Identifying and culturing undifferentiated bovine spermatogonial stem cells

Abstract

Spermatogonial stem cells (SSCs) are responsible for maintaining spermatogenesis throughout the life of an individual. These cells can be isolated and transplanted, resulting in colonization and restoration of testicular function. However, selection methods and molecular markers used for isolation of these cells do not recognize the true testicular stem cells. Moreover, the number of studies aiming to characterize these cells in bovine is smaller than in mouse. In this proposal, we will test the hypothesis that SOX2, NANOG or OCT4 are markers for bovine undifferentiated SSCs in three experiments. First, the efficiency of methods for initial selection of spermatogonial cells will be tested. Then, expression of these aforementioned genes will be verified in the testicular tissue, together with other markers used for SSC selection. Then, we intend to find genes differentially expressed in the population of testis cells that are positive for one of the markers tested. In the fourth experiment, isolated SSCs will be transplanted into mice and colony formation will be assessed. In the fifth and last experiment, MACS isolated SSCs will be cultured, in order to assess maintenance of in vitro stemness. We intend with this proposal identify markers of undifferentiated bovine SSCs and also identify cellular membrane markers that are present in this population of cells. Therefore, we aim to obtain a more efficient cellular population to colonize testis after transplantation and also capable to be cultured in vitro. (AU)