Role of phospholipase C gamma at fertilization and during mitosis in sea urchin eggs and embryos

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eg gs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the e arly embryo. How sperm induces these calcium transients is still far from b eing understood. In sea urchin eggs, both InsP(3) and ryanodine receptors c ontribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP(3) after fertilization remains an enig ma. In order to determine whether PLC gamma might be the origin of the peaks of InsP(3) production that punctuate the first mitotic cell cycles of the fer tilized sea urchin egg, we have amplified by RT-PCR several fragments of se a urchin PLC gamma containing the two SH2 domains, The sequence shares simi larities with SH2 domains of PLC gamma from mammals. One fragment was subcl oned into a bacterial expression plasmid and a GST-fusion protein was produ ced and purified. Antibodies raised to the GST fusion protein demonstrate t he presence of PLC gamma protein in eggs, Microinjection of the fragment in to embryos interferes with mitosis. A related construct made from bovine PL C gamma also delayed or prevented entry into mitosis and blocked or prolong ed metaphase, The bovine construct also blocked the calcium transient at fe rtilization, in contrast to a tandem SH2 control construct which did not in hibit either fertilization or mitosis. Our data indicate that PLC gamma pla ys a key role during fertilization and early development.