Hepes

Human embryos were cultured as described previously 30, 31. Thawed embryos were placed in a polystyrene dish containing 0.5 M sucrose solution for 10 minutes, then 0.2 M sucrose solution for a subsequent 10 minutes. Next, embryos were washed with Quinn's Advantage Medium with HEPES (Cooper Surgical, Trumbull, CT, http://www.coopersurgical.com) with the addition of 5% Quinn's Advantage Serum Protein Substitute (Cooper Surgical). Embryos were cultured in either Quinn's Advantage Cleavage or Blastocyst Medium (depending on stage) plus 10% Serum Protein Substitute (Cooper Surgical) under mineral oil (Sigma, St Louis, MO, http://www.sigmaaldrich.com) at 37°C with 6% CO₂, 5% O₂, and 89% N₂ under standard human embryo culture conditions and in agreement with current clinical IVF practice. The zona pellucida was removed by treatment with Acidified Tyrode's Solution (Millipore, Billerica, MA, http://www.millipore.com), and single blastomeres were collected by incubating in Quinn's Advantage Ca2+ and Mg2+‐free medium with HEPES (Cooper Surgical) for 5–20 minutes at 37°C with pipetting to break up into single cells. Blastomeres were tubed and flash frozen at −80°C until qRT‐PCR analysis.

Mice were maintained in accordance with the policies of the Konkuk University Institutional Animal Care and Use Committee (IACUC). This study was approved by the Konkuk University IACUC (approval number KU12081). Four-week-old ICR mice were purchased from Orient-Bio (Gyeonggi-do, Korea) and were housed in a controlled barrier facility at Konkuk University. The mouse room is equipped with an automated light-dark cycle system and mice were fed a standard rodent chow (LabDiet, St. Louis, MO, USA). Mice were superovulated by injecting 7.5 IU PMSG (Sigma-Aldrich, St. Louis, MO, USA) and 7.5 IU hCG at a 48 h intervals. After 13–14 h after hCG injection, mice were sacrificed and oviducts were collected. Each mouse typically ovulated approximately 17–20 cumulus-oocyte complexes (COCs). COCs were collected by oviduct flushing 13–14 h post-hCG injection. Cumulus cells were removed by treating COCs with hyaluronidase (300 µg/ml) for 2 min. MII oocytes were collected, transferred to Quinn’s Advantage medium with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES, Cooper Surgical, Trumbull, CT, USA) containing 20% fetal bovine serum (FBS, Gibco, Life Technologies, Grand Island, NY, USA), and cultured at 37°C.