Abstract
Leydig cell transplantation is a better alternative in the treatment of androgen-deficient males. The main purpose of this study was to investigate the effects of induced pluripotent stem cell-derived conditioned medium (iPS-CM) on the anti-apoptosis, proliferation and function of immature Leydig cells (ILCs), and illuminate the underlying mechanisms. ILCs were exposed to 200 μmol/L hydrogen peroxide (H2 O2 ) for 24 hours with or without iPS-CM treatments. Cell apoptosis was detected by flow cytometric analysis. Cell proliferation was assessed using cell cycle assays and EdU staining. The steroidogenic enzyme expressions were quantified with Western blotting. The results showed that iPS-CM significantly reduced H2 O2 -induced ILC apoptosis through down-regulation of autophagic and apoptotic proteins LC3-I/II, Beclin-1, P62, P53 and BAX as well as up-regulation of BCL-2, which could be inhibited by LY294002 (25 μmol/L). iPS-CM could also promote ILC proliferation through up-regulation of β-catenin and its target proteins cyclin D1, c-Myc and survivin, but was inhibited by XAV939 (10 μmol/L). The level of bFGF in iPS-CM was higher than that of DMEM-LG. Exogenous bFGF (20 ng/mL) or Wnt signalling agonist lithium chloride (LiCl) (20 mmol/L) added into DMEM-LG could achieve the similar effects of iPS-CM. Meanwhile, iPS-CM could improve the medium testosterone levels and up-regulation of LHCGR, SCARB1, STAR, CYP11A1, HSD3B1, CYP17A1, HSD17B3 and SF-1 in H2 O2 -induced ILCs. In conclusion, iPS-CM could reduce H2 O2 -induced ILC apoptosis through the activation of autophagy, promote proliferation through up-regulation of Wnt/β-catenin pathway and enhance testosterone production through increasing steroidogenic enzyme expressions, which might be used in regenerative medicine for future.