Crossley, Ian Brian; (1990) How the sperm triggers the fertilisation calcium wave in the sea urchin egg. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Fertilisation is characterised by an increase in cytosolic free calcium (Ca). This crucial event transforms the egg, releasing it from its state of cell cycle arrest and stimulating embryonic development. In the sea urchin egg the Ca1 increase is the result of increased polyphosphoinositide (PPI) hydrolysis and the production of the calcium mobilising internal messenger inositol 1,4,5-trisphosphate. I discuss the contribution of inositol polyphosphates to the calcium changes at fertilisation particularly their ability to stimulate calcium entry. I have used cortical granule exocytosis as an indicator of Ca1 and measured Ca1 in single eggs with the calcium sensitive fluorescent dye fura2. I show that, contrary to other reports, inositol phosphates activate eggs by a mechanism that is entirely independent of external calcium. A sperm activates an egg by triggering a regenerative response that involves calcium-stimulated PPI hydrolysis: in this way calcium release leads to further calcium release in an explosive manner. It has been suggested that the sperm triggers this response through a GTP-binding protein. However I show that an analogue of GDP, Guanosine-5'-O-(2-thiodiphosphate), that inhibits GTP-binding protein activation, does not prevent the sperm-induced increase in Ca or incorporation of the sperm into the egg. Also a GTP analogue, Guanosine-5'-O-thiotriphosphate, which stimulates GTP-binding proteins and causes egg activation, does so by a different mechanism than sperm. This data argues against the idea that a sperm triggers egg activation through a GTP-binding protein mechanism. The mobilisation of intracellular calcium through PPI hydrolysis and the production of inositol phosphates is a common signal transduction pathway present in many tissues. My data suggests that inositol phosphates act only to mobilise intracellular calcium and not to stimulate calcium influx. They also suggest that fertilisation in the sea urchin may involve a novel transduction pathway because the PPI hydrolysis is not linked to a GTP-binding protein as in other systems.

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