A flow cytometric technique using peanut agglutinin for evaluating acrosomal loss from human spermatozoa

Flow cytometric methods for the quantification of acrosome-reacted ejaculated human spermatozoa are described in which fluorescence-labeled peanut agglutinin (Arachis hypogaea) binding to the outer acrosomal membrane is used after incubation with ethidium homodimer (EHD) as vital dye and membrane permeation using methanol. Fluorescein-labeled fucoidan (F-fucoidin) was shown to bind to sperm that were also stained by propidium iodide (PI) and EHD; therefore, the use of F-fucoidin as vital dye was incorporated into the study. F-fucoidin and EHD can withstand the acrosome-staining procedure, unlike PI, which is leached out during cell processing. During incubation of spermatozoa, nonviable cells, as indicated by PI or F-fucoidin staining, appear in parallel with acrosome-reacted cells regardless of whether they are induced by the calcium ionophore A23187. The window of time in which viable, acrosome-reacted sperm cells can be detected is narrow and could depend on the vital dye as well as the acrosomal marker used.