This research group recently collected novel evidence suggesting that progesterone (P), via receptor (R)-mediated pathways, plays a key role in local control of the primate follicle/corpus luteum (CL). Further studies are planned to investigate the forms of PR mRNAs and proteins in the macaque CL and their regulation by gonadotropins (LH, CG and P throughout the luteal lifespan (Aim No. 1). Also, experiments will examine the roles of P in follicle rupture/luteinization during the periovulatory interval (Aim No. 2), in maintenance of luteal structure-function in the developed CL of the cycle (Aim No. 3) and in rescue of CL structure-function during early pregnancy (Aim No. 4). Three protocols will be employed in which luteotropic hormone support is provided by exogenous gonadotropins to eliminate steroid effects via the hypothalamic-pituitary axis during the periovulatory interval (Protocol 1; hCG bolus), at midluteal phase of the cycle (Protocol 2, hLH tid), and in stimulated early pregnancy (Protocol 3; increasing doses of hCG). Protocols will be combined with P ablation (using the 3 -hydroxysteroid dehydrogenase inhibitor, Trilostane) and P replacement (a potent, nonmetabolized progestin, R5020), followed by removal of follicular aspirates (follicular fluid and granulosa cells), the follicle or the corpus luteum for in vitro analyses. PR mRNAs will be detected by Northern analyses and quantitated by RNase protection assay. PR proteins will be analyzed by Western blotting, immunocytochemistry (ICC) and radioligand binding. Effects of gonadotropins and P ablation/replacement on tissue remodeling (protease expression, vascularization, cell proliferation and cell death) and tissue differentiation (steroidogenesis and inhibin production) will be evaluated. For example, ICC of a cell proliferation marker (Ki67), an endothelial cell marker (PECAM) and cell apoptosis (DNA end labeling) will be performed. Northern and Western analyses, well as ICC, will be employed to detect mRNA and protein for matrix metalloproteinase (MMPs) and their inhibitors (TIMPs) and for components of the steroidogenic pathway (e.g., P450SCC and 3-beta-HSD). These studies should establish that the primate corpus luteum is a target tissue for its own hormone product, P, and support the novel concept that at least part of the luteotropic action of LH/CG is mediated by the autocrine or paracrine action of P. This work will also provide unique insight into mechanisms leading to infertility (e.g., anovulation, luteinized unruptured follicle [LUF] and luteal phase defects) and suggest novel methods for controlling fertility in women.
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