Figure 3.
Sex chromosome pairing and silencing defects in Spo11P306T/P306T spermatocytes. (A) Representative pachytene chromosome surface spreads immunolabeled for indicated proteins. The third column is a merge of the first two columns. HORMAD2 labels unsynapsed regions of chromosomes, including the non-pseudoautosomal region of the XY pair. In the examples shown here, the X and Y chromosomes (yellow arrowheads) are not synapsed in the Spo11P306T/P306T spermatocytes (unstained in the PAR by the synapsis marker SYCP1; and entirely unpaired in the HORMAD2 example). Size bar = 20 μm. (B) Assessment of DNA damage and silencing in pachytene spermatocytes. Meiotic chromosome spreads were immunolabeled for γH2AX and SYCP3. Size bar = 20 μm. The mutant XY body shows normal staining for γH2AX, a marker of both DNA damage and heterochromatin, even in cases where there is XY asynapsis. For each genotype, 50 cells from each of 3 animals were examined. (C) RT-qPCR of X-linked genes from P15 testes relative to WT GAPDH. N = 3 animals for each genotype.