Isolation, Identification And Culture Of Rabbit Spermatogonial Stem Cells

Spermatogenesis is a complex process, including proliferation, differentiation and the formation of germ cells to produce a large number of sperm all male animal life long. Spermatogonial stem cell (SSCs) is the basis of spermatogenesis, and SSC is the only stem cell in male individuals that can undergo mitosis and transfer the genetic information to the progeny. However, SSCs are rare, representing only0.03%of all germ cells in testis. In2003, Kanatsu-Shinohara et al. first established a long-term culture system of mouse germ stem cells in vitro. They isolated germ stem cells from pup mouse testis, and obtained pluripotent germ stem cells through long-term culture in vitro. Rabbit (Oryctolagus cuniculus) is rodent, however, the long-term culture system for its germline stem cells has not been reported.Rodent SSC transplantation technique not only is an important new tool for the study of mammalian spermatogenesis, but also can establish a basis for the study of the use of livestock spermatogonial stem cells to conserve livestock breeds and product transgenic animal. This study lays the foundation for the establishment of rabbit spermatogonial stem cells culture system in vitro and transgenic rabbit production and is beneficial to further research of stem cell culture and its growth conditions.In this study, we collected15-20days rabbits’testis, using mechanical method and two step-enzyme digestion method, and obtained single-cell suspension of rabbit SSCs. After purified by differential adhesion method, these cells were inoculated on culture mediums using Sertoli cells as feeder layer cells. The cultured cells then tested by alkaline phosphatase staining, RT-PCR and immunocytochemistry. After passage, DMSO and Control Rate Freezer were used to cryopreservation rabbit SSCs for subsequent experiments.The results are as follows:1) After two-step enzymatic digestion processing, we obtained rabbit SSCs with75%purity from these collected rabbits’testis, and differential adhesion method performing better. Using Sertoli cells as feeder layer cells, SSCs grew faster, and the SSCs colony appeared after3-4days. After passage, these cultured cells still maintained cell proliferation in3generations and provided stable material for the following experiments.2) The cultured cells were then stained with AKP, and cells showed bluish violet. These cells is AKP positive and have pluripotency, belonging to the rabbit SSCs. RT-PCR detected the expression of gremlin stem cell-specific gene ngn3and Oct4and indicated that the cultured cells are SSCs. Immunocytochemistry identified the presence of stem cell surface marker CD9, which reconfirmed that the cultured cells are SSCs.3) The survival rate of rabbit SSCs still remained at around75%after cryopreserved and resuscitated in vitro.The established long-term culture system of rabbit SSCs in vitro lays the material foundation for the spermatogenesis study, stem cell growth environment exploration and SSC transplantation. It also supplies the technical foundation for SSC isolation and culture of other livestock.