In Vitro Propagation Of Porcine Male Germline Stem Cells And The Distribution Of Cldn6in Testes

Male germline stem cells （MGSCs）are capable of self-renewnal and differentiation.However, the scarce quantity of MGSCs （0.02%~0.03%） prevents the further study.Considering the great values of MGSCs for generations of transgenic animals and applicationin treatment of male infertility，it is essential to establish a in vitro long-term culture systemfor MGSCs. The aim of this study was to explore the in vitro culture conditions for porcineMGSCs, which will be of benefit for establishment of a long-term maintained culture systemof MGSCs in pigs. This study also detected the expression of Cldn6as a MGSC marker.The main observations are as follows:1. Differential plating was performed to enrich MGSCs from single cell suspension of3-month-old porcine testis tissue. Immunocytochemical analysis showed that the proportionof THY1+cells was improved from4.68%±0.25%to47.10%±1.65%by differential plating.2. The effects of combination of fetal bovine serum, bFGF, EGF and GDNF on MGSCproliferation in vitro were evaluated. The results showed that DMEM medium containing1%fetal bovine serum,5ng/mL bFGF,10ng/mL EGF and10ng/mL GDNF could maintainthe propagation of MGSCs for2months, and cultured cells still expressed markers of porcineMGSCs, such as UCH-L1and NANOG, suggesting that this study successfully establishedthe culture of porcine MGSCs, and layed the foundation for a long-term culture of porcineMGSCs.3. The expressions of stem cell marker Cldn6in mouse testis were examined by Western Blotand immunohistochemical staining. The data showed that Cldn6was expressed in MGSCs,but not localized in Sertoli cells, suggesting that Cldn6could be used as a surface molecularmarker for male germline stem cells in pups.