Analysis of Nucleolar Morphology and Protein Localization as an Indicator of Nuclear Reprogramming

  • Olga Østrup
  • Hanne S. Pedersen
  • Hanne M. Holm
  • Poul HyttelEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1222)


When a cell is reprogrammed to a new phenotype, the nucleolus undergoes more or less dramatic modulations, which can be used as a marker for the occurrence of the reprogramming. This phenomenon is most pronounced when differentiated cells are reprogrammed to totipotency when they are submitted to cloning by somatic cell nuclear transfer. However, when cells are reprogrammed by less fundamental means, as for example treatment by Xenopus extract or expression of pluripotency genes, more subtle nucleolar modulations can also be noted. The monitoring and understanding of the reprogramming-related nucleolar modulations are based upon detailed knowledge about the nucleolar changes that occur during normal development from the developing oocyte over oocyte maturation and fertilization to the activation of the embryonic genome in the early embryo. Below, the ultrastructural and molecular modulations of the nucleolus are summarized in this developmental context, but also as they occur in assisted reproductive technologies such as in vitro fertilization and somatic cell nuclear transfer. Moreover, detailed protocols for monitoring the nucleolar changes by transmission electron microscopy and immunocytochemistry are presented.

Key words

Nucleolus Nucleus Transmission electron microscopy Immunocytochemistry Assisted reproductive technology 


  1. 1.
    Shaw PJ, Jordan EG (1995) The nucleolus. Annu Rev Cell Dev Biol 11:93–121PubMedCrossRefGoogle Scholar
  2. 2.
    Scheer U, Hock R (1999) Structure and function of the nucleolus. Curr Opin Cell Biol 11:385–390PubMedCrossRefGoogle Scholar
  3. 3.
    Wachtler F, Stahl A (1993) The nucleolus: a structural and functional interpretation. Micron 24:473–505CrossRefGoogle Scholar
  4. 4.
    Biggiogera M, Malatesta M, Abolhassani-Dadras S et al (2001) Revealing the unseen: the organizer region of the nucleolus. J Cell Sci 114:3199–3205PubMedGoogle Scholar
  5. 5.
    Koberna K, Malinsky J, Pliss A et al (2002) Ribosomal genes in focus: new transcripts label the dense fibrillar components and form clusters indicative of “Christmas trees” in situ. J Cell Biol 157:743–748PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Flechon JE, Kopecny V (1998) The nature of the “nucleolus precursor body” in early preimplantation embryos: a review of fine-structure cytochemical, immunocytochemical and autoradiographic data related to nucleolar function. Zygote 6:183–191PubMedCrossRefGoogle Scholar
  7. 7.
    Maddox-Hyttel P, Bjerregaard B, Laurincik J (2005) Meiosis and embryo technology: renaissance of the nucleolus. Reprod Fertil Dev 17:3–14PubMedCrossRefGoogle Scholar
  8. 8.
    Hyttel P, Laurincik J, Rosenkranz C et al (2000) Nucleolar proteins and ultrastructure in pre-implantation porcine embryos developed in vivo. Biol Reprod 63:1848–1856PubMedCrossRefGoogle Scholar
  9. 9.
    Laurincik J, Thomsen PD, Hay-Schmidt A et al (2000) Nucleolar proteins and nuclear ultrastructure in pre-implantation bovine embryos produced in vitro. Biol Reprod 62:1024–1032PubMedCrossRefGoogle Scholar
  10. 10.
    Laurincik J, Schmoll F, Mahabir E et al (2003) Nucleolar proteins and ultrastructure in bovine in vivo developed, in vitro produced, and parthenogenetic cleavage-stage embryos. Mol Reprod Dev 65:73–85PubMedCrossRefGoogle Scholar
  11. 11.
    Thompson EM (1996) Chromatin structure and gene expression in the preimplantation mammalian embryo. Reprod Nutr Dev 36:619–635PubMedGoogle Scholar
  12. 12.
    Hay-Schmidt A, Viuff D, Greve T et al (2001) Transcriptional activity in in vivo developed early cleavage stage bovine embryos. Theriogenology 56:167–176PubMedCrossRefGoogle Scholar
  13. 13.
    Ogushi S, Palmieri C, Fulka H et al (2008) The maternal nucleolus is essential for early embryonic development in mammals. Science 319:613–616PubMedCrossRefGoogle Scholar
  14. 14.
    Gavrilova EV, Kuznetsova IS, Enukashivili NI et al (2009) Localization of satellite DNA and associated protein in respect to nucleolar precursor bodies in one- and two-cell mouse embryos. Tsitologiia 51:455–464PubMedGoogle Scholar
  15. 15.
    Fulka H, Fulka J Jr (2010) Nucleolar transplantation in oocytes and zygotes: challenges for further research. Mol Hum Reprod 16:63–67PubMedCrossRefGoogle Scholar
  16. 16.
    Martin C, Beaujean N, Brochard V et al (2006) Genome restructuring in mouse embryos during reprogramming and early development. Dev Biol 292:317–332PubMedCrossRefGoogle Scholar
  17. 17.
    Martin C, Brochard V, Migne C et al (2006) Architectural reorganization of the nuclei upon transfer into oocytes accompanies genome reprogramming. Mol Reprod Dev 73:1102–1111PubMedCrossRefGoogle Scholar
  18. 18.
    Ahmed K, Dehghani H, Rugg-Gunn P et al (2010) Global chromatin architecture reflects pluripotency and lineage commitment in the early mouse embryo. PLoS One 5:e10531PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Pichugin A, Le BD, Adenot P et al (2010) Dynamics of constitutive heterochromation: two contrasted kinetics of genome restructuring in early cloned bovine embryos. Reproduction 139:129–137PubMedCrossRefGoogle Scholar
  20. 20.
    Hyttel P, Laurincik J, Zakhartchenko V et al (2001) Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from embryonic cells. Cloning 3:69–81PubMedCrossRefGoogle Scholar
  21. 21.
    Svarcova O, Strejcek F, Petrovicova I et al (2008) The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos. Mol Reprod Dev 75:1095–1103PubMedCrossRefGoogle Scholar
  22. 22.
    Deshmukh RS, Østrup O, Strejcek F et al (2012) Early aberrations in chromatin dynamics in embryos produced under in vitro conditions. Cell Reprogram 14:225–234PubMedPubMedCentralGoogle Scholar
  23. 23.
    Laurincik J, Bjerregaard B, Strejcek F et al (2004) Nucleolar ultrastructure and protein allocation in in vitro produced porcine embryos. Mol Reprod Dev 86:327–334CrossRefGoogle Scholar
  24. 24.
    Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblasts cultures by defined factors. Cell 126:663–676PubMedCrossRefGoogle Scholar
  25. 25.
    Vierbuchen T, Ostermeier A, Pang ZP et al (2010) Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463:1035–1041PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Østrup O, Hyttel P, Klærke DA et al (2011) Remodeling of ribosomal genes in somatic cells by Xenopus egg extract. Biochem Biophys Res Commun 412:487–493PubMedCrossRefGoogle Scholar
  27. 27.
    Murayama K, Ohmori A, Fujimura H et al (2008) Epigenetic control of rDNA loci in response to intracellular energy status. Cell 133:627–639PubMedCrossRefGoogle Scholar
  28. 28.
    Preuss S, Pikaard CC (2007) rRNA gene silencing and nucleolar dominance: insights into a chromosome-scale epigenetic on/off switch. Biochim Biophys Acta 1769:383–392PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Santoro R, Grummt I (2005) Epigenetic mechanism of rRNA gene silencing: temporal order of NoRC-mediated histone modification chromatin remodeling and DNA methylation. Mol Cell Biol 25:2539–2546PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Glauert AM (1975) Fixation, dehydration and embedding of biological specimens. American Elsevier, New YorkGoogle Scholar
  31. 31.
    Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Olga Østrup
    • 1
  • Hanne S. Pedersen
    • 2
  • Hanne M. Holm
    • 3
  • Poul Hyttel
    • 3
    Email author
  1. 1.Department of Tumor BiologyOslo University Hospital RHOsloNorway
  2. 2.Department of Animal ScienceAarhus UniversityAarhusDenmark
  3. 3.Department of Clinical Veterinary and Animal SciencesUniversity of CopenhagenFrederiksbergDenmark

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