Mammalian females enter puberty with follicular reserves that exceed the number needed for ovulation during a single lifetime. Follicular depletion occurs throughout reproductive life and ends in menopause, or reproductive senescence, when the follicle pool is exhausted. The mechanisms regulating the production of a species-specific initial follicle pool are not well understood. However, the establishment of a follicular reserve is critical to defining the length of reproductive cyclicity. Here we show that activin A (rh-ActA), a known regulator of follicle formation and growth in vitro, increased the number of postnatal mouse primordial follicles by 30% when administered to neonatal animals during the time of germline cyst breakdown and follicle assembly. This expansion in the initial follicle pool was characterized by a significant increase in both germ cell and granulosa cell proliferation. However, the excess follicles formed shortly after birth did not persist into puberty and both adult rh-ActA- and vehicle-treated animals demonstrated normal fertility. A follicle atresia kinetic constant (k(A)) was modeled for the two groups of animals, and consistent with the empirical data, the k(A) for rh-ActA-treated was twice that of vehicle-treated animals. Kinetic constants for follicle formation, follicle loss and follicle expansion from birth to postnatal day 19 were also derived for vehicle and rh-ActA treatment conditions. Importantly, introduction of exogenous rh-ActA revealed an intrinsic ovarian quorum sensing mechanism that controls the number of follicles available at puberty. We propose that there is an optimal number of oocytes present at puberty, and when the follicle number is exceeded, it occurs at the expense of oocyte quality. The proposed mechanism provides a means by which the ovary eliminates excess follicles containing oocytes of poor quality prior to puberty, thus maintaining fertility in the face of abnormal hormonal stimuli in the prepubertal period.

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.

Activin, a member of the transforming growth factor-␤ superfamily, is an important modulator of follicle-stimulating hormone synthesis and secretion in the pituitary and plays autocrine/paracrine roles in the regulation of ovarian follicle development. From a microarray study on mouse ovarian granulosa cells, we discovered that the estrogen receptor ␤ (ER␤) is inducible by activin. We previously demonstrated that estrogen suppresses activin gene expression, suggesting a feedback relationship between these two follicle-regulating hormones. The purpose of this study was to investigate fully activin A regulation of ER expression. Real time reverse transcription-PCR assays on cultured granulosa cells showed that both ER␣ and ER␤ mRNAs were induced by activin A at 4, 12, and 24 h in a dose-responsive manner. Western blots confirmed an increase in their protein levels. Consistent with increased ER␣ and ER␤ expression, activin A stimulated estradiol-induced estrogen response element promoter activity. Activin A stimulation of ER expression was a direct effect at the level of gene transcription, as it was not abolished by cycloheximide but was abolished by actinomycin D, and in transfected granulosa cells activin A stimulated ER␣ promoter activity. To investigate the effect of activin in vivo and, thus, its biological significance, we examined ER expression in inhibin transgenic mice that have decreased activin expression and discovered that these mice had decreased ER␣ and ER␤ expression in the ovary. We also found that ER mRNA levels were decreased in Mü llerian inhibiting substance promoter (MIS)-Smad2 dominant negative mice that have impaired activin signaling through Smad2, and small interfering RNAs targeting Smad2 or Smad3 suppressed ER␣ promoter activation, suggesting that Smad2 and Smad3 are involved in regulating ER levels. Therefore, this study reveals an important role for activin in inducing the expression of ERs in the mouse ovary and suggests important interplay between activin and estrogen signaling.Activin and its functional antagonist inhibin were originally isolated from gonadal sources as endocrine factors regulating the synthesis and secretion of follicle-stimulating hormone by the pituitary gland (1-8). More recent studies have indicated that activin acts predominantly as a local paracrine and autocrine factor (9, 10). Consistent with the fact that activin is a member of the transforming growth factor-␤ (TGF-␤) 2 superfamily, activin has a variety of functions and is involved in many physiological processes, including embryonic development, wound repair, inflammation, renal tubule morphogenesis, and neuroprotection. In the reproductive system, in addition to regulating gonadotropin release, activin and inhibin have been shown to play an important role in regulating the formation and development of ovarian follicles in the female (10 -15) and development and function of the testis in the male (16). Ovarian follicle formation and development is a dynamic process finely regulated by various intrinsic ...

The question of whether estrogens or estrogen-like compounds would alter differentially the enzymatic activity of the FOF1-ATPase was addressed. Mitochondrial fractions of the liver, brain, and heart were obtained from adult male rats and solubilized by digitonin. About 85% of the adenosine triphosphate hydrolysis by these three preparations come from the mitochondrial FOF1-ATPase. The enzymatic activity differed in the following order: liver < brain < heart. A concentration of 13 nM estradiol stimulated the FOF1-ATPase activity in heart by 10% (p < 0.01), but not in liver or brain. 17beta-estradiol competed off the binding of estradiol-17beta-17-(O-carboxymethyl)oxime:125I-labeled bovine serium albumin to mitochondrial preparations of the heart, revealing two binding sites. Resveratrol inhibited the F0F1-ATPase activity in both heart and liver with an IC50 of 13-15 microM, which confirmed our previous report in preparations of brain. Lower doses (picomolar to nanomolar) of resveratrol stimulated the FOF1-ATPase activity in liver by 10% but not in heart. At 6.7 microM, diethylstilbestrol (DES) inhibited the FOF1-ATPase activity in the three preparations by 61-67%. This study demonstrates that estradiol activates rat heart mitochondrial FOF1-ATPase at physiologic concentrations and that the FOF1-ATPase activity is markedly different in rat liver, brain, and heart. In addition, estradiol, DES, and resveratrol alter the FOF1-ATPase activity selectively, probably via different mechanisms.

Activin, a member of the TGF-␤ 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-␤A 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. (Endocrinology 152: 303-312, 2011) M ammalian ovarian follicle formation and development involves establishment of the initial follicle pool, follicle growth, proper maturation of eggs, and timely production and release of hormones (1, 2). This process is critical for propagation of the species as well as for development and homeostasis. Regulation of follicle formation and development requires intrinsic and endocrine factors as well as interactions between multiple cell types within the ovary (1-5). Among the many intraovarian factors, substantial evidence indicates that activin and inhibin, members of the TGF-␤ superfamily, play important autocrine/paracrine roles in this process (6 -9). Activin and inhibin were first isolated from gonadal sources as endocrine factors that either stimulate (activin) or suppress (inhibin) the synthesis and secretion of FSH by the pituitary gland (10 -16). Later studies indicated that activin acts predominantly as a local paracrine and autocrine factor (17, 18).