Fig 2: PLK1 regulates the SGO2-dependent loading of MCAK to the inner centromeres. Double immunolabeling of MCAK (green) and SYCP3 (red), and counterstaining of the chromatin with DAPI (blue) on squashed diakinesis (Dia.) (A) and metaphase I (M I) (C) control spermatocytes, and diakinesis (B) and metaphase I (D) BI 2536-treated spermatocytes. Yellow arrowheads indicate SYCP3 agglomerates in the nucleoplasm of the control diakinesis spermatocyte. Scale bar represents 5 μm.

Fig 3: EXOSC10 localization during meiotic germ cell differentiation. (a) Dynamic localisation of EXOSC10 (red) during spermatocyte differentiation in comparison to γ-H2AX (green). Fluorescence signal intensities are plotted for XY bodies in early (panel A framed in green), and late pachytene (panel B in yellow), and diplotene (panel C in red). The tubule stage is given. A stippled line outlines the borders of seminiferous tubules of a 35dpp mouse testis. (b) Laser scanning microscopic analysis of EXOSC10 (red) and XY body chromatin (γ-H2AX, green) in diplotene XY bodies of a stage XI tubule. The sex bodies of the boxed spermatocytes are shown below as Z-projections of 3 × 1 µm image stacks (from upper left to lower right). (c) Immunofluorescence analysis of nuclear spreads at different stages of meiotic prophase I as indicated to the left. Images show DAPI-stained DNA (blue), γ-H2AX (green) and EXOSC10 (red). The rightmost column shows all signals (merge). White arrowheads point to XY bodies associated with an EXOSC10 positive aggregate. (d) A similar experiment is shown for prophase I stages that are given to the right, revealing DNA with DAPI (blue), the synaptonemal complex with the SYCP3 marker (green) and EXOSC10 (red). Typical cases of associated XY body-EXOSC10 signals are encircled in the rightmost column (merge).

Fig 4: PAR-associated RPA2 foci.(a) Loop-axis organization of the mo-2 region of chr9 in late zygonema. Compare with the PAR (Fig. 3b). Scale bars: 1 μm. Error bars: means ± SD. (b) Low mo-2 copy number correlates with less loop–axis reorganization (SIM images of late-zygotene F1-hybrid spermatocytes). Scale bars: 1 μm. The differentiation of the B6 PAR was observed in both hybrids B6 × MSM and MSM × B6 in 3 and 4 spermatocytes, respectively by SIM (1 mouse for each) and in more than 20 spermatocytes by conventional microscopy in two mice of each genotype. (c,d,e) Immuno-FISH for RPA2 and mo-2 was used to detect DSBs cytologically in wild type and the indicated mutants. To analyze Rec8 and Hormad1 mutations, we compared to mutants lacking SYCE1 (a synaptonemal complex central element component62) because Syce1−/− mutants show similar meiotic progression defects without defective RMMAI recruitment. Panel c shows representative images. Scale bars: 2 μm, inset 1 μm. Panel d shows the global counts of RPA2 foci for zygotene (zyg) or zygotene-like cells and for pachytene (pach) or pachytene-like cells. Panel e shows, for each cell, the fraction of mo-2 regions that had a colocalized RPA2 focus. Red lines: means ± SD. Statistical significance is indicated in panels c and d for comparisons (two-sided Student’s t tests) of wild type to Ankrd31−/− or of Syce1−/− to either Rec8−/− or Hormad1−/− for matched stages. Exact p values are in Data File S7. Note that the number of discretely scorable mo-2 regions in panel e varied from cell to cell depending on pairing status. (f) Frequent DSB formation at mo-2 regions in the PARs and on autosomes does not require HORMAD1. Micrograph at left shows two adjacent spermatocytes (boundary indicated by dashed line). Scale bar: 2 μm. Insets at right show higher magnification views of the numbered mo-2 regions, all of which are associated with RPA2 immunostaining of varying intensity. This picture illustrates the preferential RPA2 focus formation in mo-2 regions in a Hormad1−/− mouse; quantification is in panel 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 from leptonema to mid-pachynema, and to distinguish the X or Y PAR from chromosomes 9 and 13. Chr4 was not assayed because the mo-2 FISH signal was often barely detectable. The top panel shows the global number of RPA2 foci per cell. Black lines are means ± SD. The bottom panel shows the percentage of spermatocytes with an RPA2 focus overlapping the PAR (X, Y, or both) or overlapping chr9 or chr13. A representative image of an early pachytene spermatocyte is shown. Note that, as previously shown for the PAR2, autosomal mo-2 regions continue to accumulate RPA2 foci beyond the time when global RPA2 foci have largely or completely ceased accumulating. Scale bar: 2 μm. (h) X–Y 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, suggesting that multiple PAR DSBs can be formed during one meiosis (see also ref. 2 for further discussion). Scale bar: 1μm. 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. (j) Percentage of spermatocytes at the zygotene-pachytene transition with no (0), 1, 2 or 3 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). N indicates the number of spermatocytes analyzed. A representative picture 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.

Fig 5: SMC3 is phosphorylated at Ser1083 during prophase I.(A) Testis nuclear extracts were immunoprecipitated with the anti-SMC3 antibody, followed by treatment with (+) or without (−) phosphatase (PPase) and phosphatase inhibitors (Inhibitor). 80% of the immunoprecipitated SMC3 and the rest were separated on a gradient gel and immunoblotted with antibodies against the Ser1083-phosphorylated form of SMC3 (pS1083) and normal SMC3, respectively. (B) Testis nuclear extracts were immunoprecipitated without (Mock) or with the anti-pS1083 antibody. The immunoprecipitates were probed with antibodies against meiotic chromosome axis components. The asterisk marks a non-specific band. (C) Nuclear spreads of spermatocytes were labeled with anti-pS1083, anti-SYCP3 and anti-HORMAD1 antibodies. Arrowheads indicate the XY bivalent. Bars, 10 µm.