RNF8 and H2AX function epistatically in class switching and spermatogenesis. (A) Frequency of IgG1 expression in B cells from WT, H2AX^−/−, RNF8^−/−, and H2AX^−/−RNF8^−/− mice stimulated with LPS plus IL-4 for 4 d. One representative out of three independent experiments is shown (Fig. S3). (B) Comparison of testes size in RNF8^+/+ and RNF8^−/− mice. Bar, 3 mm. (C, top) Sections of testes stained with hematoxylin-eosin. (arrowhead 1) In RNF8^+/+ testes, mature sperm is organized in a rosette-like manner and is abundant. In RNF8^−/− testes, the amount of sperm is much reduced. RNF8 mutant testes show evidence of degeneration such as the accumulation of Leydig cells in intertubular spaces (arrowhead 2) and areas of vacuolization (arrowhead 3). Bar, 50 µm. (bottom left) Immunofluorescence of isolated WT mouse spermatocytes with antibodies against SCP3 (red) and FK2 (green). The image illustrates that the staining of FK2 is restricted to the XY body. Bar, 5 mm. (bottom right) Immunofluorescence of testes from RNF8^+/+ and RNF8^−/− animals stained with antibodies against γH2AX (green) and FK2 (red). Note that although a normal γH2AX signal was detected in both tissues, the sex body–associated FK2 signal (arrows) was not detectable in RNF8^−/− testes. Bars, 50 mm. Two mice of each genotype were analyzed in B and C.