Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes

Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclo-some (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (> 18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.

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Epithelial Cell Transforming Protein 2 (ECT2) Depletion Blocks Polar Body Extrusion and Generates Mouse Oocytes Containing Two Metaphase II Spindles

Endocrinology ◽

10.1210/en.2009-0830 ◽

2010 ◽

Vol 151 (2) ◽

pp. 755-765 ◽

Cited By ~ 15

Author(s):

Judith Elbaz ◽

Yitzhak Reizel ◽

Nava Nevo ◽

Dalia Galiani ◽

Nava Dekel

Keyword(s):

Epithelial Cell ◽

Polar Body ◽

Small Gtpase ◽

Cyclin Dependent Kinase ◽

Mouse Oocytes ◽

First Polar Body ◽

Cell Transforming ◽

Polar Body Extrusion ◽

Maturation Promoting Factor

Completion of the first meiosis in oocytes is achieved by the extrusion of the first polar body (PBI), a particular example of cell division. In mitosis, the small GTPase RhoA, which is activated by epithelial cell transforming protein 2 (ECT2), orchestrates contractile ring constriction, thus enabling cytokinesis. However, the involvement of this pathway in mammalian oocytes has not been established. To characterize the role of ECT2 in PBI emission in mouse oocytes, the small interfering RNA approach was employed. We found that ECT2 depletion significantly reduces PBI emission, induces first metaphase arrest, and generates oocytes containing two properly formed spindles of the second metaphase. Moreover, we describe, for the first time, that before PBI emission, RhoA forms a ring that is preceded by a dome-like accumulation at the oocyte cortex, next to the spindle. This unique mode of RhoA translocation failed to occur in the absence of ECT2. We further found that the Rho-dependent kinase, a main RhoA effector, is essential for PBI emission. In addition, we demonstrate herein that ECT2 is subjected to phosphorylation/dephosphorylation throughout meiosis in oocytes and further reveal that PBI emission is temporally associated with ECT2 dephosphorylation. Our data provide the first demonstration that an active cyclin-dependent kinase 1, the catalytic subunit of the maturation-promoting factor, phosphorylates ECT2 during the first meiotic metaphase and that cyclin-dependent kinase 1 inactivation at anaphase allows ECT2 dephosphorylation. In conclusion, our study demonstrates the indispensable role of the maturation-promoting factor/ECT2/RhoA pathway in PBI extrusion in mouse oocytes.

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The Aurora kinase inhibitor ZM447439 accelerates first meiosis in mouse oocytes by overriding the spindle assembly checkpoint

Reproduction ◽

10.1530/rep-10-0223 ◽

2010 ◽

Vol 140 (4) ◽

pp. 521-530 ◽

Cited By ~ 46

Author(s):

Simon I R Lane ◽

Heng-Yu Chang ◽

Phoebe C Jennings ◽

Keith T Jones

Keyword(s):

Spindle Assembly Checkpoint ◽

Kinase Inhibitor ◽

Polar Body ◽

Spindle Assembly ◽

Aurora Kinase ◽

Mitotic Cell ◽

Cyclin B1 ◽

Anaphase Promoting Complex ◽

Mouse Oocytes ◽

Polar Body Extrusion

Previous studies have established that when maturing mouse oocytes are continuously incubated with the Aurora inhibitor ZM447439, meiotic maturation is blocked. In this study, we observe that by altering the time of addition of the inhibitor, oocyte maturation can actually be accelerated by 1 h as measured by the timing of polar body extrusion. ZM447439 also had the ability to overcome a spindle assembly checkpoint (SAC) arrest caused by nocodazole and so rescue polar body extrusion. Consistent with the ability of the SAC to inhibit cyclin B1 degradation by blocking activation of the anaphase-promoting complex, we could also observe a rescue in cyclin B1 degradation when ZM447439 was added to nocodazole-treated oocytes. The acceleration of the first meiotic division by ZM447439, which has not been achieved previously, and its effects on the SAC are all consistent with the proposed mitotic role of Aurora B in activating the SAC. We hypothesize that Aurora kinase activity controls the SAC in meiosis I, despite differences to the mitotic cell cycle division in spindle architecture brought about by the meiotic mono-orientation of sister kinetochores.

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Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity

The Journal of Cell Biology ◽

10.1083/jcb.201305049 ◽

2014 ◽

Vol 204 (6) ◽

pp. 891-900 ◽

Cited By ~ 24

Author(s):

Ibtissem Nabti ◽

Petros Marangos ◽

Jenny Bormann ◽

Nobuaki R. Kudo ◽

John Carroll

Keyword(s):

Protein Kinase ◽

Spindle Assembly Checkpoint ◽

Mitogen Activated Protein Kinase ◽

Cyclin Dependent Kinase ◽

Anaphase Promoting Complex ◽

Female Meiosis ◽

Dual Mode ◽

Mitogen Activated Protein ◽

Meiosis I

Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. In this paper, we find that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates essential for meiosis I. Furthermore, inhibition of MAPK around the onset of APC/C activity at the transition from meiosis I to meiosis II led to accelerated completion of meiosis I and an increase in aneuploidy at metaphase II. These effects appear to be mediated via a Cdk1/MAPK-dependent stabilization of the spindle assembly checkpoint, which when inhibited leads to increased APC/C activity. These findings demonstrate new roles for MAPK in the regulation of meiosis in mammalian oocytes.

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Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis

The Journal of Cell Biology ◽

10.1083/jcb.200604140 ◽

2006 ◽

Vol 174 (6) ◽

pp. 791-801 ◽

Cited By ~ 103

Author(s):

Suzanne Madgwick ◽

David V. Hansen ◽

Mark Levasseur ◽

Peter K. Jackson ◽

Keith T. Jones

Keyword(s):

Fluorescent Protein ◽

Polar Body ◽

Cyclin B1 ◽

Anaphase Promoting Complex ◽

Morpholino Knockdown ◽

Short Time ◽

Mitotic Inhibitor ◽

Antisense Morpholino ◽

Meiosis I

During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1. Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.

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True Nondisjunction of Whole Bivalents in Oocytes with Attachment and Congression Defects

Cytogenetic and Genome Research ◽

10.1159/000458513 ◽

2017 ◽

Vol 151 (1) ◽

pp. 10-17 ◽

Cited By ~ 3

Author(s):

Martin Sodek ◽

Kristina Kovacovicova ◽

Martin Anger

Keyword(s):

Chromosome Segregation ◽

Developmental Disorders ◽

Polar Body ◽

Direct Consequence ◽

Early Embryos ◽

First Polar Body ◽

Polar Body Extrusion ◽

Meiosis I

Chromosome segregation in mammalian oocytes is prone to errors causing aneuploidy with consequences such as precocious termination of development or severe developmental disorders. Aneuploidy also represents a serious problem in procedures utilizing mammalian gametes and early embryos in vitro. In our study, we focused on congression defects during meiosis I and observed whole nondisjoined bivalents in meiosis II as a direct consequence, together with a substantially delayed first polar body extrusion. We also show that the congression defects are accompanied by less stable attachments of the kinetochores. Our results describe a process by which congression defects directly contribute to aneuploidy.

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Maturation Promoting Factor Governs the First Polar Body Extrusion Through the ECT2-RhoA-ROCK Signaling Pathway in Mouse Oocytes.

Biology of Reproduction ◽

10.1093/biolreprod/81.s1.24 ◽

2009 ◽

Vol 81 (Suppl_1) ◽

pp. 24-24 ◽

Cited By ~ 1

Author(s):

Judith Elbaz ◽

Nava Nevo ◽

Dalia Galiani ◽

Nava Dekel

Keyword(s):

Signaling Pathway ◽

Polar Body ◽

Mouse Oocytes ◽

First Polar Body ◽

Polar Body Extrusion ◽

Maturation Promoting Factor

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A Single Bivalent Efficiently Inhibits Cyclin B1 Degradation and Polar Body Extrusion in Mouse Oocytes Indicating Robust SAC during Female Meiosis I

PLoS ONE ◽

10.1371/journal.pone.0027143 ◽

2011 ◽

Vol 6 (11) ◽

pp. e27143 ◽

Cited By ~ 20

Author(s):

Steffen Hoffmann ◽

Bernard Maro ◽

Jacek Z. Kubiak ◽

Zbigniew Polanski

Keyword(s):

Polar Body ◽

Cyclin B1 ◽

Female Meiosis ◽

Mouse Oocytes ◽

Polar Body Extrusion ◽

Meiosis I

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Vitrification-induced activation of lysosomal cathepsin B perturbs spindle assembly checkpoint function in mouse oocytes

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaaa051 ◽

2020 ◽

Vol 26 (9) ◽

pp. 689-701

Author(s):

Ahmed Z Balboula ◽

Karen Schindler ◽

Tomoya Kotani ◽

Manabu Kawahara ◽

Masashi Takahashi

Keyword(s):

Cathepsin B ◽

Spindle Assembly Checkpoint ◽

Caspase 3 ◽

Polar Body ◽

Spindle Assembly ◽

Oocyte Quality ◽

Developmental Competence ◽

Dependent Manner ◽

Mouse Oocytes ◽

First Polar Body

Abstract As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus–oocyte complexes were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential SAC protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step toward improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.

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Cyclin B3 is required for metaphase to anaphase transition in oocyte meiosis I

The Journal of Cell Biology ◽

10.1083/jcb.201808088 ◽

2019 ◽

Vol 218 (5) ◽

pp. 1553-1563 ◽

Cited By ~ 18

Author(s):

Yufei Li ◽

Leyun Wang ◽

Linlin Zhang ◽

Zhengquan He ◽

Guihai Feng ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Cell Cycle Control ◽

Spindle Assembly ◽

Specific Cell ◽

Anaphase Promoting Complex ◽

Cyclin Dependent Kinases ◽

Oocyte Meiosis ◽

Cyclin B3 ◽

Meiosis I

Meiosis with a single round of DNA replication and two successive rounds of chromosome segregation requires specific cyclins associated with cyclin-dependent kinases (CDKs) to ensure its fidelity. But how cyclins control the distinctive meiosis is still largely unknown. In this study, we explored the role of cyclin B3 in female meiosis by generating Ccnb3 mutant mice via CRISPR/Cas9. Ccnb3 mutant oocytes characteristically arrested at metaphase I (MetI) with normal spindle assembly and lacked enough anaphase-promoting complex/cyclosome (APC/C) activity, which is spindle assembly checkpoint (SAC) independent, to initiate anaphase I (AnaI). Securin siRNA or CDK1 inhibitor supplements rescued the MetI arrest. Furthermore, CCNB3 directly interacts with CDK1 to exert kinase function. Besides, the MetI arrest oocytes had normal development after intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA), along with releasing the sister chromatids, which implies that Ccnb3 exclusively functioned in meiosis I, rather than meiosis II. Our study sheds light on the specific cell cycle control of cyclins in meiosis.

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