Oocyte stage-specific effects of MTOR determine granulosa cell fate and oocyte quality in mice.

MTOR (mechanistic target of rapamycin) is a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation. Here we show that conditional knockout (cKO) of Mtor in either primordial or growing oocytes caused infertility but differentially affected oocyte quality, granulosa cell fate, and follicular development. cKO of Mtor in nongrowing primordial oocytes caused defective follicular development leading to progressive degeneration of oocytes and loss of granulosa cell identity coincident with the acquisition of immature Sertoli cell-like characteristics. Although Mtor was deleted at the primordial oocyte stage, DNA damage accumulated in oocytes during their later growth, and there was a marked alteration of the transcriptome in the few oocytes that achieved the fully grown stage. Although oocyte quality and fertility were also compromised when Mtor was deleted after oocytes had begun to grow, these occurred without overtly affecting folliculogenesis or the oocyte transcriptome. Nevertheless, there was a significant change in a cohort of proteins in mature oocytes. In particular, down-regulation of PRC1 (protein regulator of cytokinesis 1) impaired completion of the first meiotic division. Therefore, MTOR-dependent pathways in primordial or growing oocytes differentially affected downstream processes including follicular development, sex-specific identity of early granulosa cells, maintenance of oocyte genome integrity, oocyte gene expression, meiosis, and preimplantation developmental competence.

Reproduction: Oocytes Call, Granulosa Cells Connect.

Making functional eggs requires a bidirectional conversation between oocytes and their companion somatic cells. Filopodia emanating from somatic cells carry crucial developmental information to oocytes, and a new study shows that oocytes signal elaboration of this key connection.

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.

Oocyte-dependent activation of MTOR in cumulus cells controls the development and survival of cumulus-oocyte complexes.

Communication between oocytes and their companion somatic cells promotes the healthy development of ovarian follicles, which is crucial for producing oocytes that can be fertilized and are competent to support embryogenesis. However, how oocyte-derived signaling regulates these essential processes remains largely undefined. Here, we demonstrate that oocyte-derived paracrine factors, particularly GDF9 and GDF9-BMP15 heterodimer, promote the development and survival of cumulus-cell-oocyte complexes (COCs), partly by suppressing the expression of Ddit4l, a negative regulator of MTOR, and enabling the activation of MTOR signaling in cumulus cells. Cumulus cells expressed less Ddit4l mRNA and protein than mural granulosa cells, which is in striking contrast to the expression of phosphorylated RPS6 (a major downstream effector of MTOR). Knockdown of Ddit4l activated MTOR signaling in cumulus cells, whereas inhibition of MTOR in COCs compromised oocyte developmental competence and cumulus cell survival, with the latter likely to be attributable to specific changes in a subset of transcripts in the transcriptome of COCs. Therefore, oocyte suppression of Ddit4l expression allows for MTOR activation in cumulus cells, and this oocyte-dependent activation of MTOR signaling in cumulus cells controls the development and survival of COCs.

Histone reader BRWD1 targets and restricts recombination to the Igk locus.

B lymphopoiesis requires that immunoglobulin genes be accessible to RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin, which suggests that additional mechanisms must restrict RAG-mediated DNA cleavage. Here we show that developmental downregulation of interleukin 7 (IL-7)-receptor signaling in small pre-B cells induced expression of the bromodomain-family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-B cell receptor (pre-BCR)-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5' to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings represent a paradigm in which, at any particular antigen-receptor locus, specialized mechanisms enforce lineage- and stage-specific recombination.

Transcriptomic diversification of developing cumulus and mural granulosa cells in mouse ovarian follicles.

Cumulus cells and mural granulosa cells (MGCs) have functionally distinct roles in antral follicles, and comparison of their transcriptomes at a global and systems level can propel future studies on mechanisms underlying their functional diversity. These cells were isolated from small and large antral follicles before and after stimulation of immature mice with gonadotropins, respectively. Both cell types underwent dramatic transcriptomic changes, and differences between them increased with follicular growth. Although cumulus cells of both stages of follicular development are competent to undergo expansion in vitro, they were otherwise remarkably dissimilar with transcriptomic changes quantitatively equivalent to those of MGCs. Gene ontology analysis revealed that cumulus cells of small follicles were enriched in transcripts generally associated with catalytic components of metabolic processes, while those from large follicles were involved in regulation of metabolism, cell differentiation, and adhesion. Contrast of cumulus cells versus MGCs revealed that cumulus cells were enriched in transcripts associated with metabolism and cell proliferation while MGCs were enriched for transcripts involved in cell signaling and differentiation. In vitro and in vivo models were used to test the hypothesis that higher levels of transcripts in cumulus cells versus MGCs is the result of stimulation by oocyte-derived paracrine factors (ODPFs). Surprisingly ∼48% of transcripts higher in cumulus cells than MGCs were not stimulated by ODPFs. Those stimulated by ODPFs were mainly associated with cell division, mRNA processing, or the catalytic pathways of metabolism, while those not stimulated by ODPFs were associated with regulatory processes such as signaling, transcription, phosphorylation, or the regulation of metabolism.

Mouse BRWD1 is critical for spermatid postmeiotic transcription and female meiotic chromosome stability.

Postmeiotic gene expression is essential for development and maturation of sperm and eggs. We report that the dual bromodomain-containing protein BRWD1, which is essential for both male and female fertility, promotes haploid spermatid-specific transcription but has distinct roles in oocyte meiotic progression. Brwd1 deficiency caused down-regulation of ∼300 mostly spermatid-specific transcripts in testis, including nearly complete elimination of those encoding the protamines and transition proteins, but was not associated with global epigenetic changes in chromatin, which suggests that BRWD1 acts selectively. In females, Brwd1 ablation caused severe chromosome condensation and structural defects associated with abnormal telomere structure but only minor changes in gene expression at the germinal vesicle stage, including more than twofold overexpression of the histone methyltransferase MLL5 and LINE-1 elements transposons. Thus, loss of BRWD1 function interferes with the completion of oogenesis and spermatogenesis through sexually dimorphic mechanisms: it is essential in females for epigenetic control of meiotic chromosome stability and in males for haploid gene transcription during postmeiotic sperm differentiation.

Amino acid 72 of mouse and human GDF9 mature domain is responsible for altered homodimer bioactivities but has subtle effects on GDF9:BMP15 heterodimer activities.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted paralogs of the transforming growth factor beta (TGFbeta) superfamily. In mammals, these two growth factors play critical roles in folliculogenesis. As previously reported, an arginine in the pre-helix loop of GDF5 defines the high binding specificity to its type 1 receptor. Interestingly, bioactive mouse GDF9 and human BMP15 share the conserved arginine in the pre-helix loop, but their low-activity counterparts (mouse BMP15 and human GDF9) have a glycine or a proline instead. To address the question of whether the arginine residue defines the different activities of GDF9 and BMP15 homodimers and their heterodimers in human and mouse, we used site-directed mutagenesis to change the species-specific residues in human and mouse proteins, and examined their activities in our in vitro assays. Although amino acid 72 of mature GDF9 is responsible for altered homodimer bioactivities, neither the corresponding BMP15 amino acid 62 nor the intact pre-helix loop is indispensable for BMP15 homodimer activity. However, amino acid 72 in GDF9 only has only subtle effects on GDF9:BMP15 heterodimer activity. Based on previous studies and our recent findings, we provide hypothetical models to understand the molecular mechanism to define activities of the homodimeric and heterodimeric ligands. The arginine residue in the pre-helix loop of GDF9 homodimer may prevent the inhibition from its pro-domain or directly alter receptor binding, but this residue in GDF9 does not significantly affect the heterodimer activity, because of suggested conformational changes during heterodimer formation.

Applying "gold standards" to in-vitro-derived germ cells.

Germ cells are the ultimate stem cells, and reports of their in vitro derivation generate excitement due to potential applications in reproductive medicine. To date, there is no firm evidence that meiosis, the hallmark of gametogenesis, can be faithfully replicated outside of the gonad. We propose benchmarks for evaluating in vitro derivation of germ cells, facilitating realization of their potential.

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.

Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes.

Coordinated regulation of oocyte and ovarian follicular development is essential for fertility. In particular, the progression of meiosis, a germ cell-specific cell division that reduces the number of chromosomes from diploid to haploid, must be arrested until just before ovulation. Follicular somatic cells are well-known to impose this arrest, which is essential for oocyte-follicle developmental synchrony. Follicular somatic cells sustain meiotic arrest via the natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system, and possibly also via high levels of the purine hypoxanthine in the follicular fluid. Upon activation by the ligand NPPC, NPR2, the predominant guanylyl cyclase in follicular somatic cells, produces cyclic guanosine monophosphate (cGMP), which maintains meiotic arrest after transfer to the oocyte via gap junctions. Here we report that both the NPPC/NPR2 system and hypoxanthine require the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme required for the production of guanylyl metabolites and cGMP. Furthermore, oocyte-derived paracrine factors, particularly the growth differentiation factor 9-bone morphogenetic protein 15 heterodimer, promote expression of Impdh and Npr2 and elevate cGMP levels in cumulus cells. Thus, although the somatic compartment of ovarian follicles plays an essential role in the maintenance of oocyte meiotic arrest, as has been known for many years, this function of the somatic cells is surprisingly regulated by signals from the oocyte itself.

Oocyte differentiation is genetically dissociable from meiosis in mice.

Oogenesis is the process by which ovarian germ cells undertake meiosis and differentiate to become eggs. In mice, Stra8 is required for the chromosomal events of meiosis to occur, but its role in differentiation remains unknown. Here we report Stra8-deficient ovarian germ cells that grow and differentiate into oocyte-like cells that synthesize zonae pellucidae, organize surrounding somatic cells into follicles, are ovulated in response to hormonal stimulation, undergo asymmetric cell division to produce a polar body and cleave to form two-cell embryos upon fertilization. These events occur without premeiotic chromosomal replication, sister chromatid cohesion, synapsis or recombination. Thus, oocyte growth and differentiation are genetically dissociable from the chromosomal events of meiosis. These findings open to study the independent contributions of meiosis and oocyte differentiation to the making of a functional egg.

Growth differentiation factor 9:bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions.

The TGF-ß superfamily is the largest family of secreted proteins in mammals, and members of the TGF-ß family are involved in most developmental and physiological processes. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), oocyte-secreted paralogs of the TGF-ß superfamily, have been shown genetically to control ovarian physiology. Although previous studies found that GDF9 and BMP15 homodimers can modulate ovarian pathways in vitro, the functional species-specific significance of GDF9:BMP15 heterodimers remained unresolved. Therefore, we engineered and produced purified recombinant mouse and human GDF9 and BMP15 homodimers and GDF9:BMP15 heterodimers to compare their molecular characteristics and physiological functions. In mouse granulosa cell and cumulus cell expansion assays, mouse GDF9 and human BMP15 homodimers can up-regulate cumulus expansion-related genes (Ptx3, Has2, and Ptgs2) and promote cumulus expansion in vitro, whereas mouse BMP15 and human GDF9 homodimers are essentially inactive. However, we discovered that mouse GDF9:BMP15 heterodimer is ∼10- to 30-fold more biopotent than mouse GDF9 homodimer, and human GDF9:BMP15 heterodimer is ∼1,000- to 3,000-fold more bioactive than human BMP15 homodimer. We also demonstrate that the heterodimers require the kinase activities of ALK4/5/7 and BMPR2 to activate SMAD2/3 but unexpectedly need ALK6 as a coreceptor in the signaling complex in granulosa cells. Our findings that GDF9:BMP15 heterodimers are the most bioactive ligands in mice and humans compared with homodimers explain many puzzling genetic and physiological data generated during the last two decades and have important implications for improving female fertility in mammals.

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.

Meiosis arrest female 1 (MARF1) has nuage-like function in mammalian oocytes.

Orderly regulation of meiosis and protection of germline genomic integrity from transposable elements are essential for male and female gamete development. In the male germline, these processes are ensured by proteins associated with cytoplasmic nuage, but morphologically similar germ granules or nuage have not been identified in mammalian female germ cells. Indeed, many mutations affecting nuage-associated proteins such as PIWI and tudor domain containing proteins 5 and 7 (TDRD5/7) can result in failure of meiosis, up-regulation of retrotransposons, and infertility only in males and not in females. We recently identified MARF1 (meiosis arrest female 1) as a protein essential for controlling meiosis and retrotransposon surveillance in oocytes; and in contrast to PIWI-pathway mutations, Marf1 mutant females are infertile, whereas mutant males are fertile. Here we put forward the hypothesis that MARF1 in mouse oocytes is a functional counterpart of the nuage-associated components of spermatocytes. We describe the developmental pattern of Marf1 expression and its roles in retrotransposon silencing and protection from DNA double-strand breaks. Analysis of MARF1 protein domains compared with PIWI and TDRD5/7 revealed that these functional similarities are reflected in remarkable structural analogies. Thus, functions that in the male germline require protein interactions and cooperative scaffolding are combined in MARF1, allowing a single molecule to execute crucial activities of meiotic regulation and protection of germline genomic integrity.

Luteinizing hormone reduces the activity of the NPR2 guanylyl cyclase in mouse ovarian follicles, contributing to the cyclic GMP decrease that promotes resumption of meiosis in oocytes.

In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 min, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2h, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte.

MARF1 regulates essential oogenic processes in mice.

Development of fertilization-competent oocytes depends on integrated processes controlling meiosis, cytoplasmic development, and maintenance of genomic integrity. We show that meiosis arrest female 1 (MARF1) is required for these processes in mammalian oocytes. Mutations of Marf1 cause female infertility characterized by up-regulation of a cohort of transcripts, increased retrotransposon expression, defective cytoplasmic maturation, and meiotic arrest. Up-regulation of protein phosphatase 2 catalytic subunit (PPP2CB) is key to the meiotic arrest phenotype. Moreover, Iap and Line1 retrotransposon messenger RNAs are also up-regulated, and, concomitantly, DNA double-strand breaks are elevated in mutant oocytes. Therefore MARF1, by suppressing levels of specific transcripts, is an essential regulator of important oogenic processes leading to female fertility and the development of healthy offspring.