FIGURE 5:

Conditional mutation of Smc5 does not result in abnormal meiotic prophase progression in male mice. (A–I) No differences were observed when comgparin chromatin spread preparations from juvenile control and Smc5 cKO (Stra8-Cre) germ cells staged at prophase to metaphase I. Assessments were using germ cells from mice ≥8 wk old. (A) Chromatin spreads were immunolabeled with antibodies against γH2AX (blue), the SC lateral element protein SYCP3 (red), and the SC central element protein SYCP1 (green). Scale bar: 10 μm. (B) Bar graph showing comparable distributions of prophase stages in control and Smc5 cKO testes. Black bars indicate standard error. The P values (Mann-Whitney, two-tailed) for the indicated comparisons are not significant (n.s.). (C, E) Chromatin spread preparations were immunolabeled with antibodies for the SC lateral element protein SYCP3 (red), CEN (kinetochore/centromere marker) and DNA repair proteins RAD51 or DMC1 (green). (D) Scatter dot–plot graph showing RAD51 foci counts per cell on the autosomal chromosomes or the XY chromosome of juvenile control mice (n = 74, autosomal average = 13.22, XY mean = 7.34) and Smc5 cKO mice (n = 75, autosomal mean = 15.40, XY average = 8.44). Bars indicate mean RAD51 foci number per cell with 95% confidence interval. The P values (Mann-Whitney, two-tailed test) for the indicated comparisons are not significant (n.s.). (F) Scatter dot–plot graph showing DMC1 foci counts per cell on the autosomal chromosomes or the XY chromosome of juvenile control mice (n = 18, autosomal mean = 21.22, XY mean = 9.22) and Smc5 cKO mice (n = 18, autosomal mean = 20.50, XY mean = 8.11). Bars indicate mean DMC1 foci number per cell with 95% confidence interval. The P values (Mann-Whitney, two-tailed) for the indicated comparisons are not significant (n.s.). (G) Chromatin spreads were immunolabeled with antibodies, the SC lateral element protein SYCP3 (red), and the MLH1 (crossover protein, green). (H) Scatter dot–plot graph showing no significant difference in the mean number of MLH1 foci per chromosome axis when comparing control (n = 24, mean = 1.16) and Smc5 cKO (n = 25, mean = 1.14) chromosome spread preparations. Bars indicate the average with standard error. The P value (Mann-Whitney, two-tailed test) for the indicated comparison is not significant (n.s.). (I) No differences were observed in chromatin spread preparations of control and Smc5 cKO (Stra8-Cre) germ cells at metaphase I after treatment with okadaic acid. Chromatin spreads were immunolabeled with antibodies against the SC lateral element protein SYCP3 (red) and CEN (kinetochore/centromere marker, blue) and counterstained with DAPI (DNA, blue). Scale bar: 10 μm. See Supplemental Figure S5 for additional data.