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Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and evidence for
Abstract:
These studies were undertaken to determine if the content of prostaglandin endoperoxide (PGH) synthase (PGS) was regulated in rat ovarian follicles in a time-, tissue-, and dose-dependent manner. For quantitation and localization of the enzyme by immunoblot and immunofluorescent analyses, respectively, a polyclonal antibody to highly purified (95%) ovine seminal vesicle PGS [mol wt, (Mr), 72,000] was generated in a rabbit, and an immunoglobulin G fraction of the antiserum was purified by elution from a PGS affinity resin. Soluble cell extracts were prepared from: 1) small antral (SA) and preovulatory (PO) rat follicles before and at selected time intervals after exposure to increasing doses (0.25-10.0 IU) of hCG, 2) granulosa and thecal cells of these same follicles, and 3) granulosa cells of hypophysectomized (H) rats before and after treatment with estradiol (HE), estradiol and FSH (HEF), and 10 IU hCG. Immunoblot analyses demonstrated that the content of PGS (Mr, 72,000) was low (negligible) in granulosa and thecal cells of SA and PO follicles, was induced preferentially (approximately 15-fold) in granulosa cells between 1 and 7 h after hCG treatment (0.5, 2.0, or 10 IU), and declined between 12-24 h after administration of 10 IU hCG, reaching undetectable amounts in corpora lutea isolated 48 h after hCG treatment. PGS (Mr, 72,000) was also induced by 10 IU hCG in granulosa cells of HEF rats, but was low in granulosa cells of H, HE, and HEF rats. In contrast, prostacyclin synthase was present in granulosa cells of preantral (H and HE rats), SA, and PO follicles, was not induced by hCG, and was highest in thecal cells. Immunofluorescent analyses confirmed both the tissue-specific localization and induction of PGS in granulosa cells of rat PO follicles treated with 10 IU hCG and the subcellular fractionation analyses showing that the PGS that was induced by hCG was localized primarily to a membrane (plasma membrane?) fraction of granulosa cells. Immunofluorescent data also demonstrated immunoreactive PGS in the vascular tissue of the rat corpus luteum but not in the luteal cells. Results of these studies document unequivocally that the synthesis of prostaglandins that is increased by LH/hCG in rats preceding ovulation is associated with an increased PGS content, that induction of the induced enzyme is transient, and that the enzyme is primarily localized to granulosa cell membrane fractions.