RESUMO
Gonadotropin-releasing hormone (GnRH) is a key neuropeptide of the reproductive system. However, little is known about the role of GnRH in the spotted scat (Scatophagus argus). Here, three GnRH subtypes (cGnRH-II, sGnRH, and sbGnRH) were identified in the spotted scat. cGnRH-II and sGnRH were only expressed in the brains and gonads of both male and female fish, exhibiting a tissue-specific expression pattern, while sbGnRH was expressed at different transcription levels in all examined tissues. During ovarian maturation, hypothalamus-associated sbGnRH was upregulated, while the expression of sGnRH was variable and cGnRH-II first increased and then decreased. In vivo experiments showed that sbGnRH significantly promoted the expression of fsh and lh genes in a dose-dependent manner and exhibited a desensitization effect on lh expression at high concentrations. For sGnRH and cGnRH-II, only high concentrations could induce fsh and lh expression. Furthermore, treatment with highly concentrated sbGnRH peptide also induced fsh and lh expression, whereas the sGnRH and cGnRH-II peptides only induced fsh expression in vitro. 17ß-Estradiol (E2) significantly inhibited the expression of sbGnRH mRNA in a dose-dependent manner and did not impact sGnRH and cGnRH-II mRNA levels in vivo or in vitro. The inhibitory effect of E2 on sbGnRH expression was attenuated by the estrogen receptor (ER) broad-spectrum antagonist (fulvestrant) and the ERα-specific antagonist (methyl-piperidinopyrazole), respectively, implying that the feedback regulation on sbGnRH is mediated via ERα. This study provides a theoretical basis for the reproductive endocrinology of the spotted scat by studying GnRH.
Assuntos
Estrogênios/metabolismo , Peixes/fisiologia , Hormônio Liberador de Gonadotropina/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Estradiol , Feminino , Hormônio Foliculoestimulante/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/genética , Hipotálamo , Hormônio Luteinizante/metabolismo , Ovário/crescimento & desenvolvimento , Filogenia , Receptores de Estrogênio/antagonistas & inibidores , Transcriptoma/efeitos dos fármacosRESUMO
Phoenixin (Pnx) is an endogenous peptide known to be involved in reproduction and food intake in rats, with two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleost. Here, pnx was cloned and was detected in all tissues of both male and female in spotted scat (Scatophagus argus), including growth axis, hypothalamus, pituitary, and liver. Real-time PCR analysis showed that pnx in the hypothalamus increased significantly after 2â¯d and 7â¯d fasting, while reduced significantly after re-feeding (Pâ¯<â¯0.05). When pituitary and liver fragments were cultured in vitro with Pnx-14 and Pnx-20 (10â¯nM and 100â¯nM) for 6â¯h, the expression of ghrhr (growth hormone-releasing hormone receptor) and gh (growth hormone) in the pituitary, and ghr1 (growth hormone receptor 1) in the liver increased significantly, except ghr2 (growth hormone receptor 2) incubated with 10â¯nM and 100â¯nM Pnx-20 and ghr1 incubated with 10â¯nM Pnx-20. Similarly, the expression of ghrhr and gh in the pituitary, as well as ghr1 and ghr2 in the liver, increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10â¯ng/g and 100â¯ng/g body weight). These results indicate that Pnx is likely to be involved in the regulation of food intake, and also regulates the growth of S. argus by increasing ghrhr and gh expression in the pituitary, ghr1 and ghr2 in the liver, and ghr1 directly in the liver.
Assuntos
Ingestão de Energia , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hormônios Hipotalâmicos/metabolismo , Hipotálamo/metabolismo , Hormônios Peptídicos/metabolismo , Perciformes/fisiologia , Animais , Aquicultura , China , Ingestão de Energia/efeitos dos fármacos , Feminino , Proteínas de Peixes/administração & dosagem , Proteínas de Peixes/genética , Proteínas de Peixes/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônio do Crescimento/agonistas , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Hormônios Hipotalâmicos/administração & dosagem , Hormônios Hipotalâmicos/genética , Hormônios Hipotalâmicos/farmacologia , Hipotálamo/efeitos dos fármacos , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Especificidade de Órgãos , Hormônios Peptídicos/administração & dosagem , Hormônios Peptídicos/genética , Hormônios Peptídicos/farmacologia , Perciformes/crescimento & desenvolvimento , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Isoformas de Proteínas/administração & dosagem , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Distribuição Aleatória , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/agonistas , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Receptores da Somatotropina/agonistas , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Técnicas de Cultura de Tecidos/veterinária , Aumento de PesoRESUMO
Spexin (Spx), a novel neuropeptide, composed of 14 amino acid residues, is evolutionally conserved from fish to mammals. It has been suggested that Spx has pleiotropic functions in mammals. However, reports about Spx are very limited. To clarify the roles of Spx in the regulation of reproduction and food-intake in the spotted scat, the spx (ssspx) gene was cloned and analyzed. Analysis of the tissue distribution by RT-PCR showed that ssspx expression was widespread. During ovary development, expression of ssspx was found to be highest in phase II, moderate in phase III, and at its lowest level in phase IV. Ssspx expression was significantly down-regulated in the hypothalamus after treatment with E2 both in vitro and in vivo. A significant increase of ssspx was observed after 2 and 7â¯days of food deprivation. However, the ssspx transcript levels in the 7â¯day fasting group decreased significantly after refeeding 3â¯h after the scheduled feeding time. This suggests that ssSpx may be involved in the regulation of reproduction and food-intake in the spotted scat.
Assuntos
Perfilação da Expressão Gênica , Hormônios Peptídicos/genética , Perciformes/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Estradiol/farmacologia , Jejum , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Ovário/efeitos dos fármacos , Ovário/embriologia , Ovário/metabolismo , Hormônios Peptídicos/química , Hormônios Peptídicos/metabolismo , Perciformes/metabolismo , Filogenia , Reprodução , Alinhamento de Sequência , Distribuição Tecidual/efeitos dos fármacosRESUMO
Melanocortin-4 receptor (Mc4r) function related to reproduction in fish has not been extensively investigated. Here, we report on gene expression changes by real-time PCR following treatment with Mc4r agonists and antagonists in the spotted scat (Scatophagus argus). Using in vitro incubated hypothalamus, the Mc4r nonselective agonist NDP-MSH ([Nle4, D-Phe7]-α-melanocyte stimulating hormone; 10-6 M) and selective agonist THIQ (N-[(3R)-1, 2, 3, 4-Tetrahydroisoquinolinium-3-ylcarbonyl]- (1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl) piperidin-1-yl]-2-oxoethylamine; 10-7 M) significantly increased the expression of gnrh (Gonadotropin releasing hormone), while the Mc4r nonselective antagonist SHU9119 (Ac-Nle-[Asp-His-DPhe/DNal(2')-Arg-Trp-Lys]-NH2; 10-6 M) and selective antagonist Ipsen 5i (compound 5i synthesized in Ipsen Research Laboratories; 10-6 M) significantly inhibited gnrh expression after 3 h of incubation. In incubated pituitary tissue, NDP-MSH and THIQ significantly increased the expression of fshb (Follicle-stimulating hormone beta subunit) and lhb (Luteinizing hormone beta subunit), while SHU9119 and Ipsen 5i significantly decreased fshb and lhb expression after 3 h of incubation. During the in vivo experiment, THIQ (1 mg/kg bw) significantly increased gnrh expression in hypothalamic tissue, as well as the fshb and lhb expression in pituitary tissue 12 h after abdominal injection. Furthermore, Ipsen 5i (1 mg/kg bw) significantly inhibited gnrh expression in hypothalamic tissue, as well as fshb and lhb gene expression in pituitary tissue 12 h after abdominal injection. In summary, Mc4r singling appears to stimulate gnrh expression in the hypothalamus, thereby modulating the synthesis of Fsh and Lh in the pituitary. In addition, Mc4r also appears to directly regulate fshb and lhb levels in the pituitary in spotted scat. Our study suggests that Mc4r, through the hypothalamus and pituitary, participates in reproductive regulation in fish.