During ovulation, the oocyte and associated cumulus cells are expelled from the ovary and travel to the ampulla of the oviduct where fertilization occurs. Concurrently, granulosa and theca cells of an ovulating follicle luteinize and form lutein cells of the highly vascular corpus luteum, which secrete progesterone for pregnancy maintenance. Just prior to ovulation, the gene endothelin-2 (Edn2) is highly yet transiently expressed for a two-hour window. Mature endothelin-2 protein (EDN2) is a 21 amino acid peptide that is classically described as a potent vasoconstrictor. EDN2 is produced exclusively by the granulosa cells of mature follicles, and is an important factor in ovulation; mice that are pharmacologically treated to block action of the two endothelin receptors ovulate fewer oocytes and form fewer corpora lutea. Similar effects are seen in mice that globally lack Edn2, though they also expire at a young age. While it is clear that EDN2 plays a vital role in ovulation, the exact role for EDN2 has yet to be elucidated. Previous work suggests that EDN2 may cause contraction of mature follicles during rupture, but may also be involved in the critical processes of angiogenesis and leukocyte migration. To understand the pattern of expression, an Edn2-iCre mouse was generated and Edn2 expression was characterized throughout the body; Edn2 has widespread expression, particularly throughout the skin and ovary, with additional punctate expression in the GI tract, uterus, brain, kidney, and pituitary. This mouse model will be a useful tool for examining EDN2 signaling targets. Next, to address the role of EDN2 in ovarian contraction, the effect of EDN2 on ovaries from cats, dogs, and chickens was compared; all species’ ovaries exhibit a strong and sustained contraction relative to the amount of smooth muscle present in the ovary. Thus EDN2 has an evolutionarily conserved contractile response. To further characterize EDN2 action in the ovary, Edn2 was removed from either the whole ovary, or only the granulosa cells using a novel Esr2-iCre model. In each model, loss of EDN2 prevents follicle rupture but not folliculogenesis or corpus luteum formation. RT-PCR revealed that expression of genes critical in angiogenesis and leukocyte action are not modified by Edn2-ablation, implying no significant involvement of EDN2 in these ovulatory biological processes. To determine whether endothelin receptor A (Ednra, EDNRA, ETA) mediates EDN2 contractile action during ovulation, a conditional gene knockout approach was employed to selectively ablate Ednra in smooth muscle cells through a tamoxifen-inducible Cre recombinase system. However, all mice including wild type controls treated with tamoxifen had severely impaired ovulation; while ovulation occurred, the role of EDNRA cannot yet be determined. Alternatively, available data indicate that granulosa cell-specific loss of Ednra causes subfertility, indicating that EDN2 likely exerts an effect through EDNRA receptors on granulosa cells. Lastly, temporal regulation of Edn2 expression by progesterone receptor (PGR) was confirmed using PGR-knockout mice. Overall, it is proposed based on results herein that EDN2 targets EDNRA to induce periovulatory contraction as a conserved mechanism leading to follicle rupture and subsequent corpus luteum formation, and that this action is under regulation by PGR. Therefore, EDN2 is critical for normal ovulation and fecundity.
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