Detection of Separase activity using a cleavage sensor in live mouse oocytes Detection of Separase activity using a cleavage sensor in live mouse oocytes.

International audienceSeparase proteolytically removes Cohesin complexes from sister chromatid arms, which is essential for chromosome segregation. Regulation of Separase activity is essential for proper cell cycle progression and correct chromosome segregation. Onset of Separase activity has not yet been observed in live oocytes. We describe here a method for detecting Separase activity in mouse oocytes in vivo. This method utilizes a previously described cleavage sensor made up of H2B-mCherry fused with Scc1(107-268 aa)-YFP. The cleavage sensor is loaded on the chromosomes through its H2B tag, and the signal from both mCherry and YFP is visible. Upon Separase activation the Scc1 fragment is cleaved and YFP dissociates from the chromosomes. The change in the ratio between mCherry and YFP fluorescence intensity is a readout of Separase activity

Changing Mad2 Levels Affects Chromosome Segregation and Spindle Assembly Checkpoint Control in Female Mouse Meiosis I

The spindle assembly checkpoint (SAC) ensures correct separation of sister chromatids in somatic cells and provokes a cell cycle arrest in metaphase if one chromatid is not correctly attached to the bipolar spindle. Prolonged metaphase arrest due to overexpression of Mad2 has been shown to be deleterious to the ensuing anaphase, leading to the generation of aneuploidies and tumorigenesis. Additionally, some SAC components are essential for correct timing of prometaphase. In meiosis, we and others have shown previously that the Mad2-dependent SAC is functional during the first meiotic division in mouse oocytes. Expression of a dominant-negative form of Mad2 interferes with the SAC in metaphase I, and a knock-down approach using RNA interference accelerates anaphase onset in meiosis I. To prove unambigiously the importance of SAC control for mammalian female meiosis I we analyzed oocyte maturation in Mad2 heterozygote mice, and in oocytes overexpressing a GFP-tagged version of Mad2. In this study we show for the first time that loss of one Mad2 allele, as well as overexpression of Mad2 lead to chromosome missegregation events in meiosis I, and therefore the generation of aneuploid metaphase II oocytes. Furthermore, SAC control is impaired in mad2+/− oocytes, also leading to the generation of aneuploidies in meiosis I

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

Sister chromatid segregation in meiosis II: Deprotection through phosphorylation

International audienceMeiotic divisions (meiosis I and II) are specialized cell divisions to generate haploid gametes. The first meiotic division with the separation of chromosomes is named reductional division. The second division, which takes place immediately after meiosis I without intervening S-phase, is equational, with the separation of sister chromatids, similar to mitosis. This meiotic segregation pattern requires the two-step removal of the cohesin complex holding sister chromatids together: cohesin is removed from chromosome arms that have been subjected to homologous recombination in meiosis I and from the centromere region in meiosis II. Cohesin in the centromere region is protected from removal in meiosis I, but this protection has to be removeddeprotectedfor sister chromatid segregation in meiosis II. Whereas the mechanisms of cohesin protection are quite well understood, the mechanisms of deprotection have been largely unknown until recently. In this review I summarize our current knowledge on cohesin deprotection

Separase Control and Cohesin Cleavage in Oocytes: Should I Stay or Should I Go?

The key to gametogenesis is the proper execution of a specialized form of cell division named meiosis. Prior to the meiotic divisions, the recombination of maternal and paternal chromosomes creates new genetic combinations necessary for fitness and adaptation to an ever-changing environment. Two rounds of chromosome segregation -meiosis I and II- have to take place without intermediate S-phase and lead to the creation of haploid gametes harboring only half of the genetic material. Importantly, the segregation patterns of the two divisions are fundamentally different and require adaptation of the mitotic cell cycle machinery to the specificities of meiosis. Separase, the enzyme that cleaves Rec8, a subunit of the cohesin complex constituting the physical connection between sister chromatids, has to be activated twice: once in meiosis I and immediately afterwards, in meiosis II. Rec8 is cleaved on chromosome arms in meiosis I and in the centromere region in meiosis II. This step-wise cohesin removal is essential to generate gametes of the correct ploidy and thus, embryo viability. Hence, separase control and Rec8 cleavage must be perfectly controlled in time and space. Focusing on mammalian oocytes, this review lays out what we know and what we still ignore about this fascinating mechanism

Cycling through mammalian meiosis: B-type Cyclins in oocytes

International audienceB-type cyclins in association with Cdk1 mediate key steps of mitosis and meiosis, by phosphorylating a plethora of substrates. Progression through the meiotic cell cycle requires the execution of two cell divisions named meiosis I and II without intervening S-phase, to obtain haploid gametes. These two divisions are highly asymmetric in the large oocyte. Chromosome segregation in meiosis I and sister chromatid segregation in meiosis II requires the sharp, switch-like inactivation of Cdk1 activity, which is brought about by degradation of B-type cyclins and counteracting phosphatases. Importantly and contrary to mitosis, inactivation of Cdk1 must not allow S-phase to take place at exit from meiosis I. Here, we describe recent studies on the regulation of translation and degradation of B-type cyclins in mouse oocytes, and how far their roles are redundant or specific, with a special focus on the recently discovered oocyte-specific role of cyclin B3

Protocol to measure cleavage efficiency of the meiotic cohesin subunit Rec8 by separase in mouse oocytes using a biosensor

International audienceHere we describe a biosensor to assess meiotic cohesin cleavage by live imaging in mouse oocytes. The biosensor is targeted to chromosomes and consists of two fluorophores flanking a fragment of Rec8 containing Separase cleavage sites. Cleavage of Rec8 leads to dissociation of one of the fluorophores from chromosomes which is used to estimate cleavage efficiency. We detail the use of this biosensor in mouse oocytes completing the first meiotic division in presence or absence of AuroraB/C inhibitor. For details on the application of this protocol, please refer to (Nikalayevich et al., 2022)

Spindle assembly checkpoint control in meiosis (the roles of Mad2 and Mps1 in mouse oocytes)

En mitose, les chromatides sœurs sont ségrégées tandis qu en méiose I ce sont les chromosomes homologues qui sont séparés. Des erreurs de ségrégation des chromosomes mènent à des aneuploïdies qui peuvent avoir des conséquences sévères. En mitose, la ségrégation équivalente des chromosomes dépend d un système de surveillance appelé point de contrôle du fuseau (ou SAC) qui retarde l entrée en anaphase jusqu à ce que l alignement et l orientation des chromatides sœurs sur le fuseau soient corrects. Le SAC est fonctionnel en méiose I mais la manière dont il contrôle l alignement et l orientation correcte des homologues sur le fuseau demeure inconnue. J ai analysé les rôles des protéines du SAC mitotique 1) Mad2 et 2) Mps1 en méiose I dans l ovocyte de souris par des approches génétiques. 1) Nous montrons que l haploinsuffisance aussi bien que la surexpression de Mad2 conduisent à des aneuploïdies en méiose I. Nous mettons en évidence une corrélation entre le taux d ovocytes Mad2+/- aneuploïdes et la baisse de la fertilité observée chez les femelles correspondantes. Les niveaux protéiques de Mad2 doivent donc êtres rigoureusement régulés pour assurer la ségrégation équivalente des chromosomes en méiose I. 2) Nous avons étudié le rôle encore inconnu de la kinase Mps1 en méiose I, en exprimant de manière conditionnelle une forme mutante de la protéine spécifiquement dans l ovocyte. Nos résultats prouvent que Mps1 est requise pour le SAC et la durée de la méiose I, et également pour la réparation des erreurs d attachement des homologues au fuseau. La perte de ces fonctions de Mps1 génère des aneuploïdies en méiose I et induit une baisse sévère de fertilitéPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF