Skip to main content
SpringerLink
Log in

Proliferated Leydig cells for engineered testis-like tissue regeneration with testosterone-secreting ability

  • Original Article
  • Tissue Engineering
  • Published:
Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Tissue engineering approach provides a hopeful strategy for reconstructing testis testosterone-secreting functions. However, limited source and low proliferative activity in vitro of Leydig cells (LCs, the main testosteroneproducing cells) makes testis-like tissue regeneration difficult to be achieved. This study explored the feasibility of in vitro expanding LCs and their potential application in testis-like tissue regeneration. LC lineage cells were isolated from Sprague-Dawley (SD) rats by differential adhesion method and cell composition was identified by expressions of 3β-HSD, LHR, LIFR, and c-kit. A modified expansion medium (EM) system was used to test the feasibility of in vitro expanding LC lineage. The results showed that the attached cells reached a high purification of LC lineage (>90%, indicated by positive expression of 3β-HSD) and that EM significantly enhanced proliferation of LC lineage compared to regular medium, which was testified to be related to the presence of stem LCs that was implied by positive expressions of LIFR and c-kit as well as the transition of 3β-HSD expression from negative to positive in partial cells. Importantly, the proliferated LCs showed relatively sustained testosterone-secreting ability in vitro and these cells combined with biodegradable scaffolds successfully regenerated testis-like tissue with sustained testosteronesecreting function in vivo, which was supported by the enhanced serum testosterone level in castrated rats. All these results indicated that the differential adhesion method could efficiently isolate and purify LC lineage and that EM system could efficiently promote proliferation and functional maintenance of LC lineage, providing a good cell source for testes-like tissue regeneration.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. M Beer, R Kay, Testicular prostheses, Urol Clin North Am, 16, 133 (1989).

    PubMed  CAS  Google Scholar 

  2. D Zilberman, H Winkler, N Kleinmann, et al., Testicular prosthesis insertion following testicular loss or atrophy during early childhood—technical aspects and evaluation of patient satisfaction, J Pediatr Urol, 3, 461 (2007).

    Article  PubMed  CAS  Google Scholar 

  3. CC Ho, SF Tong, WY Low, CJ Ng, et al., A randomized, double-blind, placebo-controlled trial on the effect of longacting testosterone treatment as assessed by the Aging Male Symptoms scale, BJU Int, 110, 260 (2012).

    Article  PubMed  CAS  Google Scholar 

  4. A Aynsley-Green, M Zachmann, R Illig, et al., Congenital bilateral anorchia in childhood: a clinical, endocrine and therapeutic evaluation of twenty-one cases, Clin Endocrinol (Oxf), 5, 381 (1976).

    Article  CAS  Google Scholar 

  5. F Jockenhovel. Testosterone therapy — what, when and to whom?, Aging Male, 7, 319 (2004).

    Article  PubMed  CAS  Google Scholar 

  6. A Raya-Rivera, C Baez, A Atala, et al., Tissue engineered testicular prostheses with prolonged testosterone release, World J Urol, 26, 351 (2008).

    Article  PubMed  CAS  Google Scholar 

  7. F Jockenhovel. Testosterone supplementation: what and how to give, Aging Male, 6, 200 (2003).

    Article  PubMed  CAS  Google Scholar 

  8. F Saad, A Aversa, A Isidori, et al., Onset of effects of testosterone treatment and time span until maximum effects are achieved, Eur J Endocrinol, 165, 673 (2011).

    Google Scholar 

  9. T John, M Fordman. Spontaneous ruputre of testicular prothesis with external leakage of silicone-a rare event, J Urol, 170, 1306 (2003).

    Article  PubMed  CAS  Google Scholar 

  10. M Lappe. Silicone-reactive disorde: a new autoimmune disease cuased by immunostimulation and superantigens, Med Hypotheses, 41, 348 (1993).

    Article  PubMed  CAS  Google Scholar 

  11. A Atala. Tissue engineering approaches for genital reconstruction, Adv Exp Med Biol, 511, 289 (2002).

    Article  PubMed  Google Scholar 

  12. R Langer, JP Vacanti. Tissue engineering, Science, 920, 260 (1993).

    Google Scholar 

  13. PX Ma. Biomimetic materials for tissue engineering. Adv Drug Deliv Rev, 60, 184 (2008).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  14. SM Mendis-Handagama, HB Ariyaratne. Differentiation of the adult Leydig cell population in the postnatal testis. Biol Reprod, 65, 660 (2001).

    Article  PubMed  CAS  Google Scholar 

  15. J Tai, HW Johnson, WJ Tze. Successful transplantation of Leydig cells in castrated inbred rats, Transplantation, 47, 1087 (1989).

    Article  PubMed  CAS  Google Scholar 

  16. JH van Dam, KJ Teerds, FF Rommerts. Transplantation and subsequent recovery of small amounts of isolated Leydig cells, Arch Androl, 22, 123 (1989).

    Article  PubMed  Google Scholar 

  17. J Sun, YB Xi, ZD Zhang, et al., Leydig cell transplantation restores androgen production in surgically castrated prepubertal rats, Asian J Androl, 11, 405 (2009).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  18. JP Fouquet, F Raynaud. Renewal of Leydig cells in the neonatal and adult monkey: a radioautographic study, Biol Cell, 54, 187 (1985).

    Article  PubMed  CAS  Google Scholar 

  19. P Amat, R Paniagua, M Nistal, et al., Mitosis in adult human Leydig cells. Cell Tissue Res, 243, 219 (1986).

    Article  PubMed  CAS  Google Scholar 

  20. KJ Teerds, DG De Rooij, FF Rommerts et al., The regulation of the proliferation and differentiation of rat Leydig cell precursor cells after EDS administration or daily HCG treatment, J Androl, 9, 343 (1988).

    PubMed  CAS  Google Scholar 

  21. K Shiraishi, M Ascoli. Lutropin/choriogonadotropin stimulate the proliferation of primary cultures of rat Leydig cells through a pathway that involves activation of the extracellularly regulated kinase 1/2 cascade, Endocrinology, 148, 3214 (2007).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  22. JY Browning, JJ Heindel, HE Grotjan. Primary culture of purified Leydig cells isolated from adult rat testes. Endocrinology, 112, 543 (1983).

    Article  PubMed  CAS  Google Scholar 

  23. GR Klinefelter, LL Ewing. Optimizing testosterone production by purified adult rat Leydig cells in vitro. In Vitro Cell Dev Biol, 24, 543 (1988).

    Article  Google Scholar 

  24. A Salva, GR Klinefelter, Hardy MP. Purification of rat leydig cells: increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells. J Androl, 22, 665 (2001).

    PubMed  CAS  Google Scholar 

  25. D Conner. Mouse embryonic stem (ES) cell culture. In: Struhl K, ed. Current Protocol in Molecular Biology. New York: Wiley; 2001. pp. S51.

    Google Scholar 

  26. R Ge, Q Dong, C Sottas, et al., In search of rat stem Leydig cells: Identification, isolation, and lineage-specific development. Proc Natl Acad Sci U S A, 103, 2719 (2006).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. GR Chen, RS Ge, H Lin, et al., Development of a cryopreservation protocol for Leydig cells, Hum Reprod, 22, 2160 (2007).

    Article  PubMed  CAS  Google Scholar 

  28. T Furukawa, T Kubota, A Suto, et al., Clinical usefulness of chemosensitivity testing using the MTT assay, J Surg Oncol, 48, 188 (1991).

    Article  PubMed  CAS  Google Scholar 

  29. LE Rees, S Gunasekaran, F Sipaul, et al., The isolation and characterisation of primary human laryngeal epithelial cells, Mol Immunol, 43, 725 (2006).

    Article  PubMed  CAS  Google Scholar 

  30. AW Wognum, PM Lansdorp, RK Humphries, et al., Detection and isolation of the erythropoietin receptor using biotinylated erythropoietin. Blood, 76, 697 (1990).

    PubMed  CAS  Google Scholar 

  31. R Ge, M Hardy. Variation in the end products of androgen biosynthesis and metabolism during postnatal differentiation of rat Leydig cells, Endocrinology, 139, 3787 (1998).

    PubMed  CAS  Google Scholar 

  32. K Liu, GD Zhou, W Liu et al., The dependence of in vivo stable ectopic chondrogenesis by human mesenchymal stem cells on chondrogenic differentiation in vitro, Biomaterials, 29, 2183 (2008).

    Article  PubMed  CAS  Google Scholar 

  33. D Yan, G Zhou, X Zhou, et al., The impact of low levels of collagen IX and pyridinoline on the mechanical properties of in vitro engineered cartilage, Biomaterials, 30, 814 (2009).

    Article  PubMed  CAS  Google Scholar 

  34. M Hardy, B Zirkin, L Ewing. Kinetic studies on the development of the adult population of Leydig cells in testes of the pubertal rat, Endocrinology, 124, 762 (1989).

    Article  PubMed  CAS  Google Scholar 

  35. H Ariyarante, C Mendis-Handagama. Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity, Biol Reprod, 62, 280 (2000).

    Google Scholar 

  36. EL Stanley, DS Johnston, J Fan, et al., Stem Leydig cell differentiation: gene expression during development of the adult rat population of Leydig cells, Biol Reprod, 85, 1161 (2011).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  37. LS Wright, J Li, MA Caldwell, et al., Gene expression in human neural stem cells: effects of leukemia inhibitory factor, J Neurochem, 86, 179 (2003).

    Article  PubMed  CAS  Google Scholar 

  38. C Piquet-Pellorce, I Dorval-Coiffec, MD Pham, et al., Leukemia inhibitory factor expression and regulation within the testis, Endocrinology, 141, 1136 (2000).

    Article  PubMed  CAS  Google Scholar 

  39. J Brennan, C Tilmann, B Capel. Pdgfr-alpha mediates testis cord organization and fetal Leydig cell development in the XY gonad, Genes Dev, 17, 800 (2003).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  40. S Basciani, S Mariani, M Arizzi, et al., Expression of plateletderived growth factor-A (PDGF-A), PDGF-B, and PDGF receptor-alpha and -beta during human testicular development and disease, J Clin Endocrinol Metab, 87, 2310 (2002).

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China, 200433

    Hongda Bi, Xiaoyun Wang & Xin Xing

  2. Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Tissue Engineering, National Tissue Engineering Center of China, Shanghai, China, 200011

    Wei Liu, Yilin Cao & Guangdong Zhou

Authors
  1. Hongda Bi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoyun Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xin Xing
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yilin Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Guangdong Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Xiaoyun Wang, Xin Xing or Guangdong Zhou.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bi, H., Wang, X., Xing, X. et al. Proliferated Leydig cells for engineered testis-like tissue regeneration with testosterone-secreting ability. Tissue Eng Regen Med 11, 379–386 (2014). https://doi.org/10.1007/s13770-014-0057-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13770-014-0057-0

Key words

Search

Navigation