In Vitro Culture Characteristics And Cryopreservation Of Spermatogonia In Mouse

As the stem cells of spermatogenesis, spermatogonial stem cells (spermatogonial stem cells,SSCs) are the only diploid immortal cell group which have self-renewwal and multi-differentiation potential in adult animal. For several years, the development of spermatogonial transplantion technique has provided the enormous convenience for SSCs'correlation research and make the SSCs'reseach reached further. In order to provide the theory basis and technical method for SSCs'in vitro differentiation,transgene, transplantation technique in mouse , this research mainly carried on SSCs'in vitro culture characteristics and cryopreservation in mouse. Results as follows:To investigate in vitro surviving and proliferating characteristics of mouse spermatogonia and optimize its in vitro culture system. Kunming mouse testes of 7 day-of-age were collected to isolate seminiferous epithelial cells (spermatogonia and Sertoli cells mainly) using one step of enzymatic digestion, after being plated in D-MEM, D-MEM/F12 or KSOM respectively, the spermatogonial survival and proliferation in vitro was tracked systematically.The results indicated mouse spermatogonia could survive and proliferate in D-MEM and D-MEM/F12, and they decreased gradually from 1 d to 9 d or 1 d to 8 d, increased rapidly from 9 d to 13 d or 8 d to 12 d in primary culture, the increase slowed down after 17 d or 16 d, however, mouse spermatogania couldn't proliferate in KSOM except for surviving for a short period.To search the method to store mouse spermatogomial single cell form and functional structure form (seminiferous tubes and whole testes).This experiment used 10% DMSO as cryoprotectant, seminiferous epithelial cells, seminiferous tubes and whole testes were stored at–70℃, meanwhile, seminiferous epithelial cells were stored in liquid nitrogen.The results indicated the percentage of viable cells of whole testes, seminiferous tubes and seminiferous epithelial cells were 85.75±2.24%,93.28±0.37% and 90.21±0.89% after one week's cryopreservation. the percentage of viable cells of seminiferous tubes was 91.27±0.39% after two weeks'cryopreservation, the percentage of viable cells of seminiferous epithelial cells were 91.17±1.47%,90.51±1.34% and 89.73±2.51% after two weeks',one month's and three months'cryopreservation.The recovered spermatogonia appeared extensive proliferating behavior in culture medium at the tenth day. To explore the method for purifying mouse spermatogonia and check the feasibility whether purified mouse spermatogonia could proliferate on no-feeder layers, the mechanical shaking method was used to purify mouse spermatogonial colonies which were being extensive proliferation, purified spermatogonial colonies were cultured on uncoated or glutin-coated cell culture plates, the culture characteristics of spermatogonial colonies was observed carefully.The results indacated the effect of the purifying method was ideal, spermatogonia could survive 20~25 d on no-feeder layers,while spermatogonial proliferation was not observed.In spermatogonial colonies, the shape of spermatogonia changed from round or elliptoid to sperm-like gradually, showed differentiational characteristics.