Studying Fertilization in Cell-Free Extracts
Xenopus oocytes, eggs, and embryos serve as an ideal model system to study several aspects of animal development (e.g., gametogenesis, fertilization, embryogenesis, and organogenesis). In particular, the Xenopus system has been extensively employed not only as a “living cell” system but also as a “cell-free” or “reconstitutional” system. In this chapter, we describe a protocol for studying the molecular mechanism of egg fertilization with the use of cell-free extracts and membrane/lipid rafts prepared from unfertilized, metaphase II-arrested Xenopus eggs. By using this experimental system, we have reconstituted a series of signal transduction events associated with egg fertilization, such as sperm-egg membrane interaction, activation of Src tyrosine kinase and phospholipase Cγ, production of inositol trisphosphate, transient calcium release, and cell cycle transition. This type of reconstitutional system may allow us to perform focused proteomics (e.g., rafts) as well as global protein analysis (i.e., whole egg proteome) of fertilization in a cell-free manner. As one of these proteomics approaches, we provide a protocol for molecular identification of Xenopus egg raft proteins using mass spectrometry and database mining.
Key WordsCalcium release cell-free system CSF extract egg egg activation embryogenesis expressed sequence tag fertilization gametogenesis in vitro reconstitution mass spectrometry proteome proteomics raft reproduction signal transduction sperm Src tyrosine phosphorylation unigene
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