Figure 2. Meiotic defects in rapamycin-treated spermatocytes. Immunostaining of spread nuclei of spermatocytes for the synaptonemal complex proteins SYCP3 (red) and SYCP1 (green) or recombination proteins (green). (a) Identification of leptotene, zygotene and pachytene spermatocytes by SYCP1 and SYCP3 immunostaining. (b) Proportion of meiotic spermatocyte populations in control and rapamycin-treated mice. Leptotene (L), zygotene (Z), pachytene (P) and diplotene (D). (c) Percentage of e-P (early pachytene), m-P (middle pachytene) and l-P (late pachytene) spermatocytes. (d, e) Significantly increased frequency of abnormal localization of SYCP1 to the sex chromosomes in pachytene stage spermatocytes. White arrows and white arrowheads mark the sex chromosomes in control and rapamycin-treated spermatocytes, respectively. (f, g) Immunostaining for RAD51 (green) and SYCP3 (red) in leptotene, early zygotene (Ea-Zygotene), late zygotene (La-Zygotene) and pachytene stage spermatocytes identifies similar numbers of RAD51 foci per cell in spermatocytes from control and rapamycin-treated mice. (h) Similar numbers of MLH1 foci per cell in control and rapamycin-treated spermatocytes based on MLH1 (green) and SYCP3 (red) immunostaining. Error bars represent SD (*P < 0.05, Student’s t test).