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Ovulation is the fundamental biological process during which an oocyte is expelled from the ovary, and it is an essential step toward establishing a pregnancy. Understanding regulatory mechanisms governing the ovulation process is essential for diagnosing and treating causes of infertility, identifying contraceptive targets, and developing novel contraception methods. Endothelin-2 (EDN2) is a 21 amino acid-long peptide that is transiently synthesized by granulosa cells of the ovulatory follicle prior to ovulation and plays an essential role in ovulation via promoting contraction in the myofibroblast cells of the theca layer of the follicle. This review describes the organization of the endothelin system, summarizes recent findings on the expression and synthesis of the endothelin system in the ovary, illustrates the roles that EDN2 plays in regulating ovulation, and discusses EDN2 as a potential target of contraception.

Endothelin-2 (EDN2), expressed at a narrow window during the periovulatory period, critically affects ovulation and corpus luteum (CL) formation. LH (acting mainly via cAMP) and hypoxia are implicated in CL formation; therefore, we aimed to elucidate how these signals regulate EDN2 using human primary (hGLCs) and immortalized (SVOG) granulosa-lutein cells. The hypoxiamiR, microRNA-210 (miR-210) was identified as a new essential player in EDN2 expression. Hypoxia (either mimetic compound-CoCl 2 , or low O 2 ) elevated hypoxia-inducible factor 1A (HIF1A), miR-210 and EDN2 Hypoxia-induced miR-210 was suppressed in HIF1A-silenced SVOG cells, suggesting that miR-210 is HIF1A dependent. Elevated miR-210 levels in hypoxia or by miR-210 overexpression, increased EDN2 Conversely, miR-210 inhibition reduced EDN2 levels, even in the presence of CoCl 2 , indicating the importance of miR-210 in the hypoxic induction of EDN2 A molecule that destabilizes HIF1A protein, glycerol-3-phosphate dehydrogenase 1-like gene- GPD1L , was established as a miR-210 target in both cell types. It was decreased by miR-210-mimic and was increased by miR-inhibitor. Furthermore, reducing GPD1L by endogenously elevated miR-210 (in hypoxia), miR-210-mimic or by GPD1L siRNA resulted in elevated HIF1A protein and EDN2 levels, implying a vital role for GPD1L in the hypoxic induction of EDN2 Under normoxic conditions, forskolin (adenylyl cyclase activator) triggered changes typical of hypoxia. It elevated HIF1A , EDN2 and miR-210 while inhibiting GPD1L Furthermore, HIF1A silencing greatly reduced forskolin's ability to elevate EDN2 and miR-210. This study highlights the novel regulatory roles of miR-210 and its gene target, GPD1L, in hypoxia and cAMP-induced EDN2 by human granulosa-lutein cells., (© 2018 Society for Reproduction and Fertility.)

Hypoxia-inducible factor 1 alpha (HIF1A) and endothelin 2 (EDN2) are transiently expressed during the same time window in the developing corpus luteum (CL). In this study, we sought to investigate the involvement of LH/cAMP, reactive oxygen species (ROS), and a hypoxia-mimetic compound (CoCl2) on HIF1A expression and how it affected EDN2 levels, using transformed human granulosa cells (thGCs) and primary bovine granulosa cells (GCs). CoCl2 elevated HIF1A protein levels in thGCs in a dose-dependent manner. Forskolin alone had no significant effect; however, forskolin and CoCl2 together further induced HIF1A protein and EDN2 mRNA expression in thGCs. Similarly, in primary GCs, LH with CoCl2 synergistically augmented HIF1A protein levels, which resulted in higher expression of EDN2 and another well-known hypoxia-inducible gene, VEGF (VEGFA). Importantly, LH alone elevated HIF1A mRNA but not its protein. The successful knockdown of HIF1A in thGCs using siRNA abolished hypoxia-induced EDN2 and also the additive effect of forskolin and CoCl2. We then examined the roles of ROS in thGCs: hydrogen peroxide (20 and 50 μM) elevated HIF1A protein as well as the expression of EDN2, implying that induction of HIF1A protein levels is sufficient to stimulate the expression of EDN2 (and VEGF) in normoxia. A broad-range ROS scavenger, butylated hydroxyanisole, inhibited CoCl2-induced HIF1A protein with a concomitant reduction in the mRNA expression of EDN2 and VEGF in thGCs. The results obtained in this study suggest that HIF1A, induced by various stimuli, is an essential mediator of EDN2 mRNA expression. The results may also explain the rise in the levels of HIF1A-dependent genes (EDN2 and VEGF) in the developing CL., (© 2015 Society for Reproduction and Fertility.)