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

Date Published: November 28, 2007

Publisher: Public Library of Science

Author(s): Théodora Niault, Khaled Hached, Rocío Sotillo, Peter K. Sorger, Bernard Maro, Robert Benezra, Katja Wassmann, Mikhail Blagosklonny.

Abstract: 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.

Partial Text: Chromosome missegregation events in mammalian female meiosis have severe consequences, leading to a drop in fertility due to the development of aneuploid embryos followed by spontaneous abortions. In humans, chromosome segregation errors in female meiosis I can lead to the development of trisomies, such as trisomy 21 [1]. The SAC has been shown to be required for the fidelity of chromosome segregation in mitosis, verifying the correct attachment of each pair of sister chromatids to the opposite poles of the bipolar spindle via their kinetochores [2]. The presence of a single erroneously attached kinetochore activates the SAC and leads to a metaphase arrest to permit repair of the attachment. Components of the SAC include the Mad (Mitotic Arrest Deficient) and Bub (Budding Uninhibited by Benomyl) proteins (Mad1-3, Bub1, BubR1, Bub3), as well as Mps1 (Monopolar Spindle 1), CenpE, and others [2]. Most SAC proteins are localized to unattached kinetochores in mitosis. Mad2 has a key role in that it directly inhibits the ubiquitination activity of the Anaphase Promoting Complex/Cyclosome (APC/C) in association with its activator Cdc20, and therefore anaphase onset. Once the SAC is inactivated, APC/C-Cdc20 ubiquitinates Securin and Cyclin B, targeting both proteins for degradation by the 26S proteasome. Securin is a protein inhibitor of a protease named Separase. Degradation of Securin leads to the activation of Separase which removes the cohesin complex holding sister chromatids together by cleaving one of its subunits, Scc1 [3]. Cdk1-Cyclin B complexes also inhibit Separase function and exit from mitosis, therefore Cyclin B needs to be degraded as well [4]. Hence inhibition of APC/C-Cdc20 by the SAC prevents sister chromatid separation in mitosis. In addition to its role at the metaphase-to-anaphase transition it has been shown that the SAC components Mad2 and BubR1 are implicated in the correct timing of prometaphase length. Knockdown of either protein leads to accelerated anaphase onset independent of their role in SAC control upon spindle disruption in metaphase, and independent of kinetochore attachment of both proteins [5].

To prove the importance of SAC control for female mouse meiosis I we examined the first meiotic division in oocytes derived from mad2+/− mice, compared to mad2+/+ littermates. Denuded Germinal Vesicle (GV) stage oocytes arrested in prophase of meiosis I (after chiasmata have been formed and recombination has taken place) are collected from the ovaries of adult female mice. They can be induced to undergo meiosis I in culture in a completely synchronized manner. Cell division in meiosis I is asymmetric, and visible due to the extrusion of a small Polar Body (PB). Upon completion of meiosis I oocytes progress into metaphase of meiosis II where they remain arrested until fertilization occurs (Figure 1A). Control (littermates) and mad2+/− oocytes were analyzed by time lapse video microscopy to visualize chromosome movements (DNA was stained with Hoechst) and Polar Body Extrusion (PBE) with Phase Contrast (DIC) (Figure 1B). Photon excitation to visualize Hoechst is eventually toxic for oocytes, therefore we also followed meiotic maturation of untreated oocytes by determining the time of PBE through observation with a binocular microscope. Control oocytes of this strain extrude PBs 7.5 to 9.5 hrs after GVBD (Germinal Vesicle Breakdown, corresponds to Nuclear Envelope Breakdown in mitosis), with a peak time average at 8 h50 min (530 min), whereas PBE was significantly accelerated by 33 min on average (8 h17 min–497 min) in mad2+/− oocytes (Figure 1C, D). Meiosis I is 6,3% shorter in mad2+/− oocytes, which is comparable to the 10% decrease in the duration of mitosis observed after Mad2 RNAi [5]. We therefore conclude that Mad2 is important for correct timing of prometaphase in meiosis I, just like in mitosis [5].

Our model in Figure 5D illustrates how we explain the generation of aneuploidies in meiosis I upon changing Mad2 levels in mouse oocytes: Reducing Mad2 levels leads to an acceleration of meiosis I, not leaving enough time for proper attachment of the bipolar spindle to functional kinetochores in metaphase I. SAC function is affected, and therefore oocytes with unattached kinetochores will eventually progress into meiosis II. Consistent with our observation a correlation between the concentration of transcripts of SAC components, and missegregation events in human oocytes has been described: the authors show that there seems to be an age-dependent decrease in Mad2 and Bub1 transcripts that may potentially underlie the increased incidence of aneuploidies in older women [23].