Distinct membrane fractions from mouse sperm bind different zona pellucida glycoproteins

Interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized at the molecular level. To identify sperm proteins that recognize ligand ZP3, we used sonicated sperm membrane fractions as competitors in a quantitative binding assay. Sonicated membranes were density fractionated into 4 fractions. Bands 1-3 contained membrane vesicles, and band 4 contained axonemal and midpiece fragments. In competitive binding assays, bands 1, 2, and 3 but not band 4 were able to compete with live, capacitated, intact sperm for soluble I-125-ZP binding. Affinity-purified ZP fractions consisting of a ZP3-enriched fraction (I-125-ZP3) and a fraction enriched for ligands ZP1 and ZP2 and depleted of ZP3 (I-125-ZP1/2) were obtained by antibody affinity purification of ZP3. In competitive binding assays, bands 2 and 3 competed for I-125-ZP3 binding, but band 1 did not interact with enriched I-125-ZP3. None of the membrane fractions competed for I-125-ZP1/2 binding. These results demonstrate that band 2 and band 3 contain sperm components that interact with ZP3 alone and that components in band 1 interact with ZP3 in conjunction with either ZP1 or ZP2. These data indicate that there must be at least 2 unique sperm plasma membrane components that mediate intact sperm interactions with ZP glycoproteins in mouse. Bands 2 and 3 are likely to contain a primary ZP-binding protein because they interacted directly with ZP3, whereas band 1 may contain sperm proteins involved in later interactions with the ZP, perhaps transitional interactions to maintain sperm contact with the ZP during acrosomal exocytosis.