JCS049353 Supplementary Material

Files in this Data Supplement:

• Supplemental Tables S1 -

• Supplemental Figure S1 -

  Fig. S1. Sycp1^−/− oocytes: deficient synapsis and double strand break repair. (a) Absence of Sycp1 protein results in normal chromosomal axes formation, as shown here for STAG3 protein (red). Labeling for cohesins REC8, RAD21, SMC1β and SMC1α, or axial element proteins Sycp2 and Sycp3 yielded similar results (data not shown). Centromeres are labelled by CREST antiserum (white). Sycp1^−/− pachytene oocytes show complete lack of synapsis (40 centromeres and axes vs 20 in wild-type oocytes). (b) Wild-type and Sycp1^−/− pachytene oocytes, derived from E18.5 ovaries, were stained with an antibody recognizing DMC1 and Rad51 proteins, and the number of foci on chromosomal axes (n=31 for wild type and 26 for Sycp1^−/− oocytes) were counted. The obtained values were placed in the corresponding columns; red dots indicate the average values. Number of DMC1/Rad51 foci in pachytene oocytes was significantly higher for Sycp1^−/− than wild-type oocytes (P<0.0001, Student’s t-test), indicating inefficient or delayed repair of double strand breaks. (c) Wild-type and Sycp1^−/− zygotene oocytes derived from E16.5 ovaries were labelled with BRCA1 antibody (green), STAG3 antibody (red) and CREST antiserum (white). First round of axial BRCA1 recruitment is not affected in zygotene Sycp1^−/− oocytes. Scale bars (A,C): 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Double labeling of Smc1β^−/− oocytes with antibodies against BRCA1 and γH2A.X. Smc1β^−/− oocytes with low (on the left) and high (on the right) levels of asynapsis were double labelled with BRCA1 (green) and γH2A.X (blue) antibodies. The chromosomal axes were identified using a STAG3 antibody (red). Both images were taken at the same exposure time and processed in exactly the same way to permit a direct comparison. Bright BRCA1 staining on asynapsed axes in oocytes with limited asynapsis is correlated with an intense γH2A.X signal. By contrast, a weaker dot-like BRCA1 signal is observed on asynapsed chromosomes in Smc1β^−/− oocytes that display a high level of asynapsis and is accompanied by restricted H2A.X phosphorylation. Scale bars: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. ATR in wild-type, Sycp1^−/− and Smc1β^−/− oocytes. Oocytes derived from wild-type, Sycp1^−/− and Smc1β^−/− ovaries at embryonic day 18.5 (E18.5) were probed with an antibody against ATR (white in the top row, green in merge images). Chromosomal axes were identified using an antibody against STAG3 (red) and centromeres were visualized using a CREST antiserum (white). ATR was observed as bright continuous filaments on asynapsed chromosomal axes in wild-type and Smc1β^−/− oocytes with a few asynapsed chromosomes and as fragmented, weakly stained structures in Sycp1^−/− and Smc1β^−/− oocytes with extensive asynapsis. Arrows point to the asynapsed chromosomes. Scale bars: 10 µm.

• Supplemental Figure S4 -

  Fig. S4. Silencing of asynapsed chromatin in Smc1β^−/− oocytes. (a) Smc1b^−/− oocytes were stained with anti-RNA pol II antibody (green) and co-stained with anti-γH2A.X (blue). Chromosomal axes were labelled with Sycp2 antibody (red). The insert shows a region with asynapsed chromosomes. RNA pol II staining displays reduced levels around asynapsed chromosomes, where γH2A.X signal is strong. Scale bar: 10 µm. (b) Quantification showed linear correlation between reduction of RNA pol II intensity on chromatin adjacent to asynapsed axes and γH2A.X signal intensity (correlation coefficient R=0.8). Red line shows linear fit.

• Supplemental Figure S5 -

  Fig. S5. γH2A.X staining in Sycp3^−/− and Sycp3^−/−Smc1β^−/− oocytes. Sycp3^−/− single mutant and Sycp3^−/−Smc1β^−/− double mutant pachytene oocytes were probed with antibody to γH2A.X (green). Chromosomal axes are marked by STAG3 antibody (red), and centromeres by CREST antiserum (white). Pachytene oocytes lacking Sycp3 protein are deprived of bright uniform γH2A.X domains encompassing the chromosomal axes. Scale bars: 10 µm.