Detection of acrosome‐reacted toad sperm based on specific lectin binding to the inner acrosomal membrane

AbstractWhen the sperm of the toad Bufo japonicus were treated with fluorescein isothiocyanate (FITC)‐conjugated peanut agglutinin (PNA), soybean agglutinin (SBA), or Dolichos biflorus agglutinin (DBA), a few sperm fluoresced at the acrosomal region. The number of sperm showing this lectin binding to the acrosome increased significantly upon mild sonication of the sperm suspension. Electron microscopy revealed that ferritin‐conjugated PNA bind not to the outer acrosomal and overlying plasma membranes, but specifically to the surface of the inner acrosomal membrane exposed by sonication. Both the percentage of FITC‐PNA‐labeled sperm and the activity of vitelline coat lysin released by sperm increased in good correlation with increasing sonication time, although the PNA‐labeled sperm decreased in number upon longer sonication. These results indicate that the binding of FITC‐PNA to the sperm provides a reliable measure of the acrosome reaction of Bufo sperm.