Fig. 1. Persistent sperm binding to huZP2 rescue mouse embryos after fertilization. (A) Embryos were collected 1, 2, 4 and 24 hours after insemination and stained with Hoechst 33342 and Alexa 568-conjugated-soybean trypsin inhibitor (SBTI) before viewing by differential interference contrast (DIC) and confocal microscopy: (1-8) images of in vitro fertilization with Acr3-EGFP sperm (5x10⁵) and wild-type (referred to as `normal') eggs; (9-16) same as 1-8, but after in vitro fertilization of huZP2 transgenic eggs; (17-24) same as 1-8, but after in vitro fertilization of huZP2 rescue eggs. Only the green and red channels of each image are displayed. Intact acrosomes are indicated by green (EGFP) on the anterior surface of sperm heads; reacted acrosomes are indicated by red because of Alexa 568-SBTI binding to the inner acrosomal membrane. Fertilization rates (>85%) were comparable among genotypes as judged by decondensing Hoechst-positive sperm nuclei in the egg cytoplasm (data not shown). (B) Bar graph (mean and s.e.m.) of sperm binding to normal (A, 1-8) huZP2 transgenic (A, 9-16) and huZP2 rescue (A, 17-24) embryos. Dotted lines represent linear regression of averages of number of sperm binding to normal (green), huZP2 transgenic (blue) or huZP2 rescue (red) embryos. (C) Postfertilization cleavage of ZP2 detected by immunoblot using monoclonal antibody to mouse ZP2, and normal eggs or embryos isolated 0, 1, 2, 4 and 24 hours after in vitro fertilization with normal sperm. Scale bar: 20 µm.