallis test, p < 0.05). Trends in distribution of Bsp and 5HT were comparable in the hypothalamus and preoptic area in juvenile and sexually recrudescing females. In general, density of specific [3H]ketanserin binding sites was directly related to 5HT content of brain regions in juvenile and sexually recrudescing females. 5HT concentrations (pmol/g tissue) were approximately 900-fold greater than Bsp (fmol/g tissue) in all brain regions, and approximately 300-fold greater than Bsp in the olfactory lobe. These results suggest important regulatory role(s) for 5HT in the trout preoptic-hypothalamo-hypophysial axis, which may differ from 5HT role(s) in trout olfactory lobe.     

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.