Ksom aa medium

GV oocytes were isolated by a technique described previously (Radonova et al., 2020 (link)). For MII oocytes isolation, mice were stimulated with pregnant mare serum gonadotropin (PMSG, 5 IU, Merck) followed 44–48 h by next stimulation with human chorionic gonadotropin (hCG, 5IU, Merck). MII oocytes were isolated 16–17 h later by manual rupturing of oviduct in the M2 medium (Merck) with 0.05% hyaluronidase (Merck). MII oocytes were then cultivated in the KSOM + AA medium (Caisson Laboratories) under the mineral oil (NidOil) in standard conditions (37°C, 5% CO₂).

MII oocytes were parthenogenetically activated by 4.5 min cultivation in 7% ethanol in M2 medium. After activation cells were transferred into KSOM AA medium (Caisson, USA), covered with mineral oil and cultured at 37°C, 5% CO₂ for 6–7 hours, after which cells were scored for pronuclei and microinjected. Embryos were transferred to microscope for live imaging at the 2-cell stage, approx. 47 hours after activation.

ICR/BDF1 mice were stimulated with pregnant mares serum gonadotropin (PMSG, 5 IU, Sigma Aldrich, Czech Republic) and human chorionic gonadotropin (hCG, 5 IU, Sigma Aldrich, Czech Republic) at a 44–48 hour interval. To collect embryos, mice were mated at the time of hCG administration. MII oocytes were collected 14–16 h after hCG administration from non-mated mice, 2-cell embryos were collected 40–44 h after hCG administration. MII oocytes and embryos were collect by manual rupturing of the ampulla. The cumulus cells were removed from MII oocytes by pipetting in M2 medium supplemented with hyaluronidase (150 IU/ml, Sigma Aldrich, Czech Republic). Cells were subsequently cultured in KSOM AA medium (Caisson, USA), covered with mineral oil at 37°C, 5% CO₂.