Maturation promoting factor destabilization mediates human chorionic gonadotropin induced meiotic resumption in rat oocytes

Human chorionic gonadotropin (hCG) mimics the action of luteinizing hormone (LH) and triggers meiotic maturation and ovulation in mammals. The mechanism by which hCG triggers meiotic resumption in mammalian oocytes remains poorly understood. We aimed to find out the impact of hCG surge on morphological changes, adenosine 3′,5′‐cyclic monophosphate (cAMP), guanosine 3′,5′‐cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), Wee1, early mitotic inhibitor 2 (Emi2), anaphase‐promoting complex/cyclosome (APC/C), meiotic arrest deficient protein 2 (MAD2), phosphorylation status of cyclin‐dependent kinase 1 (Cdk1), its activity and cyclin B1 expression levels during meiotic resumption from diplotene as well as metaphase‐II (M‐II) arrest in cumulus oocyte complexes (COCs). Our data suggest that hCG surge increased cyclic nucleotides level in encircling granulosa cells but decreased their level in oocyte. The reduced intraoocyte cyclic nucleotides level is associated with the decrease of Cdc25B, Thr161 phosphorylated Cdk1 and Emi2 expression levels. On the other hand, hCG surge increased Wee1, Thr14/Tyr15 phosphorylated Cdk1, APC/C as well as MAD2 expression levels. The elevated APC/C activity reduced cyclin B1 level. The changes in phosphorylation status of Cdk1 and reduced cyclin B1 level might have resulted in maturation promoting factor (MPF) destabilization. The destabilized MPF finally triggered resumption of meiosis from diplotene as well as M‐II arrest in rat oocytes.     

Wee1B,Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption

After a long period of quiescence at dictyate prophase I, termed the germinal vesicle (GV) stage, mammalian oocytes reenter meiosis by activating the Cdc2–cyclin B complex (maturation-promoting factor [MPF]). The activity of MPF is regulated by Wee1/Myt1 kinases and Cdc25 phosphatases. In this study, we demonstrate that the sequestration of components that regulate MPF activity in distinct subcellular compartments is essential for their function during meiosis. Down-regulation of either Wee1B or Myt1 causes partial meiotic resumption, and oocytes reenter the cell cycle only when both proteins are down-regulated. Shortly before GV breakdown (GVBD), Cdc25B is translocated from the cytoplasm to the nucleus, whereas Wee1B is exported from the nucleus to the cytoplasm. These movements are regulated by PKA inactivation and MPF activation, respectively. Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest. Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.     

Emi1 Class of Proteins Regulate Entry into Meiosis and the Meiosis I to Meiosis II Transition in Xenopus Oocytes

Xenopus oocytes are arrested at the G2/prophase boundary of meiosis I and enter meiosis in response to progesterone. A hallmark of meiosis is the absence of DNA replication between the successive cell division phases meiosis I (MI) and meiosis II (MII). After the MI-MII transition, Xenopus eggs are locked in metaphase II by the cytostatic factor (CSF) arrest to prevent parthenogenesis. Early Mitotic Inhibitor 1 (Emi1) maintains CSF arrest by inhibiting the ability of the Anaphase Promoting Complex (APC) to direct the destruction of cyclin B. To investigate whether Emi1 has an earlier role in meiosis, we injected Xenopus oocytes with neutralizing antibodies against Emi1 at G2/prophase and during the MI-MII transition. Progesterone-treated G2/prophase oocytes injected with anti-Emi1 antibody fail to activate Maturation Promoting Factor (MPF), a complex of cdc2/cyclin B, and the MAPK pathway, and do not undergo germinal vesicle breakdown (GVBD). Injection of purified ?90 cyclin B protein or blocking anti-Emi1 antibody with purified Emi1 protein rescues these meiotic processes in Emi1-neutralized oocytes. Acute inhibition of Emi1 in progesterone treated oocytes immediately after GVBD causes rapid loss of cdc2 activity with simultaneous loss of cyclin B levels and inactivation of the MAPK pathway. These oocytes decondense their chromosomes and enter a DNA replication phase instead of progressing to MII. Prior ablation of Cdc20, addition of methyl-ubiquitin, or addition of indestructible ?90 cyclin B rescues the MI-MII transition in Emi1 inhibited oocytes.     

Role of Greatwall kinase in release of mouse oocytes from diplotene arrest

In eukaryotes, mitosis entry is induced by activation of maturation‐promoting factor (MPF), which is regulated by a network of kinases and phosphatases. It has been suggested that Greatwall (GWL) kinase was crucial for the M‐phase entry and could maintain cyclin B–Cdc2 activity through regulation of protein phosphatase 2A (PP2A), a counteracting phosphatase of MPF. Here, the role of GWL was assessed during release of mouse oocytes from prophase I arrest. GWL was crucial for meiotic maturation in mouse oocytes. As a positive regulator for meiosis resumption, GWL was continually expressed in germinal vesicle (GV) and MII stage oocytes and two‐cell stage embryos. Additionally, GWL localized to the nucleus and dispersed into cytoplasm during GV breakdown (GVBD). Furthermore, downregulation of GWL or overexpression of catalytically‐inactive GWL inhibited partial meiotic maturation. This prophase I arrest induced by GWL depletion could be rescued by the PP2A inhibition. However, both GWL‐depleted and rescued oocytes had severe spindle defects that hardly reached MII. In contrast, oocytes overexpressing wild‐type GWL resumed meiosis and progressed to MII stage. Thus, our data demonstrate that GWL acts in a pathway with PP2A which is essential for prophase I exit and metaphase I microtubule assembly in mouse oocytes.     

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.     

A critical balance between Cyclin B synthesis and Myt1 activity controls meiosis entry in Xenopus oocytes

In fully grown oocytes, meiosis is arrested at first prophase until species-specific initiation signals trigger maturation. Meiotic resumption universally involves early activation of M phase-promoting factor (Cdc2 kinase-Cyclin B complex, MPF) by dephosphorylation of the inhibitory Thr14/Tyr15 sites of Cdc2. However, underlying mechanisms vary. In Xenopus oocytes, deciphering the intervening chain of events has been hampered by a sensitive amplification loop involving Cdc2-Cyclin B, the inhibitory kinase Myt1 and the activating phosphatase Cdc25. In this study we provide evidence that the critical event in meiotic resumption is a change in the balance between inhibitory Myt1 activity and Cyclin B neosynthesis. First, we show that in fully grown oocytes Myt1 is essential for maintaining prophase I arrest. Second, we demonstrate that, upon upregulation of Cyclin B synthesis in response to progesterone, rapid inactivating phosphorylation of Myt1 occurs, mediated by Cdc2 and without any significant contribution of Mos/MAPK or Plx1. We propose a model in which the appearance of active MPF complexes following increased Cyclin B synthesis causes Myt1 inhibition, upstream of the MPF/Cdc25 amplification loop.     

APCFZR1 prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing

FZR1 is an anaphase-promoting complex (APC) activator best known for its role in the mitotic cell cycle at M-phase exit, in G1, and in maintaining genome integrity. Previous studies also established that it prevents meiotic resumption, equivalent to the G2/M transition. Here we report that mouse oocytes lacking FZR1 undergo passage through meiosis I that is accelerated by ∼1 h, and this is due to an earlier onset of spindle assembly checkpoint (SAC) satisfaction and APC^CDC20 activity. However, loss of FZR1 did not compromise SAC functionality; instead, earlier SAC satisfaction was achieved because the bipolar meiotic spindle was assembled more quickly in the absence of FZR1. This novel regulation of spindle assembly by FZR1 led to premature bivalent attachment to microtubules and loss of kinetochore-bound MAD2. Bivalents, however, were observed to congress poorly, leading to nondisjunction rates of 25%. We conclude that in mouse oocytes FZR1 controls the timing of assembly of the bipolar spindle and in so doing the timing of SAC satisfaction and APC^CDC20 activity. This study implicates FZR1 as a major regulator of prometaphase whose activity helps to prevent chromosome nondisjunction.     

Multiple Requirements of PLK1 during Mouse Oocyte Maturation

Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1’s functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.     

SGK1 is essential for meiotic resumption in mammalian oocytes

In mammalian females, oocytes are stored in the ovary and meiosis is arrested at the diplotene stage of prophase I. When females reach puberty oocytes are selectively recruited in cycles to grow, overcome the meiotic arrest, complete the first meiotic division and become mature (ready for fertilization). At a molecular level, the master regulator of prophase I arrest and meiotic resumption is the maturation-promoting factor (MPF) complex, formed by the active form of cyclin dependent kinase 1 (CDK1) and Cyclin B1. However, we still do not have complete information regarding the factors implicated in MPF activation.In this study we document that out of three mammalian serum-glucocorticoid kinase proteins (SGK1, SGK2, SGK3), mouse oocytes express only SGK1 with a phosphorylated (active) form dominantly localized in the nucleoplasm. Further, suppression of SGK1 activity in oocytes results in decreased CDK1 activation via the phosphatase cell division cycle 25B (CDC25B), consequently delaying or inhibiting nuclear envelope breakdown. Expression of exogenous constitutively active CDK1 can rescue the phenotype induced by SGK1 inhibition. These findings bring new insights into the molecular pathways acting upstream of MPF and a better understanding of meiotic resumption control by presenting a new key player SGK1 in mammalian oocytes.     

Critical effect of pigWee1B on the regulation of meiotic resumption in porcine immature oocytes

Porcine immature oocytes require protein synthesis for meiotic resumption, thus the importance of Cdc2 inhibitory phosphorylation in their meiotic arrest remains controversial. We examined the involvement of Cdc2 phosphorylation in the meiotic arrest of porcine oocytes with a special focus on Wee1B, an oocyte-specific Wee1 family member recently reported in mouse oocytes. We cloned a Wee1B homologue of pig by RT-PCR followed by 5’- and 3’-RACE. Overexpression of pigWee1B in porcine immature oocytes by the injection of pigWee1B mRNA almost completely blocked the germinal vesicle breakdown (GVBD) under the low cAMP concentration, which could not block their spontaneous meiotic resumption by itself. The MPF activation and cyclin B synthesis were inhibited in these oocytes. Conversely, downregulation of pigWee1B expression by the injection of specific antisense mRNA induced GVBD in the oocytes, the spontaneous meiotic resumption of which was blocked by the high concentration of cAMP (dbcAMP). In these oocytes, the MPF activity was elevated and cyclin B was accumulated. Downregulation of pigMyt1, another Wee1 family member, could not induce the GVBD under the same condition. The inhibition of tyrosine phosphatase by vanadate blocked the GVBD even in the pigWee1B-downregulated oocytes. These results suggest that the inhibitory phosphorylation of CDC2, which is catalyzed by pigWee1B, but not pigMyt1, is involved in the meiotic arrest of porcine oocytes, and that the inactivation of Wee1B in combination with the phosphatase activation induces the conversion of pre-MPF to the active MPF and starts the cyclin B synthesis, follwed by a further increase of MPF and meiotic resumption.     

Essential role of protein phosphatase 2A in metaphase II arrest and activation of mouse eggs shown by okadaic acid, dominant negative protein phosphatase 2A, and FTY720

Vertebrate eggs arrest at second meiotic metaphase. The fertilizing sperm causes meiotic exit through Ca(2+)-mediated activation of the anaphase-promoting complex/cyclosome (APC/C). Although the loss in activity of the M-phase kinase CDK1 is known to be an essential downstream event of this process, the contribution of phosphatases to arrest and meiotic resumption is less apparent, especially in mammals. Therefore, we explored the role of protein phosphatase 2A (PP2A) in mouse eggs using pharmacological inhibition and activation as well as a functionally dominant-negative catalytic PP2A subunit (dn-PP2Ac-L199P) coupled with live cell imaging. We observed that PP2A inhibition using okadaic acid induced events normally observed at fertilization: degradation of the APC/C substrates cyclin B1 and securin resulting from loss of the APC/C inhibitor Emi2. Although sister chromatids separated, chromatin remained condensed, and polar body extrusion was blocked as a result of a rapid spindle disruption, which could be ameliorated by non-degradable cyclin B1, suggesting that spindle integrity was affected by CDK1 loss. Similar cell cycle effects to okadaic acid were also observed using dominant-negative PP2Ac. Preincubation of eggs with the PP2A activator FTY720 could block many of the actions of okadaic acid, including Emi2, cyclin B1, and securin degradation and sister chromatid separation. Therefore, in conclusion, we used okadaic acid, dn-PP2Ac-L199P, and FTY720 on mouse eggs to demonstrate that PP2A is needed to for both continued metaphase arrest and successful exit from meiosis.     

Characterization and expression of a maternal axolotl cyclin B1 during oogenesis and early development

The M phase promoting factor (MPF) is a dimer composed of a catalytic Cdk1 subunit and a Cyclin B regulatory subunit. We have characterized a cDNA containing the entire coding sequence of an axolotl Cyclin B1 protein that is able to promote MPF activity when added to a fraction from prophase I oocytes that contains monomeric Cdk1. The axolotl cyclin B1 gene is expressed as a maternal mRNA in oocytes and early embryos. Its poly(A) tail length increases in metaphase II oocytes and then decreases regularly during the first embryonic cell cycles. Endogenous Cyclin B1 protein is first expressed during oocyte meiotic maturation. Its level oscillates after fertilization and is coordinated to the phosphorylation level of tyrosine 15 residue of Cdk1 (pTyr15), with both maxima preceding each cell division. As expected, when translated into microinjected oocytes, axolotl Cyclin B1 induces the resumption of meiosis. In electrically activated unfertilized eggs (UFE), Cyclin B1 and pTyr15 cyclic accumulations are observed with kinetics different from those of the early embryonic cycles. The axolotl embryo and UFE provide interesting in vivo comparative models for studying events controlling Cyclin B1 regulation during development.     

Prophase I arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APC(Cdh1)

Mammalian oocytes are arrested in prophase of the first meiotic division. Progression into the first meiotic division is driven by an increase in the activity of maturation-promoting factor (MPF). In mouse oocytes, we find that early mitotic inhibitor 1 (Emi1), an inhibitor of the anaphase-promoting complex (APC) that is responsible for cyclin B destruction and inactivation of MPF, is present at prophase I and undergoes Skp1-Cul1-F-box/betaTrCP-mediated destruction immediately after germinal vesicle breakdown (GVBD). Exogenous Emi1 or the inhibition of Emi1 destruction in prophase-arrested oocytes leads to a stabilization of cyclin B1-GFP that is sufficient to trigger GVBD. In contrast, the depletion of Emi1 using morpholino oligonucleotides increases cyclin B1-GFP destruction, resulting in an attenuation of MPF activation and a delay of entry into the first meiotic division. Finally, we show that Emi1-dependent effects on meiosis I require the presence of Cdh1. These observations reveal a novel mechanism for the control of entry into the first meiotic division: an Emi1-dependent inhibition of APC(Cdh1).     

Overexpression of cyclin A1 promotes meiotic resumption but induces premature chromosome separation in mouse oocyte

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.     

Reduction of nitric oxide level leads to spontaneous resumption of meiosis in diplotene-arrested rat oocytes cultured in?vitro

The present study was aimed to investigate whether a decrease of nitric oxide (NO) level is beneficial for sponateous resumptiom of meiosis in diplotene-arrested oocytes cultured in vitro. For this purpose, diplotene-arrested oocytes were collected from ovary of immature female rats after a single subcutaneous injection of 20 IU pregnant mare’s serum gonadotropins (PMSG) for 48 h. In vitro effects of S-nitroso-l-acetyl penicillamine (SNAP; an NO donor) and aminoguanidine (AG; an inducible NOS [iNOS] inhibitor), intracellular NO, cyclic guanosine monophosphate (cGMP), Cdc25B, Thr-14/Tyr-15 and Thr-161 phosphorylated cyclin-dependent kinase-1 (CDK1), and cyclin B1 levels were analyzed. The SNAP inhibited spontaneous meiotic resumption form diplotene arrest in a concentration-dependent manner, while AG-induced meiotic resumption form diplotene in 0.1 mmol/L 3-isobutyl-1-methylxanthine (IBMX)-treated oocytes in a concentration-dependent manner. The intracellular NO as well as cGMP levels were decreased significantly during spontaneous meiotic resumption from diplotene arrest. The reduction of Cdc25B expression level was associated with the accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. However, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels were reduced significantly during meiotic resumption from diplotene arrest. Taken together, these data suggest that the inhibition of iNOS expression leads to a decrease of NO and cGMP levels thereby decreasing Cdc25B level. The reduced CDC25 B level leads to accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. As a result, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels are decreased leading to maturation-promoting factor (MPF) inactivation. The inactive MPF finally induced meiotic resumption from diplotene stage in rat oocytes cultured in vitro.     

Maintenance of sister chromatid attachment in mouse eggs through maturation-promoting factor activity

Mammalian eggs naturally arrest at metaphase of the second meiotic division, until sperm triggers a series of Ca(2+) spikes that result in activation of the anaphase-promoting complex/cyclosome (APC/C). APC/C activation at metaphase targets destruction-box containing substrates, such as cyclin B1 and securin, for degradation, and as such eggs complete the second meiotic division. Cyclin B1 degradation reduces maturation (M-phase)-promoting factor (MPF) activity and securin degradation allows sister chromatid separation. Here we examined the second meiotic division in mouse eggs following expression of a cyclin B1 construct with an N-terminal 90 amino acid deletion (Delta 90 cyclin B1) that was visualized by coupling to EGFP. This cyclin construct was not an APC/C substrate, and so following fertilization, sperm were incapable of stimulating Delta 90 cyclin B1 degradation. In these eggs, chromatin remained condensed and no pronuclei formed. As a consequence of the lack of pronucleus formation, sperm-triggered Ca(2+) spiking continued indefinitely, consistent with a current model in which the sperm-activating factor is localized to the nucleus. Because Ca(2+) spiking was not inhibited by Delta 90 cyclin B1, the degradation timing of securin, visualized by coupling it to EGFP, was unaffected. However, despite rapid securin degradation, sister chromatids remained attached. This was a direct consequence of MPF activity because separation was induced following application of the MPF inhibitor roscovitine. Similar observations regarding the ability of MPF to prevent sister chromatid separation have recently been made in Xenopus egg extracts and in HeLa cells. The results presented here show this mechanism can also occur in intact mammalian eggs and further that this mechanism appears conserved among vertebrates. We present a model in which metaphase II arrest is maintained primarily by MPF levels only.     

Checkpoint kinase 1 is essential for meiotic cell cycle regulation in mouse oocytes

Checkpoint kinase 1 (Chk1) plays key roles in all currently defined cell cycle checkpoints, but its functions in mouse oocyte meiosis remain unclear. In this study, we report the expression, localization and functions of Chk1 in mouse oocyte meiosis. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages and localized to the spindle from pro-metaphase I (pro-MI) to MII stages in mouse oocytes. Chk1 depletion facilitated the G₂/M transition while Chk1 overexpression inhibited the G₂/M transition as indicated by germinal vesicle breakdown (GVBD), through regulation of Cdh1 and Cyclin B1. Chk1 depletion did not affect meiotic cell cycle progression after GVBD, but its overexpression after GVBD activated the spindle assembly checkpoint and prevented homologous chromosome segregation, thus arresting oocytes at pro-MI or metaphase I (MI) stages. These results suggest that Chk1 is indispensable for prophase I arrest and functions in G₂/M checkpoint regulation in meiotic oocytes. Moreover, Chk1 overexpression affects meiotic spindle assembly checkpoint regulation and thus chromosome segregation.     

Combined use of dbcAMP and IBMX minimizes the damage induced by a long‐term artificial meiotic arrest in mouse germinal vesicle oocytes

Phosphodiesterase (PDE)‐mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3‐isobutyl‐1‐methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV‐stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double‐strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 μM dbcAMP and 10.0 μM IBMX efficiently inhibited meiotic resumption in GV‐stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.