The spindle checkpoint kinase bub1 and cyclin e/cdk2 both contribute to the establishment of meiotic metaphase arrest by cytostatic factor

In vertebrate unfertilized eggs, metaphase arrest in Meiosis II is mediated by an activity known as cytostatic factor (CSF). CSF arrest is dependent upon Mos-dependent activation of the MAPK/Rsk pathway, and Rsk activates the spindle checkpoint kinase Bub1, leading to inhibition of the anaphase-promoting complex (APC), an E3 ubiquitin ligase required for the metaphase/anaphase transition. However, it is not known whether Bub1 is required for the establishment of CSF arrest or whether other pathways also contribute. Here, we show that immunodepletion of Bub1 from egg extracts blocks the ability of Mos to establish CSF arrest, and arrest can be restored by the addition of wild-type, but not kinase-dead, Bub1. The appearance of CSF arrest at Meiosis II may result from coexpression of cyclin E/Cdk2 with the MAPK/Bub1 pathway. Cyclin E/Cdk2 was able to cause metaphase arrest in egg extracts even in the absence of Mos and could also inhibit cyclin B degradation in oocytes when expressed at anaphase of Meiosis I. Once it has been established, metaphase arrest can be maintained in the absence of MAPK, Bub1, or cyclin E/Cdk2 activity. Both pathways are independent of each other, but each appears to block activation of the APC, which is required for cyclin B degradation and the metaphase/anaphase transition.     

Emi2 at the Crossroads: Where CSF Meets MPF

Vertebrate eggs arrest at metaphase of meiosis II due to an activity known as cytostatic factor (CSF). CSF antagonizes the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C), preventing cyclin B destruction and meiotic exit until fertilization occurs. A puzzling feature of CSF arrest is that APC/C inhibition is leaky. Ongoing cyclin B synthesis is counterbalanced by a limited amount of APC/C-mediated cyclin B destruction; thus, cyclin B/Cdc2 activity remains at steady state. How the APC/C can be slightly active toward cyclin B, and yet restrained from ubiquitinating cyclin B altogether, is unknown. Emi2/XErp1 is the critical CSF component directly responsible for APC/C inhibition during CSF arrest. Fertilization triggers the Ca2+-dependent destruction of Emi2, releasing the APC/C to ubiquitinate the full pool of cyclin B and initiate completion of meiosis. Previously, we showed that a phosphatase maintains Emi2’s APC/C-inhibitory activity in CSF-arrested Xenopus egg extracts. Here, we demonstrate that phosphatase inhibition permits Emi2 phosphorylation at thr-545 and -551, which inactivates Emi2. Furthermore, we provide evidence that adding excess cyclin B to CSF extracts stimulates Cdc2 phosphorylation of these same residues, antagonizing Emi2-APC/C association. Our findings suggest a model wherein the pool of Emi2 acts analogously to a rheostat by integrating Cdc2 and phosphatase activities to prevent cyclin B overaccumulation and Cdc2 hyperactivity during the indefinite period of time between arrival at metaphase II and eventual fertilization. Finally, we propose that inactivation of Emi2 by Cdc2 permits mitotic progression during early embryonic cleavage cycles.     

APC/C-Cdc20-mediated degradation of cyclin B participates in CSF arrest in unfertilized Xenopus eggs

In vertebrates, unfertilized eggs are arrested at meiotic metaphase II (meta-II) by cytostatic factor (CSF), with Cdc2 activity maintained at a constant, high level. CSF is thought to suppress cyclin B degradation through the inhibition of the anaphase-promoting complex/cyclosome (APC/C)-Cdc20 while cyclin B synthesis continues in unfertilized eggs. Thus, it is a mystery how Cdc2 activity is kept constant during CSF arrest. Here, we show that the APC/C-Cdc20 can mediate cyclin B degradation in CSF-arrested Xenopus eggs and extracts, in such a way that when Cdc2 activity is elevated beyond a critical level, APC/C-Cdc20-dependent cyclin B degradation is activated and Cdc2 activity consequently declines to the critical level. This feedback control of Cdc2 activity is shown to be required for keeping Cdc2 activity constant during meta-II arrest. We have also shown that Mos/MAPK pathway is essential for preventing the cyclin B degradation from inactivating Cdc2 below the critical level required to sustain meta-II arrest. Our results indicate that under CSF arrest, Mos/MAPK activity suppresses cyclin B degradation, preventing Cdc2 activity from falling below normal meta-II levels, whereas activation of APC/C-Cdc20-mediated cyclin B degradation at elevated levels of Cdc2 activity prevents Cdc2 activity from reaching excessively high levels.     

Spindle checkpoint proteins Mad1 and Mad2 are required for cytostatic factor-mediated metaphase arrest

In cells containing disrupted spindles, the spindle assembly checkpoint arrests the cell cycle in metaphase. The budding uninhibited by benzimidazole (Bub) 1, mitotic arrest-deficient (Mad) 1, and Mad2 proteins promote this checkpoint through sustained inhibition of the anaphase-promoting complex/cyclosome. Vertebrate oocytes undergoing meiotic maturation arrest in metaphase of meiosis II due to a cytoplasmic activity termed cytostatic factor (CSF), which appears not to be regulated by spindle dynamics. Here, we show that microinjection of Mad1 or Mad2 protein into early Xenopus laevis embryos causes metaphase arrest like that caused by Mos. Microinjection of antibodies to either Mad1 or Mad2 into maturing oocytes blocks the establishment of CSF arrest in meiosis II, and immunodepletion of either protein blocked the establishment of CSF arrest by Mos in egg extracts. A Mad2 mutant unable to oligomerize (Mad2 R133A) did not cause cell cycle arrest in blastomeres or in egg extracts. Once CSF arrest has been established, maintenance of metaphase arrest requires Mad1, but not Mad2 or Bub1. These results suggest a model in which CSF arrest by Mos is mediated by the Mad1 and Mad2 proteins in a manner distinct from the spindle checkpoint.     

Translational Unmasking of Emi2 Directs Cytostatic Factor Arrest in Meiosis II

Cytostatic factor (CSF) arrests unfertilized vertebrate eggs in metaphase of meiosis II by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from mediating cyclin destruction. The APC/C inhibitor Emi2/XErp1 satisfies a number of historical criteria for the molecular identification of CSF, but the mechanism by which CSF is activated selectively in meiosis II is the remaining unexplained criterion. Here we provide an explanation by showing that Emi2 is expressed specifically in meiosis II through translational de-repression or “unmasking” of its mRNA. We find that Emi2 protein is undetectable in immature, G2/prophase-arrested Xenopus oocytes and accumulates ~90 minutes after germinal vesicle breakdown. The 3’ untranslated region of Emi2 mRNA contains cytoplasmic polyadenylation elements that directly bind the CPEB protein and confer temporal regulation of Emi2 polyadenylation and translation. Our results demonstrate that cytoplasmic polyadenylation and translational unmasking of Emi2 directs meiosis II-specific CSF arrest.     

Calcium elevation at fertilization coordinates phosphorylation of XErp1/Emi2 by Plx1 and CaMK II to release metaphase arrest by cytostatic factor

BACKGROUND: Vertebrate oocytes are arrested at second meiotic metaphase by cytostatic factor (CSF) while awaiting fertilization. Accumulating evidence has suggested that inhibition of the anaphase-promoting complex/cyclosome (APC/C) is responsible for this arrest. Xenopus polo-like kinase 1 (Plx1) is required for activation of the APC/C at the metaphase-anaphase transition, and calcium elevation, upon fertilization/activation of eggs, acting through calmodulin-dependent kinase II (CaMKII) is sufficient to activate the APC/C and terminate CSF arrest. However, connections between the Plx1 pathway and the CaMKII pathway have not been identified. RESULTS: Overexpression of Plx1 causes CSF release in the absence of calcium, and depletion of Plx1 from egg extracts blocks induction of CSF release by calcium and CaMKII. Prior phosphorylation of the APC/C inhibitor XErp1/Emi2 by CaMK II renders it a good substrate for Plx1, and phosphorylation by both kinases together promotes its degradation in egg extracts. The pathway is enhanced by the ability of Plx1 to cause calcium-independent activation of CaMKII. The results identify the targets of CaMKII and Plx1 that promote egg activation and define the first known pathway of CSF release in which an APC/C inhibitor is targeted for degradation only when both CaMKII and Plx1 are active after calcium elevation at fertilization. CONCLUSIONS: Plx1 with an intact polo-box domain is necessary for release of CSF arrest and sufficient when overexpressed. It acts at the same level as CaMKII in the pathway of calcium-induced CSF release by cooperating with CaMKII to regulate APC/C regulator(s), such as XErp1/Emi2, rather than by directly activating the APC/C itself.     

The anaphase-promoting complex/cyclosome inhibitor Emi2 is essential for meiotic but not mitotic cell cycles

Vertebrate oocytes awaiting fertilization are arrested at metaphase of meiosis II by cytostatic factor (CSF). This arrest is due to inhibition of the anaphase-promoting complex/cyclosome, in part by a newly identified protein, Emi2 (xErp1). Emi2 is required for maintenance of CSF arrest in egg extracts, but its function in CSF establishment in oocytes and the normal embryonic cell cycle is unknown. Here we show that during oocyte maturation, Emi2 appears only after metaphase I, and its level peaks at CSF arrest (metaphase II). In M phase, Emi2 undergoes a phosphorylation-dependent electrophoretic shift. Microinjection of antisense oligonucleotides against Emi2 into stage VI oocytes blocks progression through meiosis II and the establishment of CSF arrest. Recombinant Emi2 rescues CSF arrest in these oocytes and also causes CSF arrest in egg extracts and in blastomeres of two-cell embryos. Fertilization triggers rapid, complete degradation of Emi2, but it is resynthesized in the first embryonic cell cycle to reach levels 5-fold lower than during CSF arrest. However, depletion of the protein from cycling egg extracts does not prevent mitotic cell cycle progression. Thus, Emi2 plays an essential role in meiotic but not mitotic cell cycles.     

The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes

In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.     

Interplay between Cdc2 kinase and the c-Mos/MAPK pathway between metaphase I and metaphase II in Xenopus oocytes

Xenopus oocytes arrested in prophase I resume meiotic division in response to progesterone and arrest at metaphase II. Entry into meiosis I depends on the activation of Cdc2 kinase [M-phase promoting factor (MPF)]. To better understand the role of Cdc2, MPF activity was specifically inhibited by injection of the CDK inhibitor, Cip1. When Cip1 is injected at germinal vesicle breakdown (GVBD) time, Cdc25 and Plx1 are both dephosphorylated and Cdc2 is rephosphorylated on tyrosine. The autoamplification loop characterizing MPF is therefore not only required for MPF generation before GVBD, but also for its stability during the GVBD period. The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), responsible for cyclin degradation, is also under the control of Cdc2; therefore, Cdc2 activity itself induces its own inactivation through cyclin degradation, allowing the exit from the first meiotic division. In contrast, cyclin accumulation, responsible for Cdc2 activity increase allowing entry into metaphase II, is independent of Cdc2. The c-Mos/mitogen-activated protein kinase (MAPK) pathway remains active when Cdc2 activity is inhibited at GVBD time. This pathway could be responsible for the sustained cyclin neosynthesis. In contrast, during the metaphase II block, the c-Mos/MAPK pathway depends on Cdc2. Therefore, the metaphase II block depends on a dynamic interplay between MPF and CSF, the c-Mos/MAPK pathway stabilizing cyclin B, whereas in turn, MPF prevents c-Mos degradation.     

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.     

Functional interaction between p90Rsk2 and Emi1 contributes to the metaphase arrest of mouse oocytes

Vertebrate eggs arrest at metaphase of the second meiotic division before fertilization under the effect of a cytostatic factor (CSF). This arrest is established during oocyte maturation by the MAPK kinase module, comprised of Mos, MEK, MAPKs and p90Rsk. Maintenance of CSF arrest at metaphase requires inhibitors of the anaphase-promoting complex (APC) like Emi1, which sequesters the APC activator Cdc20. Although it was proposed that the Mos pathway and Emi1 act independently, neither one alone is sufficient to entirely reproduce CSF arrest. Herein we demonstrate that p90Rsk2 associates with and phosphorylates Emi1 upstream of the binding region for Cdc20, thus stabilizing their interaction. Experiments in transfected cells and two-cell embryos indicate that Emi1 and p90Rsk2 cooperate to induce the metaphase arrest. Moreover, oocyte maturation was impaired by interfering with the interaction between p90Rsk2 and Emi1 or by RNA interference of Emi1. Our results indicate that p90Rsk2 and Emi1 functionally interact during oocyte maturation and that the Mos pathway establishes CSF activity through stabilization of an APC-inhibitory complex composed by Emi1 and Cdc20 before fertilization.     

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.     

The pathway of MAP kinase mediation of CSF arrest in Xenopus oocytes.

A cytoplasmic activity in mature oocytes responsible for second meiotic metaphase arrest was identified over 30 years ago in amphibian oocytes. In Xenopus oocytes CSF activity is initiated by the progesterone-dependent synthesis of Mos, a MAPK kinase kinase, which activates the MAPK pathway. CSF arrest is mediated by a sole MAPK target, the protein kinase p90Rsk which leads to inhibition of cyclin B degradation by the anaphase-promoting complex. Rsk phosphorylates and activates the Bub1 protein kinase, which may cause metaphase arrest due to inhibition of the anaphase-promoting complex (APC) by a conserved mechanism defined genetically in yeast and mammalian cells. CSF arrest in vertebrate oocytes by p90Rsk provides a potential link between the MAPK pathway and the spindle assembly checkpoint in the cell cycle.     

XCdh1 is involved in progesterone-induced oocyte maturation

The molecular events triggered during progesterone-induced oocyte maturation in Xenopus are not well understood. One of the first events is the activation of the MAPK cascade and the maturation-promoting factor (MPF). The latter triggers meiosis I resumption and meiosis II progression until the metaphase II arrest. The release of the metaphase II is mediated by the anaphase-promoting complex (APC)-dependent degradation of cyclin B. This degradation activity requires the APC activator Cdc20 that activates ubiquitination reactions by recruiting substrates to the APC. However, recent reports in different organisms involve other APC regulators during different phases of the meiotic cycle. Therefore, we investigated the role of another APC regulator, XCdh1 during the G2/M transition in meiosis I in the Xenopus oocyte. Here, we report that XCdh1 protein is expressed in oocytes. Besides, injection of specific XCdh1 antisense inhibits progesterone-induced G2/M transition that can be rescued by adding back the purified human Cdh1 protein. On the other hand, ectopic expression of low levels of XCdh1 protein has a positive effect on the G2/M transition by facilitating this process. Moreover, the sole injection of XCdh1 mRNA triggers Mos protein synthesis and biphosphorylation of MAPK in absence of progesterone. Altogether, these data show that XCdh1 has a positive role during the G2/M transition in the oocyte. According to our results, its role could be independent of the APC.     

CaM kinase II initiates meiotic spindle depolymerization independently of APC/C activation

Altered spindle microtubule dynamics at anaphase onset are the basis for chromosome segregation. In Xenopus laevis egg extracts, increasing free calcium levels and subsequently rising calcium-calmodulin–dependent kinase II (CaMKII) activity promote a release from meiosis II arrest and reentry into anaphase. CaMKII induces the activation of the anaphase-promoting complex/cyclosome (APC/C), which destines securin and cyclin B for degradation to allow chromosome separation and mitotic exit.     

Bub1 is activated by the protein kinase p90(Rsk) during Xenopus oocyte maturation

BACKGROUND: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint. RESULTS: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. CONCLUSIONS: Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk).     

Dynamic regulation of Emi2 by Emi2-bound Cdk1/Plk1/CK1 and PP2A-B56 in meiotic arrest of Xenopus eggs

In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Mos and Emi2, an inhibitor of the APC/C ubiquitin ligase. In Xenopus, Cdk1 phosphorylates Emi2 and both destabilizes and inactivates it, whereas Mos recruits PP2A phosphatase to antagonize the Cdk1 phosphorylation. However, how Cdk1 phosphorylation inhibits Emi2 is largely unknown. Here we show that multiple N-terminal Cdk1 phosphorylation motifs bind cyclin B1-Cdk1 itself, Plk1, and CK1δ/ε to inhibit Emi2. Plk1, after rebinding to other sites by self-priming phosphorylation, partially destabilizes Emi2. Cdk1 and CK1δ/ε sequentially phosphorylate the C-terminal APC/C-docking site, thereby cooperatively inhibiting Emi2 from binding the APC/C. In the presence of Mos, however, PP2A-B56β/ε bind to Emi2 and keep dephosphorylating it, particularly at the APC/C-docking site. Thus, Emi2 stability and activity are dynamically regulated by Emi2-bound multiple kinases and PP2A phosphatase. Our data also suggest a general role for Cdk1 substrate phosphorylation motifs in M phase regulation.     

p90Rsk is required for G1 phase arrest in unfertilized starfish eggs

The cell cycle in oocytes generally arrests at a particular meiotic stage to await fertilization. This arrest occurs at metaphase of meiosis II (meta-II) in frog and mouse, and at G1 phase after completion of meiosis II in starfish. Despite this difference in the arrest phase, both arrests depend on the same Mos-MAPK (mitogen-activated protein kinase) pathway, indicating that the difference relies on particular downstream effectors. Immediately downstream of MAPK, Rsk (p90 ribosomal S6 kinase, p90(Rsk)) is required for the frog meta-II arrest. However, the mouse meta-II arrest challenges this requirement, and no downstream effector has been identified in the starfish G1 arrest. To investigate the downstream effector of MAPK in the starfish G1 arrest, we used a neutralizing antibody against Rsk and a constitutively active form of Rsk. Rsk was activated downstream of the Mos-MAPK pathway during meiosis. In G1 eggs, inhibition of Rsk activity released the arrest and initiated DNA replication without fertilization. Conversely, maintenance of Rsk activity prevented DNA replication following fertilization. In early embryos, injection of Mos activated the MAPK-Rsk pathway, resulting in G1 arrest. Moreover, inhibition of Rsk activity during meiosis I led to parthenogenetic activation without meiosis II. We conclude that immediately downstream of MAPK, Rsk is necessary and sufficient for the starfish G1 arrest. Although CSF (cytostatic factor) was originally defined for meta-II arrest in frog eggs, we propose to distinguish ;G1-CSF' for starfish from ;meta-II-CSF' for frog and mouse. The present study thus reveals a novel role of Rsk for G1-CSF.     

Involvement of Mos-MEK-MAPK pathway in cytostatic factor (CSF) arrest in eggs of the parthenogenetic insect, Athalia rosae

Extensive survey of meiotic metaphase II arrest during oocyte maturation in vertebrates revealed that the mitogen-activated protein kinase (MAPK) pathway regulated by the c-mos proto-oncogene product, Mos, has an essential role in cytostatic activity, termed cytostatic factor (CSF). In contrast, little is known in invertebrates in which meiotic arrest occurs in most cases at metaphase I (MI arrest). A parthenogenetic insect, the sawfly Athalia rosae, in which artificial egg activation is practicable, has advantages to investigate the mechanisms of MI arrest. Both the MAPK/extracellular signal-regulated protein kinase kinase (MEK) and MAPK were phosphorylated and maintained active in MI-arrested sawfly eggs, whereas they were dephosphorylated soon after egg activation. Treatment of MI-arrested eggs with U0126, an inhibitor of MEK, resulted in dephosphorylation of MAPK and MI arrest was resumed. The sawfly c-mos gene orthologue encoding a serine/threonine kinase was cloned and analyzed. It was expressed in nurse cells in the ovaries. To examine CSF activity of the sawfly Mos, synthesized glutathione S-transferase (GST)-fusion sawfly Mos protein was injected into MI-resumed eggs in which MEK and MAPK were dephosphorylated. Both MEK and MAPK were phosphorylated again upon injection. In these GST-fusion sawfly Mos-injected eggs subsequent mitotic (syncytial) divisions were blocked and embryonic development was ceased. These results demonstrated that the MEK-MAPK pathway was involved in maintaining CSF arrest in sawfly eggs and Mos functioned as its upstream regulatory molecule.     

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.