Abstract
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.
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References
Masui, Y. (2000) The elusive cytostatic factor in the animal egg. Nat. Rev. Mol. Cell. Biol. 1, 228–232.
Iwao, Y. (2000) Mechanisms of egg activation and polyspermy block in amphibians and comparative aspects with fertilization in other vertebrates. Zool. Sci. 17, 699–709.
Yanagimachi, R. (1994) Fertilization, in The Physiology of Reproduction (Knobil, E. and Neill, J. D., eds.), Raven, New York, pp. 189–317.
Runft, L. L., Jaffe, L. A., and Mehlmann, L. M. (2002) Egg activation at fertilization: where it all begins. Dev. Biol. 245, 237–254.
Sato, K., Iwasaki, T., Hirahara, S., Nishihira, Y., and Fukami, Y. (2004) Molecular dissection of egg fertilization signaling with the aid of tyrosine kinase-specific inhibitor and activator strategies. Biochim. Biophys. Ada 1697, 103–121.
Sato, K., Aoto, M., Mori, K., et al. (1996) Purification and characterization of a src-related p57 protein-tyrosine kinase from Xenopus oocytes. J. Biol. Chem. 271, 13,250–13,257.
Sato, K., Iwao, Y., Fujimura, T., et al. (1999) Evidence for the involvement of a Srcrelated tyrosine kinase in Xenopus egg activation. Dev. Biol. 209, 308–320.
Sato, K., Tokmakov, A. A., Iwasaki, T., and Fukami, Y. (2000) Tyrosine kinase-dependent activation of phospholipase Cy is required for calcium transient in Xenopus egg fertilization. Dev. Biol. 224, 453–469.
Sato, K., Ogawa, K., Iwasaki, T., Tokmakov, A. A., and Fukami, Y. (2001) Hydrogen peroxide induces Src family tyrosine kinase-dependent activation of Xenopus eggs. Dev. Growth Differ. 43, 55–72.
Sato, K., Iwasaki, T., Ogawa, K., Konishi, M., Tokmakov, A. A., and Fukami, Y. (2002) Low density detergent-insoluble membrane of Xenopus eggs: subcellular microdomain for tyrosine kinase signaling in fertilization. Development 129, 885–896.
Murray, A. W. (1991) Cell cycle extracts. Methods Cell Biol. 36, 581–605.
Pappin, D. J. C, Hojrup, P., and Bleasby, A. J. (1993) Rapid identification of proteins by peptide-mass fingerprinting. Curr. Biol. 3, 327–332
James, P., Quadroni, M., Carafoli, E., and Gonnet, G. (1994) Protein identification in DNA databases by peptide mass fingerprinting. Protein Sci. 3, 1347–1350.
Eng, J. K., McCormack, A. L., and Yates, J. R., III (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989.
Yates, J. R., III, Eng, J. K., and McCormack, A. L. (1995) Mining genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases. Anal. Chem. 67, 3202–3210.
Neubauer, G., King, A., Rappsilber, J., et al. (1998) Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat. Genet. 20, 46–50.
Choudhary, J. S., Blackstock, W. P., Creasy, D. M., and Cottrell, J. S. (2001) Interrogating the human genome using uninterpreted mass spectrometry data. Proteomics 1, 651–667.
Tokmakov, A. A., Sato, K., Iwasaki, T., and Fukami, Y. (2002) Src kinase induces calcium release in Xenopus egg extracts via PLCγ and IP3-dependent mechanism. Cell Calcium 32, 11–20.
Sato, K., Tokmakov, A. A., He, C.-L., et al. (2003) Reconstitution of Src-dependent PLCγ phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs. J. Biol. Chem. 278, 38,413–38,420.
Sakakibara, K., Sato, K., Yoshino, K., et al. (2005) Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization. J. Biol. Chem. 280, 15,029–15,037.
Mahbub Hasan, A. K. M., Sato, K., Sakakibara, K., et al. (2005) Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization. Dev. Biol. in press.
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© 2006 Humana Press Inc., Totowa, NJ
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Sato, Ki., Yoshino, Ki., Tokmakov, A.A., Iwasaki, T., Yonezawa, K., Fukami, Y. (2006). Studying Fertilization in Cell-Free Extracts. In: Liu, X.J. (eds) Xenopus Protocols. Methods in Molecular Biology™, vol 322. Humana Press. https://doi.org/10.1007/978-1-59745-000-3_28
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DOI: https://doi.org/10.1007/978-1-59745-000-3_28
Publisher Name: Humana Press
Print ISBN: 978-1-58829-362-6
Online ISBN: 978-1-59745-000-3
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