Study On The Isolation And Differentiation Of The Mouse Spermatogonia In Vitro

Differentiation of sperm cells in mammal is essential for preservation of species extension.Spermatogonia Stem Cell is the spring of generation of germ.It has important theoretical and realistic meanings to obtain mature germ cells through culture of spermatogonia in vitro.Otherwise, spermatogonia Stem Cells can be cryopreservation in vitro,genetic operation and transplantation,playing essential value for medical study, biological experiment and biological technology.In this study,isolation,purification,passage culture,staining,observation of morph,structure and biological characteristic of mice sertoli cells,leydig cells and spermatogonia were operated by combination of physics and chemistry methods . Factors affecting differentiation of spermatogonia stem cell were probed by adding different nutrition factors and co-culture of sertoli cells, leydig cells and spermatogonia. The results were as follows:1.Male Kun Ming mouse aged 16-22 d was chose as experimental animal.After step-by-step digestion by collagenase and trypsase,and hypotonic treatment by 0.05M Tris-HCL,sertoli cells of 2×106 were averagely obtained in each testis.The cell purification was up to 89.87% and viable count was account for 95.15% in total cell population.Moreover,sertoli cells has high adherence mouse,entering log phase at cultured 2 d and platform phase at 6 d.Thereafter,the mouse of cell proliferation was decreasing,but no apoptosis was appeared at 7 d.2 . Male Kun Ming mouse aged nearly 6 weeks was chose as experimental animal.Leydig cells could maintain normal growth character,which were obtained through limited time digestion by collagenase and trypsase and density gradient centrifugation of Percoll.However, cells motility mouse was decreased compared to sertoli cells,averagely up to 85.02%.3. Male Kun Ming mouse aged 7-8 d was chose as experimental animal.Spermatogonia obtained through limited time digestion by collagenase and trypsase and density gradient centrifugation of Percoll mainly distributed between Percoll gradient of 27%-35%.The purification of spermatogonia was up to 62.5% after using method of selectivity adherence.Mice spermatogonia were began to apoptosis when single culture at 3 d.4. Adding FSH or Testosterone to spermatogonia cell medium in single culture,FSH did not remarkably stimulate differentiation of spermatogonia,but Testosterone could promote differentiation of spermatogonia in vitro culture.To add testicle liquid from adult mouse to cell medium , it was significantly promoted differentiation of spermatogonia in vitro.Co-culture of sertoli cells, leydig cells and spermatogonia also could simulate the differentiation of spermatogonia,but the effect was lower than testicle liquid.Additionally,the effects of varied method of digestion and passage on proliferation,differentiation and passage count of spermatogonia were different.