Human sperm bind and penetrate humanized zonae pellucidae. (A) Ovulated eggs from huZP1–4 rescue mice expressing human ZP1, ZP2, ZP3, and ZP4 but not mouse ZP1, ZP2, or ZP3 after staining with monoclonal antibodies to individual zona proteins. Antibody binding was detected by confocal microscopy as described in Fig. 1. (B) Human sperm binding to huZP1–4 rescue eggs and two-cell embryos. Confocal z projections (top) and DIC (bottom) images after staining with Hoechst. (C) Acrosome status of human sperm within the perivitelline (PV) space of huZP1–4 rescue eggs was determined by confocal microscopy after staining with Hoechst and using SPESP1 (red) and CD46 (green) antibodies that detect acrosome-intact and -reacted sperm, respectively (Fig. S3). The localization of sperm within the perivitelline space was confirmed by x-y, x-z, and y-z orthogonal views, where the x, y, and z axes are colored green, red, and blue, respectively.