**2.2 Removal of the zona pellucida**

The cumulus oophorus of the matured COCs was removed mechanically by vortexing (8 min). Subsequently, the cumulus-denuded oocytes were incubated in 0.1% protease (P5147, Sigma-Aldrich, Bornem, Belgium) in phosphate-buffered saline (PBS) for 5 to 15 min at 37°C to dissolve their zona pellucida (ZP). Afterwards the oocytes were washed and transferred to the incubator to allow recovery of the oolemma for at least 30 min (Tanghe *et al.* 2004*a*).
