Purification and characterization of the primary acrosomal autoantigen of guinea pig epididymal spermatozoa.
Previous studies showed that sperm auto- and alloantigens participate in guinea pig (GP) fertilization. In an effort to determine how alloantibodies to GP sperm acrosomal contents (AC) inhibit fertilization, we identified acrosomal auto- and alloantigens using Western blots. The predominant autoantigens migrated with Mr = 25,000, Mr = 51,000, and Mr = 55,000 under nonreducing conditions. The primary (Mr = 25,000) acrosomal autoantigen, AA1, was purified to homogeneity from AC by gel filtration, cation-exchange chromatography, chromatofocusing, and a final gel filtration. We also purified AA1 from an acidic glycerol extract of spermatozoa by gel filtration, chromatofocusing, and high-performance liquid chromatography on hydroxylapatite. AA1 is a protein and shares at least one antigenic determinant with a 51,000 Mr acrosomal component. AA1 is acrosome-specific, as determined by immunoabsorption and by indirect immunofluorescence on testicular cells. By quantitative enzyme-linked immunosorbent assay, AA1 comprises 6.4% of acrosomal protein in GP spermatozoa. On the basis of its physiochemical properties and localization, we conclude that AA1 is a unique sperm autoantigen. Surprisingly, several antibody preparations, including allo- and heteroantibodies with high anti-AA1 titers, did not inhibit fertilization in vitro. Thus, the mechanism by which alloantibodies to AC inhibit GP fertilization in vitro is not by binding to AA1.
Hardy
D M
DM
Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque 87131.
Huang
T T
TT
Driscoll
W J
WJ
Tung
K K
KK
Wild
G C
GC
D000177:Acrosome / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D001323:Autoantibodies / Q000032:analysis; D001324:Autoantigens / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006168:Guinea Pigs; D006651:Histocytochemistry; D007118:Immunoassay; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013094:Spermatozoa / Q000276:immunology*
D001323:Autoantibodies; D001324:Autoantigens