Figure 3. Abnormal localization of sex body silencing factors in pachytene spermatocytes from rapamycin-treated mice. Immunostaining of spread nuclei for SYCP3 (red) and silencing factors (green). (a) Control pachynema with restriction of γH2AX signal to the sex chromatin. (b) Pachynema from rapamycin-treated mice with γH2AX signal in autosomal regions (spermatocytes analyzed: Control, n=210; Rapamycin, n=250. χ2 test). White arrowheads indicate the sex chromosomes. (c-e) Localization of ATR to sex body and chromosomal axes in control spermatocytes (c) contrasting with weak ATR staining to the sex body in spermatocytes from rapamycin-treated mice (d-e). White arrowheads indicate the sex chromosomes. (f, g) Percentage of pachytene spermatocytes with abnormal localization of γH2AX or ATR. (h, i) Immunostaining for H3K9me3 in early pachytene stage (h) and diplotene stage (i) spermatocytes from control and rapamycin-treated mice. White arrows mark the sex chromosomes. (j, k) Quantification of spermatocytes with defects in H3K9me3 localization at the early pachytene stage (j) and diplotene stage (k). Scale bar, 10μm.