Cdc20 (cell division cycle 20) and Cdh1 are the activating subunits of APC (anaphase-promoting complex), an E3-ubiquitin ligase that drives cells into anaphase by inducing degradation of cyclin B and the anaphase inhibitor securin. To prevent chromosome missegregation due to early degradation of cyclin B and securin, mitotic checkpoint protein complexes consisting of BubR1, Bub3 and Mad2 bind to and inhibit APCCdc20 until all chromosomes are properly attached to the mitotic spindle and aligned in the metaphase plate. The nuclear transport factors Rae1 and Nup98, which convert into mitotic checkpoint proteins in M-phase, further prevent chromosome missegregation by assembling into a complex with APCCdh1 and delaying APCCdh1-mediated ubiquitination of securin. Disruption of Mad2, BubR1, Bub3 or Rae1 in mice results in substantial aneuploidy in somatic tissues, but whether these genes are equally important for accurate chromosome segregation during meiosis has not yet been established. To address this issue, we generated cohorts of male mice in which Mad2, BubR1, Bub3, Rae1 and Nup98 were disrupted either individually or in combination. We tested the fertility of these mice and performed chromosome counts on secondary spermatocytes. We found that male fertility and accurate chromosome segregation during spermatogenesis are highly dependent on BubR1, but not Mad2, Bub3, Rae1 and Nup98. Our results suggest that the mechanisms ensuring accurate chromosome segregation differ between mitotic and meiotic cells.

Introduction

Newborns have several million oocytes that remain arrested in the first meiotic division until maturation [1,2]. Oocyte numbers gradually decline from birth to the fifth decade of life, when complete oocyte depletion triggers menopause. Both oocyte maturation and apoptosis contribute to this progressive decline. Recent studies in mice provide strong evidence for the existence of proliferative germ cells that sustain oocyte and follicle production during postnatal life [35]. Although one would expect a postnatal presence of stem cells in human ovaries, their existence has not yet been confirmed. Not only does the amount of oocytes decline over time but also the quality of oocytes produced, primarily because the fidelity of chromosome segregation declines markedly with age [1]. Increasing rates of chromosome missegregation lead to increasing numbers of aneuploid embryos and a higher risk of miscarriage or the birth of a child with a chromosomal abnormality (e.g. Down's syndrome). Although it has been well recognized that aneuploidy is a major reason for the decrease in fertility with age, the molecular basis underlying the increase in chromosome missegregation over time remains poorly defined. During mitotic divisions, the so-called spindle assembly checkpoint acts to ensure accurate chromosome segregation by delaying metaphase–anaphase transition until all chromosomes are properly attached to the mitotic spindle and aligned in the metaphase plate [6,7]. In budding yeast, loss of spindle checkpoint activity has been shown to result in increased chromosome missegregation during meiotic cell divisions [8]. However, whether and how the spindle assembly checkpoint is required for accurate meiotic chromosome segregation in mammals has not yet been established. We have generated a series of checkpoint-defective mice to test the requirement of individual mitotic checkpoint proteins for fertility and proper chromosome segregation in meiosis.

The spindle assembly checkpoint

The molecular networks that regulate orderly progression through mitosis and meiosis are very complex and only partly understood. Several key events in mitosis are regulated through the actions of APC (anaphase-promoting complex), a large multiprotein E3-ubiquitin ligase that targets key mitotic regulators for destruction by the 26 S proteasome [911]. These mitotic regulators include cyclin A and cyclin B, Nek2, securin, Cdc20 (cell division cycle 20), aurora A and aurora B and Plk1. Active APC is present from prophase to late G1 phase but its enzymatic activity is tightly regulated by various mechanisms to ensure correct timing of degradation of each of its substrates [12]. At the onset of mitosis, APC activation is accomplished through phosphorylation of various APC subunits by the mitotic kinases Cdk1 (cyclin-dependent kinase 1)/cyclin B and Plk1 [1315] and recruitment of the co-activator Cdc20 [1618]. Cdc20-activated APC targets cyclin A for degradation by the 26 S proteasome during prophase [19,20], whereas it targets cyclin B and securin for destruction at the transition from metaphase to anaphase (Figure 1). In turn, cyclin B and securin destruction triggers separase activation, sister chromatid separation and anaphase onset [6,10,21]. When cyclin B is destroyed, inhibitory phosphates are removed from the Cdc20-related activator Cdh1, which allows for the binding of Cdh1 to APC. The resulting Cdh1-activated APC complexes then target Cdc20 for destruction by the proteasome [18,22]. Several inhibitors of APC-mediated degradation have been identified. One such inhibitor is Emi1, which binds to Cdc20 at the onset of mitosis to avoid premature activation of the APC [23]. Another key inhibitor of APC activity is the spindle assembly checkpoint (Figure 1). This checkpoint is activated by kinetochores that are not yet attached to microtubules and chromosome pairs that lack tension across sister chromatids [24,25]. A variety of mitotic checkpoint proteins, including Bub3, Bub1, BubR1, Mad1 and Mad2, bind to kinetochores that lack attachment or tension. There, they generate a so-called ‘wait anaphase’ signal that diffuses into the mitotic cytosol [26]. The precise composition of ‘wait anaphase’ signal remains unclear, but complexes consisting of Bub3, BubR1 and Mad2 seem to play a central role in establishing it [2729]. It has been well documented that these complexes bind to Cdc20 to delay ubiquitination of cyclin B and securin [10,21]. Inactivation of the spindle assembly checkpoint occurs after proper alignment of mitotic chromosomes at the metaphase plate. This inactivation involves the release of Bub3–BubR1–Mad2 protein complexes from Cdc20, thereby allowing formation of Cdc20-activated APC and subsequent destruction of cyclin B and securin. Separase, which is inhibited through association with securin and also by cyclin B/Cdk1-mediated phosphorylation, subsequently triggers sister chromatid separation by cleavage of the cohesin subunit Scc1 [21].

Timing of APC-mediated degradation of securin and cyclin B at the metaphase–anaphase transition

Figure 1
Timing of APC-mediated degradation of securin and cyclin B at the metaphase–anaphase transition

In prometaphase, Cdc20-activated APC is inhibited by Mad2–Bub3–BubR1 complexes. Low levels of BubR1 result in premature degradation of cyclin B, suggesting that Cdc20-activated APC primarily ubiquitinates this substrate at the metaphase–anaphase transition after release of Mad2, Bub3 and BubR1. Cdh1-activated APC is also present in prometaphase but is inhibited by Rae1–Nup98 complexes. Low levels of these inhibitors result in premature degradation of securin, suggesting that Cdh1-activated APC primarily ubiquitinates this substrate at the metaphase–anaphase transition after release of Nup98–Rae1 complexes from Cdh1-activated APC.

Figure 1
Timing of APC-mediated degradation of securin and cyclin B at the metaphase–anaphase transition

In prometaphase, Cdc20-activated APC is inhibited by Mad2–Bub3–BubR1 complexes. Low levels of BubR1 result in premature degradation of cyclin B, suggesting that Cdc20-activated APC primarily ubiquitinates this substrate at the metaphase–anaphase transition after release of Mad2, Bub3 and BubR1. Cdh1-activated APC is also present in prometaphase but is inhibited by Rae1–Nup98 complexes. Low levels of these inhibitors result in premature degradation of securin, suggesting that Cdh1-activated APC primarily ubiquitinates this substrate at the metaphase–anaphase transition after release of Nup98–Rae1 complexes from Cdh1-activated APC.

Recently, we have reported that mutant mice with low amounts of Nup98 and Rae1 separate their sister chromatids prematurely and develop massive aneuploidy [30]. Cells with small amounts of Rae1 and Nup98 destroy securin prematurely in prometaphase instead of at the metaphase–anaphase transition. Conversely, the timing of cyclin B destruction is normal in these cells. We found that in prometaphase, Rae1 and Nup98 associate with the APC to prevent degradation of securin. Rae1 and Nup98 specifically bind to Cdh1-activated APC and have no affinity for Cdc20-activated APC (Figure 1). This was not anticipated because earlier work had indicated that phosphorylation of Cdh1 in early mitosis prevents the formation of Cdh1-activated APC [18,22]. This mechanism, however, does not fully prevent formation of APCCdh1 in early mitosis because Cdh1 co-immunoprecipitates with the APC subunit Cdc27 from prometaphase cell extracts [30]. In fact, comparative co-immunoprecipitation experiments suggest that prometaphase cell extracts contain similar amounts of APCCdc20 and APCCdh1 [31]. Dissociation of Rae1 and Nup98 from APCCdh1 coincides with the release of BubR1 from APCCdc20 [30]. Because the release of BubR1, and its co-inhibitors Bub3 and Mad2, is known to occur at the metaphase–anaphase transition to activate APCCdc20 and drive cells into anaphase [10,32], it seems that dissociation of Rae1 and Nup98 from APCCdh1 also occurs at this mitotic stage and for the same purpose. If APCCdc20 promotes anaphase by triggering destruction of both cyclin B and securin, one would expect to see premature degradation of both of these proteins in cells with low levels of BubR1. We critically tested this by using BubR1 hypomorphic cells that express only one-tenth of normal BubR1 levels [33]. Surprisingly, only cyclin B was prematurely degraded, suggesting that Cdc20 is the primary APC activator of cyclin B destruction, but not of securin destruction (Figure 1). On the other hand, Cdh1-activated APC might have a more important role in the destruction of securin.

Somatic cells with small amounts of mitotic checkpoint proteins develop severe aneuploidy

Gene knockout studies for Mad2, Bub3, BubR1, Rae1 and Nup98 indicate that most of the principal components of the mitotic checkpoint machinery are essential for early embryogenesis and cell viability themselves [30,3338]. On the other hand, heterozygous loss of one or more mitotic checkpoint genes has no detectable impact on embryogenesis and postnatal development. We have measured the degree of aneuploidy in a variety of mitotic checkpoint-defective mice by performing chromosome counts on splenocytes [30,31,33,36,39]. Typically, we collected the spleens from 5-month-old animals (or older) and prepared splenocyte suspensions. To enrich for mitotic cells, splenocytes were cultured for 4–5 h in the presence of colcemid before preparing metaphase spreads. Aneuploidy can also be measured in thymocytes of relatively young animals. We find that the percentages of aneuploid cells in spleen and thymus are generally highly comparable (results not shown). Other somatic tissues and cell types from checkpoint-defective mice have been more difficult to analyse for aneuploidy because procedures to prepare metaphase spreads require collagenase treatment and extensive culture periods during which chromosome missegregation can occur. We found that mutant mice that express approx. 10% of normal BubR1 protein levels (designated BubR1H/H mice) have no detectable aneuploidy at birth, but have accumulated 15% aneuploid splenocytes at 5 months of age (Table 1) [33]. At 5 months, mice that are haploinsufficient for Mad2 contain on average approx. 18% aneuploid splenocytes [39], whereas Rae1 and Bub3 haploinsufficient mice each have 9% aneuploid splenocytes at that age [36]. Extremely high levels of aneuploid splenocytes are present in 5-month-old Bub3/Rae1 and Nup98/Rae1 double haploinsufficient mice (aneuploidy was 36 and 32% respectively) [30,36]. Chromosome counts on passage 5 mouse embryonic fibroblasts yielded percentages of aneuploidy that are largely consistent with those found in splenocytes from 5-month-old animals [30,33,36]. This suggests that the degree of aneuploidy in splenocytes is representative for other tissues and cell types with similar mitotic indexes; however, this needs further verification.

Table 1
Differential mitotic checkpoint protein requirements in secondary spermatocytes and splenocytes
    Secondary spermatocytes with indicated number of duplicated chromosomes   
Mouse genotype Age (nMeiotic figures inspected Percentage aneuploid figures (S.D.) 18 19 20 21 Percentage meiotic figures with PMSCS (S.D.) Percentage aneuploid figures in splenocytes 
Wild-type 5 months (3) 150 0 (0)  150  0 (0) 
 27 months (3) 150 3 (1)  145 0 (0) 
 35 months (3) 150 7 (1) 139 0 (0) 
Bub3+/− 5 months (2) 100 0 (0)   50  0 (0) 
 27 months (1) 50  49  29 
Rae1+/− 5 months (2) 100 0 (0)   50  0 (0) 
 27 months (1) 50  49  30 
Mad2+/− 5 months (1) 50   50  18 
Rae1+/−/Nup98+/− 5 months (1) 50   50  32 
 16 months (1) 50   50  35 
Bub3+/−/Rae1+/− 5 months (3) 150 0 (0)   150  0 (0) 36 
 27 months (3) 150 7 (2) 139 1 (2) 47 
BubR1H/H 5 months (3) 150 5 (1) 143 15 (2) 15 
    Secondary spermatocytes with indicated number of duplicated chromosomes   
Mouse genotype Age (nMeiotic figures inspected Percentage aneuploid figures (S.D.) 18 19 20 21 Percentage meiotic figures with PMSCS (S.D.) Percentage aneuploid figures in splenocytes 
Wild-type 5 months (3) 150 0 (0)  150  0 (0) 
 27 months (3) 150 3 (1)  145 0 (0) 
 35 months (3) 150 7 (1) 139 0 (0) 
Bub3+/− 5 months (2) 100 0 (0)   50  0 (0) 
 27 months (1) 50  49  29 
Rae1+/− 5 months (2) 100 0 (0)   50  0 (0) 
 27 months (1) 50  49  30 
Mad2+/− 5 months (1) 50   50  18 
Rae1+/−/Nup98+/− 5 months (1) 50   50  32 
 16 months (1) 50   50  35 
Bub3+/−/Rae1+/− 5 months (3) 150 0 (0)   150  0 (0) 36 
 27 months (3) 150 7 (2) 139 1 (2) 47 
BubR1H/H 5 months (3) 150 5 (1) 143 15 (2) 15 

Meiosis and fertility are unaffected in most mitotic checkpoint-deficient mouse models

Both male and female BubR1H/H mice are unable to establish any pregnancies [33]. Testes of BubR1H/H males appear histologically normal and their average weight is only approx. 15% below that of wild-type testes. However, spermatozoa counts of BubR1H/H mice are approx. 4 times lower than those of control mice. Spermatozoa from BubR1H/H mice have normal motility and morphology and attach to wild-type eggs in vitro. However, they produce two-cell-stage embryos 13 times less frequently than wild-type spermatozoa. To investigate the chromosomal status of BubR1H/H germ cells, we collected testes of 5-month-old mice, prepared metaphase spreads from single cell suspensions and performed chromosome counts of secondary spermatocytes [33]. Abnormal chromosome numbers were present in approx. 5% of BubR1H/H spermatocytes compared with 0% of wild-type spermatocytes. In addition, 15% of BubR1H/H spermatocytes exhibited PMSCS (premature sister chromatid segregation). Like testes, ovaries from young BubR1H/H females appeared histologically normal. They yielded mature eggs that were arrested at metaphase of meiosis II. However, in contrast with control eggs, most of these eggs showed severe chromosome congression defects, indicating that infertility in female BubR1H/H mice can be attributed to meiotic chromosome segregation defects.

Besides BubR1 hypomorphic mice [38], none of the other mitotic checkpoint-deficient mouse models exhibits male or female sterility [3436,40]. This is surprising because several of these mouse mutants, including Bub3/Rae1 and Rae1/Nup98 double haploinsufficient mice, have >2-fold more aneuploid splenocytes than age-matched BubR1 hypomorphic mice [30,36]. One possible explanation for this discrepancy could be that aneuploidy in the germline is not a key determinant of fertility. Alternatively, the degree of aneuploidy in spleen or other somatic tissues might not correlate well with the degree of aneuploidy in the germline. To distinguish between these possibilities, we performed chromosome counts on metaphase spreads from secondary spermatocytes of 5-month-old males with various mitotic checkpoint gene defects. At 5 months, Mad2, Bub3 and Rae1 single haploinsufficient mice as well as Bub3/Rae1 and Rae1/Nup98 double haploinsufficient mice had no detectable aneuploidy in secondary spermatocytes despite significant aneuploidy in splenocytes (Table 1). In addition, although Bub3/Rae1 and Rae1/Nup98 double haploinsufficient splenocytes exhibit substantial PMSCS, spermatocytes from these mice displayed no PMSCS (Table 1). Chromosome counts on spermatocytes of 27-month-old single haploinsufficient Bub3 and Rae1 mice yielded 2% aneuploid figures, but this percentage was similar to that of 27-month-old wild-type males. However, there was a slight but significant increase in aneuploid spermatocytes in 27-month-old Bub3/Rae1 double haploinsufficient mice in comparison with age-matched wild-type mice. Taken together, these results establish that there are differential mitotic checkpoint protein requirements in somatic and germ cells and suggest that only BubR1 is a key determinant of accurate chromosome segregation in the germline. The function of Mad2, Bub3, Rae1 and Nup98 in the female germline remains to be determined. However, given that the fecundity and reproductive lifespan of Mad2+/−, Bub3+/−, Rae1+/−, Nup98+/−, Bub3+/−/Rae1+/− and Rae1+/−/Nup98+/− females appear normal, there may not be a prominent role for these mitotic checkpoint proteins in chromosome segregation during oogenesis.

Conclusion

BubR1 expression is extremely high in testes and ovaries of young animals but, as mice age, BubR1 levels progressively decline in these organs [33]. Bub3 and Rae1 levels are also high in testis and ovary, but in contrast with BubR1, these checkpoint proteins remain highly expressed until extreme old age. The kinds of chromosome segregation defects seen in oocytes of mutant mice expressing low levels of BubR1 are reminiscent of those seen in oocytes of women approaching the end of their reproductive lifespan [1,41]. This, combined with the age-dependent decline in ovarian BubR1, suggests that progressive loss of BubR1 activity might play a causal role in age-related female infertility and certain birth defects. Paternal fertility also declines with advancing age [42], and a regulatory role for BubR1 is certainly conceivable, given the decline in BubR1 levels in testes of aging normal mice and the negative impact of BubR1 deficiency on fertility of male mutant mice. It will be interesting to examine whether mice carrying a transgene that maintains high levels of BubR1 in the germline have an extended reproductive lifespan.

Meiosis and the Causes and Consequences of Recombination: Biochemical Society Focused Meeting held at University of Warwick, U.K., 29–31 March 2006. Organized by R. Borts (Leicester, U.K.), D. Charlesworth (Edinburgh, U.K.), A. Eyre-Walker (Sussex, U.K.), A. Goldman (Sheffield, U.K.), G. McVean (Oxford, U.K.), D. Monckton (Glasgow, U.K.), G. Moore (John Innes Centre, U.K.), J. Richards (Roslin Biocentre, U.K.) and M. Stark (Glasgow, U.K.). Edited by D. Monckton.

Abbreviations

     
  • APC

    anaphase-promoting complex

  •  
  • Cdc20

    cell division cycle 20

  •  
  • Cdk1

    cyclin-dependent kinase 1

  •  
  • PMSCS

    premature sister chromatid segregation

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