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 precipitation with ammonium sulfate, anion-exchange chromatography, and reverse-phase HPLC, and was characterized by electrophoretic and membrane blotting techniques. Blotting techniques, using the ECL Glycoprotein Detection System (Amersham Life Science, Buckinghamshire, UK) and wheat germ agglutinin (WGA)-peroxidase, established the presence of sialic 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-reacted with the antiserum, with only the major form reacting with WGA-peroxidase. Extracts of washed epididymal spermatozoa contained a polypeptide with the same electrophoretic mobility as the major form. Additionally, the antiserum detected cross-reacting material in seminal plasma and in extracts from ejaculated spermatozoa. When spermatozoa were incubated under conditions shown to promote capacitation, the cross-reacting material could not be detected in sperm extracts. These results are consistent with the following conclusions: 1) antiagglutinin contains sialic acid residues that may be related to its immunoreactivity and molecular heterogeneity, and 2) either sperm-bound antiagglutinin is released or its epitope recognized by the antiserum is altered after ejaculation and in vitro capacitation.
Harayama
H
H
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA. harayama@icluna.kobe-u.ac.jp
Kato
S
S
Hammerstedt
R H
RH
D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004542:Ejaculation; D015180:Electrophoresis, Gel, Two-Dimensional; D004822:Epididymis / Q000378:metabolism*; D008297:Male; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D009195:Peroxidase / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D012661:Semen / Q000737:chemistry; D013073:Sperm Agglutination / Q000187:drug effects*; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000737:chemistry; D013552:Swine; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D011506:Proteins; D014909:Wheat Germ Agglutinins; D009195:Peroxidase; D019158:N-Acetylneuraminic Acid