Plasmid Transfection of Granulosa Cells

GCs were isolated and cultured using previously described methods [12 (link)]. Briefly, fresh ovaries from adult sows were washed alternately with saline and 75% alcohol, and follicular fluid from healthy follicles was drawn, collected into centrifuge tubes, and centrifuged to collect cells. After washing with phosphate-buffered saline, the cells were resuspended using DMEM/F12 medium (containing 15% fetal bovine serum and 1% penicillin), seeded into cell culture plates, and incubated at 37 °C in a 5% CO₂ incubator. When both GC density and status reached the transfection requirement, plasmids (1000 ng per well) were transfected into GCs by using HighGene (ABclonal, Wuhan, China) according to instructions. After 24 h of transfection, GCs (5 × 10⁵) were collected for luciferase reporter assay. Luciferase activity was measured using a luciferase reporter assay kit (Vazyme, Nanjing, China) according to the instructions.