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Mice were superovulated to produce fully grown GV oocytes by injecting them with 10 IU of pregnant mare serum gonadotropin (PMSG) intraperitoneally. After 48 h, we collected GV oocytes from 6–8-week-old mice and placed them in M2 medium (Cat#M7167, Sigma) supplemented with 2.5 µM milrinone to maintain oocyte arrest at the GV stage. The oocytes were subsequently incubated in M16 medium (Cat:MR-016, Sigma) and surrounded by liquid paraffin oil at 37 °C in an atmosphere of 5% CO2 in compressed air at high humidity.
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