223 ENZYMATIC ACTIVITY LEVEL OF DIFFERENT GLYCOSIDASES IN INTACT AND ACROSOME-REACTED PORCINE SPERM

The sperm–egg interactions are species-specific forms of cell recognition and the binding event which are a necessary prerequisite for fertilization (Park et al. 2002 Anim. Reprod. Sci. 72, 83–94). Glycosidase enzymes that remove carbohydrates could play an important role in the reproductive tract, modulating decisive physiological events mediated by carbohydrates, which play a key role in sperm–oocyte recognition. The aim of this study was to analyze the presence of the glycosidases α-D-mannosidase, α-L-fucosidase, β-D-glucosaminidase, and β-D-galactosaminidase in intact and acrosome-reacted sperm from fertile matured boars. Sperm were washed three times in PBS by centrifugation at 800g for 10 min. The pelleted sperm were resuspended in the same buffer to obtain a final concentration of 250 � 106 spermatozoa mL–1. The acrosome reaction was induced by incubation of the sperm with 10 µm of calcium ionophore A23187 at 37�C for 30 min. Different enzymes were detected by incubating 8 µL (for α-Dmannosidase) or 80 αL (for the rest of the enzymes) of sperm sample with the corresponding substrate conjugated to 4-methylumbelliferil for 2 h at 37�C in PBS at pH 7.3. Fluorescences were read on a Fluostar Galaxy fluorimeter (BMG LabTech GmbH, Offenburg, Germany), using wavelengths of 340 and 450 nm for excitation and emission, respectively, and were corrected by subtracting tissue and substrate blanks. The results were analyzed using a one way ANOVA. An average of fluorescence units of 9685.86 � 1081.75, 7394.63 � 874.29, 3154.17 � 514.10, and 1666.40 � 117.86 was detected in the intact sperm sample for the α-D-mannosidase, α-L-fucosidase, β-D-glucosaminidase, and β-D-galactosaminidase, respectively. For the acrosome-reacted sperm sample (60–65% acrosome-reacted sperm in the samples measured by fluorescence microscope), an average of 9756.14 � 1011.45, 7026.93 � 771.48, 1185.70 � 277.51, and 1111.60 � 176.70 for α-D-mannosidase, α-L-fucosidase, β-D-glucosaminidase, and β-D-galactosaminidase, respectively. Statistically significant differences (P < 0.05) between intact and acrosome-reacted sperm were detected only for the β-D-glucosaminidase and β-D-galactosaminidase. These results suggest that the four different enzymes detected are mainly present in the sperm plasma membrane. Under the conditions used in this study, α-D-mannosidase is the main enzyme activity present in the sperm. Importantly, β-D-glucosaminidase and β-D-galactosaminidase activity detected in the intact sperm is decreased after the induction of the acrosome reaction.