Distinct properties of the XY pseudoautosomal region crucial for male meiosis

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.

Figure 1

Late PAR pairing during male meiosis. A. FISH assay for pairing. i–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-chr18 and distal-chr19 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.

Figure 2

Distinct temporal and structural properties of the PAR. A. Nucleus-wide RAD51/DMC1 foci in spermatocytes (bars = mean±SD). B. Assay for PAR DSB formation. IF against RAD51/DMC1 and SYCP3 (i) and FISH (ii) with probes shown in Figure 1Ai on a leptotene spermatocyte nucleus. Scale bar, 10 μm. iii, Zoom-ins of Y and X PARs from i (29), and an overlay of the PAR FISH signal with SYCP3 (left), 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 ref. 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 (grey brackets in insets), see also Table 1. Scale bar, 5 μm.

Figure 3

Genetic control of PAR recombination and pairing. A. Spo11 splice variants (see also fig. S4). i) Genomic organization and splicing. Spo11β includes exon 2, Spo11α excludes it. Y, catalytic tyrosine. ii–iii) Reverse transcriptase-PCR from flow-sorted meiocyte populations of adult mice. –RT, no reverse transcriptase; 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 knock-out allele (fig. S5D). 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; 57–65 nuclei scored/genotype. C. 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 = mean±SD). E. Nuclei (%) with PAR RAD51/DMC1 foci in mice of the indicated genotypes. 41–55 nuclei were scored per stage for each genotype. Asterisks indicate significant differences (P≤ 0.0002, two-tailed Mann-Whitney test). n.s., not significant, P=0.09.