Abstract
Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.
Highlights
At the time of fertilization, when a spermatozoon encounters an oocyte, it first binds to its membrane and both membranes fuse together
Little data have been published on the comportment and role of membrane rafts during mammalian fertilization, neither on their associated proteins such as flotillins and caveolins [12,13] or tyrosine kinases involved in oocyte activation
Cholesterol repletion experiments performed at 15 mM MbCD/cholesterol showed a recovery of both fertilization rate (FR) and fertilization index (FI) of MbCD-treated oocytes, of the FI in which reversibility was close to the control level (Fig. 2A,B)
Summary
At the time of fertilization, when a spermatozoon encounters an oocyte, it first binds to its membrane and both membranes fuse together. The basic structure of cell membranes is the lipid bilayer, composed of two apposing leaflets, forming a two-dimensional liquid with fascinating properties designed to perform the functions cells require [8] To coordinate these functions, the bilayer has evolved the propensity to segregate its constituents laterally to form specialized functional microdomains permitting membrane subcompartmentalization and the formation of signaling platforms [9]. One of the lipids known to promote raft association is the GPI anchor, and as said above at least one protein anchored to GPI is essential in gamete adhesion/fusion [5,6] Another lipid constituting the rafts is the ganglioside GM1, which is expressed on the mouse oocyte and cleaving embryos [10] showing a differential distribution with respect to monosialylGb5Cer-enriched membrane rafts in preimplantation embryos [11]. The aim of this work was to study membrane raft domains to characterize their components and evaluate their functional significance in relation to mouse oocyte fertilization
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