Fig. 5. Reversible sperm binding to wild-type, huZP2 transgenic and huZP2 rescue eggs and embryos. Wild-type (referred to as `normal') (1,2), huZP2 transgenic (5,6) and huZP2 rescue (9,10) eggs or huZP2 transgenic (13,14) and huZP2 rescue (17,18) two-cell embryos were incubated with normal sperm for 1 hour (1,5,9) or 24 hours (13,17) and stained with Alexa 568-SBTI and Hoechst before imaging by DIC (1,5,9,13,17) and confocal microscopy (2,6,10,14,18). After a brief rinse, 5x10⁵ motile, capacitated Acr3-EGFP sperm were added and incubated for an additional 1 hour. After washing to remove non-adherent sperm, eggs and embryos were stained with Alexa 568-SBTI before imaging by DIC (3,7,11,15,19) and confocal microscopy to observe SBTI and EGFP (4,8,12,16,20), reflecting acrosome-reacted and acrosome-intact sperm, respectively. Images were modified in Adobe Photoshop to remove nuclear staining from EGFP-positive sperm; thus, Hoechst-positive, EGFP-negative sperm (4,8,12,16,20) reflect those that were not displaced by EGFP-sperm. Insets (3,7,11,15,19), Zp3EGFP control two-cell mouse embryos from each incubation after washing.