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 (n8) and equatorial kinetochores (n73) 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 hours post-GVBD regardless of chromosomal position. Note the absence of Mad2 at kinetochores (arrowheads, G) of a polar- displaced bivalent (arrow, G). (H)Cold-stable microtubule content. Immunostained images depict two phenotypes after cold treatment: the ‘cold-instability phenotype’ (microtubule depolymerisation and high Mad2) and the ‘cold-stable phenotype’ (stable microtubules and low Mad2). Scale bars: 10m. DEVELOPMENT SAC in mouse oocytes RESEARCH REPORT 1943

Displacement of the bulk of Mad2 occurs in spite (Fig. 1B,BЈ) before disappearing by late anaphase I (Fig. 1C). of low K-fibre content Overall, these data are consistent with previous analyses showing We next investigated why such a protracted interval elapsed before late K-fibre formation in oocytes (Brunet et al., 1999). Importantly, Mad2 became completely displaced from bivalents that were however, by using two independent measures for K-fibres we could equatorial and bi-oriented. During mitosis, microtubules that form now quantify K-fibre content and correlate this with kinetochore stable end-on attachments with kinetochores (K-fibres) are crucial Mad2 levels specifically during the bipolar stage. for displacing Mad2 from bi-oriented chromosomes (Hoffman et Intriguingly, these data now show that by 6 hours post-GVBD, al., 2001; Putkey et al., 2002). We therefore asked whether K-fibre when the majority of kinetochore Mad2 was displaced, most content differed between 6 and 8 hours post-GVBD. K-fibres oocytes were deficient in K-fibres, pointing to an unusually high impart tension across kinetochores (Deluca et al., 2002) and are propensity for kinetochores to acquire microtubule interactions – differentially stable to cold treatment (Rieder, 1981). We found that that is, even unstable interactions sufficed for displacing Mad2. We cold treatment induced rudimentary spindles and Mad2 re- hypothesized that this unusual propensity could account for the recruitment to kinetochores in ~60% and ~10% of oocytes at 6 and lack of preferential Mad2 recruitment to the small numbers of polar 8 hours post-GVBD, respectively (Fig. 1H). Next, by measuring bivalents in wild-type oocytes, thereby compromising biased SAC inter-kinetochore distances, we found that tension became maximal activation at polar bivalents. at 8 hours post-GVBD (supplementary material Fig. S2), coincident with which, kinetochore BubR1 (Bub1b – Mouse CENP-E depletion induces polar bivalents and Genome Informatics) levels, which are well known to become unstable kinetochore-microtubule interactions depleted in response to tension (Skoufias et al., 2001; Uchida et al., In order to test our hypothesis, we sought conditions under which 2009), declined significantly (Fig. 1A,D). As with mitotic BubR1, unstable kinetochore-microtubule interactions and polar which declines but does not disappear at metaphase (Hoffman et chromosomes were prevalent, two features that characterise mitotic al., 2001), BubR1 persisted at kinetochores by early anaphase I cells deficient for the plus-end-directed kinesin-7 motor CENP-E

Fig. 2. Polar chromosomal displacement and unstable kinetochore-microtubule interactions following CENP-E depletion. (A-D)z-projections of immunostained CENP-E-depleted mouse oocytes. In the oocyte shown in A, compact (yellow arrow) and extended (white arrow) bivalents are displaced from the midline, as more clearly seen in magnified views of separate z-sections (Z1, AЈ and Z2, AЉ) (see also supplementary material Fig. S1C-E). In B, a polar compact bivalent with juxtaposed kinetochores (arrowheads) is magnified. By 8 hours (C) and 10 hours (D) post-GVBD, polewards bivalent displacement becomes increasingly severe (arrows). (E)Proportions of oocytes bearing (1) all aligned chromosomes, (2) chromosomes displaced from the main equatorial group but not severely polar-displaced (e.g. A), and (3) severely displaced polar bivalents (e.g. B-D) were determined at 6, 8 and 10 hours post- GVBD. (F)MII-arrested oocytes. Note the polar bodies (arrowheads) and tight chromosomal alignment in the wild-type oocyte (green arrow). (G)Cold-stable microtubule content after CENP-E depletion. Percentages of ‘cold-stable’ and ‘cold-instability’ phenotypes (see Fig. 1H) at 6, 8 and 10 hours post-GVBD. Depicted is a typical cold-treated CENP-E-depleted oocyte exhibiting spindle collapse and strong Mad2 recruitment. Scale bars: 10m. DEVELOPMENT 1944 RESEARCH REPORT Development 139 (11)

(Putkey et al., 2002). We found that CENP-E undergoes net synthesis and localises to kinetochores until metaphase I, before relocating to the spindle midzone at anaphase I (supplementary material Fig. S3). To evaluate CENP-E function, we depleted CENP-E using a morpholino antisense approach (supplementary material Fig. S4). We found that 93% (n73) of CENP-E-depleted oocytes assembled a bipolar spindle by 6 hours post-GVBD. During the delayed MI transit observed after CENP-E depletion (discussed below), the proportion of oocytes exhibiting severely polar-displaced chromosomes almost trebled (Fig. 2A-E), suggesting that CENP-E- depleted oocytes were unable to restrain bivalents at the equator. Also, 37% (n38) and 40% (n42) of bivalents at 6 and 8 hours post- GVBD, respectively, possessed juxtaposed kinetochores following CENP-E depletion (Fig. 2A,B), indicating that CENP-E was required for kinetochore reorientation and that failure to do so contributed to misalignment. Predictably, among CENP-E-depleted oocytes that exited MI, gross misalignment persisted at meiosis II (MII) (Fig. 2F). Notably, ~70-80% of CENP-E-depleted oocytes chronically lacked prominent cold-stable microtubules (Fig. 2G), consistent with the known role of CENP-E in stabilising kinetochore-microtubule interactions (Putkey et al., 2002). Overall, therefore, following CENP-E depletion, unstable kinetochore-microtubule interactions predominate and polar bivalents are frequent.

Mad2 is not enriched at polar bivalents and becomes completely displaced by unstable attachments after CENP-E depletion Although mean Mad2 intensity declined between 2 and 8 hours post-GVBD following CENP-E depletion, Mad2 displacement was delayed by ~2 hours compared with wild-type oocytes (Fig. 3A-I), and this was likely to reflect the reduced efficiency of kinetochore- microtubule interactions. Surprisingly, even with marked misalignment, there was no discernible difference in Mad2 fluorescence ascribable to chromosomal position at 6 hours (Fig. 3A-C) or 8 hours (Fig. 3D-F) post-GVBD. Instead, as with wild- type oocytes, Mad2 declined uniformly across kinetochores without any evidence of selective retention at polar chromosomes (Fig. 3J). Strikingly, although only about one-third of CENP-E- depleted oocytes exhibited cold-stable microtubules, kinetochores still became completely devoid of Mad2 by 10 hours post-GVBD (Fig. 3G-I). Mad2 dissociation was dependent upon kinetochore- microtubule interactions, as Mad2 was re-recruited following spindle depolymerisation with nocodazole (n12; Fig. 3K). Thus, polar kinetochores in oocytes can become saturated with non-K- fibre attachments, contrasting sharply with CENP-E-deficient mitotic cells in which compromised K-fibre formation culminates Fig. 3. CENP-E-depleted kinetochores become devoid of Mad2 in in chronic and biased Mad2 recruitment to polar-displaced spite of polar displacement. (A-C)By 6 hours post-GVBD, most kinetochores (Putkey et al., 2002). Collectively, these data strongly kinetochores retain Mad2 that is equally prominent at equatorially support the contention that oocyte kinetochores bind microtubules located bivalents (B) as at polar-displaced bivalents regardless of relatively easily, and that this promotes Mad2 dissociation and whether bivalents are compact (yellow arrow, A) or extended (white severely compromises the ability to selectively direct SAC arrows, C). (D-F)By 8 hours post-GVBD, low Mad2 levels are detectable components to polar bivalents. in some oocytes (D), but Mad2 is undetectable in others (E,F). Where Mad2 is detectable, there is no discernible difference between The presence of Mad2 at kinetochores correlates equatorial and polar bivalents (D). (G,H)By 10 hours post-GVBD, Mad2 with ongoing SAC activation in oocytes is completely undetectable, including at severely poleward-displaced We next examined whether kinetochore Mad2 retention was bivalents. (I,J)Kinetochore Mad2 levels in wild-type and CENP-E- depleted oocytes (I) and at polar and equatorial bivalents in CENP-E- physiologically relevant to SAC signalling. In mouse oocytes, depleted oocytes (J). Intensities were normalised either to values at 2 cyclin B1 destruction, a reporter for SAC inactivation (Clute and hours post-GVBD in wild-type oocytes (I) or to maximal intensities in Pines, 1999), is observed by 8 hours post-GVBD (Fig. 4B) (Homer individual oocytes (J). Data are mean ± s.e.m.; *P<0.05 by Student’s t- et al., 2009) and therefore correlates very closely with Mad2 test). (K) Mad2 becomes re-recruited in CENP-E-depleted oocytes

dissociation (Fig. 1A,D). PBE becomes maximal ~2 hours later, treated with nocodazole. Scale bars: 10m. DEVELOPMENT SAC in mouse oocytes RESEARCH REPORT 1945

Fig. 4. Exit from MI in CENP-E-depleted oocytes is mediated by Mad2 and BubR1. (A)PBE in wild-type, mock-depleted (+ControlMO), CENP- E-depleted (+CENPEMO), Mad2-depleted (+Mad2MO), CENP-E and Mad2 co-depleted (+CENPEMO+Mad2MO) oocytes, CENP-E-depleted oocytes co-expressing either BubR1 (+CENPEMO+BubR1 cRNA) or GFP (+CENPEMO+GFP cRNA), CENP-E and Mad2 co-depleted oocytes co-expressing BubR1 (+CENPEMO+Mad2MO+BubR1 cRNA) and CENP-E and mock co-depleted oocytes co-expressing BubR1 (+CENPEMO+ControlMO+BubR1 cRNA). Data are mean ± s.e.m. (B,C)Western blots showing cyclin B1 (B) and BubR1 and securin (C) (30 oocytes per lane). GAPDH and actin served as loading controls. Band intensities at 8 hours post-GVBD were normalised to values in controls. when cyclin B1 is at a nadir (Homer et al., 2005b), entirely instance in aged oocytes (Pan et al., 2008; Volarcik et al., 1998) and consistent with our current findings showing peak PBE at ~10 among recombination-deficient oocytes (Nagaoka et al., 2011) – do hours post-GVBD (Fig. 4A). By contrast, following CENP-E not prevent anaphase onset even though SAC functionality remains depletion, delayed Mad2 dissociation results in cyclin B1 grossly intact with the capacity to respond to minute inhibitory stabilisation by 8 hours post-GVBD and delayed PBE (Fig. 4A,B). signals (Hoffmann et al., 2011). Conversely, when polar bivalents Following Mad2 displacement by 10 hours post-GVBD, however, are rare, as in younger oocytes, the inability to react to polar PBE increased markedly, so that by 20 hours post-GVBD, PBE chromosomes is inconsequential and aneuploidy rates are low approached wild-type levels (Fig. 4A). Furthermore, in both wild- (Duncan et al., 2009; Homer et al., 2005b; Pan et al., 2008). We type and CENP-E-depleted oocytes, Mad2 depletion accelerated speculate that the exposed location of kinetochores at the very PBE (Fig. 4A). Altogether, these data indicate that the presence of extremities of bivalents (see supplementary material Fig. S1EЈ) and Mad2 at kinetochores is indicative of continuing SAC activation. the high microtubule density of oocyte spindles greatly augment the likelihood of microtubule capture even when stable attachments BubR1 instability contributes to delayed MI do not form. transit after CENP-E depletion These data also show that CENP-E fulfils two important roles Notably, following co-depletion of CENP-E and Mad2, the required for stable bi-orientation in oocytes: kinetochore accelerated transit typical of Mad2-depleted oocytes (Homer et al., reorientation followed by K-fibre formation, the latter being 2005b) was not observed and indeed transit through MI remained required for restraining bi-oriented bivalents at the equator. slower than in wild-type oocytes (Fig. 4A), indicating that SAC Intriguingly, whereas CENP-E relocates mitotic chromosomes activation was not solely responsible for MI delays following from the pole to equator (congression) (Kapoor et al., 2006), CENP-E depletion. CENP-E is known to interact with BubR1 during MI CENP-E prevents chromosomal drift in the opposite (Chan et al., 1998), and in oocytes BubR1 restrains securin (Pttg1 direction. These data help to explain the misalignment – Mouse Genome Informatics) overaccumulation that would phenotypes of aged oocytes, which have been found to exhibit otherwise induce MI arrest (Homer et al., 2009). Interestingly, we reduced levels of CENP-E (Pan et al., 2008; Volarcik et al., found that BubR1 was reduced and securin was stabilised in 1998). We also reveal an unexpected effect of CENP-E depletion CENP-E-depleted oocytes (Fig. 4C). Furthermore, PBE in CENP- in oocytes, that of BubR1 instability, which impacts MI E-depleted oocytes could be partially restored by expressing progression. It is interesting to speculate that oocytes might BubR1, but not GFP, from exogenous cRNA (Fig. 4A). Thus, deploy such SAC-independent delays under conditions that delayed MI transit after CENP-E depletion was in part due to predispose to polar chromosomes. BubR1 instability. Together, SAC activation and BubR1 instability accounted for MI delays after CENP-E depletion, as PBE returned Acknowledgements to wild-type levels following combined Mad2 depletion and BubR1 We thank Tim Yen, Stephen Taylor and Katja Wassmann for the very generous expression (Fig. 4A). gifts of reagents; and are grateful to the J. Carroll lab for helpful discussions. These data show that the non-K-fibre-based mode in oocytes Funding enables polar chromosomes to evade the SAC. This reconciles the This work was supported by a Wellcome Trust Fellowship [082587/Z/07/Z to

seemingly contradictory observation that polar chromosomes – for H.H.]. Deposited in PMC for release after 6 months. DEVELOPMENT 1946 RESEARCH REPORT Development 139 (11)

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Mad2 prevents aneuploidy and premature proteolysis of checkpoint to prevent aneuploidy due to single chromosome loss. J. Cell Biol. cyclin B and securin during meiosis I in mouse oocytes. Genes Dev. 19, 202-207. 162, 551-563. Homer, H., Gui, L. and Carroll, J. (2009). A spindle assembly checkpoint protein Yang, K. T., Li, S. K., Chang, C. C., Tang, C. J., Lin, Y. N., Lee, S. C. and Tang, T. functions in prophase I arrest and prometaphase progression. Science 326, 991- K. (2010). Aurora-C kinase deficiency causes cytokinesis failure in meiosis I and 994. production of large polyploid oocytes in mice. Mol. Biol. Cell 21, 2371-2383. DEVELOPMENT