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BIOLOGY Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes Ferdusy Dia1, Tierra Strange1, Jenny Liang1, Jacob Hamilton1, Karen M. Berkowitz1,2
1Department of Biochemistry & Molecular Biology, Drexel University College of Medicine, 2Department of Obstetrics & Gynecology, Drexel University College of
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SUMMARY Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.
CITE THIS ARTICLE Copy Citation | Download Citations Dia, F., Strange, T., Liang, J., Hamilton, J., Berkowitz, K. M. Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes. J. Vis. Exp. (129), e55378, doi:10.3791/55378 (2017).
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ABSTRACT Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.
INTRODUCTION Mice are used extensively as a model organism to study meiosis in mammals. Both the normal developmental processes and defects that occur during meiosis can be evaluated. The timeline and progression of salient features that occur during prophase I and substages, as well as a multitude of specific proteins involved in processes crucial to regulation of meiosis can be characterized in both wild type and mutant mice. Several specialized processes that occur during meiosis I can be studied in detail (reviewed in Handel and Schimenti 1). These include DNA double-strand break (DSB) formation and repair, recombination, synaptonemal complex formation, and chromosome segregation. An important aspect of studying meiotic processes is the ability to examine nuclei containing homologous chromosomes that are visually
PROTOCOL All methods involving mice utilized high ethical and welfare standards, and were approved by the Institutional Animal Care and Use Committee at Drexel University College of Medicine. 1. Preparation of solutions and instruments needed 1. Prepare Hypotonic Extraction Buffer (HEB) in 50 mL of ultrapure laboratory grade water, pH 8.2 - 8.4 (Table 1). Make the solution fresh each time, keep on ice, and use within 2 h of preparing spreads; this is necessary to optimize the reducing and protease inhibiting activities of DTT and PMSF, respectively. Use 10 mL of HEB for each pair of mouse testes. 2. Prepare a 100 mM sucrose solution (fresh each time) by adding 0.342
REPRESENTATIVE RESULTS The ability of this method to provide large numbers of surface spread nuclei containing intact homologs depends on three main factors: 1) appropriate incubation time of seminiferous tubules in hypotonic buffer (i.e. HEB) to obtain adequately swelled spermatocyte nuclei that burst but do not disintegrate from prolonged incubation in HEB, 2) micropipetting the sucrose droplets of cells to obtain a separated suspension of nuclei that are not clumped together, and 3) tilting each fixative coated slide in one direction at a time and not back and forth. Once prepared, surface spread nuclei can be immunostained with fluorescently labeled antibodies to proteins of interest and then imaged with microscopy (representative examples are shown in Figure 1 and Figure 2. Figure 1 shows examples of wild type pachytene nuclei that are well spread (A),
DISCUSSION To obtain high numbers of well spread, good quality spermatocyte nuclei, the most critical steps of this protocol include appropriate incubation time of seminiferous tubules in HEB, appropriate mincing of seminiferous tubules, and the technique employed to spread the sucrose cell suspension across the fixative coated slide(s). We incubate seminiferous tubules from testes of wild type males ranging from postnatal day 15 to adulthood in HEB for 45 min, while testes from comparably aged Chtf18null mice are incubated for only 30 min because testes are half the size and contain significantly fewer germ cells8. In our experience, incubation of Chtf18-null testes for longer than 30 min compromises the integrity of the cells, resulting in poorly resolved homologs. Seminiferous tubules are minced only to obtain a cloudy sucrose cell suspension, and each slide
DISCLOSURES The authors have nothing to disclose.
ACKNOWLEDGEMENTS The authors acknowledge the technical assistance of Abigail Harris. They also acknowledge Paula Cohen and Kim Holloway for helping to optimize the protocol of preparing surface-spread nuclei from mouse spermatocytes. The authors also appreciate critical reading of the manuscript by Karen Schindler. This work was supported by NIH R01 GM106262 to K.M.B.
MATERIALS Name
Company
Catalog Number
Photo-Flo 200
Kodak
1464510
Comments
16% Paraformaldehyde Electron (formaldehyde aqueous Microscopy solution) Sciences
RT 15710
Teflon printed 3 ring slides
Electron Microscopy Sciences
63418-11
Premium uncharged frosted end glass slides
Several commercial brands
Surgical scissors (sharp and blunt tips)
Fine Science 14001-12 Different brand of a similar type will work Tools
Dilute 16% solution into 4% working stocks with 1X PBS and freeze aliquots at -20 °C
Clean slides in ethyl or isopropyl alcohol and allow to drip dry prior to use; label slides on frosted end with a permanent marker or pencil depending on subsequent use of slides
Fine tip surgical scissors Fine Science 14058-11 Different brand of a similar type will work (sharp tips) Tools Straight fine tip forceps
Fine Science 11050-10 Different brand of a similar type will work Tools
Curved medium tip forceps
Fisher
Scalpel handle
Fine Science 10003-12 Different brand of a similar type will work Tools
Mouse anti-SYCP3
Abcam
Anti-centromere protein (derived from human CREST patient serum)
Antibodies 15-234Incorporated 0001
16100110 Different brand of a similar type will work
ab97672
Use at 1:300 Use at 1:50 We use Nunc square culture dishes 500 cm2/well with moistened paper towels
Humidified chamber Widefield microscope with epifluorescence
Leica microsystems
Any standard model
Coplin jars
Several commercial brands
50 ml capacity; 40 mm (1.6 in.) diameter, holds 10 slides (back to back)
Petri dishes
Several commercial brands
100 x 15 mm diameter, polystyrene sterile untreated
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REFERENCES 1. Handel, M. A., Schimenti, J. C. Genetics of mammalian meiosis: regulation, dynamics and impact on fertility. Nat Rev Genet. 11, 124136 (2010). 2. 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). 3. Counce, S. J., Meyer, G. F. Differentiation of the synaptonemal complex and the kinetochore in Locusta spermatocytes studied by whole mount electron microscopy. Chromosoma. 44, 231-253 (1973). 4. Speed, R. M. Meiosis in the foetal mouse ovary. I. An analysis at the light microscope level using surface-spreading. Chromosoma. 85, 427-437 (1982). 5. Ashley, T., et al. Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates. Chromosoma. 104, 19-28 (1995). 6. Baker, S. M., et al. Involvement of mouse mLh1 in DNA mismatch repair and meiotic crossing over. Nat Genet. 13, 336-342 (1996). 7. Plug, A. W., Xu, J., Reddy, G., Golub, E. I., Ashley, T. Presynaptic association of Rad51 protein with selected sites in meiotic chromatin. Proc Natl Acad Sci U S A. 93, 5920-5924 (1996). 8. Berkowitz, K. M., et al. Disruption of CHTF18 causes defective meiotic recombination in male mice. PLoS Genet. 8, e1002996 (2012). 9. Gomez, R., et al. Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes. EMBO Rep. 17, 695-707 (2016). 10. Brooker, A. S., Berkowitz, K. M. The roles of cohesins in mitosis, meiosis, and human health and disease. Methods Mol Biol. 1170, 229-266 (2014). 11. McNicoll, F., Stevense, M., Jessberger, R. Cohesin in gametogenesis. Curr Top Dev Biol. 102, 1-34 (2013). 12. Rankin, S. Complex elaboration: making sense of meiotic cohesin dynamics. FEBS J. 282, 2426-2443 (2015). 13. Holloway, J. K., Sun, X., Yokoo, R., Villeneuve, A. M., Cohen, P. E. Mammalian CNTD1 is critical for meiotic crossover maturation and deselection of excess precrossover sites. J Cell Biol. 205, 633-641 (2014). 14. La Salle, S., Sun, F., Handel, M. A. Isolation and short-term culture of mouse spermatocytes for analysis of meiosis. Methods Mol Biol. 558, 279-297 (2009). 15. Wiltshire, T., Park, C., Caldwell, K. A., Handel, M. A. Induced premature G2/M-phase transition in pachytene spermatocytes includes events unique to meiosis. Dev Biol. 169, 557-567 (1995).
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