Effects of N-carbamylglutamate and arginine on steroidogenesis and proliferation of porcine granulosa cells in vitro

Abstract

In vivo studies have determined that dietary N-carbamylglutamate (NCG) and arginine (ARG) can enhance reproductive performance in gilts, ewes and rats. We hypothesized that both NCG and ARG will enhance hormone-induced estradiol (E2) production by granulosa cells, explaining why these compounds could improve reproductive performance in pigs. Therefore, the objective of these studies was to evaluate the direct effects of NCG and ARG on porcine granulosa cell proliferation and steroidogenesis, using an in vitro cell culture system. Granulosa cells from small (1-5 mm) and large ( >5 mm) porcine follicles were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing 500 ng/ml of testosterone, and NCG or ARG at various doses in the presence of either follicle-stimulating hormone (FSH; 30 ng/ml), insulin-like growth factor-1 (IGF1; 30 ng/ml), or both. Numbers of granulosa cells were determined at the end of the experiment using a Coulter counter and concentrations of progesterone (P4) and E2 in culture medium were determined using radioimmunoassays. Our results indicated that large-follicle granulosa cells (LGGC) were more responsive to NCG and ARG than small-follicle granulosa cells (SMGC). Specifically, NCG and ARG significantly decreased IGF1 plus FSH induced P4 production, whereas E2 production and cell proliferation were not significantly affected in SMGC. In LGGC, NCG inhibited (P < 0.05) basal and FSH-induced P4 and E2 production but stimulated cell numbers; whereas ARG inhibited FSH-induced E2 production and cell numbers. These studies indicate that NCG and ARG directly affect follicular function in the pig.