Evaluation of human sperm acrosome reaction and viability by flow cytometry

We report in this work a quantitative procedure developed to evaluate the a crosomal reaction and vitality of human spermatozoa, with three color stain ing simultaneously. Twenty normal human sperm were labeled with GB24 monocl onal antibody, a fluorescein isothiocyanate conjugated lectin and propidium iodide (supravital stain). Four conjugated lectins were investigated: WGA, Con-A, PNA and UEA-I. Acrosome reaction was induced with calcium ionophore A-23187. Analyses were made by flow cytometry and electron microscopy. A h igh percentage of spermatozoa that stained with propidium iodide was found. The results of four lectins show an interaction between GB24 and lectin bi nding. Significant differences of fluorescence index were obtained between the samples with calcium ionophore A-23187 and the samples without it. The WGA-GB24 association shows an independent behavior and this may depend on t he fact that WGA binds to the cytoplasmic membrane of human spermatozoa and GB24 antibody bind inner acrosome membrane. Using Con-A, PNA and UEA-I a c rowded staining is likely to occur because these lectins and GB24 antibody mainly bind to acrosome membranes, and our results then show a dose relatio n.