Redox activity associated with the maturation and capacitation of mammalian spermatozoa

As rat spermatozoa undergo epididymal maturation, they acquire the ability to exhibit a spontaneous burst of luminol-peroxidase-dependent chemiluminescence when released into a simple, defined culture medium. This activity was suppressed by inhibitors of plasma membrane redox systems such as diphenylene iodonium, p-chloromercuribenzenesulfonic acid, and capsaicin, but was resistant to inhibition by resiniferatoxin and rotenone. The luminol-peroxidase signal was dependent on the presence of bicarbonate, enhanced by the substitution of fructose for glucose, and severely suppressed by desferoxamine, superoxide dimutase, and catalase. Both L- and D-arginine were stimulatory, suggesting the involvement of *NO in this spontaneous chemiluminescence activity. The L-arginine-dependent, but not the D-arginine-dependent, activity was significantly suppressed by an inhibitor of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester). L- and D-arginine could also stimulate redox activity observed in immature caput epididymal cells, but only after prolonged incubation. The inhibitory effects of uric acid and ascorbate suggested the chemiluminescence signal might be induced by peroxynitrite. This conclusion was supported by confocal imaging of the cells following treatment with 4-amino-5-methylamino-2',7'-difluorofluorescein. Stimulation or suppression of the redox activity detected by luminol-peroxidase led to corresponding changes in the ability of the spermatozoa to exhibit acrosomal exocytosis, indicating that this pathway is of fundamental biological significance.