Sertoli Cells pp 229-243 | Cite as

Regulation of Blood-Testis Barrier (BTB) Dynamics, Role of Actin-, and Microtubule-Based Cytoskeletons

  • Qing Wen
  • Elizabeth I. Tang
  • Nan Li
  • Dolores D. Mruk
  • Will M. Lee
  • Bruno Silvestrini
  • C. Yan ChengEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1748)


The blood-testis barrier (BTB) is an important ultrastructure in the testis that supports meiosis and postmeiotic spermatid development since a delay in the establishment of a functional Sertoli cell barrier during postnatal development in rats or mice by 17–20 day postpartum (dpp) would lead to a delay of the first wave of meiosis. Furthermore, irreversible disruption of the BTB by toxicants also induces infertility in rodents. Herein, we summarize recent findings that BTB dynamics (i.e., disassembly, reassembly, and stabilization) are supported by the concerted efforts of the actin- and microtubule (MT)-based cytoskeletons. We focus on the role of two actin nucleation protein complexes, namely, the Arp2/3 (actin-related protein 2/3) complex and formin 1 (or the formin 1/spire 1 complex) known to induce actin nucleation, respectively, by conferring plasticity to actin cytoskeleton. We also focus on the MT plus (+)-end tracking protein (+TIP) EB1 (end-binding protein 1) which is known to confer MT stabilization. Furthermore, we discuss in particular how the interactions of these proteins modulate BTB dynamics during spermatogenesis. These findings also yield a novel hypothetical concept regarding the molecular mechanism that modulates BTB function.


Testis Sertoli cell Blood-testis barrier Spermatogenesis Ectoplasmic specialization Tight junction Gap junction Desmosome Seminiferous epithelial cycle 



This work was supported by grants from the National Institutes of Health (NICHD R01 HD056034 to C.Y.C.; 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., and Hong Kong University Seed Funding to W.M.L.; W.Q. was supported in part from The F. Lau Memorial Fellowship, The Noopolis Foundation, and The Economic Development Council.


  1. 1.
    Cheng CY, Mruk DD (2012) The blood-testis barrier and its implication in male contraception. Pharmacol Rev 64:16–64CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Pelletier RM (2011) The blood-testis barrier: the junctional permeability, the proteins and the lipids. Prog Histochem Cytochem 46:49–127CrossRefPubMedGoogle Scholar
  3. 3.
    Stanton PG (2016) Regulation of the blood-testis barrier. Semin Cell Dev Biol 59:166–173. CrossRefPubMedGoogle Scholar
  4. 4.
    Mruk DD, Cheng CY (2015) The mammalian blood-testis barrier: its biology and regulation. Endocr Rev 36(5):564–591. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    O’Donnell L, O’Bryan MK (2014) Microtubules and spermatogenesis. Semin Cell Dev Biol 30:45–54. CrossRefPubMedGoogle Scholar
  6. 6.
    O’Donnell L (2014) Mechanisms of spermiogenesis and spermiation and how they are disturbed. Spermatogenesis 4(2):e979623. CrossRefPubMedGoogle Scholar
  7. 7.
    Tang EI, Mruk DD, Cheng CY (2013) MAP/microtubule affinity-regulating kinases, microtubule dynamics, and spermatogenesis. J Endocrinol 217:R13–R23CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Tang EI, Mruk DD, Lee WM, Cheng CY (2015) Cell-cell interactions, cell polarity, and the blood-testis barrier. In: Ebnet K (ed) Cell polarity 1. Springer International Publishing, Geneva, pp 303–326. Google Scholar
  9. 9.
    Tang EI, Mruk DD, Cheng CY (2016) Regulation of microtubule (MT)-based cytoskeleton in the seminiferous epithelium during spermatogenesis. Semin Cell Dev Biol 59:35–45. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Lie PPY, Mruk DD, Lee WM, Cheng CY (2010) Cytoskeletal dynamics and spermatogenesis. Philos Trans R Soc Lond B Biol Sci 365:1581–1592CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Vogl AW, Vaid KS, Guttman JA (2008) The Sertoli cell cytoskeleton. Adv Exp Med Biol 636:186–211CrossRefPubMedGoogle Scholar
  12. 12.
    Wen Q et al (2016) Transport of germ cells across the seminiferous epithelium during spermatogenesis—the involvement of both actin- and microtubule-based cytoskeletons. Tissue Barriers 4(4):e1265042. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Tang EI, Lee WM, Cheng CY (2016) Coordination of actin- and microtubule-based cytoskeletons supports transport of spermatids and residual bodies/phagosomes during spermatogenesis in the rat testis. Endocrinology 157(4):1644–1659. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Li N, Wong CK, Cheng CY (2016) Plastins regulate ectoplasmic specialization via its actin bundling activity on microfilaments in the rat testis. Asian J Androl 18:716–722. CrossRefPubMedGoogle Scholar
  15. 15.
    Li N, Mruk DD, Tang EI, Lee WM, Wong CK, Cheng CY (2016) Formin 1 regulates microtubule and F-actin organization to support spermatid transport during spermatogenesis in the rat testis. Endocrinology 157:2894–2908. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Li N, Lee WM, Cheng CY (2016) Overexpression of plastin 3 in Sertoli cells disrupts actin microfilament bundle homeostasis and perturbs the tight junction barrier. Spermatogenesis 6(1):e1206353. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Lie PPY, Chan AYN, Mruk DD, Lee WM, Cheng CY (2010) Restricted Arp3 expression in the testis prevents blood-testis barrier disruption during junction restructuring at spermatogenesis. Proc Natl Acad Sci USA 107:11411–11416CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Li N, Mruk DD, Wong CKC, Han D, Lee WM, Cheng CY (2015) Formin 1 regulates ectoplasmic specialization in the rat testis through its actin nucleation and bundling activity. Endocrinology 156:2969–2983CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Li N, Mruk DD, Cheng CY (2015) Actin binding proteins in blood-testis barrier function. Curr Opin Endocrinol Diabetes Obes 22:238–247CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Li N, Tang EI, Cheng CY (2016) Regulation of blood-testis barrier by actin binding proteins and protein kinases. Reproduction 151(3):R29–R41. CrossRefPubMedGoogle Scholar
  21. 21.
    Hess RA, de Franca LR (2008) Spermatogenesis and cycle of the seminiferous epithelium. Adv Exp Med Biol 636:1–15PubMedGoogle Scholar
  22. 22.
    Xiao X, Mruk DD, Wong CKC, Cheng CY (2014) Germ cell transport across the seminiferous epithelium during spermatogenesis. Physiology 29(4):286–298. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Zhou F, Leder P, Martin SS (2006) Formin-1 protein associates with microtubules through a peptide domain encoded by exon-2. Exp Cell Res 312(7):1119–1126. CrossRefPubMedGoogle Scholar
  24. 24.
    Lie PPY, Mruk DD, Lee WM, Cheng CY (2009) Epidermal growth factor receptor pathway substrate 8 (Eps8) is a novel regulator of cell adhesion and the blood-testis barrier integrity in the seminiferous epithelium. FASEB J 23:2555–2567CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Qian X, Mruk DD, Wong EWP, Lie PPY, Cheng CY (2013) Palladin is a regulator of actin filament bundles at the ectoplasmic specialization in the rat testis. Endocrinology 154:1907–1920CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Johnson KJ (2014) Testicular histopathology associated with disruption of the Sertoli cell cytoskeleton. Spermatogenesis 4:e979106. CrossRefPubMedGoogle Scholar
  27. 27.
    Heindel JJ (2006) Role of exposure to environmental chemicals in the developmental basis of reproductive disease and dysfunction. Semin Reprod Med 24:156–167CrossRefGoogle Scholar
  28. 28.
    Boekelheide K (1993) Sertoli cell toxicants. In: Russell L, Griswold M (eds) The sertoli cell. Cache River Press, Clearwater, pp 551–575Google Scholar
  29. 29.
    Boekelheide K et al (2003) 2,5-Hexanedione-induced testicular injury. Annu Rev Pharmacol Toxciol 43:125–147CrossRefGoogle Scholar
  30. 30.
    Yan HHN, Mruk DD, Wong EWP, Lee WM, Cheng CY (2008) An autocrine axis in the testis that coordinates spermiation and blood-testis barrier restructuring during spermatogenesis. Proc Natl Acad Sci USA 105:8950–8955CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Su L, Mruk DD, Lie PPY, Silvestrini B, Cheng CY (2012) A peptide derived from laminin-γ3 reversibly impairs spermatogenesis in rats. Nat Communs 3:1185. CrossRefGoogle Scholar
  32. 32.
    Gao Y, Mruk DD, Lui WY, Lee WM, Cheng CY (2016) F5-peptide induces aspermatogenesis by disrupting organization of actin- and microtubule-based cytoskeletons in the testis. Oncotarget 7:64203–64220PubMedPubMedCentralGoogle Scholar
  33. 33.
    Wong EWP, Cheng CY (2013) NC1 domain of collagen α3(IV) derived from the basement membrane regulates Sertoli cell blood-testis barrier dynamics. Spermatogenesis 3:e25465. PMID:23885308; PMCID:PMC23710226CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Gao Y, Mruk D, Chen H, Lui WY, Lee WM, Cheng CY (2017) Regulation of the blood-testis barrier by a local axis in the testis: role of laminin alpha2 in the basement membrane. FASEB J 31:584–597. CrossRefPubMedGoogle Scholar
  35. 35.
    Gao Y, Chen H, Lui WY, Lee WM, Cheng CY (2017) Basement membrane laminin α2 regulation of BTB dynamics via its effects on F-actin and microtubule (MT) cytoskeletons is mediated through mTORC1 signaling. Endocrinology 158:963–978. CrossRefPubMedGoogle Scholar
  36. 36.
    Yao PL, Lin YC, Richburg JH (2010) Mono-(2-ethylhexyl) phthalate-induced disruption of junctional complexes in the seminiferous epithelium of the rodent testis is mediated by MMP2. Biol Reprod 82:516–527CrossRefPubMedGoogle Scholar
  37. 37.
    Yao PL, Lin YC, Richburg JH (2009) TNFα-mediated disruption of spermatogenesis in response to Sertoli cell injury in rodents is partially regulated by MMP2. Biol Reprod 80:581–589CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Siu MKY, Cheng CY (2004) Interactions of proteases, protease inhibitors, and the β1 integrin/laminin γ3 protein complex in the regulation of ectoplasmic specialization dynamics in the rat testis. Biol Reprod 70:945–964CrossRefPubMedGoogle Scholar
  39. 39.
    Siu MKY, Lee WM, Cheng CY (2003) The interplay of collagen IV, tumor necrosis factor-α, gelatinase B (matrix metalloprotease-9), and tissue inhibitor of metalloprotease-1 in the basal lamina regulates Sertoli cell-tight junction dynamics in the rat testis. Endocrinology 144:371–387CrossRefPubMedGoogle Scholar
  40. 40.
    Chen H, Mruk DD, Xiao X, Cheng CY (2017) Human spermatogenesis and its regulation. In: Winters SJ, Huhtaniemi IT (eds) Male hypogonadism, contemporary endocrinology. Springer International Publishing AG, New York, pp 1–24. Google Scholar
  41. 41.
    Sharpe RM (1994) Regulation of spermatogenesis. In: Knobil E, Neill JD (eds) The physiology of reproduction. Raven Press, New York, pp 1363–1434Google Scholar
  42. 42.
    O’Donnell L, Meachem SJ, Stanton PG, McLachlan RI (2006) Endocrine regulation of spermatogenesis. In: Neill JD (ed) Physiology of reproduction, 3rd edn. Elsevier, Amsterdam, pp 1017–1069Google Scholar
  43. 43.
    O’Donnell L, Robertson KM, Jones ME, Simpson ER (2001) Estrogen and spermatogenesis. Endocr Rev 22:289–318PubMedGoogle Scholar
  44. 44.
    McLachlan RI, O’Donnell L, Meachem SJ, Stanton PG, de Kretser DM, Pratis K, Robertson DM (2002) Hormonal regulation of spermatogenesis in primates and man: insights for development of the male hormonal contraceptive. J Androl 23:149–162PubMedGoogle Scholar
  45. 45.
    Easton AS (2012) Regulation of permeability across the blood-brain barrier. Adv Exp Med Biol 763:1–19PubMedGoogle Scholar
  46. 46.
    Campbell M, Humphries P (2012) The blood-retina barrier: tight junctions and barrier modulation. Adv Exp Med Biol 763:70–84PubMedGoogle Scholar
  47. 47.
    Dore-Duffy P, Cleary K (2011) Morphology and properties of pericytes. Methods Mol Biol 686:49–68. CrossRefPubMedGoogle Scholar
  48. 48.
    Winkler EA, Bell RD, Zlokovic BV (2011) Central nervous system pericytes in health and disease. Nat Neurosci 14(11):1398–1405. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Cheng CY, Mruk DD (2010) A local autocrine axis in the testes that regulates spermatogenesis. Nature Rev Endocrinol 6:380–395CrossRefGoogle Scholar
  50. 50.
    Setchell BP (2008) Blood-testis barrier, functional and transport proteins and spermatogenesis. Adv Exp Med Biol 636:212–233CrossRefPubMedGoogle Scholar
  51. 51.
    Mruk DD, Cheng CY (2011) Desmosomes in the testis. Moving into an unchartered territory. Spermatogenesis 1:47–51CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Livne A, Geiger B (2016) The inner workings of stress fibers—from contractile machinery to focal adhesions and back. J Cell Sci 129(7):1293–1304. CrossRefPubMedGoogle Scholar
  53. 53.
    Spence EF, Soderling SH (2015) Actin out: regulation of the synaptic cytoskeleton. J Biol Chem 290(48):28613–28622. CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Pizarro-Cerda J, Chorev DS, Geiger B, Cossart P (2017) The diverse family of Arp2/3 complexes. Trends Cell Biol 27(2):93–100. CrossRefPubMedGoogle Scholar
  55. 55.
    Dominguez R (2016) The WH2 domain and actin nucleation: necessary but insufficient. Trends Biochem Sci 41(6):478–490. CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Cheng CY, Mruk DD (2011) Regulation of spermiogenesis, spermiation and blood-testis barrier dynamics: novel insights from studies on Eps8 and Arp3. Biochem J 435:553–562CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Qian X, Mruk DD, Cheng YH, Cheng CY (2013) Actin cross-linking protein palladin and spermatogenesis. Spermatogenesis 3:e23473. CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Qian X, Mruk DD, Cheng YH, Cheng CY (2013) RAI14 (retinoic acid induced protein 14) is an F-actin regulator—lesson from the testis. Spermatogenesis 3:e24824CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Li SY, Mruk DD, Cheng CY (2013) Focal adhesion kinase is a regulator of F-actin dynamics: new insights from studies in the testis. Spermatogenesis 3(3):e25385. CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Vogl AW, Young JS, Du M (2013) New insights into roles of tubulobulbar complexes in sperm release and turnover of blood-testis barrier. Int Rev Cell Mol Biol 303:319–355CrossRefPubMedGoogle Scholar
  61. 61.
    Vogl AW, Du M, Wang XY, Young JS (2014) Novel clathrin/actin-based endocytic machinery associated with junction turnover in the seminiferous epithelium. Semin Cell Dev Biol 30:55–64CrossRefPubMedGoogle Scholar
  62. 62.
    Russell LD (1979) Observations on the inter-relationships of Sertoli cells at the level of the blood-testis barrier: evidence for formation and resorption of Sertoli-Sertoli tubulobulbar complexes during the spermatogenic cycle of the rat. Am J Anat 155:259–279CrossRefPubMedGoogle Scholar
  63. 63.
    Xiao X, Mruk DD, Wong EWP, Lee WM, Han D, Wong CKC, Cheng CY (2014) Differential effects of c-Src and c-Yes on the endocytic vesicle-mediated trafficking events at the Sertoli cell blood-testis barrier: an in vitro study. Am J Physiol Endocrinol Metab 307:E553–E562CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Zeller R et al (1999) Formin defines a large family of morphorgulatory genes and functions in establishment of the polarising region. Cell Tissue Res 296:85–93CrossRefPubMedGoogle Scholar
  65. 65.
    Baarlink C, Brandt D, Grosse R (2010) SnapShot: formins. Cell 142:172CrossRefPubMedGoogle Scholar
  66. 66.
    Grikscheit K, Grosse R (2016) Formins at the junction. Trends Biochem Sci 41(2):148–159. CrossRefPubMedGoogle Scholar
  67. 67.
    Woychik RP, Maas RL, Zeller R, Vogt TF, Leder P (1990) ‘Formins’: proteins deduced from the alternative transcripts of the limb deformity gene. Nature 346:850–853CrossRefPubMedGoogle Scholar
  68. 68.
    Kobielak A, Pasolli HA, Fuchs E (2004) Mammalian formin-1 participates in adherens junctions and polymerization of linear actin cables. Nat Cell Biol 6:21–30CrossRefPubMedGoogle Scholar
  69. 69.
    Li N, Mruk DD, Tang EI, Wong CKC, Lee WM, Silvestrini B, Cheng CY (2015) Formins: actin nucleators that regulate cytoskeletal dynamics during spermatogenesis. Spermatogenesis 5:e1066476. CrossRefPubMedPubMedCentralGoogle Scholar
  70. 70.
    Goode BL, Eck MJ (2007) Mechanism and function of formins in the control of actin assembly. Annu Rev Biochem 76:593–627CrossRefPubMedGoogle Scholar
  71. 71.
    Breitsprecher D, Goode BL (2013) Formins at a glance. J Cell Sci 126(Pt 1):1–7. CrossRefPubMedPubMedCentralGoogle Scholar
  72. 72.
    Bohnert KA, Willet AH, Kovar DR, Gould KL (2013) Formin-based control of the actin cytoskeleton during cytokinesis. Biochem Soc Trans 41(6):1750–1754. CrossRefPubMedPubMedCentralGoogle Scholar
  73. 73.
    Quinlan ME, Heuser JE, Kerkhoff E, Mullins RD (2005) Drosophila spire is an actin nucleation factor. Nature 433:382–388CrossRefPubMedGoogle Scholar
  74. 74.
    Vizcarra CL et al (2011) Structure and function of the interacting domains of Spre and Fmn-family formins. Proc Natl Acad Sci U S A 108:11884–11889CrossRefPubMedPubMedCentralGoogle Scholar
  75. 75.
    Quinlan ME, Hilgert S, Bedrossian A, Mullins RD, Kerkhoff E (2007) Regulatory interactions between two actin nucleators, Spire and Cappuccino. J Cell Biol 179:117–128CrossRefPubMedPubMedCentralGoogle Scholar
  76. 76.
    Dietrich S, Weiβ S, Pleiser S, Kerkhoff E (2013) Structural and functional insights into the Spir/formin actin nucleator complex. Biol Chem 394(12):1649–1660. CrossRefPubMedGoogle Scholar
  77. 77.
    Carlier MF, Husson C, Renault L, Didry D (2011) Control of actin assembly by the WH2 domains and their multifunctional tandem repeats in Spire and Cordon-Bleu. Int Rev Cell Mol Biol 290:55–85. CrossRefPubMedGoogle Scholar
  78. 78.
    Carlier MF, Pernier J, Montaville P, Shekhar S, Kuhn S, Cytoskeleton D, Motility G (2015) Control of polarized assembly of actin filaments in cell motility. Cell Mol Life Sci 72(16):3051–3067. CrossRefPubMedPubMedCentralGoogle Scholar
  79. 79.
    Xiao X, Wong EWP, Lie PPY, Mruk DD, Wong CKC, Cheng CY (2014) Cytokines, polarity proteins and endosomal protein trafficking and signaling—the Sertoli cell blood-testis barrier in vitro as a study model. Methods Enzymol 534:181–194CrossRefPubMedPubMedCentralGoogle Scholar
  80. 80.
    Su WH, Mruk DD, Cheng CY (2013) Regulation of actin dynamics and protein trafficking during spermatogenesis—insights into a complex process. Crit Rev Biochem Mol Biol 48:153–172CrossRefPubMedPubMedCentralGoogle Scholar
  81. 81.
    Tang EI, Mok KW, Lee WM, Cheng CY (2015) EB1 regulates tubulin and actin cytoskeletal networks at the Sertoli cell blood-testis barrier in male rats—an in vitro study. Endocrinology 156:680–693CrossRefPubMedGoogle Scholar
  82. 82.
    Akhmanova A, Steinmetz MO (2015) Control of microtubule organization and dynamics: two ends in the limelight. Nat Rev Mol Cell Biol 16(12):711–726. CrossRefPubMedGoogle Scholar
  83. 83.
    Nehlig A, Molina A, Rodrigues-Ferreira S, Honore S, Nahmias C (2017) Regulation of end-binding protein EB1 in the control of microtubule dynamics. Cell Mol Life Sci 74(13):2381–2393. CrossRefPubMedGoogle Scholar
  84. 84.
    Bowne-Anderson H, Hibbel A, Howard J (2015) Regulation of microtubule growth and catastrophe: unifying theory and experiment. Trends Cell Biol 25(12):769–779. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Qing Wen
    • 1
  • Elizabeth I. Tang
    • 1
  • Nan Li
    • 1
  • Dolores D. Mruk
    • 1
  • Will M. Lee
    • 2
  • Bruno Silvestrini
    • 3
  • C. Yan Cheng
    • 1
    • 3
    Email author
  1. 1.The Mary M. Wohlford Laboratory for Male Contraceptive ResearchCenter for Biomedical Research, Population CouncilNew YorkUSA
  2. 2.School of Biological SciencesUniversity of Hong KongHong KongChina
  3. 3.S.B.M. Pharmaceuticals SrlRomeItaly

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