Endothelin-2 induce ovulation by constricting ovarian follicle via EDNRA-mediated pathway

Jongki Cho1,2, Masashi Yanagisawa3, Chemyong Ko1. 1University of Kentucky, Clinical Sciences, 40503, United States, 2Chungnam National University, College of Veterinary Medicine, 305-764, Korea, Republic of, 3University of Texas, Biophysics and Molecular Genetics,, United States.

Endothelin 2 (EDN2) induces follicular rupture for ovulation by constricting periovulatory follicles.We hypothesized that EDNRA expressed in the smooth would directly mediate the EDN2-induced contractile response.To test the hypothesis, we induced a selective null mutation of endothelin receptor type A (EDNRA) gene specifically in the smooth muscle cells of premature mice. Floxed EDNRA(EDNRAflox/flox) mice were cross bred with SMACreERT2mice that express Cre recombinase specifically in the smooth muscle cells upon tamoxifen (TAM)injection in vivo. Through this breeding strategy, we produced two genotypes, EDNRAflox/floxSMACreERT2 andEDNRAflox/flox. Upon TAM injection, null mutation was induced in the EDNRAflox/floxSMACreERT2 mice while EDNRA gene was intact in the EDNRAflox/flox mice. These mice were injected withTAM or vehicle (oil) at the age of 21 days after birth. TAM (0.5 mg/mouse/day) or oil was injected for 5 consecutive days. Then the animals were given 3-day long rest (no injection) before ovulation was induced by injecting themwith pregnant mare’s serum gonadotropin (PMSG; 5 IU/mouse) and human chorionic gonadotropin (hCG; 5 IU/mouse).Eighteen hours after hCG injection, the mice were euthanized by CO2 overdose followed byassessment of ovulatory capacity by counting the numbers ovulated oocytes and of contractile response to EDN2(50 mg/L) by measuring isometric tension.Number of ovulated oocytes were significantly lower EDNRAflox/floxSMACreERT2 mice that received TAM injection compared to oil treated ones (7.6± 3.2 oocytes/ovary in TAM group vs. 34.5±6.3 in oil group, p<0.05, n = 8). In the EDNRAflox/flox mice, TAM did not reduce ovulatory capacity (15.4±8.4 oocytes/ovary in TAM group vs. 30.8±4.1; p<0.05, n = 5). Contractile force was not increased after EDN2 treatment in the TAM-injected EDNRAflox/floxSMACreERT2 mouse ovary, not in the oil-injected mice.In conclusion, the results demonstrated that EDNRA mediates endothelin induced ovarian constriction.