Ghrelin Negatively Affects the Function of Ovarian Follicles in Mature Pigs by Direct Action on Basal and Gonadotropin-Stimulated Steroidogenesis

Abstract

We previously showed that expression of ghrelin messenger RNA is significantly increased in the ovaries of cycling pigs but not in prepubertal animals and that ghrelin stimulates estradiol (E2) secretion by ovarian follicles in prepubertal animals. The present study investigated in vitro the role of ghrelin in regulating the ovarian steroidogenesis during estrus cycle in mature pigs. Small (SFs), medium (MFs), and large (LFs) ovarian follicles were collected on days 4 to 6, 10 to 12, and 16 to 18 of the estrous cycle from cycling pigs and exposed to 20, 100, and 500 pg/mL ghrelin for 24 hours. In additional experiments, MFs were exposed to ghrelin plus 100 ng/mL follicle-stimulating hormone (FSH) or luteinizing hormone (LH). Levels of progesterone (P4), testosterone (T), and E2 in culture medium were determined by enzyme-linked immunosorbent assay, and the expression of the steroid pathway enzymes 3β hydroxysteroid dehydrogenase (HSD), 17β-HSD, and cytochrome P450 aromatase (CYP19) was evaluated by Western blotting. Ghrelin had no effect on steroid secretion when present at 20 pg/mL, its concentration in follicular fluid, whereas at 100 pg/mL and 500 pg/mL, its concentration in serum, ghrelin significantly decreased secretion of P4, T, and E2. Moreover, all concentrations of ghrelin decreased steroid secretion in FSH- and LH-stimulated follicles. Western blot analysis showed that ghrelin inhibited expression of 3β-HSD, 17β-HSD, and CYP19 proteins. These results suggest that ghrelin, by direct inhibition of 3β-HSD, 17β-HSD, and CYP19 protein expression, inhibits LH- and FSH-stimulated steroid secretion by ovarian follicles, thus negatively affecting ovarian steroidogenesis in mature pigs.