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Activin, a member of the TGF-β superfamily, is an important modulator of FSH synthesis and secretion and is involved in reproductive dysfunctions and cancers. It also regulates ovarian follicle development. To understand the mechanisms and pathways by which activin regulates follicle function, we performed a microarray study and identified 240 activin regulated genes in mouse granulosa cells. The gene most strongly inhibited by activin was Cyp26b1, which encodes a P450 cytochrome enzyme that degrades retinoic acid (RA). Cyp26b1 has been shown to play an important role in male germ cell meiosis, but its expression is largely lost in the ovary around embryonic d 12.5. This study demonstrated that Cyp26b1 mRNA was expressed in granulosa cells of follicles at all postnatal developmental stages. A striking inverse spatial and temporal correlation between Cyp26b1 and activin-βA mRNA expression was observed. Cyp26b1 expression was also elevated in a transgenic mouse model that has decreased activin expression. The Cyp26 inhibitor R115866 stimulated the proliferation of primary cultured mouse granulosa cells, and a similar effect was observed with RA and activin. A pan-RA receptor inhibitor, AGN194310, abolished the stimulatory effect of either RA or activin on granulosa cell proliferation, indicating an involvement of RA receptor-mediated signaling. Overall, this study provides new insights into the mechanisms of activin action in the ovary. We conclude that Cyp26b1 is expressed in the postnatal mouse ovary, regulated by activin, and involved in the control of granulosa cell proliferation.

In the ovary, the steroid hormone estrogen and the TGF-beta superfamily member activin are both produced by granulosa cells and they both have intraovarian functions. Emerging evidence has indicated an interaction of these two signaling pathways. Based on the fact that estrogen and activin can impact early follicle formation and development, we hypothesize that estrogen treatment may alter activin signaling in the neonatal ovary. Therefore, this study was designed to examine the effect of neonatal diethylstilbestrol (DES) and estradiol (E(2)) exposure on the mRNA and protein levels of the key factors involved in activin signaling in the mouse ovary. CD-1 mouse pups were given daily injections of DES, E(2), or oil on postnatal d 1-5, and ovaries and sera were collected on d 19. Neonatal DES or E(2) exposure decreased the number of small antral follicles, induced multioocytic follicle formation, and decreased activin beta-subunit mRNA and protein levels. Consistent with local loss of beta-subunit expression, the phosphorylation of Smad 2, a marker of activin-dependent signaling, was decreased in the estrogen-treated ovaries. The decreased beta-subunit expression resulted in a decrease in serum inhibin levels, with a corresponding increase in FSH. Estrogen also suppressed activin subunit gene promoter activities, suggesting a direct transcriptional effect. Overall, this study demonstrates that activin subunits are targets of estrogen action in the early mouse ovary.

Previous studies from our laboratory, demonstrating that suppression of serum FSH by RU486 requires a high estrogen (E) background, suggested that E-inducible progesterone receptors play a role in the regulation of FSH secretion. We demonstrated further that the type II antiprogestin RU486 and the type I antiprogestin ZK98299 both suppressed the elevated serum FSH and FSHbeta messenger RNA levels similarly on the evening of proestrus, but had divergent effects on the morning of estrus, when only RU486, but not ZK98299, lowered the elevated serum FSH level (secondary FSH surge). In the present work we used primary anterior pituitary cell culture to examine whether RU486 caused direct, E-dependent suppression of basal and recombinant human activin A (activin)-induced FSH secretion in the gonadotrope and to compare this direct effect, if any, with that of ZK98299. Primary cell cultures were prepared from anterior pituitaries collected from cycling female rats either on metestrous or proestrous morning and cultured in DMEM, supplemented with charcoal-stripped serum without or with 10 nM estradiol (E2) for 96 h; exposure to test agents occurred during the last 48 h of culture. FSH released into the medium and intracellular FSH content were determined by RIA. In cells from the anterior pituitary of metestrous rats cultured in E2-free medium, neither antiprogestin (10 nM) affected FSH release; in contrast, when cells were cultured in medium to which E2 had been added, both antiprogestins caused profound suppression of both basal and activin (10 ng/ml)-stimulated FSH release. In cell cultures from proestrous rats, both antiprogestins caused a slight, but significant, suppression of basal FSH release even in the absence of added E2; activin-stimulated FSH release, however, was not affected. Upon exposure of the cells from proestrous rats to E2, the antiprogestins potently suppressed both basal and activin-stimulated FSH secretion. Because the foregoing incubations were performed in culture medium devoid of progesterone (P4), the actions of the antiprogestins on FSH secretion were independent of the natural ligand. Addition of P4 (10 nM) to the cell cultures stimulated basal and activin-induced FSH release more in the presence than in the absence of E2. The FSH response to P4 was completely blocked by both antiprogestins in both the absence and presence of E2. Finally, both RU486 and ZK98299 blocked the stimulatory effect of corticosterone (1 microM) on FSH secretion. The observed effects of P4 and antiprogestins were specific for FSH secretion; LH secretion was not similarly suppressed by either antiprogestin, but was, in fact, stimulated by ZK98299 in E2-treated cells. We conclude that 1) E2-inducible progesterone receptors interact with activin-mediated signal transduction to regulate FSH secretion, and 2) unlike on the morning of estrus in vivo, RU486 and ZK98299 affect FSH secretion similarly in the gonadotrope in vitro.

Previous in vivo studies from our laboratory indicated that administration of the antiprogestin RU486 on proestrus suppresses both the preovulatory gonadotropin surges and the secondary FSH surge, suggesting a role for the progesterone receptor (PR) in the generation of these surges. The present study was designed to test the effects of another antiprogestin, ZK98299, which has been reported to block the PR through a mechanism different from that of RU486, on gonadotropin secretion in vivo. RU486 and ZK98299 (2 and 6 mg/kg) were administered s.c. at 1230 h on proestrus; uterine intraluminal fluid content, serum gonadotropins, and gonadotropin subunit messenger RNAs (mRNAs) were determined at 1830 h on proestrus and at 0900 h on estrus. At 1830 h on proestrus, both RU486 and ZK98299 at both doses caused equal suppression of the preovulatory FSH surge and FSHbeta mRNA. Both antiprogestins also equally attenuated the preovulatory LH surge at this time, with the higher doses causing greater suppression. In contrast, at 0900 h on estrus, the antiprogestins affected serum FSH differentially; only RU486 suppressed the secondary FSH surge despite the fact that both drugs prevented the release of uterine intraluminal fluid, confirming blockade of progesterone action at the level of the uterus. Neither drug had a significant effect on FSHbeta mRNA at 0900 h on estrus. ZK98299 at the higher dose caused a small, but significant, increase in serum LH. In a subsequent experiment, we compared the effects of RU486 and ZK98299 (6 mg/kg, s.c.), administered at 1230 h on proestrus, on serum FSH raised above the natural secondary FSH surge on the morning of estrus by passive immunization with an antiserum to inhibin-alpha (anti-I) at 1700 h on proestrus. Consistent with the results of the first experiment, both antiprogestins blocked the release of uterine intraluminal fluid, but only RU486 lowered serum FSH in both the normal sheep serum-treated controls and anti-I-treated rats; in contrast, ZK98299 actually increased serum FSH in the normal sheep serum-treated control animals. ZK98299 also increased FSHbeta mRNA in the control group; RU486, on the other hand, reduced FSHbeta mRNA only in the anti-I group. The results demonstrate unequivocally that whereas the effects of the two antiprogestins on serum FSH and FSHbeta mRNA are similar on proestrus, they are divergent on estrus. The data suggest that the functional state of the PR/transcriptional activation complex in the gonadotrope on the morning of estrus is different from that on the evening of proestrus.