Skip to main content
SpringerLink
Log in

Isolation and Short-Term Culture of Mouse Spermatocytes for Analysis of Meiosis

  • Protocol
  • First Online:
Meiosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 558))

Abstract

Understanding meiosis is facilitated by in vitro experimental approaches, but this has not been easily applicable to mammalian meiocytes. Available methods for in vitro analysis of mammalian oocytes are generally limited to experimental analysis of the late prophase period. Short-term cultures of male germ cells have been useful for analysis of earlier meiotic prophase pathways, as well as onset of the meiotic division phase, but no studies have achieved reliable spermatogenesis in vitro. Here we describe a method for preparing highly enriched pachytene spermatocytes from mouse testicular cell suspensions using cell-size fractionation by sedimentation through a bovine serum albumin gradient at unit gravity. We also provide a procedure for short-term culture of spermatocytes and the pharmacological induction of the prophase-to-division phase transition.

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

Access this chapter

Log in via an institution
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 169.99
Price excludes VAT (USA)
  • Compact, lightweight 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. Eppig, J. J. (1999) Mouse oocyte maturation, fertilization, and preimplantation development in vitro, in A Comparative Methods Approach to the Study of Oocytes and Embryos (Richter, J. D., ed.), Oxford University Press, Oxford, UK, pp. 3–9.

    Google Scholar 

  2. Handel, M. A. and Eppig, J. J. (1998) Sexual dimorphism in the regulation of mammalian meiosis. Meiosis and Gametogenesis – Curr. Topics Dev. Biol. 37, 333–358.

    CAS  Google Scholar 

  3. Staub, C. (2001) A century of research on mammalian male germ cell meiotic differentiation in vitro. J. Androl. 22, 911–926.

    PubMed  CAS  Google Scholar 

  4. Staub, C., Hue, D., Nicolle, J. C., Perrard-Sapori, M. H., Segretain, D., and Durand, P. (2000) The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells. Exp. Cell Res. 260, 85–95.

    Article  PubMed  CAS  Google Scholar 

  5. Perrard, M. H., Hue, D., Staub, C., LeVern, Y., Kerboeuf, D., and Durand, P. (2003) Development of the meiotic step in testes of pubertal rats: comparison between the in vivo situation and under in vitro conditions. Mol. Reprod. Dev. 65, 86–95.

    Article  PubMed  CAS  Google Scholar 

  6. Godet, M., Damestoy, A., Mouradian, S., Rudkin, B. B., and Durand, P. (2004) Key role for cyclin-dependent kinases in the first and second meiotic divisions of rat spermatocytes. Biol. Reprod. 70, 1147–1152.

    Article  PubMed  CAS  Google Scholar 

  7. Aponte, P. M., van Bragt, M. P., de Rooij, D. G., and van Pelt, A. M. (2005) Spermatogonial stem cells: characteristics and experimental possibilities. APMIS 113, 727–742.

    Article  PubMed  Google Scholar 

  8. Meng, J., Holdcraft, R. W., Shima, J. E., Griswold, M. D., and Braun, R. E. (2005) Androgens regulate the permeability of the blood-testis barrier. Proc. Natl. Acad. Sci. USA 102, 16696–16700.

    Article  PubMed  CAS  Google Scholar 

  9. Gow, A., Southwood, C. M., Jing, S. L., Pariali, M., Riordan, G. P., Brodie, S. E., Danias, J., Bronstein, J. M., Kachar, B., and Lazzarini, R. A. (1999) CNS myelin and Sertoli cell tight junction strands are absent in Osp/Claudin-11 null mice. Cell 99, 649–659.

    Article  PubMed  CAS  Google Scholar 

  10. Saitou, M., Furuse, M., Sasaki, H., Schulzke, J.-D., Fromm, M., Takano, H., Noda, T., and Tsukita, S. (2000) Complex phenotype of mice lacking occludin, a component of tight junction strands. Mol. Biol. Cell 11, 4131–4142.

    PubMed  CAS  Google Scholar 

  11. Russell, L. B. (1977) Movement of spermatocytes from the basal to the adluminal compartment of the rat testis. Am J. Anat. 148, 313–328.

    Article  PubMed  CAS  Google Scholar 

  12. Mruk, D. D. and Cheng, C. Y. (2004) Sertoli-Sertoli and Sertoli-germ cell interactions and their significance in germ cell movement in the seminiferous epithelium during spermatogenesis. Endocr. Rev. 25, 747–806.

    Article  PubMed  CAS  Google Scholar 

  13. Kerr, J. B., Loveland, K. L., O'Bryan, M. K., and de Kretser, D. M. (2006) Cytology of the testis and intrinsic control mechanisms, in Knobil and Neill's Physiology of Reproduction, vol. 1 (Neill, J. D., ed.), Elsevier, pp. 827–947.

    Google Scholar 

  14. Meng, X., Lindahl, M., Hyvonen, M. E., Parvinen, M., de Rooij, D. G., Hess, M. W., Raatikainen-Ahokas, A., Saino, K., Rauvala, H., Lakso, M., Pichel, J. G., Westphal, H., Saarma, M., and Sarilola, H. (2000) Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287, 1489–1493.

    Article  PubMed  CAS  Google Scholar 

  15. De Franca, L. R., Hess, R. A., Cooke, P. S., and Russell, L. D. (1996) Neonatal hypothyroidism causes delayed Sertoli cell maturation in rats treated with propylthiouracil: evidence that the Sertoli cell controls testis growth. Anat. Rec. 242, 57–69.

    Article  Google Scholar 

  16. Griswold, M. D. (1993) Protein secretion by Sertoli cells: general considerations, in The Sertoli Cell (Griswold, M. D., ed.), Cache River Press, Clearwater, FL, pp. 195–200.

    Google Scholar 

  17. Wright, W. (1993) Cellular interactions in the seminiferous epithelium, in Cell and Molecular Biology of the Testis (Desjardins, C. and Ewing, L., eds.), Oxford University Press, New York, pp. 377–399.

    Google Scholar 

  18. Griswold, M. D. (1998) The central role of Sertoli cells in spermatogenesis. Semin. Cell Dev. Biol. 9, 411–416.

    Article  PubMed  CAS  Google Scholar 

  19. Griswold, M. D. and McLean, D. (2006) The Sertoli cell, in Knobil and Neill's Physiology of Reproduction, vol. 1 (Neill, J. D., ed.), Elsevier, pp. 949–975.

    Google Scholar 

  20. Akama, T. O., Nakagawa, H., Sugihara, K., Narisawa, S., Ohyama, C., Nishimura, S. I., O'Brien, D. A., Moremen, K. W., Millan, J. L., and Fukuda, M. N. (2002) Germ cell survival through carbohydrate-mediated interaction with Sertoli cells. Science 295, 124–127.

    Article  PubMed  Google Scholar 

  21. Russell, L. D., Chiarini-Garcia, H., Korsmeyer, S. J., and Knudson, C. M. (2002) Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis. Biol. Reprod. 66, 950–958.

    Article  PubMed  CAS  Google Scholar 

  22. Jahnukainen, K., Chrysis, D., Hou, M., Parvinen, M., Eksborg, S., and Soder, O. (2004) Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis. Biol. Reprod. 70, 290–296.

    Article  PubMed  CAS  Google Scholar 

  23. Romrell, L. J., Bellvé, A. R., and Fawcett, D. W. (1976) Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization. Dev. Biol. 49, 119–131.

    Article  PubMed  CAS  Google Scholar 

  24. Bellvé, A. R., Cavicchia, J. C., Millette, C. F., O'Brien, D. A., Bhatnagar, Y. M., and Dym, M. (1977) Spermatogenic cells of the prepuberal mouse. J. Cell Biol. 74, 68–85.

    Article  PubMed  Google Scholar 

  25. Bellvé, A. R. (1993) Purification, culture and fractionation of spermatogenic cells. Methods Enzymol. 225, 84–113.

    Article  PubMed  Google Scholar 

  26. Handel, M. A., Caldwell, K. A., and Wiltshire, T. (1995) Culture of pachytene spermatocytes for analysis of meiosis. Dev. Genet. 16, 128–139.

    Article  PubMed  CAS  Google Scholar 

  27. O'Brien, D. A. (1987) Stage-specific protein synthesis by isolated spermatogenic cells throughout meiosis and early spermiogenesis in the mouse. Biol. Reprod. 37, 147–157.

    Article  PubMed  Google Scholar 

  28. Cobb, J., Miyaike, M., Kikuchi, A., and Handel, M. A. (1999) Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase II alpha localization and chromosome condensation. Chromosoma 108, 412–425.

    Article  PubMed  CAS  Google Scholar 

  29. Inselman, A., Eaker, S., and Handel, M. A. (2003) Temporal expression of cell cycle-related proteins during spermatogenesis: Establishing a timeline for the onset of the meiotic divisions. Cytogenet. Genome Res. 103, 277–284.

    Article  PubMed  CAS  Google Scholar 

  30. Wiltshire, T., Park, C., Caldwell, K. A., and Handel, M. A. (1995) Induced premature G2/M-phase transition in pachytene spermatocytes includes events unique to meiosis. Dev. Biol. 169, 557–567.

    Article  PubMed  CAS  Google Scholar 

  31. Cobb, J., Cargile, B., and Handel, M. A. (1999) Acquisition of competence to condense metaphase I chromosomes during spermatogenesis. Dev. Biol. 205, 49–64.

    Article  PubMed  CAS  Google Scholar 

  32. Cobb, J., Reddy, R. K., Park, C., and Handel, M. A. (1997) Analysis of expression and function of topoisomerase I and II during meiosis in male mice. Mol. Reprod. Dev. 46, 489–498.

    Article  PubMed  CAS  Google Scholar 

  33. Inselman, A. and Handel, M. A. (2004) Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes. Biol. Reprod. 71, 570–578.

    Article  PubMed  CAS  Google Scholar 

  34. Eaker, S., Pyle, A., Cobb, J., and Handel, M. A. (2001) Evidence for meiotic spindle checkpoint from analysis of spermatocytes from Robertsonian-chromosome heterozygous mice. J. Cell Sci. 114, 2953–2965.

    PubMed  CAS  Google Scholar 

  35. Eaker, S., Cobb, J., Pyle, A., and Handel, M. A. (2002) Meiotic prophase abnormalities and metaphase cell death in MLH1-deficient mouse spermatocytes: Insights into regulation of spermatogenic progress. Dev. Biol. 249, 85–95.

    Article  PubMed  CAS  Google Scholar 

  36. Kotaja, N., Kimmins, S., Brancorsini, S., Hentsch, D., Vonesch, J.-L., Davidson, I., Parvinen, M., and Sassone-Corsi, P. (2004) Preparation, isolation and characterization of stage-specific spermatogenic cells for cellular and molecular analysis. Nat. Methods 1, 249–254.

    Article  PubMed  CAS  Google Scholar 

  37. Kimura, Y., Tateno, H., Handel, M. A., and Yanagimachi, R. (1998) Factors affecting meiotic and developmental competence of primary spermatocyte nuclei injected into mouse oocytes. Biol. Reprod. 59, 871–877.

    Article  PubMed  CAS  Google Scholar 

  38. Tarsounas, M., Pearlman, R. E., and Moens, P. B. (1999) Meiotic activation of rat pachytene spermatocytes with okadaic acid: the behaviour of synaptonemal complex components SYN1/SCP1 and COR1/SCP3. J. Cell Sci. 112, 423–434.

    PubMed  CAS  Google Scholar 

  39. Marcon, E. and Moens, P. (2003) MLH1p and MLH3p localize to precociously induced chiasmata of okadaic-acid-treated mouse spermatocytes. Genetics 165, 2283–2287.

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Work described here is supported by NIH grants to M.A.H. (HD33816 and HD48998) and a Cancer Center Core Grant to The Jackson Laboratory (CA34196). S.L. is funded in part by a fellowship from les Fonds de la Recherche en Santé du Québec (FRSQ).

Author information

Authors and Affiliations

  1. The Jackson Laboratory, Bar Harbor, ME, USA

    Sophie La Salle, Fengyun Sun & Mary Ann Handel

Authors
  1. Sophie La Salle
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fengyun Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mary Ann Handel
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, York Ave. 1275, New York, 10021-6007, U.S.A.

    Scott Keeney

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

La Salle, S., Sun, F., Handel, M.A. (2009). Isolation and Short-Term Culture of Mouse Spermatocytes for Analysis of Meiosis. In: Keeney, S. (eds) Meiosis. Methods in Molecular Biology, vol 558. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-103-5_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-103-5_17

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-102-8

  • Online ISBN: 978-1-60761-103-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation