ELECTROPHORETIC CHARACTERIZATION OF BOAR EPIDIDYMAL ANTIAGGLUTININ

Boar epididymal antiagglutinin, previously shown to inhibit sperm head -to-head agglutination, was purified from cauda epididymal plasma by p recipitation with ammonium sulfate, anion-exchange chromatography, and reverse-phase HPLC, and was characterized by electrophoretic and memb rane blotting techniques. Blotting techniques, using the ECL Glycoprot ein Detection System (Amersham Life Science, Buckinghamshire, UK) and wheat germ agglutinin (WCA)-peroxidase, established the presence of si alic acid residues on purified antiagglutinin. Removal of sialic acid residues from antiagglutinin greatly reduced its immunoreactivity with the specific antiserum. Further purification by two-dimensional PAGE established the presence of one major and two minor forms that cross-r eacted with the antiserum, with only the major form reacting with WGA- peroxidase. Extracts of washed epididymal spermatozoa contained a poly peptide with the same electrophoretic mobility as the major form. Addi tionally, the antiserum detected cross-reacting material in seminal pl asma and in extracts from ejaculated spermatozoa. When spermatozoa wer e incubated under conditions shown to promote capacitation, the cross- reacting material could not be detected in sperm extracts. These resul ts are consistent with the following conclusions: 1) antiagglutinin co ntains sialic acid residues that may be related to its immunoreactivit y and molecular heterogeneity, and 2) either sperm-bound antiagglutini n is released or its epitope recognized by the antiserum is altered af ter ejaculation and in vitro capacitation.