Advertisement

Immunohistochemical Approaches to the Study of Human Fetal Ovarian Development

  • Jing He
  • Andrew J. Childs
  • Jieqian Zhou
  • Richard A. AndersonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 957)

Abstract

The development of primordial germ cells into oocytes within primordial follicles involves a complex sequence of proliferation, developmental commitment, entry and arrest in meiosis, and association with surrounding somatic cells. These processes occur over the first few months of development in the human, with multiple stages of development present at any one time point. Immunohistochemistry has been hugely instructive in identifying the various key stages in ovarian development, by allowing simultaneous visualization of different stages of germ cell development, and their spatial arrangement. These studies allow comparison with other species and have identified key differences between human and murine ovarian development as well as giving a basis for functional studies. In this chapter we describe the main methodologies used in immunohistochemistry, using both chromogen and fluorescence approaches, and both single and double antigen detection.

Key words

Immunohistochemistry Immunofluorescence Germ cell Detection systems Antibodies Antigen retrieval 

Notes

Acknowledgements

We are grateful to Anne Saunderson and the staff of the Bruntsfield Suite of the Royal Infirmary of Edinburgh for assistance with patient recruitment and specimen collection and to members of the Anderson Lab and Histology core facility at the MRC Centre for Reproductive Health for assistance in developing these protocols. This work was supported by the Medical Research Council (programme grant G1100357 to R.A.A.) and Medical Research Scotland (research grant 354 FRG to A.J.C.).

Funding

This work was supported by the Medical Research Council (programme grant G1100357 to R.A.A.) and Medical Research Scotland (research grant 354 FRG to A.J.C.).

References

  1. 1.
    Byskov AG (1986) Differentiation of mammalian embryonic gonad. Physiol Rev 66:71–117PubMedGoogle Scholar
  2. 2.
    Wartenburg H (1981) Differentiation and development of the testes. In: Burger H, de Kretser DM (eds) The testis, 2nd edn. Raven, New York, pp 39–81Google Scholar
  3. 3.
    Hanley NA, Hagan DM, Clement-Jones M, Ball SG, Strachan T, Salas-Cortes L, McElreavey K, Lindsay S, Robson S, Bullen P, Ostrer H, Wilson DI (2000) SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development. Mech Dev 91:403–407PubMedCrossRefGoogle Scholar
  4. 4.
    Kurilo LF (1981) Oogenesis in antenatal development in man. Hum Genet 57:86–92PubMedCrossRefGoogle Scholar
  5. 5.
    Pepling ME, Spradling AC (1998) Female mouse germ cells form synchronously dividing cysts. Development 125:3323–3328PubMedGoogle Scholar
  6. 6.
    Tingen C, Kim A, Woodruff TK (2009) The primordial pool of follicles and nest breakdown in mammalian ovaries. Mol Hum Reprod 15:795–803PubMedCrossRefGoogle Scholar
  7. 7.
    Childs AJ, Anderson RA (2012) Experimental approaches to the study of human primordial germ cells. Methods Mol Biol 825:199–210PubMedCrossRefGoogle Scholar
  8. 8.
    Anderson RA, Fulton N, Cowan G, Coutts S, Saunders PT (2007) Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis. BMC Dev Biol 7:136PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Stoop H, Honecker F, Cools M, de Krijger R, Bokemeyer C, Looijenga LH (2005) Differentiation and development of human female germ cells during prenatal gonadogenesis: an immunohistochemical study. Hum Reprod 20:1466–1476PubMedCrossRefGoogle Scholar
  10. 10.
    Bullejos M, Koopman P (2004) Germ cells enter meiosis in a rostro-caudal wave during development of the mouse ovary. Mol Reprod Dev 68:422–428PubMedCrossRefGoogle Scholar
  11. 11.
    Menke DB, Koubova J, Page DC (2003) Sexual differentiation of germ cells in XX mouse gonads occurs in an anterior-to-posterior wave. Dev Biol 262:303–312PubMedCrossRefGoogle Scholar
  12. 12.
    Duffin K, Bayne RA, Childs AJ, Collins C, Anderson RA (2009) The forkhead transcription factor FOXL2 is expressed in somatic cells of the human ovary prior to follicle formation. Mol Hum Reprod 15:771–777PubMedCrossRefGoogle Scholar
  13. 13.
    ten Dijke P, Hill CS (2004) New insights into TGF-beta-Smad signalling. Trends Biochem Sci 29:265–273PubMedCrossRefGoogle Scholar
  14. 14.
    Coutts SM, Childs AJ, Fulton N, Collins C, Bayne RA, McNeilly AS, Anderson RA (2008) Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression. Dev Biol 314:189–199PubMedCrossRefGoogle Scholar
  15. 15.
    Childs AJ, Kinnell HL, Collins CS, Hogg K, Bayne RA, Green SJ, McNeilly AS, Anderson RA (2010) BMP signaling in the human fetal ovary is developmentally regulated and promotes primordial germ cell apoptosis. Stem Cells 28:1368–1378PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Childs AJ, Bayne RA, Murray AA, Martins Da Silva SJ, Collins CS, Spears N, Anderson RA (2010) Differential expression and regulation by activin of the neurotrophins BDNF and NT4 during human and mouse ovarian development. Dev Dyn 239:1211–1219PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Childs AJ, Anderson RA (2009) Activin A selectively represses expression of the membrane-bound isoform of Kit ligand in human fetal ovary. Fertil Steril 92:1416–1419PubMedCrossRefGoogle Scholar
  18. 18.
    Anderson G, Gordon K (1996) Tissue processing, microtomy and paraffin sections. In: Bankroft JD, Stevens A (eds) Theory and practice of histological techniques, 4th edn. Churchill Livingstone, New YorkGoogle Scholar
  19. 19.
    Smith SR, Fulton N, Collins CS, Welsh M, Bayne RA, Coutts SM, Childs AJ, Anderson RA (2010) N- and E-cadherin expression in human ovarian and urogenital duct development. Fertil Steril 93:2348–2353PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Jing He
    • 1
  • Andrew J. Childs
    • 1
  • Jieqian Zhou
    • 1
  • Richard A. Anderson
    • 1
    Email author
  1. 1.MRC Centre for Reproductive Health, The Queen’s Medical Research InstituteThe University of EdinburghEdinburghUK

Personalised recommendations