MONOCLONAL-ANTIBODIES TO CANINE INTRAACROSOMAL SPERM PROTEINS RECOGNIZING ACROSOMAL STATUS DURING CAPACITATION AND ACROSOME REACTION

Monoclonal antibodies Ds-1 and Ds-2 specifically labelling dog sperm a crosome were prepared by immunization of mice with acetic acid extract s of dog spermatozoa. Electron microscopy and indirect immunofluoresce nce 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 nonreducing conditions, and 73 kDa and 54 kDa proteins after red uction (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 o f 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 a nd p40/Ds-2 form disulfide-bonded complexes. Decrease in the rate of a crosomes stained with Ds-1 and Ds-2 was correlated with the progress o f capacitation resulting in the increased rate of spontaneous acrosome reactions, as suggested by a dramatic effect of A23187. Monoclonal an tibody to boar acrosin (ACR-2) recognized dog sperm acrosin homologue. A higher rate of ACR-2-negative spermatozoa was observed after capaci tation 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 comp artment of acrosome, distinct from acrosin and possibly representing f raction of acrosomal matrix.