Abstract
Meiotic division is a dynamic process that exhibits active interactive behaviors amongst different intracellular structures and components for spindle assembly and chromosome segregation. Understanding the mechanisms of meiotic spindle assembly and chromosome segregation therefore requires a quantitative analysis of spatiotemporal relationships among different structures and components. In this chapter, we describe a method for triple-color live imaging of meiotic division in mouse oocytes. This approach combines the microinjection of RNAs encoding proteins tagged with green and red fluorescent proteins and the visualization of microtubules with the fluorogenic far-red probe SiR-Tubulin. This method enables the simultaneous spatiotemporal mapping of three different components of the spindle and chromosomes, which opens the way to quantitative analysis of their interactive behaviors.
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References
Dumont J, Petri S, Pellegrin F et al (2007) A centriole- and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes. J Cell Biol 176:295–305. https://doi.org/10.1083/jcb.200605199
Schuh M, Ellenberg J (2007) Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes. Cell 130:484–498. https://doi.org/10.1016/j.cell.2007.06.025
Tsurumi C, Hoffmann S, Geley S et al (2004) The spindle assembly checkpoint is not essential for CSF arrest of mouse. J Cell Biol 167(6):1037–1050
Lukinavičius G, Reymond L, D’Este E et al (2014) Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat Methods 11:731–733. https://doi.org/10.1038/nmeth.2972
Balboula A, Nguyen A, Gentilello A et al (2016) Haspin kinase regulates microtubule-organizing center clustering and stability through aurora kinase C in mouse oocytes. J Cell Sci 129:3648–3660. https://doi.org/10.1242/jcs.189340
Clift D, Schuh M (2015) A three-step MTOC fragmentation mechanism facilitates bipolar spindle assembly in mouse oocytes. Nat Commun 6:7217. https://doi.org/10.1038/ncomms8217
Kyogoku H, Kitajima T (2017) Large cytoplasm is linked to the error-prone nature of oocytes. Dev Cell 41:287–298.e4. https://doi.org/10.1016/j.devcel.2017.04.009
Kitajima T, Ohsugi M, Ellenberg J (2011) Complete kinetochore tracking reveals error-prone homologous chromosome Biorientation in mammalian oocytes. Cell 146:568–581. https://doi.org/10.1016/j.cell.2011.07.031
Yoshida S, Sakakibara Y, Kitajima TS (2016) Live imaging of intracellular dynamics during meiotic maturation in mouse oocytes. Methods Mol Biol 1457:241–251. https://doi.org/10.1007/978-1-4939-3795-0_18
Rabut G, Ellenberg J (2004) Automatic real-time three-dimensional cell tracking by fluorescence microscopy. J Microsc 216:131–137. https://doi.org/10.1111/j.0022-2720.2004.01404.x
Nagy A, Gertsenstein M, Vintersten K, Behringer R (2003) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 148–149
Stein P, Schultz RM (2010) ICSI in the mouse. Methods Enzymol 476:251–262
Oesterle A, Sutter Instrument Company (2015) Pipette cookbook 2015 P-97 & P-1000 micropipette pullers. Sutter Instrument Company, Novato
Acknowledgment
We thank Dr. Jan Ellenberg for a macro for automated confocal microscopy, Dr. Melina Schuh for the Cep192 construct, and the RIKEN Kobe imaging and animal facilities for support. This work was supported by the National Sustainability Programme of the Czech Ministry of Education, Youth and Sports [project number LO1609] to P.S., and by the research grants JSPS KAKENHI 26650072/16H01226/16H06161, JSPS Bilateral Joint Research projects with P.S., and RIKEN CDB intramural grant to T.S.K.
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Courtois, A., Solc, P., Kitajima, T.S. (2018). Triple-Color Live Imaging of Mouse Oocytes. In: Verlhac, MH., Terret, ME. (eds) Mouse Oocyte Development. Methods in Molecular Biology, vol 1818. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8603-3_10
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DOI: https://doi.org/10.1007/978-1-4939-8603-3_10
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