Hyaluronidase treatment

FVB/N mice were kept under 12 h light/dark cycle and had free access to food and water. 4–7 weeks old females were injected with 5 IU i.p. Pregnant Mare's Serum (Folligon, Intervet) followed 44 h later by 5 IU i.p. injection of human chorionic gonadotropin (hCG) (Chorulon, Intervet). Females were mated with male FVB/N strain studs. The females were sacrificed 19–21 h later by cervical dislocation and the oviducts were collected in M-2 medium (Millipore). Cumulus cells were removed by 0.3 mg/ml hyaluronidase treatment (Sigma-Aldrich). Oocytes or 1-cell embryos were collected 21–23 h after hCG. Embryos were cultured in KSOM medium (Millipore) under ovoil-100 (Vitrolife) until 2-cell (45–47 h after hCG) and 8-cell (71–73 h after hCG) stages.

Ovaries removed from the slaughtered animals were kept in PBS and brought to the laboratory immediately. One ovary from each animal was used for the collection of oocytes and cumulus cells. The cumulus-oocyte complexes (COCs) were aspirated by puncturing the follicles (diameter ≥2 mm) and collected under the stereomicroscope SMZ-10 (Nikon, Japan). Firmly compacted COCs with multilayered cumulus cells and a uniformly granulated cytoplasm were separated into oocytes and cumulus cells by repeated aspiration using a narrow-bore Pasteur pipette. Final denudation of the oocytes was performed by hyaluronidase treatment (Sigma-Aldrich; at a working concentration of 300 μg/mL), followed by washing thrice in PBS. The collected samples were stored at -80°C until RNA extraction.