Skip to main content

Cytological Monitoring of Meiotic Crossovers in Spermatocytes and Oocytes

Part of the Methods in Molecular Biology book series (MIMB,volume 2153)

Abstract

Crossing-over between homologous chromosomes is essential for accurate chromosome segregation at anaphase-I of meiosis. Defective crossing-over is associated with infertility, pregnancy miscarriage, and congenital disease. This chapter presents optimized protocols for the analysis of meiotic crossovers at the cytological level in spermatocytes and oocytes from mouse. The first approach employs immunocytology to detect MLH1, a DNA mismatch-repair protein that specifically marks crossover sites in the pachytene stage of meiotic prophase-I. These immunocytological methods have general utility for the analysis of other recombination steps, such as initiation and DNA strand exchange. The second approach visualizes chiasmata, the points of physical exchange between homologous chromosomes that are present during the diakinesis and metaphase-I stages. Both approaches are readily adaptable to the analysis of crossing over in other vertebrate species.

Key words

  • Meiosis
  • Homologous recombination
  • Crossover
  • Chiasma
  • Chromosome spread
  • Spermatocytes
  • Oocytes
  • Aneuploidy
  • MLH1

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

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-0644-5_19
  • Chapter length: 20 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   149.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-0644-5
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   199.99
Price excludes VAT (USA)
Hardcover Book
USD   279.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Hunter N (2015) Meiotic recombination: the essence of heredity. Cold Spring Harb Perspect Biol 7(12):a016618. https://doi.org/10.1101/cshperspect.a016618

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  2. Lake CM, Hawley RS (2016) Becoming a crossover-competent DSB. Semin Cell Dev Biol 54:117–125. https://doi.org/10.1016/j.semcdb.2016.01.008

    CrossRef  PubMed  Google Scholar 

  3. Herbert M, Kalleas D, Cooney D, Lamb M, Lister L (2015) Meiosis and maternal aging: insights from aneuploid oocytes and trisomy births. Cold Spring Harb Perspect Biol 7(4):a017970. https://doi.org/10.1101/cshperspect.a017970

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  4. Nagaoka SI, Hassold TJ, Hunt PA (2012) Human aneuploidy: mechanisms and new insights into an age-old problem. Nat Rev Genet 13(7):493–504. https://doi.org/10.1038/nrg3245

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  5. Qiao H, Prasada Rao HB, Yang Y, Fong JH, Cloutier JM, Deacon DC, Nagel KE, Swartz RK, Strong E, Holloway JK, Cohen PE, Schimenti J, Ward J, Hunter N (2014) Antagonistic roles of ubiquitin ligase HEI10 and SUMO ligase RNF212 regulate meiotic recombination. Nat Genet 46(2):194–199. https://doi.org/10.1038/ng.2858

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Reynolds A, Qiao H, Yang Y, Chen JK, Jackson N, Biswas K, Holloway JK, Baudat F, de Massy B, Wang J, Höög C, Cohen PE, Hunter N (2013) RNF212 is a dosage-sensitive regulator of crossing-over during mammalian meiosis. Nat Genet 45(3):269–278. https://doi.org/10.1038/ng.2541

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Susiarjo M, Hassold TJ, Freeman E, Hunt PA (2007) Bisphenol A exposure in utero disrupts early oogenesis in the mouse. PLoS Genet 3(1):e5. https://doi.org/10.1371/journal.pgen.0030005

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  8. Kong A, Thorleifsson G, Frigge ML, Masson G, Gudbjartsson DF, Villemoes R, Magnusdottir E, Olafsdottir SB, Thorsteinsdottir U, Stefansson K (2014) Common and low-frequency variants associated with genome-wide recombination rate. Nat Genet 46(1):11–16. https://doi.org/10.1038/ng.2833

    CAS  CrossRef  PubMed  Google Scholar 

  9. Gely-Pernot A, Saci S, Kernanec PY, Hao C, Giton F, Kervarrec C, Tevosian S, Mazaud-Guittot S, Smagulova F (2017) Embryonic exposure to the widely-used herbicide atrazine disrupts meiosis and normal follicle formation in female mice. Sci Rep 7(1):3526. https://doi.org/10.1038/s41598-017-03738-1

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  10. Baker SM, Plug AW, Prolla TA, Bronner CE, Harris AC, Yao X, Christie DM, Monell C, Arnheim N, Bradley A, Ashley T, Liskay RM (1996) Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over. Nat Genet 13(3):336–342

    CAS  CrossRef  Google Scholar 

  11. Hunter N, Borts RH (1997) Mlh1 is unique among mismatch repair proteins in its ability to promote crossing-over during meiosis. Genes Dev 11(12):1573–1582

    CAS  CrossRef  Google Scholar 

  12. Lhuissier FG, Offenberg HH, Wittich PE, Vischer NO, Heyting C (2007) The mismatch repair protein MLH1 marks a subset of strongly interfering crossovers in tomato. Plant Cell 19(3):862–876. https://doi.org/10.1105/tpc.106.049106

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  13. Anderson LK, Reeves A, Webb LM, Ashley T (1999) Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein. Genetics 151(4):1569–1579

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Gruhn JR, Rubio C, Broman KW, Hunt PA, Hassold T (2013) Cytological studies of human meiosis: sex-specific differences in recombination originate at, or prior to, establishment of double-strand breaks. PLoS One 8(12):e85075. https://doi.org/10.1371/journal.pone.0085075

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  15. Guillon H, Baudat F, Grey C, Liskay RM, de Massy B (2005) Crossover and noncrossover pathways in mouse meiosis. Mol Cell 20(4):563–573. https://doi.org/10.1016/j.molcel.2005.09.021

    CAS  CrossRef  PubMed  Google Scholar 

  16. Hulten MA (2011) On the origin of crossover interference: a chromosome oscillatory movement (COM) model. Mol Cytogenet 4:10. https://doi.org/10.1186/1755-8166-4-10

    CrossRef  PubMed  PubMed Central  Google Scholar 

  17. Jones GH, Franklin FC (2006) Meiotic crossing-over: obligation and interference. Cell 126(2):246–248. https://doi.org/10.1016/j.cell.2006.07.010

    CAS  CrossRef  PubMed  Google Scholar 

  18. de Kretser DM, Loveland KL, Meinhardt A, Simorangkir D, Wreford N (1998) Spermatogenesis. Hum Reprod 13(Suppl 1):1–8. https://doi.org/10.1093/humrep/13.suppl_1.1

    CrossRef  PubMed  Google Scholar 

  19. Hunter N (2017) Oocyte quality control: causes, mechanisms, and consequences. Cold Spring Harb Symp Quant Biol 82:235–247. https://doi.org/10.1101/sqb.2017.82.035394

    CrossRef  PubMed  Google Scholar 

  20. Cohen PE, Pollack SE, Pollard JW (2006) Genetic analysis of chromosome pairing, recombination, and cell cycle control during first meiotic prophase in mammals. Endocr Rev 27(4):398–426

    CAS  CrossRef  Google Scholar 

  21. Chambon JP, Hached K, Wassmann K (2013) Chromosome spreads with centromere staining in mouse oocytes. Methods Mol Biol 957:203–212. https://doi.org/10.1007/978-1-62703-191-2_14

    CAS  CrossRef  PubMed  Google Scholar 

  22. MacLennan M, Crichton JH, Playfoot CJ, Adams IR (2015) Oocyte development, meiosis and aneuploidy. Semin Cell Dev Biol 45:68–76. https://doi.org/10.1016/j.semcdb.2015.10.005

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  23. Zelazowski MJ, Sandoval M, Paniker L, Hamilton HM, Han J, Gribbell MA, Kang R, Cole F (2017) Age-dependent alterations in meiotic recombination cause chromosome segregation errors in spermatocytes. Cell 171(3):601–614. e613. https://doi.org/10.1016/j.cell.2017.08.042

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

M.I. was supported by a Japan Society for the Promotion of Science postdoctoral fellowship for research abroad. S.S. was supported by an A.P. Giannini Foundation postdoctoral fellowship. N.H. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Neil Hunter .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Yun, Y., Ito, M., Sandhu, S., Hunter, N. (2021). Cytological Monitoring of Meiotic Crossovers in Spermatocytes and Oocytes. In: Aguilera, A., Carreira, A. (eds) Homologous Recombination. Methods in Molecular Biology, vol 2153. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0644-5_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0644-5_19

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0643-8

  • Online ISBN: 978-1-0716-0644-5

  • eBook Packages: Springer Protocols