Molecular Human Reproduction, Vol.15, No.3 pp. 139– 147, 2009
Advanced Access publication on January 29, 2009 doi:10.1093/molehr/gap007
NEW RESEARCH HORIZON Invited Review
Control of homologous chromosome
division in the mammalian oocyte
Janet E. Holt 1 and Keith T. Jones
School of Biomedical Sciences, Faculty of Health, University of Newcastle, Callaghan, NSW 2308, Australia
1
Correspondence address. School of Biomedical Sciences, Faculty of Health, University of Newcastle, Callaghan, NSW 2308, Australia.
Tel: þ61-02-49-21-86-13; Fax: þ61-02-49-21-79-03; E-mail: [email protected]
abstract: Homologous chromosomes are segregated during the first meiotic division (meiosis I). Unfortunately, human oocytes are
particularly susceptible to mis-segregation errors, so generating aneuploid, often non-viable, embryos. Here we review the cell biology of
meiosis I and how homolog disjunction is regulated for mammalian oocytes. We focus on the activity of the anaphase-promoting
complex/cyclosome (APC/C), which is responsible for timely degradation of the cohesin component, REC8 and the cyclin B regulatory
subunit of maturation-promoting factor, both essential steps for meiosis I completion. In particular, we examine the role played by the
spindle assembly checkpoint in controlling the APC/C activity, and in so doing ensuring accurate disjunction of homologs.
Key words: oocyte / homologs / meiosis / recombination / aneuploidy
Introduction
This review examines current developments in our understanding of
how homologous chromosome segregation is controlled during the
first meiotic division (meiosis I) of mammalian oocytes. Many human
aneuploidies are derived from segregation errors in the maternal
meiotic divisions (Hassold et al., 2007). It is therefore important to
understand the control mechanism at play during meiosis. The two
meiotic divisions of the oocyte, which generate a haploid gamete,
are protracted compared with the mitotic divisions of adult cells
(Fig. 1). This produces a unique set of challenges in ensuring fidelity
of chromosome separation. For example, homologous chromosome
pairs (homologs) must remain associated for months (e.g. in mice)
or years (e.g. in humans) in the germinal vesicle (GV)-arrested
oocyte prior to the completion of meiosis.
In female meiosis, alignment of parental homologs (referred to as
bivalents since they are comprised of two chromosomes, each with
two chromatids) during fetal life allows recombinatorial exchange of
genetic material through crossovers in the homologous chromosome
arms (Fig. 2). Resolution of homologs is then only achieved in the
oocyte of the mature adult during meiosis I. Sister chromatids remain
attached and only separate during the second meiotic division
(meiosis II). Separation of both homologs during meiosis I, and sister
chromatids (sometimes called dyads) during meiosis II relies on the
pulling forces of microtubules, which attach to centromeric chromatin
at kinetochores (Fig. 2). Both in meiosis I and in meiosis II separation
is only made possible by the dissolution of cohesin complexes, which
embrace sister chromatids. Noteworthy features of meiosis I are that:
(i) sister kinetochores act as a functional unit, allowing each pair of
sister chromatids to move to the same pole (Fig. 2); (ii) centromeric
cohesin is protected from degradation, otherwise sisters would prematurely separate in meiosis I and (iii) crossovers allow homologs to stay
attached when kinetochore pulling forces are exerted across them.
There have been several developments over recent years in our
understanding of meiosis I control in mammalian oocytes: (i) in
terms of the importance of the spindle assembly checkpoint (SAC)
and its target the anaphase-promoting complex/cyclosome (APC/
C); and (ii) in terms of the molecular components, notable the Shugoshins, which ensure segregation of homologs in meiosis I and
sisters in meiosis II. Such recent advances warrant review.
Chromosome attachment and
segregation
In meiosis, it is generally assumed that the normal mitotic cohesin cohort
of RAD21/SCC1, SMC1a, SMC3, STAG1/SA1 and STAG2/SA2 are
complemented by the meiosis-specific components REC8, SMC1b and
STAG3 (Revenkova and Jessberger, 2005). Indeed, these three meiosisspecific components have been reported in mouse oocytes (Prieto et al.,
2004). However, it is interesting to note that gender-specific differences
may exist in the mix of cohesin components used, since STAG2 is
reported to be absent in oocytes, but present in sperm (Prieto et al.,
2002, 2004).Cohesin components are likely to be involved in forming
the synaptonemal complex, and thus influencing recombination events,
since they are loaded onto chromosomes before recombination and
appear to be involved in recruiting synaptonemal complex protein
(SYCP) members on to the synaptonemal complex (Prieto et al.,
& The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Holt and Jones
Figure 1 A comparison of the events and timing of somatic cell mitosis with female meiosis in the mouse.
Somatic cells (top) generally complete mitosis in a matter of minutes without pause. However, meiosis (bottom) is completed over a more protracted time period, with
two periods of arrest. Meiosis begins in fetal life in mammals and progresses to prophase I whereupon the oocyte is then arrested—so-called ‘GV arrest’—for months or
years dependent upon species until hormonal stimulation promotes resumption of meiosis in adult life. Thereafter, the progression from GV breakdown to second polar
body extrusion occurs in 20 h (mouse) which includes a period of metaphase II arrest, prior to fertilization. In contrast to mitosis where sister chromatids separate in
anaphase, in meiosis homologs separate in anaphase I and sisters in anaphase II. Ana, anaphase; GVB, germinal vesicle breakdown, Met, metaphase; NEB, nuclear envelope
breakdown; PBE, polar body extrusion; Pro, prophase; Tel, telophase.
Figure 2 A comparison of kinetochore arrangements during segregation of chromosomes in mitosis and meiosis.
(A) Mitotic sister chromatids are held together by cohesin complexes along their arms, with centromeric cohesin providing a source of tension once spindles microtubules
attach and arm cohesins have dissociated (mostly in prophase), for correct segregation to occur in anaphase. Sister kinetochores are arranged ‘back-to-back’, and are
attached to opposite spindle poles. (B) In contrast, in meiosis I crossovers between homologs and cohesin bonds between sister chromatid arms ensure maintenance
of a bivalent structure, with tension arising from crossover sites once microtubules attach to kinetochores. Sister kinetochores are attached in a ‘side-by-side’ conformation to the same spindle pole, so that each homolog of the pair is attached to opposite poles. Sister chromatids remain physically linked by centromeric cohesin,
which is protected from degradation during meiosis I, until meiosis II. In meiosis II, the sister kinetochores resume a ‘back-to-back’ conformation and attach to opposite
poles, with tension arising from centromeric cohesion bonds, similar to mitosis.
2004). Furthermore, removal of cohesin family members parallels the loss
of SYCP3 shortly after birth (Prieto et al., 2004). Removal, however, is
probably not total because REC8 staining in dictyate arrested oocytes
is readily detected associated with chromatin (Lee et al., 2006); and the
cohesin family of proteins have a well-established role in maintaining
sister chromatid cohesion in mitosis and meiosis of a number of
species (Revenkova and Jessberger, 2005; Peters et al., 2008).
The cohesin proteins hold sisters together along chromosome arms
and at the centromere (Fig. 2). In mitosis, these proteins complex to
form ring-like structures around sisters until prometaphase when most
of the arm complexes dissociate via a ‘prophase pathway’ that relies
on phosphorylation of STAG1 and STAG2 by Polo-like kinase 1
(PLK1) and mediated by Aurora kinase B (Gimenez-Abian et al.,
2004; Hauf et al., 2005; Nasmyth, 2005). Anaphase onset sees the
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Homologous chromosomes and oocytes
proteolytic degradation of remaining arm and centromeric RAD21 by
separase, allowing sisters to segregate to opposite poles (Nakajima
et al., 2007). In contrast, meiotic sister chromatids do not lose
cohesin from their arms until anaphase I onset, and this is mediated
solely by REC8 degradation through separase activity rather than dissociation (Terret et al., 2003; Kudo et al., 2006). Importantly, sister
centromere cohesin remains intact to allow attachment and movement of sisters to the same pole. Centromeric REC8 is not cleaved
until the onset of anaphase II, when sisters need to segregate in
order to create the haploid maternal genome (Fig. 2).
Regulation of cohesin
degradation
The anaphase-promoting
complex/cyclosome
The multimeric APC/C is a master regulator of mitotic and meiotic
progression, ultimately responsible for controlling the degradation of
cohesive ties between chromosomes. At the metaphase –anaphase
transition, the ubiquitin E3 ligase activity of the APC/C marks for
degradation securin (PTTG1), whose function before metaphase is
to keep the protease separase (ESP1) inactive. At anaphase onset,
the freed separase cleaves the RAD21/REC8 component of the
cohesin complex, breaking the ring-like cohesin structure, and allowing
sister chromatids (mitosis; meiosis II) or homologs (meiosis I) to be
pulled apart. The APC/C is responsible for targeting a variety of
cell-cycle-related proteins for destruction, not just securin. Also
important is the ability of the APC/C to target degradation of cyclin
B, the regulatory subunit of the heterodimer maturation-promoting
factor (MPF). Cyclin B leads to a loss in the kinase activity of its
binding partner CDK1 (CDC2a, CDC2, p34CDC2), the catalytic
subunit of MPF (Peters, 2006).
The APC/C requires the accessory proteins CDC20/p55CDC or
FZR1/CDH1/FYR for its activity. These confer a degree of substrate
specificity upon the complex (Visintin et al., 1997; Peters, 2006), and
activate it at distinct stages of the cell cycle (Fig. 3). In mitosis, at the
metaphase –anaphase transition, APC/CCDC20 is responsible for
securin and cyclin B degradation. CDC20 appears to require the
high MPF activity normally observed at metaphase in order to associate with the APC/C, whereas for FZR1, the opposite occurs (Peters,
2006 and references therein). FZR1 is negatively regulated by
MPF-mediated phosphorylation, and binding to the APC/C during
metaphase is therefore inhibited. The loss in MPF activity associated
with cyclin B degradation at anaphase onset, therefore, promotes
both APC/CCDC20 inactivation and APC/CFZR1 activation. APC/
CFZR1 is consequently active in late mitosis/G1, and is involved in
the degradation of residual cyclin B and securin, as well as other substrates such as PLK1 and Aurora kinase B. Its activity appears to be
needed to allow maintenance of the G1 phase of the cell cycle
(Peters, 2002).
APC/CFZR1 activity in meiosis I
Interestingly, APC/CFZR1 activity is observed during early meiosis in
prophase I. Here it is responsible for maintaining GV arrest by degrading cyclin B, thereby keeping low MPF levels (Fig. 3) (Reis et al., 2006;
Marangos and Carroll, 2008; Yamamuro et al., 2008). Following germinal vesicle breakdown (GVB) and until late prometaphase I, APC/
CFZR1 is not switched off by MPF (Reis et al., 2007), and this is
because MPF activity rises slowly in maturing oocytes. During prometaphase I, APC/CFZR1 is observed to switch substrate preference, so
as to degrade CDC20 (Fig. 3). The mechanism for this substrate
switch away from securin/cyclin B1 is unclear, but it has already
been established that APC/CFZR1 substrates can be degraded sequentially and at different phases of the cell cycle (Lindon and Pines, 2004;
Rape and Kirschner, 2004). APC/CFZR1 activity is eventually switched
off during meiosis I by the rising MPF activity. Therefore, CDC20
protein levels start to rise, as does APC/CCDC20 activity. In turn,
this induces both securin and cyclin B degradation (Fig. 3), and therefore, chromosome separation and anaphase onset (Yu, 2002; Reis
et al., 2007). The prometaphase I period of APC/CFZR1 activity is
important in proper passage through meiosis I, because FZR1 antisense knockdown prevents CDC20 degradation and brings forward
in time the period of APC/CCDC20-mediated cyclin B and securin
degradation. These effects of FZR1 knockdown cause the oocyte to
extrude a polar body much earlier than controls and lead to a missegregation of homologs (Reis et al., 2007).
The essential role of the APC/C to meiosis I appears evolutionary
conserved (Pesin and Orr-Weaver, 2008).The one well-cited exception is Xenopus, where APC/C activity has been reported to be dispensable (Peter et al., 2001; Taieb et al., 2001). However, in this
vertebrate (Tang et al., 2008), as it is in mouse (Madgwick et al.,
2006), the maturing oocyte needs to suppress Emi2 expression in
order not to stall during meiosis I. Emi2 being the APC/C inhibitor
used to arrest oocytes physiologically at metaphase II before fertilization. In addition, separase activity is needed for Xenopus oocytes to
progress through meiosis I (Fan et al., 2006), and this would most
likely be induced by APC/C-mediated securin degradation. Such
observations imply that some APC/C activity is needed in order to
progress through meiosis I, therefore, further work is needed to
clarify its role in this important model animal.
Separase-mediated MPF inhibition
in meiosis I
Release of active separase following securin cleavage not only
degrades the RAD21/REC8 component of cohesin to allow chromosome separation, but also appears essential for meiosis I exit through a
distinct non-proteolytic function. Separase and MPF are able to form a
complex, which nullifies both the proteolytic function of separase and
the kinase function of MPF (Gorr et al., 2005). Introduction of an antibody into oocytes to block the interaction of MPF with separase fails
to block homolog segregation but does block polar body extrusion
and the drop in MPF activity normally observed at the completion
of meiosis I (Gorr et al., 2006). These observations suggest that separase is needed to bind MPF, in order to reduce MPF activity and obtain
polar body extrusion (Gorr et al., 2006; Kudo et al., 2006). Therefore,
during meiosis I, loss of MPF may be through a combination of cyclin B
degradation and separase binding. Paradoxically, oocytes from separase knockout mice readily undergo loss of MPF in meiosis I (Kudo
et al., 2006). The proteolytic activity of separase has certainly been
knocked out in these mice as observed by the lack of any REC8 proteolysis and inhibition of homolog disjunction. However, the floxed
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Figure 3 Reversal of APC/CCDC20 and APC/CFZR1 activities during meiosis I.
Activity of APC/C during mitosis (A) and meiosis (B), illustrating the temporal activity with its co-activators CDC20 and FZR1. For the APC/C substrates: cyclin B1,
securin, and CDC20, their known periods of degradation are shown under the APC/C peaks (as determined by loss of endogenous protein). (A) At the metaphase –
anaphase transition, mitotic APC/CCDC20 degrades securin and cyclin B1 to allow chromosome segregation, this is followed by APC/CFZR1, which has a wider range of
substrates including CDC20 (Peters, 2006). (B) In meiosis APC/CFZR1 precedes that of APC/CCDC20, during GV arrest it can degrade both securin and cyclin B1 (Reis
et al., 2006; Marangos and Carroll, 2008). After GVBD, APC/CFZR1 appears to switch substrate choice, and targets CDC20 for degradation in preference to cyclin B1 and
securin. This period of activity seems to allow time for homolog congression on the metaphase I plate (Reis et al., 2007). A decrease in APC/CFZR1 activity during oocyte
maturation (through rising MPF levels and FZR1 loss) allows re-synthesis of CDC20 and then APC/CCDC20 starts degradation of securin and cyclin B1, thus allowing
homolog segregation (Reis et al., 2007). APC/CCDC20 activity increases again at fertilization, with APC/CCDH1 following extrusion of the second polar body (Chang
et al., 2004). In meiosis, as in mitosis, periods of APC/CCDC20 activity are sensitive to inhibition by the SAC, as is readily observed by introduction of spindle
poisons such as nocodazole (Kot and Handel, 1990; Nixon et al., 2002; Brunet et al., 2003; Homer et al., 2005a, b; Jones, 2005; Madgwick et al., 2005).
separase gene in this knockout only has the exons involved in proteolysis floxed, and not the exons involved in MPF binding (Wirth et al.,
2006). Therefore, it remains possible that a truncated separase is
expressed in these oocytes still capable of MPF binding.
Shugoshin-mediated inhibition of sister
separation in meiosis I
APC/CCDC20-regulated separase activity is responsible for REC8
degradation during meiosis I, but how are these cohesin ties selectively
degraded such that homologs and not sisters are allowed to separate?
The answer appears to lie in a unique protection mechanism controlled by the Shugoshin family of proteins (SGO1/SGOL1 and
SGO2/SGOL2 in mammals). Localizing to centromeres, SGO protects centromeric cohesin from degradation through recruitment of
protein phosphatase 2A (PP2A) to the centromere, which may
dephosphorylate proteins that normally require phosphorylation for
efficient separase-mediated cohesin cleavage (Kitajima et al., 2006;
Riedel et al., 2006; Lee et al., 2008; Llano et al., 2008). In mouse
oocytes, SGO2 appears to be the key protector of centromeric
REC8, as judged by experiments in which expression of Sgo2 is
knocked down by RNAi (Lee et al., 2008) or by a gene knockout
(Llano et al., 2008).With both strategies, sister chromatids appear
to have separated by the time the oocyte has entered meiosis II. Protection of sister chromatid cohesion during meiosis is regulated by
tension on the kinetochore (Lee et al., 2008). During meiosis II,
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Homologous chromosomes and oocytes
where sister chromatids are attached to opposite spindle poles by
bipolar microtubule-kinetochore attachment (Fig. 2B), the microtubule pulling tension generated across the centromere induces a relocation of SGO2/PP2A away from centromeric cohesin, and so
separase-induced cleavage of REC8 ensues. However, in meiosis I,
where tension does not develop across sister kinetochores because
they are not attached to opposite spindle poles (they are acting as
single functional units), REC8 is protected from degradation. This
neatly explains why homologs are separated in meiosis I and sister
chromatids in meiosis II (Prieto et al., 2002; Gomez et al., 2007;
Lee et al., 2008; Llano et al., 2008).
Chromosome – spindle dynamics
during segregation
At the heart of chromosome disjunction lies the formation of a stable
bipolar meiotic spindle, upon which chromosome attach, align and are
pulled toward spindle poles to allow segregation. In contrast to mitotic
cells, microtubule organizing centers (MTOCs) are responsible for the
formation of the oocyte spindle rather than the nucleating potential of
the centrioles (Brunet and Maro, 2005; Schuh and Ellenberg, 2007).
MTOCs seed the growth of microtubules and are brought in close
proximity to chromosomes shortly after GVB, whereafter they are
organized into a bipolar barrel-shaped spindle, which migrates
toward the oocyte cortex during the metaphase to anaphase transition (Dumont et al., 2007; Vogt et al., 2008).
Although chromatin interaction with microtubules appears to influence microtubule dynamics, a bipolar spindle can still form in the
absence of chromatin, such as in the case of enucleated mouse
oocytes (Brunet et al., 1998; Yang et al., 2007). However, here the
success rate of forming a bipolar spindle is lower, since often multiple
spindles are formed. It has recently been proposed that the influence
of chromatin on the spindle formation and function may be dependent
upon the small GTPase Ran. Measurements of RanGTP levels in live
mouse oocytes demonstrate accumulation around chromosomes
during all stages of meiotic maturation and closely follow them as they
migrate toward the cortex (Dumont et al., 2007). Alterations in this
RanGTP gradient still allowed a meiosis I spindle to form but with detrimental effects upon spindle length and timing of bipolar attachments.
Kinetochores
It has been proposed that dynamically unstable microtubules use a
‘search and capture’ method to locate kinetochores. However, more
recent theoretical estimates taking into account nuclear diameter and
chromosome number suggest this process must be biased in some
manner probably via recruitment by RanGTP that influences microtubule
dynamics (Odde, 2005; Wollman et al., 2005; Dumont et al., 2007).
The kinetochores themselves are the tripartite structures consisting
of a chromatin containing inner plate, protein-rich outer plate and an
outermost fibrous corona layer. The kinetochore is responsible for
chromosomal attachment to the microtubules and possesses monitoring capabilities through the chromosomal passenger complex proteins
and the SAC. Greater than 60 kinetochore proteins have been identified in yeast; however, the discovery of vertebrate homologs has
remained elusive due to lack of sequence homology (Fukagawa,
2008). Kinetochores are assembled upon centromeric regions of
chromatin which feature specialized H3 histone variant CENP-A
nucleosomes interspersed with H3 histones (Karpen and Allshire,
1997). In mouse oocytes, phosphorylated histone H3 is detectable
from prometaphase to late telophase with phosphorylation
events associated with Aurora kinase activity (Wang et al., 2006).
This histone structure forms the inner plate of the kinetochore.
The outer kinetochore plate forms at GVB stage in the oocyte and
contains the plus end microtubule attachment sites, although the outer
fibrous corona is the first region to encounter microtubules (Maiato
et al., 2004). Components of the outer plate play roles in microtubule
polymerization/depolymerization, microtubule binding and stable
attachment and translocation of kinetochores laterally to achieve
appropriate alignment or segregation. Also associated with the outer
kinetochore are regulators of kinetochore assembly and microtubule
interactions including PLK1, and players in the SAC checkpoint
BUB1, BUBR1 (BUB1b), BUB3, MAD1 (MAD1/1), MAD2 (MAD
2/1) and MPS1 (TTK protein kinase) (Skibbens et al., 1993; Maiato
et al., 2004; Cheeseman and Desai, 2008; Fukagawa, 2008). The
motor proteins and SAC components are the most dynamic
members of the kinetochore—present at high concentrations in the
absence of microtubules yet declining once attachment has occurred
[see (Maiato et al., 2004) for review].
Polar orientation: an important decision
An important feature of kinetochore arrangement specific to meiosis
is the ‘side-by-side’ arrangement of sister kinetochores in meiosis I in
contrast to their ‘back-to-back’ arrangement in mitosis and meiosis II
(Fig. 2). This is driven by a requirement for sister chromatids to attach
to microtubules arising from the same pole during meiosis I, thus the
sisters act as a functional unit, with the homologs instead being
attached to opposing spindle poles. This contrasts with mitosis,
where sisters are attached to opposite poles.
Both sister kinetochores are active in meiosis I and attach to microtubules in fission yeasts and in higher eukaryotes (Goldstein, 1981;
Parra et al., 2004; Hauf et al., 2007), however, in budding yeast it is
thought that one sister kinetochore may be inactive, such that the
sisters can only move to the one pole (Winey et al., 2005; Hauf
et al., 2007). In budding yeast, the monopolin complex interacts
with IPL/Aurora kinase B independently of cohesins to orient the
kinetochores toward the same pole; however, vertebrate monopolin
homologs have not been found (Toth et al., 2000; Yokobayashi and
Watanabe, 2005). Aurora kinase B in both fission and budding yeast
appears to play a role in orientating kinetochores in a manner that
favors attachment of microtubules via a mechanism that is conserved
between mitosis and meiosis. Disruption of the bivalent structures
results in incorrect bipolar attachment of sisters in meiosis I, like
that of sister chromatids in mitosis or meiosis II (Hauf et al., 2007;
Monje-Casas et al., 2007). Watanabe et al. (1999) proposed that
REC8 in fission yeast chromosomal arms coordinates sister chromatid
kinetochores to orient into a side-by-side conformation in meiosis I,
with REC8 loss in the arms promoting a back-to-back conformation
in meiosis II (Watanabe, 2004). The model was further refined such
that the fission yeast protein MOA1, which associates with REC8
only at the centromere, is thought to facilitate centromere cohesion
and thus mono-orient the kinetochores, as loss of MOA allows
sisters to attach to opposite poles (Yokobayashi and Watanabe,
2005).
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Regulation of homolog
separation through the SAC
The SAC and the chromosomal
passenger complex
Attachments between microtubules and kinetochores are inherently
error prone presumably as a result of the predominantly random
nature of the ‘search and capture’ process. However, as previously
discussed, in meiosis I the sisters must behave as a single unit, and
therefore, errors may occur when sisters of the same homolog
form an amphitelic (bipolar) attachment, or when both homologs
attach to the same pole in a syntelic manner. The detection of microtubule attachment errors is an essential process to ensure maintenance of correct cell ploidy, and the mitotic and meiotic cell
possesses intricate mechanisms for triggering a delay in the progression of anaphase in compromised cells, via members of the SAC
and chromosomal passenger complex systems.
The SAC inhibits APC/C activity
The underlying mechanism that ultimately allows an unperturbed
oocyte to progress through anaphase I is the loss of MPF activity
and separase release from its inhibitory partner securin. APC/
CCDC20 activity in oocytes proteolytically degrades securin and cyclin
B (Reis et al., 2007), making it the key target of the SAC and therefore
its inhibition is a key feature of meiosis I. The components of the SAC
system form an intracellular signaling network that are believed primarily to sequester CDC20 away from the APC/C. APC/C activity
is thus diminished and securin cannot be degraded, so preventing
separase-mediated separation of homologs (Yu, 2002). The MAD
and BUB proteins accumulate at kinetochores that either lack
proper attachment to microtubules or fail to develop tension across
them (Vogt et al., 2008). This generates a ‘wait-anaphase’ signal in
the progression of the cell cycle, usually in the period of prometaphase
until metaphase, when all chromosomes are aligned in the equatorial
region of the spinde and are under tension. MAD2 conformational
changes at the kinetochore occur early in the arrest signal cascade,
and are thought to allow MAD2 to bind CDC20, sequestering it
away from the APC/C and so preventing APC/C activity. Diffusion
of this complex away from the kinetochore allows further MAD2/
CDC20 interactions to occur (De Antoni et al., 2005; Vink et al.,
2006). The SAC kinases BUBR1 and BUB3 can also bind CDC20
and may act synergistically with MAD2 to inhibit the activity of
APC/C (Zhou et al., 2002; Mao et al., 2003). An interesting recent
report concluded that the major function of MAD2 may be to
promote CDC20 interaction with BUBR1 and BUB3, rather than
form any stable interaction (Nilsson et al., 2008). The interaction of
CDC20 with BUBR1 and BUB3 did not sequester it away from the
APC/C, but instead promoted its interaction, leading to CDC20 ubiquitination and degradation. It is therefore tempting to speculate that
the CDC20 degradation readily observed during meiosis I (Reis et al.,
2007) is due to activation of the SAC at this time.
Acting in concert with one another, the various chromosomal passenger complex components, including INCENP, Aurora B, Survivin
and Borealin/Dasra, allow the SAC to function in cases where microtubule tension is lessened (Carvalho et al., 2003; Ditchfield et al.,
Holt and Jones
2003; Lens et al., 2003; Ruchaud et al., 2007). chromosomal passenger
complex proteins target SAC components to the kinetochore: for
example Aurora B is required to target BUBR1, MAD2, CENP-E
(Ditchfield et al., 2003). As previously discussed, Aurora B is required
for the mono-orientation of sister in meiosis I as well as the correct
bipolar attachment of homologs through the potential building of
favorable sites for attachment. However, as a chromosomal passenger
complex component, Aurora B may directly deal with any detected
attachment errors by activating the SAC when lack of tension is
sensed, and promoting the release of incorrectly attached microtubules (Biggins and Murray, 2001; Tanaka et al., 2002; Hauf et al.,
2007). A key kinetochore-bound target is the NDC80 complex, and
upon phosphorylation by Aurora B, microtubule binding affinity of
this complex is reduced allowing rebinding events to occur until
correct attachment is achieved (Cheeseman et al., 2006).
Functionality of the SAC in oocytes
Numerous studies indicate SAC protein members are present and
functional in the mammalian oocyte (Kallio et al., 2000; Brunet
et al., 2003; Zhang et al., 2004; Homer et al., 2005b, c). Depletion
of BUB1 (via antibodies) and MAD2 (via morpholino antisense oligonucleotides) brings forward the timing of APC/C-mediated cyclin B
and/or securin degradation in mouse oocytes, and significantly
increases misaligned chromosomes and aneuploidy rates (Homer
et al., 2005c; Yin et al., 2006). Similarly, anaphase is brought
forward and chromosome alignment disrupted when anti-MAD2 antibodies are injected into rat oocytes (Zhang et al., 2004). Spindle
poison (e.g. nocodazole) treatments of post-GVBD oocytes leads to
metaphase arrest (Brunet et al., 2003; Wassmann et al., 2003) and furthermore, in functional SAC mutants or SAC-component depleted
oocytes (BUB1, MAD2) this arrest is overridden, providing further evidence that in oocytes the SAC is functional (Wassmann et al., 2003;
Homer et al., 2005b; Yin et al., 2006). It is intriguing to note,
however, that degradation of securin and cyclin B in mouse oocytes
begins 2– 3 h prior to extrusion of the first polar body indicating
APC/CCDC20 is active and the SAC now inactive (Homer et al.,
2005a), yet mature, stable end-on kinetochore/microtubule interactions are only formed just prior to polar body extrusion (Brunet
et al., 1999; Homer et al., 2005a).
The SAC and the XO mouse
Studies in oocytes from ‘XO’ mice appear to contrast with evidence
for a completely functional SAC in meiosis I. XO mice possess a univalent X chromosome, which lacks a pairing partner during recombination. Based on a functional SAC, one would predict the oocyte
would undergo metaphase I arrest, as the SAC detects a lack of
tension across the X chromosome. However, no delay in progression
through meiosis I is observed in XO oocytes, in fact the only difference noted is that XO oocytes undergo the metaphase I –anaphase
I transition faster. Video-timelapse of mouse oocytes during meiosis
I reveals that they spend the majority of time in a prometaphase I
state, with congressed chromosomes oscillating slowly about the
spindle equator (Schuh and Ellenberg, 2007). It is therefore possible
that video-timelapse of XO oocytes would reveal less time spent at
this stage of meiosis I. In XO oocytes, both ‘reductional’ (normal
meiosis I division) or ‘equational’ (normal mitosis/meiosis II division)-
145
Homologous chromosomes and oocytes
type segregation can result from syntelic or amphitelic attachments,
respectively. This suggests the oocyte does not possess a specialist
mechanism for segregating achiasmate chromosomes as seen in
some species (Koehler and Hassold, 1998).
The SAC and the SYCP3 knockout mouse
Another mouse model for univalent meiotic segregation is the SYCP3
knockout, which has reduced crossover events in response to a compromised synaptonemal complex (Yuan et al., 2000; Yang et al., 2006;
Kouznetsova et al., 2007). The achiasmate chromosomes act as univalents, and as for the XO mouse, no SAC arrest appears to be
imposed on oocytes during meiosis I. Instead, here it was elegantly
shown that the univalents form bipolar microtubule attachments,
with each sister kinetochore attaching to opposite spindle poles possibly because SYCP3 expression is needed for monopolar attachments
of sisters in meiosis I (Parra et al., 2004). In this way, tension
could develop across the sister kinetochore and therefore the SAC
would not be expected to be switched on. In molecular terms, this
would now be explained by the normal protective role of SGO2 in
meiosis I being lost as the tension across the kinetochore is developed
(Lee et al., 2008).
Future directions
The model of a microtubule-imposed tension developing across a
kinetochore to displace SGO2 in meiosis II, but not meiosis I, is an
important breakthrough in our understanding of meiotic chromosome
segregation. We now need to determine if there is absolute block to
back-to-back attachment of sister kinetochores in meiosis I, and if so
how this is ameliorated in the SYCP3 knockout. Or alternatively, if
back-to-back orientations exist to then be replaced by side-to-side
during prometaphase I. If so, the back-to-back orientation would
develop tension across it and potentially lose cohesion by loss of
SGO2. In the SYCP3 knockout, sister centromeres were observed
to be situated further apart from one another in bipolarly attached univalents (Kouznetsova et al., 2007). Although this may be a direct result
of applied tension across the spindle, it remains conceivable that the
bivalent structure may normally be crucial in imposing conformational
restraints upon sister kinetochores such that they must retain a
side-by-side configuration which is essential for monopolar attachment
(Fig. 2). Another issue still be resolved is the prevalent reductional division observed in the XO mouse during meiosis I. Here the X univalent displays an ‘intact’ reductional segregation in 45 –75% of
oocytes, a rate that is dependent on strain (Hodges et al., 2001). In
such reductional division, there could have been no release of the
SAC since tension cannot develop across the chromosomes, as
argued for the SYCP3 knockout (LeMaire-Adkins et al., 1997).
Another interesting anomaly of mammalian oocyte SAC function is
demonstrated by the knockdown of APC/C activator FZR1 during
meiosis I. Loss of FZR1 accelerates passage through meiosis I probably
by preventing loss of CDC20, also precociously activating APC/
CCDC20. This leads to premature cyclin B1 and securin degradation
(Reis et al., 2007). These FZR1 knockdown oocytes are highly aneuploid because of this accelerated meiosis I. Interestingly, the SAC
appears not to be activated under these conditions even though the
FZR1 knockdown leads to gross mis-segregation of intact bivalents.
However, in these knockdown oocytes a metaphase I arrest could
still be induced by addition of microtubule disrupting agents or overexpression of MAD2.
The data on the segregation of the XO univalent and of the behavior of homologs in the FZR1 knockdown indicate that the SAC mechanisms of the oocyte are unique; what may be important in activating
the SAC in mitosis may differ in meiosis. Factors such as the influence
of the relatively large oocyte volume upon SAC-associated signal generation and diffusion may play a role in addition to the intrinsic differences between the timing of mitosis and meiosis. The close coupling of
SAC initiation and APC/C-mediated degradation evident in mitosis is
lacking in meiosis and may be related to the protracted nature of
mammalian oocyte meiosis. These inherent differences require
further investigation to complete our understanding of how the
oocyte ensures accurate homolog disjunction and maintenance of
euploidy in the maturing gamete.
Funding
The authors acknowledge grant funding through the National Health
and Medical Research Council (NHMRC).
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Submitted on October 22, 2008; resubmitted on January 19, 2009; accepted on
January 27, 2009