In Dnmt3l-null pachytene spermatocytes, increasing levels of asynapsis attenuate the MSUC response. (a and b) A rare pachytene spermatocyte with asynapsis restricted to what are probably the X and Y axes. ATR has been recruited to the asynapsed axes and has spread to the associated chromatin, and there is phosphorylation of H2AX throughout the associated chromatin. (c–f) With increasing asynapsis, the ATR becomes more focal and axially restricted, and the γH2AX staining becomes weaker and progressively more fragmented. Arrows indicate axes with partner exchange indicative of nonhomologous synapsis. (g and h) Staining of a spermatogenic cell squash preparation for the midpachytene marker H1t and for γH2AX shows that the majority of H1t-positive cells have fragmented γH2AX staining; only rare cells have a single sex body–like γH2AX-positive domain (bottom insets). In the control (top insets), cells with fragmented γH2AX staining (presumed to be zygotene) are H1t negative, whereas all H1t-positive spermatocytes have a single γH2AX-positive sex body (the small H1t-positive cells are round spermatids). (i) Quantitation of the whole nuclear γH2AX signal for rare pachytene spermatocytes with only the X and Y axes asynapsed and for pachytene spermatocytes with increasing levels of asynapsis shows that the amount of γH2AX does not increase in response to asynapsis (fitted blue regression line). The projected increase in γH2AX signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. Bars: (a–f) 10 μm; (g and h) 15 μm.