Abstract

Apelin was believed to be an adipokine secreted from adipose tissue. However, studies demonstrate that it is a pleiotropic peptide and has several effects on the female reproductive system. In this study, the effect of different doses of IGF1 and FSH in the presence of apelin-13 on the production of estradiol and progesterone was evaluated in the follicular granulosa cells of buffalo ovaries, in addition, the effects of different doses of apelin isoforms (apelin-13 and apelin-17) on proliferation, the expression of Bax protein and total antioxidant capacity activity of the same cells were investigated. Granulosa cells of buffalo ovaries were cultured in the presence of different doses of IGF1 and FSH with or without apelin-13 (10-9M) to evaluate its effect on the secretion of estradiol and progesterone. WST-1 method was used to survey the effect of apelin on granulosa cells proliferation and cytotoxicity. In addition, the antioxidant capacity of the cells in the presence of apelin was assessed using FRAP method. mRNA and protein levels of Bax protein were measured in granulosa cells treated with apelin using real-time PCR and western blot techniques. Apelin-13 stimulated the effect of IGF1 on the production of estradiol and progesterone, and the progesterone production levels were affected by apelin-13 dose-dependently. However, it did not significantly stimulate the effect of FSH on the secretion of estradiol or progesterone. Apelin-13 (all doses) and -17 (10-8 and 10-9 M) improved the proliferation of granulosa cells. Moreover, preincubation of the cells for an hour by apelin receptor antagonist (ML221, 10 μM) did not significantly affect the proliferation of cells. Neither apelin-13 nor apelin-17 were not cytotoxic for the cells compared to the control treatment. Apelin-13 at the doses of 10-6 and 10-8 M substantially up and down-regulated Bax protein expression; however, such effects were not observed when the cells were preincubated with ML221. In addition, apelin-17 did not influence the expression amount of Bax. Furthermore, both apelin-13 and -17 improved the total antioxidant capacity of the ovarian granulosa cells, but such effects were not seen when the cells were preincubated with ML221. These findings indicate that apelin enhanced the IGF1-induced steroidogenesis and improved the cell proliferation and antioxidant capacity of follicular granulosa cells of buffalo ovaries; however, its effect on Bax expression was divergent.