Skip to main content

Dose-dependent effects of busulfan on dog testes in preparation for spermatogonial stem cell transplantation

Abstract

Successful male germ cell transplantation requires depletion of the host germ cells to allow efficient colonization of the donor spermatogonial stem cells. Although a sterilizing drug, busulfan, is commonly used for the preparation of recipient models before transplantation, the optimal dose of this drug has not yet been defined in dogs. In this study, 1-year-old mongrel dogs were intravenously injected with three different concentrations of busulfan (10, 15, or 17.5 mg/kg). Four weeks after busulfan treatment, no fully matured spermatozoa were detected in any of the busulfan-treated groups. However, small numbers of PGP9.5-positive spermatogonia were detected in all treatment groups, although no synaptonemal complex protein-3-positive spermatocytes were detected. Of note, acrosin-positive spermatids were not detected in the dogs treated with 15 or 17.5 mg/kg busulfan, but were detected in the other group. Eight weeks after busulfan treatment, the dogs treated with 10 mg/kg busulfan fully recovered, but those in the other groups did not. PGP9.5-positive spermatogonia were detected in the 10 mg/kg group, and at a similar level as in the control group, but these cells were rarely detected in the 15 and 17.5 mg/kg groups. These results suggest that a dose of 15–17.5 mg/kg is optimal for ablative treatment with busulfan to prepare the recipient dogs for male germ cell transplantation. At least eight weeks should be allowed for recovery. The results of this study might facilitate the production of recipient dogs for male germ cell transplantation and can also contribute to studies on chemotherapy.

References

  1. 1.

    Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci USA 1994; 91(24): 11298–11302.

    CAS  Article  Google Scholar 

  2. 2.

    Ogawa T, Dobrinski I, Brinster RL. Recipient preparation is critical for spermatogonial transplantation in the rat. Tissue Cell 1999; 31(3): 461–472.

    CAS  Article  Google Scholar 

  3. 3.

    Jahnukainen K, Ehmcke J, Quader MA, Saiful Huq M, Epperly MW, Hergenrother S, Nurmio M, Schlatt S. Testicular recovery after irradiation differs in prepubertal and pubertal non-human primates, and can be enhanced by autologous germ cell transplantation. Hum Reprod 2011; 26(3): 1945–1954.

    Article  Google Scholar 

  4. 4.

    Shinohara T, Avarbock MR, Brinster RL. Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models. Dev Biol 2000; 220(4): 401–411.

    CAS  Article  Google Scholar 

  5. 5.

    Jegou B, Peake RA, Irby DC, de Kretser DM. Effects of the induction of experimental cryptorchidism and subsequent orchidopexy on testicular function in immature rats. Biol Reprod 1984; 30(3): 179–187.

    CAS  Article  Google Scholar 

  6. 6.

    Bucci LR, Meistrich ML. Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations. Mutat Res 1987; 176(4): 259–268.

    CAS  Article  Google Scholar 

  7. 7.

    Wang DZ, Zhou XH, Yuan YL, Zheng XM. Optimal dose of busulfan for depleting testicular germ cells of recipient mice before spermatogonial transplantation. Asian J Androl 2010; 12(3): 263–270.

    CAS  Article  Google Scholar 

  8. 8.

    Kim JH, Jung-Ha HS, Lee HT, Chung KS. Development of a positive method for male stem cell-mediated gene transfer in mouse and pig. Mol Reprod Dev 1997; 46(3): 515–526.

    CAS  Article  Google Scholar 

  9. 9.

    Hermann BP, Sukhwani M, Winkler F, Pascarella JN, Peters KA, Sheng Y, Valli H, Rodriguez M, Ezzelarab M, Dargo G, Peterson K, Masterson K, Ramsey C, Ward T, Lienesch M, Volk A, Cooper DK, Thomson AW, Kiss JE, Penedo MC, Schatten GP, Mitalipov S, Orwig KE. Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm. Cell Stem Cell 2012; 11(3): 715–726.

    CAS  Article  Google Scholar 

  10. 10.

    Deeg HJ, Schuler US, Shulman H, Ehrsam M, Renner U, Yu C, Storb R, Ehninger G. Myeloablation by intravenous busulfan and hematopoietic reconstitution with autologous marrow in a canine model. Biol Blood Marrow Transplant 1999; 5(2): 316–321.

    CAS  Article  Google Scholar 

  11. 11.

    Lee WY, Lee R, Park HJ, Do JT, Park C, Kim JH, Jhun H, Lee JH, Hur T, Song H. Characterization of male germ cell markers in canine testis. See comment in PubMed Commons belowAnim Reprod Sci 2017; 182: 1–8.

    CAS  Google Scholar 

  12. 12.

    Hill JR, Dobrinski I. Male germ cell transplantation in livestock. Reprod Fertil Dev 2006; 18(1-2): 13–18.

    Google Scholar 

  13. 13.

    Brinster CJ, Ryu BY, Avarbock MR, Karagenc L, Brinster RL, Orwig KE. Restoration of fertility by germ cell transplantation requires effective recipient preparation. Biol Reprod 2003; 69(3): 412–420.

    CAS  Article  Google Scholar 

  14. 14.

    Hassan M, Oberg G, Björkholm M, Wallin I, Lindgren M. Influence of prophylactic anticonvulsant therapy on high-dose busulphan kinetics. Cancer Chemother Pharmacol 1993; 33(3): 181–186.

    CAS  Article  Google Scholar 

  15. 15.

    Boettger-Tong HL, Johnston DS, Russell LD, Griswold MD, Bishop CE. Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of supporting transplanted spermatogenesis. Biol Reprod 2000; 63(3):1185–1191.

    Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Hyunjhung Jhun or Won-Young Lee.

Rights and permissions

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://doi.org/creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hur, TY., Lee, SH., Ock, SA. et al. Dose-dependent effects of busulfan on dog testes in preparation for spermatogonial stem cell transplantation. Lab Anim Res 33, 264–269 (2017). https://doi.org/10.5625/lar.2017.33.3.264

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.5625/lar.2017.33.3.264

Keywords

  • Dog
  • busulfan
  • testis
  • germ cell