The NO pathway acts late during the fertilization response in sea urchin eggs.
Both the inositol 1,4,5-trisphosphate (InsP(3)) and ryanodine receptor pathways contribute to the Ca(2+) 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 InsP(3) receptor pathway has a primary role in causing Ca(2+) 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 Ca(2+) at fertilization and establish that NO levels rise after, not before, the Ca(2+) wave is initiated and that this rise is Ca(2+)-dependent. By inhibiting the increase in NO at fertilization, we find not that the Ca(2+) 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 Ca(2+) wave to regulate the duration of the fertilization Ca(2+) transient.
Leckie
Calum
C
School of Cell and Molecular Biosciences, The Medical School, Framlington Place, University of Newcastle upon Tyne, Tyne and Wear NE2 4HH, United Kingdom.
Empson
Ruth
R
Becchetti
Andrea
A
Thomas
Justyn
J
Galione
Antony
A
Whitaker
Michael
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D036563:Cyclic ADP-Ribose / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D005260:Female; D005306:Fertilization / Q000502:physiology*; D005456:Fluorescent Dyes; D009569:Nitric Oxide / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012617:Sea Urchins
D005456:Fluorescent Dyes; D019837:Ryanodine Receptor Calcium Release Channel; D036563:Cyclic ADP-Ribose; D009569:Nitric Oxide; D006152:Cyclic GMP; D002118:Calcium