A role of nitric oxide (NO) increase in sea urchin eggs at fertilization

Abstract

Previous studies on the nitric oxide (NO) increase at fertilization in sea urchin eggs indicate that the NO increase is Ca²⁺-dependent and attributed to the late Ca²⁺ rise. However, its role in fertilization still remains unclear. To investigate whether the NO increase is related to any ionic changes we made, simultaneous measurements of the activation current, by a single electrode voltage clamp, and NO, using the NO indicator, DAF-FM. Results demonstrated the NO increase occurred at the time of peak current (t_p) which corresponds to peak [Ca²⁺]_i, suggesting that NO has no relation to any other ionic changes besides [Ca²⁺]_i. To ascertain the role of the NO increase, a NO scavenger, PTIO was used to eliminate the NO increase. We attempted to measure O₂ consumption by a polarographic method to examine whether NO regulated a respiratory burst for protection as reported in other biological systems. However, measuring O₂ consumption resulted in an inconclusive output. To explore further, autofluorescence of reduced pyridine nucleotides, NAD(P)H, which is related to O₂ consumption, was measured in controls and when the NO increase was eliminated by PTIO. Surprisingly, PTIO decreased the rate of the fluorescence change in NAD(P)H and the late phase of increase in NAD(P)H was eliminated. PTIO also suppressed the production of H₂O₂ and caused high and weak fertilization envelope (FE). These results suggest that NO increase upregulates NAD(P)H and consolidates FE hardening by H₂O₂. [J Physiol Sci. 2008;58 Suppl:S200]