Maintenance of meiotic prophase arrest in vertebrate oocytes by a Gs protein-mediated pathway

Maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on an elevated level of cAMP in the oocyte. To investigate how the cAMP level is regulated, we examined whether the activity of an oocyte G protein of the family that stimulates adenylyl cyclase, Gs, is required to maintain meiotic arrest. Microinjection of a dominant negative form of Gs into Xenopus and mouse oocytes, or microinjection of an antibody that inhibits the Gs G protein into zebrafish oocytes, caused meiosis to resume. Together with previous studies, these results support the conclusion that Gs-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic prophase arrest in vertebrate oocytes.     

Meiotic arrest in human oocytes is maintained by a Gs signaling pathway

In mammalian oocytes, the maintenance of meiotic prophase I arrest prior to the surge of LH that stimulates meiotic maturation depends on a high level of cAMP within the oocyte. In mouse and rat, the cAMP is generated in the oocyte, and this requires the activity of a constitutively active, Gs-linked receptor, GPR3 or GPR12, respectively. To examine if human oocyte meiotic arrest depends on a similar pathway, we used RT-PCR and Western blotting to look at whether human oocytes express the same components for maintaining arrest as rodent oocytes. RNA encoding GPR3, but not GPR12, was expressed. RNA encoding adenylate cyclase type 3, which is the major adenylate cyclase required for maintaining meiotic arrest in the mouse oocyte, was also expressed, as was Galphas protein. To determine if this pathway is functional in the human oocyte, we examined the effect of injecting a function-blocking antibody against Galphas on meiotic resumption. This antibody stimulated meiotic resumption of human oocytes that were maintained at the prophase I stage using a phosphodiesterase inhibitor. These results demonstrate that human oocytes maintain meiotic arrest prior to the LH surge using a signaling pathway similar to that of rodent oocytes.     

Involvement of Chk1 kinase in prophase I arrest of Xenopus oocytes

Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. Here we have cloned a Xenopus homolog of Chk1, determined its developmental expression, and examined its possible role in prophase I arrest of oocytes. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. We discuss the possibility that Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes.     

α-endosulfine (ENSA) regulates exit from prophase I arrest in mouse oocytes

Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.     

Two zinc finger proteins, OMA-1 and OMA-2, are redundantly required for oocyte maturation in C. elegans.

Oocytes are released from meiotic prophase I arrest through a process termed oocyte maturation. We present here a genetic characterization of oocyte maturation, using C. elegans as a model system. We show that two TIS11 zinc finger-containing proteins, OMA-1 and OMA-2, express specifically in maturing oocytes and function redundantly in oocyte maturation. Oocytes in oma-1;oma-2 mutants initiate but do not complete maturation and arrest at a defined point in prophase I. Two maturation signal-induced molecular events, including the maintenance of activated MAP kinase, do not occur in Oma oocytes. The Oma prophase arrest is released by inactivation of a MYT-1-like kinase, suggesting that OMA-1 and OMA-2 function upstream of MYT-1 as positive regulators of prophase progression during meiotic maturation.     

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.     

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.     

Comparative analysis of meiotic progression in female mice bearing mutations in genes of the DNA mismatch repair pathway

The DNA mismatch repair (MMR) family functions in a variety of contexts to preserve genome integrity in most eukaryotes. In particular, members of the MMR family are involved in the process of meiotic recombination in germ cells. MMR gene mutations in mice result in meiotic disruption during prophase I, but the extent of this disruption often differs between male and female meiocytes. To address the role of MMR proteins specifically in female meiosis, we explored the progression of oocytes through prophase I and the meiotic divisions in mice harboring deletions in members of the MMR pathway (Mlh1, Mlh3, Exo1, and an ATPase-deficient variant of Mlh1, Mlh1(G67R)). The colocalization of MLH1 and MLH3, key proteins involved in stabilization of nascent crossovers, was dependent on intact heterodimer formation and was highly correlated with the ability of oocytes to progress through to metaphase II. The exception was Exo1(-/-) oocytes, in which normal MLH1/MLH3 localization was observed followed by failure to proceed to metaphase II. All mutant oocytes were able to resume meiosis after dictyate arrest, but they showed a dramatic decline in chiasmata (to less than 25% of normal), accompanied by varied progression through metaphase I. Taken together, these results demonstrate that MMR function is required for the formation and stabilization of crossovers in mammalian oocytes and that, in the absence of a functional MMR system, the failure to maintain chiasmata results in a reduced ability to proceed normally through the first and second meiotic divisions, despite near-normal levels of meiotic resumption after dictyate arrest.     

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.     

The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. Often this marks a period of rapid growth for the oocyte and is necessary to coordinate meiotic progression with the developmental events of oogenesis. In Drosophila, the oocyte develops within a 16-cell germline cyst. Throughout much of oogenesis, the oocyte remains in prophase of meiosis I. By contrast, its 15 mitotic sisters enter the endocycle and become polyploid in preparation for their role as nurse cells. How germline cysts establish and maintain these two independent cell cycles is unknown. We demonstrate a role for the p21(CIP)/p27(Kip1)/p57(Kip2)-like cyclin-dependent kinase inhibitor (cki) dacapo in the maintenance of the meiotic cycle in Drosophila oocytes. Our data indicate that it is through the differential regulation of the cki Dacapo that two modes of cell-cycle regulation are independently maintained within the common cytoplasm of ovarian cysts.     

A member of the Ste20/PAK family of protein kinases is involved in both arrest of Xenopus oocytes at G2/prophase of the first meiotic cell cycle and in prevention of apoptosis.

We have identified new members (X-PAKs) of the Ste20/PAK family of protein kinases in Xenopus, and investigated their role in the process that maintains oocytes arrested in the cell cycle. Microinjection of a catalytically inactive mutant of X-PAK1 with a K/R substitution in the ATP binding site, also deleted of its Nter-half that contains the conserved domains responsible for binding of both Cdc42/Rac GTPases and SH3-containing proteins, greatly facilitates oocyte release from G2/prophase arrest by progesterone and insulin. Addition of the same X-PAK1 mutant to cell cycle extracts from unfertilized eggs induced apoptosis, as shown by activation of caspases and cytological changes in in vitro-assembled nuclei. This was suppressed by adding Bcl-2 or the DEVD peptide inhibitor of caspases, and rescued by competing the dominant-negative mutant with its constitutively active X-PAK1 counterpart. Such results indicate that X-PAK1 (or another member of the Xenopus Ste20/PAK family of protein kinases) is involved in arrest of oocytes at G2/prophase and prevention of apoptosis; thus death by apoptosis and release of healthy oocytes from cell cycle arrest may be linked. That cell cycle arrest protects oocytes from apoptosis is consistent with the finding that extracts from metaphase II-arrested oocytes are less sensitive to apoptotic signals than those from activated eggs.     

Ras family proteins: new players involved in the diplotene arrest of Xenopus oocytes

Oogonia undergo numerous mitotic cell cycles before completing the last DNA replication and entering the meiotic prophase I. After chromosome pairing and chromatid exchanges between paired chromosomes, the oocyte I remains arrested at the diplotene stage of the first meiotic prophase. Oocyte growth then occurs independently of cell division; indeed, during this growth period, oocytes (4n DNA) are prevented from completing the meiotic divisions. How is the prophase arrest regulated? One of the players of the prophase block is the high level of intracellular cAMP, maintained by an active adenylate cyclase. By using lethal toxin from Clostridium sordellii (LT), a glucosyl-transferase that glucosylates and inactivates small G proteins of the Ras subfamily, we have shown that inhibition of either Ras or Rap or both proteins is sufficient to release the prophase block of Xenopus oocytes in a cAMP-dependent manner. The implications of Ras family proteins as new players involved in the prophase arrest of Xenopus oocytes will be discussed here.     

The C.elegans MAPK phosphatase LIP-1 is required for the G(2)/M meiotic arrest of developing oocytes

In the Caenorhabditis elegans hermaphrodite germline, spatially restricted mitogen-activated protein kinase (MAPK) signalling controls the meiotic cell cycle. First, the MAPK signal is necessary for the germ cells to progress through pachytene of meiotic prophase I. As the germ cells exit pachytene and enter diplotene/diakinesis, MAPK is inactivated and the developing oocytes arrest in diakinesis (G(2)/M arrest). During oocyte maturation, a signal from the sperm reactivates MAPK to promote M phase entry. Here, we show that the MAPK phosphatase LIP-1 dephosphorylates MAPK as germ cells exit pachytene in order to maintain MAPK in an inactive state during oocyte development. Germ cells lacking LIP-1 fail to arrest the cell cycle at the G(2)/M boundary, and they enter a mitotic cell cycle without fertilization. LIP-1 thus coordinates oocyte cell cycle progression and maturation with ovulation and fertilization.     

NPP-16/Nup50 Function and CDK-1 Inactivation Are Associated with Anoxia-induced Prophase Arrest in Caenorhabditis elegans

Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type–arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.     

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.     

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.     

Developmental control of oocyte maturation and egg activation in metazoan models

Production of functional eggs requires meiosis to be coordinated with developmental signals. Oocytes arrest in prophase I to permit oocyte differentiation, and in most animals, a second meiotic arrest links completion of meiosis to fertilization. Comparison of oocyte maturation and egg activation between mammals, Caenorhabditis elegans, and Drosophila reveal conserved signaling pathways and regulatory mechanisms as well as unique adaptations for reproductive strategies. Recent studies in mammals and C. elegans show the role of signaling between surrounding somatic cells and the oocyte in maintaining the prophase I arrest and controlling maturation. Proteins that regulate levels of active Cdk1/cyclin B during prophase I arrest have been identified in Drosophila. Protein kinases play crucial roles in the transition from meiosis in the oocyte to mitotic embryonic divisions in C. elegans and Drosophila. Here we will contrast the regulation of key meiotic events in oocytes.     

The APC/C activator FZR1 coordinates the timing of meiotic resumption during prophase I arrest in mammalian oocytes

FZR1, an activator of the anaphase-promoting complex/cyclosome (APC/C), is recognized for its roles in the mitotic cell cycle. To examine its meiotic function in females we generated an oocyte-specific knockout of the Fzr1 gene (Fzr1(Δ/Δ)). The total number of fully grown oocytes enclosed in cumulus complexes was 35-40% lower in oocytes from Fzr1(Δ/Δ) mice and there was a commensurate rise in denuded, meiotically advanced and/or fragmented oocytes. The ability of Fzr1(Δ/Δ) oocytes to remain prophase I/germinal vesicle (GV) arrested in vitro was also compromised, despite the addition of the phosphodiesterase milrinone. Meiotic competency of smaller diameter oocytes was also accelerated by Fzr1 loss. Cyclin B1 levels were elevated ~5-fold in Fzr1(Δ/Δ) oocytes, whereas securin and CDC25B, two other APC/C(FZR1) substrates, were unchanged. Cyclin B1 overexpression can mimic the effects of Fzr1 loss on GV arrest and here we show that cyclin B1 knockdown in Fzr1(Δ/Δ) oocytes affects the timing of meiotic resumption. Therefore, the effects of Fzr1 loss are mediated, at least in part, by raised cyclin B1. Thus, APC/C(FZR1) activity is required to repress cyclin B1 levels in oocytes during prophase I arrest in the ovary, thereby maintaining meiotic quiescence until hormonal cues trigger resumption.     

Zinc maintains prophase I arrest in mouse oocytes through regulation of the MOS-MAPK pathway

Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.