Bovine male germline stem-like cells cultured in serum- and feeder-free medium

Male germline stem cells (mGSCs) presented in male testis are responsible for spermatogenesis during their whole life. However, little information can be found on the culture of bovine mGSCs, and the current culture system needs to be improved. In this study, we compared the effects of several commercial serum-free media and different extra-cellular matrix on the enrichment and cultivation of mGSCs. To find out the best culture condition, the biological characteristics of the cultured cells were evaluated by morphological observation, RT-PCR and immunofluorescent staining. According to the cells' condition in different experiment groups, we found out an efficient cultivation system for bovine mGSCs derived from neonate testis. In this serum- and feeder-free medium, the cultured cells maintained the typical morphology, and expressed specific surface markers of both pluripotent ES cells and mGSCs, including SSEA-1, CD49f, C-MYC, PLZF, GFRα1, LIN28, NANOG, Oct4 and SOX2 in commercial human ESCs medium PeproGrow-hESC + BIO (6-bromoindirubin-3'-oxime). Embryoid bodies, derived from the bovine mGSCs, and were formed by ganging drop culture. The retinoic acid induced bovine mGSCs were positive for Stra8, SCP3, DZAL, EMA1 and VASA, and resembled spermatid cells morphologically. Thus, we found an efficient bovine mGSCs-cultivation system, which is lack in serum and feeder.