Monoclonal antibodies to canine intra-acrosomal sperm proteins recognizing acrosomal status during capacitation and acrosome reaction.
Monoclonal antibodies Ds-1 and Ds-2 specifically labelling dog sperm acrosome were prepared by immunization of mice with acetic acid extracts of dog spermatozoa. Electron microscopy and indirect immunofluorescence localized the site of Ds-1 and Ds-2 proteins inside the acrosomal vesicle. Ds-1 antibody detected 55, 76, 115, 120 and 190 kDa proteins under non-reducing conditions, and 73 kDa and 54 kDa proteins after reduction (p73/Ds-1 and p54/Ds-1). 92 kDa and 40 kDa proteins recognized by Ds-2 (p92/Ds-2 and p40/Ds-2) migrated at > 200 kDa in the absence of reducing agent. In vivo, p73/Ds-1 and p54/Ds-1 are therefore likely to be present both in free and complexed form, while all of p92/Ds-2 and p40/Ds-2 form disulfide-bonded complexes. Decrease in the rate of acrosomes stained with Ds-1 and Ds-2 was correlated with the progress of capacitation resulting in the increased rate of spontaneous acrosome reactions, as suggested by a dramatic effect of A23187. Monoclonal antibody to boar acrosin (ACR-2) recognized dog sperm acrosin homologue. A higher rate of ACR-2-negative spermatozoa was observed after capacitation and A23187 treatment compared to Ds-1 and Ds-2, indicating that proteins recognized by Ds-1 and Ds-2 are localized in a specific compartment of acrosome, distinct from acrosin and possibly representing fraction of acrosomal matrix.
Geussová
G
G
Laboratory of Biology and Biochemistry of Fertilization, Czech Academy of Sciences, Prague, Czech Republic.
Pĕknicová
J
J
Capková
J
J
Kaláb
P
P
Moos
J
J
Philimonenko
V V
VV
Hozák
P
P
D000176:Acrosin / Q000032:analysis; D000177:Acrosome / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D000911:Antibodies, Monoclonal; D000001:Calcimycin / Q000494:pharmacology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016253:Microscopy, Immunoelectron; D011506:Proteins / Q000032:analysis* / Q000276:immunology; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000648:ultrastructure*
D000911:Antibodies, Monoclonal; D011506:Proteins; D000001:Calcimycin; D000176:Acrosin