FLOW CYTOMETRIC EVALUATION OF THE ACROSOME REACTION OF HUMAN SPERMATOZOA - A NEW METHOD USING A PHOTOACTIVATED SUPRAVITAL STAIN

A flow cytometric assay using a double-stain method for the measuremen t of the acrosome reaction of human spermatozoa is described. The use of a stable photoactivated stain, ethidium monoazide, allowed evaluati on of the viability of spermatozoa. This stain was more stable in fixe d samples than propidium iodide, which is not bound covalently to DNA and is therefore removed readily during the washing procedure. The per meabilized acrosome nas labelled with Pisum sativum agglutinin conjuga ted with fluoroisothiocyanate. Since this lectin binds to the acrosome and acrosomal contents, a decrease in the fluorescence intensity allo ws the cytometric evaluation of the acrosome reaction. Microscopic ana lysis and now cytometric analysis were well correlated and cell sortin g was performed to ensure the homogeneity of each different subpopulat ion encountered.