To further confirm this observation, the immunolocalization of FLOT2 and IZUMO was studied after inducing the acrosome reaction with the calcium ionophore A23187. As shown in Figure 6 , sperm that have lost PNA staining showed a new IZUMO localization. Although both FLOT2 and IZUMO displayed a different staining pattern in acrosome-reacted cells versus intact sperm, they did not colocalize (data not shown). While FLOT2 was restricted to the equatorial segment, IZUMO distributed into adjacent regions.