Fig. 3. De novo Acr3-EGFP sperm binding to two-cell embryos. (A) Two-cell embryos derived from wild-type (referred to as `normal') (1-3), huZP2 transgenic (4-6) and huZP2 rescue (7-9) mice were incubated with 5x10⁵ motile, capacitated Acr3-EGFP sperm for 1 hour and washed; Zp3-EGFP two-cell mouse embryos were used as controls (insets 1,4,7). Embryos were stained with Alexa 568-SBTI and fixed before imaging by DIC (1,4,7) and confocal microscopy to observe EGFP (2,5,8) or SBTI binding (3,6,9), reflecting acrosome-intact and acrosome-reacted sperm, respectively. (B) Aliquots of embryos from A were incubated for an additional 23 hours, and similar numbers of sperm bound to huZP2 transgenic (1-3, 49±8 s.e.m. per embryo) and huZP2 rescue (4-6, 73±15 s.e.m. per embryo) embryos. However, sperm binding to huZP2-transgenic-derived embryos were acrosome-reacted (97%), and those binding to huZP2-rescue-derived embryos remained acrosome-intact (94%). Scale bars: 30 µm.