Accepted Article

The spindle checkpoint and chromosome segregation in meiosis

Gary J. Gorbsky Cell Cycle & Cancer Biology Oklahoma Medical Research Foundation Oklahoma City, OK 73104 Voice: 405-271-8168 Fax: 405-271-7313 Email: [email protected] Website: http://gorbsky.omrf.org/ Article Type: Review Article Running Title: The spindle checkpoint in meiosis

Keywords: microtubule, spindle, cell cycle, mitosis, anaphase-promoting complex, cyclosome, spermatocyte, oocyte, kinetochore, aneuploidy

Abstract The spindle checkpoint is a key regulator of chromosome segregation in mitosis and meiosis. Its function is to prevent precocious anaphase onset before chromosomes have achieved bipolar attachment to the spindle. The spindle checkpoint comprises a complex set of signaling pathways that integrate microtubule dynamics, biomechanical forces at the kinetochores, and intricate regulation of protein interactions and post-translational modifications. Historically, many key observations that gave rise to the initial concepts of the spindle checkpoint were carried out in meiotic systems. In contrast with mitosis, the two distinct chromosome segregation events of meiosis present a special challenge for the regulation of checkpoint signaling. Preservation of fidelity in chromosome segregation in meiosis, controlled by the spindle checkpoint, also has significant impact in human health. This review highlights the contributions from meiotic systems in understanding the spindle checkpoint as well as the role of checkpoint signaling in controlling the complex divisions of meiosis.

Introduction The current concepts of cell cycle regulation focus around the idea of a biochemical clock whose progression is regulated by a set of failsafe monitors called checkpoints. Checkpoints are signaling pathways that can detect a cellular defect, stop cell cycle progression, and often initiate specific repair pathways [1]. One of these is the spindle checkpoint, sometimes called

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/febs.13166 This article is protected by copyright. All rights reserved.

Accepted Article

the spindle assembly checkpoint, the wait-anaphase checkpoint, or the mitotic checkpoint. (The last term, mitotic checkpoint, is somewhat of a misnomer since the checkpoint also functions in meiosis.) In mitosis and meiosis, the spindle checkpoint blocks progression from metaphase to anaphase when spindle microtubules are disrupted or when their connections to the kinetochores of chromosomes are compromised. Under these circumstances cell cycle progression to anaphase is delayed to allow more time for chromosomes to achieve proper bipolar spindle attachment. Kinetochores lacking stable attachment to spindle microtubules generate the checkpoint signal. Microtubule interactions with kinetochores diminish the strength of the checkpoint signal. Once mature attachments of microtubules to kinetochores are fully formed and kinetochores come under mechanical tension, checkpoint signaling is fully inhibited, and the cell proceeds to anaphase.

Although most recent studies of the spindle checkpoint have focused on mitosis, the spindle checkpoint also plays an essential role in the more complex and lengthier chromosome segregation events of meiosis. From a medical standpoint, the meiotic spindle checkpoint has been of great interest in that checkpoint failure promotes production of aneuploid gametes that are important causes of infertility and birth defects. Interestingly, many of the seminal studies that provided the groundwork in analysis of the spindle checkpoint were carried out in meiotic systems. Understanding of the biochemistry and cell biology of spindle checkpoint function has profited by comparing studies in both mitosis and meiosis. This review will highlight direct contributions from meiotic systems and their integration with insight from mitosis in understanding the spindle checkpoint in meiosis. This review does not discuss the early chromosome pairing movements and regulatory checkpoints that function within the intact nucleus during meiotic prophase. These topics are addressed in other recent reviews [2-5]

Meiotic systems: mechanical tension and kinetochore-microtubule interaction A key aspect in elucidating the interaction of chromosome movement with checkpoint regulation is the key role of mechanical tension caused by kinetochore attachment to spindle microtubules. On its own this topic is inherently fascinating cell biology in that it involves mechanical regulation of biochemical signals that ultimately can only be fully understood at the level of how physical forces impact the structural biology of proteins and their interactions. How does pulling by spindle microtubules alter the structure and interaction of kinetochore proteins to change their signaling properties? The intellectual roots for this intriguing question lay in studies of meiosis. In 1958, studying spermatocytes in Ostracod crustaceans, Dietz first suggested that mechanical tension induced by bipolar attachment of meiotic chromosomes might stabilize kinetochore binding to the fibrillar structures then known as spindle fibers [6]. (Only later would spindle fibers be identified as bundles of dynamic microtubules [7, 8]). Working in insect spermatocyte meiosis, Nicklas and colleagues carried out groundbreaking studies exploring the role of mechanical tension by using fine glass needles to micromanipulate chromosomes within living cells. These studies demonstrated conclusively that mechanical tension led to stabilization of kinetochore attachments to microtubules [9, 10]. In the most convincing demonstrations, the researchers stabilized a syntelic attachment (both kinetochores attached to the same spindle pole) of a bivalent chromosome during prometaphase by imparting an outward, opposing tension with a microneedle. Nicklas later used calibrated glass microneedles to measure the

This article is protected by copyright. All rights reserved.

Accepted Article

stall force needed to stop a moving chromosome in a grasshopper spermatocyte [11]. This work established the maximal forces that a meiotic kinetochore could exert, which were many times that calculated to move chromosomes through a solution the viscosity of cytoplasm. It should be noted that in more recent laser trap measurements, the maximum forces exerted by meiotic chromosomes moving poleward in spermatocytes of Platyhelminth flatworms and crane flies were significantly lower than those reported from the Nicklas microneedle deflection measurements [12]. The reason for the discrepancy remains unknown.

Discovering the spindle checkpoint

Many of the micromanipulation studies of insect spermatocyte chromosomes were carried out well before the modern concepts of cell cycle regulation and cell cycle checkpoints were conceived. It was long known that the drug, colchicine, caused cells to accumulate in M phase [13]. It was found that colchicine bound to tubulin and could thereby disrupt microtubules, including those of mitotic spindles [7, 14]. This approach was applied to study mitotic chromosomes, including the karyotyping of cultured fetal cells obtained by amniocentesis, to screen for chromosome abnormalities [15, 16]. How colchicine caused cell cycle arrest in M phase was primarily thought to be dependency; namely, further progression of the cell cycle could not occur without chromosome segregation. However, even in the mid 20th century, there was clear but unappreciated evidence that this dependency model was inadequate. Researchers had reported the existence of colchicine-mitosis or c-mitosis wherein cells of various species would exit mitosis and meiosis without chromosome segregation, reconstituting interphase nuclei with double the chromosome content [17]. Many years later, an underground debate amongst cell biologists developed around the observation, which several researchers had noticed, that a cell in mitosis would delay anaphase onset for an extended period if even a single chromosome had failed to align at the metaphase plate. However, the only specific published information describing the phenomenon was a widely discussed meeting abstract that appeared in 1970 [18].

The field of cell cycle biology entered the modern era in the 1980’s, again initiated by key experiments in meiotic systems. In this case, studies in echinoderms and frogs were pivotal in the identification of a cytoplasmic activity in mature eggs termed “Maturation Promoting Factor,” or MPF, which, when injected into prophase oocytes, could induce germinal vesicle breakdown and progression through meiosis to produce mature eggs [19]. Importantly, cytoplasm from the initial injected and matured oocytes could be used to activate other prophase oocytes, indicating the factor was a self-amplifying activity or enzyme. Finally, the confluence of studies of the early cleavage stages in marine embryos, key genetic experiments in fission yeast, and a heroic biochemical fractionation of Xenopus egg extracts led to the identification of MPF, now termed Cyclin-dependent kinase 1 (Cdk1) [20-22]. Various Cyclin-dependent kinases and their activators, Cyclins, were subsequently found to be the primary regulators of cell cycle transitions.

This article is protected by copyright. All rights reserved.

Accepted Article

In 1989, from their study of cell cycle mutants in budding yeast, Hartwell and Weinert formally introduced the concept of cell cycle checkpoints [1]. Two years later McIntosh drew from the checkpoint theory and the chromosome manipulation studies in spermatocytes to propose a remarkably prescient model for the spindle checkpoint [23] (Fig. 1). This model proposed that a diffusible, inhibitory, checkpoint signal that could block anaphase onset was generated at relaxed kinetochores of unaligned chromosomes. The checkpoint signal would have a finite lifetime. Mechanical tension on sister kinetochores after bipolar attachment of spindle microtubules would halt generation of the checkpoint signal allowing anaphase onset. Li and Nicklas tested this model in a meiotic system using the unique sex chromosomes in the spermatocytes of praying mantids [24]. In this species, spermatocytes have three sex chromosomes, X1, X2, and a large Y chromosome. In meiosis each of the X chromosomes pairs with one arm of the Y to form a sex trivalent. In this way the two X’s normally segregate to the same pole while the Y goes to opposite pole during anaphase of meiosis I (MI). However, in approximately 10% of the spermatocytes, one of the X chromosomes fails to pair with the Y and monoorients to one of the poles, and the maloriented chromosome induces a strong block to the onset of anaphase I [25]. In the mantid, the arrested spermatocytes eventually die. Losing 10% of the spermatocyte population might seem extraordinarily wasteful. However, this 10% loss may be insignificant to the species survival due to the female’s proclivity toward sexual cannibalism, whereby she is prone to sever and devour her mate’s head during copulation and sometimes consume him completely afterward [26]. When transplanted into culture, mantid spermatocytes containing an unpaired X undergo a checkpoint arrest of several hours before entering anaphase. Li and Nicklas used a microneedle to impart tension on the unpaired X, pulling against the microtubule bundle connecting it to the pole (Fig. 2). When the free X was pulled with the microneedle, anaphase ensued within an hour, a stunning demonstration of how mechanical tension controls checkpoint signaling in meiosis [27].

McIntosh’s model for regulation of spindle checkpoint signaling predicted that that individual kinetochores within a single cell would have different biochemical properties, depending upon mechanical tension [23]. The first indication that individual kinetochores, even sister kinetochores, could be biochemically distinct came from studies of mitotic cells using a phosphoepitope monoclonal antibody named 3F3/2 [28]. This antibody was made by immunizing mice with extracts from meiotic Xenopus eggs and was originally thought to be specific for thio-phosphoepitopes formed in the presence of ATPγS [29]. However, it was subsequently shown to bind endogenous phosphoepitopes at kinetochores in a unique pattern. It bound with highest affinity to kinetochores of unaligned chromosomes and disappeared as the chromosomes aligned at the metaphase plate [28]. Thus expression of this phosphoepitope at kinetochores appeared to correlate positively with mitotic kinetochores that were active in checkpoint signaling. Nicklas and coworkers solidified this correlation through experiments in meiosis. They used the 3F3/2 antibody and, by micromanipulation of spermatocyte chromosomes, directly showed that kinetochore attachment and mechanical tension regulated the expression of the 3F3/2 phosphoepitope at kinetochores of living grasshopper and praying mantis spermatocytes [24, 30] (Fig. 3). Then, using spermatocytes that had been detergent lysed, they demonstrated that microneedle-induced tension could directly inhibit the kinetochore kinase that catalyzed the 3F3/2 kinetochore phosphoepitope [31]. This kinase was later shown to be Polo-like kinase 1 [32, 33]. Thus, mechanical tension could directly down regulate an enzyme activity at meiotic kinetochores and control checkpoint signaling. Subsequent studies in

This article is protected by copyright. All rights reserved.

Accepted Article

mitotic systems proved that individual kinetochores in living cells could show many other biochemical differences and confirmed the idea that microtubule attachment and tension regulate the kinetochore binding and enzymatic activities of a large number of proteins important in checkpoint signaling (reviewed in [34, 35]).

Kinetochores and the biochemistry of spindle checkpoint signaling The primary target of the spindle checkpoint is the Anaphase-Promoting Complex or Cyclosome (APC/C). The APC/C is an E3, or ubiquitin ligase. It catalyzes the addition of multiple ubiquitins on several target proteins to cause their recognition and degradation by the proteasome. APC/C activity is dependent on binding of an activator protein. During mitosis and meiosis, the most important activator protein is Cdc20, which also plays a key role in the checkpoint. In yeast and Drosophila, meiosis-specific APC/C activator proteins have been found (reviewed in [36]). The APC/C also has interphase functions and there relies on another activator, Cdh1. However, Cdh1 is also important in the early stages of mammalian oocyte meiosis [37, 38]. To block the metaphase-anaphase transition in both mitosis and meiosis, the spindle checkpoint prevents APC/C-Cdc20 from targeting two key proteins, Securin and Cyclin B. Securin is an inhibitor of the protease, Separase. In MI Separase is required at anaphase to sever the Cohesin protein complex, which holds homologs together distal to sites of crossover (see accompanying article by Rankin). The Cohesin near sister centromeres is protected during anaphase I to hold sister chromatids together. After alignment of chromosomes at metaphase of meiosis II (MII) and in mitosis, Separase is activated to cleave the remaining Cohesin between sister chromatids. The other important target of APC/C-Cdc20, Cyclin B, is the activator of the master M phase kinase, Cyclin-dependent kinase 1 (Cdk1). In species where it has been analyzed, partial destruction of Cyclin B during anaphase of MI allows cytokinesis and resculpting of the cytoplasm to prepare for MII. However, Cdk1 activity is not completely extinguished in order to prevent relicensing of DNA replication. Anaphase of MII is accompanied by complete destruction of Cyclin B and full entry into interphase.

Primarily from studies of mitotic cells in yeast and other organisms, a set of checkpoint signaling proteins have been identified. These include the Mitotic arrest deficient (Mad) proteins, Mad1, Mad2 and Mad3, the Budding uninhibited by benzimidizole (Bub) proteins, Bub1 and Bub3, and the Monopolar spindle 1(Mps1) protein (reviewed in [34, 35]). Kinase cascades are important in checkpoint signaling. Bub1 and Mps1 are serine/threonine kinases. Other serine/threonine kinases involved in checkpoint signaling are Aurora B (Ipl1 in yeast) and Polo-like kinase 1 (Plk1, Cdc5 in yeast). The Mad3 homolog in vertebrates is a large protein called Bub-Related 1 (BubR1), which unlike Mad3, contains a kinase domain. It remains controversial whether BubR1 is a true kinase or is a pseudokinase, in which the kinase domain contributes other functions such as protein stability [39, 40]. In metazoans, the RZZ complex, composed of the proteins Rod, ZW10 and Zwilch, is also required for checkpoint signaling, in part by helping to recruit Mad1/Mad2 to kinetochores [41, 42].

This article is protected by copyright. All rights reserved.

Accepted Article

Kinetochores lacking microtubule attachment and mechanical tension accumulate high concentrations of the checkpoint signaling proteins. This accumulation appears to provide a catalytic platform for the production of APC/C inhibitors (Fig. 4A). The first is formed by Mad1 and Mad2. Mad2 can take two conformations, an open or closed form. In the closed form, Mad2 can bind and inhibit Cdc20. One copy of Mad2 binds to kinetochore-bound Mad1 in the closed conformation. The Mad1-bound Mad2 can then convert other copies of Mad2 from the open to closed form to bind to and inhibit Cdc20. In a simultaneous or sequential reaction, Cdc20-Mad2 binds to BubR1-Bub3 to form the mitotic checkpoint complex (MCC). The MCC is a highly potent inhibitor of M phase APC/C [43, 44] (Fig. 3A).

Although it has been argued that the spindle checkpoint is an all-or-nothing signal [45], recent studies in mitotic cells suggest that the checkpoint signal can vary in strength and is proportional to the number of unaligned or unattached chromosomes [46, 47]. Additional controversy has swirled around whether the checkpoint monitors microtubule attachment and/or mechanical tension at kinetochores and whether attachment and tension signal through the same or distinct pathways (reviewed in [48, 49]). Biologically, it is clear that microtubule stability, mechanical tension, and checkpoint signaling are interwoven through feedback loops. Indeed many checkpoint signaling proteins and modifiers have additional roles, modifying the stability of kinetochore-microtubule attachments and promoting chromosome alignment at the metaphase plate.

Once chromosomes are aligned at metaphase, spindle checkpoint signaling is silenced to allow APC/C-Cdc20 to ubiquitylate Securin and Cyclin B. Multiple mechanisms of silencing are used (Fig. 4B). One consequence of bipolar attachment of chromosomes is mechanical tension on kinetochores. As described above, mechanical tension stabilizes microtubule-kinetochore connections. This is important because in metazoans, certain checkpoint proteins such as Mad1, Mad2 and BubR1 are depleted from kinetochores stably attached to microtubules through active transport along the microtubules by the motor protein dynein (Fig. 4B) [50]. But tension also appears to affect checkpoint signaling in other, more direct ways. One idea was that the checkpoint might be regulated by stretching of the chromatin located between kinetochores (reviewed in [51]). However, other work suggests that this aspect of mechanical tension may be unimportant in spindle checkpoint signaling [52]. Studies using super resolution light microscopy support the idea that mechanical tension imparted by microtubules induces rearrangement of proteins within individual kinetochores [53, 54]. Thus mechanical tension within the kinetochore may induce structural deformations that alter enzyme activities, for example displacing kinases from their substrates.

As another aspect of checkpoint silencing, stable kinetochore microtubule interactions result in recruitment of phosphatases to oppose checkpoint signaling kinases such as Mps1 and Aurora B [55, 56] (Fig 3B). Finally, in higher organisms, the protein p31comet is a Mad2-binding protein that aids in destabilizing the MCC and releasing Cdc20 to activate the APC/C, although how p31comet action is regulated before and after bipolar attachment of chromosomes is not certain [57, 58].

This article is protected by copyright. All rights reserved.

Accepted Article

The meiotic spindle checkpoint in non-vertebrates Despite their genetic tractability, few studies of meiotic spindle checkpoint signaling have been carried out in budding yeast. The yeast Securin, Pds1, is degraded at anaphase onset in both MI and II [59]. In mitosis, loss of checkpoint signaling in budding yeast does not induce strong segregation defects. However, in MI, deletion of Mad1 or Mad2, but not Mad3, significantly increases segregation defects [60, 61]. MII is much less affected. The segregation defects can be rescued by delaying anaphase onset through expression of a degradation-resistant form of yeast Securin. Long chromosomes with crossovers near their distal tips showed more dependence on Mad2 for proper segregation [62] (see accompanying article by Pezza and Sansom). Such chromosomes are more likely to have initially oriented both kinetochores to the same spindle pole and thus show a greater requirement for error correction. This evidence suggests that Mad1 and Mad2 may play important roles in correcting improper chromosome orientation in MI. All three Mad proteins were required to arrest meiosis after treatment with a microtubule drug. Mad1 and Mad2 were also required for the metaphase delay induced by the presence of non-exchange chromosomes (chromosomes that cannot form crossovers) but Mad3 mediated a delay during prophase that occurred in every meiosis. [63] (see accompanying article by Kurdzo and Dawson). Human BubR1 could complement the yeast Mad3 deletion, indicating conservation of function. In yeast lacking Mad2, the APC/C becomes prematurely active in prometaphase [64]. This does not happen in Mad3 deleted cells, again indicative of separate functions for Mad3 in budding yeast. Meiosis is similarly regulated by spindle checkpoint signaling in fission yeast [65, 66].

Studies of the meiotic spindle checkpoint have also been carried out in invertebrate metazoans. As noted above, many key studies on the abilities of individual chromosomes to arrest meiosis, the importance of mechanical tension to control the checkpoint, and to control kinetochore biochemistry were carried out by Nicklas and coworkers using spermatocytes of grasshoppers and praying mantids. In Drosophila spermatocytes, where homologs pair but not through crossovers, the meiotic spindle checkpoint is relatively weak, causing a short delay in anaphase onset in MI when spindles were disrupted with a microtubule drug or in the presence of four univalent chromosomes [67]. However, when only two univalents were present, MI timing was not significantly altered. Segregation in spermatocytes was impaired in Bub1 mutant flies [68]. Remarkably, in Drosophila, inactivation of spindle checkpoint signaling with null alleles of Mad2 and alleles of BubR1 specifically designed to disrupt its checkpoint function result in successful mitotic and meiotic divisions, producing flies that are both viable and fertile [69, 70]. The lethality in Drosophila mutants of other checkpoint genes such as Rod, Zw10, Bub3, and null alleles of BuR1 [71-74] likely reflects the combined consequences of checkpoint loss with compromised chromosome movements. As is the case for budding yeast Mad3, BubR1 in flies also plays an important role in meiotic prophase [75]. Deletion of Mps1 in Drosophila oocytes accelerates entry into anaphase in MI and is particularly detrimental to proper distribution of the small fourth chromosome, which unlike the other chromosomes, does not form crossovers but instead relies upon an alternative segregation pathway called the “distributive” system [76]. The distributive system requires more time for accurate segregation and thus is more sensitive to premature anaphase onset [77] (see accompanying article by Kurdzo and Dawson). Finally, while deletion of canonical checkpoint proteins lead to accelerated anaphase in meiosis, one study in Drosophila oocytes reported that this was not accompanied by increased degradation of Cyclin

This article is protected by copyright. All rights reserved.

Accepted Article

B protein, suggesting that the meiotic spindle checkpoint may not function by regulation of APC/C activity in this situation [78]. Drosophila oocytes undergo a normal physiological arrest at metaphase I, where they remain until fertilization. This oocyte-specific arrest may impact conventional pathways of checkpoint function. Spindle checkpoint proteins have been examined to a lesser degree in other non-vertebrate animals, such as nematodes and in plants, where to differing degrees they appear to play similar roles in regulating chromosome segregation events [79-82]. One notable exception is that Mps1 kinase, an essential component of kinetochore assembly and checkpoint signaling in other organisms, is not present in C. elegans [83].

The meiotic spindle checkpoint in vertebrates By far, most studies of the spindle checkpoint in meiosis have been carried out in vertebrate oocytes, primarily in mouse with a significant contribution from frog. The reasons are both practical and medical. Mature vertebrate oocytes can be isolated in the germinal vesicle stage (prophase of meiosis 1). These can then be hormonally stimulated in vitro to mature through MI and the initial stages of MII. In most vertebrate species, oocytes then physiologically arrest at metaphase of MII to await fertilization and are termed eggs. This metaphase II arrest is enforced by an activity termed “Cytostatic Factor.” An important component of Cytostatic Factor is a protein called Early mitotic inhibitor-2 (Emi2), which binds and inhibits the APC/C [84, 85]. At fertilization or after artificial activation with calcium ionophore or electrical stimulation, release of calcium leads to the degradation of Emi2 and induction of anaphase II.

In oocyte meiosis, after breakdown of the germinal vesicle (aka prophase nucleus) the time to anaphase I onset requires an extended period, from about 2 hours in Xenopus to several hours in mouse. In vertebrate mitosis, the time from nuclear envelope breakdown to anaphase is much shorter, usually 0.5 to 1 hour. In mouse oocytes, MI is prolonged through the activity of APC/C-Cdh1, which normally is thought to function in interphase cells. In mammalian oocytes after germinal vesicle breakdown, APC/C-Cdh1 targets Cdc20 for degradation but not Cyclin B or Securin [38]. If Cdh1 is depleted with inhibitory morpholinos, APC/C-Cdc20 becomes active early targeting Securin and Cyclin B and resulting in premature anaphase and faulty segregation of homologous chromosomes. In oocytes, Map kinase and Cdk1 both participate in promoting progression through meiosis [86-88]. Recent studies inhibiting these kinases suggest that both kinases synergize to block APC/C-mediated degradation of Cyclin B and Securin in early MI [89].

Spindle assembly in vertebrate oocyte meiosis also occurs through a process distinct from that in mitosis. Unlike other cell types, vertebrate oocytes lack centrioles and highly focused spindle poles. Instead bipolar spindles form from coalescence of microtubule nucleating centers that are focused by microtubules, which assemble near the chromosomes [90]. In mouse oocytes, immediately after germinal vesicle breakdown, kinetochores do not initially form end-on attachments but associate laterally with the spindle microtubules and congress to the spindle equator. Only during the latter half of prometaphase do kinetochore-microtubule interactions mature into end-on attachments apparently driven by a continuing rise in Cdk1 activity [91, 92]. Three dimensional kinetic tracking chromosome movements in MI of mouse oocytes shows that

This article is protected by copyright. All rights reserved.

Accepted Article

most chromosomes undergo one or more rounds of correcting inappropriate kinetochore attachments before attaining stable bipolar attachment to spindle microtubules [93]. Interestingly, this same behavior of multiple rounds of error correction is also seen in budding yeast meiosis [94].

Numerous experiments have demonstrated the existence of functional spindle checkpoint signaling in mouse oocyte meiosis. The arrest in response to microtubule drugs and for normal timing of chromosome segregation are dependent on canonical checkpoint signaling proteins [95-103], for review see [104]. A recent study demonstrated that inhibition of the spindle checkpoint through several different perturbations during MI increases basal APC/C activity, accelerates anaphase onset by approximately 2 hours, and results in increased aneuploidy [102]. Most studies have centered on checkpoint control during MI, but evidence indicates that checkpoint signaling can also suppress anaphase onset in MII in mouse oocytes [97].

In Xenopus oocytes it was initially thought that APC/C activity was not required for the MI to MII transition [105, 106], although later work is consistent with typical APC/C-mediated degradation of Securin and Cyclin B at the MI to MII transition [107, 108]. However, Xenopus oocytes do not arrest or delay the MI to MII transition when spindle microtubules are disrupted, indicating that they lack spindle checkpoint regulation [109]. One difference is that normal meiotic maturation in Xenopus oocytes occurs much more rapidly, approximately 2 hours, compared with mouse oocytes where it requires 7 or more hours. Potentially, a critical difference in the two systems may simply be the larger size and the greater ratio of cytoplasm to chromatin in Xenopus oocytes. Crude cytoplasmic extracts prepared from activated Xenopus eggs can recapitulate several rounds of the cell cycle where Cyclin B is degraded and resynthesized. In the first round of Cyclin B degradation, which is equivalent to anaphase of MII, microtubule drugs added to the extract have no effect unless the extract is supplemented with a large number of sperm nuclei. When the sperm are added, the spindle checkpoint blocks progression [110]. When placed into the MII egg extract, the sperm nuclei immediately form unpaired, kinetochore-containing chromatids. Thus, adding sperm nuclei to reduce the cytoplasm to chromatin ratio (or more specifically the cytoplasm to kinetochore ratio) causes the MII extract to become sensitive to inhibition by the spindle checkpoint.

In contrast to oocytes, mouse spermatocytes show strong checkpoint signaling, comparable to that in somatic cell mitosis. Spermatocytes will block anaphase onset in the presence one or more mis-attached or misaligned chromosomes [111]. Oocytes arrest MI only when treated at concentrations of microtubule disruptors, such as nocodazole, that completely depolymerize the spindle (Fig. 5A and 5B). At lower drug concentrations, which allow spindle microtubules to form but compromise normal kinetochore-microtubule interactions, oocytes can still show a delay of anaphase onset [100] (Fig. 5C). The mouse oocyte spindle checkpoint appears relatively insensitive to chromosomes that are simply misaligned but bound to spindle microtubules, and checkpoint protein accumulation at the kinetochores of misaligned chromosomes is weak. [103, 112, 113]. One instructive model is the XO mouse. In mouse spermatocytes engineered to have a XO karyotype, the unpaired X induces a strong meiotic metaphase arrest that eventually induces apoptosis [114, 115]. This is remarkably parallel with the praying mantid spermatocytes

This article is protected by copyright. All rights reserved.

Accepted Article

described above. In contrast, XO oocytes undergo anaphase without delay, whether both chromatids of the unpaired X move to the same pole or separate precociously in MI [116]. In the situation where an unpaired X orients to one pole, the two sister kinetochores are associated with microtubules but not under normal tension. There meiotic spindle checkpoint signaling is unable to induce a delay or arrest (Fig. 5D). In another example, depletion of the spindle pole protein, NUMA, results in a reduction of interkinetochore tension and chromosome alignment defects in MI that fail to activate the spindle checkpoint and delay anaphase [117]. In mouse oocytes surgically bisected with a glass needle, half oocytes lacking chromosomes undergo accelerated degradation of GFP-Cyclin B [118]. This can be not be blocked by application of the microtubule drug, nocodazole, which is effective in blocking Cyclin B degradation in half oocytes containing chromosomes. However, a single univalent containing four kinetochores can prevent the premature degradation of GFP-Cyclin B that would occur in a half oocyte lacking any chromosomes. The Mlh1 gene is required for meiotic recombination (see accompanying article by Pezza and Sansam). In oocytes from mlh1-/- mice, the univalents result in a checkpoint arrest. In some oocytes, the sister kinetochores on the univalent chromosomes attach to opposite poles and achieve some level of “loose” alignment at the metaphase plate. After a substantial delay these oocytes and undergo anaphase onset [119]. Examination of the resulting MII oocytes indicate that checkpoint signaling is overridden when an average of 4 univalents remain that have not yet achieved bipolar attachment of sister kinetochores (Fig. 5E).

The above observations suggest that spindle checkpoint arrest in mouse oocytes requires strong checkpoint signaling from the combined output of several kinetochores lacking microtubule attachment and tension. Dilution of checkpoint signals in the large volume of oocyte cytoplasm likely contributes to the apparent reduced ability of oocytes to block progression in the presence of a small number of improperly attached chromosomes, particularly in comparison to the stringent checkpoint signaling characteristic of spermatocyte meiosis or somatic cell mitosis.

Meiotic aneuploidy and maternal age In humans, meiotic aneuploidy is a major source of infertility, miscarriage, and congenital birth defects. The majority of meiotic aneuploidies in humans are due to errors in oogenesis. Combined data for detectable human trisomy after fertilization in humans indicates that most segregation errors occur during MI, though a substantial fraction also occur in MII [120]. However, analysis of first and second polar bodies obtained during in vitro fertilization therapy from women in their late 30’s or early 40’s reveals that anomalous events are common in both MI and MII [121, 122]. Most aneuploidies result in inviable embryos, but some autosomal trisomies and some sex chromosome aneuploidies produce fetuses that survive to term. It has long been recognized that the risk for trisomy rises sharply for women near the end of their reproductive lifespan [123].

Multiple genetic, environmental, and age-related factors likely contribute to the production of aneuploid gametes [124, 125]. The fact that most errors occur in oogenesis implicates the relaxed spindle checkpoint of oocytes as a potential contributing cause. Analysis of oocytes

This article is protected by copyright. All rights reserved.

Accepted Article

from older women has revealed diminished levels of message for several checkpoint proteins including Bub1, BubR1, Bub3 and Mad2 [126, 127]. In at least some strains, mouse oocytes from older mice also show increased aneuploidy [128-130]. Mice engineered to express reduced levels of functional checkpoint proteins BubR1, Bub1, and Mad2 exhibit increased oocyte aneuploidy and spindle defects [101, 131, 132]. Older female mice also show lower levels of checkpoint message or protein expression in their oocytes or ovaries [128, 131]. Despite this evidence, some studies have reported that oocytes from older mice do not exhibit a weaker spindle checkpoint since they do not show accelerated timing of anaphase onset in MI, and they arrest when treated with the microtubule drug, nocodazole [133, 134]. However, a more recent study showed that oocytes from older females are significantly impaired in their ability to arrest anaphase onset when a low concentration of nocodazole is applied [135]. Kinetochores in oocytes from older mice have also been reported to show reduced ability to form stable bipolar attachments in MI [135, 136]. As discussed above, mechanical tension from correct bipolar attachment is an important contributor to stabilizing kinetochore-microtubule attachment and to checkpoint silencing. The studies described may point to a general degradation of kinetochore function in oocytes of aged females that could contribute to higher rates of aneuploidy.

Another proposed source of meiotic aneuploidy and its increased incidence with advanced maternal age, is loss of chromosome cohesion [137, 138] (see accompanying article by Rankin). In mammals, oocytes are formed during the mother’s fetal life. In S phase, cohesin is loaded to hold sister chromatids together. The fetal oocytes then enter an extended prophase of meiosis until ovulation. Experiments in mice indicate that cohesin proteins loaded during S phase in the fetus are not turned over or replaced before ovulation [139, 140]. If applied to humans these observations indicate that the cohesin proteins laid down during a woman’s fetal life persist for up to five decades. Interestingly, in Drosophila oocytes a recently identified cohesin “rejuvenation” system operates to add functional cohesin during prophase of meiosis [141]. However, nothing comparable in vertebrates is known, so it is still believed that no fresh cohesin is loaded after S phase. Diminution of levels of Cohesin complex proteins and increased susceptibility to pathways that remove Cohesin accompany increased aneuploidy in the oocytes of older mice [133, 134, 142-144]. Immunohistochemical analysis also demonstrates reduced cohesin protein expression in the human oocytes of women over 40 [145]. Interestingly, a recent study provides evidence for a link between the cohesin complex and the spindle checkpoint in oocyte meiosis [146]. As described above, in mlh1-/- oocytes, a checkpoint arrest occurs if a sufficient number of univalents remain where sister kinetochores have not achieved bipolar attachment. Researchers engineered mlh1-/- oocytes to express a form of the meiotic cohesin subunit, Rec8, that could be artificially cleaved by TEV protease. Controls that did not receive TEV protease remained arrested. Introduction of TEV protease cleaved cohesin to produce unpaired sister chromatids and anaphase occurred after a short delay. Thus during MI a strong checkpoint arrest was induced by unpaired univalents, whereas a much weaker one was induced by unpaired chromatids. Interestingly, generating unpaired sister kinetochores in a mitotic cell, the one cell zygote induced a strong checkpoint arrest. This was done through TEV cleavage of the mitotic cohesin subunit, Rad21/Scc1. This mitotic arrest occurred in a cytoplasm of similar volume to that of the oocyte suggesting that cytoplasmic volume was not the difference. The authors interpreted this work to suggest a specific role for intact cohesin in meiotic but not mitotic checkpoint signaling.

This article is protected by copyright. All rights reserved.

Accepted Article

The spindle checkpoint in meiosis: lessons now and for the future Despite the fervent hopes of research scientists and desires of journal editors for a complete mechanistic story to publish, perhaps the most important lesson learned in studying the spindle checkpoint in meiosis is that biological pathways cannot be neatly catalogued into discrete, fully traceable branches. Instead the pathways converge, intersect, and diverge in knots that defy simple untangling. The pathways of spindle checkpoint signaling, kinetochore-microtubule attachment, and chromosome cohesion cannot be fully separated, in part, because the proteins involved play interlocking roles. Many of the canonical checkpoint signaling proteins such as Bub1, BubR1 and Mad2 also impact the stability of kinetochore-microtubule interactions and thus feed back on the control of checkpoint signaling by mechanical tension. A particularly strong lesson on the interrelatedness of meiotic pathways is provided by the protein, Shugoshinlike 2 (Sgol2). In mammals, Sgol2 is essential in meiosis. Sgol2 knockout mice are viable without noticeable defects, except infertility in both males and females caused by premature loss of sister chromatid cohesion during anaphase I [147]. Sgol2 maintains cohesion by binding and recruiting protein phosphatase 2A (PP2A) and thereby protects cohesin near the kinetochores during MI. But Sgol2 has other interactions. Sgol2 also binds to the checkpoint protein, Mad2, and through a mechanism involving Aurora kinases, it regulates the kinetochore activity of the important microtubule depolymerase [148]. Through binding of PP2A and Mad2, Sgol2 appears to participate in silencing the meiotic spindle checkpoint [148]. Thus, Sgol2 may function as a nexus for synchronizing pathways of chromosome alignment, checkpoint silencing, and cohesion maintenance in meiosis. Complex feedback regulation appears to be the norm in cell cycle regulation. Mapping these interactions in dissection of the spindle checkpoint pathway poses an exciting challenge to future research.

Acknowledgements The author’s laboratory is supported by grant R01GM111731 from the National Institute of General Medical Sciences, grant HR12-177 from the Oklahoma Center for the Advancement of Science and Technology, a grant from the Oklahoma Center for Adult Stem Research, and by the McCasland Foundation.

References

1. Hartwell, L. H. & Weinert, T. A. (1989) Checkpoints: controls that ensure the order of cell cycle events, Science. 246, 629-34. 2. Subramanian, V. V. & Hochwagen, A. (2014) The meiotic checkpoint network: step-by-step through meiotic prophase, Cold Spring Harb Perspect Biol. 6, a016675. 3. Woglar, A. & Jantsch, V. (2014) Chromosome movement in meiosis I prophase of Caenorhabditis elegans, Chromosoma. 123, 15-24. 4. Yamamoto, A. (2014) Gathering up meiotic telomeres: a novel function of the microtubuleorganizing center, Cell Mol Life Sci. 71, 2119-34. 5. Kracklauer, M. P., Link, J. & Alsheimer, M. (2013) LINCing the nuclear envelope to gametogenesis, Curr Top Dev Biol. 102, 127-57. 6. Dietz, R. (1958) [Multiple sex chromosomes in Ostracoda cypria, their evolution and division characteristics], Chromosoma. 9, 359-440.

This article is protected by copyright. All rights reserved.

Accepted Article

7. Inoue, S. & Sato, H. (1967) Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement, J Gen Physiol. 50, Suppl:259-92. 8. Ledbetter, M. C. & Porter, K. R. (1963) A "Microtubule" in Plant Cell Fine Structure, J Cell Biol. 19, 239-50. 9. Ault, J. G. & Nicklas, R. B. (1989) Tension, microtubule rearrangements, and the proper distribution of chromosomes in mitosis, Chromosoma. 98, 33-9. 10. Nicklas, R. B. & Koch, C. A. (1969) Chromosome micromanipulation. 3. Spindle fiber tension and the reorientation of mal-oriented chromosomes, J Cell Biol. 43, 40-50. 11. Nicklas, R. B. (1983) Measurements of the force produced by the mitotic spindle in anaphase, J Cell Biol. 97, 542-8. 12. Ferraro-Gideon, J., Sheykhani, R., Zhu, Q., Duquette, M. L., Berns, M. W. & Forer, A. (2013) Measurements of forces produced by the mitotic spindle using optical tweezers, Mol Biol Cell. 24, 1375-86. 13. Eigsti, O. J., Dustin, P., Jr. & Gay-Winn, N. (1949) On the discovery of the action of colchicine on mitosis in 1889, Science. 110, 692. 14. Weisenberg, R. C., Borisy, G. G. & Taylor, E. W. (1968) The colchicine-binding protein of mammalian brain and its relation to microtubules, Biochemistry. 7, 4466-79. 15. Steele, M. W. & Breg, W. R., Jr. (1966) Chromosome analysis of human amniotic-fluid cells, Lancet. 1, 383-5. 16. Thiede, H. A., Creasman, W. T. & Metcalfe, S. (1966) Antenatal analysis of the human chromosomes, Am J Obstet Gynecol. 94, 589-90. 17. Fankhauser, G. & Humphrey, R. R. (1952) The Rare Occurrence of Mitosis Without Spindle Apparatus ("Colchicine Mitosis") Producing Endopolyploidy in Embryos of the Axolotl, Proc Natl Acad Sci U S A. 38, 1073-82. 18. Zirkle, R. E. (1970) Invovlement of the prometaphase kinetochore in prevention of precocious anaphase, J Cell Biol. 47, 235a. 19. Wasserman, W. J. & Masui, Y. (1976) A cytoplasmic factor promoting oocyte maturation: its extraction and preliminary characterization, Science. 191, 1266-8. 20. Lohka, M. J., Hayes, M. K. & Maller, J. L. (1988) Purification of maturation-promoting factor, an intracellular regulator of early mitotic events, Proc Natl Acad Sci U S A. 85, 3009-13. 21. Gould, K. L. & Nurse, P. (1989) Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis, Nature. 342, 39-45. 22. Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D. & Hunt, T. (1983) Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division, Cell. 33, 389-96. 23. McIntosh, J. R. (1991) Structural and mechanical control of mitotic progression, Cold Spring Harb Symp Quant Biol. 56, 613-9. 24. Li, X. & Nicklas, R. B. (1997) Tension-sensitive kinetochore phosphorylation and the chromosome distribution checkpoint in praying mantid spermatocytes, J Cell Sci. 110 ( Pt 5), 537-45. 25. Callan, H. G. & Jacobs, P. A. (1957) The meiotic process in Mantis religiosa L. males, J Genetics. 55, 200-217. 26. Roeder, K. D. (1935) An experimental analysis of the sexual behavior of the praying mantis (Mantis religiosa L.), Biol Bull. 69, 203-220. 27. Li, X. & Nicklas, R. B. (1995) Mitotic forces control a cell-cycle checkpoint, Nature. 373, 630-2. 28. Gorbsky, G. J. & Ricketts, W. A. (1993) Differential expression of a phosphoepitope at the kinetochores of moving chromosomes, J Cell Biol. 122, 1311-21. 29. Cyert, M. S., Scherson, T. & Kirschner, M. W. (1988) Monoclonal antibodies specific for thiophosphorylated proteins recognize Xenopus MPF, Dev Biol. 129, 209-16.

This article is protected by copyright. All rights reserved.

Accepted Article

30. Nicklas, R. B., Ward, S. C. & Gorbsky, G. J. (1995) Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint, J Cell Biol. 130, 929-39. 31. Nicklas, R. B., Campbell, M. S., Ward, S. C. & Gorbsky, G. J. (1998) Tension-sensitive kinetochore phosphorylation in vitro, J Cell Sci. 111 ( Pt 21), 3189-96. 32. Ahonen, L. J., Kallio, M. J., Daum, J. R., Bolton, M., Manke, I. A., Yaffe, M. B., Stukenberg, P. T. & Gorbsky, G. J. (2005) Polo-like kinase 1 creates the tension-sensing 3F3/2 phosphoepitope and modulates the association of spindle-checkpoint proteins at kinetochores, Curr Biol. 15, 1078-89. 33. Wong, O. K. & Fang, G. (2005) Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores, J Cell Biol. 170, 709-19. 34. Jia, L., Kim, S. & Yu, H. (2013) Tracking spindle checkpoint signals from kinetochores to APC/C, Trends Biochem Sci. 38, 302-11. 35. Sacristan, C. & Kops, G. J. (2014) Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling, Trends Cell Biol. 36. Pesin, J. A. & Orr-Weaver, T. L. (2008) Regulation of APC/C activators in mitosis and meiosis, Annu Rev Cell Dev Biol. 24, 475-99. 37. Holt, J. E., Lane, S. I., Jennings, P., Garcia-Higuera, I., Moreno, S. & Jones, K. T. (2012) APC(FZR1) prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing, Mol Biol Cell. 23, 3970-81. 38. Reis, A., Madgwick, S., Chang, H. Y., Nabti, I., Levasseur, M. & Jones, K. T. (2007) Prometaphase APCcdh1 activity prevents non-disjunction in mammalian oocytes, Nat Cell Biol. 9, 1192-8. 39. Suijkerbuijk, S. J., van Dam, T. J., Karagoz, G. E., von Castelmur, E., Hubner, N. C., Duarte, A. M., Vleugel, M., Perrakis, A., Rudiger, S. G., Snel, B. & Kops, G. J. (2012) The vertebrate mitotic checkpoint protein BUBR1 is an unusual pseudokinase, Dev Cell. 22, 1321-9. 40. Guo, Y., Kim, C., Ahmad, S., Zhang, J. & Mao, Y. (2012) CENP-E--dependent BubR1 autophosphorylation enhances chromosome alignment and the mitotic checkpoint, J Cell Biol. 198, 205-17. 41. Buffin, E., Lefebvre, C., Huang, J., Gagou, M. E. & Karess, R. E. (2005) Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex, Curr Biol. 15, 856-61. 42. Kops, G. J., Kim, Y., Weaver, B. A., Mao, Y., McLeod, I., Yates, J. R., 3rd, Tagaya, M. & Cleveland, D. W. (2005) ZW10 links mitotic checkpoint signaling to the structural kinetochore, J Cell Biol. 169, 49-60. 43. Sudakin, V., Chan, G. K. & Yen, T. J. (2001) Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2, J Cell Biol. 154, 925-36. 44. Han, J. S., Holland, A. J., Fachinetti, D., Kulukian, A., Cetin, B. & Cleveland, D. W. (2013) Catalytic assembly of the mitotic checkpoint inhibitor BubR1-Cdc20 by a Mad2-induced functional switch in Cdc20, Mol Cell. 51, 92-104. 45. Khodjakov, A. & Rieder, C. L. (2009) The nature of cell-cycle checkpoints: facts and fallacies, J Biol. 8, 88. 46. Dick, A. E. & Gerlich, D. W. (2013) Kinetic framework of spindle assembly checkpoint signalling, Nat Cell Biol. 15, 1370-7. 47. Collin, P., Nashchekina, O., Walker, R. & Pines, J. (2013) The spindle assembly checkpoint works like a rheostat rather than a toggle switch, Nat Cell Biol. 15, 1378-85. 48. Maresca, T. J. & Salmon, E. D. (2010) Welcome to a new kind of tension: translating kinetochore mechanics into a wait-anaphase signal, J Cell Sci. 123, 825-35. 49. Wang, Y., Jin, F., Higgins, R. & McKnight, K. (2014) The current view for the silencing of the spindle assembly checkpoint, Cell Cycle. 13, 1694-701. 50. Howell, B. J., McEwen, B. F., Canman, J. C., Hoffman, D. B., Farrar, E. M., Rieder, C. L. & Salmon, E. D. (2001) Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation, J Cell Biol. 155, 1159-72.

This article is protected by copyright. All rights reserved.

Accepted Article

51. Musacchio, A. & Salmon, E. D. (2007) The spindle-assembly checkpoint in space and time, Nat Rev Mol Cell Biol. 8, 379-93. 52. O'Connell, C. B., Loncarek, J., Hergert, P., Kourtidis, A., Conklin, D. S. & Khodjakov, A. (2008) The spindle assembly checkpoint is satisfied in the absence of interkinetochore tension during mitosis with unreplicated genomes, J Cell Biol. 183, 29-36. 53. Maresca, T. J. & Salmon, E. D. (2009) Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity, J Cell Biol. 184, 37381. 54. Uchida, K. S., Takagaki, K., Kumada, K., Hirayama, Y., Noda, T. & Hirota, T. (2009) Kinetochore stretching inactivates the spindle assembly checkpoint, J Cell Biol. 184, 383-90. 55. Rosenberg, J. S., Cross, F. R. & Funabiki, H. (2011) KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint, Curr Biol. 21, 942-7. 56. Vanoosthuyse, V. & Hardwick, K. G. (2009) A novel protein phosphatase 1-dependent spindle checkpoint silencing mechanism, Curr Biol. 19, 1176-81. 57. Xia, G., Luo, X., Habu, T., Rizo, J., Matsumoto, T. & Yu, H. (2004) Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint, EMBO J. 23, 3133-43. 58. Eytan, E., Wang, K., Miniowitz-Shemtov, S., Sitry-Shevah, D., Kaisari, S., Yen, T. J., Liu, S. T. & Hershko, A. (2014) Disassembly of mitotic checkpoint complexes by the joint action of the AAA-ATPase TRIP13 and p31comet, Proc Natl Acad Sci U S A. 111, 12019-24. 59. Salah, S. M. & Nasmyth, K. (2000) Destruction of the securin Pds1p occurs at the onset of anaphase during both meiotic divisions in yeast, Chromosoma. 109, 27-34. 60. Shonn, M. A., McCarroll, R. & Murray, A. W. (2000) Requirement of the spindle checkpoint for proper chromosome segregation in budding yeast meiosis, Science. 289, 300-3. 61. Shonn, M. A., Murray, A. L. & Murray, A. W. (2003) Spindle checkpoint component Mad2 contributes to biorientation of homologous chromosomes, Curr Biol. 13, 1979-84. 62. Lacefield, S. & Murray, A. W. (2007) The spindle checkpoint rescues the meiotic segregation of chromosomes whose crossovers are far from the centromere, Nat Genet. 39, 1273-7. 63. Cheslock, P. S., Kemp, B. J., Boumil, R. M. & Dawson, D. S. (2005) The roles of MAD1, MAD2 and MAD3 in meiotic progression and the segregation of nonexchange chromosomes, Nat Genet. 37, 756-60. 64. Tsuchiya, D., Gonzalez, C. & Lacefield, S. (2011) The spindle checkpoint protein Mad2 regulates APC/C activity during prometaphase and metaphase of meiosis I in Saccharomyces cerevisiae, Mol Biol Cell. 22, 2848-61. 65. Yamamoto, A., Kitamura, K., Hihara, D., Hirose, Y., Katsuyama, S. & Hiraoka, Y. (2008) Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation, J Cell Biol. 182, 277-88. 66. Yamaguchi, S., Decottignies, A. & Nurse, P. (2003) Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint, EMBO J. 22, 1075-87. 67. Rebollo, E. & Gonzalez, C. (2000) Visualizing the spindle checkpoint in Drosophila spermatocytes, EMBO Rep. 1, 65-70. 68. Basu, J., Bousbaa, H., Logarinho, E., Li, Z., Williams, B. C., Lopes, C., Sunkel, C. E. & Goldberg, M. L. (1999) Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila, J Cell Biol. 146, 13-28. 69. Buffin, E., Emre, D. & Karess, R. E. (2007) Flies without a spindle checkpoint, Nat Cell Biol. 9, 565-72. 70. Rahmani, Z., Gagou, M. E., Lefebvre, C., Emre, D. & Karess, R. E. (2009) Separating the spindle, checkpoint, and timer functions of BubR1, J Cell Biol. 187, 597-605.

This article is protected by copyright. All rights reserved.

Accepted Article

71. Basto, R., Gomes, R. & Karess, R. E. (2000) Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila, Nat Cell Biol. 2, 939-43. 72. Lopes, C. S., Sampaio, P., Williams, B., Goldberg, M. & Sunkel, C. E. (2005) The Drosophila Bub3 protein is required for the mitotic checkpoint and for normal accumulation of cyclins during G2 and early stages of mitosis, J Cell Sci. 118, 187-98. 73. Williams, B. C., Karr, T. L., Montgomery, J. M. & Goldberg, M. L. (1992) The Drosophila l(1)zw10 gene product, required for accurate mitotic chromosome segregation, is redistributed at anaphase onset, J Cell Biol. 118, 759-73. 74. Perez-Mongiovi, D., Malmanche, N., Bousbaa, H. & Sunkel, C. (2005) Maternal expression of the checkpoint protein BubR1 is required for synchrony of syncytial nuclear divisions and polar body arrest in Drosophila melanogaster, Development. 132, 4509-20. 75. Malmanche, N., Owen, S., Gegick, S., Steffensen, S., Tomkiel, J. E. & Sunkel, C. E. (2007) Drosophila BubR1 is essential for meiotic sister-chromatid cohesion and maintenance of synaptonemal complex, Curr Biol. 17, 1489-97. 76. Gilliland, W. D., Wayson, S. M. & Hawley, R. S. (2005) The meiotic defects of mutants in the Drosophila mps1 gene reveal a critical role of Mps1 in the segregation of achiasmate homologs, Curr Biol. 15, 672-7. 77. Gilliland, W. D., Hughes, S. E., Cotitta, J. L., Takeo, S., Xiang, Y. & Hawley, R. S. (2007) The multiple roles of mps1 in Drosophila female meiosis, PLoS Genet. 3, e113. 78. Batiha, O. & Swan, A. (2012) Evidence that the spindle assembly checkpoint does not regulate APC(Fzy) activity in Drosophila female meiosis, Genome. 55, 63-7. 79. Moyle, M. W., Kim, T., Hattersley, N., Espeut, J., Cheerambathur, D. K., Oegema, K. & Desai, A. (2014) A Bub1-Mad1 interaction targets the Mad1-Mad2 complex to unattached kinetochores to initiate the spindle checkpoint, J Cell Biol. 204, 647-57. 80. Kitagawa, R. & Rose, A. M. (1999) Components of the spindle-assembly checkpoint are essential in Caenorhabditis elegans, Nat Cell Biol. 1, 514-21. 81. Zamariola, L., Tiang, C. L., De Storme, N., Pawlowski, W. & Geelen, D. (2014) Chromosome segregation in plant meiosis, Front Plant Sci. 5, 279. 82. Yu, H. G., Muszynski, M. G. & Kelly Dawe, R. (1999) The maize homologue of the cell cycle checkpoint protein MAD2 reveals kinetochore substructure and contrasting mitotic and meiotic localization patterns, J Cell Biol. 145, 425-35. 83. Vleugel, M., Hoogendoorn, E., Snel, B. & Kops, G. J. (2012) Evolution and function of the mitotic checkpoint, Dev Cell. 23, 239-50. 84. Hansen, D. V., Tung, J. J. & Jackson, P. K. (2006) CaMKII and polo-like kinase 1 sequentially phosphorylate the cytostatic factor Emi2/XErp1 to trigger its destruction and meiotic exit, Proc Natl Acad Sci U S A. 103, 608-13. 85. Shoji, S., Yoshida, N., Amanai, M., Ohgishi, M., Fukui, T., Fujimoto, S., Nakano, Y., Kajikawa, E. & Perry, A. C. (2006) Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20, EMBO J. 25, 834-45. 86. Posada, J. & Cooper, J. A. (1992) Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes, Science. 255, 212-5. 87. Shibuya, E. K., Boulton, T. G., Cobb, M. H. & Ruderman, J. V. (1992) Activation of p42 MAP kinase and the release of oocytes from cell cycle arrest, EMBO J. 11, 3963-75. 88. Verlhac, M. H., Kubiak, J. Z., Clarke, H. J. & Maro, B. (1994) Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes, Development. 120, 1017-25. 89. Nabti, I., Marangos, P., Bormann, J., Kudo, N. R. & Carroll, J. (2014) Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity, J Cell Biol. 204, 891-900. 90. Messinger, S. M. & Albertini, D. F. (1991) Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte, J Cell Sci. 100 ( Pt 2), 289-98.

This article is protected by copyright. All rights reserved.

Accepted Article

91. Brunet, S., Maria, A. S., Guillaud, P., Dujardin, D., Kubiak, J. Z. & Maro, B. (1999) Kinetochore fibers are not involved in the formation of the first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase, J Cell Biol. 146, 1-12. 92. Davydenko, O., Schultz, R. M. & Lampson, M. A. (2013) Increased CDK1 activity determines the timing of kinetochore-microtubule attachments in meiosis I, J Cell Biol. 202, 2219. 93. Kitajima, T. S., Ohsugi, M. & Ellenberg, J. (2011) Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes, Cell. 146, 568-81. 94. Meyer, R. E., Kim, S., Obeso, D., Straight, P. D., Winey, M. & Dawson, D. S. (2013) Mps1 and Ipl1/Aurora B act sequentially to correctly orient chromosomes on the meiotic spindle of budding yeast, Science. 339, 1071-4. 95. Brunet, S., Pahlavan, G., Taylor, S. & Maro, B. (2003) Functionality of the spindle checkpoint during the first meiotic division of mammalian oocytes, Reproduction. 126, 443-50. 96. Eichenlaub-Ritter, U. & Boll, I. (1989) Nocodazole sensitivity, age-related aneuploidy, and alterations in the cell cycle during maturation of mouse oocytes, Cytogenet Cell Genet. 52, 1706. 97. Tsurumi, C., Hoffmann, S., Geley, S., Graeser, R. & Polanski, Z. (2004) The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes, J Cell Biol. 167, 103750. 98. Homer, H. A., McDougall, A., Levasseur, M., Yallop, K., Murdoch, A. P. & Herbert, M. (2005) Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes, Genes Dev. 19, 202-7. 99. Homer, H. A., McDougall, A., Levasseur, M., Murdoch, A. P. & Herbert, M. (2005) Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes, Reproduction. 130, 829-43. 100. Wassmann, K., Niault, T. & Maro, B. (2003) Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes, Curr Biol. 13, 1596-608. 101. Niault, T., Hached, K., Sotillo, R., Sorger, P. K., Maro, B., Benezra, R. & Wassmann, K. (2007) Changing Mad2 levels affects chromosome segregation and spindle assembly checkpoint control in female mouse meiosis I, PLoS One. 2, e1165. 102. Lane, S. I. & Jones, K. T. (2014) Non-canonical function of spindle assembly checkpoint proteins after APC activation reduces aneuploidy in mouse oocytes, Nat Commun. 5, 3444. 103. Lane, S. I., Yun, Y. & Jones, K. T. (2012) Timing of anaphase-promoting complex activation in mouse oocytes is predicted by microtubule-kinetochore attachment but not by bivalent alignment or tension, Development. 139, 1947-55. 104. Polanski, Z. (2013) Spindle assembly checkpoint regulation of chromosome segregation in mammalian oocytes, Reprod Fertil Dev. 25, 472-83. 105. Peter, M., Castro, A., Lorca, T., Le Peuch, C., Magnaghi-Jaulin, L., Doree, M. & Labbe, J. C. (2001) The APC is dispensable for first meiotic anaphase in Xenopus oocytes, Nat Cell Biol. 3, 83-7. 106. Taieb, F. E., Gross, S. D., Lewellyn, A. L. & Maller, J. L. (2001) Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes, Curr Biol. 11, 508-13. 107. Zhang, X., Ma, C., Miller, A. L., Katbi, H. A., Bement, W. M. & Liu, X. J. (2008) Polar body emission requires a RhoA contractile ring and Cdc42-mediated membrane protrusion, Dev Cell. 15, 386-400. 108. Fan, H. Y., Sun, Q. Y. & Zou, H. (2006) Regulation of Separase in meiosis: Separase is activated at the metaphase I-II transition in Xenopus oocytes during meiosis, Cell Cycle. 5, 198204. 109. Shao, H., Li, R., Ma, C., Chen, E. & Liu, X. J. (2013) Xenopus oocyte meiosis lacks spindle assembly checkpoint control, J Cell Biol. 201, 191-200.

This article is protected by copyright. All rights reserved.

Accepted Article

110. Minshull, J., Sun, H., Tonks, N. K. & Murray, A. W. (1994) A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts, Cell. 79, 475-86. 111. Eaker, S., Pyle, A., Cobb, J. & Handel, M. A. (2001) Evidence for meiotic spindle checkpoint from analysis of spermatocytes from Robertsonian-chromosome heterozygous mice, J Cell Sci. 114, 2953-65. 112. Gui, L. & Homer, H. (2012) Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes, Development. 139, 1941-6. 113. Sebestova, J., Danylevska, A., Novakova, L., Kubelka, M. & Anger, M. (2012) Lack of response to unaligned chromosomes in mammalian female gametes, Cell Cycle. 11, 3011-8. 114. de Boer, P., de Jong, J. H. & van der Hoeven, F. A. (1991) Meiosis in a sterile male mouse with an isoYq marker chromosome, Cytogenet Cell Genet. 56, 36-9. 115. Sutcliffe, M. J., Darling, S. M. & Burgoyne, P. S. (1991) Spermatogenesis in XY, XYSxra and XOSxra mice: a quantitative analysis of spermatogenesis throughout puberty, Mol Reprod Dev. 30, 81-9. 116. LeMaire-Adkins, R., Radke, K. & Hunt, P. A. (1997) Lack of checkpoint control at the metaphase/anaphase transition: a mechanism of meiotic nondisjunction in mammalian females, J Cell Biol. 139, 1611-9. 117. Kolano, A., Brunet, S., Silk, A. D., Cleveland, D. W. & Verlhac, M. H. (2012) Error-prone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension, Proc Natl Acad Sci U S A. 109, E1858-67. 118. Hoffmann, S., Maro, B., Kubiak, J. Z. & Polanski, Z. (2011) A single bivalent efficiently inhibits cyclin B1 degradation and polar body extrusion in mouse oocytes indicating robust SAC during female meiosis I, PLoS One. 6, e27143. 119. Nagaoka, S. I., Hodges, C. A., Albertini, D. F. & Hunt, P. A. (2011) Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors, Curr Biol. 21, 651-7. 120. Hassold, T., Hall, H. & Hunt, P. (2007) The origin of human aneuploidy: where we have been, where we are going, Hum Mol Genet. 16 Spec No. 2, R203-8. 121. Fragouli, E., Alfarawati, S., Goodall, N. N., Sanchez-Garcia, J. F., Colls, P. & Wells, D. (2011) The cytogenetics of polar bodies: insights into female meiosis and the diagnosis of aneuploidy, Mol Hum Reprod. 17, 286-95. 122. Kuliev, A., Zlatopolsky, Z., Kirillova, I., Spivakova, J. & Cieslak Janzen, J. (2011) Meiosis errors in over 20,000 oocytes studied in the practice of preimplantation aneuploidy testing, Reprod Biomed Online. 22, 2-8. 123. Hassold, T. & Hunt, P. (2001) To err (meiotically) is human: the genesis of human aneuploidy, Nat Rev Genet. 2, 280-91. 124. Jones, K. T. & Lane, S. I. (2012) Chromosomal, metabolic, environmental, and hormonal origins of aneuploidy in mammalian oocytes, Exp Cell Res. 318, 1394-9. 125. Nagaoka, S. I., Hassold, T. J. & Hunt, P. A. (2012) Human aneuploidy: mechanisms and new insights into an age-old problem, Nat Rev Genet. 13, 493-504. 126. Steuerwald, N., Cohen, J., Herrera, R. J., Sandalinas, M. & Brenner, C. A. (2001) Association between spindle assembly checkpoint expression and maternal age in human oocytes, Mol Hum Reprod. 7, 49-55. 127. Steuerwald, N. M., Bermudez, M. G., Wells, D., Munne, S. & Cohen, J. (2007) Maternal age-related differential global expression profiles observed in human oocytes, Reprod Biomed Online. 14, 700-8. 128. Pan, H., Ma, P., Zhu, W. & Schultz, R. M. (2008) Age-associated increase in aneuploidy and changes in gene expression in mouse eggs, Dev Biol. 316, 397-407. 129. Merriman, J. A., Jennings, P. C., McLaughlin, E. A. & Jones, K. T. (2012) Effect of aging on superovulation efficiency, aneuploidy rates, and sister chromatid cohesion in mice aged up to 15 months, Biol Reprod. 86, 49.

This article is protected by copyright. All rights reserved.

Accepted Article

130. Selesniemi, K., Lee, H. J., Muhlhauser, A. & Tilly, J. L. (2011) Prevention of maternal aging-associated oocyte aneuploidy and meiotic spindle defects in mice by dietary and genetic strategies, Proc Natl Acad Sci U S A. 108, 12319-24. 131. Baker, D. J., Jeganathan, K. B., Cameron, J. D., Thompson, M., Juneja, S., Kopecka, A., Kumar, R., Jenkins, R. B., de Groen, P. C., Roche, P. & van Deursen, J. M. (2004) BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice, Nat Genet. 36, 744-9. 132. Leland, S., Nagarajan, P., Polyzos, A., Thomas, S., Samaan, G., Donnell, R., Marchetti, F. & Venkatachalam, S. (2009) Heterozygosity for a Bub1 mutation causes female-specific germ cell aneuploidy in mice, Proc Natl Acad Sci U S A. 106, 12776-81. 133. Duncan, F. E., Chiang, T., Schultz, R. M. & Lampson, M. A. (2009) Evidence that a defective spindle assembly checkpoint is not the primary cause of maternal age-associated aneuploidy in mouse eggs, Biol Reprod. 81, 768-76. 134. Lister, L. M., Kouznetsova, A., Hyslop, L. A., Kalleas, D., Pace, S. L., Barel, J. C., Nathan, A., Floros, V., Adelfalk, C., Watanabe, Y., Jessberger, R., Kirkwood, T. B., Hoog, C. & Herbert, M. (2010) Age-related meiotic segregation errors in mammalian oocytes are preceded by depletion of cohesin and Sgo2, Curr Biol. 20, 1511-21. 135. Yun, Y., Holt, J. E., Lane, S. I., McLaughlin, E. A., Merriman, J. A. & Jones, K. T. (2014) Reduced ability to recover from spindle disruption and loss of kinetochore spindle assembly checkpoint proteins in oocytes from aged mice, Cell Cycle. 13, 1938-47. 136. Shomper, M., Lappa, C. & FitzHarris, G. (2014) Kinetochore microtubule establishment is defective in oocytes from aged mice, Cell Cycle. 13, 1171-9. 137. Hodges, C. A., Revenkova, E., Jessberger, R., Hassold, T. J. & Hunt, P. A. (2005) SMC1beta-deficient female mice provide evidence that cohesins are a missing link in agerelated nondisjunction, Nat Genet. 37, 1351-5. 138. Wolstenholme, J. & Angell, R. R. (2000) Maternal age and trisomy--a unifying mechanism of formation, Chromosoma. 109, 435-8. 139. Revenkova, E., Herrmann, K., Adelfalk, C. & Jessberger, R. (2010) Oocyte cohesin expression restricted to predictyate stages provides full fertility and prevents aneuploidy, Curr Biol. 20, 1529-33. 140. Tachibana-Konwalski, K., Godwin, J., van der Weyden, L., Champion, L., Kudo, N. R., Adams, D. J. & Nasmyth, K. (2010) Rec8-containing cohesin maintains bivalents without turnover during the growing phase of mouse oocytes, Genes Dev. 24, 2505-16. 141. Weng, K. A., Jeffreys, C. A. & Bickel, S. E. (2014) Rejuvenation of Meiotic Cohesion in Oocytes during Prophase I Is Required for Chiasma Maintenance and Accurate Chromosome Segregation, PLoS Genet. 10, e1004607. 142. Liu, L. & Keefe, D. L. (2008) Defective cohesin is associated with age-dependent misaligned chromosomes in oocytes, Reprod Biomed Online. 16, 103-12. 143. Chiang, T., Schultz, R. M. & Lampson, M. A. (2011) Age-dependent susceptibility of chromosome cohesion to premature separase activation in mouse oocytes, Biol Reprod. 85, 1279-83. 144. Yun, Y., Lane, S. I. & Jones, K. T. (2014) Premature dyad separation in meiosis II is the major segregation error with maternal age in mouse oocytes, Development. 141, 199-208. 145. Tsutsumi, M., Fujiwara, R., Nishizawa, H., Ito, M., Kogo, H., Inagaki, H., Ohye, T., Kato, T., Fujii, T. & Kurahashi, H. (2014) Age-related decrease of meiotic cohesins in human oocytes, PLoS One. 9, e96710. 146. Tachibana-Konwalski, K., Godwin, J., Borsos, M., Rattani, A., Adams, D. J. & Nasmyth, K. (2013) Spindle assembly checkpoint of oocytes depends on a kinetochore structure determined by cohesin in meiosis I, Curr Biol. 23, 2534-9. 147. Llano, E., Gomez, R., Gutierrez-Caballero, C., Herran, Y., Sanchez-Martin, M., VazquezQuinones, L., Hernandez, T., de Alava, E., Cuadrado, A., Barbero, J. L., Suja, J. A. & Pendas,

This article is protected by copyright. All rights reserved.

Accepted Article

A. M. (2008) Shugoshin-2 is essential for the completion of meiosis but not for mitotic cell division in mice, Genes Dev. 22, 2400-13. 148. Rattani, A., Wolna, M., Ploquin, M., Helmhart, W., Morrone, S., Mayer, B., Godwin, J., Xu, W., Stemmann, O., Pendas, A. & Nasmyth, K. (2013) Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes, Elife. 2, e01133.

Figure 1 The spindle checkpoint signal is catalyzed at the kinetochores of unaligned chromosomes and decays with time in the cytoplasm. Checkpoint generation may be regulated by both microtubule attachment and mechanical tension. In the first panel, depicting paired homologous chromosomes in MI, the signal from lower kinetochore on the unaligned chromosome is both unattached to spindle microtubules and lacks tension and thus generates a stronger signal than the upper kinetochore. The checkpoint signal diffuses into the cytoplasm where it undergoes a time-dependent decay. In the second panel all chromosomes have achieved bipolar attachment. The interval required for complete decay of the checkpoint signal allows sufficient time for the last attaching chromosome to align at the metaphase plate. In the third panel the checkpoint signal has fully decayed allowing anaphase onset.

This article is protected by copyright. All rights reserved.

Accepted Article

Figure 2 Mechanical tension overcomes the spindle checkpoint arrest in praying mantid spermatocytes. Praying mantid spermatocytes contain a sex trivalent consisting of a y chromosome and two x chromosomes. In approximately 10% of spermatocytes one of the x chromosomes fails to pair with the y. The univalent x generates a strong spindle checkpoint arrest. Using a glass microneedle to apply tension stabilizes the kinetochore attachment of the univalent x and extinguishes checkpoint signaling, allowing anaphase to occur. The microneedle does not penetrate the cell membrane but merely pushes it into the chromosome.

This article is protected by copyright. All rights reserved.

Accepted Article

Figure 3 The phosphoepitope recognized by the 3F3/2 monoclonal antibody shows stronger expression on a univalent X chromosome lacking tension in a praying mantid spermatocyte. (A) Normal metaphase I configuration showing bioriented sex trivalent (y and both x chromosomes labeled). Immunofluorescence labeling with the 3F3/2 antibody shows equal labeling of all kinetochores. (B) The X1 chromosome failed to pair with the Y and is univalent. It shows stronger labeling with the 3F3/2 antibody. An overlay of phase and fluorescence labeling is shown on the left and fluorescence alone on the right. The inset in B shows a side-by-side comparison of the indicated kinetochores. Figure reproduced from [24].

This article is protected by copyright. All rights reserved.

Accepted Article

Figure 4 The unattached kinetochore is a catalytic platform for assembly of the mitotic checkpoint complex (MCC), which inhibits the APC/C ubiquitin ligase. (A) There are likely more than 100 different proteins that contribute to the function of the kinetochore. Depicted are the major players for spindle checkpoint signaling at kinetochores. Through mechanisms that are only partially understood (see text) and through the functions of several mitotic kinases including Mps1, Bub1, Plk1 and Aurora B, the APC/C activator, Cdc20, is joined with Mad2 and BubR1/Bub3 to form the MCC, which inhibits APC/C activity. (B) Stable microtubule binding after bipolar attachment of chromosomes on the spindle silences the spindle checkpoint and inhibits MCC catalysis in several ways. One is the dynein-dependent stripping of checkpoint signaling components from the kinetochores along the microtubules. Another, less well understood, is the tension-dependent rearrangement of kinetochore components (depicted by the change in shape of the kinetochore protein complex). One aspect of this rearrangement may be to alter the access of kinases to their substrates. Another consequence of stable microtubule attachment is the increased recruitment of protein phosphatases that oppose checkpoint signaling kinases. Finally, the protein p31comet binds Mad2 to contribute to MCC disassembly.

This article is protected by copyright. All rights reserved.

Accepted Article

Figure 5 Spindle checkpoint effectiveness in mouse oocytes during MI depends on the number of kinetochores that are unattached or lack bipolar tension. (A) Bipolar attachment of bivalent chromosomes allows normal timing of oocyte meiosis. (B) A high concentration of the microtubule drug, nocodazole, disrupts the meiotic spindle producing strong checkpoint signaling and meiotic arrest. (C) Lower concentrations of nocodazole allow spindle assembly but partially disrupts normal microtubule kinetochore interactions producing a weaker checkpoint signal that partially delays anaphase onset. (D) A single X chromosome univalent in XO oocytes does not significantly delay anaphase onset. (E) In mlh1-/- mutant oocytes, few crossovers form and most chromosomes are univalent. Many but not all achieve bipolar attachment of sister kinetochores. It requires about 4 univalent chromosomes that lack bipolar attachment of sister kinetochores to delay anaphase.

This article is protected by copyright. All rights reserved.