McDougall, Alexander David; (1993) Mechanisms underlying the generation and propagation of the fertilization calcium wave in sea urchin eggs. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Fertilization in sea urchins has a characteristic latent period of about 25 seconds (from the time of sperm-egg fusion) before the Ca++i increases (first at the point of sperm-egg fusion) and sweeps across the egg in turn activating it. No model can faithfully explain either what happens during the latent period to trigger this local Ca++i increase or how the Ca++i wave propagates across the egg. There is evidence that shows that the second messenger inositol 1,4,5-trisphosphate (InsP3) does not solely control both events. Indeed, recent evidence shows that the sea urchin egg is also likely to contain another Ca++i release mechanism-the ryanodine receptor. Results presented here will show that there may indeed be another form of Ca++i release distinct from either the InsP3 or ryanodine receptors. From confocal microscopy I have found that during the latent period a small spherical Ca++i wave may travel inwards from the plasma membrane. This wave is insensitive to inhibitors to either the InsP3 or ryanodine Ca++ release channels (namely heparin and ruthenium red) and may propagate through some other mechanism. More evidence in favour of the existence of another Ca++i release channel (which may be involved during the latent period) came through experiments with cGMP. cGMP may contribute to the local Ca++i release that occurs during the latent period since it specifically releases Ca++i and given that the sperm have a guanylate cyclase. It is shown here that cGMP releases Ca++i through a mechanism insensitive to heparin and only partly sensitive to ruthenium red. This novel Carelease channel may be sensitive to some sulfhydryl reagents since the sulfhydryl oxidizing reagent thimerosal too unmasks a novel Ca++i release mechanism. Here eggs bathed in 1 mM thimerosal seawater spontaneously activate through a mechanism insensitive to either heparin or ruthenium red. This channel may have a role at fertilization since thimerosal also reduces both the duration of the latent period and the time to peak Ca++i at fertilization. Not all sulfhydryl reagents have the same effect - oxidized glutathione releases Ca++ from sea urchins, but through a heparin sensitive mechanism. At the end of the latent period a Ca++i wave sweeps across the egg at an almost constant velocity [4 µm/sec (taking around 25 seconds to reach the opposite pole)]. Whole cell Ca++i data has indicated that this wave is not sensitive to heparin, thus suggesting a mechanism independent of InsP3. Experiments shown here with ruthenium red and heparin indicate that the wave may propagate through both the InsP3 and ryanodine receptors. Additionally there remains a release mechanism which is insensitive to either heparin or ruthenium red at fertilization. Here again it is likely that there is another type of Ca++i release mechanism in the sea urchin egg, which may play a role during propagation of the fertilization Ca++i wave. This Ca++i wave does a number of things. It is shown here that one function may be to trigger the second phase of inward current, which is known to travel across the egg surface as a band preceding the wave of exocytosis.

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