The Establishment Of Mouse Testis Cell 3D Culture In Vitro And Spermatogonial Stem Cell Enrichment From Adult Mice

The spermatogenetic abnormalities with the symptoms of oligospermia, azoospermia or sperm deformity are the major causes of male infertility. To investigate the mechanisms of the problems of spermatogenesis and provide a solution to the infertilities, in this study we established a mouse testis cell 3D culture system in vitro with the scaffolds made from mouse testis. We further developed a protocol of enrichment of spermatogonial stem cells from adult mouse testis. The main results as follows:1. The preparation of mouse testis scaffolds:The testis obtained from 4-5w old Kun Ming mouse were treated with different concentrations of SDS for different lengths of time. Then the morphology and biochemical components of the scaffold were analyzed. Base on the analysis results we developed an optimized condition to prepare for the mouse testicular scaffolds.2. Te preparation of mouse testicular cells:The testicular cells were isolated from 5 to 7 day old male testis. After injection of cells into the testis scaffolds, the scaffolds were cultured in vitro for different lengths of time. Finally, we employed H&E staining, immunohistochemistry, flow cytometry, RT-PCR techniques thoroughly examine the cells in the scaffolds. Our results demonstrated the scaffolds can support the differentiation of spermatogonial stem cells in vitro.3. The enrichment of the adult mouse spermatogonial stem cells:A two-step enzymatic digestion method was used to isolate testis cells from 6-8w old Kun Ming mouse testis. Then the cells were cultured in vitro for 3 days before being subjected to different methods of examinations.The results showed that testicular cells isolated from adult testes being straightly cultured for 3 days can significant improve the numbers of spermatogonial stem cells.We have for the first time employed natural testis scaffolds to culture mouse testis cells in vitro. The method has further developed the testis cells culture technology, which would provide idealy technical platform to study spermatogenesis and a solution of the male infertility.