A Novel Pathway Involving Progesterone Receptor, Endothelin-2, and Endothelin Receptor B Controls Ovulation in Mice

Abstract

The steroid hormone progesterone (P) plays a pivotal role during ovulation. Mice lacking P receptor (Pgr) gene fail to ovulate due to a defect in follicular rupture. The P receptor (PGR)-regulated pathways that modulate ovulation, however, remain poorly understood. To identify these pathways, we performed gene expression profiling using ovaries from mice subjected to gonadotropin-induced superovulation in the presence and in the absence of CDB-2914, a synthetic PGR antagonist. Prominent among the genes that were down-regulated in response to CDB-2914 was endothelin (ET)-2, a potent vasoactive molecule. ET-2 mRNA was transiently induced in mural granulosa cells of the preovulatory follicles immediately preceding ovulation. This induction was absent in the ovaries of PGR null mice, indicating a critical role of this receptor in ET-2 expression. To investigate the functional role of ET-2 during ovulation, we employed selective antagonists of endothelin receptors, ETR-A and ETR-B. Mice treated with an ETR-B antagonist exhibited a dramatic (>85%) decline in the number of released oocytes. Strong expression of ETR-B was observed in the mural and cumulus granulosa cells of the preovulatory follicles as well as in the capillaries lining the inner border of the theca interna. We also identified cGMP-dependent protein kinase II, a previously reported PGR-regulated gene, as a downstream target of ET-2 during ovulation. Collectively, our studies uncovered a unique pathway in which ET-2, produced by PGR in mural granulosa cells, acts in a paracrine or autocrine manner on multiple cell types within the preovulatory follicle to control the final events leading to its rupture.

Production and binding of endothelin-2 (EDN2) in the rat ovary: endothelin receptor subtype A (EDNRA)-mediated contraction

“…Furthermore, the role of alternative contractile factors such as prostaglandins (Priddy and Killick 1993), histamines (Schmidt et al 1987) and bradykinin (Smith and Perks 1984) should be considered inclusive to the process of ovulation. Palanisamy et al (2006) reported that a dose of 10 mg per kg bodyweight of the EDNRB antagonist, BQ788, administered intra-peritoneally to immature gonadotrophin-primed mice resulted in a dramatic decrease in ovulation. They treated mice with BQ788 at 4, 6 or 8 h after hCG and reported increasing effectiveness of BQ788 in the ability to inhibit ovulation the closer the antagonist was administered towards the time of ovulation.…”

“…Greater than 85% inhibition of ovulation was reported when BQ788 was administered at hCG + 8 h. In contrast, the isometric tension analysis led us to hypothesise that treatment with the EDNRA antagonist BQ123, but not BQ788, would inhibit EDN2-induced ovarian contraction and reduce or delay ovulation. We used the same dose (by bodyweight) as Palanisamy et al (2006) when administering the antagonists by intra-peritoneal injection and a dose of 10 µL of 1 mM antagonists (∼90 µg per kg bodyweight) when administering the antagonists directly into the ovarian stroma. We chose to use this alternative technique as it (1) reduced the potential for non-specific effects of treatment with these vaso-regulatory antagonists and (2) was successful in reducing the rate of ovulation when tezosentan was administered in a previous experiment (Ko et al 2006).…”

“…5a, b). This was an unexpected finding given that (1) intra-ovarian treatment of rats with the dual endothelin receptor antagonist tezosentan decreases ovulation rate (Ko et al 2006) and (2) intra-peritoneal treatment of mice with BQ788, but not JKC-301, is also reported to reduce the rate of ovulation (Palanisamy et al 2006). We therefore sought to reexamine the effectiveness of using peptide-based receptor-specific antagonists on the rate of ovulation using mice as the experimental model.…”

“…A dose of 10 mg per kg body weight was chosen for i.p. treatments based upon the use of these antagonists in mice (Palanisamy et al 2006), whereas 10 µL of a 1 mM solution of each antagonist was chosen for i.o. treatments to give an estimated total ovarian concentration ∼10-fold higher than that used for the isometric tension experiments in vitro.…”

In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation

“…Because VSM is required for vasoconstriction, the mice provided a model to test whether failure of vasoconstriction contributes to anovulation. In additional experiments with wild-type mice, we blocked the increase in the expression of endothelin 2 (Edn2) by granulosa cells that normally occurs within hours before follicle rupture (10,11). Because EDN2 is a potent vasoconstrictor, this approach allowed us to test the effect on follicle rupture of inhibiting vasoconstriction versus treatment with exogenous compounds to restore vasoconstriction.…”