Estradiol

Bovine ovaries were transported from the slaughterhouse to the laboratory within 2 h at 35 °C. Follicles of 2–8 mm were selected to obtain cumulus–oocyte complexes (COCs), and COCs wrapped in at least three intact layers of cumulus cells (CCs) were cultured separately in the IVM solution supplemented with NMN, BER, or COR. For IVM, at least 50 COCs were put into 500 μL IVM solution covered by oil at 38.5 °C, 5% CO₂, for 22–24 h. The IVM solution was comprised of M199 (Gibco BRL, Grand Island, NY, USA), luteinizing hormone (10 μg/mL), estradiol (1 μg/mL), follicle-stimulating hormone (10 μg/mL), heparin (10 μg/mL), penicillin and streptomycin (0.4 mg/mL) (Gibco BRL, Grand Island, NY, USA), and fetal bovine serum (FBS, 10% v/v). After IVM, COCs were incubated with hyaluronidase (0.1%, w/v) to isolate CCs, and the oocytes exhibiting even distribution of cytoplasm and at the MII stage were selected for subsequent experiments.

Mouse OGCs were isolated and cultured according to the protocols in our previous study.⁵ Ten‐week‐old female C57BL/6 mice (n = 10) were purchased from the Experimental Animal Centre of Shanghai University of Traditional Chinese Medicine. The mice were sacrificed by cervical dislocation. Ovarian tissues were isolated under sterile conditions and incubated in ice‐cold (4°C) phosphate‐buffered saline (PBS). Next, the ovarian tissues were minced and digested with 2.0 mL of hyaluronidase (0.1%, Sigma‐Aldrich) for 1 min at 37°C. The digested sample was gently pipetted and incubated with 200 μL of foetal calf serum (Gibco) to terminate digestion. The suspension was then filtered through a 200‐mesh cell strainer. The filtrate was mixed with 5.0 mL of PBS and centrifuged at 300 g and 10°C for 5 minutes. The supernatant was discarded, and the pellet resuspended in 5.0 mL of PBS and centrifuged at 1500 rpm and 10°C for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in Dulbecco's Modified Eagle's Medium:Ham's F‐12 medium (1:1) supplemented with 10% foetal bovine serum, 10 ng/mL basic fibroblast growth factor, 10 ng/mL epidermal growth factor, 2 mmol/L l‐glutamine, 10 ng/mL growth hormone and 15 ng/mL estradiol (Gibco). The cell suspension was seeded in six‐well cell culture plates and cultured at 37°C in 5% CO₂ until 80% confluency.