Isolation and Preparation of Rat and Human Gametes

Rat cumulus–oocytes complex (COC) collection. Female superovulation was induced in prepubescent rats by an intraperitoneal injection of pregnant mare serum gonadotropin (20 U.I. PMSG) on day one and human chorionic gonadotropin (40 U.I. HCG) on day three. Twelve hours later, we collected oviducts containing oocytes surrounded by follicle cells after cervical dislocation euthanasia [51 (link)]. Once the cells from each oviduct were recovered, we left them equilibrate in Ferticult^® medium at 37 °C and CO₂ 5% for 1 h [46 (link)].
Rat sperm cell collection. Male rats were previously anesthetized (Sevoflurane, vol % 8) and then euthanized with a 10 mL injection of Dolethal. After sacrifice, we collected and cut the epididymis to allow the exit of sperm into HTF-BSA culture medium (Human Tubal Fluid, Millipore, St-Quentin-en-Yvelines, France, with 0.4% BSA: Bovine Serum Albumin, Sigma-Aldrich, St. Quentin-Fallavier, France) for 1 h at 37 °C and CO₂ 5% under mineral oil (Sigma-Aldrich^®, France) [30 (link)].
Human sperm collection. We used frozen human sperm from healthy fertile donors. After thawing, we aliquoted the preparation and centrifuged it for 10 minutes at 420× g. The supernatants were discarded, and the pellets were exposed to various exposure conditions [30 (link)].