Cloning And Expression Of Gdf9,Alk5 And Bmpr2 And Preliminary Studies On Its Function In Schizothorax Prenanti

The TGF-βsuperfamily and its receptors played an important role in the development of gonads.To explore the function of growth differentiation factor-9（gdf9）and its receptors,bone morphogenetic protein receptor 2（bmpr2）and activin receptor-like kinase 5（alk5）,during the gonadal development of Schizothorax prenanti,in the present study,the full-length of gdf9,bmpr2 and alk5 cDNA were cloned,their expression patterns were analyzed in gonad and exogenous E2-fed juvenile fish.The main results were as follows.1.The full-length cDNA of gdf9 was 1958 bp,encoding 413 amino acids.There was a typical TGF-βdomain in the deduced amino acid sequence,and its molecular weight was approximately 47.6 k Da.Phylogenetic analysis showed that it was close to Cyprinus carpio,gibel carp and Danio rerio.Expression pattern analysis shown that the expression of gdf9 in gonads was significantly higher than that in other tissues;in ovary,the expression of gdf9 significantly increased at perinucleolar stage ovary,and then decreased at mid-vitellogenic stage ovary;while the expression in testis was significantly decreased in mid-spermatogenic testis,and then increased significant at the late-spermatogenesis.2.The bmpr2 cDNA of S.prenanti was 3704 bp,which encoded 1070 amino acids residues with 116.4 k Da.The amino acid sequence contained six N-glycosylation sites and a transmembrane domain.Relation analysis found that the bmpr2 was closest to D.rerio.Compared with other tissues,highest expression was detected in gonads,and the expression levels in the gonad significant increased at perinucleolar stage ovary and early-spermatogenic testis,and then decreased at mid-vitellogenic stage ovary and late-spermatogenesis.3.The full-length 1952 bp of alk5 cDNA sequence were obtained.The deduced amino acid had 501 amino acids with about 56.5 k Da,including two N-glycosylation sites,a transmembrane domain and one typical SSGSGG domain.The phylogenetic tree results showed that S.prenanti and D.rerio were clustered together.The expression of alk5 in gonads was significantly higher than that in other tissues;the expression of alk5 in ovary increased firstly and then decreased at perinucleolar stage ovary and early-spermatogenic testis.4.Gdf9,Bmpr2 and Alk5 proteins was both localized in ovary and testis.The results of immunohistochemistry showed that Gdf9 positive signal was detected in primary oocytes,cortical alveoli stage oocytes,vitellogenic stage oocytes and granulosa cells.The positive signal of Bmpr2 was detected in the theca and cytoplasm of oocytes and granulosa cells.Alk5 was mainly expressed in the theca and cytoplasm of oocytes and granulosa cells.In the testis,Gdf9,Bmpr2 and Alk5 signals were located in permatogonia,spermatocyte,spermatogenesis and Sertoli cells.5.There was no significant effect on the survival of the juvenile with different concentrations of E₂-fed.With the dose increased and time prolonged,the expression of gdf9,bmpr2 and alk5 in gonads showed an increased trend,indicating that the expression had time-and dose-dependent effect on E₂.In this study,it was found that gdf9,bmpr2 and alk5 were expressed in the gonads of S.prenanti at different development stages,which indicated that gdf9,bmpr2 and alk5 regulated the development of oocytes and sperm cells by the means of autocrine or paracrine.