Angiogenesis in the Primate Ovulatory Follicle

排卵是成功繁殖的关键。排卵性黄体生成素（LH）峰刺激排卵期卵泡产生前列腺素E2（PGE 2）。阻止PGE 2的产生会导致排卵失败。LH刺激的卵泡新血管形成对排卵也是必需的，因为抑制卵泡血管生成会阻止排卵。这一建议提出了一个总体假设，即PGE 2介导排卵LH峰调节排卵卵泡内血管生成的能力。血管生成是 通过血管生长因子调节。在目的1中，我们将确定LH是否作用于卵泡颗粒细胞，以PGE 2依赖的方式调节血管生长因子的产生。在给予促排卵剂量LH样激素hCG之前和之后获得具有多个卵泡的猴卵巢;一些猴还将接受前列腺素（PG）合成抑制剂的伴随给药。 将通过qPCR评估颗粒细胞、卵泡液和整个卵巢的血管生长因子mRNA，并通过蛋白质印迹、ELISA和组织切片的免疫荧光检测评估蛋白质。将进行体外研究以确定PGE 2是否直接作用于颗粒细胞以调节血管生长因子mRNA和蛋白。血管生长因子作用于内皮细胞以促进新血管的形成。在目标2中，我们将确定PGE 2和血管生长因子是否作用于血管内皮细胞。 卵泡刺激血管生成。使用一种新的猴子卵巢内皮细胞增殖的人口，我们将确定是否PGE 2刺激内皮细胞增殖，迁移和新的毛细血管的形成。类似地，由颗粒细胞、颗粒细胞条件培养基和与颗粒细胞共培养产生的血管生长因子将用于确定血管生长因子是否刺激血管生成所必需的内皮细胞功能。最后，对用hCG、hCG+PG合成抑制剂或hCG+PG合成抑制剂+PGE 2体内处理后获得的猴卵巢进行组织学评估，以确定排卵卵泡的新血管形成。拟议的研究可能会证明，前列腺素E2和血管生长因子直接作用于内皮细胞，以促进灵长类动物排卵卵泡的新血管形成。卵泡PGE 2水平或PGE 2受体活性的调节可能为不孕症提供有效的治疗或为开发新的避孕药提供靶点。

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.