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The NO pathway acts late during the fertilization response in sea urchin eggs
Calum Leckie1, Ruth Empson, Andrea Becchetti
1School of Cell and Molecular Biosciences, The Medical School, Framlington Place, University of Newcastle upon Tyne, Tyne and Wear NE2 4HH, United Kingdom.
Abstract:
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.
Insights
Nitric oxide (NO) does not initiate the calcium transient during sea urchin egg fertilization. Instead, NO acts downstream of the calcium wave to regulate its duration, clarifying the roles of NO and inositol trisphosphate pathways.
Area of Science:
- Reproductive biology
- Cellular signaling
- Developmental biology
Background:
- Fertilization in sea urchin eggs involves complex calcium (Ca2+) signaling.
- Both inositol 1,4,5-trisphosphate (InsP3) and ryanodine receptor pathways are known to contribute to the Ca2+ transient.
- The precise role of nitric oxide (NO) as a primary egg activator has been recently questioned.
Purpose of the Study:
- To investigate the hypothesis that NO is a primary activator of sea urchin eggs.
- To define the role of the NO/cGMP/cyclic ADP-ribose (cADPR) pathway in fertilization.
- To elucidate the temporal relationship between NO production and Ca2+ release during egg activation.
Main Methods:
- Measurement of NO and Ca2+ levels using fluorescence indicators during sea urchin egg fertilization.
- Inhibition of NO increase at fertilization to assess its impact on the Ca2+ transient.
- Assessment of the effects of inhibiting cGMP and cADPR signaling on fertilization dynamics.
Main Results:
- NO levels were found to rise after the initiation of the Ca2+ wave and are Ca2+-dependent.
- Inhibition of NO increase reduced the duration of the Ca2+ transient but did not affect latency or rise time.
- cGMP and cADPR signaling inhibition mirrored the effects of NO inhibition, with cADPR generated at fertilization.
Conclusions:
- NO is unlikely to be a primary egg activator in sea urchin fertilization.
- NO acts downstream of the initial Ca2+ wave to regulate the duration of the fertilization Ca2+ transient.
- The NO/cGMP/cADPR pathway plays a modulatory role in fertilization rather than initiating the Ca2+ release.