Angiogenesis in the Primate Ovulatory Follicle

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Ovulation is essential for successful reproduction. The ovulatory luteinizing hormone (LH) surge stimulates periovulatory follicles to produce prostaglandin E2 (PGE2). Blockade of PGE2 production causes ovulation failure. LH-stimulated neovascularization of the follicle is also essential for ovulation as inhibition of follicular angiogenesis prevents ovulation. This proposal puts for the overall hypothesis that PGE2 mediates the ability of the ovulatory LH surge to regulate angiogenesis within the ovulatory follicle. Angiogenesis is regulated via vascular growth factors. In Aim 1, we will determine if LH acts at follicular granulosa cells to regulate production of vascular growth factors in a PGE2-dependent manner. Monkey ovaries with multiple follicles will be obtained before and after administration of an ovulatory dose of the LH-like hormone hCG; some monkeys will also receive concomitant administration of a prostaglandin (PG) synthesis inhibitor. Granulosa cells, follicular fluid, and whole ovaries will be assessed for vascular growth factor mRNAs by qPCR and proteins by western blotting, ELISA, and immunofiuorescent detection on tissue sections. In vitro studies will be performed to determine if PGE2 acts directiy at granulosa cells to regulate vascular growth factor mRNA and proteins. Vascular growth factors act at endothelial cells to promote the formation of new blood vessels. In Aim 2, we will determine if PGE2 and vascular growth factors act at endothelial cells of the follicle to stimulate angiogenesis. Using a novel proliferating population of monkey ovarian endothelial cells, we will determine if PGE2 stimulates endothelial cell proliferation, migration, and formation of new capillaries. Similarly, vascular growth factors produced by granulosa cells, granulosa cell-conditioned media, and coculture with granulosa cells will be used to determine if vascular growth factors stimulate endothelial cell functions necessary for angiogenesis. Finally, monkey ovaries obtained after treatment in vivo with hCG, hCG+PG synthesis inhibitor, or hCG+PG synthesis inhibitor+PGE2 will be assessed histologically for neovascularization of the ovulatory follicle. The proposed studies will likely demonstrate that PGE2 and vascular growth factors act directly at endothelial cells to promote the neovascularization of the primate ovulatory follicle. Modulation of follicular PGE2 levels or PGE2 receptor activity may provide effective treatments for infertility or suggest targets for the development of novel contraceptives.