EVALUATION OF STIMULUS-INDUCED ACROSOME REACTION BY 2-COLOR FLOW CYTOMETRIC ANALYSIS

Acrosome status in human spermatozoa from 20 normozoospermic men was e valuated by flow cytometry following the induction of the acrosome rea ction with the ionophore A23187. Dual fluorescence staining of methano l fixed spermatozoa incubated with and without (control) the ionophore A23187 was performed with probes which targeted the outer acrosomal m embrane (OAM) (rhodamine-labelled Arachis hypogaea agglutinin) or cons tituents of the acrosomal vesicle (fluorescein-labelled Pisum sativum agglutinin). Flow cytometry analysis revealed two major subpopulations of cells: acrosome-intact and acrosome-reacted spermatozoa after indu ction of the acrosome reaction. The intensity of green and red fluores cence in acrosome-reacted spermatozoa was significantly lower than tha t of the acrosome-intact control spermatozoa (P < 0.0001), The intensi ty of green fluorescence in the acrosome-intact subpopulation of sperm atozoa was significantly higher than that of the control population (P < 0.002), Exposure of spermatozoa to the ionophore A23187 resulted in reliable enhancement of the number of spermatozoa with very high inte nsity of green and/or red fluorescence compared with the control (P < 0.03). An inverse correlation between the number of acrosome-reacted s permatozoa and spermatozoa with a very high intensity of green and/or red fluorescence was demonstrated (r = -0.631, P < 0.01), This method provides an objective and efficient procedure for quantitative estimat ion of the acrosomal status of human spermatozoa.