Culture media and supplements affect proliferation, colony-formation, and potency of porcine male germ cells

Abstract

Gonocytes are germline stem cells in the neonatal testis with important potential applications in fertility restoration and transgenesis. Using stepwise experiments, we examined the effects of different media combined with fetal bovine serum (FBS) and/or knockout serum replacement (KSR) on the in vitro proliferation, colony-formation, ultrastructure, and expression of pluripotency markers of porcine gonocytes. Testis cells from 1-wk-old piglets were cultured for 28 days in 6 different culture media (DMEM, DMEM/F12, GMEM, α-MEM, StemPro, and RPMI), each supplemented with 5%, 10%, or 15% FBS and/or 5%, 10%, or 15% KSR. The media and FBS/KSR combination leading to the maximum number of gonocytes, and their colonies were selected for further analyses. KSR supplementation resulted in a reduced somatic cell propagation and increased gonocyte colony formation (P < 0.001). Culturing in DMEM+15%FBS led to the greatest number of gonocytes (P < 0.001), while the largest diameter and greatest number of colonies were formed in DMEM+5%FBS+10%KSR cultures (P < 0.001). Gonocytes and their colonies in DMEM+15%FBS expressed all the examined gonocyte and pluripotency markers. KSR alone did not support gonocyte propagation, likely due to a reduced somatic cell proliferation; however, the combination of FBS and KSR increased gonocyte colony formation and their size.

Keywords: Gonocytes; Pig; Pluripotency; Two-dimensional culture; Ultrastructure.