Abstract
The starfish oocyte has proven useful for studies involving microinjection because it is relatively large (190 μm) and optically clear. These oocytes are easily obtained from the ovary arrested at prophase of meiosis I, making them useful as a model system for the study of cell cycle-related events. In this chapter, a method for combining microinjection with immunoblotting of single cells is described. Individual starfish oocytes are injected, removed from the microinjection chamber, and analyzed by immunoblotting for the dual-phosphorylated form of mitogen-activated protein kinase (MAPK). This method will allow for experiments testing the regulation of MAPK in single cells and for the manipulation of these cells by a quantitative microinjection technique.
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References
Haccard, O., and Jessus, C. (2006) Oocyte maturation, Mos, and cyclins. Cell Cycle 5(11), 1152–1159.
Liang, C-G., Su, Y-Q., Fan, H-Y., Schatten, H., and Sun, Q-Y. (2007) Mechanisms regulating oocyte meiotic resumption: roles of mitogen-activated protein kinase. Mol. Endocrinology 21, 2037–2055.
Avruch, J. (2007) MAP kinase pathways: the first twenty years. Biochim. Biophys. Acta 1773, 1150–1160.
Shaul, Y.D., and Seger, R. (2007). The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim. Biophys. Acta 1773, 1213–1226.
Caffrey, D.R., O’Neill, L.A.J., Shields, D.C. (1999) The evolution of the MAP kinase pathways: coduplication of interacting proteins leads to new signaling cascades. J. Mol. Evol. 49, 567–582.
Posada, J., and Cooper, J.A. (1992) Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes. Science 255, 212–215.
Kishimoto, T. (2004) More than G1 or G2 arrest: useful starfish oocyte system for investigating skillful MAP kinase. Biol. Cell 96, 241–244.
Philipova, R., and Whitaker, M. (1998) MAP kinase activity increases during mitosis in early sea urchin embryos. J. Cell Sci. 111, 2497–2505.
Kumano, M., Carroll, D.J., Denu, J.M., and Foltz, K.R. (2001) Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization. Dev. Biol. 236, 244–257.
Smythe, T.L., Stricker, S.A. (2005) Germinal vesicle breakdown is not fully dependent on MAPK activation in maturing oocytes of marine nemertean worms. Mol. Reprod. Dev. 70, 91–102.
Miller, M.A., Nguyen, V.Q., Lee, M.H., Kosinski, M., Schedl, T., Caprioli, R.M., and Greenstein, D. (2001) A sperm cytoskeletal protein that signals oocyte meiotic maturation and ovulation. Science 291, 2144–2147.
Eckberg, W.R. (1997) MAP and cdc2 kinase activities at germinal vesicle breakdown in Chaetopterus. Dev. Biol. 191, 182–190.
Tachibana, K., Machida, T., Nomura, Y., and T. Kishimoto (1997) MAP kinase links the fertilization signal transduction pathway to the G1/S-phase transition in starfish eggs. EMBO J. 16, 4333–4339.
Posada, J., Yew, N., Ahn, N.G., Vande Woude, G.F., and Cooper, J.A. (1993) Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro. Mol. Biol. Cell. 13, 2546–2553.
Matten, W.T., Copeland, T.D., Ahn, N.G., and Vande Woulde, G.F. (1996) Positive feedback between MAP kinase and Mos during Xenopus oocyte maturation. Dev. Biol. 179, 485–492.
Lutz, L.B., Cole, L.M., Gupta, M.K., Kwist, K.W., Auchus, R.J. (2001) Evidence that androgens are the primary steroids produced by Xenopus laevis ovaries and may signal through the classical androgen receptor to promote oocyte maturation. Proc. Natl. Acad. Sci. USA 98, 13728–13733.
Jaffe, L.A. and Terasaki, M. (2004). Quantitative microinjection of oocytes, eggs, and embryos. Meth. Cell Biol. 74, 219–242.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
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Carroll, D.J., Hua, W. (2009). Combining Microinjection and Immunoblotting to Analyze MAP Kinase Phosphorylation in Single Starfish Oocytes and Eggs. In: Carroll, D. (eds) Microinjection. Methods in Molecular Biology, vol 518. Humana Press. https://doi.org/10.1007/978-1-59745-202-1_5
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DOI: https://doi.org/10.1007/978-1-59745-202-1_5
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