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ISOLATION AND CHARACTERIZATION OF A NOVEL OVARIAN ISOFORM OF SOLUBLE EPOXIDE HYDROLASE.
Hennebold, Jon1, Stouffer, Richard 1, Tanaka, Mamoru 2, Kirkman, Nikki 2, Macfarlane, Jane 2, Adashi, Eli 2, 1 2
ABSTRACT- The technique suppression subtractive hybridization (SSH) was used to systematically isolate ovary-selective genes (Hennebold et al., Endocrinology 141:2725, 2000). The aim of the present study was to characterize a novel ovary-selective cDNA isolated from the SSH-derived library. This cDNA was largely restricted in its expression to the ovary with limited expression observed in the epididymis. Ovarian expression of this novel cDNA was limited to the periovulatory phase of a simulated estrous cycle (0 to 12 hrs post-hCG injection in PMSG-primed mice). As SSH yields partial cDNA fragments, the full-length sequence of the gene was determined by Rapid Amplification of cDNA Ends (RACE) to be a novel ovary-selective isoform of the enzyme soluble epoxide hydrolase (Ov-sEH). Expression of Ov-sEH was restricted to the mural granulosa cells of large periovulatory antral follicles as determined by in situ hybridization. The expression of hepatic sEH (Hp-sEH) in the liver is known to be under the control of the steroid receptor superfamily member Peroxisome Proliferater-Activated Receptor-
(PPAR ). Accordingly, treatment of mice with clofibrate, a selective agonist for PPAR, increased the expression of Hp-sEH in the liver. Ovarian expression of Ov-sEH, however, was not regulated by clofibrate. Expression of the Hp-sEH isoform was detected in the mouse ovary during the luteal phase of a simulated estrous cycle but was also refractory to PPAR-dependent transcriptional regulation. Notably, sEH activity in ovarian homogenates was observed to vary throughout a simulated estrous cycle in a similar manner to the sEH mRNA expression data. Ovarian sEH activity increased 2-fold at 6 hrs (p<0.05) and 12 hrs (p<0.05) post-hCG injection relative to controls. Maximal activity (3-fold increase relative to controls, p<0.05) was observed 48 hrs post-hCG injection. These studies suggest that Ov-sEH is important for ovulation and/or luteinization of the follicle, whereas Hp-sEH may be associated with the structure-function of the corpus luteum. The Ov-sEH and Hp-sEH isoforms may regulate ovarian processes via their ability to metabolize arachidonate-derived epoxides (epoxyeicosatrienoic acids), known modulators of prostaglandin production. Supported by NIH Grants HD 30288, HD 37845, HD 20869; and U54 Center HD 18185.
KEY WORDS: ovary-selective expression, epoxide hydrolase, eicosanoids