Markers of Ovarian Antral Follicular Development in Sheep: Differences Between Final (FW) and Penultimate (PW) Wave Pre-Ovulatory Follicles.

Abstract

Treatment of non-prolific Western white-faced (WWF) ewes with prostaglandin F2alpha (PGF2alpha) and medroxy progesterone acetate (MAP) increased ovulation rate. The increase in ovulation rate was achieved largely by the addition of the ovulation of follicles from the penultimate (PW) follicular wave of the cycle to the expected ovulation of follicles from the final (FW) wave of the cycle. However, the number of lambs born after this treatment was not enhanced, suggesting that the PW follicles ovulating were not functionally viable. We were interested to compare the functional viability of PW and FW follicles. The objective of this study was to evaluate the expression of markers of vascularization and/or angiogenesis: vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS) and Factor VIII; a marker of gap junctional communication, connexin (Cx) 43; and a marker of cellular proliferation, proliferating cell nuclear antigen (PCNA) in preovulatory follicles obtained from the PW and FW in WWF ewes treated as described above. On day 8 of the estrous cycle, 15 ewes were given a single injection of PGF2alpha (15 mg i.m) and an intravaginal MAP sponge, which remained in place for 6 days. Thirty six to forty eight hours after sponge removal, ewes were ovariectomized by mid-ventral laparotomy. Ovaries that contain FW and/PW follicles from 7 ewes were fixed in Carnoy's solution for immunohistochemical detection of selected proteins. Computerized image analysis was used for quantification of protein expression. Granulosa and theca were isolated from FW and PW follicles from the remaining 8 ewes, suspended in PBS and stored at -80°C until RNA extraction. Messenger RNA levels were quantified using real-time PCR. Prostaglandin F2alpha and MAP treatment gave rise to preovulatory follicles emerging both from the FW and PW of the cycle; PW follicles had a longer (P<0.05) lifespan compared to FW follicles (7.1 ± 0.2 vs 3.0 ± 0.2). Protein expression for VEGF, eNOS, Cx43, PCNA and Factor VIII was greater in FW than in PW preovulatory follicles (P<0.05). For granulosa cells, Cx43 mRNA expression was greater (P<0.05) in FW than in PW preovulatory follicles. For theca cells, VEGF mRNA expression was greater (P<0.05) in FW than PW preovulatory follicles. In conclusion, expression of some markers of vascularization/ angiogenesis, gap junctional communication and cell proliferation appeared to be decreased in PW compared to FW preovulatory follicles. This suggests that although the treatment with PGF2alpha and MAP enhanced ovulation rate by extending the lifespan of PW follicles, these follicles were not as functionally viable as FW follicles. Therefore, this treatment provides a useful model to study aged antral follicles. (poster)