A zona pellucida is formed with ZP4 in the absence of ZP2, and female mice are sterile. (A) Phylogeny of mouse and human zona proteins indicate two clades; one composed of ZP1, ZP2, and ZP4 and the other of ZP3. There is no mouse ZP4 protein because of multiple stop and missense codons in the cognate gene. Mya, million years ago. (B) Schematic representation of the four zona pellucida proteins with 8 or 10 conserved cysteine residues. The resultant disulfide bonds differ in the zona domains of the ZP1/2/4 and ZP3 clades and are indicated as A and B, respectively. The postfertilization cleavage site is marked on ZP2, and both ZP1 and ZP4 contain trefoil domains. (C) Glutaraldehyde-fixed, plastic-embedded ovarian sections (3 µm) from 8–10-wk-old normal, Zp2^Null, moQuad^(huZP4), and moQuad-Zp2^Null mice were stained with periodic acid Schiff’s reagent to highlight the zona pellucida (arrows) and counterstained with hematoxylin. (D) Formaldehyde-fixed moQuad-Zp2^Null eggs stained with protein-specific monoclonal antibodies. Fluorescent and DIC images were merged and faux colored. (E) Mouse sperm binding to moQuad^(huZP4) and moQuad-Zp2^Null eggs. Inset, 2.5× magnification. Zp3^EGFP mouse eggs (green zona) and mouse two-cell embryos were positive and negative sperm-binding controls, respectively. Schematics to the left reflect protein composition of the zonae pellucidae with the source of sperm below.