Catecholestrogens stimulate progestin secretion by cultured porcine granulosa cells

Abstract

The enzymatic metabolism of estradiol (E 2) to the catecholestrogens, 2-hydroxyestradiol (2-OH-E 2) and 4-hydroxyestradiol (4-OH-E 2) in granulosa cells has been reported. Therefore, we evaluated the effects of these compounds and compared them to those of E 2 on porcine granulosa cells cultured in serum-free medium. Cultures of granulosa cells were exposed to various treatments of E 2, 2-OH-E 2, 4-OH-E 2 and(or) follicle-stimulating hormone (FSH) for 4 days and concentrations of progesterone in medium and cell numbers were determined. After 4 days of treatment, 2-OH-E 2 and 4-OH-E 2 stimulated basal progesterone production by granulosa cells, but 4-OH-E 2 was less effective than 2-OH-E 2. 2-OH-E 2 (1 μg/ml) stimulated progesterone production by 3.3 ± 0.6-fold ( n = 6 experiments), whereas E 2 (1 μg/ml) stimulated progesterone production 9.9 ± 1.7-fold ( n = 6 experiments). 2-OH-E 2 at 4 μg/ml further stimulated progesterone production to 10.7 ± 2.2-fold above controls ( n = 9 experiments), whereas 4 μg/ml of E 2 did not cause further stimulation of progesterone production. Thus, the average potency of 2-OH-E 2 was less than E 2. Concurrent treatment with 2-OH-E 2 (4 μg/ml) and saturating concentrations of E 2 resulted in further significant increases in progesterone production above the effects of either single treatment both in the absence and presence of FSH (200 ng/ml). In the presence of FSH, potency of 2-OH-E 2 and E 2 was similar. Progesterone production stimulated by E 2 was significantly reduced by addition of the antiestrogens nafoxidine and CI-628, whereas the action of 2-OH-E 2 was unaffected. In addition, the responses of granulosa cells to 2-OH-E 2 and E 2 in terms of progesterone production was not significantly correlated across several experiments ( r= −0.29). The present studies show that 2-OH-E 2 enhances progesterone secretion by porcine granulosa cells and that the mechanisms of action of E 2 and 2-OH-E 2 may be different. Apparently, the effects of 2-OH-E 2 are exerted through a mechanism other than the E 2 receptor. Thus, it seems unlikely that effects of E 2 on granulosa cell function are mediated totally by 2-OH-E 2. Instead, catecholestrogen production could provide an intraovarian autocrine control mechanism whereby metabolites of E 2 could independently cause further stimulation of granulosa cell differentiation.