In Vitro Differentiation And Cryopreservation Of Bovine Male Germ-line Stem Cells

Male germ-line stem cells(mGSCs)have capabilitty of self-renewal and latent capabilitty of differentiation. mGSC is the unique diploid immortal cell which can transfer genetic information to filial generation. The combination of transgenic technology and mGSCs heterotraspanting will supply some new opportunities and pathes to cloning animal, transgenic animal and gene therapy of some human hereditary disease. The experiment studied the isolation, cultivation and cryopreservation of mGSC that isolated and purified from from 5-6 month old bovine fetal tetisis, new born bovine tetisis by adopting mixed enzymes digestion and different attaching velocities methods. The results as follow:1. In vitro culture of Sertoli cells isolated and purified from bovine testisis. The results showed: bovine testis in this stage is suitable for isolating and purifying Sertoli cells; 0.25% trypsin+0.02%EDTA is an efficient way to digest bovine fetal sertli cells; we also found that, the optimal period for in vitro study of Sertoli cells is 3-20 days, bovine fetal sertoli cells in log phase can obviously promote the development of bovine IVF embro in vitro.2. mGSC isolated, purified and co-culture with Sertoli cells. The results showed: Sertoli cells is indispensable to mGSC's proliferation and differentiation in vitro.The Sertoli cells in logarithmic phase has a significant on mGSC's attaching , proliferation and differentiation. After 16 days co-culture with Sertoli cells, mGSC differentiated to long sperm successfully. A preliminary system for mGSC's inducing differentiation is established.3. Cryopreservation and refrigeration of mGSC. The result showed: the upper limit time of bovine fetal's testis preservation in 4℃is 48h. Those methods are not suitable for cryopreservation of mGSC, which are program freezing of Bovine embryo ,cryopreservation of Bovine semen and vitrificated cryopreservation of Bovine embryo. Cryopreservation of mGSC method should refer to normal freezing method. It is suggested that Cryopreserved mGSC seperately.