The pseudo–sex body domain of Spo11-null pachytene spermatocytes is transcriptionally silenced. (a and b) Cot1 RNA FISH staining detects nascent transcripts within the nucleus; the pseudo–sex body marked by γH2AX is a transcriptionally repressed domain. (c and d) In Spo11-null pachytene spermatocytes, as a result of asynapsis/nonhomologous synapsis, the two autosomal Atr loci are nearly always well separated. In the cell shown, there is a single Atr RNA FISH signal (arrows); DNA FISH shows that the second non-transcribing Atr locus lies within the pseudo–sex body domain (white outline). (e and f) A pachytene spermatocyte showing transcription of the Y chromosomal gene Zfy2 when it is not located in the pseudo–sex body. (g and h) A nontranscribing Zfy2 locus lying within the pseudo–sex body. (i–n) RNA/DNA FISH for the Ddx3x/Usp9x X chromosomal BAC showing two transcribing pachytene spermatocytes with the locus outside the pseudo–sex body and one nontranscribing pachytene spermatocyte in which the locus lies within the pseudo–sex body. (a–n) White outlines indicate the extent of the γH2AX domain before DNA FISH, and arrows point to the FISH signals (either RNA FISH or DNA FISH). Bar, 5 μm.