Extraction and quantification of acrosin, beta-N-acetylglucosaminidase, and arylsulfatase-A from equine ejaculated...

Acrosin, Arysulfatase A, and beta-N-acetylglucosaminidase are three key enzymes localized within the mammalian acrosome that play a pivotal role in the penetration of the oocyte. The objectives of this study were to compare two methods of enzyme extraction based on the activities of these enzymes from equine spermatozoa. Method A utilized a 0.5 M Tris-maleate buffer containing 0.1% Triton X-100 and Hyamine 2389. Method B used 0.05 M Tris-HCl, 0.05 M MgCl2 in 0.05 M Tris-maleate, followed by 0.05 M Tris-maleate containing 0.1% Triton X-100. Results indicated that acrosin was initially bound in an acrosin-acrosin inhibitor complex; this complex was dissociated after incubating the extract in 2 mM HCl. Significant (P < 0.001) increases in acrosin activity were found after-acid extraction from 0.076 U/mg after Method B to 0.327 U/mg after Method A. Arylsulfatase A activity was found to have a higher mean activity (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 have 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 semen, and provide additional insight into biochemical alterations between normal vs. abnormal semen.