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Whole-Mount Immunohistochemistry to Study Spermatogonial Stem Cells and Spermatogenic Lineage Development in Mice, Monkeys, and Humans

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Stem Cells and Tissue Repair

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1210))

Abstract

Spermatogonial stem cells (SSCs) and undifferentiated progenitor spermatogonia in mammalian seminiferous tubules are organized in chains, connected by intracellular bridges. Clone size is generally related to stem cell potential, with shorter chains containing the majority of the stem cell population. Immunofluorescence detection of spermatogonia-specific proteins in whole-mount seminiferous tubule preparations is the only method that allows researchers to relate clone size with the molecular phenotype in spermatogenic lineage development. Here we describe in detail the method used to detect nuclear, cytoplasmic, and cell surface molecules in seminiferous tubules isolated from mouse, monkey, and human testes.

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References

  1. Clermont Y, Leblond CP (1953) Renewal of spermatogonia in the rat. Am J Anat 93(3):475–501

    Article  CAS  PubMed  Google Scholar 

  2. Huckins C (1971) The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat Rec 169(3):533–557

    Article  CAS  PubMed  Google Scholar 

  3. Oakberg EF (1971) Spermatogonial stem-cell renewal in the mouse. Anat Rec 169(3):515–531

    Article  CAS  PubMed  Google Scholar 

  4. Brinster RL, Zimmermann JW (1994) Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 91(24):11298–11302

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Phillips BT, Gassei K, Orwig KE (2010) Spermatogonial stem cell regulation and spermatogenesis. Philos Trans R Soc Lond B Biol Sci 365(1546):1663–1678

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Yoshida S, Nabeshima Y, Nakagawa T (2007) Stem cell heterogeneity: actual and potential stem cell compartments in mouse spermatogenesis. Ann N Y Acad Sci 1120:47–58

    Article  PubMed  Google Scholar 

  7. Morimoto H et al (2009) Phenotypic plasticity of mouse spermatogonial stem cells. PLoS One 4(11):e7909

    Article  PubMed Central  PubMed  Google Scholar 

  8. Hara K et al (2014) Mouse spermatogenic stem cells continually interconvert between equipotent singly isolated and syncytial states. Cell Stem Cell 14(5):658–672

    Google Scholar 

  9. Heller CG, Clermont Y (1963) Spermato-genesis in man: an estimate of its duration. Science 140:184–186

    Article  CAS  PubMed  Google Scholar 

  10. Clermont Y (1963) The cycle of the seminiferous epithelium in man. Am J Anat 112:35–51

    Article  CAS  PubMed  Google Scholar 

  11. Clermont Y (1966) Renewal of spermatogonia in man. Am J Anat 118(2):509–524

    Article  CAS  PubMed  Google Scholar 

  12. Clermont Y (1966) Spermatogenesis in man. A study of the spermatogonial population. Fertil Steril 17(6):705–721

    CAS  PubMed  Google Scholar 

  13. Clermont Y (1969) Two classes of spermatogonial stem cells in the monkey (Cercopithecus aethiops). Am J Anat 126(1):57–71

    Article  CAS  PubMed  Google Scholar 

  14. Nakagawa T et al (2010) Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science 328(5974):62–67

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Huckins C, Oakberg EF (1978) Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules. I The normal testes. Anat Rec 192(4):519–528

    Article  CAS  PubMed  Google Scholar 

  16. Oakberg EF (1971) A new concept of spermatogonial stem-cell renewal in the mouse and its relationship to genetic effects. Mutat Res 11(1):1–7

    Article  CAS  PubMed  Google Scholar 

  17. Clermont Y, Antar M (1973) Duration of the cycle of the seminiferous epithelium and the spermatogonial renewal in the monkey Macaca arctoides. Am J Anat 136(2):153–165

    Article  CAS  PubMed  Google Scholar 

  18. Clermont Y (1972) Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 52(1):198–236

    CAS  PubMed  Google Scholar 

  19. Gassei K, Orwig KE (2013) SALL4 expression in gonocytes and spermatogonial clones of postnatal mouse testes. PLoS One 8(1):e53976

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Costoya JA et al (2004) Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 36(6):653–659

    Article  CAS  PubMed  Google Scholar 

  21. Oatley MJ et al (2011) Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells in mice. Biol Reprod 85(2):347–356

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. van Bragt MP et al (2008) Expression of the pluripotency marker UTF1 is restricted to a subpopulation of early A spermatogonia in rat testis. Reproduction 136(1):33–40

    Article  PubMed  Google Scholar 

  23. Ballow D et al (2006) Sohlh1 is essential for spermatogonial differentiation. Dev Biol 294(1):161–167

    Article  CAS  PubMed  Google Scholar 

  24. Ballow DJ et al (2006) Sohlh2 is a germ cell-specific bHLH transcription factor. Gene Expr Patterns 6(8):1014–1018

    Article  CAS  PubMed  Google Scholar 

  25. Zheng K et al (2009) The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse. BMC Dev Biol 9:38

    Article  PubMed Central  PubMed  Google Scholar 

  26. Suzuki A, Tsuda M, Saga Y (2007) Functional redundancy among Nanos proteins and a distinct role of Nanos2 during male germ cell development. Development 134(1):77–83

    Article  CAS  PubMed  Google Scholar 

  27. Meng X et al (2000) Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287(5457):1489–1493

    Article  CAS  PubMed  Google Scholar 

  28. Seandel M et al (2008) Niche players: spermatogonial progenitors marked by GPR125. Cell Cycle 7(2):135–140

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Tokuda M et al (2007) CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes. Biol Reprod 76(1):130–141

    Article  CAS  PubMed  Google Scholar 

  30. Suzuki H et al (2009) The heterogeneity of spermatogonia is revealed by their topology and expression of marker proteins including the germ cell-specific proteins Nanos2 and Nanos3. Dev Biol 336(2):222–231

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Kyle E. Orwig .

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Gassei, K., Valli, H., Orwig, K.E. (2014). Whole-Mount Immunohistochemistry to Study Spermatogonial Stem Cells and Spermatogenic Lineage Development in Mice, Monkeys, and Humans. In: Kioussi, C. (eds) Stem Cells and Tissue Repair. Methods in Molecular Biology, vol 1210. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1435-7_15

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  • DOI: https://doi.org/10.1007/978-1-4939-1435-7_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1434-0

  • Online ISBN: 978-1-4939-1435-7

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