Hepes buffered medium 199

Oviducts were collected from a local slaughterhouse and transported to the laboratory on ice. After removal of surrounding tissue, the oviducts were washed three times in PBS supplemented with penicillin (10 µg/mL) and streptomycin (10 µg/mL) (Gibco). Oviduct luminal epithelial cells were collected from the ampullary end of the oviducts by squeezing. The cells were washed twice in HEPES-buffered Medium 199 (Gibco) supplemented with penicillin (100 µg/mL), streptomycin (100 µg/mL), and gentamicin (50 µg/mL, Sigma, G1272) and centrifuged at 500× g for 5 min at 25 °C. The cells were then cultured for 24 h at 37 °C with 5% CO₂ in HEPES-buffered Medium 199 supplemented with penicillin (100 µg/mL), streptomycin (100 µg/mL), gentamicin (50 µg/mL), and 10% fetal calf serum (FCS; Bovogen Biologicals, Melbourne, Australia).
HEK293 (human embryonic kidney) cells were grown in DMEM/F12 supplemented with penicillin (100 µg/mL), streptomycin (100 µg/mL), gentamicin (50 µg/mL), and 10% FCS at 37 °C with 5% CO₂.

The cattle, from which we obtained their ovaries, had been slaughtered for the public edible meat. Those ovaries were discarded without any utilization. Hence, an ethics statement in our paper was not required to use ovary for experiments as previously described [22 (link)–24 ]. Bovine ovaries were obtained from an abattoir, placed in saline containing a 0.1% solution of antibiotic/antimycotic (Gibco Laboratories, Grand Island, NY, USA: AB), and transported to the laboratory within 1–3 h. Oocyte collection and in vitro maturation of oocytes were carried out as previously described [25 ]. More than 30 ovaries from Japanese black and holstein cows were collected to permit appropriate an experimental replication. Oocytes with intact cumulus cells and evenly granulated cytoplasm were selected, washed and cultured in modified TCM-199 (m-TCM199) for 22–25 h at 39.0°C under 5% CO₂ in air with high humidity. m-TCM199 consisted of hepes-buffered medium 199 (Gibco) supplemented with 0.1% (w/v) polyvinyl alcohol (PVA;Sigma Chemical Co, St. Louis,MO.), 0.5 mM sodium pyruvate (Nacalai Tesque Inc., Kyoto, Japan), 1% AB, 0.02 AU/ml FSH (Antrin, Denka), and 1 μg/ml estradiol-17β (Sigma). The rates of in vitro maturation after 22–24 hrs were more than 95% in all experiments.