Detection and Measurement of Membrane-Bound Protein Tyrosine Kinases in the Zebrafish Egg

  • Wenjun Wu
  • William H. Kinsey
Part of the Methods in Molecular Biology™ book series (MIMB, volume 253)

Abstract

The study of the biochemical events involved in fertilization and egg activation has historically been directed toward marine invertebrate and amphibian eggs (1), although recent progress has been made in the analysis of a few enzymes that are abundant in mammalian eggs (2,3). The zebrafish system is also a promising model that shares several advantages with marine invertebrate eggs, yet has the advantage of being a vertebrate. For example, the zebrafish eggs are reasonably clear optically and can be obtained in quantities suitable for biochemical analysis. They can be fertilized synchronously and will develop rapidly (4). The zebrafish system also benefits from the fact that the DNA sequence homology with mammals is very high, so that tools developed in mammalian systems can be applied to zebrafish eggs with a reasonable expectation of success. Finally, the potential for genetic analysis of the fertilization process could ultimately provide novel insights into the signaling mechanisms used at fertilization.

Keywords

Sucrose HPLC MgCl2 Vanadate CaCl2 

References

  1. 1.
    Whitaker, M. and Swann, K. (1993) Lighting the fuse at fertilization. Development 117, 1–12.Google Scholar
  2. 2.
    Moore, H. D. (2001) Molecular biology of fertilization. J. Reprod. Fertil. 57(Suppl.), 105–110.Google Scholar
  3. 3.
    Carroll, J. (2001) The initiation and regulation of Ca2+ signalling at fertilization in mammals. Semin. Cell Dev. Biol. 12, 37–43.PubMedCrossRefGoogle Scholar
  4. 4.
    Hart, N. H. (1990) Fertilization in teleost fishes: mechanisms of sperm-egg interactions. Int. Rev. Cytol. 121, 1–66.PubMedCrossRefGoogle Scholar
  5. 5.
    Kamsteeg, E. J. and Deen, P. M. (2001) Detection of aquaporin-2 in the plasma membranes of oocytes: a novel isolation method with improved yield and purity. Biochem. Biophys. Res. Commun. 282, 683–690.PubMedCrossRefGoogle Scholar
  6. 6.
    Wu, W. and Kinsey, W. H. (2001) Fertilization triggers activation of Fyn kinase in the zebrafish egg. Int. J. Dev. Biol. 44, 837–841.Google Scholar
  7. 7.
    Lee, K. W., Webb, S. E., and Miller, A. L. (1999) A wave of free cytosolic calcium traverses zebrafish eggs on activation. Dev. Biol. 214, 168–180.PubMedCrossRefGoogle Scholar
  8. 8.
    Westerfield, M. (1993) The Zebrafish Book, University of Oregon Press, Eugene.Google Scholar
  9. 9.
    Hart, N. H. and Yu, S. F. (1980) Cortical granule exocytosis and cell surface reorganization in eggs of Brachydanio. J. Exp. Zool. 213, 137–159.PubMedCrossRefGoogle Scholar
  10. 10.
    Beams, H. W., Kessel, R. G., Shih, C., et al. (1985) Scanning electron microscopy on blastodisc formation in zebrafish. J. Morphol. 184, 41–49.CrossRefGoogle Scholar
  11. 11.
    Zheng, X. M. and Shalloway, D. (2001) Two mechanisms activate PTPa during mitosis. EMBO J. 20, 6037–6049.PubMedCrossRefGoogle Scholar
  12. 12.
    Wu, W. and Kinsey, W. H. (2002) Role of PTPase(s) in regulating Fyn kinase at fertilization of the zebrafish egg. Devel. Biol. 247, 286–294.CrossRefGoogle Scholar
  13. 13.
    Su, J., Muranjan, M., and Sap, J. (1999) Receptor protein tyrosine phosphatase α activates Src-family kinases and controls integrin-mediated responses in fibroblasts. Curr. Biol. 9, 505–511.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Wenjun Wu
    • 1
  • William H. Kinsey
    • 1
  1. 1.Department of Anatomy and Cell BiologyUniversity of Kansas Medical CenterKansas City

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