Effects of glucose on glycolysis and steroidogenesis as well as related gene expression in ovine granulosa cells in vitro

Abstract

Our previous studies indicate that short-term dietary supplementation of ewes during the luteal phase can increase fertility due to elevated glucose and steroid hormone concentrations in follicles. However, the regulatory relationship between glucose and steroidogenesis during follicular development has not been clarified. The present study was conducted to determine the direct effects of different glucose levels on glycolysis and steroid secretion in ovarian granulosa cells (GCs) obtained from ewes, using an in vitro cell culture system. The GCs were collected from small follicles (1–3 mm) and treated with various doses of glucose (0, 2.1, 4.2, 8.4, 16.8 and 33.6 mM) in serum-free medium for 24 h. We observed that different dose of glucose increased the concentrations of lactic acid (LA) (linear and quadratic, P < 0.05), pyruvic acid (PA) (linear and quadratic, P < 0.05), oestradiol (E2) (linear and quadratic, P < 0.05) and progesterone (P4) (linear and quadratic, P < 0.05). The concentrations of PA, E2 and P4 all reached the highest levels in the 8.4 mM glucose group. Further study found that the mRNA and protein expression levels of glycolysis- related (LDHA and PKM2) and steroidogenesis- related (STAR, CYP11A1 and CYP19A1) genes increased with increasing glucose concentration up to 8.4 mM and then decreased at higher concentrations. There were positive correlations of glycolytic metabolites with steroidogenesis (P < 0.001, the range of r is: 0.746–0.929). This study showed that glucose dose had a significant effect on glycolysis synthesis and steroidogenesis of GCs, and 8.4 mM represented an optimum concentration for the secretion of steroid hormones by ovine GCs in vitro.