Eight- to ten-week-old ICR female mice (Japan SLC, Hamamatsu, Japan) were superovulated by injection of 5 IU of equine chorionic gonadotropin (eCG; ASUKA Pharmaceutical, Tokyo, Japan), followed by 5 IU of human chorionic gonadotropin (hCG; ASUKA Pharmaceutical) 48 h later. Unfertilized eggs were harvested 14 h after hCG injection and placed in a 90-μl droplet of HTF supplemented with 4 mg/ml BSA (A3311; Sigma-Aldrich, St. Louis, MO, USA) [13 (link)]. Spermatozoa were collected from the cauda epididymis of 11- to 15-week-old ICR male mice (Japan SLC) and cultured for 2 h in 100-μl of HTF supplemented with 4 mg/ml BSA. After preincubation, sperm were introduced into fertilization droplets at a final concentration of 1 × 106 cells/ml. After a 3-h incubation, fertilized 1-cell embryos were collected and washed 3 times in KSOM supplemented with amino acids [14 (link)] and 4 mg/ml BSA and then used for
microinjection [15 (link)].