Primary Spermatogonial Cell Isolation

Primary SCs were isolated according to previously described procedures [18 (link)–21 (link)]. In brief, trypsin (HyClone, Rockford, IL, USA) and collagenase IV (Sigma-Aldrich, St. Louis, MO, USA) were used to digest the scissored testes tissue blocks for 30–60 min at 37°C. After stopping the digestion by addition of 10% FBS (Gibco, USA) containing Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12, 1 : 1) medium (pH 7.2) (HyClone, Thermo Fisher Scientific, Rockford, IL, USA), the mixture was sieved through sterilized 100 meshes. Newly separated cells were cultured for 48 hours in 10 cm diameter dishes (Corning, New York, USA) with DMEM/F12, 10% FBS, and 1x penicillin-streptomycin (Beyotime Institute of Biotechnology, Haimen, China) in 5% CO₂ at 35°C. Then, cells were treated with 20 mM Tris-HCl (pH 7.4) for 2.5 min.