Membrane raft integrity is necessary to efficiently accomplish fertilization in mouse oocytes

BUSCHIAZZO J.; IALY-RADIO C; WOLF J.P; SERRES C; LEFÈVRE B.; ZIYYAT A.

Montevideo

Congreso; VI International Meeting of the Latin American Society of Developmental Biology (LASDB); 2012

Latin American Society of Developmental Biology (LASDB)

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Until now, CD9 tetraspanin and one or several non identified glycosylphosphatidylinositol-anchored proteins (GPI-APs) are the only egg membrane proteins known to be essential for fertilization. Recently, it has been proposed a link between tetraspanins and GPI-APs at fertilization. While tetraspanins have the ability to laterally associate in tetraspanin-enriched microdomains, GPI-APs are enriched in membrane rafts. In this study, methyl-beta-cyclodextrin (MbCD) was used for cholesterol modulation in order to assess membrane raft involvement in mouse fertilization. The effect of MbCD treatment on cholesterol content and behavior was analyzed in living oocytes using the fluorescent probe BODIPY-cholesterol. A fluorescent cholera toxin was used to determine the presence of the raft marker lipid, ganglioside GM1. We also evaluated the identification of the raft proteins flotillin-2, a marker of planar microdomains, and caveolin-1, the structural protein of caveolae. Functionality of rafts was assessed by disruption of these microdomains and evaluation of Gas1 (GPI-AP) and Src kinase staining in nystatin- and MbCD-treated oocytes. Cholesterol depleted oocytes were fertilized with capacitated spermatozoa. The fertilization rate underwent a significant decrease in depleted oocytes. Cholesterol removal also decreased the fertilization index and delayed the extrusion of the second polar body. Cholesterol repletion induced a recovery of fertilization. A relevant finding concerns the presence of the raft markers flotillin-2, caveolin-1 and GM1 on the oocyte plasma membrane. Raft disruption either by MbCD or by nystatin affected the membrane distribution of Gas1 generating its aggregation. On the contrary, cholesterol removal disturbed membrane localization of c-Src. In conclusion, our results highlight the functional importance of membrane rafts for mouse fertilization and its dependence on cholesterol. Further investigations remain necessary to precise the role of raft domains on mouse fertilization.