Role of Hypoxia in the Regulation of Periovulatory Endothelin-2 (EDN2) Expression in the Mouse Ovary.

Abstract

Endothelin-2 (EDN2) was recently proposed as a final contractile facilitator of ovulation (Ko et al., Endocrinology 147(4):1770-1779, 2006). Spatially, expression of mRNA for EDN2 is restricted to granulosa cells of periovulatory follicles. Temporally, mRNA for this contractile peptide is expressed immediately prior to follicular rupture. Evidence suggests the induction of hypoxic stress within the periovulatory follicle prior to ovulation and the primary objective of this study was to test the hypothesis that hypoxia mediates END2 expression in granulosa cells at ovulation. Immature mice were treated with 5 IU PMSG followed 48 h later by 5 IU hCG. Granulosa cells were isolated at 9 h after hCG, cultured under normal or hypoxic conditions and the expression level of mRNA for EDN2 was compared. After 3 and 6 h of culture, expression of mRNA for EDN2 was increased when granulosa cells were cultured in a hypoxic environment (p < 0.05). Subsequent promoter analysis found that the 5 upstream region of the EDN2 promoter was responsible for hypoxia-mediated changes in EDN2 expression. This promoter region contains multiple sites for potential transcriptional regulation including that by hypoxia-inducible factor 1 (HIF-1; ACGTG) at -1297 bp. The second objective of this study was to examine the potential regulation of EDN2 expression by the progesterone receptor (PR) or cyclooxygenase-2 (COX-2), two key mediators of periovulatory events. Gonadotropin-primed mice were treated with vehicle, a PR antagonist (RU-486) or COX inhibitor (indomethacin) and expression of mRNA for EDN2 was determined in ovaries collected at 12 h after hCG. Treatment with RU-486 or indomethacin did not affect expression of mRNA for EDN2 (p > 0.05). Taken together, it is believed that hypoxia, but not the PR or COX-2, regulates gonadotropin-induced expression of EDN2 in the periovulatory follicle.