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

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

Arpp19 et ENSA, deux inhibiteurs de la phosphatase PP2A-B55 contrôlent de manière différentielle le cycle cellulaire

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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

Ensa controls S-phase length by modulating Treslin levels.

The Greatwall/Ensa/PP2A-B55 pathway is essential for controlling mitotic substrate phosphorylation and mitotic entry. Here, we investigate the effect of the knockdown of the Gwl substrate, Ensa, in human cells. Unexpectedly, Ensa knockdown promotes a dramatic extension of S phase associated with a lowered density of replication forks. Notably, Ensa depletion results in a decrease of Treslin levels, a pivotal protein for the firing of replication origins. Accordingly, the extended S phase in Ensa-depleted cells is completely rescued by the overexpression of Treslin. Our data herein reveal a new mechanism by which normal cells regulate S-phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.The Greatwall/Ensa/PP2A-B55 pathway controls mitotic substrate phosphorylation and mitotic entry. Here the authors show that cells regulate S phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway

Ensa controls S-phase length by modulating Treslin levels

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Anaphase onset is accelerated in <i>mad2+/−</i> mice.

<p>(A) Entry into the first meiotic division in mouse oocytes is induced by removing dbcAMP from the culture medium. Synchronized oocytes undergo GVBD around 1,5 hours after release, and extrude a PB around 8–9 hours after GVBD. MPF (M-phase promoting factor) activity increases from GVBD until metaphase I, drops when the first PB is extruded, and increases again as oocytes progress into meiosis II. (B) Time lapse video microscopy of oocytes with the indicated phenotype undergoing the first meiotic division. Chromosomes were labelled with Hoechst. Anaphase onset and PBE were observed. Only movies with at least 80% of oocytes extruding a PB at times comparable to control oocytes in the incubator without exposure to Hoechst excitation light were used. (C) Cumulative times of PBE and (D) distribution of PBE (same data set) in <i>mad2+/+</i> (n = 53) and <i>mad2+/−</i> (n = 70) oocytes. The peak time average of PBE in <i>mad2+/−</i> oocytes is significantly earlier (33 min) than in <i>mad2+/+</i> oocytes (497 min and 530 min respectively, p<0,01 with both the T and the U test, p value of the T test (2 tail, type2) = 0,00232, p value of the U test (2 tail) = 0,00572). The results of three independent experiments are shown.</p

Cyclin B and Securin are degraded before PBE in <i>mad2+/−</i> oocytes.

<p>(A) Time lapse video microscopy to establish the time of anaphase onset and PBE. Anaphase onset can sometimes be observed in the timepoint before PBE. (B) Quantitation of GFP-Cyclin B in <i>mad2+/+</i> and <i>mad2+/−</i> oocytes relative to anaphase onset and PBE. Live video analysis shows that GFP-Cyclin B (quantifications of the fluorescence signal are shown as a graph, arbitrary units set to maximal levels of 100) reaches its lowest levels before PBE. Timepoints were taken every 20 minutes, chromosomes were labelled with Hoechst. A representative oocyte is shown and the number of successfully analyzed oocytes is indicated. No differences between anaphase onset, PBE and GFP-Cyclin B degradation were detected between control and <i>mad2+/−</i> oocytes. (C) Quantifications of Securin-YFP fluorescence signal intensities in <i>mad2+/+</i> and <i>mad2+/−</i> oocytes relative to anaphase onset and PBE, as described in (B). No differences between anaphase onset, PBE and Securin-YFP degradation were detected. (D) Kinase assays to assess MPF activity during meiosis I in <i>mad2+/+</i> and <i>mad2+/−</i> oocytes. Histone H1 (H1) was used as a substrate. Control (ctrl): GVBD+8 h (-PB) without substrate addition. (E) Endogenous Securin levels in <i>mad2+/+</i> and <i>mad2+/−</i> oocytes 3 hours after GVBD. 20 oocytes each were used.</p