The MSUC initiator proteins BRCA1 and ATR are recruited to the pseudo–sex body domain of Spo11-null pachytene spermatocytes. (a–c) SYCP3 and BRCA1 staining of control spermatocytes showing the appearance of BRCA1 foci on the forming axial elements in leptotene, their loss after synapsis during zygotene, and BRCA1 accumulation on the asynapsed X and Y axes in pachytene. (d) The kinase ATR appears throughout the chromatin of the asynapsed X and Y axes in pachytene of control males, and there is an associated phosphorylation of H2AX (γH2AX), which defines the sex body domain (inset). (e–g) In Spo11-null spermatocytes, which lack meiotic DSBs, BRCA1 foci still appear on the axial elements in leptotene, are retained throughout the asynaptic zygotene stage (magnified in insets), and are lost in conjunction with the nonhomologous synapsis apparent in pachytene (highlighted in insets, which show synapsed and asynapsed axes with BRCA1 staining offset). There is concurrent BRCA1 accumulation on a subset of asynapsed axes (arrow) that mark the location of the pseudo–sex body. (h) Staining with ATR or γH2AX (inset) marks the entire chromatin domain of the pseudo–sex body. (i and j) In Brca1^Δ11/Δ11,Trp53^+/− leptotene and zygotene spermatocytes, no foci are detected, confirming the specificity of the BRCA1 antibody. (k and l) A Spo11-null pachytene spermatocyte stained with SYCP3 and SYCP1. The synapsed regions appear light blue because of colocalization; the pseudo–sex body domain marked with ATR is restricted to the chromatin of asynapsed axes but only encompasses a small proportion of such chromatin. Bars,10 μm.