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. 2010 Jul 7;24(8):1529–1542. doi: 10.1210/me.2010-0141

β-Catenin (CTNNB1) Promotes Preovulatory Follicular Development but Represses LH-Mediated Ovulation and Luteinization

Heng-Yu Fan 1, Annalouise O'Connor 1, Manami Shitanaka 1, Masayuki Shimada 1, Zhilin Liu 1, JoAnne S Richards 1
PMCID: PMC2940463  PMID: 20610534

Abstract

Wingless-type mouse mammary tumor virus integration site family (WNT)/β-catenin (CTNNB1) pathway components are expressed in ovarian granulosa cells, direct female gonad development, and are regulated by the pituitary gonadotropins. However, the in vivo functions of CTNNB1 during preovulatory follicular development, ovulation, and luteinization remain unclear. Using a mouse model Ctnnb1(Ex3)fl/fl;Cyp19-Cre (Ctnnb1(Ex3)gc−/−), expressing dominant stable CTNNB1 in granulosa cells of small antral and preovulatory follicles, we show that CTNNB1 facilitates FSH-induced follicular growth and decreases the follicle atresia (granulosa cell apoptosis). At the molecular level, WNT signaling and FSH synergistically promote the expression of genes required for cell proliferation and estrogen biosynthesis, but decrease FOXO1, which negatively regulates proliferation and steroidogenesis. Conversely, dominant stable CTNNB1 represses LH-induced oocyte maturation, ovulation, luteinization, and progesterone biosynthesis. Specifically, granulosa cells in the Ctnnb1(Ex3)gc−/− mice showed compromised responses to the LH surge and decreased levels of the epidermal growth factor-like factors (Areg and Ereg) that in vivo and in vitro mediate LH action. One underlying mechanism by which CTNNB1 prevents LH responses is by reducing phosphorylation of cAMP-responsive element-binding protein, which is essential for the expression of Areg and Ereg. By contrast, depletion of Ctnnb1 using the Ctnnb1fl/fl;Cyp19-Cre mice did not alter FSH regulation of preovulatory follicular development or female fertility but dramatically enhanced LH induction of genes in granulosa cells in culture. Thus, CTNNB1 can enhance FSH and LH actions in antral follicles but overactivation of CTNNB1 negatively effects LH-induced ovulation and luteinization, highlighting the cell context-dependent and developmental stage-specific interactions of WNT/CTNNB1 pathway and G protein-coupled gonadotropin receptors in female fertility.

The WNT signaling pathway component β-Catenin can enhance the physiological effects of FSH in growing follicles whereas over-activation of β-catenin blocks LH-induced ovulation and luteinization.

The ovary is a multicompartmental organ in which follicles grow, nurture the maturation of fertilizable oocytes, and release them at the time of ovulation. The ovary is also an endocrine tissue that secretes steroid hormones such as estradiol and progesterone required for the preparation of the reproductive tract for implantation and establishment of pregnancy (1). For these functions to occur, a highly coordinated series of events supporting follicular development must take place.

The growth and development of the ovarian follicle from the preantral to the antral and ovulatory stages involves a complex process orchestrated by several factors including the pituitary gonadotropins FSH and LH (2), as well as intraovarian growth-regulatory factors such as IGF-I (3,4), and steroids such as estradiol and progesterone (5,6,7). A host of additional signaling pathways including the TGF-β family (8,9,10), the phosphatidylinositol 3-kinase pathway (11,12,13), and the MAPK pathway (14,15,16), are required at specific stages of follicular development and ovulation. These studies have documented the stage-specific expression of numerous genes that control follicular development [Fshr (FSH receptor), Cyp19 (aromatase), Ccnd2 (cyclin D2)], ovulation [Areg (amphiregulin), Ereg (Epiregulin), Btc (β-cellulin), Ptgs2 (prostaglandin H synthese 2)], and luteinization [Lhcgr (LH/hCG receptor), Cyp11a1 (cytochrome P450 side chain cleavage enzyme), Star (steroidogenic acute regulatory protein), Sfrp4 (Secreted frizzle related protein-4)].

In addition to these factors, several members of the complex wingless-type mouse mammary tumor virus integration site family (WNT) signaling pathway are expressed in granulosa cells. They are regulated by gonadotropins and presumed to be functional during follicle development, ovulation, and luteinization (17,18,19,20). WNTs are secreted signaling molecules that act locally to control diverse developmental processes such as cell fate specification, cell proliferation, and cell differentiation (21,22,23,24,25). WNTs transduce their signals by binding to their Frizzled (FZD) family receptors to activate distinct signaling cascades. A key effector of the canonical WNT/FZD pathway is β-catenin (CTNNB1), a protein that not only mediates cell-cell adhesion but also can act as a transcription factor. In the latter context, CTNNB1 protein is phosphorylated and subsequently degraded by a large multiprotein complex that includes glycogen synthase kinase (GSK)3-β, adenomatous polyposis coli, and Axins. However, when WNT activates the FZD cascade, CTNNB1 dissociates from this complex and translocates to the nucleus where it acts as a transcriptional cofactor of T-cell factor (TCF) and modulates the expression of a wide range of genes (21). However, CTNNB1 is also known to partner other transcription factors such as SMADs (Sma- and Mad-related proteins), FOXO1 (26), steroidogenic factor-1 (27), and other nuclear receptors (23).

WNT signaling plays a critical role in the development of the embryonic gonad where WNT4 directs and specifies ovarian formation by suppressing testis development (28,29). The impact of WNT4 at this critical stage of ovarian development led to additional studies describing the expression of WNT signaling components (Wnt2, Wnt4, Sfrp4, Fzd1, and Fzd4) in granulosa cells and luteal cells of mouse and rat ovaries (18). Although potential functional redundancy of WNTs and their receptors, as well as the defects of early embryonic development in the corresponding gene knockout mice, have restricted studies in the ovary, recent generation of mice with a conditional knockout of Wnt4 in granulosa cells document that they are subfertile and exhibit impaired antral follicle development (30). These results provide evidence that WNT4 can impact functions in the adult as well as in the embryonic gonad. A recent report by Wang et al. (20) indicates that WNT2 is also expressed and functional in cultured granulosa cells and that CTNNB1 plays a key role in mediating its ability to promote DNA synthesis and proliferation in this context.

The extent to which the canonical vs. noncanonical pathways are involved in WNT2 and WNT4 signaling at later stages in follicular development remains to be clearly documented. Efforts have also been made to study the ovarian function of CTNNB1 in vivo using targeted expression of a dominant stable CTNNB1 (31,32) and targeted disruption of the Ctnnb1 gene (33). The Ctnnb1(Ex3)fl/+;Amhr2-Cre mice develop follicle-like lesions by 6 wk of age. These lesions progressed to granulosa cell tumors by 7.5 months of age (31). The early onset of follicular lesions in this mouse strain is attributed to the expression of the Amhr2 in the somatic cells of developing ovaries as early as embryonic d 14 (34). Nilson and colleagues (35) generated a Ctnnb1 conditional knockout mouse strain using the Amhr2-Cre mice. The depletion of Ctnnb1 in granulosa cells by Amhr2-Cre was not efficient, and no obvious ovarian functional defect was observed. However, CTNNB1 activity can facilitate FSH-stimulated aromatase expression and estrogen biosynthesis in cultured rat granulosa cells (35) as well as proliferation (20) supporting the role of the canonical pathway in mediating WNT4 and WNT2 actions in antral follicles (30). The Ctnnb1 cKO mice were infertile largely due to the uterine defects caused by expression of Amhr2-Cre, and hence the disruption of Ctnnb1, in oviductal and uterine cells (33).

To target the expression and disruption of Ctnnb1 more precisely to the ovary during later stages of follicular development and luteinization and thereby analyze the effects of the canonical pathway more precisely at these stages of follicular development, we have used our Cyp19-Cre mice in which expression of Cre recombinase is high and limited to granulosa cells of antral and preovulatory follicles (36,37). Using both CTNNB1 gain-of-function (Ctnnb1(Ex3)fl/fl;Cyp19-Cre) and loss-of-function (Ctnnb1fl/fl;Cyp19-Cre) mouse models, we provide novel evidence that CTNNB1 facilitates FSH-stimulated follicle growth in vivo but blocks LH-induced ovulation and luteinization.

Results

Equine chorionic gonadotropin (eCG)/FSH increases CTNNB1 in granulosa cells of growing follicles

To study the function of CTNNB1 in granulosa cells, we employed both the gain-of-function and loss-of-function approaches. We generated the mouse strain that expresses dominant stable CTNNB1 in the granulosa cells of growing follicles by crossing the previously reported Ctnnb1(Ex3)fl/fl mice with Cyp19-Cre mice (37). We depleted the Ctnnb1 expression specifically in granulosa cells of growing follicles by crossing the Ctnnb1fl/fl mice with Cyp19-Cre mice (termed as Ctnnb1gc−/−). Expression of the dominant stable CTNNB1 (CTNNB1ΔEx3) and the disruption of the endogenous Ctnnb1 were confirmed in granulosa cells by immunofluorescent staining (Fig. 1, A–I) and Western blot analyses (Fig. 1, J–K). Specifically, in wild-type (WT) 23- to 25-d-old immature mice, CTNNB1 is expressed in granulosa cells of growing follicles at the transition from the preantral to antral stage, but its level is relatively low in smaller preantral follicles (Fig. 1, A and C). At 24–48 h after eCG treatment that stimulates follicular development to the preovulatory stage, CTNNB1 is highly expressed in Granulosa cells of the large antral follicles (Fig. 1B). In the ovaries of Ctnnb1(Ex3)gc−/− mice (Fig. 1, D–F) that express dominant stable CTNNB1, the levels of CTNNB1 protein in Granulosa cells are higher than those in the WT ovaries (Fig. 1, A–C). In contrast, in the Ctnnblfl/fl;Cyp19-Cre (Ctnnb1gc−/−) mouse strain, CTNNB1 was depleted in Granulosa cells of preantral/antral follicles (Fig. 1, G and I), preovulatory follicles (Fig. 1H), and in luteal cells after ovulation (data not shown). Western blots show expression of the mutant CTNNB1 protein in granulosa cells isolated from Ctnnb1(Ex3)gc+/− mice (Fig. 1J) as well as reduced levels of CTNNB1 in granulosa cells of the Ctnnb1gc−/− mice (Fig. 1K). Neither FSH nor pharmacological agents that mimic LH action (forskolin and PMA) alter the levels of CTNNB1 in WT or mutant granulosa cells in culture (Fig. 1, J–K).

Figure 1.

Figure 1

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Expression of CTNNB1 in ovarian granulosa cells. A–I, Expression of CTNNB1 in ovaries of WT (A–C), Ctnnb1(Ex3)gc+/− (D–F), and Ctnnb1gc−/− (G–I) mice at postnatal d 25 without or after eCG treatment for 48 h. Scale bar, 150 μm. J and K, Western blots of cell extracts prepared from granulosa cells of 23-d-old immature WT, Ctnnb1(Ex3)gc−/−, and Ctnnb1gc−/− mice show the expression levels of WT and mutant CTNNB1 proteins. Some granulosa cells were cultured with FSH (100 ng/ml) or FSH plus forskolin (For) (10 μm)/PMA (20 nm) for 24 h. DAPI, 4′,6-Diamidino-2-phenylindole.

CTNNB1 facilitates the FSH-induced follicle growth and target gene expression in granulosa cells in vivo

Mutation of one copy of Ctnnb1 (Ctnnb1(Ex3)gc+/−) or both Ctnnb1 alleles (Ctnnb1(Ex3)gc−/−) leads to similar phenotypes, but the latter is more pronounced. The ovaries of Ctnnb1(Ex3)gc−/− mice were slightly larger than those of WT mice at postnatal d 23. This effect was augmented to an approximately 2-fold increase after treatment with eCG that stimulates follicular development Ctnnb1(Ex3)gc−/− ovaries (Fig. 2A). When the proliferation of granulosa cells was analyzed using bromodeoxyuridine (BrdU) incorporation and immunostaining, signals in granulosa cells of preantral/early antral follicles were not visibly different between WT and Ctnnb1(Ex3)gc−/− mice (Fig. 2C). However, there were more growing small antral follicles (Fig. 2, B and C). Additionally, apoptosis as detected by the terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay (Fig. 2C) was reduced significantly in granulosa cells of the mutant ovaries. Real-time RT-PCR and immunofluorescence show that the expression of several FSH target genes known to be important for granulosa cell proliferation and follicle growth were markedly increased in granulosa cells isolated from Ctnnb1(Ex3)gc−/− mice compared with those from WT mice, at 44 h post-eCG treatment. These genes include those encoding the Fshr (FSH receptor), Cyp19 (aromatase), Ccnd2 (cyclin D2), Nr5a1 (steroidogenic factor 1, SF1) (Fig. 2D) and cyclin A (Fig. 2C). Lhcgr (LH receptor) mRNA levels showed no difference whereas a known CTNNB1 target gene, Axin2, was elevated markedly even before eCG treatment (Fig. 2D), indicating the increased CTNNB1 activity. FOXO1, a transcription factor known to repress granulosa cell proliferation and steroidogenesis (38,39), was highly expressed in small follicles of WT mice but was down-regulated by FSH/eCG in granulosa cells of preovulatory follicles. Foxo1 mRNA and protein were reduced significantly in granulosa cells of Ctnnb1(Ex3)gc−/− mice, even before FSH/eCG-treatments (Fig. 2, C and E).

Figure 2.

Figure 2

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The dominant stable CTNNB1 facilitates FSH-stimulated effects in growing follicles. A, Overactivation of CTNNB1 leads to an increase of ovarian weight, measured on postnatal d 23 before or after eCG treatment for 24 h. (Ovaries from six mice were measured in each treatment, n = 6). B, Ovaries were collected from immature WT and Ctnnb1(Ex3)gc−/− mice treated with eCG for 24 h. The total numbers of antral follicles were counted on series ovarian sections (n = 6). C, Proliferation, apoptosis, cyclin A expression, and FOXO1 expression in ovaries of WT and Ctnnb1(Ex3)gc−/− mice were analyzed by a BrdU incorporation assay, TUNEL assay, and immunofluorescence, respectively. Scale bar, 150 μm. D, Real-time RT-PCR shows the expression of indicated genes in granulosa cells isolated from WT and Ctnnb1(Ex3)gc−/− mice (n =3 for each group) with or without eCG treatment (44 h). E, Real-time RT-PCR and Western blots show the expression of FOXO1 in granulosa cells isolated from WT and Ctnnb1(Ex3)gc−/− mice (n = 3 for each group) with or without eCG treatment (44 h). DAPI, 4′,6-Diamidino-2-phenylindole.

CTNNB1 enhances the FSH target gene expression in cultured granulosa cells

To study the direct effects of FSH on Ctnnb1 mutant granulosa cells, we isolated undifferentiated granulosa cells from immature WT, Ctnnb1(Ex3)gc−/− and Ctnnb1gc−/− mice and cultured them with FSH for 24 h. In granulosa cells expressing CTNNB1ΔEx3, FSH induction of Fshr and Cyp19 mRNA expression was more dramatic than in WT granulosa cells (Fig. 3A). In contrast, the induction of these two genes by FSH was reduced in CTNNB1-depleted granulosa cells (Fig. 3A). To confirm FSH activation of CTNNB1, granulosa cells were isolated from growing follicles, transfected with TCF-luciferase reporter constructs (Topflash and Fopflash), and treated with or without FSH (Fig. 3B). A basal level of CTNNB1/TCF activity was detected in untreated cells transfected with Topflash but not Fopflash (control). FSH treatment increased the transcriptional activity of CTNNB1/TCF-responsive promoter (Topflash) in granulosa cells (Fig. 3B).

Figure 3.

Figure 3

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FSH/cAMP signals stimulate CTNNB1/TCF activity in granulosa cells and induce the expression of FSH target genes. A, Granulosa cells were isolated from eCG-primed (24 h) 23-d-old immature mice (WT, Ctnnb1(Ex3)gc−/−, and Ctnnb1gc−/−) and treated with FSH for 24 h. Expression of Fshr and Cyp19 mRNA was analyzed by real-time PCR. B, CTNNB1/TCF activity was determined in cultured granulosa cells transfected with a TCF-luciferase reporter plasmid (Topflash) or its control plasmid containing mutated TCF sites (Fopflash) and treated with or without FSH (100 ng/ml, 4 h). CTNNB1/TCF activity was also determined in granulosa cells isolated from 23-d-old WT, Ctnnb1(Ex3)gc−/−, and Ctnnb1gc−/− mice. C, Immunofluorescent localization of CTNNB1. Granulosa cells were isolated from eCG-primed (24 h) 23-d-old WT, Ctnnb1(Ex3)gc−/−, and Ctnnb1gc−/− mice and cultured overnight. Cells were fixed at 4 h after FSH treatment and immunostained with an antibody against total CTNNB1. Scale bar, 5 μm. D, Luciferase assays and real-time RT-PCR show the effects of WNT4/FSH cotreatment on CTNNB1/TCF activity and gene expression in cultured granulosa cells. E, Immunofluorescent localization of CTNNB1. Nontreated or FSH-treated (4 h) WT granulosa cells were fixed and immunostained with antibodies against total, inactive (phosphorylated), and active (nonphosphorylated) CTNNB1. Scale bar, 5 μm. F, Western blots show the FSH-induced phosphorylation of AKT, GSK3β, and CREB in cultured granulosa cells (WT and Ctnnb1(Ex3)gc−/−). G, Immunofluorescent localization of pCREB-S133 in FSH-treated granulosa cells (WT and Ctnnb1(Ex3)gc−/−). DAPI, 4′,6-Diamidino-2-phenylindole.

FSH dramatically induced the nuclear accumulation of nonphosphorylated, active CTNNB1 in WT cells compared with untreated cells (Fig. 3E). No overall changes were observed in phosphorylated CTNNB1 that was localized to cytoskeletons and plasma membranes (Fig. 3E). Granulosa cells expressing the CTNNB1ΔEx3 mutant exhibit strong nuclear localization even without FSH treatment (Fig. 3C, middle panel) compared with WT cells (Fig. 3C, upper panel). In contrast, minimal immunostaining was observed in the CTNNB1-depleted cells (Fig. 3C, lower panel). Consistent with the patterns of CTNNB1 localization in the WT and mutant cells, luciferase assays using the Topflash vector showed increased CTNNB1/TCF activity in CTNNB1ΔEx3-expressing granulosa cells when compared with WT granulosa cells, before and after FSH stimulation (Fig. 3B). No activity was observed in the CTNNB1-depleted cells obtained from the Ctnnb1gc−/− mice (Fig. 3B). Collectively, these results confirm that FSH can stimulate the CTNNB1/TCF activity in granulosa cells, that this is dependent on the nuclear translocation of CTNNB1, and that CTNNB1 activation facilitates the certain physiological actions of FSH in granulosa cells.

Consistent with the results obtained from Ctnnb1(Ex3)gc−/− mice and cultured granulosa cells, recombinant WNT4 [which is the most abundant form of WNTs expressed in the ovary (18)] and FSH synergistically activated CTNNB1/TCF activity at low concentrations (Fig. 3D). In addition, cotreatment of WNT4 and FSH had a more dramatic effect on the induction of Cyp19 and Axin2, and the repression of Foxo1, than did FSH treatment alone (Fig. 3D). In both WT and Ctnnb1(Ex3)gc−/−mutant granulosa cells, FSH stimulated the phosphatidylinositol 3-kinase/AKT pathway (Fig. 3F and Ref. 36), leading to increased phosphorylation and presumed inactivation of GSK3β, a negative regulator of CTNNB1 (Fig. 3F). FSH also activates protein kinase A (PKA) leading to the phosphorylation (at S133) and activation of cAMP-responsive element-binding protein (CREB) in both WT and Ctnnb1(Ex3)gc−/− mutant granulosa cells (Fig. 3, F and G), indicating that dominant stable CTNNB1 does not impair these FSH-mediated events in granulosa cells of antral follicles.

Dominant stable CTNNB1 represses the LH/human (h)CG-induced oocyte maturation, cumulus expansion, and ovulation

To determine the reproductive consequences of either depleting or overexpressing CTNNB1 in granulosa cells, 6-month mating studies and superovulation experiments, using eCG and hCG, were done in the mutant and WT mice. The Ctnnb1gc−/− mice were fertile and delivered the same number of pups and ovulated an equal number of oocytes (Fig. 4A) as did WT controls. These results indicate that despite greater depletion of CTNNB1in granulosa cells than reported in previous studies (33), CTNNB1 is not overtly essential for antral follicular development, ovulation, or luteinization.

Figure 4.

Figure 4

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Expression of dominant stable CTNNB1 in granulosa cells blocks ovulation and luteinization induced by LH. A, Fertility was examined by breeding 6-wk-old WT, Ctnnb1(Ex3)gc+/− Ctnnb1(Ex3)gc−/−, and Ctnnb1gc−/− females with fertile WT males for 6 months (n = 6 for each genotype) and by treating immature mice of each gene with a superovulatory regimen of homones. Ovulated oocytes were collected from oviducts and counted at 16 h after hCG injections. (n = 8∼10 for each genotype.) B, Rates of oocyte GVBD were counted on series ovarian sections of WT (hCG 8 h) and Ctnnb1(Ex3)gc+/− mice (hCG,8 h and 16 h). For WT mice treated with hCG for 16 h, GVBD was determined in ovulated oocytes collected from oviducts. (n = 6∼8 for each group). C, Real-time RT-PCR shows the mRNA expression of ovulation-related LH-target genes in granulosa cells isolated from WT and Ctnnb1(Ex3)gc+/− mice before and after hCG treatment (4 h) (n = 3∼4 for each group). D, Hematoxylin and eosin staining shows the ovarian histology of 23-d-old WT and Ctnnb1(Ex3)gc−/− mice treated with eCG (48 h) or eCG (48 h) + hCG (8 h, 16 h, and 48 h), as well as the ovarian histology of 8-wk-old cycling mice (WT and Ctnnb1(Ex3)gc−/−). Scale bar, 70 μm for images of hCG,8 h; Scale bar, 150 μm for other images.

By contrast to the Ctnnb1gc−/− mice, fertility was compromised in the mice carrying one copy of dominant stable Ctnnb1 (Ctnnb1(Ex3)gc+/−) and to a greater extent in the Ctnnb1(Ex3)gc−/− mice. They produced fewer pups per litter and ovulated fewer oocytes than the control mice at 16 h post-hCG (Fig. 4A). Histological studies of hCG-treated Ctnnb1(Ex3)gc−/− ovaries showed that germinal vesicle breakdown (GVBD; resumption of meiosis) was blocked in approximately 50% of the follicles (Fig. 4, B and D, hCG8 h). Cumulus cell-oocyte complex (COC) expansion was also impaired in the preovulatory follicles (Fig. 4D, hCG8 h). The unruptured follicles of the Ctnnb1(Ex3)gc−/−mice persisted at 16 h after hCG (Fig. 4D). LH/hCG induce the expression of three epidermal growth factor (EGF)-like factors (amphiregulin, epiregulin, and β-cellulin) in preovulatory follicles, and these EGF-like factors function as intrafollicular effectors of LH in triggering oocyte GVBD and cumulus expansion (15,40,41). Therefore, real-time RT-PCR was done to analyze the expression of these and other genes (Ptgs2, Tnfaip6, and Ptx3) known to be required for ovulation. LH induced these genes in granulosa cells of WT mice (Fig. 4C) and Ctnnb1gc−/− mice (data not shown), but to a lesser degree in cells collected from Ctnnb1(Ex3)gc−/− mice (Fig. 4C). Consistent with the ovulation defects, corpora lutea (CL) were absent in most ovaries of Ctnnb1(Ex3)gc−/− mice (Fig. 4D, either at 48 h post-hCG in the superovulation regimen or in 8-wk-old adult mice).

Dominant stable CTNNB1 represses the expression of EGF-like factors Areg and Ereg that are required for cumulus expansion

The reduced expression levels of Areg, Ereg, and Btc in the Ctnnb1(Ex3)gc−/− mice indicated that the decreased levels of these EGF-like factors might be the primary defect mediating impaired ovulation and COC expansion in these mutant mice. To test this, we performed COC expansion assays in vitro by isolating COCs from preovulatory follicles of WT, Ctnnb1(Ex3)gc+/−, and Ctnnb1gc−/− mice and culturing them with agonists known to stimulate expansion. As shown, FSH and AREG induced expansion in COCs isolated from WT and Ctnnb1gc−/− mice. However, FSH failed to induce expansion in COCs isolated from Ctnnb1(Ex3)gc+/− mice (Fig. 5A). The poor response of the Ctnnb1(Ex3)gc+/− COCs to FSH appears to be related to impaired activation of critical genes. Specifically, FSH treatment induces expression of the EGF-like factors and expansion-related genes (Ptgs2 as the example) in WT and Ctnnb1gc−/− COCs, but not the Ctnnb1(Ex3)gc+/− COCs (Fig. 5B). These results further suggest that the ovarian defects observed in Ctnnb1(Ex3)gc+/− mice might be caused by decreased gonadotropins (LH/hCG in vivo and FSH in vitro) and their induction of the EGF-like factors. Indeed, AREG treatment rescued the defects of COC expansion and Ptgs2 expression in Ctnnb1(Ex3)gc+/− COCs (Fig. 5C), although the expression levels of Areg and Ereg mRNA remained low (Fig. 5C).

Figure 5.

Figure 5

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Dominant stable CTNNB1 represses the expression of EGF-like factors Areg and Ereg that are required for COC expansion. A, In vitro COC expansion assay. Fully grown COCs were isolated from WT, Ctnnb1(Ex3)gc−/−, and Ctnnb1gc−/− mice at 44–48 h after eCG treatment and cultured in COC medium (30 COCs/100 μl medium). Cumulus expansion was induced by an overnight treatment of FSH (100 ng/ml) or amphiregulin (AREG, 100 ng/ml). B and C, Real-time RT-PCR shows the expression of indicated genes in cultured COCs treated with FSH (B) or AREG (C). In each experimental group, total RNA was isolated from 100 COCs.

Dominant stable CTNNB1 repression of Areg and Ereg expression involves CREB

Because LH induction of Areg and Ereg is initiated by PKA but is maintained by ERK1/2 (14), we determined whether CTNNB1 altered either the PKA- or ERK1/2-signaling cascades. For this, whole-cell extracts were prepared from granulosa cells isolated from eCG-primed WT and Ctnnb1(Ex3)gc+/− mice at 0, 2, and 4 h post-hCG. Western blot analyses show that hCG activation of the MEK/ERK1/2/RSK cascade, which is essential for ovulation and luteinization, was not affected by dominant stable CTNNB1 at 2 h post-hCG, but was significantly decreased at 4 h (Fig. 6, A and B). In addition, levels of phospho-CREB were reduced in the Ctnnb1(Ex3)gc+/− ovaries compared with WT (Fig. 6, A and B), especially in granulosa cells of preovulatory follicles, indicating that CTNNB1 alters signaling via a CREB-mediated pathway at this later stage of follicular development.

Figure 6.

Figure 6

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Dominant stable CTNNB1 represses the expression of EGF-like factors Areg and Ereg via a CREB-mediated pathway. A, Western blots show the expression/phosphorylation levels of indicated proteins in ovarian lysates prepared from hCG-treated WT and Ctnnb1(Ex3)gc+/− mice. B, Immunofluorescent staining shows the phosphorylation of ERK1/2 and CREB in ovaries of hCG-treated WT and Ctnnb1(Ex3)gc+/− mice. C, Luciferase assays shows the activity of the Areg promoter [wild type and CRE-site mutated (ΔCRE)]-luciferase constructs in cultured granulosa cells (WT) treated with LH (500 ng/ml) or forskolin/PMA (4 h). D, Luciferase assay shows the activity of Areg-luciferase construct in cultured granulosa cells (WT, Ctnnb1(Ex3)gc+/−, and Ctnnb1gc−/−) treated with forskolin/PMA (4 h). E, EMSA shows the interaction of phospho-CREB with the Areg promoter CRE site. A double-stand DNA probe derived from the consensus CRE site in mouse Areg promoter was labeled with 32P isotope. The DNA probe was incubated with whole-cell lysates prepared from ovaries of hCG-treated mice (WT and Ctnnb1(Ex3)gc−/−) or lysates preincubated with anti-pCREB IgG and then subjected to nondenatured PAGE. DAPI, 4′,6-Diamidino-2-phenylindole.

Because there is a consensus cAMP response element (CRE) site in Areg promoter, we further studied the reason for decreased Areg expression in granulosa cells of Ctnnb1(Ex3)gc+/− mice. For this, we transfected mouse granulosa cells with an Areg-luciferase promoter-reporter construct (provided by Dr. Franco DeMayo, Baylor College of Medicine). When the cells were stimulated with either LH or forskolin/PMA, luciferase activity was increased significantly (Fig. 6C). However, the induction of Areg promoter activity was abolished when the CRE site was mutated (Fig. 6C). In agreement with the loss of Areg mRNA expression in cells expressing the dominant CTNNB1, the activity of Areg promoter was compromised in CTNNB1(Ex3)gc+/− mutant granulosa cells compared with WT or Ctnnb1-depleted cells (Fig. 6D). Direct binding between phospho-CREB and the CRE site of Areg promoter was confirmed by EMSA (Fig. 6E). In WT ovaries, granulosa cell extracts prepared after hCG treatment showed increased binding of phospho-CREB to the Areg promoter, but this event was inhibited in extracts prepared from Ctnnb1(Ex3)gc+/− ovaries.

CTNNB1 prevents luteinization of granulosa cells and the expression of LH target genes essential for progesterone biosynthesis

In addition to ovulation failure, luteinization of granulosa cells was also impaired in the ovaries of Ctnnb1(Ex3)gc−/− mice, when examined at 48 h after hCG treatment (Fig. 4D, hCG 48 h). To determine the molecular changes underlying the defects in luteinization, real-time RT-PCR and in situ hybridization analyses were done for specific luteal cell marker genes (Fig. 7, A and C). Expression of Star, Cyp11a1, Sfrp4, and Lhcgr were significantly compromised in the ovaries of Ctnnb1(Ex3)gc+/− mice (Fig. 7, A and C). In the WT mice, serum progesterone levels increased dramatically at 48 h after hCG injection, due to the formation of CLs. However, the serum progesterone levels were much lower in hCG-treated Ctnnb1(Ex3)gc+/− mice (Fig. 7B).

Figure 7.

Figure 7

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Dominant stable CTNNB1 blocks the expression of LH target genes required for luteinization. A, In situ hybridization shows the expression of mRNAs encoding Cyp11a1 and Sfrp4 in WT and Ctnnb1(Ex3)gc+/− ovaries at 48 h after hCG treatment. Histology of the ovaries is shown by hematoxylin staining (bright-field images); localization of Cyp11a1 or Sfrp4 mRNA is shown by dark-field images captured with Zeiss Axioplan microscope. Scale bar, 150 μm. B, Serum progesterone levels in 25-day-old WT and Ctnnb1(Ex3)gc+/− mice before and after hCG treatment (48 h). C, Real-time RT-PCR shows the expression of indicated LH target genes in luteal cells isolated from WT and Ctnnb1(Ex3)gc+/− mice before and after hCG treatment (48 h) (n = 3 for each group). D, Granulosa cells were isolated from eCG-primed (24 h) 23-d-old immature mice (WT, Ctnnb1(Ex3)gc+/−, and Ctnnb1gc−/−) and treated with or without forskolin/PMA-treatment (24 h). Expression of indicated luteal cell marker genes was detected by real-time RT-PCR.

These same defects were observed when granulosa cells were cultured and treated with forskolin and PMA (the pharmacological reagents that mimic LH effects in cultured granulosa cells) (42). Robust expression of luteal cell marker genes was observed in WT granulosa cells, whereas the effects of forskolin and PMA were dramatically compromised in granulosa cells expressing CTNNB1ΔEx3 (Fig. 7D, upper panels). In striking contrast, Ctnnb1-depleted granulosa cells isolated from Ctnnb1gc−/− mice respond to forskolin and PMA with even stronger expression of these LH target genes than in WT cells (Fig. 7D, lower panels). These results further confirm that CTNNB1 activity negatively affects the LH-induced ovulation and luteinization in vivo as well as in vitro.

Discussion

Follicular growth, ovulation, and luteinization are regulated not only by the pituitary hormones but also by intraovarian growth-regulatory factors. Moreover, the pituitary hormones modulate the activity of many intraovarian pathways including components of the WNT/FZD signaling cascade (18,19). Of relevance to the studies presented herein, FSH was shown to phosphorylate GSK3β via the AKT pathway (43), providing early evidence that FSH might directly impact the functional activity of CTNNB1 in granulosa cells. Additionally, CTNNB1 has been shown to enhance FSH-mediated induction of Cyp19a1 in rat granulosa cells in culture (35). FSH and LH also regulate the expression of several members of the WNT/FZD signaling cascade in the adult mouse ovary, especially in preovulatory and ovulating follicles. Expression of Wnt4, Fzd1, Fzd4, and Sfrp4 are each selectively increased (18). Therefore, understanding the functional relevance of these signaling molecules at defined stages of follicular development and luteinization in vivo is critical for understanding ovarian development.

Although Wnt2-null mice do not appear to exhibit ovarian defects (44), disrupting Wnt2 in cultured granulosa cells reduced their proliferation via CTNNB1 (20). Conditional disruption of Wnt4 in granulosa cells in vivo impairs follicular growth at the antral stage, and the mice are subfertile and express altered gene expression profiles including Cyp19a1 (30). Fzd4-null mice exhibit impaired luteinization (17). These few studies indicate that WNT/FZD signaling may be important in the adult ovary as well as the embryonic ovary (45). Because the WNT/FZD canonical pathway is dependent on the activation of CTNNB1, one would predict major changes in granulosa cell functions if expression of this factor was either disrupted or overexpressed. Attempts to understand the function of CTNNB1in granulosa cells have been made using both gain-of-function and loss-of-function approaches by generating the Ctnnb1(Ex3)fl/fl;Amhr2-Cre mice (31) and Ctnnb1fl/fl;Amhr2-Cre mice (33), respectively. However, because Amhr2-Cre is expressed at early stages of follicular development, the Ctnnb1(Ex3)fl/fl;Amhr2-Cre mice developed premature follicle lesions that eventually became granulosa cell tumors (31). Thus, in this mouse strain, analyzing the selective effects of CTNNB1 in preovulatory follicles was not possible. In addition, the Amhr2-Cre recombinase activity in granulosa cells did not appear sufficient to provide pronounced effects of depleting Ctnnb1 (33). Moreover, because Amhr2-Cre is also expressed in uterine cells, the effects of CTNNB1 on reproductive outcomes were not restricted to the ovary.

Therefore, to overcome these limitations and caveats, we generated the gain-of-function and loss-of-function mutants by mating the Ctnnb1(Ex3)fl/fl and Ctnnb1fl/fl mice to our Cyp19-Cre mice (36,37). The Cyp19-Cre mice have the advantage that the expression of Cre recombinase is granulosa cell specific and is enhanced in preovulatory follicles in a manner similar to the endogenous Cyp19a1 gene (14,36,37). Our results show that depleting Ctnnb1 in granulosa cells of growing and preovulatory follicles does not cause any overt defects in follicular development, ovulation, or luteinization, confirming the studies of Nilson and co-workers (35). Moreover, the Ctnnb1fl/fl;Cyp19-Cre mice are fertile because there is no uterine defect as observed in the Ctnnb1fl/fl;Amhr2-Cre mice (33). Although overt signs of altered ovarian cell function in vivo were not evident in the Ctnnb1gc−/− mice, the depletion of Ctnnb1 did reduce the response of granulosa cells to FSH in culture. This was shown by the impaired induction of Fshr and Cyp19a1 mRNAs and reduced activation of the Topflash-luciferase promoter-reporter vector, supporting the studies of Nilson and colleagues (33). Conversely, the responses of the Ctnnb1-depleted granulosa cells to the LH mimetics, forskolin and PMA, were markedly enhanced, as shown by the elevated induction of Lhcgr, Star, Cyp11a1, and Sfrp4. Thus, endogenous, activated CTNNB1 has the potential to enhance not only granulosa cell proliferation (20) but also FSH-mediated program of granulosa cell differentiation but reduce the LH-mediated program. This duality of CTNNB1 functions at different stages of follicular development may explain why the disruption of CTNNB1 alone does not mimic the results of the Wnt4 conditional knockout mice. Alternatively, there is the possibility that, despite the approximately 90% reduction of CTNNB1 protein in the Ctnnb1gc−/− granulosa cells (Fig. 1K), a sufficient amount of CTNNB1 remains to prevent a detectable phenotypic change in this mouse strain.

The ability of CTNNB1 to enhance preovulatory follicular development but to block luteinization was revealed more potently in the gain-of-function context. Specifically, overexpression of CTNNB1 in the Ctnnb1(Ex3)gc−/− mice promoted increased numbers of growing follicles, reduced apoptosis, and increased expression of genes involved in the growth of preovulatory follicles, including Cyp19a1, Fshr, Nr5a1, and cyclin A. Moreover, overexpression of CTNNB1 at this stage of follicular development did not alter FSH activation of PKA and CREB phosphorylation. However, FSH induction of Lhcgr was not enhanced, indicating that the effects of CTNNB1 are gene specific and not directed toward all FSH-regulated genes. In addition, CTNNB1 induced expression of a known CTNNB1 target gene Axin2 (32), showing that CTNNB1 was active and that conversion of granulosa cells to the CTNNB1 program had been initiated in these cells. Despite the enhancement of follicular functions, the Ctnnb1(Ex3)gc−/− mice are subfertile due to severely impaired responses to LH/hCG and reduced expression of genes required for ovulation and COC expansion (Areg, Ereg, Ptgs2) as well as luteinization (Cyp11a1, Star, Sfrp4, and Lhcgr).

The ability of CTNNB1 to block the action of LH appears to be mediated, at least in part, by changes in the PKA/CREB pathway and the disruption of the RAS/MEK/ERK1/2 pathway. Levels of phospho-CREB were lower in the mutant Ctnnb1(Ex3)gc−/− granulosa cells compared with WT granulosa cells isolated 2 h after hCG in vivo, and this was associated with reduced CREB-binding activity in the mutant cell extracts compared with WT in EMSAs. Because previous studies suggested that Areg and Ereg are induced by LH/hCG in granulosa cells through PKA/CREB pathway (14,46), and because Areg and Ereg were reduced in the Ctnnb1(Ex3)gc−/− granulosa cells (Fig. 4C), we sought to determine the activity of the Areg promoter in the Ctnnb1(Ex3)gc−/− granulosa cells. When intact and CRE-mutant Areg promoter-luciferase constructs were transfected into WT granulosa cells, LH or forskolin/PMA induced Areg-luciferase activity, and this required a functional CRE site that binds CREB. When the same constructs were transfected into WT, Ctnnb1gc−/− mice and Ctnnb1(Ex3)gc−/− granulosa cells, luciferase activity was reduced markedly only in the Ctnnb1(Ex3)gc−/− cells overexpressing CTNNB1. The mechanisms by which CTNNB1 disrupts CREB phosphorylation and binding remain to be resolved but could involve direct binding of CTNNB1 to CREB or competition for CBP/p300 signaling. Consistent with the decreased expression of EGF-like factors, the activity of ERK1/2 pathway failed to be maintained in the preovulatory follicles of Ctnnb1(Ex3)gc−/− mice. It has been shown recently that the sustained ERK1/2 signaling is essential for oocyte maturation and COC expansion (14,47). Therefore, the reduced activity of ERK1/2 by 4 h after hCG is the most likely cause of the ovulation and luteinization defects observed in the Ctnnb1(Ex3)gc−/− mice. However, we cannot rule out that the dominant stable CTNNB1 may cause ovulation failure by affecting unidentified targets in granulosa cells.

In summary, FSH and WNT signaling pathways can intersect to activate CTNNB1 in granulosa cells to enhance FSH action and promote preovulatory follicular growth and survival (Fig. 8A). However, CTNNB1, at least in excess, suppresses the downstream responses to LH-induced terminal differentiation of granulosa cells (Fig. 8B). The duality of CTNNB1-regulated events may contribute to the phenotype of the Wnt4 conditional knockout mice (30). The potent ability of CTNNB1 to enhance proliferation and block differentiation likely explains its ability to favor granulosa cell proliferation in cultured granulosa cells (20) as well as granulosa cell tumor formation in vivo (31,48). CTNNB1 appears to impact proliferation vs. differentiation in other systems as well, including adipogenesis (23,49) and hematopoeisis (50). The effects of CTNNB1 appear to be gene and context specific, indicating that CTNNB1 has specific interacting partners on the promoters of specific genes (26). In the case of Areg promoter in granulosa cells, CTNNB1 appears to block the binding and phosphorylation of CREB. In the case of the Cyp19a1 promoter, CTNNB1 appears to enhance NR5A1 activity (35) even though the Cyp19a1 promoter contains a functional CRE site (51,52). Remarkably and still inexplicitly, granulosa cells in normal growing follicles of adult mice do not appear to be highly dependent on CTNNB1, suggesting that other pathways downstream in the WNT/FZD pathway may be critical for mediating the effects of WNTs.

Figure 8.

Figure 8

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A schematic of CTNNB1 actions in granulosa cells. A, In proliferating granulosa cells, nuclear CTNNB1 facilitates the expression of FSH target genes required for follicle growth and estrogen biosynthesis, probably by interacting with the orphan nuclear receptor SF1 (present study and Ref. 35). In addition, FSH also promotes the nuclear accumulation of CTNNB1 and activates the CTNNB1/TCF complex, possibly by regulating GSK3β activity (55). B, During luteinization, the preovulatory LH surge stimulates the activation of cAMP/PKA/CREB pathway, and the rapid expression of EGF-like factors, especially Areg and Ereg, which function as intrafollicular effectors of LH and further induce the expression of genes essential for ovulation and luteinization, by activating the ERK1/2-C/EBPβ cascade (14,15). The dominant stable CTNNB1 prevented the LH-induced CREB phosphorylation and Areg/Ereg expression and thereby caused ovulation and luteinization defects in the mutant animals. BTC, β-catenin; EGFR, EGF receptor; FSHR, FSH receptor.

Materials and Methods

Animals and hormone treatments

WT C57BL/6 female mice were obtained from Harlan Sprague Dawley, Inc. (Chicago, IL). Mice expressing dominant stable CTNNB1 (Ctnnb1(Ex3)gc+/− and Ctnnb1(Ex3)gc−/−) or lacking CTNNB1 in granulosa cells (Ctnnb1gc−/−) were generated by crossing Cyp19-Cre (37) mice with previously reported Ctnnb1(Ex3)flox/flox (53) mice or Ctnnb1flox/flox (54) mice. Animals were housed under a 14-h light, 10-h dark schedule, provided food and water ad libitum, and were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

To study ovarian responses to exogenous gonadotropins, 21-d-old immature females were analyzed to avoid the complexity of ovarian functions associated with estrous cycles and endogenous surges of gonadotropins. Immature mice were injected ip with 4 IU eCG (Calbiochem, La Jolla, CA) to stimulate preovulatory follicle development followed 48 h later with 5 IU hCG (American Pharmaceutical Partners, Schaumburg, IL) to stimulate ovulation and luteinization.

In situ hybridization

The riboprobe in vitro transcription systems kit (Promega Corp., Madison, WI) was used to make [35S]UTP-labeled antisense and sense probes of mouse Sfrp4 (19) and Cyp11a1 (31) cDNAs. Ovaries were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections were rehydrated, treated with 20 μg/ml proteinase K, and incubated with radiolabeled riboprobe overnight at 55 C. The next day, slides were washed and dipped in photographic NTB-2 emulsion, exposed at 4 C for an appropriate length of time, developed with D-19 developer and fixer (Eastman Kodak, Rochester, NY), and stained with hematoxylin. Tissue histology was observed by light-field illumination, and dark-field illumination was used to visualize the mRNA probe.

RNA isolation and real-time RT-PCR

Total RNA was isolated from granulosa cells of at least three mice (WT or Ctnnb1 mutant). Reverse transcription was done using the SuperScript One-Step RT-PCR system with Platinum Taq kit (Invitrogen, Carlsbad, CA). The real-time PCR was performed using the Rotor-Gene 3000 thermocycler (Corbett Research, Sydney, Australia). Relative levels of mRNAs were calculated using Rotor-Gene 6.0 software and normalized to the levels of endogenous β-actin in the same samples. For each indicated gene, the relative transcript level of the control sample (left-hand bar of each graph) was set as 1. The relative transcript levels of other samples were compared with the control, and the fold changes are shown in the graph. For each experiment, quantitative PCRs were done in triplicate. Primer sequences are available upon request to the authors.

BrdU incorporation assay

Mice received an ip injection of 50 mg/kg of BrdU, and were killed 1 h after treatment. Ovaries were isolated and fixed with 4% paraformaldehyde. Incorporated BrdU was detected by immunohistochemistry using a BrdU antibody (Sigma Chemical Co., St. Louis, MO) according to manufacturer’s instructions. For direct comparison, WT and Ctnnb1(Ex3)gc−/− ovary sections from at least four individual females were processed together.

TUNEL assay

Analysis of apoptosis in WT and mutant mouse ovaries was carried out by TUNEL assay using the ApopTag Plus in situ apoptosis detection kit according to the manufacturer’s instructions (Chemicon International, Temecula, CA).

Granulosa cell culture, plasmid transfection, and luciferase assay

Granulosa cells were harvested from eCG-primed (24 h), 23-d-old mice as described previously (41). Briefly, undifferentiated granulosa cells were released from antral follicles by puncturing with a 26.5-gauge needle. Cells were cultured at a density of 1 × 106 cells in DMEM/F12 medium (Invitrogen) containing 5% fetal bovine serum (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin in 24-well culture dishes. After overnight culture, cells were washed and cultured in serum-free medium before any further treatments.

To induce the expression of FSH target genes, undifferentiated granulosa cells were treated with FSH (100 ng/ml) for 24 h. To induce the expression of LH target genes and luteinization in vitro, FSH-primed granulosa cells were treated with forskolin (10 μm) plus PMA (20 nm) for 24 h. After treatment, cells were harvested for RNA isolation or Western blots.

The plasmids for CTNNB1/TCF luciferase assay (Topflash and Fopflash) were described previously. The plasmid for Areg luciferase assay was obtained from Dr. Franco DeMayo. Primary granulosa cells cultured overnight in 24-well dishes were transfected with these plasmids (500 ng/well) using lipofectamine (Invitrogen) according to manufacturer’s instructions. At 24 h after transfection, some cells were further treated with LH (500 ng/ml) or forskolin (10 μm) plus PMA (20 nm) for 4 h before being harvested. Firefly luciferase activity was normalized to Renilla luciferase activity (cotransfected as an internal control).

Serum analysis

Mice were anesthetized and blood was collected by cardiac puncture. Progesterone and estradiol levels were analyzed by the University of Virginia Ligand Core Facility (Specialized Cooperative Centers Program in Reproduction and Infertility Research).

Immunofluorescence

Ovaries were fixed in 4% paraformaldehyde, embedded in optimal cutting temperature compound (Sakura Finetek USA, Inc., Torrance, CA) and stored at −80 C before the preparation of 7-μm sections. Sections were sequentially probed with primary antibodies as indicated in the text and Alexa Fluor 594- or 488-conjugated secondary antibodies (Molecular Probes, Sunnyvale, CA) as previously described (37). Slides were mounted using VectaShield with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Digital images were captured using an Axiphot microscope with 5×–63× objectives. For all the experiments, exposure time was kept the same for control and mutant samples.

In vitro COC expansion assay

Fully grown, nonexpanded COCs were collected from ovaries of eCG-primed immature WT or Ctnnb1 mutant mice. COCs (∼30) were plated in 100 μl of defined COC medium (MEM with Earles Salts, 25 mm HEPES, 0.25 mm sodium pyruvate, 3 mm l-glutamine, 1 mg/ml BSA, 100 U/ml penicillin, 100 μg/ml streptomycin) with 1% fetal bovine serum under the cover of mineral oil and treated with FSH (100 ng/ml) or amphiregulin (AREG, 100 ng/ml). The expansion status was checked by microscopic examination after overnight culturing.

Western blot analyses

Cell extracts containing 30 μg protein were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). After probing with primary antibodies the membranes were incubated with horseradish peroxidase-linked antirabbit antibodies (Cell Signaling Technologies, Danvers, MA) and washed, and the bound antibodies were visualized using the ECL Substrate. The primary antibodies used were: CREB, ERK1/2, CTNNB1, cyclin A (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibodies against FOXO1, phospho-AKT, phospho-MEK1/2, phospho-RSK1/2/3, phospho-ERK1/2, phospho-CTNNB1, and phospho-CREB1 were also used (1:1000 dilution, Cell Signaling Technologies).

Statistical analyses

The data for real-time RT-PCR assays, breeding experiments, hormone levels, and superovulation tests are represented as means ± sd. Data were analyzed by using GraphPad Prism Programs (ANOVA or t test; GraphPad Prism, San Diego, CA) to determine significance. Values were considered significantly different if P ≤ 0.05 or P ≤ 0.01.

Acknowledgments

We thank Dr. Michael Mancini and members of the Integrated Microscopy Core, Baylor College of Medicine, Houston, Texas, for their assistance and Dr. Francesco DeMayo, Baylor College of Medicine, Houston, Texas for the Areg-luciferase construct. Dr. You-Qiang Su, The Jackson Laboratory, Bar Harbor, Maine provided helpful suggestions and Noritaka Noma, Hiroshima University, Hiroshima, Japan provided experimental assistance. We thank the University of Virginia Ligand Assay Core Laboratory for the analyzing serum levels of progesterone and estradiol [Specialized Cooperative Centers Program in Reproduction and Infertility Research (SCCPIR)].

Footnotes

This work is supported by National Institutes of Health (NIH) Grants NIH-HD16229, NIH-HD07495 (SCCPIR), Project II (to J.S.R.), Grant-in-Aid for Scientific Research (No. 21688019, No. 21028014, and No. 21248032) from the Japan Society for the Promotion of Science (to M.S.), and NIH Postdoctoral Training Grant NIH-HD07165 (to H.Y.F.) and NIH-U54 HD28934 (SCCPIR) at the University of Virginia.

Disclosure Summary: The authors have nothing to disclose.

First Published Online July 7, 2010

Abbreviations: BrdU, Bromodeoxyuridine; CG, chorionic gonadotropin; CL, corpora lutea; COC, cumulus cell-oocyte complex;CRE, cAMP-responsive element; CREB, cAMP-responsive element-binding protein; CTNNB1, β-catenin; FZD, Frizzled; GSK, glycogen synthase kinase GVBD, germinal vesicle breakdown; PKA, protein kinase A; PMA, phorbol-12-myristate 13-acetate; TCF, T-cell factor; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick end labeling; WNT, wingless-type mouse mammary tumor virus integration site family; WT, wild type.

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