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The dynamics of plasma membrane PtdIns(4,5)P₂ at fertilization of mouse eggs

¹ Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK
² Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
³ Endocrinology and Reproduction Research Branch, National Institutes of Health, Bethesda, MD 20892, USA

^* Author for correspondence (e-mail: j.carroll{at}ucl.ac.uk )

Accepted 5 March 2002

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)P₃], which results from the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂]. Using confocal imaging to simultaneously monitor Ca²⁺ and plasma membrane PtdIns(4,5)P₂ in single living mouse eggs we have sought to establish the relationship between the kinetics of PtdIns(4,5)P₂ metabolism and the Ca²⁺ oscillations at fertilization. We report that there is no detectable net loss of plasma membrane PtdIns(4,5)P₂ 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 PtdIns(4,5)P₂ is detected in half the eggs studied. Although we were unable to detect a widespread loss of PtdIns(4,5)P₂, we found that fertilization triggers a net increase in plasma membrane PtdIns(4,5)P₂ that is localized to the vegetal cortex. The fertilization-induced increase in PtdIns(4,5)P₂ follows the increase in Ca²⁺, is blocked by Ca²⁺ buffers and can be mimicked, albeit with slower kinetics, by photoreleasing Ins(1,4,5)P₃. Inhibition of Ca²⁺-dependent exocytosis of cortical granules, without interfering with Ca²⁺ transients, inhibits the PtdIns(4,5)P₂ increase. The increase appears to be due to de novo synthesis since it is inhibited by micromolar wortmannin. Finally, there is no increase in PtdIns(4,5)P₂ in immature oocytes that are not competent to extrude cortical granules. These studies suggest that fertilization does not deplete plasma membrane PtdIns(4,5)P₂ and that one of the pathways for increasing PtdIns(4,5)P₂ at fertilization is invoked by exocytosis of cortical granules.

Key words: Phosphatidylinositol 4,5-bisphosphate, Oocyte, GFP

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PtdIns(4,5)P₂ dynamics at fertilization

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