Previous studies indicate that the nitric oxide (NO) increase at fertilization in sea urchin eggs is Ca2+-dependent and attributed to the late Ca2+ rise. However, its role in fertilization still remains unclear. Simultaneous measurements of the activation current, by a single electrode voltage clamp, and NO, using the NO indicator DAF-FM, showed that the NO increase occurred at the time of peak current (tp) which corresponds to peak [Ca2+]i, suggesting that NO is not related to any other ionic changes besides [Ca2+]i. We measured O2 consumption by a polarographic method to examine whether NO regulated a respiratory burst for protection as reported in other biological systems. Our results suggested NO increased O2 consumption. The fluorescence of reduced pyridine nucleotides, NAD(P)H was measured in controls and when the NO increase was eliminated by PTIO, a NO scavenger. Surprisingly, PTIO decreased the rate of the fluorescence change and the late phase of increase in NAD(P)H was eliminated. PTIO also suppressed the production of H2O2 and caused weak and high fertilization envelope (FE). Our results suggest that NO increase upregulates NAD(P)H and H2O2 production and consolidates FE hardening by H2O2.
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