Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos

Nature Protocols volume 6, pages 270–284 (2011)Cite this article

Subjects

Abstract

Dynamic reprogramming of the genome takes place during the gamete-to-embryo transition. This transition defines a period of continuous and global change but has been difficult to study because of extremely limited material and varying degrees of chromatin compaction. Improved methods of detecting chromatin and gene expression changes in the germ line and in the preimplantation embryo would greatly enhance the understanding of this crucial developmental transition. Here we describe a protocol developed and used by us that improves the sensitivity of existing fluorescence in situ hybridization (FISH) methods; the protocol described here has enabled us to visualize single-copy DNA targets and corresponding nascent RNA transcripts in preimplantation embryos and during spermatogenesis. Major improvements over alternative methods involve fixation and permeabilization steps. Chromatin epitopes can be visualized simultaneously by combining FISH with immunofluorescence; multicopy and repetitive element expression can also be reliably measured. This procedure (sample preparation and staining) requires 1–1.5 d to complete and will facilitate detailed examination of spatial relationships between chromatin epitopes, DNA and RNA during the dynamic transition from gamete to embryo.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Hypotonic treatment compromises nuclear architecture.
Figure 2: Order of fixation and permeabilization affects FISH analysis of preimplantation embryos.
Figure 3
Figure 4: Preparation of male germ cell and preimplantation embryo samples.
Figure 5: Combined RNA-immunoFISH experiment.

Similar content being viewed by others

References

  1. Youngson, N.A. & Whitelaw, E. Transgenerational epigenetic effects. Annu. Rev. Genomics Hum. Genet. 9, 233–257 (2008).

    Article  CAS  Google Scholar 

  2. Carrell, D.T. & Hammoud, S.S. The human sperm epigenome and its potential role in embryonic development. Mol. Hum. Reprod. 16, 37–47 (2010).

    Article  CAS  Google Scholar 

  3. Cooper, D.W. Directed genetic change model for X chromosome inactivation in eutherian mammals. Nature 230, 292–294 (1971).

    Article  CAS  Google Scholar 

  4. Lyon, M.F. Imprinting and X chromosome inactivation In Results and Problems in Cell Differentiation (ed. Ohlsson, R.) 73–90 (Springer-Verlag, 1999).

  5. McCarrey, J.R. X-chromosome inactivation during spermatogenesis: the original dosage compensation mechanism in mammals? In Gene Families: Studies of DNA, RNA, Enzymes, and Proteins (eds. Xue, G. et al.) 59–72 (World Scientific Publishing Co., 2001).

  6. Huynh, K.D. & Lee, J.T. Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos. Nature 426, 857–862 (2003).

    Article  CAS  Google Scholar 

  7. Huynh, K.D. & Lee, J.T. X-chromosome inactivation: a hypothesis linking ontogeny and phylogeny. Nat. Rev. Genet. 6, 410–418 (2005).

    Article  CAS  Google Scholar 

  8. Hammoud, S.S. et al. Distinctive chromatin in human sperm packages genes for embryo development. Nature 460, 473–478 (2009).

    Article  CAS  Google Scholar 

  9. Arpanahi, A. et al. Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences. Genome Res. 19, 1338–1349 (2009).

    Article  CAS  Google Scholar 

  10. Brykczynska, U. et al. Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. Nat. Struct. Mol. Biol. 17, 679–687 (2010).

    Article  CAS  Google Scholar 

  11. Darzacq, X. et al. Imaging transcription in living cells. Annu. Rev. Biophys. 38, 173–196 (2009).

    Article  CAS  Google Scholar 

  12. Clemson, C.M. et al. An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol. Cell 33, 717–726 (2009).

    Article  CAS  Google Scholar 

  13. Cremer, T. & Cremer, M. Chromosome territories. Cold Spring Harb. Perspect Biol. 2, a003889 (2010).

    Article  Google Scholar 

  14. Zhao, R., Bodnar, M.S. & Spector, D.L. Nuclear neighborhoods and gene expression. Curr. Opin. Genet. Dev. 19, 172–179 (2009).

    Article  CAS  Google Scholar 

  15. Namekawa, S.H. et al. Postmeiotic sex chromatin in the male germline of mice. Curr. Biol. 16, 660–667 (2006).

    Article  CAS  Google Scholar 

  16. Namekawa, S.H., Payer, B., Huynh, K.D., Jaenisch, R. & Lee, J.T. Two-step imprinted X inactivation: repeat versus genic silencing in the mouse. Mol. Cell Biol. 30, 3187–3205 (2010).

    Article  CAS  Google Scholar 

  17. Turner, J.M., Mahadevaiah, S.K., Ellis, P.J., Mitchell, M.J. & Burgoyne, P.S. Pachytene asynapsis drives meiotic sex chromosome inactivation and leads to substantial postmeiotic repression in spermatids. Dev. Cell 10, 521–529 (2006).

    Article  CAS  Google Scholar 

  18. Okamoto, I. et al. Evidence for de novo imprinted X-chromosome inactivation independent of meiotic inactivation in mice. Nature 438, 369–373 (2005).

    Article  CAS  Google Scholar 

  19. Okamoto, I., Otte, A.P., Allis, C.D., Reinberg, D. & Heard, E. Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303, 644–649 (2004).

    Article  CAS  Google Scholar 

  20. Panning, B. X inactivation in mouse ES cells: histone modifications and FISH. Methods Enzymol. 376, 419–428 (2004).

    Article  CAS  Google Scholar 

  21. Erwin, J.A. & Lee, J.T. Characterization of X-chromosome inactivation status in human pluripotent stem cells. Curr. Protoc. Stem Cell Biol. Chapter 1 Unit 1B 6 (2010).

  22. Peters, A.H., Plug, A.W., van Vugt, M.J. & de Boer, P. A drying-down technique for the spreading of mammalian meiocytes from the male and female germline. Chromosome Res. 5, 66–68 (1997).

    Article  CAS  Google Scholar 

  23. Barlow, A.L., Benson, F.E., West, S.C. & Hulten, M.A. Distribution of the Rad51 recombinase in human and mouse spermatocytes. EMBO J. 16, 5207–5215 (1997).

    Article  CAS  Google Scholar 

  24. Hall, L.L. et al. An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells. Proc. Natl. Acad. Sci. USA 99, 8677–8682 (2002).

    Article  CAS  Google Scholar 

  25. Namekawa, S.H., VandeBerg, J.L., McCarrey, J.R. & Lee, J.T. Sex chromosome silencing in the marsupial male germ line. Proc. Natl. Acad. Sci. USA 104, 9730–9735 (2007).

    Article  CAS  Google Scholar 

  26. Greaves, I.K., Rangasamy, D., Devoy, M., Marshall Graves, J.A. & Tremethick, D.J. The X and Y chromosomes assemble into H2A.Z, containing facultative heterochromatin, following meiosis. Mol. Cell Biol. 26, 5394–5405 (2006).

    Article  CAS  Google Scholar 

  27. Khalil, A.M., Boyar, F.Z. & Driscoll, D.J. Dynamic histone modifications mark sex chromosome inactivation and reactivation during mammalian spermatogenesis. Proc. Natl. Acad. Sci. USA 101, 16583–16587 (2004).

    Article  CAS  Google Scholar 

  28. Okamoto, I. et al. Evaluating the role of inducible nitric oxide synthase using a novel and selective inducible nitric oxide synthase inhibitor in septic lung injury produced by cecal ligation and puncture. Am. J. Respir. Crit. Care Med. 162 (2 Pt 1): 716–722 (2000).

    Article  CAS  Google Scholar 

  29. Ohno, M., Aoki, N. & Sasaki, H. Allele-specific detection of nascent transcripts by fluorescence in situ hybridization reveals temporal and culture-induced changes in Igf2 imprinting during pre-implantation mouse development. Genes Cells 6, 249–259 (2001).

    Article  CAS  Google Scholar 

  30. Silva, J. et al. Establishment of histone h3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes. Dev. Cell 4, 481–495 (2003).

    Article  CAS  Google Scholar 

  31. Patrat, C. et al. Dynamic changes in paternal X-chromosome activity during imprinted X-chromosome inactivation in mice. Proc. Natl. Acad. Sci. USA 106, 5198–5203 (2009).

    Article  CAS  Google Scholar 

  32. Nagy, A., Gertsenstein, M., Vintersen, K. & Behringer, R. Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, 2003).

  33. Beatty, B., Mai, S. & Squire, J. FISH. A Practical Approach (Oxford University Press, 2002).

  34. Zhang, L.F., Huynh, K.D. & Lee, J.T. Perinucleolar targeting of the inactive X during S phase: evidence for a role in the maintenance of silencing. Cell 129, 693–706 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Hyunh, B. Payer and L.-F. Zhang for their assistance to optimize experimental conditions during the initial phases of these works. The studies have been supported by National Institutes of Health grant RO1-GM58839 to J.T.L. S.H.N. was supported by the research fellowships of the Japan Society for the Promotion of Science (JSPS) and the Charles King Trust. J.T.L. is an Investigator of the Howard Hughes Medical Institute.

Author information

Author notes

  1. Satoshi H Namekawa

    Present address: Present address: Division of Reproductive Sciences, Department of Pediatrics, Perinatal Institute, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.,

Authors and Affiliations

  1. Department of Molecular Biology, Massachusetts General Hospital, Howard Hughes Medical Institute, Boston

    Satoshi H Namekawa & Jeannie T Lee

  2. Massachusetts, USA

    Satoshi H Namekawa & Jeannie T Lee

  3. Department of Genetics, Harvard Medical School, Boston

    Satoshi H Namekawa & Jeannie T Lee

  4. Massachusetts, USA

    Satoshi H Namekawa & Jeannie T Lee

Authors
  1. Satoshi H Namekawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeannie T Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.H.N. and J.T.L. wrote the manuscript. S.H.N. designed the protocols, with guidance from lab protocols, and J.T.L. and S.H.N. conducted the experiments. J.T.L. supervised the projects.

Corresponding authors

Correspondence to Satoshi H Namekawa or Jeannie T Lee.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Namekawa, S., Lee, J. Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos. Nat Protoc 6, 270–284 (2011). https://doi.org/10.1038/nprot.2010.195

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.195

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing