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Distinct Properties of the XY Pseudoautosomal Region Crucial for Male Meiosis

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Science  18 Feb 2011:
Vol. 331, Issue 6019, pp. 916-920
DOI: 10.1126/science.1195774

Abstract

Meiosis requires that each chromosome find its homologous partner and undergo at least one crossover. X-Y chromosome segregation hinges on efficient crossing-over in a very small region of homology, the pseudoautosomal region (PAR). We find that mouse PAR DNA occupies unusually long chromosome axes, potentially as shorter chromatin loops, predicted to promote double-strand break (DSB) formation. Most PARs show delayed appearance of RAD51/DMC1 foci, which mark DSB ends, and all PARs undergo delayed DSB-mediated homologous pairing. Analysis of Spo11β isoform–specific transgenic mice revealed that late RAD51/DMC1 foci in the PAR are genetically distinct from both early PAR foci and global foci and that late PAR foci promote efficient X-Y pairing, recombination, and male fertility. Our findings uncover specific mechanisms that surmount the unique challenges of X-Y recombination.

Meiotic recombination, initiated by programmed double-strand breaks (DSBs), promotes homologous chromosome (homolog) pairing during prophase I (1). A subset of DSBs matures into crossovers that physically connect homologs so that they orient properly on the first meiotic spindle. Because sex chromosome recombination and pairing are restricted to the PAR (2), at least one DSB must form within this small region, and the homologous PAR must be located and engaged in recombination to lead to a crossover. Accordingly, the PAR in males exhibits high crossover frequency (2, 3), but sex chromosomes also missegregate more frequently than autosomes (4). Nevertheless, X-Y nondisjunction is rare, which suggests that there are mechanisms that ensure successful X-Y recombination.

X-Y pairing is more challenging than autosomal pairing, as it cannot be mediated by multiple DNA interactions along the length of the chromosomes. We used fluorescence in situ hybridization (FISH) (5) to compare timing of meiotic X-Y and autosomal pairing in mice (Fig. 1). At leptonema, when DSBs begin to form and only short chromosome axis segments are present, PAR and autosomal FISH probes were mostly unpaired. By early to mid-zygonema, when axes elongate and homologs become juxtaposed, distal ends of chr 18 and 19 were paired in ~50% of nuclei; by late zygonema, these regions were paired in nearly all nuclei (Fig. 1B and fig. S1). In contrast, the X and Y PARs were rarely paired before pachynema (Fig. 1B); hence, X-Y pairing is delayed compared with that of autosomes.

Fig. 1

Late PAR pairing during male meiosis. (A) FISH assay for pairing. (i and ii) Example of immunofluorescence (IF) and two sequential rounds of FISH on a late zygotene spermatocyte nucleus. Nuclei stained with an antibody against axis protein SYCP3 were subjected first to PAR FISH (i), then to distal chr 18 and distal chr 19 FISH (ii). Scale bar, 10 μm. (B) Nuclei (%) with unpaired and paired (≤2 μm apart) FISH signals. Chromosome synapsis status was also recorded at sites of paired signals.

DSBs precede and are required for efficient homolog pairing in mouse meiosis (6, 7). Nucleus-wide (“global”) foci of DSB markers RAD51/DMC1 peak in number at early to mid-zygonema (Fig. 2A) (8, 9). Because stable X-Y pairing occurs late, we asked whether PAR DSB kinetics is also delayed (Fig. 2B and fig. S2). More than half of cells had no RAD51/DMC1 focus in the PAR before late zygonema (Fig. 2C), distinct from global patterns. Only when global foci were already declining did the majority of cells (~70%) display PAR foci (Fig. 2C and fig. S2i). We interpret the lack of PAR foci to indicate that DSBs have not yet formed. Thus, we propose that PAR DSB formation and/or turnover are under distinct temporal control. We cannot exclude the alternative possibility that PAR DSBs have formed but are cytologically undetectable, for example, because RAD51/DMC1 have not yet been loaded onto DSB ends or because foci have already turned over. In either case, DSB dynamics and/or processing differs on the PAR.

Fig. 2

Distinct temporal and structural properties of the PAR. (A) Nucleus-wide RAD51/DMC1 foci in spermatocytes (bars show means ± SD). (B) Assay for PAR DSB formation. IF against RAD51/DMC1 and SYCP3 (i) and FISH (ii) with probes shown in Fig. 1Ai on a leptotene spermatocyte nucleus. Scale bar, 10 μm. (iii) Magnified views of Y and X PARs from frames in (i) (5) and an overlay of the PAR FISH signal with SYCP3 (right), here with a RAD51/DMC1 focus only on the X PAR. (C) Nuclei (%) with one or two PAR RAD51/DMC1 foci. (D) Axis/loop segments as a determinant of DSB potential [after (15)]. Only one homolog is shown. DNA organized on a longer axis into more and smaller loops (i) has more DSB potential than if the same DNA is organized on a shorter axis into fewer and larger loops (ii). (E) Examples of chromatin extension (gray brackets in insets); see also Table 1. Scale bar, 5 μm.

Most sites marked by PAR RAD51/DMC1 foci appeared incapable of mediating stable pairing before early pachynema (~70% of late zygotene nuclei had foci, but <20% showed PAR pairing) (Figs. 1B and 2C). The number of PAR foci per cell also increased over time. In leptonema and early to mid-zygonema, most cells with a PAR RAD51/DMC1 focus had only one (typically on X), whereas by late zygonema, two PAR foci were often present (both X and Y) (Fig. 2C). Foci on both PARs could represent two independent DSBs. If so, then having more than one X-Y recombination interaction may stabilize pairing, similar to multiple interactions that stabilize pairing of autosomes (10). Alternatively, foci on both PARs could represent the two, separated ends of a single DSB (11, 12)—with one focus marking the broken PAR and the second focus marking the other PAR (fig. S3A). In this “ends-apart” model, nuclei that have two PAR foci are those in which the X and Y PARs have successfully engaged each other. However, we found that most such nuclei showed no evidence of a preferential X-Y spatial relationship (fig. S3B), and most PAR pairing occurred abruptly at the zygonema-to-pachynema transition, i.e., after the stage when many cells displayed two PAR foci (compare Figs. 1B and 2C). Sex body formation (13) may facilitate this sudden completion of X-Y pairing by providing homology-independent X-Y proximity that simplifies the homology search.

The haploid mouse genome averages fewer than one DSB per 10 Mb (Fig. 2A), whereas the <1 Mb PAR (14) undergoes one or two DSBs (Fig. 2C), which is 10 to 20 times the genome average. We speculated that distinct higher-order chromosome structure could render the PAR more conducive to DSB formation. Meiotic recombination is proposed to occur within DNA segments residing in chromatin loops that become transiently tethered to chromosome axes (15). Loop density per micrometer of axis is constant (16) and produces an inverse relation between loop size and axis length (17). DNA arranged into smaller loops may have higher DSB potential (Fig. 2D) (18); indeed, autosomal crossover frequency in male mice correlates with axis length (19). We found that PAR axes were disproportionately long relative to DNA length and incorporated ~1 Mb per μm of axis (Table 1A). At the distal ends of chr 18 and 19 (regions with relatively frequent crossing-over) (19), DNA content was 10 to 13 Mb per μm and correlated well with axis length, i.e., the distal ~10% of DNA occupied ~10% total axis length (Table 1A). The ≥10-fold difference between PAR and autosome axes is of the magnitude expected for a region that experiences more than 10 times as many DSBs. Axes of non-PAR portions of the X and Y had a DNA content more like autosomes (≥14 Mb per μm) (fig. S4).

Table 1

Chromosome axis lengths and chromatin extension in PARs and distal ends of chr 18 and 19. Axis lengths are means ± SD and FISH signal extensions are means ± SD, for the number of observations in parentheses. Probe size is the size of the bacterial artificial chromosome (5).

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Long PAR axes predict short chromatin loops. As a proxy for loop size, we measured FISH signal extension from axes for probes in the PAR and autosomal subtelomeric regions (Fig. 2E and Table 1B). PAR FISH signals were substantially more compact at all stages (about one-third to one-seventh as extended), consistent with smaller loops. Thus, chromosome structure could be one factor that facilitates high-frequency DSB formation in the PAR.

The distinct temporal and structural features outlined above raised the possibility that mechanisms ensuring efficient PAR recombination and pairing may be under different genetic control from autosomes. Characterization of a variant of SPO11, the evolutionarily conserved meiotic DSB catalyst (1), validated this hypothesis (Fig. 3). Two major mRNA splicing isoforms in mice and humans are Spo11α and Spo11β (7, 2022) (Fig. 3Ai and fig. S5). Spo11β is expressed early in meiosis, when most DSBs are formed, whereas Spo11α is expressed later (7, 20, 23) (Fig. 3Aii and fig. S6A). Thus, SPO11β is likely responsible for most DSB formation.

Fig. 3

Genetic control of PAR recombination and pairing. (A) Spo11 splice variants (see also fig. S5). (i) Genomic organization and splicing. Spo11β includes exon 2, Spo11α excludes it. Y, catalytic tyrosine. (ii and iii) Reverse transcription polymerase chain reaction from flow-sorted meiocyte populations of adult mice. –RT, no reverse transcription; L/Z, leptonema/zygonema; P/D, pachynema/diplonema; S, spermatids. (iv) SPO11 protein levels in adult testis extracts. Asterisk, a lower-mobility protein likely originating from the knockout allele (fig. S6D). (B) IF of SYCP1 and SYCP3 on pachytene nuclei (i) and of SYCP3 plus whole-chromosome FISH of early metaphase I spermatocyte nuclei (ii) from mice of the indicated genotypes. Inset in (i), schematic of X and Y chromosomes. Scale bars, 10 μm. (iii) Quantification of X-Y association; 57 to 65 nuclei scored per genotype. (C) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL)–stained testis sections; apoptotic cells stain brown. Elongating spermatids (arrows) are rare in Spo11β-only mice. Inset shows a lagging chromosome (arrowhead) in a TUNEL-positive cell. (D) RAD51/DMC1 focus counts in spermatocytes from control and Spo11β-only mice (bars show means ± SD). (E) Nuclei (%) with PAR RAD51/DMC1 foci in mice of the indicated genotypes; for each genotype, 41 to 55 nuclei were scored per stage. *P ≤ 0.0002 (two-tailed Mann-Whitney test); n.s., not significant (P = 0.09).

We generated transgenic mice expressing Spo11βB cDNA (fig. S5) from a meiosis-specific promoter (24) (fig. S6B). Tg(Xmr-Spo11βB) transcript expression overlapped with Spo11β mRNA appearance in wild type (Fig. 3Aii and fig. S6, A and C). In testis extracts of Spo11–/– Tg(Xmr-Spo11βB)+/+ (hereafter, “Spo11β-only”) mice, SPO11βB protein approximated the total level of SPO11 in wild type (Fig. 3Aiii). The transgene did not cause obvious meiotic phenotypes in mice heterozygous at the endogenous Spo11 locus [i.e., Spo11+/– Tg(Xmr-Spo11βB)+/+], and these mice were used as controls. The profound meiotic defects of Spo11–/– mice [no recombination, failure of homolog pairing and synapsis, and infertility (6, 7, 25)] were mostly rescued by Tg(Xmr-Spo11βB) in both sexes: Autosomal homologous pairing, synapsis, and MLH1 focus formation (a crossover marker) appeared normal (Fig. 3Bi and fig. S7A). Moreover, ovaries of Spo11β-only mice contained abundant primordial follicles (fig. S7B), and Spo11β-only females were fully fertile with normal litter sizes. Thus SPO11βB supports autosomal crossing-over, pairing, and synapsis, and (in females) full meiotic progression and accurate chromosome segregation. Male meiosis was not fully rescued, however. Although sex bodies formed (fig. S7C), the X and Y failed to pair and synapse in ~70% of spermatocytes (Fig. 3B). Spo11β-only testis sections showed numerous apoptotic metaphase I cells (Fig. 3C), many with a lagging chromosome (Fig. 3C, inset, and fig. S7D), consistent with spindle checkpoint-induced apoptosis (9, 26, 27), triggered by the failure of nonrecombinant X and Y to orient properly on the metaphase I spindle. Few postmeiotic cells were formed, and testis sizes were reduced (Fig. 3C and fig. S7, D and E), so that although some Spo11β-only males produced offspring, most were infertile.

Nucleus-wide numbers and timing of RAD51/DMC1 foci were indistinguishable between Spo11β-only and control males (Fig. 3D and fig. S7F), which indicated that the X-Y pairing defect cannot be attributed to reduced global DSB levels. Similarly, the frequency of PAR RAD51/DMC1 foci in leptonema was not affected (Fig. 3E). In contrast, the percentage of late zygotene nuclei with a PAR focus was reduced in Spo11β-only males, consistent with a defect in a late-forming DSB population (PAR-specific, or possibly including a small subset of autosomal DSBs). About 70% of late zygotene nuclei lacked PAR foci (Fig. 3E), which was similar to the percentage of cells with X-Y pairing failure (Fig. 3Biii). Thus, the few PAR foci that form early in both wild-type and Spo11β-only males seem to persist until late zygonema (fig. S4, discussion), at which time recombination-mediated X-Y pairing occurs. We propose that a lack of late PAR DSBs is the cause of infertility in Spo11β-only males. In females, two fully homologous X chromosomes make PAR recombination dispensable.

Spo11α is the only splice variant missing from Spo11β-only mice that is known to be developmentally regulated, and its expression in wild type correlates with the timing of late PAR DSBs as inferred from the appearance of RAD51/DMC1 foci. It is thus possible that SPO11α, by itself or in combination with SPO11β, is needed for DSB formation in late zygonema. In this scenario, late-forming PAR DSBs are genetically separable from both global DSBs and early-forming PAR DSBs, and the surge of late-forming PAR DSBs is crucial for efficient X-Y pairing and fertility. PAR recombination occasionally fails in humans, as evidenced by paternally inherited sex chromosome aneuploidies [e.g., Klinefelter’s or Turner syndromes (28)]. Because Spo11 isoforms are conserved, we speculate that variation in Spo11 splicing patterns may be a human X-Y nondisjunction susceptibility trait.

Supporting Online Material

www.sciencemag.org/cgi/content/full/331/6019/916/DC1

SOM Text

Materials and Methods

Figs. S1 to S7

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. This work was supported by NIH grant R01 HD040916 (M.J. and S.K); International Grants in Cancer Research (AIRC) [My First AIRC Grant (MFAG) grant 4765], Italian Ministry for Education, University and Research (MIUR), the Lalor Foundation, and the American-Italian Cancer Foundation (AICF) (M.B.); and the Charles H. Revson Foundation (F.B.). We thank M. Leversha [Memorial Sloan-Kettering Cancer Center (MSKCC)], P. Bois (Scripps Florida), and K. Manova (MSKCC) for valuable advice and protocols. We are grateful to Keeney and Jasin lab members, especially I. Roig, E. de Boer, and F. Cole, and to N. Hunter (University of California, Davis) for insightful comments.
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