“…Overall, 2,980 oocytes (1,470 from diestrus phase and 1,510 from anestrus) with the zona pellucida were transferred to culture plates with 0.2% hyaluronidase solution for removal of cumulus cells. Subsequently, they were washed several times with PBS and placed into cell lysis buffer composed of 20 mM TRIS, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X‐100, 2,5 mM Sodium phosphate, 1 mM β‐glycerophosphate, 1 mM Na 3 VO 4 , 1 mg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride (Suzukamo et al, ). The lysis solution was added to the sample at the ratio of 1:1, that is, 1 oocyte/ 1 μl of solution.…”