Identification Of Gonadotropin-inhibitory Hormone And Kisspeptin Systems And Their Physiological Roles In The Half-smooth Tongue Sole(Cynoglossus Semilaevis)

Gonadotropin-inhibitory hormone(GnIH)and kisspeptin(Kiss)have been found to play key roles in the control of reproduction in mammals.However,not much information is available regarding the precise physiological roles of these two novel hypothalamic neuropeptides in teleosts.To this end,we first obtained the full-length cDNAs of gnih and its receptor gnihr,kiss2 receptor(kiss2r)and a partial-length cDNA of kiss2 from the brain of the half-smooth tongue sole(Cynoglossus semilaevis)by molecular cloning.The Gn IH precursor encoded two putative peptides(Gn IH-1 and Gn IH-2),while the Kiss2 precursor encoded the core decapeptide(Kiss2-10).Subsequently,the tissue distribution of gnih,gnihr,kiss2 and kiss2 r mRNAs was analyzed in female tongue sole by quantitative real-time PCR.Moreover,the expression patterns of gnih mRNA during ovarian maturation were also investigated.Finally,the effects of Gn IH and Kiss2 peptides on the control of tongue sole reproduction were evaluated using the hypothalamus and pituitary culture systems.The main results are as follows: 1.Molecular identification of GnIH/GnIHR system 1.1 cDNA cloning and tissue distribution of gnih and gnihr in tongue soleThe full-length cDNA of gnih was 918 bp in size with an open reading frame(ORF)of 585 bp that encoded a 194 amino acids preprohormone with a calculated molecular mass and isoelectric point of 21.73 kDa and 6.52,respectively.The GnIH precursor encoded two putative peptide sequences that included –MPMRF or –MPQRF motifs at the C-terminal.Tissue distribution analyses showed that gnih transcripts could be detected at high levels in the brain,to a lesser extent in the ovary,heart and stomach,and at low levels in the pituitary and other peripheral tissues.The full-length cDNA of gnihr was 2201 bp in size with 5' UTR of 60 bp,3'UTR of 776 bp and ORF of 1365 bp that encoded 454 amino acids.The GnIHR had seven hydrophobic transmembrane domains structure,which is a characteristic of GPCRs.In addition,Gn IHR had seven phosphorylation sites and five glycosylation sites.Tissue distribution showed that gnihr transcripts could be detected at high levels in the brain,to a lesser extent in the pituitary,and at low levels in the ovary and other peripheral tissues.1.2 Expression profiles of gnih mRNA during ovarian maturationIn the brain,the mRNA expression of gnih increased significantly at stage III,declined at stage V and reached a maximum at stage VI.In the pituitary,the levels of gnih mRNA remained stable during ovarian maturation and increased significantly to the top level at stage V and then declined back to basal levels.In contrast,the ovarian gnih mRNA levels declined sharply at stage III and remained depressed over the course of ovarian maturation.1.3 Role of GnIH on the hypothalamus functions of tongue soleWe evaluated the effects of GnIH on the reproduction-related gene expression in the hypothalamus of tongue sole for the first time.Our results showed that GnIH-1 increased the expression of gnrh2 and gnih mRNAs,but had no effects on gnrh3 and kiss2 mRNA expression.On the other hand,Gn IH2 significantly inhibited gnrh3 mRNA expression.However,Gn IH2 did not alter gnrh2,kiss2 and gnih mRNA levels.1.4 Role of GnIH on the pituitary functions of tongue soleIn addition,we examined the effects of GnIH on gonadotropin(GTH)and growth hormone(GH)synthesis and release from primary pituitary cultures.GnIH-1 increased the amounts of gh mRNA.A similar trend was observed in lh? and fsh? mRNA levels.In contrast,GnIH-2 did not alter gh,lh? and fsh? m RNA levels.Moreover,no significant changes were observed in the abundance of gth? mRNA after exposure to GnIH-1 and Gn IH-2.Slightly but significantly,both Gn IH-1 and GnIH-2 inhibited LH release,but they had no effects on the release of GH and FSH.2 Molecular identification of Kiss/KissR system 2.1 cDNA cloning and tissue distribution of kiss2 and kiss2 r in tongue soleA partial-length cDNA of kiss2 was identified from the brain of tongue sole.The kiss2 cDNA was composed of 274 bp encoding a 90 amino acid preprohormone with a calculated molecular mass and isoelectric point of 9.99 kDa and 11.08,respectively.The prepropeptide contained the conserved decapeptide,followed by GKR that corresponded to a potential site of terminal cleave and amidation.The expression pattern of kiss2 in various tissues was determined and kiss2 transcripts were clearly most abundant in the ovary,to a lesser extent in the brain and pituitary.However,other tissues examined had lower but detectable levels of kiss2.The full-length cDNA of kiss2 r was 1966 bp with 5' UTR of 342 bp,3'UTR of 481 bp and ORF of 1143 bp that encoded 380 amino acids.Kiss2 R also belongs to the GPCR superfamily,and it has eight phosphorylation sites and three glycosylation sites.Kiss2 r transcripts were most abundant in the brain,to a lesser extent in the heart,gill and pituitary.However,expression levels were close to detection limits in the other tissues.2.2 Role of Kiss2 on the hypothalamus functions of tongue soleIn order to clarify the potential physiological role of Kiss2 in tongue sole,we evaluated the effects of Kiss2 decapeptide on the reproduction-related gene expression in the hypothalamus of tongue sole.Our results showed that Kiss2 did not alter the expression of gnrh2 and gnrh3 mRNAs.However,Kiss2 significantly stimulated both gnih and kiss2 mRNA levels,while gnihr and kiss2 r mRNA levels were evidently suppressed by Kiss2.2.3 Role of Kiss2 on the pituitary functions of tongue soleSimilarly,we also evaluated the effects of Kiss2 decapeptide on the synthesis and release of GTH and GH using a primary pituitary culture system.Our results showed that neither gh nor gth subunits mRNA levels were modified by Kiss2.In addition,Kiss2 did not alter GH release,whereas Kiss2 inhibited the release of LH and FSH.