Isolation, culture, and identification of rat ovarian granulosa cells

The in vitro experiments were conducted according to the protocol described previously. 17 , 18 The successfully modeled rats were injected intraperitoneally (50 IU) with pregnant mare serum gonadotropin (PMSG, hor‐272, Prospec). The rats were fed routinely for 48 h and then killed. Under sterile conditions, the bilateral ovaries were removed with sterilized instruments, and the adipose tissue wrapped around the ovaries was removed. The ovaries were cleaned three times with sterile saline solution. Then, the ovaries were transferred to 3 mL serum‐free DMEM/F‐12 medium (HyClone), and the follicles were punctured with a 1‐mL syringe needle to release the granulosa cells. The granulosa cells were cultured in sterile bottles (2 × 10⁶ cells/bottle) using DMEM/F12 medium supplemented with 10% fetal bovine serum protein (FBS) at 37°C with 5% CO₂. After 24 h of preculture, when the cells reached approximately 70% confluence, the medium was replaced with fresh media every 2 days. When the cells reached approximately 80% confluence, they were digested with 0.25% trypsin. The cells were fixed using 4% paraformaldehyde for 10 min; then, they were stained using H&E for the identification of the rat ovarian granulosa cells.