Chromosome Spread Analysis of Spermatocytes

Chromosome spreads of prophase I spermatocytes were performed as previously described (Peters et al., 1997 (link); Kolas et al., 2005 (link)). In brief, seminiferous tubules were separated, cut into pieces with scissors, and incubated in hypotonic buffer (0.45% NaCl) for 40 min. Cell suspensions were fixed with 1% paraformaldehyde containing 0.15% Triton X-100 and 0.5 M sodium borate and then air-dried on slides. Samples were blocked with 1x ADB (1% normal donkey serum, 0.03% BSA, and 0.05% Triton X-100) for 1 h and then incubated with primary antibodies at 37°C for 12–16 h. After being blocked with 1x ADB for 5–6 h at room temperature, the samples incubated in fluorescently labeled secondary antibodies at 37 °C for 1.5 h. The slides were viewed under a LSM700 confocal microscope (Zeiss, Germany). Primary antibodies were as follows: SYCP1 (1:100, NB300-229; Novus Biologicals), SYCP3 (1:500, ab97672; Abcam), γH2AX (1:500, ab2893; Abcam).