Figure 2.

Many PAR DSBs form later than nucleus-wide DSBs, and are under different genetic control. A) Assay for nucleus-wide and PAR DSB formation. i-ii) Example of immunofluorescent (IF) and FISH staining of a spermatocyte in late zygonema. Nuclei were first stained with antibodies against RAD51 and SYCP3 and photographed (i), then FISH was performed using PAR, X and Y probes (as cartooned above the FISH image) and the same nuclei were photographed again (ii).⁴ Under the FISH conditions used in these experiments, some IF signal remains visible. The presence or absence of a RAD51 focus in the X and Y PAR was scored by comparing the IF image to an IF+FISH image overlay (iii). In this example, both the X and Y PAR (inside white squares in i-ii) display a RAD51 focus (white arrows in iii). Scale bar, 10 µm. B) Mean nucleus-wide RAD51 focus numbers (bars ± 95% confidence interval of the mean; left Y axis) and percentage of nuclei with a PAR RAD51 focus on the X and/or Y PAR (right Y axis) as prophase I progresses from leptonema to late zygonema, in mice of the indicated genotypes (see text). C) Percentage of nuclei with paired FISH signals for autosomal and PAR probes as prophase I progresses from leptonema to pachynema.