Isolation and characterisation of two sperm membrane proteins recognised by sperm-associated antibodies in infertile men.
Antisperm antibodies eluted from the surface of spermatozoa obtained from infertile men recognised several common epitopes. We tested whether these epitopes were relevant to fertility by isolating the immunodominant 37/36 and 19/18 protein zones. These protein zones were cut out of preparative slab gels and electro-eluted. The isolated proteins, P36 and P18, were used for biochemical characterisation and to produce specific antibodies in rabbits. The specific reactivity of P36 and P18 with WGA and AAL lectins, respectively, indicated the presence of lactosaminyl structures with sialic acid termini in P36 and of fucosylated residues in P18. Isoelectric focusing showed that the two proteins consist of several polypeptides. Some of these polypeptides were recognised by both human and rabbit antibodies: the pl of these epitopes was around 5.5 for P36 and 8.3-10.3 for P18. Rabbit antibodies detected the corresponding proteins on the sperm heads of methanol-fixed and of live acrosome-reacted spermatozoa. Anti-P36 antibodies bound mainly to the equatorial segment. They reduced the binding and, consequently, the penetration of zona-free hamster oocytes by human spermatozoa. Anti-P18 antibodies gave more diffuse staining of the acrosomal and post-acrosomal regions and reduced sperm-oocyte penetration without a significant effect on sperm binding. These results suggest that P36 and P18 antigens located in different compartments of the sperm head may participate in the sperm-oolemma interaction. We are currently investigating the physiological role of these antigens by sequencing the proteins isolated from the gels.
Auer
J
J
Service de Biologie de la Reproduction, CHU Cochin-Port Royal, AP-HP-Université Paris 5, Paris, France.
Senechal
H
H
Desvaux
F X
FX
Albert
M
M
De Almeida
M
M
D000818:Animals; D000954:Antigens, Surface / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western / Q000379:methods; D002462:Cell Membrane / Q000276:immunology; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007248:Infertility, Male / Q000276:immunology*; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins / Q000276:immunology* / Q000302:isolation & purification; D009865:Oocytes / Q000502:physiology; D011506:Proteins / Q000276:immunology* / Q000302:isolation & purification; D011817:Rabbits; D013094:Spermatozoa / Q000276:immunology*
D000954:Antigens, Surface; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011506:Proteins; C093735:acrosomal protein p18