Catecholestrogens, formed locally, have been proposed to serve as local regulators of ovarian function. However, to date little or no activity of the critical biosynthetic enzyme, estrogen-2/4-hydroxylase, has been identified in the ovary. In the present study we have established the presence of this enzyme in porcine ovarian cells and characterized some of its biochemical features as well as its intraovarian distribution during the reproductive cycle. Homogenates of ovarian follicular tissue converted [3H]-17β-estradiol to the catecholestrogens 2- and 4-hydroxyestradiol (2-OH-E2 and 4-OH-E2) in a linear fashion for up to 60 min, with protein concentrations of equal to or less than 1 mg. The principal product, 2-OH-E2, was stable under the conditions of the assay. The reaction exhibited a dependence on nicotinamide cofactors, a pH optimum of 7.8, and an apparent Michaelis constant (Km) of approximately 10 HM for the production of 2-OH-E2 and 4-OH-E2. The activity of different ovarian preparations varied dramatically as a function of the reproductive cycle. Assayed at saturating substrate concentrations, immature follicular tissue and luteal tissue produced 50 or less pmol 2-OH-E2/mg protein-40 min, while preovulatory follicles produced approximately 600 pmol 2-OH-E2/mg protein ⋅ 40 min: Even within the population of large presumptively preovulatory follicles, a variation in activity of more than 10- fold was encountered. Estrogen-2/4-hydroxylase activity of large preovulatory follicles correlated with the concentration of 17β-estradiol in the same follicles (r = +0.89, P < 0.001). In large preovulatory follicles enzyme activity was present in both granulosa and theca layers. However, approximately 80% of the follicular activity was localized in the membrana granulosa. Under all conditions tested and in all ovarian compartments the formation of 2-OH-E2 was favored over that of 4-OH-E2. These studies show, for the first time, significant estrogen-2/4-hydroxylase enzyme activity within ovarian tissue. The striking increase in activity in the preovulatory follicle suggests physiological control of catecholestrogen synthesis. In conjunction with other data demonstrating stimulatory actions of catecholestrogens on ovarian cells, these observations are consistent with an intraovarian autocrine or paracrine regulatory function for these metabolites.
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