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A Conserved Checkpoint Monitors Meiotic Chromosome Synapsis in Caenorhabditis elegans

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Science  09 Dec 2005:
Vol. 310, Issue 5754, pp. 1683-1686
DOI: 10.1126/science.1117468

Abstract

We report the discovery of a checkpoint that monitors synapsis between homologous chromosomes to ensure accurate meiotic segregation. Oocytes containing unsynapsed chromosomes selectively undergo apoptosis even if a germline DNA damage checkpoint is inactivated. This culling mechanism is specifically activated by unsynapsed pairing centers, cis-acting chromosome sites that are also required to promote synapsis in Caenorhabditis elegans. Apoptosis due to synaptic failure also requires the C. elegans homolog of PCH2, a budding yeast pachytene checkpoint gene, which suggests that this surveillance mechanism is widely conserved.

Meiosis requires two successive cell divisions: one in which homologous chromosomes separate and a second that partitions sister chromatids. Accurate segregation depends on the establishment of physical linkages (chiasmata) between homologous chromosomes during meiotic prophase. Chromosome pairing, the polymerization of the synaptonemal complex between paired homologs (synapsis), and crossover recombination are all required to generate chiasmata, which enable proper chromosome alignment on the meiotic spindle.

Defects in these early meiotic events can lead to cell cycle arrest or apoptosis, indicating that the events are monitored by checkpoints. In budding yeast, a “pachytene checkpoint” responds to defects in homolog synapsis and/or recombination [reviewed in (1)]. Mammalian meiosis may have two distinct checkpoints, one that responds to synaptic failure and one that responds to DNA damage (24). Because synapsis and recombination are obligately coupled in both Saccharomyces cerevisiae (5) and mice (3, 6), it has been ambiguous whether these checkpoints are triggered by recombination defects or asynapsis. Here, we have exploited the knowledge that synapsis can be completely uncoupled from meiotic recombination in C. elegans (7) to test whether defects in synapsis can directly trigger a meiotic checkpoint.

Each chromosome in C. elegans has a unique region called a pairing center (PC) that stabilizes meiotic pairing and promotes synapsis between homologs (8). The deficiency meDf2 removes the PC from the X chromosome. meDf2 homozygotes display X chromosome asynapsis in 90% of meiocytes (8) (Fig. 1A) and high rates of meiotic missegregation, as revealed by the frequency of male (XO) progeny (9). In meDf2/+ hermaphrodites, 60% of meiotic nuclei exhibit unsynapsed X chromosomes (8), but only 5 to 7% of their self-progeny are males (9). This discrepancy suggested that nuclei with unsynapsed X chromosomes might be culled selectively during meiosis.

Fig. 1.

Apoptosis preferentially eliminates meiocytes with unsynapsed chromosomes. (A) Unsynapsed X chromosomes in meDf2 pachytene nuclei are visualized as segments of HTP-3 (red) staining devoid of SYP-1 (green). mnDp66 is a duplication required for the viability of meDf2 homo- and hemizygotes (30). Scale bars, 2 μm. (B) X chromosome nondisjunction in meDf2/+ hermaphrodites is elevated by mutations in core apoptotic components but not a DNA damage checkpoint gene. Differences between meDf2/+ and ced-3;meDf2/+ or ced-4;meDf2/+ are significant (P < 0.0001). (C) Achiasmate chromosomes in mature oocytes. Six DAPI (4′,6′-diamidino-2-phenylindole)–stained bodies in the oocyte in the top image represent the six recombinant bivalents, whereas the lower image shows five autosomal bivalents and two achiasmate X chromosomes (arrows). (D) Culling of nuclei with unsynapsed chromosomes precedes oocyte maturation. Mutation of ced-3 or ced-4 in meDf2/+ hermaphrodites results in a higher fraction of mature oocytes with achiasmate X chromosomes. Differences between meDf2/+ and ced-3;meDf2/+ ced-4;meDf2/+ are significant (P < + or 0.0001).

About half of the oocytes in wild-type gonads undergo apoptosis (10). To explore the possibility that apoptosis may cull defective nuclei, we introduced mutations in the proapoptotic genes ced-3 (11) or ced-4 (12) into meDf2/+ hermaphrodites. ced-3;meDf2/+ animals produced twice as many male self-progeny as meDf2/+ hermaphrodites (Fig. 1B), indicating that apoptosis can enrich for normal gametes. We also quantified the fraction of oocytes that contained univalent X chromosomes during late meiotic prophase (Fig. 1C). ced-3;meDf2/+ hermaphrodites exhibit four times as many oocytes with achiasmate X chromosomes (Fig. 1D) as do meDf2/+ animals, providing additional evidence that apoptosis “filters” meiocytes to enrich for recombinant X chromosomes. When a ced-4 mutation was introduced into meDf2/+ hermaphrodites, the frequencies of male self-progeny (21%) (Fig. 1B) and achiasmate X chromosomes (43%) (Fig. 1D) were even more dramatically enhanced, consistent with evidence that some apoptosis is independent of ced-3 (13). Thus, meiotic nuclei with unsynapsed X chromosomes are preferentially eliminated prior to oocyte maturation.

In C. elegans, defects in synapsis can activate a germline DNA damage/meiotic recombination checkpoint (14, 15). To determine whether this checkpoint is responsible for the death of asynaptic nuclei in meDf2/+ hermaphrodites, we introduced a hus-1 (16) mutation into meDf2/+ animals (hus-1;meDf2/+). In contrast to ced-3 and ced-4, mutation of hus-1 did not enhance the incidence of male self-progeny (Fig. 1B) or achiasmate X chromosomes (Fig. 1D) in meDf2/+ hermaphrodites.

We tested whether apoptosis in meDf2/+ animals might result from hus-1–independent activation of the DNA damage/recombination checkpoint by eliminating spo-11, which is required for double-strand breaks (DSBs) (7). spo-11 mutants produce very few viable progeny (7), precluding their analysis by the assays described above. Therefore, we directly quantified apoptosis in germ lines of age-matched adult hermaphrodites using a ced-1::gfp transgene (Fig. 2A) (17). Wild-type animals exhibit an average of seven apoptotic nuclei per gonad arm (Fig. 2B) as a result of physiological apoptosis that occurs during unperturbed meiosis (10). Mutation of spo-11 or hus-1 slightly reduced the number of apoptotic nuclei. Compared with wild-type, meDf2/+ hermaphrodites exhibited an elevated number of apoptotic nuclei (13 per gonad arm), consistent with activation of apoptosis by unsynapsed chromosomes. Moreover, this high level of apoptosis requires neither DSBs (spo-11;meDf 2/+) nor an essential DNA damage checkpoint component (hus-1; meDf2/+). Elevated apoptosis in meDf2/+ hermaphrodites was also independent of the worm p53 homolog cep-1 (18, 19). These results reveal the activity of a previously undescribed meiotic checkpoint that activates apoptosis in response to defects in synapsis (Fig. 2C).

Fig. 2.

Activation of apoptosis in meDf2/+ hermaphrodites is independent of DSBs and the DNA damage check-point. (A) CED-1-GFP identifies germline apoptotic nuclei. Differential interference contrast (DIC) and fluorescent images of apoptotic nuclei in the germline of wild-type animals expressing a CED-1-GFP fusion protein. (B) Mutation of spo-11, hus-1, or cep-1 does not affect the number of apoptotic nuclei in meDf2/+ hermaphrodites. Error bars, ±2 × SEM. (C) Asynapsis of a single pair of chromosomes causes apoptosis. Meiotic nuclei in a meDf2/+ hermaphrodite stained for SYP-1 (green), HTP-3 (red), and GFP (white). The nucleus encircled by CED-1-GFP contains a pair of unsynapsed chromosomes. Surrounding nuclei show complete synapsis and lack CED-1-GFP.

Because meDf2 homozygotes show even higher levels of X chromosome asynapsis than meDf2/+ hermaphrodites, we were not surprised to detect elevated apoptosis in these animals (Fig. 3A). However, when mutations in spo-11 or hus-1 were introduced into meDf2 homozygotes, germline apoptosis was restored to wild-type levels (Fig. 3A). Thus, in meDf2 homozygotes, elevated apoptosis requires the DNA damage checkpoint, which indicates that the synapsis checkpoint is not activated. This suggested that the checkpoint might be specifically triggered by unsynapsed PCs, because half of the unsynapsed chromosomes in meDf2/+ hermaphrodites carry functional PCs, whereas meDf2 homozygotes lack unsynapsed PCs. We therefore investigated the effects of him-8 mutations. HIM-8 localizes to the X chromosome PC and is required for its roles in homolog pairing and synapsis (20). Like meDf2 homozygotes, him-8 mutants show high levels of apoptosis that strictly depend on the DNA damage check-point (Fig. 3A). Furthermore, in him-8;meDf2/+ hermaphrodites, elevated apoptosis requires hus-1 (Fig. 3A). HIM-8 is therefore required for unsynapsed X chromosomes to activate the DNA-damage–independent synapsis check-point, supporting the hypothesis that the PC plays an essential role in this mechanism. The complementary functions of these sites in both promoting synapsis and detecting defects in this process are evocative of the involvement of centromeres in both microtubule attachment and the spindle assembly checkpoint.

Fig. 3.

The synapsis checkpoint monitors the synapsis of all chromosomes through their PCs. (A) An unsynapsed PC is required to activate the synapsis checkpoint. (B) The synapsis checkpoint monitors synapsis of autosomes as well as the X chromosome.

The X chromosome in C. elegans has many distinct features (2124), including its lack of a meiotic partner in males. We therefore tested whether unsynapsed autosomes can also trigger the synapsis checkpoint. Mutation of the synaptonemal complex genes syp-1 and syp-2 results in asynapsis of all chromosomes and very high levels of apoptosis (14, 15); CED-1-GFP revealed an average of 23 corpses per gonad arm in syp-1 mutants (Fig. 3B). When DSBs are eliminated (i.e., spo-11;syp-1), apoptosis is reduced, but not to wild-type levels (14) (Fig. 3B). We detected elevated levels of apoptosis in spo-11;syp-1;meDf2 triple-mutant hermaphrodites (Fig. 3B), indicating that the synapsis checkpoint is activated in the absence of DSBs and the X chromosome PC. Thus, the synapsis checkpoint monitors the synapsis of all chromosomes, likely through their PCs.

These experiments also illustrate that the synapsis checkpoint is fully activated whether one pair (meDf2/+) or all homolog pairs (spo-11;syp-1) are unsynapsed. A similar phenomenon has been observed in mice (4). Our experiments also suggest that this check-point is saturable, unlike the DNA damage/recombination checkpoint, which exhibits clear dose-dependence (25). The checkpoint responds similarly whether all the nuclei in the germline exhibit homolog asynapsis or only 60% have unsynapsed chromosomes.

PCH2 encodes an AAA–adenosine triphosphatase (AAA-ATPase) essential for the pachytene checkpoint in budding yeast (26). The predicted gene F10B5.5 encodes a candidate worm ortholog, based on its sequence similarity and elevated germline expression (27). We analyzed a deletion allele, pch-2(tm1458), to evaluate the role of this gene during meiosis. In pch-2;meDf2/+ hermaphrodites, we observed wild-type levels of apoptotic nuclei per gonad arm (Fig. 4A), consistent with an essential role for this gene in the synapsis checkpoint. In contrast, we found that pch-2 does not reduce the number of apoptotic nuclei in either him-8 or meDf2 homozygous animals (Fig. 4A), indicating that it is dispensable for the germline DNA damage/recombination checkpoint. We also combined the pch-2(tm1458) allele with a syp-1 mutation. Like spo-11;syp-1 double mutants, pch-2;syp-1 double mutants exhibit intermediate levels of apoptosis (Fig. 4B), suggesting a defect in one of the two meiotic checkpoints but not both. In pch-2;spo-11;syp-1 triple mutants, apoptosis falls to wild-type levels (Fig. 4B), reinforcing the conclusion that pch-2 is specifically required for spo-11–independent checkpoint activation. These results support a role for the pch-2 gene in the synapsis checkpoint, but not the recombination checkpoint, in C. elegans (fig. S3).

Fig. 4.

The C. elegans gene pch-2 is required for the synapsis checkpoint but not the DNA damage/recombination checkpoint. (A) Mutation of pch-2 reduces apoptosis in meDf2/+ hermaphrodites but not in meDf2 or him-8 homozygotes. (B) Elimination of both pch-2 and spo-11 function restores apoptosis to wild-type levels in syp-1 mutants.

It is perplexing that meDf2/+ hermaphrodites exhibit persistent recombination intermediates on their unsynapsed chromosomes (8), yet they fail to activate the DNA damage/recombination checkpoint (Fig. 1, B and D, and Fig. 2B), even in the absence of pch-2 (Fig. 4 and fig. S1). We propose that an unsynapsed PC can inhibit activation of the DNA damage/recombination checkpoint but that this can be overcome by sufficient levels of unprocessed DSBs, e.g., in syp-1 mutants with fully unsynapsed chromosomes.

SPO11 function is necessary to activate the pachytene checkpoint in budding yeast, which has led to the idea that the checkpoint may monitor persistent recombination intermediates (1). However, Spo11p plays additional, nonenzymatic roles during meiosis (28), raising the possibility that SPO11-dependent chromosomal features, rather than DSBs per se, may be required for checkpoint activation. Moreover, mutation of PCH2 alleviates the meiotic arrest of some mutants (zip1 and dmc1) but not others (hop2)(1), suggesting that the yeast “pachytene checkpoint” may actually be an amalgamation of checkpoints. In light of our evidence for conservation of pch-2 function from yeast to worms, defects in synapsis may also directly trigger meiotic arrest in budding yeast.

Unsynapsed sex chromosomes can activate a p53-independent meiotic checkpoint in mammals (2). Moreover, Spo11–/– mutant mice still exhibit spermatocyte death (3) and oocyte loss that is distinguishable from the apoptosis induced by mutations of DSB processing enzymes (4). Although direct experimental evidence is still lacking, it is likely that some loss of gametes in Spo11–/– mutant mice may result from their synaptic failures.

Our results demonstrate that synapsis can be monitored independently of recombination defects to ensure the accuracy of the meiotic divisions and prevent the production of aneuploid gametes. Further elucidation of this mechanism in C. elegans will likely shed light on the basis of human infertility, particularly in males, which has been linked to synaptic defects during meiotic prophase (29).

Supporting Online Material

www.sciencemag.org/cgi/content/full/310/5754/1683/DC1

Materials and Methods

Figs. S1 to S3

References

References and Notes

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