Maintenance Of Meiotic Arrest Research Articles

PRC1-mediated epigenetic programming is required to generate the ovarian reserve

The ovarian reserve defines the female reproductive lifespan, which in humans spans decades due to robust maintenance of meiotic arrest in oocytes residing in primordial follicles. Epigenetic reprogramming, including DNA demethylation, accompanies meiotic entry, but the chromatin changes that underpin the generation and preservation of ovarian reserves are poorly defined. We report that the Polycomb Repressive Complex 1 (PRC1) establishes repressive chromatin states in perinatal mouse oocytes that directly suppress the gene expression program of meiotic prophase-I and thereby enable the transition to dictyate arrest. PRC1 dysfuction causes depletion of the ovarian reserve and leads to premature ovarian failure. Our study demonstrates a fundamental role for PRC1-mediated gene silencing in female reproductive lifespan, and reveals a critical window of epigenetic programming required to establish ovarian reserve.

Protein phosphatase 2A is essential to maintain meiotic arrest, and to prevent Ca2+ burst at spawning and eventual parthenogenesis in the larvacean Oikopleura dioica

Unfertilized eggs of most animals are arrested at a certain point in the meiotic cell cycles. Reinitiation of meiosis and the start of embryogenesis are triggered by fertilization. This arrest is essential for preventing parthenogenetic activation and for promoting proper initiation of development by fertilization. In the larvacean Oikopleura dioica, which is a simple model organism for studies of chordate development, the unfertilized egg is arrested at metaphase of meiosis I. We show here that protein phosphatase 2A (PP2A) is essential for maintenance of meiotic arrest after spawning of oocytes. Knockdown (KD) of the maternal PP2A catalytic subunit, which was found in functional screening of maternal factors, caused unfertilized eggs to spontaneously release polar bodies after spawning, and then start pseudo-cleavages without fertilization, namely, parthenogenesis. Parthenogenetic embryos failed to undergo proper mitosis and cytokinesis because of lack of a centrosome, which is to be brought into the egg by a sperm. Activation of the KD oocytes was triggered by possible rise of ambient and intracellular pH upon their release from the gonad into seawater at spawning. Live recording of intracellular calcium level of the KD oocytes indicated that the pH rise caused an aberrant Ca2+ burst, which mimicked the Ca2+ burst that occurs at fertilization. Then, the aberrant Ca2+ burst triggered meiosis resumption through Calcium/calmodulin-dependent protein kinase (CaMK II). Therefore, PP2A is essential for maintenance of meiotic arrest and prevention of parthenogenesis by suppressing the aberrant Ca2+ burst at spawning.

Expression of nitric oxide synthase (NOS) in Anabas testudineus ovary and participation of nitric oxide-cyclic GMP cascade in maintenance of meiotic arrest

Participation of cyclic nucleotide-mediated signaling in nitric oxide/soluble guanylate cyclase (NO/sGC) regulation of oocyte maturation (OM) in perch (Anabas testudineus) follicle-enclosed oocytes has been investigated. Congruent with sharp decline in follicular cyclic GMP (cGMP) level, nitric oxide synthase (NOS)-inhibitor (L-NAME) attenuates protein kinase A (PKA) phosphorylation but promotes p-ERK1/2 and p-p34Cdc2 (Thr-161) in maturing oocytes. Conversely, NO donor (SNP) prevents OM, potentially through elevated cGMP synthesis. Expression and localization of Nos2 and Nos3 immunoreactivity in perch ovary varied considerably at progressively higher stages of folliculogenesis. While sGC inhibitor (ODQ) alone could induce OM, 8-bromo-cGMP attenuates 17,20β-P-induced OM indicating functional significance of NO/sGC/cGMP in perch ovary. Interestingly, high NO/cGMP inhibition of OM shows positive relation with elevated cAMP level. MIS induced OM is more susceptible to the oocyte-specific phosphodiesterase (PDE) 3 than PDE4 inhibition. Collectively, high NO/cGMP attenuation of OM potentially involves PDE3 inhibition, cAMP accumulation and PKA activation.

SNAP23 is required for constitutive and regulated exocytosis in mouse oocytes†.

Mammalian oocytes are stored in the ovary for prolonged periods, and arrested in meiotic prophase. During this period, their plasma membranes are constantly being recycled by endocytosis and exocytosis. However, the function of this membrane turnover is unknown. Here, we investigated the requirement for exocytosis in the maintenance of meiotic arrest. Using Trim-away, a newly developed method for rapidly and specifically depleting proteins in oocytes, we have identified the SNARE protein, SNAP23, to be required for meiotic arrest. Degradation of SNAP23 causes premature meiotic resumption in follicle-enclosed oocytes. The reduction in SNAP23 is associated with loss of gap junction communication between the oocyte and surrounding follicle cells. Reduction of SNAP23 protein also inhibits regulated exocytosis in response to a Ca2+ stimulus (cortical granule exocytosis), as measured by lectin staining and cleavage of ZP2. Our results show an essential role for SNAP23 in two key processes that occur in mouse oocytes and eggs.

Valosin-containing protein is associated with maintenance of meiotic arrest in mouse oocytes†.

Valosin-containing protein (VCP) is a member of the highly conserved AAA (ATPase associated with a variety of cellular activities) superfamily. A previous study has shown that targeted deletion of Vcp in mice results in early embryonic lethality. The aim of the present study was to analyze the expression and localization of VCP and its function in meiotic arrest of mouse oocytes. Vcp mRNA and protein were expressed in multiple mouse tissues. In the ovary, VCP protein was mainly expressed in oocytes and granulosa cells. After ovulation and fertilization, Vcp mRNA and protein were detected in oocytes and preimplantation embryos. Furthermore, VCP protein was localized in both the cytoplasm and nucleus of germinal vesicle (GV)-stage oocytes and preimplantation embryos. Moreover, knockdown of Vcp in GV-stage oocytes led to a significantly increased rate of germinal vesicle breakdown (GVBD). In addition, inhibition of VCP protein improved the GVBD rate in mouse GV-stage oocytes. When VCP inhibition was reversed, the final GVBD rate returned to normal. These results provide the first evidence for a novel function of VCP in meiotic arrest of mouse oocytes.

Recent Advances in Understanding the Regulation of Oogenesis and Its Recapitulation in vitro: Mouse and Bovine Models

Abstract: The regulation of mammalian oogenesis in vivo is complicated because of numerous constantly changing events caused by ovarian cells interacting with or influencing each other. One of the most intractable questions for nearly the last 80 years has been the mechanism controlling the maintenance of meiotic arrest and the resumption of oocyte meiosis in a pre-ovulatory follicle. The question is now mostly resolved, as the regulatory mechanisms of cGMP, cAMP, and the NPPC/NPR2 system in the follicle, have recently been uncovered. Oocyte growth in vitro has also been the subject of extensive research utilizing growing oocytes at various stages in several species, including mice, cattle, pig, sheep, goat, and horse. Remarkably, the first reconstitution of the entire process of mammalian oogenesis in vitro from primordial germ cells (PGCs) was recently achieved in mice. Furthermore, even PGC-like cells, originally produced from mouse embryonic stem cells and induced pluripotent stem cells, can develop i...

Role of Cyclic Nucleotide Phosphodiesterases During Meiotic Resumption From Diplotene Arrest in Mammalian Oocytes.

Cyclic nucleotide phosphodiesterases (PDEs) are group of enzymes that hydrolyze cyclic nucleotides in wide variety of cell types including encircling granulosa cells as well as associated oocytes. One group of PDEs are located in encircling granulosa cells and another group get expressed in the oocyte, while few other PDEs are expressed in both compartments. The PDE1A, PDE4D, PDE5A, PDE8A, and PDE8B are granulosa cell specific PDEs that hydrolyze adenosine 3',5'-cyclic monophosphate (cAMP) as well as guanosine 3',5'-cyclic monophosphate (cGMP) with different affinities. PDE3A, PDE8A as well as PDE9A are expressed in oocyte and specifically responsible for the cyclic nucleotide hydrolysis in the oocyte itself. Few other PDEs such as PDE7B, PDE10A, and PDE11A are either detected in granulosa cells or oocytes. Activation of these PDEs either in encircling granulosa cells or in oocyte directly or indirectly reduces intraoocyte cAMP level. Reduction of intraoocyte cAMP level modulates phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and triggers cyclin B1 degradation that destabilizes maturation promoting factor (MPF) and/or increases Cdk1 activity. The destabilized MPF and/or increased Cdk1 activity leads to resumption of meiosis, which initiates the achievement of meiotic competency in preovulatory follicles of several mammalian species. Use of specific PDEs inhibitors block cyclic nucleotides hydrolysis that results in increase of intraoocyte cyclic nucleotides level, which leads to maintenance of meiotic arrest at diplotene stage in vivo as well as in vitro. Thus, cyclic nucleotide PDEs play important role in the achievement of meiotic competency by reducing intraoocyte cyclic nucleotides level in mammalian oocytes. J. Cell. Biochem. 118: 446-452, 2017. © 2016 Wiley Periodicals, Inc.

Immature Oocytes from Unprimed Juvenile Mice Become a Valuable Source for Embryo Production When Using C-Type Natriuretic Peptide as Essential Component of Culture Medium.

C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor 2 (NPR2) play a paramount role in the maintenance of oocyte meiotic arrest in antral follicles via the regulation of the intra-oocyte levels of cyclic guanosine monophosphate and cyclic adenosine monophosphate. We investigated the potential of CNP 1) to maintain oocyte meiotic arrest during a prolonged prematuration culture and 2) to sustain acquisition of developmental competence of immature cumulus-oocyte complexes (COCs). Compact COCs were collected from small antral follicles of prepubertal unprimed mice and placed in prematuration culture under different CNP-supplemented media conditions. A preliminary analysis showed a dose-dependent effect of CNP on the maintenance of meiotic arrest. A dose of 25 nM maintained oocytes under meiotic arrest for 24 h, and this period was extended to 48 h in the presence of estradiol. Analysis of transzonal projections of COCs cultured with CNP indicated that oocyte-cumulus connections were well preserved after the prolonged prematuration culture. Furthermore, CNP medium supplemented with FSH and GDF9 promoted oocyte growth and induced a shift in oocyte chromatin configuration from a predominantly dispersed to a condensed configuration. Following in vitro maturation, oocytes cultured under CNP were capable of extruding the first polar body at a high rate (around 80%). Blastocyst formation was significantly improved when oocytes were cultured under CNP-supplemented medium containing FSH and GDF9. This study reports for the first time a prolonged prematuration culture system, with CNP as the pivotal factor, that can efficiently maintain oocytes retrieved from unprimed prepubertal mice under meiotic arrest while promoting their acquisition of developmental competence.

Role of Pgrmc1 in estrogen maintenance of meiotic arrest in zebrafish oocytes through Gper/Egfr.

The regulation of receptor trafficking to the cell surface and its effect on responses of target cells to growth factors and hormones remain poorly understood. Initial evidence has been recently obtained using cancer cells that surface expression of the epidermal growth factor receptor (EGFR) is dependent on its association with progesterone receptor membrane component 1 (PGRMC1). Estrogen inhibition of oocyte maturation (OM) in zebrafish is mediated through G-protein-coupled estrogen membrane receptor 1 (Gper1) and involves activation of Egfr. Therefore, in this study, the potential roles of Pgrmc1 in the cell surface expression and functions of Egfr in normal cells were investigated in this in vitro OM model of Egfr action using an inhibitor of PGMRC1 signaling, AG205. A single ∼60 kDa protein band, which corresponds to the size of the Pgrmc1 dimer, was detected on plasma membranes of fully grown oocytes by western blotting. Co-treatment with the PGRMC1 inhibitor AG205 (20 μM) blocked the inhibitory effects of 100 nM estradiol-17β and the GPER agonist, G-1, on spontaneous maturation of denuded zebrafish oocytes. Moreover, reversal of these estrogen effects on OM by the EGFR inhibitors AG1478 and AG825 (50 μM) was prevented by co-incubation with the PGRMC1 inhibitor. Inhibition of Pgrmc1 signaling with AG205 also caused a decrease in Egfr-dependent signaling and Egfr expression on oocyte cell membranes. These results indicate that maintenance of Pgrmc1 signaling is required for Egfr expression on zebrafish oocyte cell membranes and for conserving the functions of Egfr in maintaining meiotic arrest through estrogen activation of Gper.

Differential regulation of kit ligand A (kitlga) expression in the zebrafish ovarian follicle cells – Evidence for the existence of a cyclic adenosine 3′, 5′ monophosphate-mediated binary regulatory system during folliculogenesis

Kit ligand (Kitl) is an important paracrine factor involved in the activation of primordial follicles from the quiescent pool and in the maintenance of meiotic arrest before germinal vesicle breakdown (GVBD). It has been reported that follicle-stimulating hormone (FSH) stimulates but luteinizing hormone (LH) suppresses the expression of Kitl in the granulosa cells in mammals. Considering that both gonadotropins signal in the follicle cells mainly by activating cyclic adenosine 3′, 5′-monophosphate (cAMP) pathway, we are intrigued by how cAMP differentially regulates Kitl expression. In the present study, we demonstrated that both human chorionic gonadotropin (hCG) and pituitary adenylate cyclase activating polypeptide (PACAP) inhibited insulin-like growth factor I (IGF-I)-induced Akt phosphorylation and kitlga expression in the zebrafish follicle cells. Further experiments showed that cAMP was involved in regulating the expression of kitlga. However, two cAMP-activated effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), had converse effects. PKA promoted whereas Epac inhibited the expression of kitlga, as demonstrated by the respective activators. Interestingly, cAMP also appeared to exert differential effects on kitlga expression at different stages of follicle development during folliculogenesis, significantly stimulating kitlga expression at the early growth stage but suppressing it at the full-grown stage before final oocyte maturation, implying a potential mechanism for differential effects of the same pathway at different stages. The inhibitory effect of forskolin (activator of adenylate cyclase) and H89 (inhibitor of PKA) on IGF-I-induced expression of kitlga suggested cross-talk between the cAMP and IGF-I-activated PI3K-Akt pathways. This study, together with our previous findings on IGF-I regulation of kitlga expression, provides important clues to the underlying mechanism that regulates Kit ligand expression during folliculogenesis in the ovary.

Cytoplasmic Anchoring of cAMP-Dependent Protein Kinase (PKA) by A-Kinase Anchor Proteins (AKAPs) Is Required for Meiotic Arrest of Porcine Full-Grown and Growing Oocytes1

Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. We previously hypothesized that the meiotic incompetence of porcine GOs was attributed to complex spatial-temporal regulation of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs), but found that AKAP1 is not involved in the meiotic incompetence of porcine GOs. In the present study, we cloned porcine cDNAs of AKAP5 and AKAP7alpha, and found that inhibiting the expression of these AKAPs induced PKA translocation into the nucleus and promoted meiotic resumption of porcine GOs without affecting the total PKA activity of GOs, whereas overexpressing these AKAPs had no effect. Because AKAPs regulate PKA localization through binding with regulatory subunits of PKA (PKA-Rs), PKA-R binding with AKAPs was inhibited by AKAP-binding inhibition peptides or PKA-R expression inhibition by antisense RNAs. We found that the expression inhibition and binding inhibition of PRKAR1A, an isoform of mammalian PKA-R, promoted meiotic resumption of porcine GOs, whereas these inhibitions of PRKAR2A, another PKA-R isoform, had no effect. In contrast, the expression inhibition and binding inhibition of PRKAR2A had higher effects than those of PRKAR1A on meiotic resumption of porcine full-grown oocytes. These results suggest that cytoplasmic anchoring of PKA by AKAPs is required for meiotic arrest of oocytes and that the PKA-R isoform working for the maintenance of meiotic arrest changed from PRKAR1A to PRKAR2A during the acquisition of meiotic competence.

Differential Regulation of Kit Ligand A Expression in the Ovary by IGF-I via Different Pathways

Kit ligand (KITL) plays indispensable roles both in primordial follicle activation and in the maintenance of meiotic arrest of the oocyte. The regulation of KITL expression in the ovary, however, remains largely unknown. In the zebrafish, there are 2 paralogues of KITL, kitlga and kitlgb, and 2 Kit receptors, kita and kitb. Consistent with the situation in mammals, kitlga is only expressed in the ovarian follicle cells, and its cognate receptor kita is expressed in the oocyte. In the present study, we demonstrated that the expression of kitlga was promoted by IGF-I through its receptor IGF-IR. The stimulation involved transcription but not translation, suggesting that the kitlga gene is likely a direct downstream target of IGF-I signaling. Further experiments showed that the stimulatory effect of IGF-I was mediated by phosphatidyl inositol 3-kinase (PI3K)-Akt pathway. IGF-I also activated MEK-ERK pathway; however, this pathway suppressed kitlga expression. The regulation of kitlga expression by IGF-I appeared to depend on the stage of follicle development with a greater induction at early stage than late stage. This may be related to changes in IGF-I signaling pathways and/or local paracrine environment. In support of this were the differential expression of IGF-I receptors (igf1ra and igf1rb) and responsiveness of IGF-I signaling pathways, especially the PI3K-Akt pathway. Furthermore, the IGF-I-induced kitlga expression was inhibited by epidermal growth factor, an oocyte-derived paracrine factor in the zebrafish follicle. This study provides evidence for a controlling mechanism underlying the regulation of KITL expression in the ovary.

Follicular cells of the amphibian ovary: Origin, structure, and functions

Formation of the follicular envelopes surrounding oocytes in the developing ovary and their subsequent morphological differentiation go hand-in-hand with succession of the steroidogenesis stages, arrest of meiosis and its maintenance, establishment of the conditions necessary for vitellogenesis, oocyte growth, and maturation. Metabolites are exchanged via gap junctions and receptor-mediated transport through the perioocytic space. The ion transport in follicular cells (FCs) regulates the plasma membrane potential, creating the conditions for efficient directed transport through gap junctions. Manifold biologically active substances accepted by follicular cells are an additional adjusting lever for regulating the state of follicle system. In this review, we have attempted to emphasize the amphibian FCs as key players in the follicle system; the more so as we have failed to find any review that would bring together the data on the origin of amphibian FCs, their morphology, as well as regulation of oocyte growth and development. As a rule, recent works in this field focus on the molecular mechanisms providing for regulation of individual stages in oocyte development. This review describes the origin and changes in the morphology of follicular cells during the development of Xenopus laevis oocyte as well as the data on their regulatory functions in vitellogenesis and their involvement in steroidogenesis, maintenance of meiotic arrest, and subsequent maturation.

The Role of 14-3-3ε Interaction with Phosphorylated Cdc25B at Its Ser321 in the Release of the Mouse Oocyte from Prophase I Arrest

The protein kinase A (PKA)/Cdc25B pathway plays a critical role in maintaining meiotic arrest in mouse oocytes. However, the molecular mechanism underlying this interchange is not known. In this study, we assessed the role of 14-3-3ε interaction with phosphorylated Cdc25B at its Ser321 as the mouse oocyte is released from prophase I arrest. The 14-3-3ε isoform is a highly conserved protein with various regulatory roles, including maintenance of meiotic arrest. Cdc25B phosphatase is also a key cell cycle regulator. 14-3-3ε binds to Cdc25B-WT, which was abrogated when Ser321 of Cdc25B was mutated to Ala. In addition, we found that 14-3-3ε and Cdc25B were co-localized. Cdc25B was translocated from the cytoplasm to the nucleus shortly before germinal vesicle breakdown (GVBD) during the primary oocyte stage of oogenesis. However, mutation of Ser321 to Ala completely abolished the cytoplasmic localization of Cdc25B. Furthermore, oocytes co-expressing of Cdc25B-WT or Cdc25B-Ser321D and 14-3-3ε were unable to undergo GVBD. In contrast, co-expression of 14-3-3ε and Cdc25B-Ser321A induced GVBD and allowed the process to continue. Down-regulation of 14-3-3ε caused partial meiotic resumption. Taken together, these data indicate that Ser321 of Cdc25B is the specific binding site for 14-3-3ε binding, and that 14-3-3ε is the significant factor in Cdc25B regulation during meiotic resumption of GV stage.

Molecular control of oocyte meiotic arrest and resumption

Mammalian oocytes within Graafian follicles are arrested at prophase I by factors from surrounding follicle cells, and resume meiosis after an LH surge from the pituitary. The maintenance of meiotic arrest requires high levels of cAMP, resulting from G-protein-coupled receptor (GPR) 3 and/or GPR12 activation of adenylyl cyclase within the oocyte. Recent studies indicate that natriuretic peptide precursor C (NPPC), acting via its cognate receptor NPR2, increases cGMP levels in granulosa cells; the cGMP then diffuses into oocytes and inhibits phosphodiesterase 3A activity and cAMP hydrolysis. Meiotic resumption is induced by LH via the generation of epidermal growth factor (EGF)-like growth factors in mural granulosa cells that activate EGF receptors in cumulus cells. However, the exact mechanisms underlying the actions of these growth factors on oocyte maturation are unclear. Herein we summarise the regulatory functions of NPPC and NPR2 in maintaining oocyte meiotic arrest and discuss the possibility that LH could stimulate meiotic resumption by decreasing NPPC content and NPR2 activity.

The G Protein Coupled Receptor 3 Is Involved in cAMP and cGMP Signaling and Maintenance of Meiotic Arrest in Porcine Oocytes

The arrest of meiotic prophase in mammalian oocytes within fully grown follicles is dependent on cyclic adenosine monophosphate (cAMP) regulation. A large part of cAMP is produced by the Gs-linked G-protein-coupled receptor (GPR) pathway. In the present study, we examined whether GPR3 is involved in the maintenance of meiotic arrest in porcine oocytes. Expression and distribution of GPR3 were examined by western blot and immunofluorescence microscopy, respectively. The results showed that GPR3 was expressed at various stages during porcine oocyte maturation. At the germinal vesicle (GV) stage, GPR3 displayed a maximal expression level, and its expression remained stable from pro-metaphase I (MI) to metaphase II (MII). Immunofluorescence staining showed that GPR3 was mainly distributed at the nuclear envelope during the GV stage and localized to the plasma membrane at pro-MI, MI and MII stages. RNA interference (RNAi) was used to knock down the GPR3 expression within oocytes. Injection of small interfering double-stranded RNA (siRNA) targeting GPR3 stimulated meiotic resumption of oocytes. On the other hand, overexpression of GPR3 inhibited meiotic maturation of porcine oocytes, which was caused by increase of cGMP and cAMP levels and inhibition of cyclin B accumulation. Furthermore, incubation of porcine oocytes with the GPR3 ligand sphingosylphosphorylcholine (SPC) inhibited oocyte maturation. We propose that GPR3 is required for maintenance of meiotic arrest in porcine oocytes through pathways involved in the regulation of cAMP and cGMP.

Involvement of GPR12 in the regulation of cell proliferation and survival

GPR12, a member of the orphan G-protein-coupled receptor family, constitutively activates the Gs protein and increases intracellular cyclic AMP concentrations. GPR12 can be activated by its known ligand-sphingosylphosphorylcholine, which regulates cellular physiological activities, including proliferation, neurite extension, cell clustering, and maintenance of meiotic arrest. However, signaling pathways involved in the GPR12-mediated physiological and biochemical changes are still not clearly illustrated. In the present study, heterologous GPR12 expression was demonstrated to promote proliferation and survival in human embryonic kidney 293 cells. Immunochemical analysis showed that Ki67, a prototypic cell cycle-related nuclear protein, might participate in the regulation of GPR12-mediated cell proliferation. Activation of extracellular signal-regulated protein kinase signaling and increased total Erk1/2 and B-cell lymphoma/leukemia-2 expression were also observed in HEK293 cells overexpressing human GPR12. In addition, we found that GPR12 promoted cell survival under serum deprivation, indicating that GPR12 may play a role in cell proliferation and survival.

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.

Cows are not mice: The role of cyclic AMP, phosphodiesterases, and adenosine monophosphate‐activated protein kinase in the maintenance of meiotic arrest in bovine oocytes

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures.

Endocytosis in the mouse oocyte and its contribution to cAMP signaling during meiotic arrest

Mammalian oocytes are arrested at prophase I of meiosis until a preovulatory surge of LH stimulates them to resume meiosis. Prior to the LH surge, high levels of cAMP within the oocyte maintain meiotic arrest; this cAMP is generated in the oocyte through the activity of the constitutively active, G(s)-coupled receptor, G-protein-coupled receptor 3 (GPR3) or GPR12. Activated GPRs are typically targeted for desensitization through receptor-mediated endocytosis, but a continuously high level of cAMP is needed for meiotic arrest. The aim of this study was to examine whether receptor-mediated endocytosis occurs in the mouse oocyte and whether this could affect the maintenance of meiotic arrest. We found that constitutive endocytosis occurs in the mouse oocyte. Inhibitors of receptor-mediated endocytosis, monodansylcadaverine and dynasore, inhibited the formation of early endosomes and completely inhibited spontaneous meiotic resumption. A red fluorescent protein-tagged GPR3 localized in the plasma membrane and within early endosomes in the oocyte, demonstrating that GPR3 is endocytosed. However, overexpression of G-protein receptor kinase 2 and β-arrestin-2 had only a modest effect on stimulating meiotic resumption, suggesting that these proteins do not play a major role in GPR3 endocytosis. Inhibition of endocytosis elevated cAMP levels within oocytes, suggesting that there is an accumulation of GPR3 at the plasma membrane. These results show that endocytosis occurs in the oocyte, leading to a decrease in cAMP production, and suggest that there is a balance between cAMP production and degradation in the arrested oocyte that maintains cAMP levels at an appropriate level during the maintenance of meiotic arrest.