SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes.

SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre-driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females.

Cooperative effects of 17ß-estradiol and oocyte-derived paracrine factors on the transcriptome of mouse cumulus cells.

Oocyte-derived paracrine factors (ODPFs) and estrogens are both essential for the development and function of ovarian follicles in mammals. Cooperation of these two factors was assessed in vitro using intact cumulus-oocyte complexes, cumulus cells cultured after the removal of oocytes [oocytectomized (OOX) cumulus cells], and OOX cumulus cells cocultured with denuded oocytes, all in the presence or absence of 17ß-estradiol (E2). Effects on the cumulus cell transcriptome were assessed by microarray analysis. There was no significant difference between the cumulus cell transcriptomes of either OOX cumulus cells cocultured with oocytes or intact cumulus-oocyte complexes. Therefore, oocyte-mediated regulation of the cumulus cell transcriptome is mediated primarily by ODPFs and not by gap junctional communication between oocytes and cumulus cells. Gene ontology analysis revealed that both ODPFs and E2 strongly affected the biological processes associated with cell proliferation in cumulus cells. E2 had limited effects on ODPF-regulated biological processes. However, in sharp contrast, ODPFs significantly affected biological processes regulated by E2 in cumulus cells. For example, only in the presence of ODPFs did E2 significantly promote the biological processes related to phosphorylation-mediated signal transduction in cumulus cells, such as the signaling pathways of epidermal growth factor, vascular endothelial growth factor, and platelet-derived growth factor. Therefore, ODPFs and E2 cooperate to regulate the cumulus cell transcriptome and, in general, oocytes modulate the effects of estrogens on cumulus cell function.

Hormonal coordination of natriuretic peptide type C and natriuretic peptide receptor 3 expression in mouse granulosa cells.

Natriuretic peptide type C (NPPC) and its receptor natriuretic peptide receptor 2 (NPR2) regulate cGMP in ovarian follicles and participate in maintaining oocyte meiotic arrest. We investigated the regulation of Nppc expression in mouse granulosa cells in vivo and in vitro. In mural granulosa cells (MGCs) in vivo, eCG caused an increase in Nppc mRNA, and subsequent human chorionic gonadotropin (hCG) treatment caused a decrease. A culture system was established for MGCs isolated from follicles not stimulated with equine chorionic gonadotropin to further define the mechanisms controlling Nppc expression. In this system, expression of Nppc mRNA was increased by estradiol (E2), with augmentation by follicle-stimulating hormone (FSH), but FSH or luteinizing hormone (LH) alone had no effect. Thus, estrogens are important for regulating Nppc expression, probably by feedback mechanisms enhancing the action of gonadotropins. In MGCs treated with E2 plus FSH in vitro, subsequent treatment with EGF, but not LH, decreased Nppc mRNA. MGCs express higher levels of both Nppc and Lhcgr mRNAs than cumulus cells. Oocyte-derived paracrine factors suppressed cumulus cell Lhcgr but not Nppc expression. Thus, higher Nppc expression by MGCs is not the result of oocyte suppression of expression in cumulus cells. Another possible regulator of the LH-induced NPPC decrease is NPR3, an NPPC clearance receptor. Human chorionic gonadotropin increased Npr3 expression in vivo and LH increased Npr3 mRNA in cultured MGCs, independently of EGF receptor activation. Interestingly, despite the increase in Npr3 mRNA, the hCG-induced decrease in ovarian NPPC occurred normally in an Npr3 mutant (lgj), thus NPR3 probably does not participate in regulation of ovarian NPPC levels or oocyte development.

Estradiol promotes and maintains cumulus cell expression of natriuretic peptide receptor 2 (NPR2) and meiotic arrest in mouse oocytes in vitro.

Natriuretic peptide type C (NPPC) and its cognate receptor natriuretic peptide receptor 2 (NPR2) are essential for maintaining meiotic arrest in mouse oocytes residing in Graafian follicles. Cumulus cells, which are associated with the oocyte, express the receptor NPR2, a guanylyl cyclase, whereas mural granulosa cells express ligand NPPC. This study determined the temporal expression of Npr2 and the hormonal factors that participate in regulating its expression and, thereby, in oocyte meiotic arrest. Stimulation of follicular development in vivo with equine chorionic gonadotropin (eCG) promoted expression of Npr2 mRNA by cumulus cells and some periantral mural granulosa cells. However, FSH did not elevate the levels of Npr2 mRNA in cultured cumulus-oocyte complexes (COCs) isolated from mice not stimulated in vivo with eCG. Nevertheless, estradiol elevated expression of this transcript in vitro to the same steady-state level found in COCs isolated from eCG-stimulated follicles in vivo. Expression of Npr2 mRNA was rapidly reduced in COCs in vitro after isolation from eCG-primed mice unless maintained in culture with estradiol. The ability of NPPC to maintain meiotic arrest in cultured COCs was transient unless culture was in estradiol-containing medium. Ability of cumulus cells to produce cyclic GMP, which is required for the maintenance of meiotic arrest, was also lost in the absence of estradiol, indicating that estradiol is required to maintain functional NPR2 receptors on cumulus cells in vitro. It is concluded that estradiol promotes and maintains expression of NPR2 in cumulus cells and participates in NPPC-mediated maintenance of oocyte meiotic arrest in vitro.

A-MYB (MYBL1) transcription factor is a master regulator of male meiosis.

The transcriptional regulation of mammalian meiosis is poorly characterized, owing to few genetic and ex vivo models. From a genetic screen, we identify the transcription factor MYBL1 as a male-specific master regulator of several crucial meiotic processes. Spermatocytes bearing a novel separation-of-function allele (Mybl1(repro9)) had subtle defects in autosome synapsis in pachynema, a high incidence of unsynapsed sex chromosomes, incomplete double-strand break repair on synapsed pachytene chromosomes and a lack of crossing over. MYBL1 protein appears in pachynema, and its mutation caused specific alterations in expression of diverse genes, including some translated postmeiotically. These data, coupled with chromatin immunoprecipitation (ChIP-chip) experiments and bioinformatic analysis of promoters, identified direct targets of MYBL1 regulation. The results reveal that MYBL1 is a master regulator of meiotic genes that are involved in multiple processes in spermatocytes, particularly those required for cell cycle progression through pachynema.

Estrogen promotes the development of mouse cumulus cells in coordination with oocyte-derived GDF9 and BMP15.

The differentiation and function of cumulus cells depend upon oocyte-derived paracrine factors, but studies on the estrogen receptor knockout mice suggested that estrogen also participates in these processes. This study investigates the possible coordination of estrogen and oocytes in the development and function of cumulus cells using cumulus expansion and the expression of transcripts required for expansion as functional endpoints. Preantral granulosa cell-oocyte complexes developed in vitro with 17ß-estradiol (E2) exhibited increased levels of cumulus expansion and Has2 transcripts, encoding hyaluronan synthase 2, compared with those developed without E2. Moreover, cumulus cell-oocyte complexes (COCs) isolated from antral follicles and maintained in culture without E2 exhibited reduced cumulus expansion and Has2 mRNA levels compared with freshly isolated COCs. Exogenous E2, provided during the maintenance culture, alleviated these deficiencies. However, when oocytes were removed from COCs, E2 supplementation did not maintain competence to undergo expansion; the presence in culture of either fully grown oocytes or recombinant growth differentiation factor 9 (GDF9) was required. Recombinant bone morphogenetic protein 15, but not fibroblast growth factor 8, augmented the GDF9 effect. Oocytes or GDF9 suppressed cumulus cell levels of Nrip1 transcripts encoding nuclear receptor-interacting protein 1, a potential inhibitor of estrogen receptor signals. Therefore, E2 and oocyte-derived paracrine factors GDF9 and bone morphogenetic protein 15 coordinate to promote the development of cumulus cells and maintain their competence to undergo expansion. Furthermore, suppression of Nrip1 expression in cumulus cells by oocyte may be one mechanism mediating cross talk between oocyte and E2 signals that promotes follicular development.

Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes.

Granulosa cells of mammalian Graafian follicles maintain oocytes in meiotic arrest, which prevents their precocious maturation. We show that mouse mural granulosa cells, which line the follicle wall, express natriuretic peptide precursor type C (Nppc) messenger RNA (mRNA), whereas cumulus cells surrounding oocytes express mRNA of the NPPC receptor NPR2, a guanylyl cyclase. NPPC increased cGMP levels in cumulus cells and oocytes and inhibited meiotic resumption in vitro. Meiotic arrest was not sustained in most Graafian follicles of Nppc or Npr2 mutant mice, and meiosis resumed precociously. Oocyte-derived paracrine factors promoted cumulus cell expression of Npr2 mRNA. Therefore, the granulosa cell ligand NPPC and its receptor NPR2 in cumulus cells prevent precocious meiotic maturation, which is critical for maturation and ovulation synchrony and for normal female fertility.

Mouse oocytes enable LH-induced maturation of the cumulus-oocyte complex via promoting EGF receptor-dependent signaling.

LH triggers the maturation of the cumulus-oocyte complex (COC), which is followed by ovulation. These ovarian follicular responses to LH are mediated by epidermal growth factor (EGF)-like growth factors produced by granulosa cells and require the participation of oocyte-derived paracrine factors. However, it is not clear how oocytes coordinate with the EGF receptor (EGFR) signaling to achieve COC maturation. The aim of the present study was to test the hypothesis that oocytes promote the expression of EGFR by cumulus cells, thus enabling them to respond to the LH-induced EGF-like peptides. Egfr mRNA and protein expression were dramatically reduced in cumulus cells of mutant mice deficient in the production of the oocyte-derived paracrine factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). Moreover, microsurgical removal of oocytes from wild-type COCs dramatically reduced expression of Egfr mRNA and protein, and these levels were restored by either coculture with oocytes or treatment with recombinant GDF9 or GDF9 plus recombinant BMP15. Blocking Sma- and Mad-related protein (SMAD)2/3 phosphorylation in vitro inhibited Egfr expression in wild-type COCs and in GDF9-treated wild-type cumulus cells, and conditional deletion of Smad2 and Smad3 genes in granulosa cells in vivo resulted in the reduction of Egfr mRNA in cumulus cells. These results indicate that oocytes promote expression of Egfr in cumulus cells, and a SMAD2/3-dependent pathway is involved in this process. At least two oocyte-derived growth factors, GDF9 and BMP15, are required for EGFR expression by cumulus cells.

Targeted suppression of Has2 mRNA in mouse cumulus cell-oocyte complexes by adenovirus-mediated short-hairpin RNA expression.

RNA interference (RNAi) is an effective tool for studying gene function in oocytes, but no studies have targeted somatic cells of primary cultured cumulus cell-oocyte complexes (COCs). This is probably due to difficulty in introducing RNAi-inducing molecules, such as a short-hairpin RNA (shRNA) gene, into COCs by commonly used transfection reagents. We therefore tested whether a developmental process of intact COCs could be suppressed by adenovirus-mediated shRNA expression. Has2, encoding hyaluronan synthase 2, was selected as the target transcript, because the process of cumulus expansion depends upon expression of Has2 mRNA and this process is easily evaluated in vitro. Intact COCs were infected with replication-incompetent adenoviruses containing an expression sequence of shRNA targeting either Has2 (Has2 shRNA) or a control transcript not expressed in cumulus cells, and the effects on epidermal growth factor (EGF)-stimulated cumulus expansion were determined. Has2 shRNA expression suppressed Has2 mRNA levels in COCs by more than 70%, without affecting expression levels of Ptgs2, Ptx3, Tnfaip6 mRNAs, which are also required for cumulus expansion, or other transcripts not related to expansion. Interestingly, levels of Areg and Ereg mRNAs were decreased in COCs expressing Has2 shRNA when compared with those in controls, while Btc mRNA levels remained unaffected. Furthermore, the degree of cumulus expansion by Has2 shRNA-expressing COCs was significantly less than that of controls. Thus adenovirus-mediated introduction of shRNA produces specific gene silencing and a phenotype in intact COCs, providing proof of principle that this method will be a helpful tool for understanding mechanisms of COC development.

Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop.

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa cell regulatory loop.

Identification of Gasz, an evolutionarily conserved gene expressed exclusively in germ cells and encoding a protein with four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper.

To discover causes of infertility and potential contraceptive targets, we used in silico subtraction and genomic database mining to identify conserved genes with germ cell-specific expression. In silico subtraction identified an expressed sequence tag (EST) present exclusively in a newborn mouse ovary library. The full-length cDNA sequence corresponding to this EST encodes a novel protein containing four ankyrin (ANK) repeats, a sterile-alpha motif (SAM), and a putative basic leucine zipper (bZIP) domain. Northern blot and semiquantitative RT-PCR analyses demonstrated that the mRNA is exclusively expressed in the mouse testis and ovary. The expression sites were localized by in situ hybridization to pachytene spermatocytes in the testis and oocytes in the ovary. Immunohistochemistry showed that the novel protein is localized to the cytoplasm in pachytene spermatocytes and early spermatids, oocytes at all stages of oogenesis, and in early preimplantation embryos. Based on its germ cell-specific expression and the presence of ANK, SAM, and basic leucine zipper domains, we have termed this novel protein GASZ. The mouse Gasz gene, which consists of 13 exons and spans 60 kb, is located on chromosome 6 between the Wnt2 and cystic fibrosis transmembrane conductance regulator (Cftr) genes. Using genomic database mining, orthologous genes encoding GASZ were identified in the rat, cow, baboon, chimpanzee, and human. Phylogenetic analyses reveal that the GASZ proteins are highly conserved among these species. Human and mouse GASZ proteins share 85.3% amino acid identity, and human and chimpanzee GASZ proteins differ by only 3 out of 475 amino acids. In humans, the GASZ gene resides on chromosome 7 and is similarly composed of 13 exons. Because both ANK repeats and the SAM domain function as protein-protein interaction modules that mediate signal transduction cascades in some systems, GASZ may represent an important cytoplasmic signal transducer that mediates protein-protein interactions during germ cell maturation in both males and females and during preimplantation embryogenesis.

Mitogen-activated protein kinase activity in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse.

This study investigated the participation of MAPK in the resumption of meiosis [germinal vesicle breakdown (GVB)] in oocytes and cumulus expansion using oocyte-cumulus cell complexes (OCC) from Mos-null mice (Mos(tm1Ev)/Mos(tm1Ev), hereafter Mos(-/-)). MAPK activity was not detected in Mos(-/-) oocytes whether they matured in vivo or in vitro, with or without gonadotropin stimulation. Therefore, there are no pathways independent of MOS that activate MAPK during gonadotropin-induced maturation. In contrast, MAPK activity was always detected coincident with GVB in Mos(+/+) oocytes. Moreover, MAPK activity was detected in cumulus cells before gonadotropin-induced GVB in OCC regardless of genotype. A specific inhibitor (U0126) of MEK, a MAPKK required for MAPK activity, inhibited gonadotropin-induced GVB in OCC of both Mos(+/+) and Mos(-/-) mice. Activation of MAPK was downstream of elevation of cAMP. U0126 also inhibited cumulus expansion stimulated by FSH, epidermal growth factor, 8-bromo-cAMP, and recombinant growth differentiation factor-9. It is concluded that under the in vitro conditions used here, gonadotropin-induced GVB requires the participation of MAPK activity in the cumulus cells, but not in the oocyte. Moreover, the induction of cumulus expansion also requires the participation of MAPK, and this action is downstream of both elevation of cAMP and growth differentiation factor-9.