Studies Related To Spermatogonial Stem Cells Of BAMA Mini-Pig

Spermatogonial stem cells (SSCs), which locate on the basement membrane of the seminiferous tubule in testis, are the unique stem cells which can differentiate and produce spermatids. Isolation, purification and proliferation of the SSCs in vitro will provide necessary material to study SSCs in vitro. Induction of SSCs and production of spermatid in vitro can show us the mechanism underlying the spermatogenesis. Fertilization with the spermatids induced from transgenic SSC will be an efficient approach to produce transgenic animals.This study focused on isolation, purification, identification, long-term proliferation and transfection of Bama mini-pig SSC and the results showed below:1. Morphology observation of testis tissue and identification of SSC in the tissue.Morphology observation of testis tissue showed that no spermatocyte, spermatid was observed in the slices of the testis at the age of 1 month, while they were observed in the slices at the age of 2 months, this result shows that the testis at 1 month of age is suitable to isolate SSC. Immunohistochemical staining showed that the SSCs in testis at 1 month of age expressed UCHL1, CDH1 and could bind with lectin DBA (Dolichos biflorus agglutinin).Thus lectin DBA and antibodies including UCHL1 and CDH1 were employed to identify SSCs of Bama mini-pig cultured in vitro.2. Isolation and long-term culture of SSC.The testis tissue was digested and dissociated with enzyme, SSCs were purified using differential plating and cultured on STO feeder layer in serum-free medium. At the first two passages, no cluster was observed, at the third passage, the cells aggregated together and formed clusters with unclear border. The SSCs have been cultured for 30 passages to date and kept cluster formation ability.3. Identification of the SSCs cultured in vitro.Immunocytochemical staining showed that the clusters could bind with DBA and expressed UCHL1 and CDH1, some cells in the colonies expressed OCT4, The result of PT-PCR showed that the clusters expressed nanog, sox2、vasa、uchll and thyl, which showed that the clusters were formed with SSCs and named spermatogonial stem cells cluster. Meanwhile, different sizes of UCHL1 positive cells were observed in the cultured cells, which showed that the size of SSCs was various. Ultrastructure observation showed that cristaes of mitochondria in SSCs cultured in vitro were more abundant than those in tissue. Haploid peak was found in DNA content analysis of the cultured cells in different passages.4. Transfection of SSCs cultured in vitroIn this study, two methods were tried to transfer exogenous gene into SSCs. Firstly, lipofectamine LTX was used to transfer the plasmid into SSCs, while the result showed that this method was not effective. Lentivirus, which expressed EGFP, was also used to infect SSCs, the result showed that the SSCs could be transduced effectively when MOI was set at 50 and transgenic SSCs were observed.In conclusion, SSCs of Bama mini-pig were isolated and long-term cultured in vitro, which provided sufficient cells for transgenic research or exploring the mechanism of the spermatogenesis. In this study, Haploid peak was found in DNA content analysis of the cultured cells in different passages, transgenic SSCs were yielded by lentivirus infection, which established a platform to produce transgenic Bama mini-pig and provided experience for SSCs study of other domestic animal.