Molecular mechanism of N-acetylcysteine regulating proliferation and hormone secretion of granulosa cells in sheep

Abstract

Granulosa cells are not only important supporting cells in follicular development, but also the main cells secreting steroids. The proliferation of GCs and steroid hormone synthesis play a key role in follicular development and atresia. In this experiment, GCs were isolated by follicular fluid aspiration, and identified by immunofluorescence technique. The effects of different concentrations of NAC (50, 100, 500, 1000 µmol/L) on sheep GCs with regards to the antioxidant levels, proliferation, apoptosis, and steroid hormone secretion were investigated. PI3K/AKT inhibitor LY294002 was used to explore the molecular mechanism of NAC on GCs proliferation and steroid hormone secretion in sheep. The results showed that in sheep GCs, all concentration of NAC group could promote the proliferation of GCs and inhibit their apoptosis. Among them, 100 µmol/L NAC had the most significant promote on the proliferation of sheep GCs for 48 h. The expression of CCND1, CDK4 and Bcl-2 in all NAC concentration group was significantly increased, and the expression of Bax was significantly decreased. All concentrations of NAC significantly decreased reactive oxygen species (ROS) concentration and increased the expression of CAT and SOD1. NAC significantly increased the expression of CYP19A1, 3β-HSD and the secretion of estradiol (E2) and progesterone (P4) in GCs. In conclusion, NAC activates the PI3K/AKT signaling pathway to promote the proliferation of GCs, E2 and P4 secretion of sheep GCs in vitro.