Fig. 2. The N terminus of moZP2 decoys sperm in vitro and prevents mouse fertilization. The N terminus of moZP2 decoys sperm in vitro and prevents mouse.

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Fig. 2. The N terminus of moZP2 decoys sperm in vitro and prevents mouse fertilization. The N terminus of moZP2 decoys sperm in vitro and prevents mouse fertilization. (A) Schematic of in vitro fertilization, in which mouse eggs in cumulus mass were incubated overnight with 1 × 105 progressive motile mouse sperm in HTF/BSA (500 μl) alone, or in the presence of beads alone or moZP235–154 peptide beads (100 μl). (B) In vitro fertilization (%) after co-incubation of eggs in cumulus mass with medium (no beads) (1), beads alone (2), or ZP2 peptide beads (3) (three biological replicates). Statistical difference from control is indicated by a letter different than a; (b) Ρ = 0.027; (c) Ρ < 0.001. (C) Sperm from AcrmCherry (red acrosome) mice were incubated with moZP235–149 peptide before (left) or after (right) acrosome exocytosis, cross-linked with formaldehyde, immunostained with a monoclonal antibody (mAb) to the N-terminal ZP2 peptide (green), DNA-stained with Hoechst (blue), and imaged by confocal microscopy and DIC. Asterisk, acrosome; Eq, equatorial segment; arrows, postacrosomal region. Scale bar, 5 μm. (D) Progressive motility of unbound mouse sperm over 8 hours. Sperm were isolated either immediately after release from the epididymis or after 1 hour of incubation in HTF/BSA before incubation with ZP2 peptide beads. Control sperm were incubated with beads alone (three biological replicates). Statistical difference from control is indicated by a letter different than a (details in table S3). (E) Sperm were prepared as in (D), and the percentage of acrosome-intact sperm that remained bound to ZP2 peptide beads was determined over 8 hours (three biological replicates). (F) Electron microscopy of acrosome-intact (left panel) and acrosome-reacted (right panel) sperm that remain bound to the ZP2 peptide beads 8 hours after insemination. IAM, inner acrosomal membrane. OAM, outer acrosomal membrane; PAR, postacrosomal region; P, perforatorium. Scale bar, 0.5 μm. Matteo A. Avella et al., Sci Transl Med 2016;8:336ra60 Copyright © 2016, American Association for the Advancement of Science