Brain derived neurotrophic factor (bdnf)

This research received approval from the Ethics Committee of Jilin University and was conducted in compliance with animal welfare guidelines (SY202311100). Ovaries from prepubertal Large White gilts were obtained from a local slaughterhouse and transported to our laboratory in a 0.9% saline solution at 37 °C. The ovaries underwent sequential washings with a dilute bromogeramine solution, 70% alcohol for disinfection, and a 0.9% saline solution. Follicular fluid from 3–5 mm diameter follicles was collected using the aspiration method, then centrifuged at 600× g for 5 min to isolate GCs. These GCs were cultured in DMEM (High Glucose) medium (Bio-Channel, Nanjing, China) supplemented with 10% (v/v) foetal bovine serum (FBS) (Gibco, NY, USA) and maintained at 37 °C in a humidified atmosphere containing 5% CO₂. When GC confluency reached 50–70%, the cells were treated with serum-free DMEM (High Glucose) containing brain-derived neurotrophic factor (BDNF) (Novoprotein, Suzhou, China) for 24 h. The cells were also exposed to 10 µM PD98059, a mitogen-activated protein kinase (MAPK-ERK1/2) activity inhibitor (Beyotime Biotechnology, Shanghai, China). After 30 min, this medium was replaced with fresh DMEM (High Glucose) containing 10% serum for an additional 24 h. Subsequently, the GCs were harvested for RT-qPCR and Western blot analyses.