Localization by indirect immunofluorescence of nitric oxide synthase in mouse and human spermatozoa

The localization of nitric oxide synthase was studied in mouse epididymal spermatozoa and freshly ejaculated human sperm. A rabbit antiserum against the neuronal isoform of the enzyme was used, and antibody binding was detected with a fluorescein isothiocyanate-conjugated polyclonal antibody specific for rabbit IgG. In mouse spermatozoa, the percentage of cells staining specifically ranged from 88% to 98%. Samples were examined after 0-, 90- and 150-min incubations in vitro. Three different patterns of staining were observed: (a) Pattern I, intense fluorescent staining localized in the acrosome and in a segment of the tail; (b) Pattern II, fluorescent staining localized only in the tail; and (c) Pattern III, faint fluorescent staining localized in the acrosomal cap and in the tail. The potential physiological significance of these patterns is discussed. Nitric oxide synthase was also localized in the acrosome of freshly ejaculated human sperm.