Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

Abstract

Critical developmental periods, such as fertilization, involve metabolic activation, membrane fusion events such as sperm-egg or plasma membrane-cortical granule merger, and production and hydrolysis of phospholipids. However, there has been no large-scale quantification of phospholipid changes during fertilization. Using an enzymatic assay, traditional FA analysis by TLC and gas chromatography, along with a new method of phospholipid measurement involving HPLC separation and evaporative light-scattering detection, we report lipid levels in eggs, sperm, and during fertilization in Xenopus laevis. Sperm were found to contain different amounts of phospholipids as compared with eggs. During fertilization, total phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, and phosphatidylserine decreased, and ceramide increased, whereas there was no change in phosphatidylcholine, cardiolipin, or phosphatidylethanolamine. FA analysis of phospholipids found numerous changes during fertilization. Because there is an increase in sn-1,2-diacylglycerol at fertilization, the FAs associated with this increase and the source of the increase in this neutral lipid were examined. Finally, activation of phospholipase C, phospholipase D, phospholipase A2, autotoxin, and sphingomyelinase at fertilization is discussed. Major goals of the LIPID Metabolites and Pathways Strategy (LIPID MAPS) are to "separate and detect all of the lipids in a specific cell," "to quantitate each of the lipid metabolites present and determine the changes in their levels," and to study lipid metabolic pathways during biological events (1). Characterizing changes in the lipid composition during defined biological events is part of the concept of "functional lipidomics" (2). These fundamental, descriptive studies will facilitate our understanding of how lipids regulate membrane fluidity and fusion, lipid rafts, the cytoskeleton, kinases, and protein binding to membranes (e.g., 3-5) to induce pleiotropic cellular effects.About 15 years ago, quantification of lipid mass changes during maturation of the oocyte to the fertilizable egg, and during fertilization of the egg by sperm, was begun in Xenopus laevis (6-9). Critical developmental events can be isolated in vitro with this model system, and large numbers of gametes can be obtained to provide sufficient amounts of second messengers and lipids. During fertilization, membrane bending and fusion are associated with the acrosome reaction of sperm (wherein the large sperm acrosome undergoes exocytosis upon interaction with layers that cover the egg), with sperm-egg merger, and with subsequent fertilization events such as cortical granule exocytosis (10, 11). Furthermore, both Xenopus and human eggs are arrested in meiosis II (12).Compared with oocytes, one disadvantage of studying eggs is that they have a short life, much less than the estimated time for lipid precursor incorporation to near equilibrium levels (6, 7). Thus, lipid analysis based on label incorporation or turnover is not reliable, and ...

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MscS-Like10 is a stretch-activated ion channel from Arabidopsis thaliana with a preference for anions

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