The PP2A inhibitor I2PP2A is essential for sister chromatid segregation in oocyte meiosis II.

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

Tension-Induced Error Correction and Not Kinetochore Attachment Status Activates the SAC in an Aurora-B/C-Dependent Manner in Oocytes

International audienceCell 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, 2, 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

Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II

International audiencePartitioning 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

Mouse oocytes depend on BubR1 for proper chromosome segregation but not for prophase I arrest

International audienceMammalian 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

Aurora B/C-dependent phosphorylation promotes Rec8 cleavage in mammalian oocytes

International audienceTo generate haploid gametes, cohesin is removed in a step-wise 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 detecting 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 activity, and identified an aminoacid residue that is phosphorylated in vivo. Accordingly, inhibition of Aurora B/C kinase during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation

Cyclin B3 implements timely vertebrate oocyte arrest for fertilization

Summary 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. Yet, how this arrest is implemented exclusively in meiosis II and not prematurely in meiosis I 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 providing 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 controlling oocyte arrest for fertilization at the correct cell cycle stage, essential for embryo viability

Cyclin B3 implements timely vertebrate oocyte arrest for fertilization

International audienceTo 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