Preparation of Testicular Samples for Histology and Immunohistochemistry

  • Barbara BilinskaEmail author
  • Anna Hejmej
  • Malgorzata Kotula-Balak
Part of the Methods in Molecular Biology book series (MIMB, volume 1748)


One approach to visualize internal structures of the testis is histological sectioning of the material. The use of testicular samples allows a detailed analysis of the structure of both seminiferous tubules and the interstitial space. It is worth noting that key role in the control of germ cell development is assigned to Sertoli cells. Thus, in this chapter the special reference is made on visualization of Sertoli cells in the seminiferous epithelium in which they create a specialized microenvironment to support the germ cell development through the formation of the blood-testis barrier (BTB). The use of transmission electron microscopy (TEM) allows a deeper insight into the BTB morphology, especially the organization of the basal ectoplasmic specialization (ES) and coexisting intercellular junctions.

Equally important, immunohistochemistry (IHC) is an appropriate technique to detect the localization of various proteins in paraffin-embedded and fixed tissues, i.e. testicular samples. A proper fixation allows to stabilize structure of the seminiferous tubules and preserve cells against irreversible damage. As such localization of various junction proteins connecting adjoined Sertoli cells and present in germ cell-Sertoli cell interfaces is possible. Also immunofluorescence (IF) is helpful to detect the distribution and relative abundance of the junctional proteins, while immunocytochemistry (ICC) is a valuable technique to show a protein distribution within a single cell (e.g. in Sertoli cell culture).


Testis Histology Ultrastructure Immunohistochemistry Immunocytochemistry Sertoli cell Blood–testis barrier Intercellular junctions 



The authors would like to acknowledge Dr. Katarzyna Chojnacka, Dr. Marta Zarzycka, Dr. Ilona Kopera-Sobota, and Dr. Ewelina Gorowska-Wojtowicz (former PhD students of B.B.) for their work and involvement in improving methods presented herein. Special thanks are expressed to Dr. Ewelina Gorowska-Wojtowicz for illustrations. Several methods described here were obtained from the studies supported by a HARMONIA 3 grant (2012/06/M/NZ4/00146) from the National Science Centre (to B.B.)


  1. 1.
    Su L, Kopera-Sobota IA, Bilinska B et al (2013) Germ cells contribute to the function of the Sertoli cell barrier. Spermatogenesis 3:e26460Google Scholar
  2. 2.
    O'Hara L, Smith LB (2015) Androgen receptor roles in spermatogenesis and infertility. Best Pract Res Clin Endocrinol Metab 29:595–605CrossRefPubMedGoogle Scholar
  3. 3.
    França LR, Hess RA, Dufour JM et al (2016) The Sertoli cell: one hundred fifty years of beauty and plasticity. Andrology 4:189–212CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Mruk DD, Cheng CY (2004) Sertoli-Sertoli and Sertoli-germ cell interactions and their significance in germ cell movement in the seminiferous epithelium during spermatogenesis. Endocr Rev 25:747–806CrossRefPubMedGoogle Scholar
  5. 5.
    Kopera I, Bilinska B, Cheng CY et al (2010) Sertoli-germ cell junctions in the testis – a review of recent data. Philos Trans R Soc Lond Ser B Biol Sci 365:1593–1605CrossRefGoogle Scholar
  6. 6.
    Mruk DD, Cheng CY (2015) The mammalian blood–testis barrier: its biology and regulation. Endocr Rev 36:564–591CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Stanton PG (2016) Regulation of the blood-testis barrier. Semin Cell Dev Biol 59:166–173CrossRefPubMedGoogle Scholar
  8. 8.
    Chojnacka K, Zarzycka M, Hejmej A et al (2016) Hydroxyflutamide affects connexin 43 via the activation of PI3K/Akt- dependent pathway but has no effect on the crosstalk between PI3K/Akt and ERK1/2 pathways at the Raf-1 kinase level in primary rat Sertoli cells. Toxicol In Vitro 31:146–157CrossRefPubMedGoogle Scholar
  9. 9.
    Cheng CY, Mruk DD (2002) Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development. Physiol Rev 82:825–874CrossRefPubMedGoogle Scholar
  10. 10.
    Mruk DD, Cheng CY (2011) An in vitro system to study Sertoli cell blood-testis barrier dynamics. Methods Mol Biol 763:237–252CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hejmej A, Kotula-Balak M, Galas J et al (2011) Effects of 4-tert-octylphenol on the testes and seminal vesicles in adult male bank voles. Reprod Toxicol 31:95–105CrossRefPubMedGoogle Scholar
  12. 12.
    Kopera I, Durlej M, Hejmej A et al (2011) Differential expression of connexin 43 in adult pig testes during normal spermatogenic cycle and after flutamide treatment. Reprod Domest Anim 46:1050–1060CrossRefPubMedGoogle Scholar
  13. 13.
    Hejmej A, Bilinska B (2014) A role of junction-mediated interactions in cells of the male reproductive tract: impact of prenatal, neonatal, and prepubertal exposure to anti-androgens on adult reproduction. Histol Histopathol 29:815–830PubMedGoogle Scholar
  14. 14.
    Hopwood D (1996) Fixation and fixatives. In: Bancroft J, Stevens A (eds) Theory and practice of histological techniques. Churchill Livingstone, New YorkGoogle Scholar
  15. 15.
    Chojnacka K, Hejmej A, Zarzycka M et al (2016) Flutamide induces alterations in the cell-cell junction ultrastructure and reduces the expression of Cx43 at the blood-testis barrier with no disturbance in the rat seminiferous tubule histology. Reprod Biol Endocrinol 14:14Google Scholar
  16. 16.
    Russell LD, Burguet S (1977) Ultrastructure of Leydig cells as revealed by secondary tissue treatment with a ferrocyanide-osmium mixture. Tissue Cell 9:751–766CrossRefGoogle Scholar
  17. 17.
    Perreira GG, Melo RCN, Russell LD (2002) Relationship of Sertoli-Sertoli tight junctions to ectoplasmic specialization in conventional and en face views. Biol Reprod 67:1232–1241CrossRefGoogle Scholar
  18. 18.
    Hejmej A, Wiszniewska B, Kosiniak-Kamysz K et al (2006) The presence of androgen receptors in the epididymis and prostate of the stallion and cryptorchid horse - a preliminary study. Vet J 171:373–379CrossRefPubMedGoogle Scholar
  19. 19.
    Hejmej A, Kotula-Balak M, Sadowska J et al (2007) Expression of connexin 43 protein in testes, epididymides and prostates of stallions. Equine Vet J 39:122–127CrossRefPubMedGoogle Scholar
  20. 20.
    Hejmej A, Kopera I, Kotula-Balak M et al (2009) Age-dependent pattern of connexin43 expression in testes of European bison (Bison bonasus, L.) J Exp Zool A 311A:667–675CrossRefGoogle Scholar
  21. 21.
    Lydka M, Kotula-Balak M, Kopera-Sobota I et al (2011) Vimentin expression in testes of Arabian stallions. Equine Vet J 43:184–189CrossRefPubMedGoogle Scholar
  22. 22.
    Hejmej A, Kopera I, Kotula-Balak M et al (2012) Are expression and localization of tight and adherens junction proteins in testes of adult boar affected by fetal and neonatal exposure to flutamide? Int J Androl 35:340–352CrossRefPubMedGoogle Scholar
  23. 23.
    Hejmej A, Kotula-Balak M, Chojnacka K et al (2013) Photoperiod-dependent effects of 4-tert-octylphenol on adherens and gap junction proteins in bank vole seminiferous tubules. Int J Endocrinol 2013:134589Google Scholar
  24. 24.
    Zarzycka M, Chojnacka K, Mruk D et al (2015) Flutamide alters distribution of c-Src and affects cell adhesion function in the adult rat seminiferous epithelium. Andrology 3:569–581CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Barbara Bilinska
    • 1
    Email author
  • Anna Hejmej
    • 1
  • Malgorzata Kotula-Balak
    • 1
  1. 1.Department of Endocrinology, Institute of Zoology and Biomedical ResearchJagiellonian UniversityKrakowPoland

Personalised recommendations