Skip to main content

Progress on the study of the mechanism of busulfan cytotoxicity

Abstract

The preparation of spermatogonial stem cell (SSC) transplant recipients laid the technical foundation for SSC transplant technology and the understanding of spermatogenesis mechanisms. Busulfan is commonly used to prepare recipients for mouse SSC transplantation; however, its safety and efficiency have been questioned. This review summarizes the relationship between SSCs and Sertoli cells (SCs), and the mechanism of busulfan toxicity against sperm cells. We concluded that the proliferation, differentiation, and apoptosis of SSCs are regulated by SCs. The endogenous spermatogenic cells are depleted by busulfan treatment via alkylation of DNA, destruction of vimentin filament distribution, disruption of SSC differentiation, promotion of SSC dormancy, and generation of oxidative stress. However, the mechanisms require further exploration. The recent establishment of a model in vitro culture system has provided a good technical foundation to further explore these mechanisms, which will help us to find more efficient methods of recipient preparation and optimal transplantation times.

This is a preview of subscription content, access via your institution.

References

  • Amann RP (2008) The cycle of the seminiferous epithelium in humans: a need to revisit? J Androl 29:469–487

    Article  Google Scholar 

  • Amlani S, Vogl AW (1988) Changes in the distribution of microtubules and intermediate filaments in mammalian Sertoli cells during spermatogenesis. Anat Rec 220:143–160

    CAS  Article  Google Scholar 

  • Cai Y, Liu T, Fang F, Shen S, Xiong C (2016) Involvement of ICAM-1 in impaired spermatogenesis after busulfan treatment in mice. Andrologia 48:37–44

    CAS  Article  Google Scholar 

  • Carlomagno G, van Bragt MP, Korver CM, Repping S, de Rooij DG, van Pelt AM (2010) BMP4-induced differentiation of a rat spermatogonial stem cell line causes changes in its cell adhesion properties. Biol Reprod 83:742–749

    CAS  Article  Google Scholar 

  • Choi YJ, Ok DW, Kwon DN, Chung JI, Kim HC, Yeo SM, Kim T, Seo HG, Kim JH (2004) Murine male germ cell apoptosis induced by busulfan treatment correlates with loss of c-kit-expression in a Fas/FasL- and p53-independent manner. FEBS Lett 575:41–51

    CAS  Article  Google Scholar 

  • de Rooij DG (2009) The spermatogonial stem cell niche. Microsc Res Tech 72:580–585

    Article  Google Scholar 

  • DeLeve LD, Wang X (2000) Role of oxidative stress and glutathione in busulfan toxicity in cultured murine hepatocytes. Pharmacology 60:143–154

    CAS  Article  Google Scholar 

  • Ebata KT, Yeh JR, Zhang X, Nagano MC (2011) Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro. Exp Cell Res 317:1319–1329

    CAS  Article  Google Scholar 

  • ElGhamrawy TA, Helmy D, Elall HF (2014) Cadherin and vimentin immunoexpression in the testis of normal and induced infertility models of albino rats. Folia Morphol (Warsz) 73:339–346

    CAS  Article  Google Scholar 

  • Feng LX, Ravindranath N, Dym M (2000) Stem cell factor/c-kit up-regulates cyclin D3 and promotes cell cycle progression via the phosphoinositide 3-kinase/p70 S6 kinase pathway in spermatogonia. J Biol Chem 275:25572–25576

    CAS  Article  Google Scholar 

  • Franca LR, Hess RA, Dufour JM, Hofmann MC, Griswold MD (2016) The Sertoli cell: one hundred fifty years of beauty and plasticity. Andrology 4:189–212

    CAS  Article  Google Scholar 

  • Furukawa S, Usuda K, Abe M, Hayashi S, Ogawa I (2007) Busulfan-induced apoptosis in rat placenta. Exp Toxicol Pathol 59:97–103

    CAS  Article  Google Scholar 

  • Ganguli N, Wadhwa N, Usmani A, Kunj N, Ganguli N, Sarkar RK, Ghorai SM, Majumdar SS (2016) An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation. Stem Cell Res Ther 7:142

    Article  Google Scholar 

  • Gupta S, Agrawal A, Agrawal S, Su H, Gollapudi S (2006) A paradox of immunodeficiency and inflammation in human aging: lessons learned from apoptosis. Immun Ageing 3:5

    Article  Google Scholar 

  • Hai Y, Hou J, Liu Y, Liu Y, Yang H, Li Z, He Z (2014) The roles and regulation of Sertoli cells in fate determinations of spermatogonial stem cells and spermatogenesis. Semin Cell Dev Biol 29:66–75

    CAS  Article  Google Scholar 

  • Hassan Z, Hellstrom-Lindberg E, Alsadi S, Edgren M, Hagglund H, Hassan M (2002) The effect of modulation of glutathione cellular content on busulphan-induced cytotoxicity on hematopoietic cells in vitro and in vivo. Bone Marrow Transpl 30:141–147

    CAS  Article  Google Scholar 

  • Hofmann MC (2008) Gdnf signaling pathways within the mammalian spermatogonial stem cell niche. Mol Cell Endocrinol 288:95–103

    CAS  Article  Google Scholar 

  • Iwamoto T, Hiraku Y, Oikawa S, Mizutani H, Kojima M, Kawanishi S (2004) DNA intrastrand cross-link at the 5′-GA-3′ sequence formed by busulfan and its role in the cytotoxic effect. Cancer Sci 95:454–458

    CAS  Article  Google Scholar 

  • Kanatsu-Shinohara M, Toyokuni S, Morimoto T, Matsui S, Honjo T, Shinohara T (2003) Functional assessment of self-renewal activity of male germline stem cells following cytotoxic damage and serial transplantation. Biol Reprod 68:1801–1807

    CAS  Article  Google Scholar 

  • Kanatsu-Shinohara M, Morimoto H, Shinohara T (2016) Fertility of male germline stem cells following spermatogonial transplantation in infertile mouse models. Biol Reprod 94:112

    Google Scholar 

  • Kissel H, Timokhina I, Hardy MP, Rothschild G, Tajima Y, Soares V, Angeles M, Whitlow SR, Manova K, Besmer P (2000) Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses. EMBO J 19:1312–1326

    CAS  Article  Google Scholar 

  • Kopecky M, Semecky V, Nachtigal P (2005) Vimentin expression during altered spermatogenesis in rats. Acta Histochem 107:279–289

    CAS  Article  Google Scholar 

  • Li Y, Zhang Y, Zhang X, Sun J, Hao J (2014) BMP4/Smad signaling pathway induces the differentiation of mouse spermatogonial stem cells via upregulation of Sohlh2. Anat Rec (Hoboken) 297:749–757

    CAS  Article  Google Scholar 

  • Li B, He X, Zhuang M, Niu B, Wu C, Mu H, Tang F, Cui Y, Liu W, Zhao B, Peng S, Li G, Hua J (2017) Melatonin ameliorates busulfan-induced spermatogonial stem cell oxidative apoptosis in mouse testes. Antioxid Redox Signal 28:385–400

    Article  Google Scholar 

  • Lin Z, Bao J, Kong Q, Bai Y, Luo F, Songyang Z, Wu Y, Huang J (2017) Effective production of recipient male pigs for spermatogonial stem cell transplantation by intratesticular injection with busulfan. Theriogenology 89:e362

    Article  Google Scholar 

  • Lindsten T, Ross AJ, King A, Zong WX, Rathmell JC, Shiels HA, Ulrich E, Waymire KG, Mahar P, Frauwirth K, Chen Y, Wei M, Eng VM, Adelman DM, Simon MC, Ma A, Golden JA, Evan G, Korsmeyer SJ, MacGregor GR, Thompson CB (2000) The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell 6:1389–1399

    CAS  Article  Google Scholar 

  • Liu S, Tang Z, Xiong T, Tang W (2011) Isolation and characterization of human spermatogonial stem cells. Reprod Biol Endocrinol 9:141

    Article  Google Scholar 

  • Luo XMZC, Yang SX, Wang LL (2010) Murine model of busulfan-induced spermatogenesis regeneration: a quantitative evaluation. Natl J Androl 16:395–399

    CAS  Google Scholar 

  • Mirhoseini M, Saki G, Hemadi M, Khodadadi A, Mohammadi Asl J (2014) Melatonin and testicular damage in busulfan treated mice. Iran Red Crescent Med J 16:e14463

    Article  Google Scholar 

  • 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

    CAS  Article  Google Scholar 

  • Muzio M, Stockwell BR, Stennicke HR, Salvesen GS, Dixit VM (1998) An induced proximity model for caspase-8 activation. J Biol Chem 273:2926–2930

    CAS  Article  Google Scholar 

  • Naruse T, Takahara M, Takagi M, Oberg KC, Ogino T (2007) Busulfan-induced central polydactyly, syndactyly and cleft hand or foot: a common mechanism of disruption leads to divergent phenotypes. Dev Growth Differ 49:533–541

    Article  Google Scholar 

  • Oatley JM, Brinster RL (2012) The germline stem cell niche unit in mammalian testes. Physiol Rev 92:577–595

    CAS  Article  Google Scholar 

  • Oatley JM, Avarbock MR, Brinster RL (2007) Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on Src family kinase signaling. J Biol Chem 282:25842–25851

    CAS  Article  Google Scholar 

  • Otsuji M, Takahara M, Naruse T, Guan D, Harada M, Zhe P, Takagi M, Ogino T (2005) Developmental abnormalities in rat embryos leading to tibial ray deficiencies induced by busulfan. Birth Defects Res A Clin Mol Teratol 73:461–467

    CAS  Article  Google Scholar 

  • Probin V, Wang Y, Zhou D (2007) Busulfan-induced senescence is dependent on ROS production upstream of the MAPK pathway. Free Radic Biol Med 42:1858–1865

    CAS  Article  Google Scholar 

  • Qin Y, Liu L, He Y, Ma W, Zhu H, Liang M, Hao H, Qin T, Zhao X, Wang D (2016a) Testicular injection of busulfan for recipient preparation in transplantation of spermatogonial stem cells in mice. Reprod Fertil Dev 28:1916–1925

    CAS  Article  Google Scholar 

  • Qin Y, Liu L, He Y, Wang C, Liang M, Chen X, Hao H, Qin T, Zhao X, Wang D (2016b) Testicular busulfan injection in mice to prepare recipients for spermatogonial stem cell transplantation is safe and non-toxic. PLoS ONE 11:e0148388

    Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  • Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Korsmeyer SJ (2001) Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292:727–730

    CAS  Article  Google Scholar 

  • Wu X, Goodyear SM, Tobias JW, Avarbock MR, Brinster RL (2011) Spermatogonial stem cell self-renewal requires ETV5-mediated downstream activation of Brachyury in mice. Biol Reprod 85:1114–1123

    CAS  Article  Google Scholar 

  • Wu C, Zhang Y, Shen Q, Zhou Z, Liu W, Hua J (2016) Resveratrol changes spermatogonial stem cells (SSCs) activity and ameliorates their loss in busulfan-induced infertile mouse. Oncotarget 7:82085–82096

    Google Scholar 

  • Xiao X, Cheng CY, Mruk DD (2012) Intercellular adhesion molecule-1 is a regulator of blood-testis barrier function. J Cell Sci 125:5677–5689

    CAS  Article  Google Scholar 

  • Xiong T, Tang W, Liu S-X, He Y-F, Tang S-W, Li J-B (2010) Both juxtacrine and paracrine signaling indispensable in spermatogonial stem cell cultures. J Reprod Contracept 21:193–202

    Article  Google Scholar 

  • Zhivotovsky B, Kroemer G (2004) Apoptosis and genomic instability. Nat Rev Mol Cell Biol 5:752–762

    CAS  Article  Google Scholar 

  • Zohni K, Zhang X, Tan SL, Chan P, Nagano MC (2012) The efficiency of male fertility restoration is dependent on the recovery kinetics of spermatogonial stem cells after cytotoxic treatment with busulfan in mice. Hum Reprod 27:44–53

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 31772595) and the Beijing Dairy Industry Innovation Team (BAIC06-2017).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dong Wang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chen, X., Liang, M. & Wang, D. Progress on the study of the mechanism of busulfan cytotoxicity. Cytotechnology 70, 497–502 (2018). https://doi.org/10.1007/s10616-018-0189-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-018-0189-5

Keywords

  • Mice
  • Busulfan
  • Spermatogenic cells
  • Spermatogonial stem cells
  • Sertoli cells