Primary Establishment Of Co-Culture System Of Ovine Germ Cell In Vitro And Identification Of Different Stages Of Germ Cells

Mammalian spermatogenesis has become a hot research field in reproductive biology,and has made a great progress.However,the molecular mechanism of the renewal of spermatogonial stem cells,signaling pathway and maturation of the spermatids after meiosis remain to be elucidated.It has not been reported how spermatids differentiate into mature spermatozoa in vitro so far.Progress in tissue culture of germ cells in vitro affords possibility for observision,treatment,and analysis of different stages of germ cells.Further more,based on tissue culture,the related factors and genes involved in mechanism of spermatogenesis can be studied easily.Therefore,it is necessary to establish a steady and effective tissue culture system for study of ovine germ cells.A basic in vitro co-culture system for ovine male germ cells has been established in this study.The specific marker genes of different stages of ovine male germ cells have also been cloned for identification during the culture,such as E-cadherin gene (cdh1),synaptonemal complex protein-3 gene(scp3),transition protein-1 gene(tnp1) and the cDNA sequence of the transition protein-2 gene(tnp2).Survival of the different germ cells in co-culture system has been identified with these marker genes according to their transcription levels.Moreover,a prokaryotic expression vector for cdh1,a specific expressional gene in spermatogonia,has been constructed and the antibody of the CDH1 has been prepared for further study.1.Cloning the ovine genes of cdh1,scp3,tnp1 and tnp2Ovine cdh1,scp3,tnp1 and tnp2 genes have been cloned and the cDNA sequences of them in lengths of 2799bp,855bp,246bp and 340bp have been obtained, respectively.Among them,the complete CDS of cdh1,scp3 and tnp1 have been cloned in length of 2652bp,708bp and 168bp and the partial CDS of tnp2 has been cloned in length of 340bp.After nucleotide homology analysis,it is suggested that cdh1,scp3 and tnp1 are highly and tnp2 is comparatively conservative in evolution grade.2.Establishment and identification of a basic co-culture system for ovine germ cellsIt was the first time to establish a basic co-culture system for ovine germ cells in this study.The ovine testicular pieces from four-month old lamb was cultured in DMEM/F12 with 10%FBS for 1 to 2 weeks and the Sertoli cells were observed attaching to the surface of the culture dish from seminiferous tubules.From 2^nd week. The spermatocytes and spermatids were able to be observed and,it suggested that the spermatogonial stem cells kept the potency of differentiation into the spermatocytes and spermitids.Without any additional growth factors,the co-culture system with 10%FBS could support the spermatogolia,spermatocytes and round spermatids for survival over 10 weeks.After co-culture of the ovine germ cells for 10 weeks,the transcriptions of cdh1,scp3,tnp2,wt1 and gapdh could still be detected.The results showed that the spermatogenesis had been going well in this co-culture system.These transcriptions can be considered the complementary evidences for the morphological observation of germ cell cultivation in our laboratory.3.Construction of prokaryotic expression vector for preparation of the antigen of the CDH1In order to identify the spermatogonial stem cells from the cultured germ cells, the antigen protein of CDH1 was prepared and purified from a prokaryotic expression vector transferred into E.coli BL21(DE3).The target fragment of the cdh1 was amplified by PCR and recovered by restriction endonuclease.Then the fragment was ligated with the prokaryotic vector pET-44a(+) and transferred into E.coli BL21(DE3).The expressed protein was isolated and purified with Ni-NTA affinity column chromatography.Western blot analysis demonstrated that the vector pET-44a(+)-CDH1 was constructed well and the target protein was highly expressed as much as 22%of total bacteria proteins.The purified target protein will be used for preparation of CDH1 antibody.