Preliminary studies conducted by the PI indicate that the ovulatory gonadotropin surge initiates prostaglandin (PG) production by the primate periovulatory follicle and the injection of a PG synthesis inhibitor directly into the follicle prevents follicle rupture without altering normal luteal progression. The proposed studies will address the hypothesis that PGs produced by the preovulatory follicle act locally to regulate specific molecular events related to follicular rupture, but not luteinization, of the primate follicle. Experiments are proposed to examine the locations and gonadotropin regulation of PG synthetic enzymes within the primate periovulatory follicle (Specific Aim 1). In vitro protocols will be used to determine if gonadotropin stimulation of granulosa and/or theca cells of the follicle is sufficient to initiate PG synthesis (Specific Aim 2). PG receptors will be localized to cells of the periovulatory follicle to determine the site(s) of PG action (specific Aim 3). PG-stimulated expression of molecular markers of follicle rupture (matrix metalloproteinases and their inhibitors [TIMPs], plasminogen activator and its inhibitor and luteinization (3beta hydroxydehydrogenase, steroidogenic acute regulatory protein [STAR], vascular endothelial growth factor (VEGF], angiopoietin-1 and 2) will be assessed (specific Aim 4). Immunocytochemistry and in situ hybridization will be used to localize PG synthetic enzymes and PG receptors to the cells of the preovulatory follicle. Gonadotropin regulation of mRNAs for PG synthetic enzymes will be determined by semi-quantitative RT-PCR based assays. PG production of cultured ovarian cells and tissues will be measured using EIA kits. In vitro expression of markers of follicular rupture/luteinization will be assessed using RT-PCR based assays for mRNAs, ELISA kits for enzyme proteins and angiogenic factors, and RIA for media progesterone. Intrafollicular injection of PG synthesis inhibitor will be used to determine if PGs regulate specific markers of follicular rupture and luteinization in vivo. These studies will likely demonstrate that the mid cycle gonadotropin surge initiates PG synthesis by primate ovarian follicle through the induction of specific enzymes and support the hypothesis that PGs act locally to trigger specific processes related to ovulation, but not luteinization, of the periovulatory follicle. This may provide insight into the mechanisms leading to infertility (e.g., leuteinized unruptured follice syndrome) and support the developoment of PG synthesis inhibitors of PG synthesis inhibitors for use as contraceptives.
