Skip to main content
SpringerLink
Log in

Monitoring the Integrity of the Blood-Testis Barrier (BTB): An In Vivo Assay

  • Protocol
  • First Online:
Sertoli Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1748))

Abstract

The blood-testis barrier is a unique ultrastructure in the mammalian testis, located near the basement membrane of the seminiferous tubule that segregates the seminiferous epithelium into the basal and the adluminal (apical) compartment. Besides restricting paracellular and transcellular passage of biomolecules (e.g., paracrine factors, hormones), water, electrolytes, and other substances including toxicants and/or drugs to enter the adluminal compartment of the epithelium, the BTB is an important ultrastructure that supports spermatogenesis. As such, a sensitive and reliable assay to monitor its integrity in vivo is helpful for studying testis biology. This assay is based on the ability of an intact BTB to exclude the diffusion of a small molecule such as sulfo-NHS-LC-biotin (C20H29N4NaO9S2, Mr. 556.59, a water-soluble and membrane-impermeable biotinylation reagent) from the basal to the apical compartment of the seminiferous epithelium. Herein, we summarize the detailed procedures on performing the assay and to obtain semiquantitative data to assess the extent of BTB damage when compared to positive controls, such as treatment of rats with cadmium chloride (CdCl2) which is known to compromise the BTB integrity.

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 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover 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. Setchell BP (2008) Blood-testis barrier, functional and transport proteins and spermatogenesis. Adv Exp Med Biol 636:212–233

    Article  CAS  PubMed  Google Scholar 

  2. Cheng CY, Mruk DD (2012) The blood-testis barrier and its implication in male contraception. Pharmacol Rev 64:16–64

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Aoki A, Fawcett DW (1975) Impermeability of sertoli cell junctions to prolonged exposure to peroxidase. Andrologia 7:63–76

    Article  CAS  PubMed  Google Scholar 

  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 25:747–806

    Article  CAS  PubMed  Google Scholar 

  5. Pelletier RM (2011) The blood-testis barrier: the junctional permeability, the proteins and the lipids. Prog Histochem Cytochem 46:49–127

    Article  PubMed  Google Scholar 

  6. Franca LR, Auharek SA, Hess RA, Dufour JM, Hinton BT (2012) Blood-tissue barriers: morphofunctional and immunological aspects of the blood-testis and blood-epididymal barriers. Adv Exp Med Biol 763:237–259

    CAS  PubMed  Google Scholar 

  7. Kaur G, Thompson LA, Dufour JM (2014) Sertoli cells–immunological sentinels of spermatogenesis. Semin Cell Dev Biol 30:36–44

    Article  CAS  PubMed  Google Scholar 

  8. Stanton PG (2016) Regulation of the blood-testis barrier. Semin Cell Dev Biol 59:166–173. https://doi.org/10.1016/j.semcdb.2016.06.018

    Article  CAS  PubMed  Google Scholar 

  9. Li N, Tang EI, Cheng CY (2016) Regulation of blood-testis barrier by actin binding proteins and protein kinases. Reproduction 151(3):R29–R41. https://doi.org/10.1530/REP-15-0463

    Article  CAS  PubMed  Google Scholar 

  10. Lie PPY, Cheng CY, Mruk DD (2013) Signalling pathways regulating the blood-testis barrier. Int J Biochem Cell Biol 45:621–625

    Article  CAS  PubMed  Google Scholar 

  11. Xiao X, Mruk DD, Wong CKC, Cheng CY (2014) Germ cell transport across the seminiferous epithelium during spermatogenesis. Physiology 29(4):286–298. https://doi.org/10.1152/physiol.00001.2014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chen H, Cheng CY (2016) Planar cell polarity (PCP) proteins and spermatogenesis. Semin Cell Dev Biol 59:99–109. https://doi.org/10.1016/j.semcdb.2016.04.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Smith BE, Braun RE (2012) Germ cell migration across Sertoli cell tight junctions. Science 338:798–802

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Govero J et al (2016) Zika virus infection damages the testes in mice. Nature 540:438–442

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jenabian MA et al (2016) Immune tolerance properties of the testicular tissue as a viral sanctuary site in ART-treated HIV-infected adults. AIDS 30(18):2777–2786. https://doi.org/10.1097/QAD.0000000000001282

    Article  CAS  PubMed  Google Scholar 

  16. Darcis G, Coombs RW, Van Lint C (2016) Exploring the anatomical HIV reservoirs: role of the testicular tissue. AIDS 30(18):2891–2893. https://doi.org/10.1097/QAD.0000000000001281

    Article  PubMed  PubMed Central  Google Scholar 

  17. Eisele E, Siliciano RF (2012) Redefining the viral reservoirs that prevent HIV-1 eradication. Immunity 37:377–388

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Siu ER, Mruk DD, Porto CS, Cheng CY (2009) Cadmium-induced testicular injury. Toxicol Appl Pharmacol 238(3):240–249

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wan HT, Mruk DD, Wong CKC, Cheng CY (2014) Perfluorooctanesulfonate (PFOS) perturbs male rat Sertoli cell blood-testis barrier function by affecting F-actin organization via p-FAK-Tyr407–an in vitro study. Endocrinology 155:249–262

    Article  PubMed  Google Scholar 

  20. Cai H et al (2011) Scrotal heat stress causes a transient alteration in tight junctions and induction of TGF-beta expression. Int J Androl 34:352–362

    Article  PubMed  Google Scholar 

  21. Hou WG, Zhao J, Li Z, Li W, Li T, Xiong LZ, Zhang YQ (2012) Effects of electromagnetic pulse irradiation on the mouse blood-testicle barrier. Urology 80(1):225 e221–225 e226

    Article  Google Scholar 

  22. Smith LB, Walker WH (2014) The regulation of spermatogenesis by androgens. Semin Cell Dev Biol 30:2–13. https://doi.org/10.1016/j.semcdb.2014.02.012

    Article  CAS  PubMed  Google Scholar 

  23. Meng J, Holdcraft RW, Shima JE, Griswold MD, Braun RE (2005) Androgens regulate the permeability of the blood-testis barrier. Proc Natl Acad Sci U S A 102:16696–16700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Xiao X et al (2014) N-wasp is required for structural integrity of the blood-testis barrier. PLoS Genet 10:e1004447

    Article  PubMed  PubMed Central  Google Scholar 

  25. Mruk DD, Cheng CY (2015) The mammalian blood-testis barrier: its biology and regulation. Endocr Rev 36:564–591

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li MW et al (2006) Tumor necrosis factor {alpha} reversibly disrupts the blood-testis barrier and impairs Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testes. J Endocrinol 190:313–329

    Article  CAS  PubMed  Google Scholar 

  27. Sarkar O, Mathur PP, Cheng CY, Mruk DD (2008) Interleukin 1 alpha (IL1A) is a novel regulator of the blood-testis barrier in the rat. Biol Reprod 78:445–454

    Article  CAS  PubMed  Google Scholar 

  28. Furuse M et al (2002) Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 156:1099–1111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wong CH, Mruk DD, Lui WY, Cheng CY (2004) Regulation of blood-testis barrier dynamics: an in vivo study. J Cell Sci 117:783–798

    Article  CAS  PubMed  Google Scholar 

  30. Lui WY, Lee WM, Cheng CY (2001) Transforming growth factor-b3 perturbs the inter-Sertoli tight junction permeability barrier in vitro possibly mediated via its effects on occludin, zonula occludens-1, and claudin-11. Endocrinology 142:1865–1877

    Article  CAS  PubMed  Google Scholar 

  31. Lui WY, Wong CH, Mruk DD, Cheng CY (2003) TGF-b3 regulates the blood-testis barrier dynamics via the p38 mitogen activated protein (MAP) kinase pathway: an in vivo study. Endocrinology 144:1139–1142

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the National Institutes of Health, NICHD R01 HD056034 to C.Y.C., and U54 HD029990 Project 5 to C.Y.C.; Hong Kong Research Grants Council (RGC)/National Natural Science Foundation of China Joint Research Scheme (N_HKU 717/12) to W.M.L., Hong Kong RGC Grant GRF17100816 to W.Y.L. and GRF 771513 to W.M.L., Hong Kong University Seed Funding to W.Y.L. and W.M.L.; NSFC Grant 81730042 to R.S.G. and NSFC Grant 31371176 to X.X., and Zhejiang Province Department of Science Technology Funding 2016F10010 to X.X.; H.C. was supported in part by The S.Y. Law Memorial Fellowship and The F. Lau Memorial Fellowship.

Author information

Authors and Affiliations

  1. The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, NY, USA

    Haiqi Chen, Dolores D. Mruk & C. Yan Cheng

  2. School of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong, China

    Wing-yee Lui & Will M. Lee

  3. Department of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang, China

    Xiang Xiao

  4. Institute of Reproductive Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China

    Renshan Ge & Qingquan Lian

  5. SBM Pharmaceuticals Srl, Rome, Italy

    Bruno Silvestrini

Authors
  1. Haiqi Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wing-yee Lui
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dolores D. Mruk
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiang Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Renshan Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qingquan Lian
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Will M. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Bruno Silvestrini
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. C. Yan Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. Yan Cheng .

Editor information

Editors and Affiliations

  1. Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal

    Marco G. Alves

  2. Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal

    Pedro F. Oliveira

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Chen, H. et al. (2018). Monitoring the Integrity of the Blood-Testis Barrier (BTB): An In Vivo Assay. In: Alves, M., Oliveira, P. (eds) Sertoli Cells. Methods in Molecular Biology, vol 1748. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7698-0_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7698-0_17

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7697-3

  • Online ISBN: 978-1-4939-7698-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation