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Free-Hand Bisection of Mouse Oocytes and Embryos

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Book cover Mammalian Oocyte Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 957))

Abstract

Mouse oocytes and zygotes are semitransparent and large cells approximately 80 μm in diameter. Bisection is one of the easiest ways for performing micromanipulations on such cells. It allows living sister halves or smaller fragments to be obtained, which can be cultured and observed for long periods of time. Bisection can be used for different kinds of experiments such as analysis of nucleo-cytoplasmic interactions, the relationship between different cellular structures or between different parts of embryos, eventually for analyzing the developmental potential of embryonic fragments. Oocyte or embryo halves can be examined by immunostaining, by measuring different cellular functions and by Western blot and genetic analysis (e.g., RT-PCR). Here we describe a detailed protocol for the free-hand bisection of mouse zona pellucida-free oocytes and embryos on an agar layer using a glass needle.

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References

  1. Tarkowski AK, Rossant J (1976) Haploid mouse blastocysts developed from bisected zygotes. Nature 259:663–665

    Article  CAS  PubMed  Google Scholar 

  2. Tarkowski AK (1977) In vitro development of haploid mouse embryos produced by bisection of one-cell fertilized eggs. J Embryol Exp Morphol 38:187–202

    Google Scholar 

  3. Bałakier H, Tarkowski AK (1980) The role of germinal vesicle karyoplasm in the development of male pronucleus in the mouse. Exp Cell Res 128:79–85

    Article  PubMed  Google Scholar 

  4. Tarkowski AK (1980) Fertilization of nucleate and anucleate egg fragments in the mouse. Exp Cell Res 128:73–77

    Article  CAS  PubMed  Google Scholar 

  5. Borsuk E, Mańka R (1988) Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid. Gamete Res 20:365–376

    Article  CAS  PubMed  Google Scholar 

  6. Borsuk E, Tarkowski AK (1989) Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs. Gamete Res 24:471–481

    Article  CAS  PubMed  Google Scholar 

  7. Balakier H, Czolowska R (1977) Cytoplasmic control of nuclear maturation in mouse oocytes. Exp Cell Res 110:466–469

    Article  CAS  PubMed  Google Scholar 

  8. Bałakier H, Masui Y (1986) Chromosome condensation activity in the cytoplasm of anucleate and nucleate fragments of mouse oocytes. Dev Biol 113:155–159

    Article  PubMed  Google Scholar 

  9. Clarke HJ, Masui Y (1987) Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes. J Cell Biol 104:831–840

    Article  CAS  PubMed  Google Scholar 

  10. Borsuk E (1991) Anucleate fragments of parthenogenetic eggs and of maturing oocytes contain complementary factors required for development of a male pronucleus. Mol Reprod Dev 29:150–156

    Article  CAS  PubMed  Google Scholar 

  11. Fulka J Jr, Ouhibi N, Fulka J, Kanka J, Moor RM (1995) Chromosome condensation activity (CCA) in bisected C57BL/6JxCBA mouse oocytes. Reprod Fertil Dev 7:1123–1127

    Article  PubMed  Google Scholar 

  12. Hoffmann S, Tsurumi C, Kubiak JZ, Polanski Z (2006) Germinal vesicle material drives meiotic cell cycle of mouse oocyte through the 3′UTR-dependent control of cyclin B1 synthesis. Dev Biol 292:46–54

    Article  CAS  PubMed  Google Scholar 

  13. Kárníková L, Urban F, Moor R, Fulka J Jr (1998) Mouse oocyte maturation: the effect of modified nucleocytoplasmic ratio. Reprod Nutr Dev 38:665–670

    Article  PubMed  Google Scholar 

  14. Kubiak JZ, Weber M, de Pennart H, Winston NJ, Maro B (1993) The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF. EMBO J 12:3773–3778

    CAS  PubMed  Google Scholar 

  15. Polanski Z (1997) Strain difference in the timing of meiosis resumption in mouse oocytes: involvement of a cytoplasmic factor(s) acting presumably upstream of the dephosphorylation of p34cdc2 kinase. Zygote 5:105–109

    Article  CAS  PubMed  Google Scholar 

  16. Polanski Z, Ledan E, Brunet S, Louvet S, Verlhac MH, Kubiak JZ, Maro B (1998) Cyclin synthesis controls the progression of meiotic maturation in mouse oocytes. Development 125:4989–4997

    CAS  PubMed  Google Scholar 

  17. Zernicka-Goetz M, Ciemerych MA, Kubiak JZ, Tarkowski AK, Maro B (1995) Cytostatic factor inactivation is induced by a calcium-dependent mechanism present until the second cell cycle in fertilized but not in parthenogenetically activated mouse eggs. J Cell Sci 108:469–474

    CAS  PubMed  Google Scholar 

  18. Kubiak JZ, Tarkowski AK (1985) Electrofusion of mouse blastomeres. Exp Cell Res 157:561–566

    Article  CAS  PubMed  Google Scholar 

  19. Ciemerych MA, Tarkowski AK, Kubiak JZ (1998) Autonomous activation of histone H1 kinase, cortical activity and microtubule organization in one- and two-cell mouse embryos. Biol Cell 90:557–564

    Article  CAS  PubMed  Google Scholar 

  20. Petzoldt U (1990) Survival of maternal mRNA in anucleate and unfertilized mouse eggs. Eur J Cell Biol 52:123–128

    CAS  PubMed  Google Scholar 

  21. Petzoldt U, Muggleton-Harris A (1987) The effect of the nucleocytoplasmic ratio on protein synthesis and expression of a stage-specific antigen in early cleaving mouse embryos. Development 99:481–491

    CAS  PubMed  Google Scholar 

  22. Brunet S, Polanski Z, Verlhac MH, Kubiak JZ, Maro B (1998) Bipolar meiotic spindle formation without chromatin. Curr Biol 8:1231–1234

    Article  CAS  PubMed  Google Scholar 

  23. Pahlavan G, Polanski Z, Kalab P, Golsteyn R, Nigg EA, Maro B (2000) Characterization of polo-like kinase 1 during meiotic maturation of the mouse oocyte. Dev Biol 220:392–400

    Article  CAS  PubMed  Google Scholar 

  24. Hoffmann S, Maro B, Kubiak JZ, Polanski Z (2011) A single bivalent efficiently inhibits cyclin B1 degradation and polar body extrusion in mouse oocytes indicating robust SAC during female meiosis I. PLoS One 6:e27143

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Ciemerych MA, Mesnard D, Zernicka-Goetz M (2000) Animal and vegetal poles of the mouse egg predict the polarity of the embryonic axis, yet are nonessential for development. Development 127:3467–3474

    CAS  PubMed  Google Scholar 

  26. Zernicka-Goetz M (1998) Fertile offspring derived from mammalian eggs lacking either animal or vegetal poles. Development 125:4803–4808

    CAS  PubMed  Google Scholar 

  27. Modlinski JA (1978) Transfer of embryonic nuclei to fertilised mouse eggs and development of tetraploid blastocysts. Nature 273:466–467

    Article  CAS  PubMed  Google Scholar 

  28. McGrath J, Solter D (1983) Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 220:1300–1302

    Article  CAS  PubMed  Google Scholar 

  29. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385:810–813

    Article  CAS  PubMed  Google Scholar 

  30. Vajta G, Lewis IM, Hyttel P, Thouas GA, Trounson AO (2001) Somatic cell cloning without micromanipulators. Cloning 3:89–95

    Article  CAS  PubMed  Google Scholar 

  31. De Felici M, Siracusa G (1982) Spontaneous hardening of the zona pellucida of mouse oocytes during in vitro culture. Gamete Res 6:107–113

    Article  Google Scholar 

  32. Komorowski S, Szczepanska K, Maleszewski M (2003) Distinct mechanisms underlie sperm-induced and protease-induced oolemma block to sperm penetration. Int J Dev Biol 47:65–69

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Malgorzata Kloc for valuable discussions and critical reading of the manuscript. This work was supported by grants from ARC and LCC to JZK.

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Authors and Affiliations

  1. Department of Genetics and Evolution, Institute of Zoology, Jagiellonian University, Cracow, Poland

    Zbigniew Polanski

  2. Cell Cycle Group, CNRS, UMR 6290, Institute of Genetics and Development of Rennes (IGDR), Rennes, France

    Jacek Z. Kubiak

  3. Faculty of Medicine, University Rennes 1, UEB, UMS 3480, Rennes, France

    Jacek Z. Kubiak

Authors
  1. Zbigniew Polanski
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  2. Jacek Z. Kubiak
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Corresponding author

Correspondence to Jacek Z. Kubiak .

Editor information

Editors and Affiliations

  1. Dept. of Cell & Developmental Biology, University College London Institute for Women's Health, London, United Kingdom

    Hayden A. Homer

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Polanski, Z., Kubiak, J.Z. (2013). Free-Hand Bisection of Mouse Oocytes and Embryos. In: Homer, H. (eds) Mammalian Oocyte Regulation. Methods in Molecular Biology, vol 957. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-191-2_18

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  • DOI: https://doi.org/10.1007/978-1-62703-191-2_18

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-190-5

  • Online ISBN: 978-1-62703-191-2

  • eBook Packages: Springer Protocols

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