Partial purification and characterization of a trypsin-like serine protease from bovine sperm.

ANDREINA CESARI; CACCIATO, C.; SÁNCHEZ, J. J.

International Journal of Andrology

Año: 2004 vol. 27 p. 311 - 318

0105-6263

Sperm proteolytic activities are important in the chemical mechanism of fertilization. Acrosin is the best characterized protease described in mammalian spermatozoa and its not believed to be the unique one. There are several evidences that suggest the presence of other serin proteases in mice and human spermatozoa. In this work we describe the purification and characterization of two proteins with proteolytic activity: BSp120 and BSp66.  Purification of BSp120 from frozen semen was developed by DEAE-chromatography followed by affinity chromatography Benzamidine Sepharose 6B. BSp66 was purified from fresh semen by a distinct protocol using a gel filtration chromatography followed by affinity chromatography Bacitracine Sepharose 4B. Zymograms and gel filtration chromatography of both proteins showed that their molecular masses were respectively 120 kDa and 66 kDa. They both digest synthetic peptides with cleavage sites specific to trypsin. Polyclonal antibodies against acrosin or proacrosin from different species, did not cross-react with BSp120 and BSp66. Antibodies raised in our laboratory against these proteins did not recognize acrosin or proacrosin, suggesting that were not related proteins. The cellular localization of these proteins by optical immunocytochemistry using specific antibodies revealed a thin signal in the apical portion of the sperm head suggesting acrosomal or membrane localization. The evidences presented in this work suggest the finding of two serine proteases with a putative rol in fertilization.