Fig. 1. Mouse and human sperm bind to the N terminus of ZP2.

(A) Schematic of the transgene used to establish the Acr^mCherry transgenic mice. SV40 poly(A), SV40 simian virus polyadenylation signal. (B) Epididymal sperm isolated from Acr^mCherry mice were fixed on polylysine-coated slides, stained with Hoechst, and imaged by confocal alone (left) or merged with differential interference contrast (DIC) microscopy (center). Two frames, 1.5-s apart, of a video before (top right) and after (bottom right) induction of acrosome exocytosis with calcium ionophore A23187. Arrows, acrosome; asterisk, nucleus. Scale bars, 10 μm. (C) Binding of sperm to cumulus-free mouse eggs using Acr^EGFP sperm freshly released from the epididymis (green, left) or Acr^mCherry sperm after 1 hour of incubation in HTF/BSA (red, middle) alone or mixed (1:1) with the freshly released sperm (right). DIC (upper) and confocal Z projection (lower) images were obtained after fixation and staining with Hoechst (three biological replicates). Scale bar, 20 μm. (D) Schematic of moZP2^35–149 (blue) and huZP2^39–154 (gray) peptides at the N terminus of ZP2 (tan) that mediate sperm-egg binding in mice and humans, respectively. Inverted triangle, moZP2 postfertilization cleavage site (¹⁶⁷LA^↓DE¹⁷⁰); zona domain, ZP2^365–630; yellow vertical bars in lower flyout, cysteine residues. (E) Model of moZP2^35–149 or huZP2^39–154 peptide beads interacting with sperm freshly released from the epididymis (green) and incubated for 1 hour (red) in HTF/BSA before insemination. (F) Mouse sperm freshly released from the epididymis (left) and thawed human sperm (right) bound to moZP2^35–149 and huZP2^39–154 peptide beads, respectively, in comparable numbers when assayed immediately or after 1 hour (hr) of incubation in HTF/BSA before insemination. Neither human nor mouse sperm bound to beads alone control (three biological replicates) (fig. S1E). Box plots reflect the median (horizontal line) number of mouse or human sperm binding to peptide beads and data points within the 10th and 90th percentiles (error bars). Boxes include the middle two quartiles, and outliers are indicated by dots.