The Characteristic Analysis Of AMH And Its Functional Research In Sertoli Cell Of Yak (Bos Grunniens)

Sertoli cells(SCs)are the only somatic cell in seminiferous Tubules,which can support the development of spermatogenic cells,secrete many kinds of cytokines,regulate the differentiation of spermatogonial stem cells,maintain the smooth progress of spermatogenesis and secrete testicular fluid.Anti-Mullerian Hormone(AMH)is a dimeric glycoprotein of Transforming Growth Factor Beta(TGF-?)superfamily.In the testis,AMH is secreted by Sertoli cells.However,there is no report about the yak AMH gene and its effect on yak Sertoli cells.The aim of this study was to elucidate the characteristics of yak AMH gene and its regulatory role in Sertoli cells.First,the c DNA sequence of yak AMH gene was cloned by Reverse Transcription-Polymerase Chain Reaction(RT-PCR)and analyzed by bioinformatics.Quantitative Real-time PCR(q RT-PCR)was used to analyze the expression of tissues and testis at different developmental stages.The function of AMH gene in yak Sertoli cells was studied by liposome-mediated RNA interference(RNAi).The research results showed that:1)The full-length of c DNA sequence the yak AMH gene was 2 017 bp(Gen Bank No.MN872668).The Open Reading Frame(ORF)was 1 728 bp,5?UTR was 211 bp,3?UTR was 78 bp and encoded 575 amino acids.Homology analysis showed that yak AMH gene had the highest homology with cattle.The structure prediction showed that the yak AMH protein was a fat-soluble hydrophilic protein with a AMH?N protein domain and a conserved TGF-?(AMH?C)domain.2)The expression analysis results in different tissues showed that the AMH was expressed in various tissues of yak,among which was most highest expressed in testis(P<0.01)and was most lowest expressed in ovary and spleen(P<0.01).3)The expression analysis results in the yak testis at different developmental stages showed that the AMH was expressed in the yak testis at different developmental stages.The expression level in yak testis increased first and then decreased with age,and was highest in juvenile stage(1 year old)(P<0.01).4)After interfering with the AMH gene,the proliferating rate of Sertoli cells at 48 h,60 h and 72 h were significantly lower than that of the negative control group(P<0.05),the expression of proliferating-related gene PCNA was significantly down-regulated than that of the negative control group(P<0.01).The expression of pro-apoptotic genes BAX and CASP3 were significantly up-regulated(P<0.01),the expression of anti-apoptotic gene BCL2 was significantly down-regulated(P<0.01).The m RNA expression of Sertoli cell development related genes ABP,SCF and AMHR2 were significantly down-regulated(P<0.01).The results showed that AMH gene promoted the expression of proliferation-related gene PCNA,inhibited the expression of apoptosis-related genes BAX and CASP3,and regulated the expression of Sertoli cell development related genes ABP,SCF and AMHR2.The results of this study provides some data for further study of the regulation mechanism of AMH gene in yak Sertoli cells.