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
MATERIALS AND METHODS
Oocyte collection and drug treatment
Oocytes were isolated from 4- to 6-week-old pregnant mare serum
gonadotropin (PMSG)-primed MF1 mice and cultured as described (Homer
et al., 2009). Nocodazole (Sigma) was used at 5 M (Homer et al., 2005a).
Experiments involving animals conformed to the relevant regulatory
standards.
1
Cell and Developmental Biology, 2Institute for Women’s Health, University College
London, London WC1E 6BT, UK.
*Author for correspondence ([email protected])
Accepted 2 April 2012
Morpholino and cRNA injection
For CENP-E depletion, germinal vesicle stage oocytes were microinjected
with a morpholino designed to target mouse Cenpe (NM_173762)
designated CENPEMO (5⬘-CAGCCACTGAAGCCTCCTCGGCCAT-3⬘;
Gene Tools) and maintained for 20-24 hours in isobutylmethylxanthine
(IBMX)-treated medium. Mad2MO, ControlMO (for mock depletions) and
human BUBR1 cRNA were described previously (Homer et al., 2009;
Homer et al., 2005b). Morpholinos were microinjected at 2 mM. For
double depletions, combinations of morpholinos (4 mM stock) were
microinjected.
Immunocytochemistry
Immunofluorescence and cold treatment (4°C for 10 minutes) were
performed as described previously (Homer et al., 2009). Primary antibodies
included -tubulin (Sigma); ACA (ImmunoVision); g-tubulin (Abcam);
CENP-E (Dr T. Yen, Fox Chase Cancer Center, USA); BubR1 (Dr Stephen
Taylor, University of Manchester, UK) and Mad2 (Dr K. Wassmann,
CNRS UMR7622, France). Secondary antibodies (Invitrogen) included
Alexa Fluor 488- or 546-labelled goat anti-human; Alexa Fluor 633- or
488-labelled goat anti-mouse; Alexa Fluor 488- or 546-labelled goat antirabbit; and Alexa Fluor 488-labelled goat anti-sheep. DNA was stained
using Hoechst 33342 (10 g/ml; Sigma). Images were captured using a
Zeiss LSM510 META confocal microscope configured as follows: for
Hoechst 33342, 364 nm UV laser excitation combined with a 385-470 nm
band-pass emission filter; for Alexa Fluor 488, 488 nm argon laser line
combined with a 505-550 nm band-pass emission filter; for Alexa Fluor
546, 543 nm helium/neon1 laser combined with a 560-615 nm band-pass
emission filter; and for Alexa Fluor 633, a 633 nm helium/neon2 laser with
a 650 nm long-pass emission filter.
Western blotting
Pre-cast 3-8% Tris-acetate gels (Invitrogen) and a mouse monoclonal antiCENP-E antibody (Abcam) were used for CENP-E. BubR1, securin,
GAPDH and actin immunoblotting were performed as described (Homer
et al., 2009; Homer, 2011). HRP-conjugated antibodies were detected using
ECL Plus (GE Healthcare) and protein bands were semi-quantitatively
assayed (Homer et al., 2009).
DEVELOPMENT
INTRODUCTION
Mad2 recruitment to improperly attached kinetochores is crucial
for generating the inhibitory spindle assembly checkpoint (SAC)
signal that prevents anaphase-promoting complex/cyclosome
(APC/C) activation and anaphase onset (Musacchio and Salmon,
2007). During mitosis, kinetochores of polar chromosomes, which
are at greatest risk of mis-segregating, recruit the highest levels of
Mad2 (Howell et al., 2000; Waters et al., 1998), thereby tightly
coupling SAC activation to aneuploidy risk.
Paradoxically, in spite of possessing an SAC (Hached et al.,
2011; Homer et al., 2005b; McGuinness et al., 2009), female
meiosis I (MI) remains notoriously error prone (Hassold and Hunt,
2009). An important cause for this vulnerability is the inability of
small numbers of polar chromosomes to prevent anaphase onset in
oocytes (Nagaoka et al., 2011). Exactly why polar chromosomes
should evade the SAC remains unknown, especially as recent data
indicate that the oocyte SAC has the capacity to react to even a
single unattached chromosome (Hoffmann et al., 2011). Here we
address the key issue of how the SAC in oocytes responds to polar
chromosomes.
RESULTS AND DISCUSSION
Mad2 is not overtly enriched at the kinetochores
of polar bivalents in wild-type oocytes
In mouse oocytes, MI lasts ~8-10 hours, beginning with germinal
vesicle breakdown (GVBD) and concluding with first polar body
extrusion (PBE), when recombined homologous chromosomes
(bivalents) segregate (Homer et al., 2009; Homer et al., 2005b).
During this time, numerous microtubule-organising centres nucleate
a spindle, which is gradually remodelled into a bipolar form (Schuh
and Ellenberg, 2007). As polar chromosomes constitute an important
focus of the SAC, we first determined when bipolarity was
established. Using strict criteria, we found that bipolarity was not
established until ~6 hours post-GVBD (supplementary material Fig.
S1A,B). Also, consistent with recent data (Kitajima et al., 2011), we
found that kinetochores reoriented to face in opposite directions by
6 hours post-GVBD (supplementary material Fig. S1C-E), beyond
which time less than 5% of oocytes (n>150) possessed severely
displaced polar bivalents, entirely in keeping with previous results
(Kitajima et al., 2011; Yang et al., 2010).
Development 139 (11)
Consistent with previous results (Kitajima et al., 2011), we found
that kinetochore levels of Mad2 (Mad2l1 – Mouse Genome
Informatics) declined during MI (Fig. 1A,D). Additionally, by
focusing on the stage during which the spindle was bipolar, we were
now able to compare Mad2 recruitment to equatorial and polar
bivalents. Significantly, low levels of Mad2 were retained at many
equatorial bivalents by 6 hours post-GVBD (when the spindle first
becomes bipolar), and there was an additional ~2 hours before
complete Mad2 displacement (Fig. 1A,D). Strikingly, among the few
oocytes with severely polar-displaced bivalents at 6 hours postGVBD, Mad2 decorated kinetochores of both equatorial and polar
bivalents to a similar degree (Fig. 1E-E⵮). Furthermore, by 8 hours
post-GVBD, all kinetochores completely lacked Mad2 (Fig. 1A,F),
even when polar bivalents were present (Fig. 1G). Collectively, this
represented a marked departure from the mitotic template in which
polar-displaced kinetochores retain high levels of Mad2 and the
attainment of an equatorial chromosomal position is promptly
followed by complete loss of Mad2 (Hoffman et al., 2001; Howell
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 anticentromere 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 kinetochorecontaining region as illustrated. Backgroundcorrected 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-E⵮) An
oocyte at 6 hours post-GVBD shows a polardisplaced bivalent (arrow) with levels of
Mad2 (E⬘) that are comparable to those at
equatorial bivalents (E⬙). (E⵮)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 polardisplaced 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
1942 RESEARCH REPORT
Displacement of the bulk of Mad2 occurs in spite
of low K-fibre content
We next investigated why such a protracted interval elapsed before
Mad2 became completely displaced from bivalents that were
equatorial and bi-oriented. During mitosis, microtubules that form
stable end-on attachments with kinetochores (K-fibres) are crucial
for displacing Mad2 from bi-oriented chromosomes (Hoffman et
al., 2001; Putkey et al., 2002). We therefore asked whether K-fibre
content differed between 6 and 8 hours post-GVBD. K-fibres
impart tension across kinetochores (Deluca et al., 2002) and are
differentially stable to cold treatment (Rieder, 1981). We found that
cold treatment induced rudimentary spindles and Mad2 rerecruitment to kinetochores in ~60% and ~10% of oocytes at 6 and
8 hours post-GVBD, respectively (Fig. 1H). Next, by measuring
inter-kinetochore distances, we found that tension became maximal
at 8 hours post-GVBD (supplementary material Fig. S2),
coincident with which, kinetochore BubR1 (Bub1b – Mouse
Genome Informatics) levels, which are well known to become
depleted in response to tension (Skoufias et al., 2001; Uchida et al.,
2009), declined significantly (Fig. 1A,D). As with mitotic BubR1,
which declines but does not disappear at metaphase (Hoffman et
al., 2001), BubR1 persisted at kinetochores by early anaphase I
RESEARCH REPORT 1943
(Fig. 1B,B⬘) before disappearing by late anaphase I (Fig. 1C).
Overall, these data are consistent with previous analyses showing
late K-fibre formation in oocytes (Brunet et al., 1999). Importantly,
however, by using two independent measures for K-fibres we could
now quantify K-fibre content and correlate this with kinetochore
Mad2 levels specifically during the bipolar stage.
Intriguingly, these data now show that by 6 hours post-GVBD,
when the majority of kinetochore Mad2 was displaced, most
oocytes were deficient in K-fibres, pointing to an unusually high
propensity for kinetochores to acquire microtubule interactions –
that is, even unstable interactions sufficed for displacing Mad2. We
hypothesized that this unusual propensity could account for the
lack of preferential Mad2 recruitment to the small numbers of polar
bivalents in wild-type oocytes, thereby compromising biased SAC
activation at polar bivalents.
CENP-E depletion induces polar bivalents and
unstable kinetochore-microtubule interactions
In order to test our hypothesis, we sought conditions under which
unstable kinetochore-microtubule interactions and polar
chromosomes were prevalent, two features that characterise mitotic
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, A⬘ and Z2, A⬙) (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 postGVBD. (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
SAC in mouse oocytes
1944 RESEARCH REPORT
Development 139 (11)
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 kinetochoremicrotubule 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 wildtype 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-Edepleted 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 kinetochoremicrotubule 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-Kfibre attachments, contrasting sharply with CENP-E-deficient
mitotic cells in which compromised K-fibre formation culminates
in chronic and biased Mad2 recruitment to polar-displaced
kinetochores (Putkey et al., 2002). Collectively, these data strongly
support the contention that oocyte kinetochores bind microtubules
relatively easily, and that this promotes Mad2 dissociation and
severely compromises the ability to selectively direct SAC
components to polar bivalents.
The presence of Mad2 at kinetochores correlates
with ongoing SAC activation in oocytes
We next examined whether kinetochore Mad2 retention was
physiologically relevant to SAC signalling. In mouse oocytes,
cyclin B1 destruction, a reporter for SAC inactivation (Clute and
Pines, 1999), is observed by 8 hours post-GVBD (Fig. 4B) (Homer
et al., 2009) and therefore correlates very closely with Mad2
dissociation (Fig. 1A,D). PBE becomes maximal ~2 hours later,
Fig. 3. CENP-E-depleted kinetochores become devoid of Mad2 in
spite of polar displacement. (A-C)By 6 hours post-GVBD, most
kinetochores retain Mad2 that is equally prominent at equatorially
located bivalents (B) as at polar-displaced bivalents regardless of
whether bivalents are compact (yellow arrow, A) or extended (white
arrows, C). (D-F)By 8 hours post-GVBD, low Mad2 levels are detectable
in some oocytes (D), but Mad2 is undetectable in others (E,F). Where
Mad2 is detectable, there is no discernible difference between
equatorial and polar bivalents (D). (G,H)By 10 hours post-GVBD, Mad2
is completely undetectable, including at severely poleward-displaced
bivalents. (I,J)Kinetochore Mad2 levels in wild-type and CENP-Edepleted oocytes (I) and at polar and equatorial bivalents in CENP-Edepleted oocytes (J). Intensities were normalised either to values at 2
hours post-GVBD in wild-type oocytes (I) or to maximal intensities in
individual oocytes (J). Data are mean ± s.e.m.; *P<0.05 by Student’s ttest). (K) Mad2 becomes re-recruited in CENP-E-depleted oocytes
treated with nocodazole. Scale bars: 10m.
DEVELOPMENT
(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-Edepleted oocytes were unable to restrain bivalents at the equator.
Also, 37% (n38) and 40% (n42) of bivalents at 6 and 8 hours postGVBD, 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.
SAC in mouse oocytes
RESEARCH REPORT 1945
when cyclin B1 is at a nadir (Homer et al., 2005b), entirely
consistent with our current findings showing peak PBE at ~10
hours post-GVBD (Fig. 4A). By contrast, following CENP-E
depletion, delayed Mad2 dissociation results in cyclin B1
stabilisation by 8 hours post-GVBD and delayed PBE (Fig. 4A,B).
Following Mad2 displacement by 10 hours post-GVBD, however,
PBE increased markedly, so that by 20 hours post-GVBD, PBE
approached wild-type levels (Fig. 4A). Furthermore, in both wildtype and CENP-E-depleted oocytes, Mad2 depletion accelerated
PBE (Fig. 4A). Altogether, these data indicate that the presence of
Mad2 at kinetochores is indicative of continuing SAC activation.
BubR1 instability contributes to delayed MI
transit after CENP-E depletion
Notably, following co-depletion of CENP-E and Mad2, the
accelerated transit typical of Mad2-depleted oocytes (Homer et al.,
2005b) was not observed and indeed transit through MI remained
slower than in wild-type oocytes (Fig. 4A), indicating that SAC
activation was not solely responsible for MI delays following
CENP-E depletion. CENP-E is known to interact with BubR1
(Chan et al., 1998), and in oocytes BubR1 restrains securin (Pttg1
– Mouse Genome Informatics) overaccumulation that would
otherwise induce MI arrest (Homer et al., 2009). Interestingly, we
found that BubR1 was reduced and securin was stabilised in
CENP-E-depleted oocytes (Fig. 4C). Furthermore, PBE in CENPE-depleted oocytes could be partially restored by expressing
BubR1, but not GFP, from exogenous cRNA (Fig. 4A). Thus,
delayed MI transit after CENP-E depletion was in part due to
BubR1 instability. Together, SAC activation and BubR1 instability
accounted for MI delays after CENP-E depletion, as PBE returned
to wild-type levels following combined Mad2 depletion and BubR1
expression (Fig. 4A).
These data show that the non-K-fibre-based mode in oocytes
enables polar chromosomes to evade the SAC. This reconciles the
seemingly contradictory observation that polar chromosomes – for
instance in aged oocytes (Pan et al., 2008; Volarcik et al., 1998) and
among recombination-deficient oocytes (Nagaoka et al., 2011) – do
not prevent anaphase onset even though SAC functionality remains
grossly intact with the capacity to respond to minute inhibitory
signals (Hoffmann et al., 2011). Conversely, when polar bivalents
are rare, as in younger oocytes, the inability to react to polar
chromosomes is inconsequential and aneuploidy rates are low
(Duncan et al., 2009; Homer et al., 2005b; Pan et al., 2008). We
speculate that the exposed location of kinetochores at the very
extremities of bivalents (see supplementary material Fig. S1E⬘) and
the high microtubule density of oocyte spindles greatly augment
the likelihood of microtubule capture even when stable attachments
do not form.
These data also show that CENP-E fulfils two important roles
required for stable bi-orientation in oocytes: kinetochore
reorientation followed by K-fibre formation, the latter being
required for restraining bi-oriented bivalents at the equator.
Intriguingly, whereas CENP-E relocates mitotic chromosomes
from the pole to equator (congression) (Kapoor et al., 2006),
during MI CENP-E prevents chromosomal drift in the opposite
direction. These data help to explain the misalignment
phenotypes of aged oocytes, which have been found to exhibit
reduced levels of CENP-E (Pan et al., 2008; Volarcik et al.,
1998). We also reveal an unexpected effect of CENP-E depletion
in oocytes, that of BubR1 instability, which impacts MI
progression. It is interesting to speculate that oocytes might
deploy such SAC-independent delays under conditions that
predispose to polar chromosomes.
Acknowledgements
We thank Tim Yen, Stephen Taylor and Katja Wassmann for the very generous
gifts of reagents; and are grateful to the J. Carroll lab for helpful discussions.
Funding
This work was supported by a Wellcome Trust Fellowship [082587/Z/07/Z to
H.H.]. Deposited in PMC for release after 6 months.
DEVELOPMENT
Fig. 4. Exit from MI in CENP-E-depleted oocytes is mediated by Mad2 and BubR1. (A)PBE in wild-type, mock-depleted (+ControlMO), CENPE-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.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.078352/-/DC1
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