Meiotic Arrest In Mouse Oocytes Research Articles

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.

DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint.

Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule–kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis.

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.

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.

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.

Wee1B and Myt1 Function Cooperatively in Maintaining Meiotic Arrest in Mouse Oocytes.

Wee1B and Myt1 Function Cooperatively in Maintaining Meiotic Arrest in Mouse Oocytes.

The Xenopus laevis Isoform of G Protein-Coupled Receptor 3 (GPR3) Is a Constitutively Active Cell Surface Receptor that Participates in Maintaining Meiotic Arrest in X. laevis Oocytes

Oocytes are held in meiotic arrest in prophase I until ovulation, when gonadotropins trigger a subpopulation of oocytes to resume meiosis in a process termed "maturation." Meiotic arrest is maintained through a mechanism whereby constitutive cAMP production exceeds phosphodiesterase-mediated degradation, leading to elevated intracellular cAMP. Studies have implicated a constitutively activated Galpha(s)-coupled receptor, G protein-coupled receptor 3 (GPR3), as one of the molecules responsible for maintaining meiotic arrest in mouse oocytes. Here we characterized the signaling and functional properties of GPR3 using the more amenable model system of Xenopus laevis oocytes. We cloned the X. laevis isoform of GPR3 (XGPR3) from oocytes and showed that overexpressed XGPR3 elevated intraoocyte cAMP, in large part via Gbetagamma signaling. Overexpressed XGPR3 suppressed steroid-triggered kinase activation and maturation of isolated oocytes, as well as gonadotropin-induced maturation of follicle-enclosed oocytes. In contrast, depletion of XGPR3 using antisense oligodeoxynucleotides reduced intracellular cAMP levels and enhanced steroid- and gonadotropin-mediated oocyte maturation. Interestingly, collagenase treatment of Xenopus oocytes cleaved and inactivated cell surface XGPR3, which enhanced steroid-triggered oocyte maturation and activation of MAPK. In addition, human chorionic gonadotropin-treatment of follicle-enclosed oocytes triggered metalloproteinase-mediated cleavage of XGPR3 at the oocyte cell surface. Together, these results suggest that GPR3 moderates the oocyte response to maturation-promoting signals, and that gonadotropin-mediated activation of metalloproteinases may play a partial role in sensitizing oocytes for maturation by inactivating constitutive GPR3 signaling.

Oocyte-specific G-protein–coupled receptor 3 (GPR3): no perturbations found in 82 women with premature ovarian failure (first report)

The oocyte-specific G-protein-coupled receptor 3 (GPR3) gene is essential in maintaining meiotic arrest in mouse oocytes. Disruption of GPR3 results in early depletion of oocytes and thus premature ovarian aging. To determine if mutations of the GPR3 gene were present in 82 predominantly North American caucasian women with premature ovarian failure (POF), we used denaturing high-performance liquid chromatography and DNA sequencing to detect sequence variants. None of the 82 POF samples showed perturbations of significance. We conclude that perturbations in GPR3 are not a common explanation for POF in this population.

A Gs-linked receptor maintains meiotic arrest in mouse oocytes, but luteinizing hormone does not cause meiotic resumption by terminating receptor-Gs signaling

The maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on the activity of a Gs G-protein that activates adenylyl cyclase and elevates cAMP, and in the mouse oocyte, Gs is activated by a constitutively active orphan receptor, GPR3. To determine whether the action of luteinizing hormone (LH) on the mouse ovarian follicle causes meiotic resumption by inhibiting GPR3-Gs signaling, we examined the effect of LH on the localization of Gαs. Gs activation in response to stimulation of an exogenously expressed β2-adrenergic receptor causes Gαs to move from the oocyte plasma membrane into the cytoplasm, whereas Gs inactivation in response to inhibition of the β2-adrenergic receptor causes Gαs to move back to the plasma membrane. However, LH does not cause a change in Gαs localization, indicating that LH does not act by terminating receptor-Gs signaling.

P-703: Human oocytes express Gs G-protein and the G-protein-coupled receptor Gpr3, key components in the maintenance of meiotic arrest in mouse oocytes

Although researchers have begun to elucidate the mechanisms that regulate meiosis in some mammals, relatively little is known about meiotic arrest and resumption in response to LH in the human oocyte. It has previously been shown that the Gs-linked, G-protein-coupled receptor GPR3 plays a role in the maintenance of meiotic arrest in the mouse oocyte by maintaining high intracellular levels of cyclic AMP. Previous studies have shown that oocytes from Gpr3 knockout mice resume meiosis within antral follicles independent of an increase in LH.

Oocyte-specific expression of Gpr3 is required for the maintenance of meiotic arrest in mouse oocytes

The maintenance of meiotic prophase arrest in mouse oocytes within fully grown follicles, prior to the surge of luteinizing hormone (LH) that triggers meiotic resumption, depends on a high level of cAMP within the oocyte. cAMP is produced within the oocyte, at least in large part, by the Gs-linked G-protein-coupled receptor, GPR3. Gpr3 is localized in the mouse oocyte but is also present throughout the follicle. To investigate whether Gpr3 in the follicle cells contributes to the maintenance of meiotic arrest, RNA interference (RNAi) was used to reduce the amount of Gpr3 RNA within follicle-enclosed oocytes. Follicle-enclosed oocytes injected with small interfering double-stranded RNA (siRNA) targeting Gpr3, but not control siRNAs, stimulated the resumption of meiosis in the majority of oocytes following a 3-day culture period. Reduction of RNA was specific for Gpr3 because an unrelated gene was not reduced by microinjection of siRNA. Meiotic resumption was stimulated in isolated oocytes injected with the same siRNA and cultured for 1 to 2 days, but at a much lower rate than in follicle-enclosed oocytes that could be cultured for longer. These results demonstrate that GPR3 specifically in the oocyte, rather than in the follicle cells, is responsible for maintenance of meiotic arrest in mouse oocytes. Furthermore, the method developed here for specifically reducing RNA in follicle-enclosed oocytes, which can be cultured for a sufficient time to reduce the level of endogenous protein, should be generally useful for targeting a wide range of other proteins that may be involved in meiotic arrest, the resumption of meiosis, fertilization, or early embryonic development.

Glutamine and the maintenance of meiotic arrest in mouse oocytes: influence of culture medium, glucose, and cumulus cells.

The selection of culture media and supplements therein has a tremendous impact on the regulation of oocyte maturation in vitro. In the present study, we have evaluated how altering the levels of glutamine in the presence or absence of glucose affects meiotic arrest in cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) when cultured in either the simple medium M16 or the more complex Eagle's minimum essential medium (MEM). We have also tested the effectiveness of follicle-stimulating hormone (FSH) in triggering germinal vesicle breakdown (GVB) and purine de novo synthesis in differing MEM culture conditions. When DO were cultured 17-18 hr in hypoxanthine (HX)- or dbcAMP-supplemented M16 medium, neither glucose nor glutamine had any effect on oocyte maturation, with dbcAMP the more effective inhibitor. In the absence of glutamine, cumulus cells promoted meiotic resumption, since significantly lower levels of meiotic arrest were maintained in CEO than in DO by either HX or dbcAMP, but addition of the amino acid dose-dependently decreased the maturation percentage in CEO below that observed in DO. In MEM, glutamine and glucose again had little effect on the maturation of DO, although the percentage of maturing DO in HX-supplemented medium was about 20% lower than that in M16 medium. In the absence of glucose, high levels of maturation were observed in CEO in glutamine-free medium that were dose-dependently lowered by the amino acid. However, when glucose was present, CEO were as effectively arrested as DO when glutamine was absent, with no further effect of the amino acid. This inhibitory action of glucose was dependent on the essential amino acids present in MEM. The effects of glutamine were not due to changes in metabolic coupling between the oocyte and cumulus cells. Measurement of purine de novo synthesis indicated that the maintenance of meiotic arrest as well as FSH induction of meiotic resumption were associated with increases in purine synthesis. We conclude that glucose and glutamine act cooperatively to promote the synthesis of new purine compounds within the somatic compartment and that the timing and duration of such synthesis determines whether meiotic resumption will be suppressed or promoted.

Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G‐proteins

Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.

Maintenance of meiotic arrest in mouse oocytes by purines: Modulation of cAMP levels and cAMP phosphodiesterase activity

Hypoxanthine and adenosine are present in preparations of mouse ovarian follicular fluid, and these purines maintain mouse oocytes in meiotic arrest in vitro (Eppig et al.: Biology of Reproduction 33:1041-1049. 1985). The first hypothesis tested in this study is that purines which maintain meiotic arrest act by maintaining meiosis-arresting levels of cyclic adenosine monophosphate (cAMP) in the oocyte. Oocyte-cumulus cell complexes were incubated in control medium (no added purines), or medium containing 0.75 mM adenosine, 4 mM hypoxanthine, or both for 3 hr and the percentage of the oocytes that underwent germinal vesicle breakdown (GVB) and the cAMP content of the intact complexes and the oocytes were determined. Adenosine alone had little inhibitory effect on GVB at this time point but sustained higher levels of cAMP in the oocytes. Hypoxanthine maintained 80% of cumulus cell-enclosed oocytes in meiotic arrest and also sustained higher cAMP levels in the oocytes. The addition of adenosine to hypoxanthine-containing medium increased the percentage of oocytes maintained in meiotic arrest, and increased the amount of cAMP in the oocytes above that maintained by either hypoxanthine or adenosine alone. Neither hypoxanthine, adenosine, nor hypoxanthine plus adenosine altered the cAMP content of intact complexes when assayed after 3 hr culture. Microinjection of an inhibitor of the catalytic subunit of cAMP-dependent protein kinase induced GVB in denuded oocytes cultured in medium containing hypoxanthine. This purine, therefore, maintained meiotic arrest by sustaining elevated cAMP levels within the oocytes. The second hypothesis tested in this study is that purines maintain meiosis-arresting levels of cAMP, at least in part, by inhibiting cAMP phosphodiesterase activity. In descending order of potency, 3-isobutyl-1-methylxanthine (IBMX), guanosine, hypoxanthine, adenosine, and xanthosine inhibited cAMP phosphodiesterase in oocyte lysates. Moreover, like the potent phosphodiesterase inhibitor IBMX, hypoxanthine augmented the meiotic arrest and cAMP accumulation mediated by follicle-stimulating hormone (FSH) in intact complexes. Therefore, inhibition of oocyte phosphodiesterase appears to be one mechanism by which the purines could maintain meiosis-arresting levels of cAMP.

Maintenance of murine oocyte meiotic arrest: Uptake and metabolism of hypoxanthine and adenosine by cumulus cell-enclosed and denuded oocytes

To analyze the potential mechanisms by which hypoxanthine and adenosine maintain meiotic arrest in mouse oocytes this study focused on: (1) the uptake and metabolism of hypoxanthine and adenosine; (2) the effect of inhibitors of inosine monophosphate (IMP) dehydrogenase on purine-mediated meiotic arrest; and (3) the role of adenosine metabolism on the maintenance of meiotic arrest. Although the denuded oocyte can take up radiolabeled hypoxanthine and adenosine, an intact cumulus oophorus greatly augments uptake of these molecules (and/or metabolites). Both of these compounds were completely metabolized during incubation in vitro: hypoxanthine was apparently metabolized to uric acid and adenosine was metabolized to ADP; a small amount of each compound was also converted to inosine by cumulus cells and transferred to the oocyte. The IMP dehydrogenase inhibitors, bredinin and mycophenolic acid (MA), induced, in a dose-dependent manner, the resumption of maturation in cumulus cell-enclosed oocytes maintained in meiotic arrest by hypoxanthine but had no effect on denuded oocytes. MA did not induce maturation when meiotic arrest was maintained by guanosine. Nor did MA alter the uptake of hypoxanthine by cumulus cell-enclosed oocytes. The poorly metabolized analog of adenosine, 2-chloroadenosine, was as effective as adenosine in its synergistic action with hypoxanthine in maintaining meiotic arrest. It is concluded that (1) hypoxanthine and adenosine are metabolized within the oocyte-cumulus cell complex; (2) xanthyl and/or guanyl compounds are produced by oocyte-cumulus cell complexes in the presence of hypoxanthine and play an important role in the maintenance of meiotic arrest; and (3) adenosine need not be metabolized to act synergistically with hypoxanthine in maintaining meiotic arrest.

A follicular fluid component prevents gonadotropin reversal of cyclic adenosine monophosphate‐dependent meiotic arrest in murine oocytes

AbstractThe effects of the putative maturation inhibitor in porcine follicular fluid on gonadotropinstimulated reversal of cyclic adenosine monophosphate (cAMP)‐maintained meiotic arrest in mouse oocytes in vitro were assessed in this study. When cumulus cell‐enclosed oocytes were cultured in a suboptimal inhibitory concentration of dibutyryl cAMP (dbcAMP), the effect of follicle‐stimulating hormone (FSH) on oocyte maturation was initially inhibitory at 3 hr, but stimulatory at 6 hr. Supplementation of the medium with an ultrafiltrate of porcine follicuiar fluid (PM10‐filtrate) completely suppressed FSH‐promoted reversal of inhibition at 6 hr. Charcoal extraction eliminated this effect of the PM10‐filtrate. FSH reversed the inhibition of maturation of cumulus cell‐enclosed oocytes maintained by a high concentration of dbcAMP and suboptimal concentrations of the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methyl xanthine (IBMX), during a 21–22‐hr culture period. However, the effect of a completely inhibitory concentration of IBMX was not reversed by gonadotropin. A component of serum was also found to inhibit FSH reversal of dbcAMP‐maintained meiotic arrest, and this activity was removed by charcoal extraction. In addition, when oocytes were cultured in medium containing a suboptimal concentration of dbcAMP plus a low molecular weight fraction (< 1,000) of porcine follicular fluid, porcine serum, or fetal bovine serum, a synergistic inhibition of maturation was observed. Experiments with highly purified gonadotropins revealed that reversal of dbcAMP‐maintained meiotic arrest occurred only in response to FSH; neither highly purified luteinizing hormone nor human chorionic gonadotropin could mimic this action of FSH. Also, this effect was mediated by the cumulus cells, since FSH could not reverse dbcAMP‐maintained meiotic arrest in denuded oocytes. Furthermore, elevating cAMP levels in denuded oocytes augmented, rather than reversed, the inhibitory action of dbcAMP on oocyte maturation. These data therefore suggest that dbcAMP‐ or IBMX‐maintained meiotic arrest in vitro is reversed by an FSH‐stimulated, cAMP‐dependent process mediated by the cumulus cells and demonstrate that a factor present both in follicular fluid and serum prevents this action of the gonadotropin.