Asymmetric SC disassembly is coupled to meiotic recombination

As the aforementioned events suggested a coupling between chiasma formation and asymmetric SC disassembly, we investigated SC disassembly in the absence of crossing over. We examined the spo-11(ok79) mutant (Dernburg et al., 1998), which assembles SC between fully aligned homologues but lacks the double-strand break (DSB)–forming enzyme that initiates recombination.

The spo-11 mutant is severely impaired in achieving orderly asymmetric departure of SYP-1,2 from meiotic prophase chromosomes (Figs. 3 and ​and44 A). This is particularly evident at diplonema, when SYP-1,2 fail to become concentrated to a single short stretch per homologue pair. Instead, spo-11 diplotene nuclei exhibit several types of anomalies. In some nuclei, SYP-1 is retained along the full lengths of coiled axes, and DAPI-staining shows that the homologues are still largely synapsed; this differs from wild-type meiosis, where axis coiling is usually seen only in nuclei exhibiting extensive desynapsis. In other nuclei, SYP-1,2 have been lost from the entire lengths of most of the chromosomes within the nucleus, but a few bright chromosome-associated SYP-1,2 signals are present; the appearance of the HIM-3 signals and DAPI-stained chromatin in such nuclei indicates that homologues are largely desynapsed. Finally, some nuclei exhibit a mixture of these two phenotypes. In summary, meiotic chromosomes in the spo-11 mutant have not lost either the ability to retain or to eject SYP-1,2; rather, they have lost the capacity to coordinate retention and loss in a manner that results in reliable asymmetric differentiation of chromosomal subdomains.

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Figure 3.

Immunolocalization of SYP-2 after pachytene exit. α-SYP-2 staining and DAPI-stained chromatin in wild-type, spo-11, and msh-5 germ lines; portions shown extend from the region of pachytene exit (left) through late diplonema/early diakinesis (right). *, somatic nucleus. Bar, 10 μm.

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Figure 4.

Localization of SYP-1 and HIM-3 after pachytene exit in untreated and γ-irradiated spo-11 mutants. (A) Portions of germ lines extend from late pachynema (left) through mid/late diplonema (right). Closed arrowheads indicate diplotene nuclei in which SYP-1 is retained along the lengths of coiled axes; open arrowheads indicate nuclei in which SYP-1 has been lost from the majority of the chromosomes but is retained on some chromosomes; arrows indicate nuclei in which each desynapsing bivalent retains a single robust stretch of SYP-1, reflecting restoration of orderly asymmetric departure of SYP-1 by γ-irradiation. (B, top) Mid-diakinesis oocyte from an untreated spo-11 worm showing lack of chiasmata and SYP-1 distributed along the lengths of the axes. (bottom) Late diplotene oocyte from a γ-irradiated spo-11 worm showing SYP-1 concentrated on a region of each bivalent in the vicinity of and/or distal to the emerging chiasma. (C) Late diakinesis oocyte from a γ-irradiated spo-11 worm showing restoration of chiasmata. Bars, 5 μm.

Failure to differentiate distinct subdomains is also evident at early/mid diakinesis in the spo-11 mutant (Figs. 4 B and 5 A). Chromosomes are fully resolved into 12 univalents, indicating eventual desynapsis, and SYP-1 signals are found distributed along the entire lengths of the axes rather than being concentrated on axis subdomains as in wild type. SYP-1 signal intensities are variable among different univalents within a given nucleus, suggesting a stochastic aspect to SYP-1 localization.

As DNA breaks induced by γ-irradiation can bypass the requirement for SPO-11, rescuing the crossover and chiasma formation defects in a spo-11 mutant (Dernburg et al., 1998), we tested whether or not γ-irradiation could also restore asymmetry of SYP-1 departure. Fig. 4 (A and B) and Fig. 5 B show that asymmetry is indeed substantially restored in the spo-11 mutant after γ-irradiation. In irradiated spo-11 worms, we observed diplotene nuclei with six robust SYP-1 stretches, each concentrated over a limited subset of axis staining corresponding to the still-synapsed regions of the desynapsing bivalents. Restoration of localized SYP-1 retention by γ-irradiation indicates that some aspect of the recombination event by itself, rather than the SPO-11 protein, is responsible for eliciting asymmetry in SC disassembly.

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Figure 5.

Dynamic localization of SYP-1 and AIR-2. (A) Immunolocalization of SYP-1 and AIR-2 in wild-type and spo-11 nuclei at the indicated stages. For diakinesis nuclei, −3, −2, and −1 indicate position relative to the spermatheca, with the oocyte in the −1 position (closest to the spermatheca) being the most mature. In A and B, detection thresholds for α-AIR-2 signals were lower for the pachytene and diplotene panels than for the diakinesis panels to permit imaging of both the lower levels of chromosome-associated AIR-2 at the earlier stages and the subchromosomal localization of the higher levels of AIR-2 in late diakinesis. DAPI-stained chromatin is shown in blue in the merged images. (B) SYP-1 and AIR-2 localization after γ-irradiation. Bars, 5 μm.

In both irradiated wild-type and spo-11 worms, some late prophase bivalents did not appear completely normal (Fig. 5 B and not depicted). Diplotene SYP-1 stretches appeared more variable in length than in unirradiated wild-type, and some late diplotene bivalents had a more symmetrical Y-shaped structure than is typical for wild-type meiosis. Furthermore, in some diakinesis bivalents the homologues were connected at extreme terminal positions. Such abnormalities likely reflect formation of crossovers at unusual chromosomal locations, as noted in Dernburg et al. (1998).

We also examined chromosome dynamics following pachytene exit in msh-5(me23) and him-14/msh-4(it21) mutants, which form DSBs and load RAD-51 but fail to complete initiated recombination events as crossovers (Zalevsky et al., 1999; Kelly et al., 2000; Colaiácovo et al., 2003). These mutants are also highly defective in achieving orderly asymmetric disassembly of SC, failing to concentrate SYP-1,2 to a single robust stretch per bivalent (Fig. 3 and not depicted). This result indicates that DSB formation and RAD-51 loading are not sufficient to trigger orderly asymmetric SC disassembly.

Together, our observations suggest that crossovers (or precursor events in the crossover pathway) serve as symmetry-breaking events during differentiation of meiotic prophase bivalents, triggering asymmetric SC disassembly and localized retention of SC central region proteins. We suggest that the single maturing or completed crossover per homologue pair subdivides the bivalent into two separate domains. Because crossovers usually occur at clearly off-center positions, these domains will be inherently unequal in length. We can envision how this asymmetry in length might set up an unstable equilibrium between association and dissociation of SYP-1,2 modules that results in localized retention and/or concentration of SYP-1,2 on the shorter segment: if the initial probability of dissociation increases with distance from the crossover, and if dissociation increases the likelihood of dissociation in adjacent intervals (e.g., by removing constraints that inhibit changes in chromosome geometry driven by chromatin expansion forces [Kleckner et al., 2004]), the observed concentration of SYP-1,2 on the shorter segment is expected to ensue.

SYP-1 guides AIR-2 localization during bivalent differentiation

The subregion of the bivalent to which SYP-1,2 localize from diplonema through mid-diakinesis corresponds to the region where Aurora-like kinase AIR-2 will become localized at the end of diakinesis (Kaitna et al., 2002; Rogers et al., 2002) and where REC-8 will be lost at the metaphase to anaphase transition of meiosis I (Pasierbek et al., 2001). Moreover, AIR-2 can phosphorylate REC-8 in vitro and is required for loss of REC-8 distal to the chiasma at meiosis I (Rogers et al., 2002). As asymmetric SC disassembly and localized SYP-1 retention after SC disassembly presage these later aspects of bivalent differentiation, we investigated the connection between SYP-1 and AIR-2 localization. Although earlier papers had suggested that AIR-2 first localizes to meiotic chromosomes at late diakinesis (in the −1 or −2 oocyte), our imaging and that of Chan et al. (2004) revealed much earlier chromosomal localization of AIR-2. We detected AIR-2 colocalized with SYP-1 during pachynema and diplonema, and dynamic changes in AIR-2 localization during the transition between these stages closely paralleled those of SYP-1 (Fig. 5). Chromosomal localization of AIR-2 in pachytene and diplotene regions of the germline is lost in syp-1 mutants (unpublished data), indicating that AIR-2 localization at these stages is SYP-1 dependent. Furthermore, AIR-2 localization tracked with SYP-1 in pachytene through diplotene stages both in the unirradiated spo-11 mutant and in γ-irradiated spo-11 mutant nuclei in which SYP-1 asymmetry had been restored. Together these observations suggest that pachytene and diplotene localization of SYP-1 dictates where AIR-2 will be localized at these stages.

AIR-2 staining diminished to nearly undetectable levels in early diakinesis, and in wild-type worms robust AIR-2 staining was later detected in the −1 and −2 oocytes, concentrated at the midbivalents on the short axis segments distal to the chiasmata (Kaitna et al., 2002; Rogers et al., 2002). In contrast, AIR-2 staining in the −1 and −2 oocytes of spo-11 mutants was less intense, was variable among different univalents within nuclei, and was distributed along the length of the univalents. Restoration of chiasmata by γ-irradiation in spo-11 mutants was accompanied by restoration of robust AIR-2 staining, usually concentrated at the midbivalent on the shorter axis segments (Fig. 5 B). These observations support a model in which asymmetry in SYP-1 localization is an early feature of asymmetric bivalent differentiation that in turn guides a later feature of bivalent differentiation, concentration of AIR-2 to the region now defined as distal to the chiasma.

We thank M. Zetka, J. Loidl, and J. Schumacher for reagents and J. Bessler, N. Miley, and K. Reddy for comments on the manuscript.

This work was supported by National Institutes of Health grant R01GM53804 to A.M. Villeneuve, by training funds from Osaka University to K. Nabeshima, and by National Research Service Award Fellowship F32HD41329 and institutional support from Harvard Medical School to M.P. Colaiácovo.