Regulates Granulosa Cell Proliferation Research Articles

Anti-Müllerian hormone (AMH) receptor type II expression and AMH activity in bovine granulosa cells

Anti-Müllerian hormone (AMH) produced by granulosa cells has previously been proposed to play a role in regulating granulosa cell differentiation and follicle selection. Although AMH receptor type II (AMHR2) dimerizes with a type I receptor to initiate AMH signaling, little is known about the regulation of AMHR2 expression in bovine granulosa cells and the role of AMH in follicle development. The primary objectives of this study were to: (1) characterize AMHR2 expression in granulosa cells during follicle development; (2) identify factors that regulate AMHR2 mRNA expression in granulosa cells; and (3) examine the role of AMH signaling in granulosa cell differentiation and proliferation. Bovine granulosa cells were isolated from 5- to 8-mm follicles before selection and deviation, as well as from 9- to 12-mm and 13- to 24-mm follicles after selection. Analyses revealed that expression of AMHR2 was greater in 5- to 8-mm follicles compared with 13- to 24-mm follicles (P < 0.05). Granulosa cells treated with bone morphogenetic protein 6 (BMP6) or BMP15, but not BMP2, significantly increased AMHR2 expression when compared with control cultured cells (P < 0.05). In addition, expression of AMH was greater in granulosa cells cultured with BMP2, BMP6, or BMP15 when compared with controls (P < 0.05). Finally, treatment with recombinant human AMH, in vitro, inhibited CYP19A1 expression in a dose-related (10–100 ng/mL) fashion, and reduced granulosa cell proliferation at 48 and 72 hours (P < 0.05). Results from these studies indicate that AMH signaling plays a role in both regulating granulosa cell proliferation and preventing granulosa cells from 5- to 8-mm follicles from undergoing premature differentiation before follicle selection.

Interaction of the transforming growth factor-β and Notch signaling pathways in the regulation of granulosa cell proliferation.

The Notch and transforming growth factor (TGF)-β signalling pathways play an important role in granulosa cell proliferation. However, the mechanisms underlying the cross-talk between these two signalling pathways are unknown. Herein we demonstrated a functional synergism between Notch and TGF-β signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-β signalling increased hairy/enhancer-of-split related with YRPW motif 2 gene (Hey2) expression (one of the target genes of the Notch pathway) in PAGCs, and suppression of TGF-β signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT), an inhibitor of Notch signalling, was rescued by both the addition of ActA and overexpression of Smad3, indicating an interaction between the TGF-β and Notch signalling pathways. Co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays were performed to identify the point of interaction between the two signalling pathways. CoIP showed direct protein-protein interaction between Smad3 and Notch2 intracellular domain (NICD2), whereas ChIP showed that Smad3 could be recruited to the promoter regions of Notch target genes as a transcription factor. Therefore, the findings of the present study support the idea that nuclear Smad3 protein can integrate with NICD2 to form a complex that acts as a transcription factor to bind specific DNA motifs in Notch target genes, such as Hey1 and Hey2, and thus participates in the transcriptional regulation of Notch target genes, as well as regulation of the proliferation of PAGCs.

Signalling pathways involved in the synergistic effects of human growth differentiation factor 9 and bone morphogenetic protein 15.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) act synergistically to regulate granulosa cell proliferation and steroid production in several species. Several non-Sma and mothers against decapentaplegic (SMAD) signalling pathways are involved in the action of murine and ovine GDF9 and BMP15 in combination, with the pathways utilised differing between the two species. The aims of this research were to determine if human GDF9 and BMP15 also act in a synergistic manner to stimulate granulosa cell proliferation and to identify which non-SMAD signalling pathways are activated. Human GDF9 with BMP15 (GDF9+BMP15) stimulated an increase in (3)H-thymidine incorporation (P<0.001), which was greater than the increase with BMP15 alone, while GDF9 alone had no effect. The stimulation of (3)H-thymidine incorporation by GDF9+BMP15 was reduced by the addition of inhibitors to the SMAD2/3, nuclear factor-KB (NF-KB) and c-Jun N-terminal kinase (JNK) signalling pathways. Inhibitors to the SMAD1/5/8, extracellular signal-regulated kinase mitogen-activated protein kinase (ERK-MAPK) or p38-MAPK pathways had no effect. The addition of the BMP receptor 2 (BMPR2) extracellular domain also inhibited stimulation of (3)H-thymidine incorporation by GDF9+BMP15. In conclusion, human GDF9 and BMP15 act synergistically to stimulate granulosa cell proliferation, a response that also involves species-specific non-SMAD signalling pathways.

Expression of Cyclins and Cyclin‐Dependent Kinase Inhibitors in Granulosa Cells from Bovine Ovary

The regulation of granulosa cell proliferation is complex, and it is essential for normal follicular development in mammals. The aim of this study was to examine the expression of cyclins and their inhibitors in the granulosa cells of follicles at different developmental stages. Follicles were classified into three groups: oestrogen-inactive dominant follicles (EIDs), oestrogen-active dominant follicles (EADs) and pre-ovulatory follicles (POs). The expression of CCND2 (cyclin D2) mRNA was significantly higher in granulosa cells from EADs and POs than in those from EIDs. The expression of CCND3 (cyclin D3) mRNA was significantly higher in granulosa cells from EADs than in those from other follicles. CCND1 (cyclin D1), CCNE1 (cyclin E1) and CCNE2 (cyclin E2) mRNA expression did not differ among the different follicular stages. The expression of CDKN1A (p21(cip1) ) and CDKN1B (p27(kip1) ) mRNA was significantly higher in granulosa cells from EIDs and POs, respectively, than in those from other follicles. Expression of CDKN2D (p19(INK4d) ) mRNA did not differ among the different follicular stages. Taken together, our study suggested that cyclins and their inhibitors are associated with granulosa cell proliferation at specific follicular developmental stages.

MicroRNA-181a Suppresses Mouse Granulosa Cell Proliferation by Targeting Activin Receptor IIA

Activin, a member of the transforming growth factor-β superfamily, promotes the growth of preantral follicles and the proliferation of granulosa cells. However, little is known about the role of microRNAs in activin-mediated granulosa cell proliferation. Here, we reported a dose- and time-dependent suppression of microRNA-181a (miR-181a) expression by activin A in mouse granulosa cells (mGC). Overexpression of miR-181a in mGC suppressed activin receptor IIA (acvr2a) expression by binding to its 3′-untranslated region (3′-UTR), resulting in down-regulation of cyclin D2 and proliferating cell nuclear antigen expression, leading to inhibition of the cellular proliferation, while overexpression of acvr2a attenuated the suppressive effect of miR-181a on mGC proliferation. Consistent with the inhibition of acvr2a expression, miR-181a prevented the phosphorylation of the activin intracellular signal transducer, mothers against decapentaplegic homolog 2 (Smad2), leading to the inactivation of activin signaling pathway. Interestingly, we found that miR-181a expression decreased in ovaries of mice at age of 8, 12, and 21 days, as compared with that in ovaries of 3-day old mice, and its level was reduced in preantral and antral follicles of mice compared with that in primary ones. Moreover, the level of miR-181a in the blood of patients with premature ovarian failure was significantly increased compared with that in normal females. This study identifies an interplay between miR-181a and acvr2a, and reveals an important role of miR-181a in regulating granulosa cell proliferation and ovarian follicle development.

The Extracellular Matrix Protein, Spondin 1, Regulates Granulosa Cell Function and May Regulate Vascularization of the Corpus Luteum.

Dramatic changes in the composition of the ovarian extracellular matrix (ECM) and cell-cell adhesion proteins occur throughout folliculogenesis. The ECM protein, Spondin 1 (SPON1), is a secreted adhesion protein best known for its ability to regulate patterning of the developing nervous system by promoting outgrowth and adhesion of neural cells. SPON1 has been previously identified in bovine follicular fluid, and was found to promote the proliferation of rat vascular smooth muscle cells. Although SPON1 is highly expressed in the human ovary, and dramatically overexpressed in ovarian cancer, a function for SPON1 in the ovary has never been studied. The objectives of our study were to: a) characterize the expression and localization of SPON1 in the mouse ovary, and b) identify what effect SPON1 may have on granulosa cell behaviour and function. Using qPCR, we found that SPON1 mRNA levels increased 5-fold in granulosa cells isolated from mice treated with PMSG for 48h compared to vehicle-treated controls, indicating that FSH increases SPON1 expression. Immunofluorescence studies localized SPON1 to regions between and surrounding both theca and granulosa cells of follicles of various sizes, from small secondary to preovulatory follicles. In the corpus luteum (CL), the pattern of SPON1 immunoreactivity was strikingly similar to that of platelet endothelial cell adhesion molecule (PECAM-1), which localizes to endothelial cells of the vasculature, suggesting that SPON1 may regulate vascularization during formation of the CL. Finally, we found that SPON1 has diverse effects when added as a recombinant protein to both granulosa cell lines and mouse primary granulosa cells. SPON1 enhanced the proliferation of KGN human granulosa cells, and in primary mouse granulosa cells, SPON1 enhanced granulosa cell viability by up to 50% compared to vehicle-treated controls, suggesting that SPON1 may regulate granulosa cell viability during folliculogenesis. Lastly, SPON1 dramatically induced aggregation of primary mouse granulosa cells, suggesting a role for SPON1 in granulosa cell adhesion, as has been reported in SPON1-treated neural cells. In summary, we have shown that the ECM protein, SPON1, regulates granulosa cell proliferation, survival, and cell-cell adhesion, and may also regulate vascularization of the corpus luteum. Given the diversity of its expression and effects on granulosa cells, we hypothesize that SPON1 plays a previously-unidentified important role in the regulation of various stages of folliculogenesis.

The mechanism of mTOR (mammalian target of rapamycin) in a mouse model of polycystic ovary syndrome (PCOS)

Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder affecting 5-10% of women in reproductive age that is characterized by hyperandrogenism, oligo- or anovulation and infertility. However the pathophysiology of PCOS still remains unknown. The mammalian target of rapamycin (mTOR) is a central component that regulates various processes including cell growth, proliferation, metabolism, and angiogenesis. mTOR signaling cascade has recently been examined in ovarian follicles where it regulates granulosa cell proliferation and differentiation. mTOR functions as two complexes, mTOR complex 1 and 2. Therefore, we hypothesized that mTORC1 and/or 2 may have important role in proliferation of theca and granulosa cells in PCOS. In the present study, we sought to determine the mTOR signaling pathway in PCOS mouse ovary. We designed 3 groups: Control (C, no treatment), PCOS (P, The injection of DHEA (6 mg/100 g BW in 0.1 ml of sesame oil) (s.c) for 20 consecutive days), Vehicle (V, daily (s.c) sesame oil alone injection). Our results showed that mTORC1 and mTORC2-mediated signaling may play a role in PCOS mouse ovary. These findings provide evidence that mTORC1 and mTORC2 may have responsibility in increased ovarian follicular cell proliferation and growth in PCOS. Consequently, these results suggest that the mTOR signaling pathways (mTORC1 and mTORC 2) may create new clinical strategies to optimize developmental competence of PCOS should target correction of the entire follicle growth, oocyte development process and anovulatory infertility in PCOS.

MTOR Controls Ovarian Follicle Growth by Regulating Granulosa Cell Proliferation

We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed.

Notch Signaling Is Involved in Ovarian Follicle Development by Regulating Granulosa Cell Proliferation

Notch signaling is an evolutionarily conserved pathway, which regulates cell proliferation, differentiation, and apoptosis. It has been reported that the members of Notch signaling are expressed in mammalian ovaries, but the exact functions of this pathway in follicle development is still unclear. In this study, primary follicles were cultured in vitro and treated with Notch signaling inhibitors, L-658,458 and N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). We found that the cultured follicles completely stopped developing after L-658,458 and DAPT treatment, most of the granulosa cells were detached, and the oocytes were also degenerated with condensed cytoplasma. Further studies demonstrated that the proliferation of granulosa cells was dependent on the Notch signaling. L-658,458 and DAPT treatment inhibited proliferation of in vitro cultured primary granulosa cells and decreased the expression of c-Myc. Lentivirus mediated overexpression of Notch intracellular domain 2, and c-Myc could promote the proliferation of granulosa cells and rescue the growth inhibition induced by L-658,458 and DAPT. In conclusion, Notch signaling is involved in follicular development by regulating granulosa cell proliferation.

Synergistic Activation of Akt by IGF-1 and cAMP Is Correlated with Altered Expression of Paracrine Factors That Regulate Follicle Progression and Ovulation in Murine Granulosa Cells.

Folliculogenesis and granulosa cell function are regulated, in part, by gonadotropin stimulation of cAMP-dependent signal transduction. For example, follicle stimulating hormone (FSH) regulates granulosa cell steroidogenesis, oocyte maturation, and the expression of paracrine factors which coordinate oocyte-granulosa cell growth and development. IGF-1 also contributes to follicular development via regulation of granulosa cell proliferation, apoptosis, and steroid production. These actions of IGF-1 are mediated through its interaction with the IGF-1 receptor, and in some cases, requires simultaneous gonadotropin-dependent signaling. Recent epidemiological studies demonstrate that IGF-1 levels decrease with increasing age, body mass index, and waist circumference. Given that advanced maternal age and obesity are the most common cause of anovulatory infertility, the objective of this study was to identify mechanisms by which IGF-1 regulates the expression of genes in granulosa cells which are crucial for follicle growth, follicle maturation, and ovulation. To achieve this objective, granulosa cells were isolated from the ovary of 23-day-old CF-1 mice and maintained in short-term culture. After 5 days, cells were exposed to no treatment, 1mM cAMP, 100ng/mL IGF-1, or a combination of cAMP and IGF-1 for 2 or 8 hours. The mRNA abundance of several paracrine factors which are produced by the granulosa cells and contribute to the coordination of oocyte and follicular growth and regulate the process of ovulation were examined using quantitative real-time PCR (QPCR). Genes associated with transzonal projections, which facilitate bi-directional communication between the granulosa cells and oocyte, were also examined. IGF-1 increased cAMP-dependent stimulation of kit ligand (Kitl), growth differentiation 9 (Gdf9), amphiregulin (Areg), and interleukin-6 (Il6) expression in both the 2 and 8 hour treatment groups. IGF-1 also increased cAMP-dependent stimulation of epiregulin (Ereg) expression 8 hours post-treatment. Conversely, anti-mullerian hormone (Amh) was downregulated upon combined treatment of granulosa cells with IGF-1 and cAMP for 2 or 8 hours. IGF-1 and cAMP alter gene expression through activation of the downstream signaling proteins Akt and Erk1/2. To assess the activity of these proteins, granulosa cells were maintained in short-term culture and exposed to no treatment, 1 mM cAMP, 100 ng/mL IGF-1, or a combination of cAMP and IGF-1 for 30 or 60 minutes. Western blot analyses demonstrated that cAMP alone or in combination with IGF-1 increased Erk1/2 phosphorylation compared to untreated cells 30 and 60 minutes post-treatment. IGF-1 or cAMP treatment alone also increased Akt phosphorylation in the granulosa cells. Interestingly, the combined treatment of IGF-1 and cAMP resulted in a synergistic increase in Akt phosphorylation indicating cross-talk between the cAMP and IGF-1 signaling pathways. Taken together, these data suggest that IGF-1 and cAMP may have an additive effect on paracrine factor gene expression due to the synergistic activation of Akt. Given that these genes are associated with follicle development and ovulation, these data also provide a plausible mechanism for age and obesity-dependent anovulatory infertility. (platform)

Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells

Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation.

Luteinizing Hormone Stimulates Mammalian Target of Rapamycin Signaling in Bovine Luteal Cells via Pathways Independent of AKT and Mitogen-Activated Protein Kinase: Modulation of Glycogen Synthase Kinase 3 and AMP-Activated Protein Kinase

LH stimulates the production of cAMP in luteal cells, which leads to the production of progesterone, a hormone critical for the maintenance of pregnancy. The mammalian target of rapamycin (MTOR) signaling cascade has recently been examined in ovarian follicles where it regulates granulosa cell proliferation and differentiation. This study examined the actions of LH on the regulation and possible role of the MTOR signaling pathway in primary cultures of bovine corpus luteum cells. Herein, we demonstrate that activation of the LH receptor stimulates the phosphorylation of the MTOR substrates ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1. The actions of LH were mimicked by forskolin and 8-bromo-cAMP. LH did not increase AKT or MAPK1/3 phosphorylation. Studies with pathway-specific inhibitors demonstrated that the MAPK kinase 1 (MAP2K1)/MAPK or phosphatidylinositol 3-kinase/AKT signaling pathways were not required for LH-stimulated MTOR/S6K1 activity. However, LH decreased the activity of glycogen synthase kinase 3Beta (GSK3B) and AMP-activated protein kinase (AMPK). The actions of LH on MTOR/S6K1 were mimicked by agents that modulated GSK3B and AMPK activity. The ability of LH to stimulate progesterone secretion was not prevented by rapamycin, a MTOR inhibitor. In contrast, activation of AMPK inhibited LH-stimulated MTOR/S6K1 signaling and progesterone secretion. In summary, the LH receptor stimulates a unique series of intracellular signals to activate MTOR/S6K1 signaling. Furthermore, LH-directed changes in AMPK and GSK3B phosphorylation appear to exert a greater impact on progesterone synthesis in the corpus luteum than rapamycin-sensitive MTOR-mediated events.

Effect of plasma from cyclic versus nutritionally induced anovulatory beef heifers on proliferation of granulosa cells in vitro

The effect of plasma from cyclic versus nutritionally induced anovulatory beef heifers was evaluated on proliferation of bovine granulosa cells in vitro. Granulosa cells were obtained from small (1–5 mm) follicles of cattle and cultured for 4 days. During the last 2 days of culture, cells were exposed to medium containing 0, 1 or 10% plasma from cyclic or anovulatory heifers in the presence or absence of IGF-I (100 ng/ml). Cell numbers were determined. Regardless of source, increasing percentage of plasma to culture medium increased cell numbers. However, the plasma-induced increase was greater in granulosa cells exposed to cyclic heifer plasma versus anovulatory heifer plasma. In addition, concomitant treatment with IGF-I dramatically improved cell proliferation induced by anovulatory heifer plasma. These results indicate that plasma from cyclic heifers contain factors that are a greater stimulus to granulosa cell proliferation than plasma from anovulatory heifers. Systemic factors such as IGF-I may play a role in directly regulating granulosa cell proliferation in cattle.

Epidermal Growth Factor (EGF) Receptor Ligands in the Chicken Ovary: I. Evidence for Heparin-Binding EGF-Like Growth Factor (HB-EGF) as a Potential Oocyte-Derived Signal to Control Granulosa Cell Proliferation and HB-EGF and Kit Ligand Expression

There is increasing evidence that epidermal growth factor (EGF) receptor (EGFR) ligand and Kit ligand (KL) play critical roles in controlling follicular development in mammals. Because little is known about their expressions in the ovary of nonmammalian vertebrate, our study aimed to examine the expression, hormonal regulation, and interaction of HB-EGF and KL in the chicken ovary. Using semiquantitative RT-PCR, we demonstrated that ovarian HB-EGF expression increased dramatically with the posthatching ovarian growth. In line with this finding, HB-EGF was shown to be produced primarily by the growing oocytes and capable of stimulating the proliferation of granulosa cells in prehierarchal (3 mm) and preovulatory follicles (F5 and F1). Although HB-EGF expression is mainly restricted to the oocytes, its expression in cultured granulosa cells could be transiently yet strongly induced by HB-EGF and other EGFR ligands including EGF and TGF-alpha. And the inducing effect of HB-EGF was completely abolished by AG1478 (10 microM) or PD98059 (100 microM), indicating that the action of HB-EGF is mediated by EGFR and intracellular MAPK/ERK signaling pathway. Unlike mammals, only KL-1, not the other three isoforms identified (KL-2, -3, and -4), was detected to be predominantly expressed in the chicken ovary. Interestingly, KL expression in undifferentiated and differentiated granulosa cells could be transiently down-regulated by HB-EGF, implying an intrafollicular communication between growing oocyte and surrounding granulosa cells through the interplay of EGFR ligand and KL. Collectively, our data suggest that HB-EGF is likely a paracrine signal from the oocyte to regulate granulosa cell proliferation and HB-EGF and KL expression during ovarian follicular development.

Regulation of granulosa cell proliferation and EGF-like ligands during the periovulatory interval in monkeys

This study seeks to clarify cell cycle dynamics of granulosa cells following hCG and elucidate the expression of epidermal growth factor (EGF)-like ligands during luteinization. Granulosa cells were obtained from rhesus macaques undergoing controlled ovarian stimulation protocols before or after an ovulatory hCG bolus. Cell cycle characteristics were determined by flow cytometry and levels of EGF receptor (EGFR), amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC) mRNAs were measured by real-time RT-PCR. The proportion of cells in S-phase was 7.5% prior to hCG and did not decline until 24 h after hCG (3.1%). EGFR protein and BTC mRNA did not change following hCG, whereas AREG and EREG mRNA increased starting at 3 and 12 h post-hCG, respectively, and remained elevated thereafter. Cell cycle transit of macaque granulosa cells does not change until 24 h after an ovulatory stimulus, whereas the EGF-like ligands EREG and AREG are increased rapidly. This suggests that luteinizing granulosa cells are refractory to mitogenic stimulation by EGFR ligands.

Transcriptional Regulation of Cyclin D2 by the PKA Pathway and Inducible cAMP Early Repressor in Granulosa Cells1

Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis.

Cell type- and stage-specific changes in HOXA7 protein expression in human ovarian folliculogenesis: possible role of GDF-9

Homeobox (HOX) genes are important transcriptional regulators in development and in adult tissues. A major obstacle to the understanding of their roles in humans has been the lack of well-defined anti-human HOX antibodies. We generated a thoroughly characterized polyclonal rabbit antibody against human HOXA7 and used it to study the distribution, role, and regulation of HOXA7 in human ovarian folliculogenesis and in granulosa cell tumors. Immunohistochemically, follicles were strongly HOXA7-positive compared with stroma. Oocytes expressed little HOXA7. Granulosa cells were predominantly negative in primordial follicles, had uniformly HOXA7-positive nuclei in primary follicles, and, as follicles matured, the subcellular localization of HOXA7 changed from nuclear to predominantly cytoplasmic. HOXA7 was mainly cytoplasmic in theca interna, but completely absent in theca externa. Granulosa cell tumors were mainly HOXA7 positive and, like in preovulatory follicles and theca interna, staining was predominantly cytoplasmic. The change in HOXA7 expression from negative primordial to positive primary follicles suggested a relationship with granulosa cell proliferation. To test this hypothesis, SV40 Tag-immortalized human granulosa cells (SVOG) were double stained with anti-HOXA7 antibody and with Ki-67 as proliferation marker. HOXA7 expression was highest in mitotic cells. In addition, growth differentiation factor-9 (GDF-9), known to be secreted by oocytes in primary human follicles, up-regulated HOXA7 protein, and stimulated proliferation of SVOG, while TGF-β1 inhibited HOXA7 expression and proliferation. This is the first report on the expression of any HOX gene in human ovarian follicles and granulosa cell tumors. It shows that HOXA7 undergoes cell type- and stage-specific changes during ovarian folliculogenesis, likely regulates granulosa cell proliferation, and in subcellular location differs between proliferating and secretory cells. The increase in HOXA7 protein in response to GDF-9 represents the first demonstration of a possible regulatory role of oocytes in ovarian follicular HOX gene expression.

Tumor necrosis factor alpha (TNF) increases granulosa cell proliferation: dependence on c-Jun and TNF receptor type 1.

TNF alpha has significant in vitro effects on steroidogenesis and folliculogenesis and reproductive alterations occur in TNF receptor type 1 (TNFR1) knockout mice. The present study investigated the effect of in vitro TNF on granulosa cell proliferation from immature mice at 28 d of age, with emphasis on intracellular signaling that regulates granulosa cell proliferation. TNF dose dependently increased granulosa cell proliferation and the proto-oncogene c-Jun protein. However, other Jun family members such as JunD was expressed constitutively and JunB was not expressed. In vitro TNF did not increase c-Jun and proliferation in granulosa cells from TNFR1 knockout mice. The time course of TNF-induced c-Jun revealed biphasic patterns of short-term (3 h) and long-term (24 h) induction. The time courses of Ser63- and Ser73-phospho c-Jun coincided with changes in total c-Jun. Among MAPK cascades, stress-activated protein kinase/c-Jun-NH(2)-teminal kinase signaling was increased transiently in TNF-treated cells, whereas p38MAPK and ERK1 and 2 were not changed. In addition, overexpression of nuclear factor-kappa B and addition of ceramide and 8-bromo-cAMP did not increase c-Jun or proliferation. Antisense oligonucleotides for c-Jun blocked cell proliferation induced by TNF. In conclusion, the above results demonstrate that TNF increased c-Jun by activating stress-activated protein kinase/c-Jun-NH(2)-teminal kinase signaling via TNFR1 in mouse granulosa cells, and the induced c-Jun resulted in increased cell proliferation.

Cyclin D2 and p27 are tissue-specific regulators of tumorigenesis in inhibin alpha knockout mice.

Inhibins are heterodimeric (alpha:betaA and alpha:betaB) endocrine, paracrine, and autocrine factors of the TGFbeta superfamily that are produced predominantly by ovarian granulosa cells in females and testicular Sertoli cells in males. Control of granulosa and Sertoli cell proliferation is lost in the inhibin alpha (Inhalpha) knockout mouse model, leading to gonadotropin-dependent gonadal tumors of the granulosa/Sertoli cell lineage in both females and males. Castrate Inhalpha knockout mice develop sex steroidogenic tumors of the adrenal cortex. Physiological control of granulosa/Sertoli cell cycle progression depends on p27Kip1 and cyclin D2, which function in the G1-->S phase transition. To study the cell cycle-regulatory factors involved in ovarian, testicular, and adrenal tumor development in vivo, we have bred Inhalpha mutant mice to mice with targeted disruptions of the p27 and cyclin D2 genes. Our previous studies demonstrated that inhibins act cooperatively with p27 to negatively regulate granulosa cell proliferation, as double mutant mice lacking inhibins and p27 develop and succumb to ovarian tumors more rapidly than Inhalpha knockout mice. Here, we report that cyclin D2 antagonizes this inhibition and is key in promoting gonadal growth and tumor development, and tumor development is markedly suppressed in double-mutant mice. We found that double-knockout females lacking cyclin D2 and Inhalpha lived longer than mice lacking inhibins alone; the majority of these double-knockout mice lived longer than 17 wk, as opposed to inhibin alpha single-knockout females with 50% survival at between 12 and 13 wk of age. Moreover, 95% of inhibin alpha knockout males succumb to testicular tumor development by 12 wk of age, whereas double knockouts were protected from early signs of tumor development and had a 50% survival of 40 wk. Interestingly, the results of these studies reflect tissue-specific consequences of loss of these cell cycle regulators. In castrate mice, loss of p27 has little effect on adrenal cortical tumor progression in the absence of inhibins, whereas loss of cyclin D2 prolongs the lifespan of cyclin D2, Inhalpha double knockouts. After gonadectomy, 50% of cyclin D2, Inhalpha double-knockout males live to more than 46 wk of age, 10 wk longer than 50% of littermates lacking only inhibins. Similarly, 50% of female cyclin D2, inhibin alpha double knockouts live to 47 wk of age before succumbing to adrenal tumor development, in contrast to the 50% survival of Inhalpha single-knockout females at between 27 and 28 wk. Thus, identification of genetic modifiers of the Inhalpha knockout tumor phenotype has led us to a better appreciation of how specific components of the cell cycle machinery contribute to tumorigenesis in the ovary, testis, and adrenal gland.

Gonadotropin and steroid control of granulosa cell proliferation during the periovulatory interval in rhesus monkeys.

Progesterone produced in response to the midcycle gonadotropin surge is essential for ovulation and luteinization of the primate follicle. Because cell-cycle arrest is associated with the initiation of luteinization, this study was designed to determine the dynamics and regulation of granulosa cell proliferation by gonadotropin and progesterone during the periovulatory interval in the primate follicle. Granulosa cells or ovaries were obtained from macaques undergoing controlled ovarian stimulation either before (0 h) or as long as 36 h following the administration of an ovulatory hCG bolus with or without a 3beta-hydroxysteroid dehydrogenase inhibitor with or without a nonmetabolizable progestin. The percentage of cells staining positive for Ki-67, a nuclear marker for cell proliferation, decreased (P < 0.05) within 12 h of hCG administration in a steroid-independent manner. Levels of cyclin D2 and E mRNA did not decline during the periovulatory interval; however, cyclin B1 mRNA was reduced significantly by 12 h. Steroid depletion increased (P < 0.05) cyclin B1 mRNA at both 12 and 36 h post-hCG and was reversible by progestin replacement at 36 h. The cyclin-dependent kinase inhibitor p21(Cip1) was transiently increased 12 h post-hCG, whereas p27(Kip1) mRNA levels increased at 36 h in a steroid-independent fashion. These data suggest that a gonadotropin bolus inhibits mitosis in granulosa cells early (12 h) in the periovulatory interval, whereas progesterone may play a later, antiproliferative role in luteinized cells of primates.