Collection of MII oocytes and in vitro fertilization

ICR mice were purchased from the Vital River Laboratories (Beijing, China), and maintained in a special pathogen-free facility at the Inner Mongolia University. The mice were kept in a constant light and temperature controlled environment (22–24 °C, 12 h light/dark cycle), and had free access to sufficient chow and water. Female ICR mice aged 8 weeks were superovulated by intraperitoneal injection of 10 IU pregnant mare serum gonadotropin (PMSG, Ningbo second hormone factory, Ningbo, China). 46–48 h after PMSG injection, each female mouse was injected with 10 IU human chorionic gonadotropin (HCG, Ningbo second hormone factory, Ningbo, China). Metaphase (M) II-stage oocytes were collected from the ampullae 14 h after HCG injection and treated with 1% hyaluronidase (Sigma, H3506) to remove the surrounding cumulus cells. Sperm cells were obtained from the cauda epididymis of male ICR mice and placed in HTF for capacitation. Gametes were co-incubated in HTF for 2 h for in vitro fertilization. For assessment of embryonic development, the fertilized oocytes, 2-cell, 4-cell, morula and blastocyst stage embryos were elevated morphologically at 10 hpi, 24 hpi, 48 hpi, 72 hpi and 96 hpi under a dissection microscope, and the corresponding developmental ratio was determined relative to the number of fertilized oocytes confirmed by 2^nd polar body extrusion and clear parental pronuclei formation. Embryos were cultured in KSOM (Sigma, 3453308) covered by mineral oil under condition of 37 °C, 5% CO₂, and saturated humidity. All studies referring to experimental animals were performed according to the experimental protocols and standards, and approved by the Institutional Animal Care and Use Committee at the Inner Mongolia University.