Two simple methods for detecting acrosome‐reacted human sperm

Abstract

Abstract We describe two methods for detecting acrosome reactions of human sperm at the light microscopic level. The techniques include the use of a supravital stain to detect dead sperm in order to differentiate between “physiological” and “degenerative” acrosome reactions. Sperm are incubated with the supravital stain Hoechst 33258 (a fluorescent DNA‐binding dye with limited membrane permeability), washed, suspended in 95% ethanol for fixation and permeabilization, and dried onto slides. The sperm are then labeled either by indirect immunofluorescence using rabbit anti‐human sperm antiserum or with fluoresceinated Pisum sativum agglutinin (PSA). Both probes intensely label the acrosomal region of acrosome‐intact sperm. Electron microscopy revealed the major site of PSA binding to be the acrosomal contents. Acrosome‐reacted sperm have diminished acrosomal labeling by both probes; sperm with nuclei labeled by Hoechst stain are considered nonviable, and are excluded from the assay. Both assays are rapid, give similar results, and detect an increase in acrosome reactions following exposure to the ionophore A23187.