Activation of Notch Signaling by Oocytes and Jag1 in Mouse Ovarian Granulosa Cells.

The Notch pathway plays diverse and complex roles in cell signaling during development. In the mammalian ovary, Notch is important for the initial formation and growth of follicles, and for regulating the proliferation and differentiation of follicular granulosa cells during the periovulatory period. This study seeks to determine the contribution of female germ cells toward the initial activation and subsequent maintenance of Notch signaling within somatic granulosa cells of the ovary. To address this issue, transgenic Notch reporter (TNR) mice were crossed with Sohlh1-mCherry (S1CF) transgenic mice to visualize Notch-active cells (EGFP) and germ cells (mCherry) simultaneously in the neonatal ovary. To test the involvement of oocytes in activation of Notch signaling in ovarian somatic cells, we ablated germ cells using busulfan, a chemotherapeutic alkylating agent, or investigated KitWv/Wv (viable dominant white-spotting) mice that lack most germ cells. The data reveal that Notch pathway activation in granulosa cells is significantly suppressed when germ cells are reduced. We further demonstrate that disruption of the gene for the Notch ligand Jag1 in oocytes similarly impacts Notch activation and that recombinant JAG1 enhances Notch target gene expression in granulosa cells. These data are consistent with the hypothesis that germ cells provide a ligand, such as Jag1, that is necessary for activation of Notch signaling in the developing ovary.

Notch Signaling Regulates Differentiation and Steroidogenesis in Female Mouse Ovarian Granulosa Cells.

The Notch pathway is a highly conserved juxtacrine signaling mechanism that is important for many cellular processes during development, including differentiation and proliferation. Although Notch is important during ovarian follicle formation and early development, its functions during the gonadotropin-dependent stages of follicle development are largely unexplored. We observed positive regulation of Notch activity and expression of Notch ligands and receptors following activation of the luteinizing hormone-receptor in prepubertal mouse ovary. JAG1, the most abundantly expressed Notch ligand in mouse ovary, revealed a striking shift in localization from oocytes to somatic cells following hormone stimulation. Using primary cultures of granulosa cells, we investigated the functions of Jag1 using small interfering RNA knockdown. The loss of JAG1 led to suppression of granulosa cell differentiation as marked by reduced expression of enzymes and factors involved in steroid biosynthesis, and in steroid secretion. Jag1 knockdown also resulted in enhanced cell proliferation. These phenotypes were replicated, although less robustly, following knockdown of the obligate canonical Notch transcription factor RBPJ. Intracellular signaling analysis revealed increased activation of the mitogenic phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways following Notch knockdown, with a mitogen-activated protein kinase kinase inhibitor blocking the enhanced proliferation observed in Jag1 knockdown granulosa cells. Activation of YB-1, a known regulator of granulosa cell differentiation genes, was suppressed by Jag1 knockdown. Overall, this study reveals a role of Notch signaling in promoting the differentiation of preovulatory granulosa cells, adding to the diverse functions of Notch in the mammalian ovary.

The role of Notch signaling in the mammalian ovary.

The Notch pathway is a contact-dependent, or juxtacrine, signaling system that is conserved in metazoan organisms and is important in many developmental processes. Recent investigations have demonstrated that the Notch pathway is active in both the embryonic and postnatal ovary and plays important roles in events including follicle assembly and growth, meiotic maturation, ovarian vasculogenesis and steroid hormone production. In mice, disruption of the Notch pathway results in ovarian pathologies affecting meiotic spindle assembly, follicle histogenesis, granulosa cell proliferation and survival, corpora luteal function and ovarian neovascularization. These aberrations result in abnormal folliculogenesis and reduced fertility. The knowledge of the cellular interactions facilitated by the Notch pathway is an important area for continuing research, and future studies are expected to enhance our understanding of ovarian function and provide critical insights for improving reproductive health. This review focuses on the expression of Notch pathway components in the ovary, and on the multiple functions of Notch signaling in follicle assembly, maturation and development. We focus on the mouse, where genetic investigations are possible, and relate this information to the human ovary.

Change in the Gastro-Intestinal Tract by Overexpressed Activin Beta A.

Originally, activins were identified as stimulators of FSH release in reproduction. Other activities, including secondary axis formation in development, have since been revealed. Here, we investigated the influence of activin ßA on the body, including the gastro-intestinal (GI) tract. Initially, the activin ßA protein was detected in the serum proportional to the amount of pCMV-rAct plasmid injected. The induced level of activin ßA in muscle was higher in female than male mice. Subsequent results revealed that stomach and intestine were severely damaged in pCMV-rAct-injected mice. At the cellular level, loss of parietal cells was observed, resulting in increased pH within the stomach. This phenomenon was more severe in male than female mice. Consistent with damage of the stomach and intestine, activin ßA often led to necrosis in the tip of the tail or foot, and loss of body weight was observed in pCMV-rAct-injected male but not female mice. Finally, in pCMV-rAct-injected mice, circulating activin ßA led to death at supraphysiological doses, and this was dependent on the strain of mice used. Taken together, these results indicate that activin ßA has an important role outside of reproduction and development, specifically in digestion. These data also indicate that activin ßA must be controlled within a narrow range because of latent lethal activity. In addition, our approach can be used effectively for functional analysis of secreted proteins.

Inhibin at 90: from discovery to clinical application, a historical review.

When it was initially discovered in 1923, inhibin was characterized as a hypophysiotropic hormone that acts on pituitary cells to regulate pituitary hormone secretion. Ninety years later, what we know about inhibin stretches far beyond its well-established capacity to inhibit activin signaling and suppress pituitary FSH production. Inhibin is one of the major reproductive hormones involved in the regulation of folliculogenesis and steroidogenesis. Although the physiological role of inhibin as an activin antagonist in other organ systems is not as well defined as it is in the pituitary-gonadal axis, inhibin also modulates biological processes in other organs through paracrine, autocrine, and/or endocrine mechanisms. Inhibin and components of its signaling pathway are expressed in many organs. Diagnostically, inhibin is used for prenatal screening of Down syndrome as part of the quadruple test and as a biochemical marker in the assessment of ovarian reserve. In this review, we provide a comprehensive summary of our current understanding of the biological role of inhibin, its relationship with activin, its signaling mechanisms, and its potential value as a diagnostic marker for reproductive function and pregnancy-associated conditions.

Notch signaling regulates ovarian follicle formation and coordinates follicular growth.

Ovarian follicles form through a process in which somatic pregranulosa cells encapsulate individual germ cells from germ cell syncytia. Complementary expression of the Notch ligand, Jagged1, in germ cells and the Notch receptor, Notch2, in pregranulosa cells suggests a role for Notch signaling in mediating cellular interactions during follicle assembly. Using a Notch reporter mouse, we demonstrate that Notch signaling is active within somatic cells of the embryonic ovary, and these cells undergo dramatic reorganization during follicle histogenesis. This coincides with a significant increase in the expression of the ligands, Jagged1 and Jagged2; the receptor, Notch2; and the target genes, Hes1 and Hey2. Histological examination of ovaries from mice with conditional deletion of Jagged1 within germ cells (J1 knockout [J1KO]) or Notch2 within granulosa cells (N2 knockout [N2KO]) reveals changes in follicle dynamics, including perturbations in the primordial follicle pool and antral follicle development. J1KO and N2KO ovaries also contain multi-oocytic follicles, which represent a failure to resolve germ cell syncytia, and follicles with enlarged oocytes but lacking somatic cell growth, signifying a potential role of Notch signaling in follicle activation and the coordination of follicle development. We also observed decreased cell proliferation and increased apoptosis in the somatic cells of both conditional knockout lines. As a consequence of these defects, J1KO female mice are subfertile; however, N2KO female mice remain fertile. This study demonstrates important functions for Jagged1 and Notch2 in the resolution of germ cell syncytia and the coordination of somatic and germ cell growth within follicles of the mouse ovary.

Mechanism of CREB recognition and coactivation by the CREB-regulated transcriptional coactivator CRTC2.

Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.

DNA methylation and histone modifications are associated with repression of the inhibin α promoter in the rat corpus luteum.

The transition from follicle to corpus luteum after ovulation is associated with profound morphological and functional changes and is accompanied by corresponding changes in gene expression. The gene encoding the α subunit of the dimeric reproductive hormone inhibin is maximally expressed in the granulosa cells of the preovulatory follicle, is rapidly repressed by the ovulatory LH surge, and is expressed at only very low levels in the corpus luteum. Although previous studies have identified transient repressors of inhibin α gene transcription, little is known about how this repression is maintained in the corpus luteum. This study examines the role of epigenetic changes, including DNA methylation and histone modification, in silencing of inhibin α gene expression. Bisulfite sequencing reveals that methylation of the inhibin α proximal promoter is low in preovulatory and ovulatory follicles but is elevated in the corpus luteum. Increased methylation during luteinization is observed within the cAMP response element in the promoter, and EMSA demonstrate that methylation of this site inhibits cAMP response element binding protein binding in vitro. Chromatin immunoprecipitation reveals that repressive histone marks H3K9 and H3K27 trimethylation are increased on the inhibin α promoter in primary luteal cells, whereas the activation mark H3K4 trimethylation is decreased. The changes in histone modification precede the alterations in DNA methylation, suggesting that they facilitate the recruitment of DNA methyltransferases. We show that the DNA methyltransferase DNMT3a is present in the ovary and in luteal cells when the inhibin α promoter becomes methylated and observe recruitment of DNMT3a to the inhibin promoter during luteinization.

The LIM domain protein FHL2 interacts with the NR5A family of nuclear receptors and CREB to activate the inhibin-α subunit gene in ovarian granulosa cells.

Nuclear receptor transcriptional activity is enhanced by interaction with coactivators. The highly related nuclear receptor 5A (NR5A) subfamily members liver receptor homolog 1 and steroidogenic factor 1 bind to and activate several of the same genes, many of which are important for reproductive function. To better understand transcriptional activation by these nuclear receptors, we sought to identify interacting proteins that might function as coactivators. The LIM domain protein four and a half LIM domain 2 (FHL2) was identified as interacting with the NR5A receptors in a yeast two-hybrid screen of a human ovary cDNA library. FHL2, and the closely related FHL1, are both expressed in the rodent ovary and in granulosa cells. Small interfering RNA-mediated knockdown of FHL1 and FHL2 in primary mouse granulosa cells reduced expression of the NR5A target genes encoding inhibin-α and P450scc. In vitro assays confirmed the interaction between the FHL and NR5A proteins and revealed that a single LIM domain of FHL2 is sufficient for this interaction, whereas determinants in both the ligand binding domain and DNA binding domain of NR5A proteins are important. FHL2 enhances the ability of both liver receptor homolog 1 and steroidogenic factor 1 to activate the inhibin-α subunit gene promoter in granulosa cells and thus functions as a transcriptional coactivator. FHL2 also interacts with cAMP response element-binding protein and substantially augments activation of inhibin gene expression by the combination of NR5A receptors and forskolin, suggesting that FHL2 may facilitate integration of these two signals. Collectively these results identify FHL2 as a novel coactivator of NR5A nuclear receptors in ovarian granulosa cells and suggest its involvement in regulating target genes important for mammalian reproduction.

Gene expression profiling reveals Cyp26b1 to be an activin regulated gene involved in ovarian granulosa cell proliferation.

Activin, a member of the TGF-ß 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-ßA 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.

The interactions between the stimulatory effect of follicle-stimulating hormone and the inhibitory effect of estrogen on mouse primordial folliculogenesis.

The murine primordial follicle pool develops largely within 3 days after birth through germline nest breakdown and enclosure of oocytes within pregranulosa cells. The mechanisms that trigger primordial follicle formation likely are influenced by a transition from the maternal to fetal hormonal milieu at the time of birth. High levels of maternal estrogen maintain intact germline nest in fetal ovary, and decrease of estrogen after birth is permissive of follicle formation. In the present study, we measured an increase in neonatal serum follicle-stimulating hormone (FSH), which corresponded to falling estradiol (E(2)) levels during the critical window of primordial follicle formation (Postnatal Days 1-3). To determine whether fetal hormones contribute in an active manner to primordial follicle formation, mouse fetal ovaries (17.5 days postcoitus) were cultured in vitro at two concentrations of E(2) (meant to reflect maternal and fetal levels of E(2)) and FSH for 6 days. High levels of E(2) (10(-6) M) inhibited germline nest breakdown, and this effect was significantly reduced when fetal ovaries were cultured in the low E(2) concentration (10(-10) M). FSH facilitated germline nest breakdown and primordial follicle formation under both high and low E(2) culture conditions. Low E(2) was identified as being more permissive for the effects of FSH on primordial follicle formation by stimulating the up-regulation of Fshr and activin beta A subunit (Inhba) expression, pregranulosa cell proliferation, and oocyte survival. The decrease of E(2) plus the presence of FSH after birth are critical for primordial follicle formation and the expression of oocyte-specific transcription factors (Figla and Nobox) in that inappropriate exposure to FSH or E(2) during follicle formation resulted in premature or delayed primordial folliculogenesis. In conclusion, with the drop of E(2) level after birth, FSH promotes primordial follicle formation in mice by stimulating local activin signaling pathways and the expression of oocyte-specific transcription factors.

Use of reporter genes to study the activity of promoters in ovarian granulosa cells.

Use of reporter genes provides a convenient way to study the activity and regulation of promoters and examine the rate and control of gene transcription. Many reporter genes and transfection methods can be efficiently used for this purpose. To investigate gene regulation and signaling pathway interactions during ovarian follicle development, we have examined promoter activities of several key follicle-regulating genes in the mouse ovary. In this chapter, we describe use of luciferase and beta-galactosidase genes as reporters and a cationic liposome mediated cell transfection method for studying regulation of activin subunit- and estrogen receptor alpha (ERalpha)-promoter activities. We have demonstrated that estrogen suppresses activin subunit gene promoter activity while activin increases ERalpha promoter activity and increases functional ER activity, suggesting a reciprocal regulation between activin and estrogen signaling in the ovary. We also discuss more broadly some key considerations in the use of reporter genes and cell-based transfection assays in endocrine research.

Roles for transforming growth factor beta superfamily proteins in early folliculogenesis.

Primordial follicle formation and the subsequent transition of follicles to the primary and secondary stages encompass the early events during folliculogenesis in mammals. These processes establish the ovarian follicle pool and prime follicles for entry into subsequent growth phases during the reproductive cycle. Perturbations during follicle formation can affect the size of the primordial follicle pool significantly, and alterations in follicle transition can cause follicles to arrest at immature stages or result in premature depletion of the follicle reserve. Determining the molecular events that regulate primordial follicle formation and early follicle growth may lead to the development of new fertility treatments. Over the last decade, many of the growth factors and signaling proteins that mediate the early stages of folliculogenesis have been identified using mouse genetic models, in vivo injection studies, and ex vivo organ culture approaches. These studies reveal important roles for the transforming growth factor beta (TGF-beta) superfamily of proteins in the ovary. This article reviews these roles for TGF-beta family proteins and focuses in particular on work from our laboratories on the functions of activin in early folliculogenesis.

Activins regulate 17beta-hydroxysteroid dehydrogenase type I transcription in murine gonadotrope cells.

Activins are pleiotropic members of the TGFbeta superfamily and were initially characterized based on their abilities to stimulate FSH synthesis and secretion by gonadotrope cells of the anterior pituitary gland. Here, we identified the gene encoding the steroidogenic enzyme, 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD1; Hsd17b1), as an activin-responsive gene in immortalized gonadotrope cells, LbetaT2. 17beta-HSD1 catalyzes the conversion of estrone to the more active 17beta-estradiol, and activin A stimulated an increase in this enzymatic activity in these cells. We demonstrated that activins signaled via the type I receptor, activin receptor-like kinase (ALK4), and the intracellular signaling protein, SMAD2, to regulate Hsd17b1 transcription in immediate-early fashion. Critical cis-elements, including a minimal SMAD-binding element, were mapped to within 100 bp of the start of transcription. Activin/ALK4 signaling also regulated Hsd17b1 transcription in both immortalized and primary cultured murine granulosa cells. The promoter regions mediating basal and activin/ALK4-regulated promoter activity were generally conserved across the different cell types. The data show that activin A rapidly regulates Hsd17b1 transcription in gonadotrope and granulosa cells and may thereby regulate local 17beta-estradiol synthesis.

Suppression of Notch signaling in the neonatal mouse ovary decreases primordial follicle formation.

Notch signaling directs cell fate during embryogenesis by influencing cell proliferation, differentiation, and apoptosis. Notch genes are expressed in the adult mouse ovary, and roles for Notch in regulating folliculogenesis are beginning to emerge from mouse genetic models. We investigated how Notch signaling might influence the formation of primordial follicles. Follicle assembly takes place when germ cell syncytia within the ovary break down and germ cells are encapsulated by pregranulosa cells. In the mouse, this occurs during the first 4-5 d of postnatal life. The expression of Notch family genes in the neonatal mouse ovary was determined through RT-PCR measurements. Jagged1, Notch2, and Hes1 transcripts were the most abundantly expressed ligand, receptor, and target gene, respectively. Jagged1 and Hey2 mRNAs were up-regulated over the period of follicle formation. Localization studies demonstrated that JAGGED1 is expressed in germ cells prior to follicle assembly and in the oocytes of primordial follicles. Pregranulosa cells that surround germ cell nests express HES1. In addition, pregranulosa cells of primordial follicles expressed NOTCH2 and Hey2 mRNA. We used an ex vivo ovary culture system to assess the requirement for Notch signaling during early follicle development. Newborn ovaries cultured in the presence of gamma-secretase inhibitors, compounds that attenuate Notch signaling, had a marked reduction in primordial follicles compared with vehicle-treated ovaries, and there was a corresponding increase in germ cells that remained within nests. These data support a functional role for Notch signaling in regulating primordial follicle formation.

Changes in the reproductive function and developmental phenotypes in mice following intramuscular injection of an activin betaA-expressing plasmid.

BACKGROUND: The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model. METHODS: To this end, an expression plasmid, pCMV-rAct, with the activin betaA cDNA fused to the cytomegalovirus promoter, was introduced into muscle of the female adult mice by direct injection. RESULTS: Activin betaA mRNA was detected in the muscle by RT-PCR and subsequent Southern blot analysis. Activin betaA was also detected, and western blot analysis revealed a relatively high level of serum activin with correspondingly increased FSH. In the pCMV-rAct-injected female mice, estrus stage within the estrous cycle was extended. Moreover, increased numbers of corpora lutea and a thickened granulosa cell layer with a small antrum in tertiary follicles within the ovary were observed. When injected female mice were mated with males of proven fertility, a subset of embryos died in utero, and most of those that survived exhibited increased body weight. CONCLUSION: Taken together, our data reveal that activin betaA can directly influence the estrous cycle, an integral part of the reproduction in female mice and activin betaA can also influence the embryo development as an endocrine fashion.

Activin regulates estrogen receptor gene expression in the mouse ovary.

Activin, a member of the transforming growth factor-beta 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 beta (ERbeta) 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 ERalpha and ERbeta 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 ERalpha and ERbeta 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 ERalpha 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 ERalpha and ERbeta expression in the ovary. We also found that ER mRNA levels were decreased in Müllerian inhibiting substance promoter (MIS)-Smad2 dominant negative mice that have impaired activin signaling through Smad2, and small interfering RNAs targeting Smad2 or Smad3 suppressed ERalpha 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.

Pituitary-specific expression and Pit-1 regulation of the rat growth hormone-releasing hormone receptor gene.

The GHRH receptor is expressed in the somatotroph cell of the anterior pituitary, where it functions to mediate GHRH-stimulated GH release. To study pituitary and somatotroph cell-specific expression of this gene, a transgenic mouse model and complementary cell culture experiments were developed. The activity of the 1.6-kb proximal rat GHRH receptor promoter was examined in vivo by generating transgenic mice with the promoter directing expression of a luciferase reporter. The promoter directs tissue-specific expression; luciferase is highly expressed in the pituitary but absent from 14 other tissues. Immunocytochemistry experiments show that transgene expression is targeted to GH-expressing somatotroph cells. The transgene is 5-fold more highly expressed in males than females, and there is an increase in transgene expression leading up to the onset of puberty. The 1.6-kb promoter was further examined in cell culture experiments, which revealed that the promoter is selectively activated in pituitary cells and that promoter-reporter expression in nonpituitary cells can be enhanced by the pituitary-specific transcription factor Pit-1. EMSAs identified 10 short regions that specifically bind Pit-1 with highly variable relative affinities. The highest affinity site was previously identified and is required for Pit-1 activation of the promoter. Four additional sites contribute to Pit-1 regulation of the promoter and are important to achieving full activation of the gene. The results show that the 1.6-kb promoter is sufficient to direct tissue- and cell-specific expression in vivo and is regulated by Pit-1.

Ovarian epithelial inclusion cysts in chronically superovulated CD1 and Smad2 dominant-negative mice.

Chronic ovulation as a contributing factor for the development of epithelial ovarian cancer in women has long been an outstanding hypothesis. To test the incessant ovulation hypothesis, mice were superovulated using weekly ip injections of pregnant mare serum gonadotropin (5 IU/animal), followed 48 h later by human chorionic gonadotropin (5 IU/animal). Wild-type CD1 mice were used along with CD1 mice expressing a Smad2 dominant-negative (Smad2DN) transgene under the control of the Müllerian inhibiting substance promoter that targets expression to the ovary and enhances cyst formation. After chronic injections, ovaries were analyzed from animals 6 months of age for the total adjusted number of cysts, cyst area, cyst location, and key signaling pathways. All observed cysts were confirmed to be of epithelial origin. The number of cysts was not significantly different between superovulated and control mice in either the wild-type or Smad2DN groups. However, the combination of the Smad2DN transgene and superovulation resulted in an increase in cyst formation compared with normal littermates that were unstimulated. Rapid proliferation of the cells lining the cysts was detected using bromodeoxyuridine and phospho-histone 3 immunohistochemistry but was not different in the ovarian surface epithelium or in the cyst lining between groups. These data suggest that chronic superovulation in Smad2DN mice results in a higher incidence of cyst formation compared with unstimulated controls, but the epithelial lined cysts did not progress to cancer over the course of this study.

Neonatal exposure to estrogens suppresses activin expression and signaling in the mouse ovary.

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.