Endothelin-2 in Ovarian Follicle Rupture

Abstract

The ovulatory process is activated by a surge of LH, a pituitary gonadotropin, which initiates a cohort of dramatic changes in biochemical, physical, and gene expression in the ovary, leading to follicle rupture and oocyte release. Here we report the identification of endothelin-2 (EDN2) as a last moment-trigger of follicle rupture. In the ovary, EDN2 is exclusively and transiently expressed in the granulosa cells immediately before ovulation. Administration of EDN2 to the ovarian tissue induced rapid contraction, whereas addition of tezosentan, an endothelin receptor antagonist, diminishes the EDN2 effect. In vivo, treatment of tezosentan before ovulation substantially decreases gonadotropin-induced superovulation. As a target tissue of EDN2 action, we identified a layer of smooth muscle cells in the follicular wall of each follicle. Taken together, our data indicate that EDN2 induces follicular rupture by constricting periovulatory follicles.

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

“…Changes in intra-follicular pressure are observed as early as midway through the periovulatory cascade (Matousek et al 2001), coincident with increases in factors affecting vascular permeability (Gómez et al 2003;Kitajima et al 2004;Miyabayashi et al 2005) and the array of proteolytic enzymes known to be integral to the breakdown of the extracellular matrix before ovulation (reviewed in Curry and Smith 2006). In contrast, expression of mRNA for Edn2 is only increased very late in the periovulatory period, ∼12 h after the induction of ovulation with hCG in the rat (Ko et al 2006), well after the first observed changes in intra-follicular pressure. Therefore, we propose that the contractile action of EDN2 would be imposed at the time of ovulation itself, upon an already weakened follicular structure, resulting in no change or an actual decrease in intra-follicular pressure (as the follicle ruptures), consistent with the follicular pressure profiles described over 40 years ago by Rondell (1964).…”

“…Granulosa cells were isolated from ovaries collected before rats were treated with PMSG, 48 h after PMSG and 12 h after hCG (n = 3 animals per time point), as described previously (Ko et al 2006). Total RNA was extracted from granulosa cells with TRIzol (Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer's directions and the integrity verified by visualisation of distinct 18S and 28S rRNA bands after ethidium bromide staining in an agarose gel.…”

“…Total RNA was extracted from granulosa cells with TRIzol (Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer's directions and the integrity verified by visualisation of distinct 18S and 28S rRNA bands after ethidium bromide staining in an agarose gel. Complementary DNA was reverse-transcribed from each sample of total RNA as described previously (Ko et al 2006). Oligonucleotide primers corresponding to cDNA for rat Ece1 (accession number BC072504; forward 5 -TTT GGG TGG TTG TGA GCA-3 , reverse 5 -ACA AGC TCC TTT CTC GAC CA-3 ), Ece2 (accession number NM_001002815; forward 5 -TGA CGC CTC AGA CTG TGA AC-3 , reverse 5 -GCC CAG GTT GTT TTC TGT GT-3 ) and L32 as an endogenous reference gene (accession number BC061562; forward 5 -GAA GCC CAA GAT CGT CAA AA-3 , reverse 5 -AGG ATC TGG CCC TGG CCC TTG AAT CT-3 ) were designed using PRIMER3 software and the specificity for each primer set was confirmed by both running the PCR products on a 2.0% agarose gel and analysing the melting (dissociation) curve in the MxPro real-time PCR analysing program (Stratagene, La Jolla, CA, USA) after each PCR reaction.…”

“…Using rats as the animal model, Matousek et al (2001) applied new techniques to the paradigm of changing intra-follicular pressure during the ovulatory cascade and we proposed that endothelin-2 (EDN2) was the final contractile signal facilitating ovulation (Ko et al 2006). Evidence suggests that these two seemingly intertwined processes are, however, independent factors within the ovulatory cascade.…”

“…Expression of mRNA for Edn2 was localised to the granulosa cells of periovulatory follicles by in situ hybridisation and blockade of the action of EDN2 within the ovary by administration of a dual endothelin receptor antagonist (tezosentan) inhibited or delayed ovulation (Ko et al 2006), providing strong evidence of the importance of this peptide to the process of follicular rupture. However, the increase in levels of mRNA for Edn2 during the ovulatory cascade were observed 12 h after treatment with hCG, raising the question as to whether translation of mRNA to functional peptide could occur within sufficient time for EDN2 to exert its proposed contractile effect on the periovulatory ovary.…”