Limited published reports and preliminary experiments conducted by the PI suggest that the rodent and primate ovary possess the capacity to produce and metabolize epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid (AA). Preliminary experiments also suggest that EETs play a role in ovulation as an inhibitor of EET metabolism significantly increased the number of oocytes released in mice following injection of an ovulatory dose of gonadotropin. Therefore, studies are designed using rodents and primates to test the hypothesis that EETs are synthesized (Aim number 1), inactivated (Aim number 2), and directly affect molecular processes (Aim number 3) and oocyte release (Aim number 4) in the ovulatory follicle. Expression of EET generating cytochrome P450 (CYP) epoxygenases during the periovulatory interval and their cellular localization will be determined by real-time PCR and in situ hybridization, respectively. The specific EET isomer(s) produced during the periovulatory interval will be determined by gas chromatography/mass spectrometry. Recently, the PI has cloned a novel ovary-selective isoform of soluble (cytoplasmic) epoxide hydrolase (sEH), an enzyme that converts EETs to their inactive diols (dihydroxyeicosatrienoic acids; DiHETEs). The expression of the ovary-selective sEH isoform was restricted to the periovulatory period. Biochemical properties of the novel ovary- selective sEH will be determined, including substrate specificity, KM/Vmax, capacity to metabolize EETs and subcellular localization. The presence of a homologous primate isoform(s) will be analyzed by rapid amplification of cDNA ends (RACE). Recent studies have demonstrated that in non-ovarian cells EETs increase the expression of prostaglandin H synthase-2 (PGHS-2), a prostaglandin (PG) synthetic enzyme critical for optimal ovulatory efficiency. The major EET species produced in the ovary during the periovulatory interval will be analyzed in vitro with respect to their ability to directly induce PGHS-2 expression. In vivo effects on PGHS-2 expression, PG production, and ovulation will be determined by using inhibitors of EET generation and metabolism. These studies will provide insight into the action and regulation of EETs in the ovulatory follicle and may yield novel approaches for the regulation of fertility.
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