RESEARCH REPORT 1941 Development 139, 1941-1946 (2012) doi:10.1242/dev.078352 © 2012. Published by The Company of Biologists Ltd Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes Liming Gui1 and Hayden Homer1,2,* SUMMARY The spindle assembly checkpoint (SAC) averts aneuploidy by coordinating proper bipolar chromosomal attachment with anaphase-promoting complex/cyclosome (APC/C)-mediated securin and cyclin B1 destruction required for anaphase onset. The generation of a Mad2-based signal at kinetochores is central to current models of SAC-based APC/C inhibition. During mitosis, kinetochores of polar-displaced chromosomes, which are at greatest risk of mis-segregating, recruit the highest levels of Mad2, thereby ensuring that SAC activation is proportionate to aneuploidy risk. Paradoxically, although an SAC operates in mammalian oocytes, meiosis I (MI) is notoriously error prone and polar-displaced chromosomes do not prevent anaphase onset. Here we find that Mad2 is not preferentially recruited to the kinetochores of polar chromosomes of wild-type mouse oocytes, in which polar chromosomes are rare, or of oocytes depleted of the kinesin-7 motor CENP-E, in which polar chromosomes are more abundant. Furthermore, in CENP-E-depleted oocytes, although polar chromosomal displacement intensified during MI and the capacity to form stable end-on attachments was severely compromised, all kinetochores nevertheless became devoid of Mad2. Thus, it is possible that the ability of the SAC to robustly discriminate chromosomal position might be compromised by the propensity of oocyte kinetochores to become saturated with unproductive attachments, thereby predisposing to aneuploidy. Our data also reveal novel functions for CENP-E in oocytes: first, CENP-E stabilises BubR1, thereby impacting MI progression; and second, CENP-E mediates bi-orientation by promoting kinetochore reorientation and preventing chromosomal drift towards the poles. KEY WORDS: Aneuploidy, CENP-E, Mad2, Meiosis I, Mouse oocytes, Spindle assembly checkpoint INTRODUCTION Morpholino and cRNA injection Mad2 recruitment to improperly attached kinetochores is crucial For CENP-E depletion, germinal vesicle stage oocytes were microinjected for generating the inhibitory spindle assembly checkpoint (SAC) with a morpholino designed to target mouse Cenpe (NM_173762) signal that prevents anaphase-promoting complex/cyclosome designated CENPEMO (5Ј-CAGCCACTGAAGCCTCCTCGGCCAT-3Ј; (APC/C) activation and anaphase onset (Musacchio and Salmon, Gene Tools) and maintained for 20-24 hours in isobutylmethylxanthine (IBMX)-treated medium. Mad2MO, ControlMO (for mock depletions) and 2007). During mitosis, kinetochores of polar chromosomes, which human BUBR1 cRNA were described previously (Homer et al., 2009; are at greatest risk of mis-segregating, recruit the highest levels of Homer et al., 2005b). Morpholinos were microinjected at 2 mM. For Mad2 (Howell et al., 2000; Waters et al., 1998), thereby tightly double depletions, combinations of morpholinos (4 mM stock) were coupling SAC activation to aneuploidy risk. microinjected. Paradoxically, in spite of possessing an SAC (Hached et al., 2011; Homer et al., 2005b; McGuinness et al., 2009), female Immunocytochemistry meiosis I (MI) remains notoriously error prone (Hassold and Hunt, Immunofluorescence and cold treatment (4°C for 10 minutes) were performed as described previously (Homer et al., 2009). Primary antibodies 2009). An important cause for this vulnerability is the inability of included -tubulin (Sigma); ACA (ImmunoVision); g-tubulin (Abcam); small numbers of polar chromosomes to prevent anaphase onset in CENP-E (Dr T. Yen, Fox Chase Cancer Center, USA); BubR1 (Dr Stephen oocytes (Nagaoka et al., 2011). Exactly why polar chromosomes Taylor, University of Manchester, UK) and Mad2 (Dr K. Wassmann, should evade the SAC remains unknown, especially as recent data CNRS UMR7622, France). Secondary antibodies (Invitrogen) included indicate that the oocyte SAC has the capacity to react to even a Alexa Fluor 488- or 546-labelled goat anti-human; Alexa Fluor 633- or single unattached chromosome (Hoffmann et al., 2011). Here we 488-labelled goat anti-mouse; Alexa Fluor 488- or 546-labelled goat anti- address the key issue of how the SAC in oocytes responds to polar rabbit; and Alexa Fluor 488-labelled goat anti-sheep. DNA was stained chromosomes. using Hoechst 33342 (10 g/ml; Sigma). Images were captured using a Zeiss LSM510 META confocal microscope configured as follows: for MATERIALS AND METHODS Hoechst 33342, 364 nm UV laser excitation combined with a 385-470 nm Oocyte collection and drug treatment band-pass emission filter; for Alexa Fluor 488, 488 nm argon laser line Oocytes were isolated from 4- to 6-week-old pregnant mare serum combined with a 505-550 nm band-pass emission filter; for Alexa Fluor gonadotropin (PMSG)-primed MF1 mice and cultured as described (Homer 546, 543 nm helium/neon1 laser combined with a 560-615 nm band-pass et al., 2009). Nocodazole (Sigma) was used at 5 M (Homer et al., 2005a). emission filter; and for Alexa Fluor 633, a 633 nm helium/neon2 laser with Experiments involving animals conformed to the relevant regulatory a 650 nm long-pass emission filter. standards. Western blotting 1 2 Pre-cast 3-8% Tris-acetate gels (Invitrogen) and a mouse monoclonal anti- Cell and Developmental Biology, Institute for Women’s Health, University College CENP-E antibody (Abcam) were used for CENP-E. BubR1, securin, London, London WC1E 6BT, UK. GAPDH and actin immunoblotting were performed as described (Homer *Author for correspondence ([email protected]) et al., 2009; Homer, 2011). HRP-conjugated antibodies were detected using ECL Plus (GE Healthcare) and protein bands were semi-quantitatively Accepted 2 April 2012 assayed (Homer et al., 2009). DEVELOPMENT 1942 RESEARCH REPORT Development 139 (11) RESULTS AND DISCUSSION Consistent with previous results (Kitajima et al., 2011), we found Mad2 is not overtly enriched at the kinetochores that kinetochore levels of Mad2 (Mad2l1 – Mouse Genome of polar bivalents in wild-type oocytes Informatics) declined during MI (Fig. 1A,D). Additionally, by In mouse oocytes, MI lasts ~8-10 hours, beginning with germinal focusing on the stage during which the spindle was bipolar, we were vesicle breakdown (GVBD) and concluding with first polar body now able to compare Mad2 recruitment to equatorial and polar extrusion (PBE), when recombined homologous chromosomes bivalents. Significantly, low levels of Mad2 were retained at many (bivalents) segregate (Homer et al., 2009; Homer et al., 2005b). equatorial bivalents by 6 hours post-GVBD (when the spindle first During this time, numerous microtubule-organising centres nucleate becomes bipolar), and there was an additional ~2 hours before a spindle, which is gradually remodelled into a bipolar form (Schuh complete Mad2 displacement (Fig. 1A,D). Strikingly, among the few and Ellenberg, 2007). As polar chromosomes constitute an important oocytes with severely polar-displaced bivalents at 6 hours post- focus of the SAC, we first determined when bipolarity was GVBD, Mad2 decorated kinetochores of both equatorial and polar established. Using strict criteria, we found that bipolarity was not bivalents to a similar degree (Fig. 1E-Eٞ). Furthermore, by 8 hours established until ~6 hours post-GVBD (supplementary material Fig. post-GVBD, all kinetochores completely lacked Mad2 (Fig. 1A,F), S1A,B). Also, consistent with recent data (Kitajima et al., 2011), we even when polar bivalents were present (Fig. 1G). Collectively, this found that kinetochores reoriented to face in opposite directions by represented a marked departure from the mitotic template in which 6 hours post-GVBD (supplementary material Fig. S1C-E), beyond polar-displaced kinetochores retain high levels of Mad2 and the which time less than 5% of oocytes (n>150) possessed severely attainment of an equatorial chromosomal position is promptly displaced polar bivalents, entirely in keeping with previous results followed by complete loss of Mad2 (Hoffman et al., 2001; Howell (Kitajima et al., 2011; Yang et al., 2010). et al., 2000; Waters et al., 1998). Fig. 1. Equatorial location and K-fibre formation are not prerequisites for Mad2 displacement. (A-C)z-projections of mouse oocytes immunostained for BubR1, Mad2 and -tubulin. DNA was stained using Hoechst 33342. (D)Quantification of kinetochore Mad2 and BubR1. Oocytes were double labelled for Mad2 or BubR1 plus anti- centromere antibody (ACA) at all four time points on the same day and z-stacks were acquired using identical settings within a subvolume spanning the entire kinetochore- containing region as illustrated. Background- corrected total integrated fluorescence intensity for a region of interest (ROI) was determined at ACA foci and for the corresponding ROI in the Mad2 and BubR1 channels. Mad2:ACA and BubR1:ACA ratios were determined for more than 200 kinetochores per time point (three experiments) and normalised to the intensity at 2 hours post-GVBD. Data are mean ± s.e.m.; *P<0.05 by Student’s t-test. (E-Eٞ) An oocyte at 6 hours post-GVBD shows a polar- displaced bivalent (arrow) with levels of Mad2 (EЈ) that are comparable to those at equatorial bivalents (EЉ). (Eٞ)Mad2 fluorescence intensities at polar kinetochores (n8) and equatorial kinetochores (n73) from four oocytes at 6 hours post-GVBD. Data were normalised to the maximal intensity within each oocyte. Data are mean ± s.e.m. (F,G)Mad2 is undetectable by 8