Meiotic chromatin spread assays were performed to determine the meiotic progression in human SSCs by 3D-I system according to the method described previously [24 (link)]. Cells were lysed by a hypotonic solution and spread evenly over slides layered with 1% PFA and 0.15% Triton X-100. Slides were dried for 24 h at room temperature in a humid chamber. Cells were treated with 0.04% photoflo for 5 min and blocked with 4% goat serum. Triple or duplicate immunostaining was performed in these cells using primary antibodies, including SYCP3 (Abcam, 1:100), CREST (Immunovision, 1:100), and MLH1 (Abcam, 1: 50), γH2AX (Abcam, 1:100), at 37 °C for 2 h in a humid chamber. Goat anti-rabbit Alexa Fluor 594 (Invitrogen) and goat anti-human Alexa Fluor 488 secondary antibodies (Invitrogen) were applied at 1:200 dilution and incubated for 90 min at 37 °C. Cells were washed three times with PBS, and images were captured with a fluorescence microscope (Leica).