LSM14B is an Oocyte-Specific RNA-Binding Protein Indispensable for Maternal mRNA Metabolism and Oocyte Development in Mice.

Mammalian oogenesis features reliance on the mRNAs produced and stored during early growth phase. These are essential for producing an oocyte competent to undergo meiotic maturation and embryogenesis later when oocytes are transcriptionally silent. The fate of maternal mRNAs hence ensures the success of oogenesis and the quality of the resulting eggs. Nevertheless, how the fate of maternal mRNAs is determined remains largely elusive. RNA-binding proteins (RBPs) are crucial regulators of oogenesis, yet the identity of the full complement of RBPs expressed in oocytes is unknown. Here, a global view of oocyte-expressed RBPs is presented: mRNA-interactome capture identifies 1396 RBPs in mouse oocytes. An analysis of one of these RBPs, LSM family member 14 (LSM14B), demonstrates that this RBP is specific to oocytes and associated with many networks essential for oogenesis. Deletion of Lsm14b results in female-specific infertility and a phenotype characterized by oocytes incompetent to complete meiosis and early embryogenesis. LSM14B serves as an interaction hub for proteins and mRNAs throughout oocyte development and regulates translation of a subset of its bound mRNAs. Therefore, RNP complexes tethered by LSM14B are found exclusively in oocytes and are essential for the control of maternal mRNA fate and oocyte development.

The oocyte cumulus complex regulates mouse sperm migration in the oviduct.

As the time of ovulation draws near, mouse spermatozoa move out of the isthmic reservoir, which is a prerequisite for fertilization. However, the molecular mechanism remains unclear. The present study revealed that mouse cumulus cells of oocytes-cumulus complexes (OCCs) expressed transforming growth factor-ß ligand 1 (TGFB1), whereas ampullary epithelial cells expressed the TGF-ß receptors, TGFBR1 and TGFBR2, and all were upregulated by luteinizing hormone (LH)/human chorionic gonadotropin (hCG). OCCs and TGFB1 increased natriuretic peptide type C (NPPC) expression in cultured ampullae via TGF-ß signaling, and NPPC treatment promoted spermatozoa moving out of the isthmic reservoir of the preovulatory oviducts. Deletion of Tgfb1 in cumulus cells and Tgfbr2 in ampullary epithelial cells blocked OCC-induced NPPC expression and spermatozoa moving out of the isthmic reservoir, resulting in compromised fertilization and fertility. Oocyte-derived paracrine factors were required for promoting cumulus cell expression of TGFB1. Therefore, oocyte-dependent and cumulus cell-derived TGFB1 promotes the expression of NPPC in oviductal ampulla, which is critical for sperm migration in the oviduct and subsequent fertilization.

MTOR-mediated interaction between the oocyte and granulosa cells regulates the development and function of both compartments in mice†.

Coordinated development of the germline and the somatic compartments within a follicle is an essential prerequisite for creating a functionally normal oocyte. Bi-directional communication between the oocyte and the granulosa cells enables the frequent interchange of metabolites and signals that support the development and functions of both compartments. Mechanistic target of rapamycin (MTOR), a conserved serine/threonine kinase and a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation, is emerging as a major player that regulates many facets of oocyte and follicle development. Here, we summarized our recent observations on the role of oocyte- and granulosa cell-expressed MTOR in the control of the oocyte's and granulosa cell's own development, as well as the development of one another, and provided new data that further strengthen the role of cumulus cell-expressed MTOR in synchronizing oocyte and follicle development. Inhibition of MTOR induced oocyte meiotic resumption in cultured large antral follicles, as well as cumulus expansion and the expression of cumulus expansion-related transcripts in cumulus-oocyte complexes in vitro. In vivo, the activity of MTOR in cumulus cells was diminished remarkably by 4 h after hCG administration. These results thus suggest that activation of MTOR in cumulus cells contributes to the maintenance of oocyte meiotic arrest before the LH surge. Based on the observations made by us here and previously, we propose that MTOR is an essential mediator of the bi-directional communication between the oocyte and granulosa cells that regulates the development and function of both compartments.

The piRNA pathway is essential for generating functional oocytes in golden hamsters.

Piwi-interacting RNAs (piRNAs) are predominantly expressed in germ cells and function in gametogenesis in various species. However, Piwi-deficient female mice are fertile and mouse oocytes express a panel of small RNAs that do not appear to be widely representative of mammals. Thus, the function of piRNAs in mammalian oogenesis remains largely unclear. Here, we generated Piwil1- and Mov10l1-deficient golden hamsters and found that all female and male mutants were sterile, with severe defects in embryogenesis and spermatogenesis, respectively. In Piwil1-deficient female hamsters, the oocytes and embryos displayed aberrant transposon accumulation and extensive transcriptomic dysregulation, and the embryos were arrested at the two-cell stage with impaired zygotic genome activation. Moreover, PIWIL1-piRNAs exert a non-redundant function in silencing endogenous retroviruses in the oocytes and embryos. Together, our findings demonstrate that piRNAs are indispensable for generating functional germ cells in golden hamsters and show the value of this model species for piRNA studies in gametogenesis, especially those related to female infertility.

mTOR Inhibition Promotes Pneumonitis through Inducing Endothelial Contraction and Hyperpermeability.

Compromised endothelial-cell (EC) barrier function is a hallmark of inflammatory diseases. mTOR inhibitors, widely applied as clinical therapies, cause pneumonitis through mechanisms that are not yet fully understood. This study aimed to elucidate the EC mechanisms underlying the pathogenesis of pneumonitis caused by mTOR inhibition (mTORi). Mice with EC-specific deletion of mTOR complex components (Mtor, Rptor or Rictor) were administered LPS to induce pulmonary injury. Cultured ECs were treated with pharmacologic inhibitors, siRNA, or overexpression plasmids. EC barrier function was evaluated in vivo with Evans blue assay and in vitro by measurement of transendothelial electrical resistance and albumin flux. mTORi increased basal and TNFα-induced EC permeability, which was caused by myosin light chain (MLC) phosphorylation-dependent cell contraction. Inactivation of mTOR kinase activity by mTORi triggered PKCδ/p38/NF-κB signaling that significantly upregulated TNFα-induced MLCK (MLC kinase) expression, whereas Raptor promoted the phosphorylation of PKCα/MYPT1 independently of its interaction with mTOR, leading to suppression of MLCP (MLC phosphatase) activity. EC-specific deficiency in mTOR, Raptor or Rictor aggravated lung inflammation in LPS-treated mice. These findings reveal that mTORi induces PKC-dependent endothelial MLC phosphorylation, contraction, and hyperpermeability that promote pneumonitis.

Echinoderm Microtubule Associated Protein Like 1 Is Indispensable for Oocyte Spindle Assembly and Meiotic Progression in Mice.

Completion of the first meiosis is an essential prerequisite for producing a functionally normal egg for fertilization and embryogenesis, but the precise mechanisms governing oocyte meiotic progression remains largely unclear. Here, we report that echinoderm microtubule associated protein (EMAP) like 1 (EML1), a member of the conserved EMAP family proteins, plays a crucial role in the control of oocyte meiotic progression in the mouse. Female mice carrying an ENU-induced nonsense mutation (c.1956T > A; p.Tyr652∗) of Eml1 are infertile, and the majority of their ovulated oocytes contain abnormal spindles and misaligned chromosomes. In accordance with the mutant oocyte phenotype, we find that EML1 is colocalized with spindle microtubules during the process of normal oocyte meiotic maturation, and knockdown (KD) of EML1 by specific morpholinos in the fully grown oocytes (FGOs) disrupts the integrity of spindles, and delays meiotic progression. Moreover, EML1-KD oocytes fail to progress to metaphase II (MII) stage after extrusion of the first polar body, but enter into interphase and form a pronucleus containing decondensed chromatins. Further analysis shows that EML1-KD impairs the recruitment of γ-tubulin and pericentrin to the spindle poles, as well as the attachment of kinetochores to microtubules and the proper inactivation of spindle assembly checkpoint at metaphase I (MI). The loss of EML1 also compromises the activation of maturation promoting factor around the time of oocyte resumption and completion of the first meiosis, which, when corrected by WEE1/2 inhibitor PD166285, efficiently rescues the phenotype of oocyte delay of meiotic resumption and inability of reaching MII. Through IP- mass spectrometry analysis, we identified that EML1 interacts with nuclear distribution gene C (NUDC), a critical mitotic regulator in somatic cells, and EML1-KD disrupts the specific localization of NUDC at oocyte spindles. Taken together, these data suggest that EML1 regulates acentrosomal spindle formation and the progression of meiosis to MII in mammalian oocytes, which is likely mediated by distinct mechanisms.

Coordinated Formation of IMPDH2 Cytoophidium in Mouse Oocytes and Granulosa Cells.

Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme catalyzing de novo biosynthesis of guanine nucleotides, aggregates under certain circumstances into a type of non-membranous filamentous macrostructure termed "cytoophidium" or "rod and ring" in several types of cells. However, the biological significance and underlying mechanism of IMPDH assembling into cytoophidium remain elusive. In mouse ovaries, IMPDH is reported to be crucial for the maintenance of oocyte-follicle developmental synchrony by providing GTP substrate for granulosa cell natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system to produce cGMP for sustaining oocyte meiotic arrest. Oocytes and the associated somatic cells in the ovary hence render an exciting model system for exploring the functional significance of formation of IMPDH cytoophidium within the cell. We report here that IMPDH2 cytoophidium forms in vivo in the growing oocytes naturally and in vitro in the cumulus-enclosed oocytes treated with IMPDH inhibitor mycophenolic acid (MPA). Inhibition of IMPDH activity in oocytes and preimplantation embryos compromises oocyte meiotic and developmental competences and the development of embryos beyond the 4-cell stage, respectively. IMPDH cytoopidium also forms in vivo in the granulosa cells of the preovulatory follicles after the surge of luteinizing hormone (LH), which coincides with the resumption of oocyte meiosis and the reduction of IMPDH2 protein expression. In cultured COCs, MPA-treatment causes the simultaneous formation of IMPDH cytoopidium in cumulus cells and the resumption of meiosis in oocytes, which is mediated by the MTOR pathway and is prevented by guanosine supplementation. Therefore, our results indicate that cytoophidia do form in the oocytes and granulosa cells at particular stages of development, which may contribute to the oocyte acquisition of meiotic and developmental competences and the induction of meiosis re-initiation by the LH surge, respectively.

DPAGT1-Mediated Protein N-Glycosylation Is Indispensable for Oocyte and Follicle Development in Mice.

Post-translational modification of proteins by N-linked glycosylation is crucial for many life processes. However, the exact contribution of N-glycosylation to mammalian female reproduction remains largely undefined. Here, DPAGT1, the enzyme that catalyzes the first step of protein N-glycosylation, is identified to be indispensable for oocyte development in mice. Dpagt1 missense mutation (c. 497A>G; p. Asp166Gly) causes female subfertility without grossly affecting other functions. Mutant females ovulate fewer eggs owing to defective development of growing follicles. Mutant oocytes have a thin and fragile zona pellucida (ZP) due to the reduction in glycosylation of ZP proteins, and display poor developmental competence after fertilization in vitro. Moreover, completion of the first meiosis is accelerated in mutant oocytes, which is coincident with the elevation of aneuploidy. Mechanistically, transcriptomic analysis reveals the downregulation of a number of transcripts essential for oocyte meiotic progression and preimplantation development (e.g., Pttgt1, Esco2, Orc6, and Npm2) in mutant oocytes, which could account for the defects observed. Furthermore, conditional knockout of Dpagt1 in oocytes recapitulates the phenotypes observed in Dpagt1 mutant females, and causes complete infertility. Taken together, these data indicate that protein N-glycosylation in oocytes is essential for female fertility in mammals by specific control of oocyte development.

Participation of EML6 in the regulation of oocyte meiotic progression in mice.

The generation of a high-quality egg for reproduction requires faithful segregation of chromosome during oocyte meiosis. Here, we report that echinoderm microtubule-associated protein like 6 (EML6) is highly expressed in oocytes, and responsible for accurate segregation of homologous chromosomes in mice. Quantitative real-time RT-PCR and immunohistochemistry analyses revealed that EML6 was predominantly expressed by oocytes in the ovaries. Whole mount immunofluorescent staining showed that EML6 was colocalized with spindle microtubules in oocytes at various stages after meiotic resumption. This specialized localization was disrupted by nocodazole, the microtubule destabilizer, while enhanced by Taxol, a microtubule stabilizing reagent. In vivo knockdown of Eml6 expression by the specific siRNA resulted in chromosome misalignment and alteration in spindle dimension at both metaphase Ⅰ and Ⅱ stages, as well as the increased aneuploidy in the mature oocytes. Thus, these data suggest that EML family proteins participate in the control of oocyte meiotic division.

Insulin Reduces Reaction of Follicular Granulosa Cells to FSH Stimulation in Women With Obesity-Related Infertility During IVF.

CONTEXT: Women with obesity usually need larger doses of FSH for ovarian stimulation, resulting in poor outcomes; however, the mechanism is still unclear. OBJECTIVE: To investigate the molecular regulation of FSH receptor (FSHR) expression associated with obesity. DESIGN: Case-control study to improve in vitro fertilization (IVF) outcomes. PATIENTS: Women with obesity (82) and women who were overweight (457) undergoing IVF and 1790 age-matched controls with normal weight from our reproductive medicine center. MAIN OUTCOME MEASURES: FSHR expression was decreased in parallel with body mass index (BMI), whereas the estradiol (E2) level on the human chorionic gonadotropin (hCG) trigger day was significantly lower. RESULTS: FSHR expression in human granulosa cells (hGCs), both mRNA (P = 0.02) and protein (P = 0.001) levels, was decreased in women who were overweight or obese. Both insulin (P < 0.001) and glucose (P = 0.0017) levels were positively correlated with BMI in fasting blood and follicle fluids (FFs) but not with FFs leptin level. We treated human granulosa-like tumor cells (KGN) cells with insulin; E2 production was compromised; the level of phosphorylated (p)-protein kinase B (p-Akt2) decreased, whereas p-glycogen synthase kinase 3 (GSK3) increased; and there were similar changes in hGCs from women with obesity. Stimulated hGCs from women with obesity with compound 21 (CP21), an inhibitor of GSK3ß, resulted in upregulated ß-catenin activation and increased FSHR expression. CP21 also increased the expression of insulin receptor substrate 1 and phosphatidylinositol 3-kinase (PI3K), as well as p-Akt2. CONCLUSIONS: Women with obesity in IVF were associated with reduced FSHR expression and E2 production caused by a dysfunctional insulin pathway. Decreased FSHR expression in hGCs from women with obesity and insulin-treated KGN cells could be rescued by an inhibitor of GSK3ß, which might be a potential target for the improvement of the impaired FSH-stimulation response in women with obesity.

Ribonuclease activity of MARF1 controls oocyte RNA homeostasis and genome integrity in mice.

Producing normal eggs for fertilization and species propagation requires completion of meiosis and protection of the genome from the ravages of retrotransposons. Mutation of Marf1 (meiosis regulator and mRNA stability factor 1) results in defects in both these key processes in mouse oocytes and thus in infertility. MARF1 was predicted to have ribonuclease activity, but the structural basis for the function of MARF1 and the contribution of its putative ribonuclease domain to the mutant oocyte phenotype was unknown. Therefore, we resolved the crystal structures of key domains of MARF1 and demonstrated by biochemical and mutagenic analyses that the ribonuclease activity of MARF1 controls oocyte meiotic progression and retrotransposon surveillance. The N-terminal NYN domain of MARF1 resembles the nuclease domains of Vpa0982, T4 RNase H, and MCPIP1 and contains four conserved aspartate residues, D178, D215, D246, and D272. The C-terminal LOTUS domain of MARF1 adopts a winged helix-turn-helix fold and binds ssRNA and dsRNA. Purified MARF1 cleaved ssRNAs in vitro, but this cleavage activity was abolished by mutations of conserved aspartates in its NYN domain and truncation of the LOTUS domain. Furthermore, a point mutation in the D272 residue in vivo caused a female-only infertile phenotype in mice, with failure of meiotic resumption and elevation of Line1 and Iap retrotransposon transcripts and DNA double-strand breaks in oocytes. Therefore, the ribonuclease activity of MARF1 controls oocyte meiosis and genome integrity. This activity depends upon conserved aspartic residues in the catalytic NYN domain and the RNA-binding activity of the LOTUS domain.

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.

Interference with the C-terminal structure of MARF1 causes defective oocyte meiotic division and female infertility in mice.

Meiosis-arrest female 1 (MARF1) is a recently identified key oogenic regulator essential for the maintenance of female fertility and genome integrity in mice. However, the detailed functions and the underlying mechanisms of MARF1 remain elusive. Here, in an attempt to create a mouse model expressing fluorescent protein-tagged MARF1 to facilitate further exploration of the roles of MARF1 in oocytes, we produced a Marf1-eGFP knockin (KI) mouse line in which the C-terminal structure and function of MARF1 were interfered by its fusing eGFP peptide. Using these Marf1-eGFP-KI mice, we revealed, unexpectedly, the functions of MARF1 in the control of oocyte meiotic division. We found that the Marf1-eGFP-KI females ovulated mature oocytes with severe meiotic and developmental defects, and thus were infertile. Moreover, meiotic reinitiation was delayed while meiotic completion was accelerated in the KI-oocytes, which was coincident with the increased incidence of oocyte aneuploidy. Therefore, MARF1 is indispensable for maintaining the fidelity of homolog segregation during oocyte maturation, and this function relies on its C-terminal domains.

The RNA-binding protein MARF1 promotes cortical neurogenesis through its RNase activity domain.

Cortical neurogenesis is a fundamental process of brain development that is spatiotemporally regulated by both intrinsic and extrinsic cues. Although recent evidence has highlighted the significance of transcription factors in cortical neurogenesis, little is known regarding the role of RNA-binding proteins (RBPs) in the post-transcriptional regulation of cortical neurogenesis. Here, we report that meiosis arrest female 1 (MARF1) is an RBP that is expressed during neuronal differentiation. Cortical neurons expressed the somatic form of MARF1 (sMARF1) but not the oocyte form (oMARF1). sMARF1 was enriched in embryonic brains, and its expression level decreased as brain development progressed. Overexpression of sMARF1 in E12.5 neuronal progenitor cells promoted neuronal differentiation, whereas sMARF1 knockdown decreased neuronal progenitor differentiation in vitro. We also examined the function of sMARF1 in vivo using an in utero electroporation technique. Overexpression of sMARF1 increased neuronal differentiation, whereas knockdown of sMARF1 inhibited differentiation in vivo. Moreover, using an RNase domain deletion mutant of sMARF1, we showed that the RNase domain is required for the effects of sMARF1 on cortical neurogenesis in vitro. Our results further elucidate the mechanisms of post-transcriptional regulation of cortical neurogenesis by RBPs.

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.

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.

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.

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.

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.