The NO pathway acts late during the fertilization response in sea urchin eggs

Both the inositol 1,4,5-trisphosphate (InsP3) and ryanodine receptor pathways contribute to the Ca2+ transient at fertilization in sea urchin eggs. To date, the precise contribution of each pathway has been difficult to ascertain. Evidence has accumulated to suggest that the InsP3 receptor pathway has a primary role in causing Ca2+ release and egg activation. However, this was recently called into question by a report implicating NO as the primary egg activator. In the present study we pursue the hypothesis that NO is a primary egg activator in sea urchin eggs and build on previous findings that an NO/cGMP/cyclic ADP-ribose (cADPR) pathway is active at fertilization in sea urchin eggs to define its role. Using a fluorescence indicator of NO levels, we have measured both NO and Ca2+ at fertilization and establish that NO levels rise after, not before, the Ca2+ wave is initiated and that this rise is Ca2+-dependent. By inhibiting the increase in NO at fertilization, we find not that the Ca2+ transient is abolished but that the duration of the transient is significantly reduced. The latency and rise time of the transient are unaffected. This effect is mirrored by the inhibition of cGMP and cADPR signaling in sea urchin eggs at fertilization. We establish that cADPR is generated at fertilization, at a time comparable to the time of the rise in NO levels. We conclude that NO is unlikely to be a primary egg activator but, rather, acts after the initiation of the Ca2+ wave to regulate the duration of the fertilization Ca2+ transient.