Abstract
Xenopus oocytes are naturally arrested at G2/M in prophase I of meiosis. Stimulation with progesterone initiates a nontranscriptional signaling pathway that culminates in the activation of Cdc2/cyclin B and reentry into meiosis. This pathway presents a paradigm for nongenomic signaling by steroid hormones and for the G2/M cell cycle transition. It has been extensively studied using intact oocytes, which are amenable to microinjection and biochemical analyses described elsewhere in this book. However, there are several experimental advantages in using in vitro systems consisting of cytosolic fractions of prophase-arrested oocytes. Because of their homogeneous nature, extracts avoid the difficulties of signaling asynchrony between individual oocytes. They are also amenable to biochemical manipulations such as protein immunodepletions, and proteins and pharmacological agents can be added easily. Despite these features, oocyte extracts have yet to achieve the widespread utility of Xenopus egg extracts, which can proceed through rounds of deoxyribonucleic acid (DNA) replication and mitosis in vitro. Here, we review the historical development of oocyte extracts and discuss the factors most crucial to success in reproducing the signaling pathway and the G2/M transition in vitro.
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References
Schmitt, A. and Nebreda, A. R. (2002) Signalling pathways in oocyte meiotic maturation. J. Cell Sci. 115, 2457–2459.
Maller, J. L. (2001) The elusive progesterone receptor in Xenopus oocytes. Proc. Natl. head. Sci. USA 98, 8–10.
Tian, J., Kim, S., Heilig, E., and Ruderman, J. V. (2000) Identification of XPR-1, a progesterone receptor required for Xenopus oocyte activation. Proc. Natl. Acad. Sci. USA 97, 14,358–14,363.
Bayaa, M, Booth, R. A., Sheng, Y., and Liu, X. J. (2000) The classical progesterone receptor mediates Xenopus oocyte maturation through a nongenomic mechanism. Proc. Natl. Acad. Sci. USA 97, 12,607–12,612.
Bagowski, C. P., Myers, J. W., and Ferrell, J. E., Jr. (2001) The classical progesterone receptor associates with p42 MAPK and is involved in phosphatidylinositol 3-kinase signaling in Xenopus oocytes. J. Biol. Chem. 276, 37,708–37,714.
Zhu, Y., Bond, J., and Thomas, P. (2003) Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor. Proc. Natl. Acad. Sci. USA 100, 2237–2242.
Lohka, M. J. and Maller, J. L. (1985) Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J. Cell Biol. 101, 518–523.
Murray, A. W. (1991) Cell cycle extracts. Methods Cell Biol. 36, 581–605.
Desai, A., Murray, A., Mitchison, T. J., and Walczak, C. E. (1999) The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. Methods Cell Biol. 61, 385–412.
Qian, Y. W., Erikson, E., Taieb, F. E., and Maller, J. L. (2001) The polo-like kinase Plxl is required for activation of the phosphatase Cdc25C and cyclin B-Cdc2 in Xenopus oocytes. Mol. Biol. Cell 12, 1791–1799.
Shibuya, E. K., Polverino, A. J., Chang, E., Wigler, M., and Ruderman, J. V. (1992) Oncogenic ras triggers the activation of 42-kDa mitogen-activated protein kinase in extracts of quiescent Xenopus oocytes. Proc. Natl. Acad. Sci. USA 89, 9831–9835.
Shibuya, E. K. and Ruderman, J. V. (1993) Mos induces the in vitro activation of mitogen-activated protein kinases in lysates of frog oocytes and mammalian somatic cells. Mol. Biol. Cell 4, 781–790.
Nebreda, A. R. and Hunt, T. (1993) The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs. EMBO J. 12, 1979–1986.
Huang, C. Y. and Ferrell, J. E., Jr. (1996) Dependence of Mos-induced Cdc2 activation on MAP kinase function in a cell-free system. EMBO J. 15, 2169–2173.
VanRenterghem, B., Browning, M. D., and Maller, J. L. (1994) Regulation of mitogen-activated protein kinase activation by protein kinases A and C in a cell-free system. J. Biol. Chem. 269, 24,666–24,672.
Shibuya, E. K., Morris, J., Rapp, U. R., and Ruderman, J. V. (1996) Activation of the Xenopus oocyte mitogen-activated protein kinase pathway by Mos is independent of Raf. Cell Growth Differ. 7, 235–241.
Hattori, S., Fukuda, M, Yamashita, T., Nakamura, S., Gotoh, Y., and Nishida, E. (1992) Activation of mitogen-activated protein kinase and its activator by ras in intact cells and in a cell-free system. J. Biol. Chem. 267, 20,346–20,351.
VanRenterghem, B., Gibbs, J. B., and Maller, J. L. (1993) Reconstitution of p21ras-dependent and-independent mitogen-activated protein kinase activation in a cell-free system. J. Biol. Chem. 268, 19,935–19,938.
Yang, J., Winkler, K., Yoshida, M., and Kornbluth, S. (1999) Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import. EMBO J. 18, 2174-2183.
Duckworth, B. C, Weaver, J. S., and Ruderman, J. V. (2002) G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A. Proc. Natl. Acad. Sci. USA 99, 16,794–16,799.
Mendez, R., Hake, L. E., Andresson, T., Littlepage, L. E., Ruderman, J. V., and Richter, J. D. (2000) Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature 404, 302–307.
Mendez, R., Murthy, K. G., Ryan, K., Manley, J. L., and Richter, J. D. (2000) Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex. Mol. Cell 6, 1253–1259.
Palmer, A., Gavin, A. C, and Nebreda, A. R. (1998) A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Mytl. EMBO J. 17, 5037–5047.
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© 2006 Humana Press Inc., Totowa, NJ
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Crane, R.F., Ruderman, J.V. (2006). Using Xenopus Oocyte Extracts to Study Signal Transduction. 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_31
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DOI: https://doi.org/10.1007/978-1-59745-000-3_31
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