Extended Data Fig. 7: PAR-associated RPA2 foci.

a, Loop–axis organization of the mo-2 region of chromosome 9 in late zygonema. Compare with the PAR (Fig. 3b). Error bars are mean ± s.d. Scale bars, 1 μm. b, Low mo-2 copy number correlates with less loop–axis reorganization (SIM images of late-zygotene F₁ hybrid spermatocytes). The differentiation of the B6 PAR was observed in both hybrids (B6 × MSM and MSM × B6) in three and four spermatocytes, respectively, by SIM (one mouse for each), and in more than 20 spermatocytes by conventional microscopy (two mice of each genotype). Scale bars, 1 μm. c–e, Immuno-FISH for RPA2 and mo-2 was used to detect DSBs cytologically in wild-type mice and the indicated mutants. To analyse Rec8 and Hormad1 mutations, we compared to mutants lacking SYCE1 (a synaptonemal complex central element component⁵³) because Syce1^–/– mutants show similar meiotic progression defects without defective RMMAI recruitment. c, Representative images. Scale bars, 2 μm (main image); 1 μm (inset). d, Global counts of RPA2 foci for zygotene (zyg) or zygotene-like cells and for pachytene (pach) or pachytene-like cells. e, Fraction of mo-2 regions, for each cell, that had a colocalized RPA2 focus. Red lines indicate mean ± s.d. Statistical significance is indicated in d, e for comparisons (two-sided Student’s t-tests) of wild type to Ankrd31^−/− or of Syce1^−/− to either Rec8^−/− or Hormad1^−/− for matched stages; for exact P values, see the associated Source Data. The number of discretely scorable mo-2 regions in e varied from cell to cell depending on pairing status. f, Frequent formation of DSBs at mo-2 regions in the PAR and on autosomes does not require HORMAD1. Left, micrograph showing two adjacent spermatocytes (boundary indicated by dashed line). Scale bar, 2 μm. Right, insets show higher-magnification views of the numbered mo-2 regions, all of which are associated with RPA2 immunostaining of varying intensity. This image illustrates the preferential formation of RPA2 foci in mo-2 regions in a Hormad1^−/− mouse; quantification is in e. g, Autosomal mo-2 regions often form DSBs late. Immuno-FISH for RPA2, mo-2 and PARb was used to detect DSBs cytologically in wild-type mice from leptonema to mid-pachynema, and to distinguish the X or Y chromosome PAR from chromosomes 9 and 13. Chromosome 4 was not assayed because the mo-2 FISH signal was often barely detectable. Top, global number of RPA2 foci per cell. Black lines indicate mean ± s.d. Bottom, percentage of spermatocytes with an RPA2 focus overlapping the PAR (X, Y or both) or overlapping chromosome 9 or 13. A representative image of an early-pachytene spermatocyte is shown. As previously shown for the PAR², autosomal mo-2 regions continue to accumulate RPA2 foci beyond the time point at which global RPA2 foci have largely or completely ceased accumulating. Scale bar, 2 μm. h, X–Y chromosome pairing status, quantified by immuno-FISH for SYCP3 and the PARd probe. i, Montage of SIM images from a B6 male, showing that multiple, distinct RPA2 foci can be detected from late zygonema to mid-pachynema. This suggests that multiple PAR DSBs can be formed during one meiosis². The presence of multiple RPA2 foci in the PAR was observed by SIM in more than 20 spermatocytes from late zygonema to mid-pachynema in one mouse. Scale bar, 1 μm. j, Percentage of spermatocytes at the zygotene–pachytene transition with no (0), one, two or three distinguishable RPA2 foci on the unsynapsed Y chromosome PAR of MSM and B6 mice. The difference between the strains is statistically significant (negative binomial regression, P = 7.2 × 10⁻⁵). The number of spermatocytes analysed is indicated. A representative image is shown for each genotype, with one RPA2 focus on the MSM PAR and two apparent sites of RPA2 accumulation on the B6 PAR. The detection of multiple foci is consistent with reported double crossovers⁶. Scale bar, 1 μm.

Source Data