Research Paper: Effects of Different Doses and Time-Dependency of Busulfan on Testes Parameters and Spermatogenesis in a Rat Model: A Quantitative Stereological Study

Document Type : Original Articles


1 Department of Anatomy, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.

2 Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

3 Endocrinology and Metabolism Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.



Objectives: The current study aimed at evaluating testis parameters and spermatogenesis changes in male rats administrated by different busulfan doses and time to construct a subfertile animal model by stereological methods.Materials and Methods: In the present study, 150 male Wistar rats randomly divided into 5 groups. All experimental groups were treated by different concentrations of busulfan (0.0, 2.5, 5, 10, and 15 mg/kg). Rats were sacrificed 1, 15, and 30 days after busulfan treatment. The tissue processing was done for stereological study and the results were analyzed by the one-way ANOVA followed by the Duncan test. Results: The most stereological parameters such as testes weight and volume, tubules volume density, interstitial tissue (P<0.05), and germinal epithelium (P<0.01) were significantly reduced by busulfan treatment. Also, at different busulfan doses, the number of spermatogenic cells including spermatogonia (P<0.05), spermatocyte, round and elongated spermatid, and the Sertoli and Leydig cells (P<0.01) significantly decreased, compared with those of the control group. The decline was more obvious in higher busulfan doses and time (from the day 15 to 30) (P<0.05). Conclusion: Most of testicular stereological parameters reduced during 15 days onwards after busulfan treatment in a dose-dependent manner.


  1. Nudell DM, Monoski MM, Lipshultz LI. Common medications and drugs: How they affect male fertility. Urologic Clinics of North America. 2002; 29(4):965–73. doi: 10.1016/s0094-0143(02)00079-4
  2. Howell SJ. Spermatogenesis after cancer treatment: Damage and recovery. Journal of the National Cancer Institute Monographs. 2005; 2005(34):12–7. doi: 10.1093/jncimonographs/lgi003
  3. Puscheck E, Philip PA, Jeyendran RS. Male fertility preservation and cancer treatment. Cancer Treatment Reviews. 2004; 30(2):173–80. doi: 10.1016/j.ctrv.2003.07.005
  4. PhO A, Knoben JE, Troutman WG. Handbook of clinical data. London: MCGraw Hill; 2002.
  5. Trevor AJ, Katzung BG, Masters SB, Kruidering Hall M. Pharmacology examination & board review. New York: McGraw Hill Medical; 2010.
  6. Westerhof GR, Ploemacher RE, Boudewijn A, Blokland I, Dillingh JH, McGown AT, et al. Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor-type chimerism in murine bone marrow transplant recipients. Cancer Research. 2000; 60(19):5470-8.
  7. Galaup A, Paci A. Pharmacology of dimethanesulfonate alkylating agents: Busulfan and treosulfan. Expert Opinion on Drug Metabolism & Toxicology. 2012; 9(3):333–47. doi: 10.1517/17425255.2013.737319
  8. Molenaar R, de Rooij DG, Rommerts FFG, Reuvers PJ, van der Molen HJ. Specific destruction of leydig cells in mature rats after In-Vivo administration of ethane dimethyl sulfonate. Biology of Reproduction. 1985; 33(5):1213–22. doi: 10.1095/biolreprod33.5.1213
  9. Silver RT, Woolf SH, Hehlmann R, Appelbaum FR, Anderson J, Bennett C, et al. An evidence-based analysis of the effect of busulfan, hydroxyurea, interferon, and allogeneic bone marrow transplantation in treating the chronic phase of chronic myeloid leukemia: Developed for the American Society of Hematology. Blood. 1999; 94(5):1517-36. PMID: 10477676
  10. Sanders JE, Buckner CD, Amos D, Levy W, Appelbaum FR, Doney K, et al. Ovarian function following marrow transplantation for aplastic anemia or leukemia. Journal of Clinical Oncology. 1988; 6(5):813–8. doi: 10.1200/jco.1988.6.5.813
  11. Boujrad N, Reviers MTH, Kamtchouing P, Perreau C, Carreau S. Evolution of somatic and germ cell populations after busulfan treatment in utero or neonatal cryptorchidism in the rat. Andrologia. 2009; 27(4):223–8. doi: 10.1111/j.1439-0272.1995.tb01097.x
  12. Jansz GF, Pomerantz DK. The effect of prenatal treatment with busulfan on in vitro androgen production by testes from rats of various ages. Canadian Journal of Physiology and Pharmacology. 1985; 63(9):1155–8. doi: 10.1139/y85-189
  13. Panahi M, Keshavarz S, Rahmanifar F, Tamadon A, Mehrabani D, Karimaghai N, et al. Busulfan induced azoospermia: Stereological evaluation of testes in rat. Veterinary Research Forum. 2015; 6(4):273.
  14. Grigg A, McLachlan R, Zajac J, Szer J. Reproductive status in long-term bone marrow transplant survivors receiving busulfan-cyclophosphamide (120 mg/kg). Bone Marrow Transplantation. 2000; 26(10):1089–95. doi: 10.1038/sj.bmt.1702695
  15. Afify Z, Shaw P, Clavano Harding A, Cowell C. Growth and endocrine function in children with acute myeloid leukaemia after bone marrow transplantation using busulfan/cyclophosphamide. Bone Marrow Transplantation. 2000; 25(10):1087–92. doi: 10.1038/sj.bmt.1702384
  16. Yang ZW, Guo Y, Lin L, Wang XH, Tong JS, Zhang GY. Quantitative (stereological) study of incomplete spermatogenic suppression induced by testosterone undecanoate injection in rats. Asian Journal of Andrology. 2004; 6(4):291-7.
  17. Aich S, Manna CK. Histophysiological changes of the testicular tissue due to busulphan administration in the wild Indian house rat (Rattus rattus). Acta Biologica Hungarica. 2001; 52(1):105-16. doi: 10.1556/abiol.52.2001.1.10
  18. Haddad S, Lamano Carvalho TL, Anselmo Franci JA, Petenusci SO, Favaretto ALV. Ultrasound stimulation of rat testes damaged by busulfan. Ultrasound in Medicine & Biology. 1997; 23(9):1421–5. doi: 10.1016/s0301-5629(97)00185-3
  19. Bucci LR, Meistrich ML. Effects of busulfan on murine spermatogenesis: Cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 1987; 176(2):259–68. doi: 10.1016/0027-5107(87)90057-1
  20. Osinubi A, Noronha C, Okanlawon A. Morphometric and stereological assessment of the effects of long-term administration of quinine on the morphology of rat testis. West African Journal of Medicine. 2006; 24(3):200-5. doi: 10.4314/wajm.v24i3.28198
  21. Mandarim de Lacerda CA. Stereological tools in biomedical research. Anais da Academia Brasileira de Ciências. 2003; 75(4):469–86. doi: 10.1590/s0001-37652003000400006
  22. Elias H, Hyde DM. An elementary introduction to stereology (quantitative microscopy). American Journal of Anatomy. 1980; 159(4):411–46. doi: 10.1002/aja.1001590407
  23. Cruz Orive LM, Weibel ER. Recent stereological methods for cell biology: A brief survey. American Journal of Physiology Lung Cellular and Molecular Physiology. 1990; 258(4):L148-56. PMID: 2185653
  24. Gundersen H, Bendtsen T, Korbo L, Marcussen N, Møller A, Nielsen K, et al. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS. 1988; 96(1-6):379–94. doi: 10.1111/j.1699-0463.1988.tb05320.x
  25. Mattfeldt T, Mall G, Gharehbaghi H, Möller P. Estimation of surface area and length with the orientator. Journal of Microscopy. 1990; 159(3):301–17. doi: 10.1111/j.1365-2818.1990.tb03036.x
  26. SØRensen FB, Kristensen IB, Grymer F, Jakobsen A. DNA-index and stereological estimation of nuclear volume in primary and metastatic malignant melanomas: A comparative study with analysis of heterogeneity. APMIS. 1990; 98(1-6):61–70. doi: 10.1111/j.1699-0463.1990.tb01003.x
  27. Dehghani F, Hassanpour A, Poost pasand A, Noorafshan A, Karbalay Doust S. Protective effects of L-carnitine and homogenized testis tissue on the testis and sperm parameters of busulfan-induced infertile male rats. Iranian Journal of Reproductive Medicine. 2013; 11(9):693-704. PMCID: PMC3941323
  28. Aitken R, Smith T, Jobling M, Baker M, De Iuliis G. Oxidative stress and male reproductive health. Asian Journal of Andrology. 2014; 16(1):31. doi: 10.4103/1008-682x.122203
  29. Bar Shira Maymon B, Yogev L, Marks A, Hauser R, Botchan A, Yavetz H. Sertoli cell inactivation by cytotoxic damage to the human testis after cancer chemotherapy. Fertility and Sterility. 2004; 81(5):1391–4. doi: 10.1016/j.fertnstert.2003.09.078
  30. Kanatsu Shinohara M, Toyokuni S, Morimoto T, Matsui S, Honjo T, Shinohara T. Functional assessment of self-renewal activity of male germline stem cells following cytotoxic damage and serial transplantation. Biology of Reproduction. 2003; 68(5):1801–7. doi: 10.1095/biolreprod.102.012575
  31. Choi YJ, Ok DW, Kwon DN, Chung J, Kim HC, Yeo SM, et al. 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 Letters. 2004; 575(1-3):41–51. doi: 10.1016/j.febslet.2004.08.034
  32. Krause VW. Teratogenic effect of busulfan on testis cells of the rat (postnatal development). Arzneimittel Forschung. 1975; 25(4):644-5.