The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini‐pig

The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini‐pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line‐derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha‐1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo‐derived thioguanine‐ and ouabain‐resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT‐PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1‐, DBA‐ and CDH1‐positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.