Sperm nitric oxide and motility: the effects of nitric oxide synthase stimulation and inhibition

Nitric oxide (NO) is synthesized from L-arginine by a family of enzymesknown as the nitric oxide synthases (NOS). We have recently shown a NOSsimilar to constitutive brain NOS (bNOS) and endothelial NOS (ecNOS) to bepresent in spermatozoa. The aim of this study is to investigate NOproduction by human spermatozoa and the effects of stimulation andinhibition of NOS. This was carried out using the Iso-NO, an isolated NOmeter and sensor, which provides rapid, accurate and direct measurements ofNO. Semen samples with normozoospermic and asthenozoospermic profiles wereprepared using a direct swim-up technique. Basal concentrations of NO andstimulated NO production were measured after exposure to the calciumionophore (A23187; 0.01-10 microM) a potent activator of constitutive NOS.NO production in human spermatozoa was significantly increased by theaddition of A23187 30 seconds after stimulation. Furthermore, this responsewas greatly diminished by pre-incubating the samples with competitiveinhibitors of L-arginine, the substrate for NOS, before treatment withcalcium ionophore. In the presence of N(G)-nitro-L-arginine methyl ester(L- NAME), N(G)-nitro-L-arginine (L-NA) or N(G)-methyl-L-arginine (L-NMMA;all at 10 microM), NO production was inhibited with a rank order of potencyL-NAME > L-NMMA > L-NA which is in accordance with the inhibition ofan endothelial type of constitutive NOS.