Mechanism of Action of 2-Hydroxyestradiol on Steroidogenesis in Ovarian Granulosa Cells: Interactions with Catecholamines and Gonadotropins Involve Cyclic Adenosine Monophosphate1

Previously we have shown that 2-bydroxyestradiol (2-OH-E2) synergizes with catecbolamines to enhance progesterone production by porcine granulosa cells in vitro. The present studies were undertaken to determine if the synergistic effects of 2-OH-E2and catecbolamines were 1) modulated by gonadotropins, 2) unique to catecholamines, and 3) mediated by cyclic adenosine monophosphate (cAMP). Undifferentiated granulosa cells from 1- to 3-mm porcine follicles were cultured in serum free medium for periods of 6–9 days. A 3-day pretreatment plus a 4-day cotreatment versus a 4-day cotreatment of granulosa cell cultures with follicle-stimulating hormone (FSH) did not significantly alter progesterone production stimulated by a saturating concentration of epinephrine (EPI; 2 μg/m1) but significantly reduced the effect of 4 μg/ml 2-0H-E2on Day 7 of culture. Four-day cotreatment of either FSH or luteinizing hormone (LH) from Day 3 to 7 of culture dramatically enhanced progesterone production stimulated by 2-OH-E2and estradiol (E2) but not by EPI when measured on Day 7 of culture. Progesterone production (expressed as “-fold of controls”) stimulated by 4-day treatment of EPI, 2-OH-E2, or EPI-plus-2-OH-E2was 1.4 ± 0.2, 8.2 ± 2.2, and 10.7 ± 1.0, respectively, in the presence of LH (n = 5 experiments), and 1.9 ± 0.1, 7.8 ± 1.4, and 10.6 ± 1.8, respectively, in the presence of FSH (n = 3 experiments). Similar to E2, 2-OH-E2significantly enhanced the stimulating effect of the cAMP analog 8-bromo-cAMP (0.5 mM) on progesterone production. The synergism between saturating concentrations of 2-OH-E2and EPI on progesterone production was not observed in the presence of 8-bromo-cAMP. However, the synergism between 2-OH-E2and EPI persisted in the presence of the phospbodiesterase inhibitor 3-isobuty1-1-methylxanthine (IBMX, 0.5 mM). In addition, cAMP accumulation over 24 h (medium plus cell extracts) stimulated by EPI or by FSH was significantly elevated in the presence of 2-OH-E2but not E2. Collectively, these results suggest that 1) 2-OH-E2can dramatically enhance LH- and FSH-stimulated progesterone production; 2) the synergism between 2-OH-E2and gonadotropins was at least as great as that of the synergism between 2-OH-E2and catecbolamines; 3) in contrast to studies with rat granulosa cells, pretreatment or concurrent treatment of porcine granulosa cells with gonadotropins and catecbolamines exerts, at best, an additive effect on the catecholaminergic response; and 4) a major locus of 2-OH-E2(but not E2)-potentiated progesterone production stimulated by EPI or FSH is exerted at the level of the generation of cAMP.