Role of endothelin 1 in follicular and luteal function in the sheep

Abstract

Reproductive activity in mammals is characterized by the recurrent presence of follicles and corpora lutea (CL) in the ovaries. In addition to the roles of follicles and corpora lutea in regulating the activity of the hypothalamic-hypophyseal-ovarian axis, those ovarian structures are responsible to generate a viable female gamete and to provide the appropriate uterine physiological conditions to sustain the embryo/fetus development during pregnancy. If fertilization of the oocyte does not occur or pregnancy fails, a new ovarian cycle starts a few days later, assuring further opportunity for onset of a new gestation. Numerous locally produced ovarian factors modulate function of follicles and CL. Endothelin 1, a 21-amino acid peptide produced in the ovary, modulates steroid production by granulosal cells and mediates luteolytic actions of PGF 2. Three experiments were carried out in order to examine the role of endothelin in follicular steroid secretion in vitro and in spontaneous and PGF 2-induced luteolysis in sheep. In experiment 1, it was hypothesized that endothelin 1 (END1) acting through its receptors ENDRA and/or ENDRB, on granulosal cells of ovine preovulatory follicles would inhibit steroid production, and therefore, prevent the premature luteinization of granulosal cells of those follicles. On day 14 of the estrous cycle (standing estrus = day 0), 17 nonpregnant Suffolk ewes were injected with 2 doses of PGF 2 (5 mg) 3 h apart. Thirty six to 40 hours after the second dose of PGF 2 , ovaries were removed by mid-ventral laparotomy. The 2 or 3 largest follicles in the ovaries were dissected and granulosal cells were harvested under sterile conditions. Aliquots of 15 L of granulosal cell suspension from each presumed preovulatory follicle were cultured in 96-well plates containing a solution of 185 L of Ham's F12 supplemented medium and treatments (single and combined effects of LH, END1, ENDRA and ENDRB). Data from estrogen-inactive 20 follicles collected from 13 ewes were analyzed. Accumulation of E 2 and P 4 by cultured granulosal cells was not affected by either LH or END1 or the END receptor blockers. Granulosal cells from follicles with the greatest concentration of E 2 in FF produced on the average significantly more E 2 and P 4 than follicles with lower concentrations of E 2 in FF. In conclusion, END1 did not affect steroid production by granulosal cells from preovulatory follicles in sheep, and therefore the working hypothesis was not supported. In the second experiment, the ENDRA antagonist BQ-610 was delivered into the CL via an osmotic minipump to examine the expression of genes related with P4 production and structural luteolysis at 6 and 24 h after exogenous PGF 2. It was hypothesized that sustained blockade of ENDRA would prevent upregulation of genes stimulated by PGF 2 during structural luteolysis. Minipumps loaded with either vehicle (2:1 methanol/saline solution) or 2.5 mg of BQ-610, were implanted in the mesovarium, with a catheter into a CL, on day 7.5 to 9 of the estrous cycle. A dose...