Enzyme immunoassay

The volume of each well in the GM was 200 μL, which was incubated for about 4 h with 5% CO₂ in air and 100% humidity at 37°C. Each follicle was mechanically isolated from the ovaries in the operating medium and placed individually in the ultra-low attachment 96-well plate. After that, follicles were cultured for 8 d in an incubator with 5% CO₂ in air and 100% humidity at 37°C. Day 0 represents the day on which the culture was started. On day 1 of culture, 100 μL of the culture medium was added to each well. On day 8, follicles were further cultured in the mature medium (MM), which contained of GM supplemented with 1.5 IU/mL human chorionic gonadotropin (hCG; ShuSheng) and 10 ng/mL of mouse epidermal growth factor (PeproTech, USA) for another 16 h for ovulation. Next, the cumulus-oocyte complexes (COCs) were expelled from follicles. The COCs were digested in 0.5 mg/mL of hyaluronidase (Sigma) within 2 min. The oocytes were measured for their ability to fertilize. Half of the medium was refreshed, and the medium was collected every day after day 2 of culture and pooled at −20°C. The concentration of estradiol (E₂) was measured using the enzyme immunoassay (Elabscience Biotechnology Co., Ltd, USA). Diameters and morphological structures of cultured follicles were observed using an inverted microscope every day from day 2 of culture.