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1.
Dev Cell ; 57(19): 2305-2320.e6, 2022 10 10.
Article in English | MEDLINE | ID: mdl-36182686

ABSTRACT

To ensure successful offspring ploidy, vertebrate oocytes must halt the cell cycle in meiosis II until sperm entry. Emi2 is essential to keep oocytes arrested until fertilization. However, how this arrest is implemented exclusively in meiosis II and not prematurely in meiosis I has until now remained enigmatic. Using mouse and frog oocytes, we show here that cyclin B3, an understudied B-type cyclin, is essential to keep Emi2 levels low in meiosis I. Direct phosphorylation of Emi2 at an evolutionarily highly conserved site by Cdk1/cyclin B3 targets Emi2 for degradation. In contrast, Cdk1/cyclin B1 is inefficient in Emi2 phosphorylation, and this provides a molecular explanation for the requirement of different B-type cyclins for oocyte maturation. Cyclin B3 degradation at exit from meiosis I enables Emi2 accumulation and thus timely arrest in meiosis II. Our findings illuminate the evolutionarily conserved mechanisms that control oocyte arrest for fertilization at the correct cell-cycle stage, which is essential for embryo viability.


Subject(s)
F-Box Proteins , Animals , Cyclin B/metabolism , Cyclin B1 , Cyclins/metabolism , F-Box Proteins/genetics , Fertilization , Male , Meiosis , Mice , Oocytes/metabolism , Semen/metabolism , Vertebrates/metabolism
2.
Curr Biol ; 32(10): 2281-2290.e4, 2022 05 23.
Article in English | MEDLINE | ID: mdl-35385691

ABSTRACT

To generate haploid gametes, cohesin is removed in a stepwise manner from chromosome arms in meiosis I and the centromere region in meiosis II to segregate chromosomes and sister chromatids, respectively. Meiotic cohesin removal requires cleavage of the meiosis-specific kleisin subunit Rec8 by the protease separase.1,2 In yeast and C. elegans, Rec8 on chromosome arms has to be phosphorylated to be cleaved in meiosis I,3-7 whereas Rec8 at the centromere is protected from cleavage by the action of PP2A-B56.8-10 However, in mammalian meiosis, it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and if so, the identity of the relevant kinase(s). This is due to technical challenges, as Rec8 is poorly conserved, preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals. Additionally, there is no turnover of Rec8 after cohesion establishment, preventing phosphomutant analysis of functional Rec8. To address the very basic question of whether Rec8 cleavage requires its phosphorylation in mammals, we adapted a biosensor that detects separase activity to study Rec8 cleavage in single mouse oocytes by live imaging. Crucially, through phosphomutant analysis, we identified phosphorylation sites in Rec8 promoting cleavage. We found that Rec8 cleavage depends on Aurora B/C kinase activities and identified an aminoacid residue that is phosphorylated in vivo. Accordingly, inhibition of Aurora B/C kinases during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation.


Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Cell Cycle Proteins/metabolism , Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , Mammals/genetics , Meiosis , Mice , Oocytes/metabolism , Phosphorylation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Separase/metabolism
3.
EMBO J ; 40(7): e106797, 2021 04 01.
Article in English | MEDLINE | ID: mdl-33644892

ABSTRACT

Partitioning of the genome in meiosis occurs through two highly specialized cell divisions, named meiosis I and meiosis II. Step-wise cohesin removal is required for chromosome segregation in meiosis I, and sister chromatid segregation in meiosis II. In meiosis I, mono-oriented sister kinetochores appear as fused together when examined by high-resolution confocal microscopy, whereas they are clearly separated in meiosis II, when attachments are bipolar. It has been proposed that bipolar tension applied by the spindle is responsible for the physical separation of sister kinetochores, removal of cohesin protection, and chromatid separation in meiosis II. We show here that this is not the case, and initial separation of sister kinetochores occurs already in anaphase I independently of bipolar spindle forces applied on sister kinetochores, in mouse oocytes. This kinetochore individualization depends on separase cleavage activity. Crucially, without kinetochore individualization in meiosis I, bivalents when present in meiosis II oocytes separate into chromosomes and not sister chromatids. This shows that whether centromeric cohesin is removed or not is determined by the kinetochore structure prior to meiosis II.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Kinetochores/metabolism , Meiosis , Animals , Cells, Cultured , Chromatids/genetics , Chromatids/metabolism , Female , Mice , Mice, Inbred C57BL , Oocytes/metabolism , Cohesins
4.
Curr Biol ; 28(1): 130-139.e3, 2018 01 08.
Article in English | MEDLINE | ID: mdl-29276128

ABSTRACT

Cell division with partitioning of the genetic material should take place only when paired chromosomes named bivalents (meiosis I) or sister chromatids (mitosis and meiosis II) are correctly attached to the bipolar spindle in a tension-generating manner. For this to happen, the spindle assembly checkpoint (SAC) checks whether unattached kinetochores are present, in which case anaphase onset is delayed to permit further establishment of attachments. Additionally, microtubules are stabilized when they are attached and under tension. In mitosis, attachments not under tension activate the so-named error correction pathway depending on Aurora B kinase substrate phosphorylation. This leads to microtubule detachments, which in turn activates the SAC [1-3]. Meiotic divisions in mammalian oocytes are highly error prone, with severe consequences for fertility and health of the offspring [4, 5]. Correct attachment of chromosomes in meiosis I leads to the generation of stretched bivalents, but-unlike mitosis-not to tension between sister kinetochores, which co-orient. Here, we set out to address whether reduction of tension applied by the spindle on bioriented bivalents activates error correction and, as a consequence, the SAC. Treatment of oocytes in late prometaphase I with Eg5 kinesin inhibitor affects spindle tension, but not attachments, as we show here using an optimized protocol for confocal imaging. After Eg5 inhibition, bivalents are correctly aligned but less stretched, and as a result, Aurora-B/C-dependent error correction with microtubule detachment takes place. This loss of attachments leads to SAC activation. Crucially, SAC activation itself does not require Aurora B/C kinase activity in oocytes.


Subject(s)
Kinetochores/physiology , M Phase Cell Cycle Checkpoints/physiology , Meiosis/physiology , Oocytes/physiology , Animals , Cell Division/drug effects , Cell Division/physiology , Cysteine/analogs & derivatives , Cysteine/pharmacology , Female , Kinesins/antagonists & inhibitors , Kinetochores/drug effects , M Phase Cell Cycle Checkpoints/drug effects , Mice , Oocytes/drug effects , Paclitaxel/pharmacology , Pyrimidines/pharmacology , Thiones/pharmacology , Tubulin Modulators/pharmacology
5.
Nat Commun ; 8(1): 694, 2017 09 25.
Article in English | MEDLINE | ID: mdl-28947820

ABSTRACT

A key feature of meiosis is the step-wise removal of cohesin, the protein complex holding sister chromatids together, first from arms in meiosis I and then from the centromere region in meiosis II. Centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage, in order to maintain sister chromatids together until their separation in meiosis II. Failures in step-wise cohesin removal result in aneuploid gametes, preventing the generation of healthy embryos. Here, we report that kinase activities of Bub1 and Mps1 are required for Sgo2 localisation to the centromere region. Mps1 inhibitor-treated oocytes are defective in centromeric cohesin protection, whereas oocytes devoid of Bub1 kinase activity, which cannot phosphorylate H2A at T121, are not perturbed in cohesin protection as long as Mps1 is functional. Mps1 and Bub1 kinase activities localise Sgo2 in meiosis I preferentially to the centromere and pericentromere respectively, indicating that Sgo2 at the centromere is required for protection.In meiosis I centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage ensuring that sister chromatids are kept together until their separation in meiosis II. Here the authors demonstrate that Bub1 and Mps1 kinase activities are required for Sgo2 localisation to the centromere region.


Subject(s)
Cell Cycle Proteins/metabolism , Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Meiosis/physiology , Oocytes/cytology , Protein Serine-Threonine Kinases/physiology , Animals , Cell Cycle Proteins/analysis , Centromere/ultrastructure , Mice , Oocytes/metabolism , Oocytes/ultrastructure , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Cohesins
6.
Methods ; 115: 17-27, 2017 02 15.
Article in English | MEDLINE | ID: mdl-27826080

ABSTRACT

In the presented work we aimed at improving confocal imaging to obtain highest possible resolution in thick biological samples, such as the mouse oocyte. We therefore developed an image processing workflow that allows improving the lateral and axial resolution of a standard confocal microscope. Our workflow comprises refractive index matching, the optimization of microscope hardware parameters and image restoration by deconvolution. We compare two different deconvolution algorithms, evaluate the necessity of denoising and establish the optimal image restoration procedure. We validate our workflow by imaging sub resolution fluorescent beads and measuring the maximum lateral and axial resolution of the confocal system. Subsequently, we apply the parameters to the imaging and data restoration of fluorescently labelled meiotic spindles of mouse oocytes. We measure a resolution increase of approximately 2-fold in the lateral and 3-fold in the axial direction throughout a depth of 60µm. This demonstrates that with our optimized workflow we reach a resolution that is comparable to 3D-SIM-imaging, but with better depth penetration for confocal images of beads and the biological sample.


Subject(s)
Image Processing, Computer-Assisted/methods , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Oocytes/ultrastructure , Spindle Apparatus/ultrastructure , Algorithms , Animals , Female , Fluorescent Dyes/chemistry , Image Processing, Computer-Assisted/statistics & numerical data , Meiosis , Mice , Microscopy, Confocal/instrumentation , Microscopy, Fluorescence/instrumentation , Microspheres , Primary Cell Culture , Workflow
7.
Nat Commun ; 6: 6946, 2015 Apr 21.
Article in English | MEDLINE | ID: mdl-25897860

ABSTRACT

Mammalian female meiosis is error prone, with rates of meiotic chromosome missegregations strongly increasing towards the end of the reproductive lifespan. A strong reduction of BubR1 has been observed in oocytes of women approaching menopause and in ovaries of aged mice, which led to the hypothesis that a gradual decline of BubR1 contributes to age-related aneuploidization. Here we employ a conditional knockout approach in mouse oocytes to dissect the meiotic roles of BubR1. We show that BubR1 is required for diverse meiotic functions, including persistent spindle assembly checkpoint activity, timing of meiosis I and the establishment of robust kinetochore-microtubule attachments in a meiosis-specific manner, but not prophase I arrest. These data reveal that BubR1 plays a multifaceted role in chromosome segregation during the first meiotic division and suggest that age-related decline of BubR1 is a key determinant of the formation of aneuploid oocytes as women approach menopause.


Subject(s)
Cell Cycle Checkpoints/physiology , Cell Cycle Proteins/metabolism , Chromosome Segregation/physiology , Meiotic Prophase I/physiology , Oocytes/physiology , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Cycle Proteins/genetics , Female , Leviviridae , Mice , Mice, Inbred Strains , Protein Serine-Threonine Kinases/genetics
8.
Curr Biol ; 23(6): 485-90, 2013 Mar 18.
Article in English | MEDLINE | ID: mdl-23434280

ABSTRACT

Haploid gametes are generated through two consecutive meiotic divisions, with the segregation of chromosome pairs in meiosis I and sister chromatids in meiosis II. Separase-mediated stepwise removal of cohesion, first from chromosome arms and later from the centromere region, is a prerequisite for maintaining sister chromatids together until their separation in meiosis II [1]. In all model organisms, centromeric cohesin is protected from separase-dependent removal in meiosis I through the activity of PP2A-B56 phosphatase, which is recruited to centromeres by shugoshin/MEI-S332 (Sgo) [2-5]. How this protection of centromeric cohesin is removed in meiosis II is not entirely clear; we find that all the PP2A subunits remain colocalized with the cohesin subunit Rec8 at the centromere of metaphase II chromosomes. Here, we show that sister chromatid separation in oocytes depends on a PP2A inhibitor, namely I2PP2A. I2PP2A colocalizes with the PP2A enzyme at centromeres at metaphase II, independently of bipolar attachment. When I2PP2A is depleted, sister chromatids fail to segregate during meiosis II. Our findings demonstrate that in oocytes I2PP2A is essential for faithful sister chromatid segregation by mediating deprotection of centromeric cohesin in meiosis II.


Subject(s)
Ciona intestinalis/physiology , Meiosis , Protein Phosphatase 2/metabolism , Sister Chromatid Exchange , Transcription Factors/metabolism , Animals , Ciona intestinalis/genetics , Male , Mice , Oocytes/physiology , Polymerase Chain Reaction
9.
Cell Rep ; 2(5): 1077-87, 2012 Nov 29.
Article in English | MEDLINE | ID: mdl-23122964

ABSTRACT

In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis.


Subject(s)
Cell Cycle Proteins/metabolism , Chromatids/metabolism , Cyclin A2/metabolism , Endopeptidases/metabolism , Meiosis , Oocytes/metabolism , Anaphase , Animals , Carrier Proteins/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cells, Cultured , Centromere/metabolism , Chromosome Segregation , Cyclin A2/antagonists & inhibitors , Cyclin A2/genetics , Cyclin B1/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Kinetochores/metabolism , Metaphase , Mice , Oocytes/cytology , Securin , Separase
10.
Development ; 138(11): 2261-71, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21558374

ABSTRACT

In female meiosis, chromosome missegregations lead to the generation of aneuploid oocytes and can cause the development of trisomies or infertility. Because mammalian female meiosis I is error prone, the full functionality of control mechanisms, such as the spindle assembly checkpoint (SAC), has been put into question. The SAC monitors the correct orientation, microtubule occupancy and tension on proteinaceous structures named kinetochores. Although it has been shown previously that the SAC exists in meiosis I, where attachments are monopolar, the role of microtubule occupancy for silencing the SAC and the importance of certain essential SAC components, such as the kinase Mps1, are unknown in mammalian oocytes. Using a conditional loss-of-function approach, we address the role of Mps1 in meiotic progression and checkpoint control in meiosis I. Our data demonstrate that kinetochore localization of Mps1 is required for the proper timing of prometaphase and is essential for SAC control, chromosome alignment and aurora C localization in meiosis I. The absence of Mps1 from kinetochores severely impairs chromosome segregation in oocyte meiosis I and, therefore, fertility in mice. In addition, we settle a long-standing question in showing that kinetochore-microtubule attachments are present in prometaphase I at a time when most of the SAC protein Mad2 disappears from kinetochores.


Subject(s)
Cell Cycle Proteins/metabolism , Kinetochores/metabolism , Meiosis/physiology , Protein Serine-Threonine Kinases/metabolism , Animals , Chromosome Segregation , Female , Fluorescent Antibody Technique , Mad2 Proteins , Mice , Mice, Transgenic , Microtubules/metabolism , Oocytes/cytology , Oocytes/metabolism , Polymerase Chain Reaction , Spindle Apparatus/metabolism
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