EXTRACTION AND QUANTIFICATION OF ACROSIN, BETA-N-ACETYLGLUCOSAMINIDASE, AND ARYLSULFATASE-A FROM EQUINE EJACULATED SPERMATOZOA

Acrosin, Arysulfatase A, and beta-N-acetylglucosaminidase are three ke y enzymes localized within the mammalian acrosome that play a pivotal role in the penetration of the oocyte. The objectives of this study we re to compare two methods of enzyme extraction based on the activities of these enzymes from equine spermatozoa. Method A utilized a 0.5M Tr is-maleate buffer containing 0.1% Triton X-100 and Hyamine 2389. Metho d B used 0.05M Tris-HCl, 0.05M MgCl2 in 0.05M Tris-maleate, followed b y 0.05M Tris-maleate containing 0.1% Triton X-100. Results indicated t hat acrosin was initially bound in an acrosin-acrosin inhibitor comple x; this complex was dissociated after incubating the extract in 2 mM H Cl. Significant (P < 0.001) increases in acrosin activity were found a fter acid extraction from 0.076 U/mg after Method B to 0.327 U/mg afte r Method A. Arylsulfatase A activity was found to have a higher mean a ctivity (P < 0.03) after Method A (0.012 U/mg) as opposed to Method B (0.007 U/mg). Similarly, beta-N-acetylglucosaminidase was found to hav e a higher mean specific activity (P < 0.001) after Method A (0.037 U/ mg) as compared to Method B (0.008 U/mg). This is the first report of the quantification of these enzymes from equine spermatozoa which can ultimately be used as an index of acrosomal damage in cryopreserved se men, and provide additional insight into biochemical alterations betwe en normal vs. abnormal semen. (C) 1997 Wiley-Liss, Inc.