Evaluation of stimulus-induced acrosome reaction by two-colour flow cytometric analysis

Acrosome status in human spermatozoa from 20 normozoospermic men wasevaluated by flow cytometry following the induction of the acrosomereaction with the ionophore A23187. Dual fluorescence staining of methanolfixed spermatozoa incubated with and without (control) the ionophore A23187was performed with probes which targeted the outer acrosomal membrane (OAM)(rhodamine-labelled Arachis hypogaea agglutinin) or constituents of theacrosomal vesicle (fluorescein- labelled Pisum sativum agglutinin). Flowcytometry analysis revealed two major subpopulations of cells:acrosome-intact and acrosome-reacted spermatozoa after induction of theacrosome reaction. The intensity of green and red fluorescence inacrosome-reacted spermatozoa was significantly lower than that of theacrosome-intact control spermatozoa (P < 0.0001). The intensity of greenfluorescence in the acrosome-intact subpopulation of spermatozoa wassignificantly higher than that of the control population (P < 0.002).Exposure of spermatozoa to the ionophore A23187 resulted in reliableenhancement of the number of spermatozoa with very high intensity of greenand/or red fluorescence compared with the control (P < 0.03). An inversecorrelation between the number of acrosome-reacted spermatozoa andspermatozoa with a very high intensity of green and/or red fluorescence wasdemonstrated (r = -0.631, P < 0.01). This method provides an objectiveand efficient procedure for quantitative estimation of the acrosomal statusof human spermatozoa.