2.3. Isolation of Sertoli cells and Leydig cells

Sertoli cells and Leydig cells were isolated from the testes of 21‐day‐old mice. Briefly, the testes denuded of tunica albuginea were incubated with 1 mg/mL collagenase type IV in DMEM (Gibco) for 15 minutes at 37°C on a shaker and then filtered through 100 μm cell strainer (Falcon) to isolate Leydig cells. The filtered seminiferous tubules were further digested with 0.25% trypsin‐EDTA (Gibco) for 15 minutes at 37°C on a shaker and then filtered through 40 μm cell strainer (Falcon). Cells in the filtrate were collected by centrifugation (250 g, 5 minutes) and resuspended in DMEM medium (Gibco) with 10% FBS (Life Technologies). Subsequently, the cell suspensions containing primary Sertoli cells and spermatogonium were cultured in cell culture flasks at 37°C with 5% CO₂. After 24 hours, the culture supernatant was collected to obtain spermatogonium and the adherent cells were treated with a hypotonic solution (20 mmol/L Tris, pH 7.4) for 2 minutes to obtain pure Sertoli cells. Sertoli cells and Leydig cells were cultured in DMEM supplemented with 10% FBS at 37°C with 5% CO₂.