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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.

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, porcine 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 μg/ml 2-OH-E2, 0.5 mM L-ascorbate or 10 μM 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 μM U-0521 in the presence of 0.5 mM ascorbate had no effect on 1 μg/ml 2-OH-E2-stimulated progesterone production, but it increased (p<0.05) the response to 4 μg/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 0-methyl derivatives.

Changes in concentrations of LH and progesterone in serum and specific binding of 125I-hCG to crude membrane preparations of luteal cells were examined during spontaneous regression (Group 1), prostaglandin-induced regression (Group 2), and growth (Group 3) of bovine corpora lutea (CL). Specific binding of 125I-hCG (presumably by the LH receptor) to luteal homogenates was validated using Scatchard analysis and shown to be specific for LH/hCG with high affinity (Ka= 2.18 ± 0.76 × 1010M−1). All cattle had estrous cycles synchronized with a synthetic prostaglandin F2α, cloprostenol (PG). Group 1 had ovaries removed on Day 17, 18, 19, on estrus, or 24 h after estrus. Group 2 had ovaries removed at 0, 12, 24, 48, 60, or 72 h after PG. Group 3 had ovaries removed at 5, 7, 9, 11, 13, or 15 days after PG. Blood samples were taken from all animals at 8 h intervals up to time of ovariectomy for measurement of serum progesterone and LH and at 2 h intervals around estrus to determine time of preovulatory LH surge. Data from heifers in Group 3 were grouped for analysis according to days after preovulatory LH surges.During spontaneous regression (Group 1), Day 19 to Day 21 of the estrous cycle, serum progesterone, specific binding of hCG, and CL weight decreased (P<0.05) concomitantly. No significant change in LH occurred prior to Day 21. During PG-induced regression of corpora lutes (Group 2), 0 to 36 h after PG, the amount of hCG binding, weights of corpora lutea and concentrations of serum progesterone decreased significantly and remained at low levels through 60 h. However, serum progesterone decreased to basal levels 12 h earlier than did hCG binding. In contrast, serum LH increased by 12 h (P<0.05) and remained elevated through 60 h after PG. Correlations between variables measured during spontaneous and induced luteal regression were similar.During CL development (Group 3), 1.9 to 4.5 days after the preovulatory LH surge, serum progesterone, specific binding of hCG, and CL weight increased coincidently (P<0.05). Maximum levels of serum progesterone and binding of hCG were reached at Day 8.3. However, between Days 8.3 and 12.4 post-LH surge, serum progesterone and CL weight did not change (P>0.05), while binding of hCG to corpora lutea decreased (P<0.05).From these data we suggest that synchronization of estrus using two injections of prostaglandin results in short-term endocrine changes that are similar to changes seen during the late luteal phase of the estrous cycle. Since serum progesterone declined to basal levels prior to binding of hCG to corpora lutea during PG-induced regression, other events such as decreased adenylate cyclase activity may be more closely associated with decreased function of corpora lutea than decreases in LH receptor during regression of corpora lutea. In addition, we conclude that changes in numbers of LH receptors may be more closely associated to progesterone secretion in early luteal development (<4.5 days post-LH surge) than during late luteal development (4.5 to 12.4 days post-LH surge). This suggests that with an increase in LH binding sites, the early developing corpus luteum may become more responsive to basal levels of serum LH causing an increase in progesterone secretion.