Skip to main content

Sperm and Oocyte Isolation Methods for Biochemical and Proteomic Analysis

  • Protocol
C. elegans

Part of the book series: Methods in Molecular Biology ((MIMB,volume 351))

  • 3586 Accesses

Abstract

The Caenorhabditis elegans gonad is a simple model to investigate molecular mechanisms that regulate fundamental cell and developmental processes. The strength of the model is that C. elegans is amenable to genetic manipulation. The complete genome sequence, advances in mass spectrometry, and RNA-mediated interference (RNAi) are now providing a technical infrastructure that complements biochemical and proteomic approaches. This chapter describes simple methods for sperm and oocyte isolation and gonad liberation that can be performed routinely in the lab, without expensive equipment. These methods are ideal for biochemical and proteomic applications, including those aiming to identify proteins based on affinity or biological activity. Germline mRNA expression profiles, RNAi feeding clones, and Gateway®-engineered ORFeome vectors are available to help validate experimental results.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Johnson, R. S., Davis, M. T., Taylor, J. A., and Patterson, S. D. (2005) Informatics for protein identification by mass spectrometry. Methods 35, 223–236.

    Article  PubMed  CAS  Google Scholar 

  2. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858.

    Article  PubMed  CAS  Google Scholar 

  3. Miller, M. A., Nguyen, V. Q., Lee, M. H., et al. (2001) A sperm cytoskeletal protein that signals oocyte meiotic maturation and ovulation. Science 291, 2144–2177.

    Article  PubMed  CAS  Google Scholar 

  4. Cheeseman, I. M., Niessen, S., Anderson, S., et al. (2004) A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension. Genes Dev. 18, 2255–2268.

    Article  PubMed  CAS  Google Scholar 

  5. Krijgsveld, J., Ketting, R. F., Mahmoudi, T., et al. (2003) Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics. Nat. Biotechnol. 21, 927–931.

    Article  PubMed  CAS  Google Scholar 

  6. Praitis, V., Casey, E., Collar, D., and Austin, J. (2001) Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 1217–1226.

    PubMed  CAS  Google Scholar 

  7. Reinke, V., Smith, H. E., Nance, J., et al. (2000) A global profile of germline gene expression in C. elegans. Mol. Cell. 6, 605–616.

    Article  PubMed  CAS  Google Scholar 

  8. Reinke, V., Gil, I. S., Ward, S., and Kazmer, K. (2004) Genome-wide germline enriched and sex-biased expression profiles in Caenorhabditis elegans. Development 131, 311–323.

    Article  PubMed  CAS  Google Scholar 

  9. Kamath, R. S., Fraser, A. G., Dong, Y., et al. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237.

    Article  PubMed  CAS  Google Scholar 

  10. Li, S., Armstrong, C. M., Bertin, N., Ge, H., Milstein, S., Boxem, M., Vidalain, P.O., Han, J. D., Chesneau, A., et al. (2004) A map of the interactome network of the metazoan C. elegans. Science 303, 540–543.

    Article  PubMed  CAS  Google Scholar 

  11. Reboul, J., Vaglio, P., Rual, J. F., et al. (2003) C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteomescale protein expression. Nat. Genet. 34, 35–41.

    Article  PubMed  Google Scholar 

  12. Klass, M. R. and Hirsh, D. (1981) Sperm isolation and biochemical analysis of the major sperm protein from C. elegans. Dev. Biol. 84, 299–312.

    Article  PubMed  CAS  Google Scholar 

  13. Schedl, T. and Kimble, J. (1988) fog-2, a germ-line-specific sex determination gene required for hermaphrodite spermatogenesis in Caenorhabditis elegans. Genetics 119, 43–61.

    PubMed  CAS  Google Scholar 

  14. Stroeher, V. L., Kennedy, B. P., Millen, K. J., et al. (1994) DNA-protein interactions in the Caenorhabditis elegans embryo: oocyte and embryonic factors that bind to the promoter of the gut-specific ges-1 gene. Dev. Biol. 163, 367–380.

    Article  PubMed  CAS  Google Scholar 

  15. Aroian, R. V., Field, C., Pruliere, G., Kenyon, C., and Alberts, B. M. (1997) Isolation of actin-associated proteins from Caenorhabditis elegans oocytes and their localization in the early embryo. Embo. J. 16, 1541–1549.

    Article  PubMed  CAS  Google Scholar 

  16. Ward, S. and Miwa, J. (1978) Characterization of temperature-sensitive, fertilization-defective mutants of the nematode Caenorhabditis elegans. Genetics 88, 285–303.

    PubMed  CAS  Google Scholar 

  17. Doniach, T. and Hodgkin, J. (1984) A sex-determining gene, fem-1, required for both male and hermaphrodite development in Caenorhabditis elegans. Dev. Biol. 106, 223–235.

    Article  PubMed  CAS  Google Scholar 

  18. Beanan, M. J. and Strome, S. (1992) Characterization of a germ-line proliferation mutation in C. elegans. Development 116, 755–766.

    PubMed  CAS  Google Scholar 

  19. Barton, M. K., Schedl, T. B., and Kimble, J. (1987) Gain-of-function mutations of fem-3, a sex-determination gene in Caenorhabditis elegans. Genetics 115, 107–119.

    PubMed  CAS  Google Scholar 

  20. Ward, S., Hogan, E., and Nelson, G. A. (1983) The initiation of spermiogenesis in the nematode Caenorhabditis elegans. Dev. Biol. 98, 70–79.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Humana Press Inc.

About this protocol

Cite this protocol

Miller, M.A. (2006). Sperm and Oocyte Isolation Methods for Biochemical and Proteomic Analysis. In: Strange, K. (eds) C. elegans. Methods in Molecular Biology, vol 351. Humana Press. https://doi.org/10.1385/1-59745-151-7:193

Download citation

  • DOI: https://doi.org/10.1385/1-59745-151-7:193

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-597-2

  • Online ISBN: 978-1-59745-151-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics