Isolation and culture of rat ovarian granulosa cells (GCs)

For the isolation of the GCs, six immature (age, 21–25 days) female rats were administered a subcutaneous injection of PMSG (150 IU/kg), and their ovaries collected after 48 h. The follicular puncture approach (23) was then used to isolate ovarian GCs, which were then pooled. The cells were filtered, then centrifuged at 200 × g for 5 min at 4°C. Following centrifugation, the supernatant was discarded and the GCs were resuspended in Dulbecco's modified Eagles medium (Thermo Fisher Scientific, Inc.) supplemented with 0.05 mg/ml gentamicin, transferrin, selenium and INS. The isolated GCs were seeded into a 6-well cell culture plate (5×10⁴/ml) and incubated in a 5% CO₂ atmosphere at 37°C in a humidified atmosphere for 24 h.