Metabolism of [3H]2-Hydroxyestradiol by Cultured Porcine Granulosa Cells: Evidence for the Presence of a Catechol-O-Methyltransferase Pathway and a Direct Stimulatory Effect of 2-Methoxyestradiol on Progesterone Production1

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Abstract
Porcine granulosa cells synthesize and respond to catecholestrogens, but the stimulatory potency of catecholestrogens on progesterone production is much less than that of estradiol (E2). Therefore, to determine if metabolism of catecholestrogens by granulosa cells could account for the reduced potency of 2-hydroxyestradiol (2-OH-E2) observed in vitro, procine granulosa cells were cultured with [3H]2-OH-E2 and medium collected at 0, 0.5, 1, 2, 4, 6, or 12 h in the presence or absence of 1 .mu.g/ml 2-OH-E2, 0.5 mM L-ascorbate or 10 .mu.M U-0521 (a specific catechol-O-methyltransferase inhibitor). Metabolism of [3H]2-OH-E2 was very rapid with only 16% of the original [3H]2-OH-E2 remaining after 4 h exposure to cells. The main metabolite comigrated with 2-methoxyestradiol (2-MeO-E2) on thin-layer chromatography. Although appreciable degradation of [3H]2-OH-E2 occurred with time in the absence of cells, formation of the O-methyl derivative was minimal. Rather, formation of polar metabolites occurred in the absence of cells. Ascorbate dramatically reduced this noncellular degradation. Ascorbate added to cell cultures had no effect on the rate of formation of O-methyl products but slowed the formation of polar compounds as well as the overall rate of degradation of [3H]-2-OH-E2 by nearly 2-fold. U-0521 completely blocked the formation of O-methyl products, slowed the overall rate of degradation of [3H]2-OH-E2 by half and resulted in an increase in polar metabolites. The effects of U-0521 and ascorbate on 2-OH-E2-stimulated progesterone production in vitro was also examined. Ascorbate (0.5 mM) enhanced the effect of 2-OH-E2 (but not E2) on progesterone production by 2-fold (p < 0.05). The addition of 10 .mu.M U-0521 in the presence of 0.5 mM ascorbate had no effect on 1 .mu.g/ml 2-OH-E2-stimulated progesterone production, but it increased (p < 0.05) the response to 4 .mu.g/ml 2-OH-E2. The effects of 2-MeO-E2, 2-OH-E2, and E2 on progesterone production by cultured granulosa cells were then compared. The ED50 of E2 was 6- to 8-fold lower than that of 2-OH-E2 and 2-MeO-E2, whereas the ED50 of 2-OH-E2 was 15% lower than that of 2-MeO-E2. In the presence of ascorbate (0.5 mM), the maximal effect of E2 and 2-OH-E2 was approximately equal, whereas 2-OH-E2 was nearly 2-fold more efficacious than 2-MeO-E2. On the basis of these results, we conclude that in porcine granulosa cells 1) 2-OH-E2 is rapidly metabolized, and 2) the principal metabolites are biologically inactive polar compounds and partially active O-methyl derivatives.

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