Cytological Monitoring of Meiotic Crossovers in Spermatocytes and Oocytes

  • Yan Yun
  • Masaru Ito
  • Sumit Sandhu
  • Neil HunterEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2153)


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 



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.


  1. 1.
    Hunter N (2015) Meiotic recombination: the essence of heredity. Cold Spring Harb Perspect Biol 7(12):a016618. Scholar
  2. 2.
    Lake CM, Hawley RS (2016) Becoming a crossover-competent DSB. Semin Cell Dev Biol 54:117–125. Scholar
  3. 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. Scholar
  4. 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. Scholar
  5. 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. Scholar
  6. 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. Scholar
  7. 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. Scholar
  8. 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. Scholar
  9. 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. Scholar
  10. 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):336–342CrossRefGoogle Scholar
  11. 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):1573–1582CrossRefGoogle Scholar
  12. 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. Scholar
  13. 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):1569–1579PubMedPubMedCentralGoogle Scholar
  14. 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. Scholar
  15. 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. Scholar
  16. 16.
    Hulten MA (2011) On the origin of crossover interference: a chromosome oscillatory movement (COM) model. Mol Cytogenet 4:10. Scholar
  17. 17.
    Jones GH, Franklin FC (2006) Meiotic crossing-over: obligation and interference. Cell 126(2):246–248. Scholar
  18. 18.
    de Kretser DM, Loveland KL, Meinhardt A, Simorangkir D, Wreford N (1998) Spermatogenesis. Hum Reprod 13(Suppl 1):1–8. Scholar
  19. 19.
    Hunter N (2017) Oocyte quality control: causes, mechanisms, and consequences. Cold Spring Harb Symp Quant Biol 82:235–247. Scholar
  20. 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):398–426CrossRefGoogle Scholar
  21. 21.
    Chambon JP, Hached K, Wassmann K (2013) Chromosome spreads with centromere staining in mouse oocytes. Methods Mol Biol 957:203–212. Scholar
  22. 22.
    MacLennan M, Crichton JH, Playfoot CJ, Adams IR (2015) Oocyte development, meiosis and aneuploidy. Semin Cell Dev Biol 45:68–76. Scholar
  23. 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. Scholar

Copyright information

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

Authors and Affiliations

  • Yan Yun
    • 1
  • Masaru Ito
    • 1
  • Sumit Sandhu
    • 1
  • Neil Hunter
    • 1
    Email author
  1. 1.Howard Hughes Medical Institute, Department of Microbiology and Molecular Genetics, DavisUniversity of CaliforniaDavisUSA

Personalised recommendations