M2 medium

Eight-week-old female Mfn2^-/- and WT mice were used to collect oocytes, embryos, and follicles. To collect germinal vesicle (GV) stage oocytes, 5 IU PMSG (Sigma, St. Louis, MO) was injected intraperitoneally and ovaries were extracted 44-48 h after the injection. To retrieve the oocytes, ovaries were punctured with a 26-gauge needle. Collected oocytes were placed in M2 medium (Sigma, St. Louis, MO) and 10 μM milrinone (Sigma, St. Louis, MO) to prevent meiotic resumption. To obtain mature oocytes, an additional injection of 5 IU of human chorionic gonadotrophin (hCG; Sigma, St. Louis, MO) to induce oocyte maturation and ovulation was given 48 h after the PMSG injection. Unfertilized oocytes at metaphase of the second meiotic division (MII) were collected from oviducts 14 h after the hCG injection. To collect fertilized embryos, females were mated with WT males immediately after the hCG injection. The following morning, mating was confirmed by the presence of a vaginal plug. Two-cell embryos were collected 44 h after hCG injection from the oviducts in KSOM medium (Millipore, Darmstadt, Germany). Blastocysts were collected 92 h after hCG injection from uterus into M2 medium (Sigma, St. Louis, MO). Secondary follicles were collected by digesting the ovaries with 1.5 mg/mL collagenase type V (Sigma, St. Louis, MO) for 1 h at 37°C in M2 medium (Sigma, St. Louis, MO) [16 (link)].

Fully-grown GV oocytes were obtained from the ovaries of superovulated female mice 48 h after intraperitoneal injection of 7.5 IU of pregnant mare serum gonadotropin (PMSG) as described previously^{37 (link)}. Ovaries were placed in a Petri dish with M2 medium (Sigma-Aldrich) supplemented with IBMX (Sigma-Aldrich) to prevent oocytes from undergoing GVBD. GV oocytes were released by puncturing antral follicles with a fine needle under a dissecting microscope. To collect MII-stage oocytes, 7.5 IU of human chorionic gonadotropin (hCG) was administered 48 h after PMSG injection to induce ovulation. Mice were sacrificed 15–16 h post hCG by cervical dislocation and cumulus-oocyte complexes (COCs) were released from the oviducts. COCs were briefly incubated in M2 medium containing 0.3 mg/mL hyaluronidase (Merck Millipore) to remove cumulus cells. For in vitro maturation, oocytes were washed and cultured in IBMX-free M16 medium (Sigma-Aldrich) for various periods of time at 37 °C in 5% CO₂ atmosphere.

Ovaries were excised and transferred into M2 medium (Merck) containing 0.1 mM 3-isobutyl-1-methylxanthine (IBMX, Merck) to keep immature oocytes. Germinal vesicle (GV) stage oocytes were isolated and subsequently cultured at 37 °C in 5% CO₂ in M16 medium (Merck) with IBMX, covered with mineral oil (Merck or YBUX) for at least 1 hour prior to microinjection. Zygotes and 2-cell embryos were isolated from oviducts 18–21 and 45–47 hours post hCG stimulation in M2 medium (Merck) and subsequently cultured in KSOM + AA (Caisson Laboratories) covered with mineral oil (Merck or YBUX) at 37 °C, 5% CO₂. For removing of the cumulus cells was used 0.05% hyaluronidase from bovine testes (Merck). Microinjection was performed in M2 medium with (oocytes) or without (embryos) IBMX inhibitor using I10 Narishige microinjector on a Leica DM IL inverted microscope. After microinjection were oocytes (M16 medium + IBMX, Merck) and embryos (KSOM + AA, Caisson Laboratories) cultured for cRNA expression for 1 to 3 hours prior to live cell imaging assay (37 °C, 5% CO₂).