The dynamics of plasma membrane PtdIns(4,5)P-2 at fertilization of mouse eggs

摘要

A series of intracellular Ca2+ oscillations are responsible for triggering egg activation and cortical granule exocytosis at fertilization in mammals. These Ca2+ oscillations are generated by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3], which results from the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2]. Using confocal imaging to simultaneously monitor Ca2+ and plasma membrane Ptdlns(4,5)P-2 in single living mouse eggs we have sought to establish the relationship between the kinetics of Ptdlns(4,5)P-2 metabolism and the Ca2+ oscillations at fertilization. We report that there is no detectable net loss of plasma membrane Ptdlns(4,5)P-2 either during the latent period or during the subsequent Ca2+ oscillations. When phosphatidylinositol 4-kinase is inhibited with micromolar wortmannin a limited decrease in plasma membrane Ptdlns(4,5)P-2 is detected in half the eggs studied. Although we were unable to detect a widespread loss of PtdIns(4,5)P-2, we found that fertilization triggers a net increase in plasma membrane PtdIns(4,5)P-2 that is localized to the vegetal cortex. The fertilization-induced increase in PtdIns(4,5)P-2 follows the increase in Ca2+, is blocked by Ca2+ buffers and can be mimicked, albeit with slower kinetics, by photoreleasing Ins(1,4,5)P-3. Inhibition of Ca2+-dependent exocytosis of cortical granules, without interfering with Ca2+ transients, inhibits the PtdIns(4,5)P-2 increase. The increase appears to be due to de novo synthesis since it is inhibited by micromolar wortmannin. Finally, there is no increase in Ptdlns(4,5)P-2 in immature oocytes that are not competent to extrude cortical granules. These studies suggest that fertilization does not deplete plasma membrane Ptdlns(4,5)P-2 and that one of the pathways for increasing Ptdlns(4,5)P-2 at fertilization is invoked by exocytosis of cortical granules. [References: 62]