A novel function for CDK2 activity at meiotic crossover sites
Fig 4
γH2AX analysis of meiotic DSB repair in WT, Cdk2T160, and Cdk2−/− spermatocytes.
Chromosome spread preparations from adult (postnatal day 40) testes immunostained with the DNA damage marker γH2AX (green) and SYCP3 (red) are shown for WT (A–D), Cdk2T160A (E–H), and Cdk2−/− (I–K) spermatocytes, for selected stages of meiotic prophase I. γH2AX can be detected as a panchromatin stain in leptotene through zygotene stage in all genotypes and all stages for the Cdk2−/− mutant. At early-pachytene, γH2AX localizes to the chromatin surrounding the X–Y bivalent (sex body)—outlined with dashed lines—in WT (C) and Cdk2T160A (G), indicating normal MSCI. This pattern is retained into mid-pachytene (D and H) but does not occur in the Cdk2−/− mutant (K). Percentages of nuclei retaining γH2AX signal specifically on autosomes, are quantified for all genotypes from leptotene to early-pachytene (L). Data are presented as a mean percentage of cells ± SD determined from 3 biological replicates (N = 60 for the overall nuclei counted for WT leptotene, zygotene, and early-pachytene stages [orange bars]; N = 60 for the overall nuclei counted for Cdk2T160A leptotene, zygotene, and early-pachytene stages [blue bars]; and N = 60 for the overall nuclei counted for Cdk2−/− leptotene, zygotene, and early-pachytene stages [gray bars]). Additional staining is shown for P40 chromosome spread preparations immunostained for RAD51 (green) and SYCP3 (red). WT (M–P), Cdk2T160A spermatocytes (Q–T), and Cdk2−/− (U–W) images are shown for selected stages of meiotic prophase I. During leptotene and zygotene stages in WT (M–N), Cdk2T160A (Q–R), and Cdk2−/− (U–V), RAD51 focus formation precedes the synapsis of homologs visualized using SYCP3. From the early-pachytene stage, fully paired homologs from WT and Cdk2T160A spermatocytes lose RAD51 foci (O–P and S–T, respectively). In pachytene-like Cdk2−/− spermatocytes, only a pachytene-like arrest state is achieved (W). Here, RAD51 foci are observed to remain bound to stretches chromosomal axes despite extensive nonhomologous synapsis. These are sites of presumed failed strand invasion events. All images are representative of at least 20 images taken for specified stages. Similar staining patterns were confirmed in at least 3 biological replicates. In all main panels, scale bars are representative of 5 μm. RAD51 foci were quantified specifically for leptotene, zygotene, and early-pachytene stages (X) by counting the average numbers of RAD51 foci per nucleus. Data are presented as individual foci counts for WT (orange bars, N = 90 for leptotene, zygotene, and early-pachytene stages), Cdk2T160A (blue bars, N = 90 for leptotene, zygotene, and early-pachytene stages) and Cdk2−/− (gray bars, N = 90 for leptotene, zygotene, and early-pachytene stages). For panels L and X, error bars are indicative of the mean and SD. All data were assumed to be non-normally distributed. Statistical significance between genotypes was determined by unpaired t test. Significance and P-values are reported directly over each comparison. The underlying data for (L, X) can be found in S1 Data. CDK2, cyclin-dependent kinase 2; DSB, double-strand break; MSCI, meiotic sex chromosome inactivation; RAD51, RAD51 recombinase; SD, standard deviation; SYCP, synaptonemal complex protein; WT, wild-type; γH2AX, phosphoserine 139 H2AX.