Fahimeh Mohammadghasemi Corresponding Author
- Department of Anatomy, Faculty of Medicine, Gilan University of Medical Sciences, Rasht, Iran
Jafar Soleimanirad
- Electron Microscopy Laboratory, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Ahmad Ali Ghanbari
- Anatomy Department, Faculty of Medicine, Tabriz Medical Sciences University, Tabriz, Iran

Received: 1/1/2008 Accepted: 1/1/2008 - Publisher : Avicenna Research Institute

Related Articles


Other Format



Introduction: Chemotherapy has adverse effects on spermatogenesis and results in reversible or irreversible oligozoospermia or azoospermia. Little is known about the ultrastructural alterations of male germ cells following busulfan administration, as an anti-cancer drug. The present study aimed to study the ultrastructural alterations or apoptotic features of male mouse germ cells, following administration of various doses of busulfan. Materials & Methods: This study was performed on thirty-two 8-week-old adult male mice. Animals were divided into four groups. The mice on the control group were treated with DMSO and in the second, third and forth groups were treated with 10mg/kg, 20mg/kg and 40mg/kg busulfan, respectively. Busulfan was injected as a single dose intraperitoneally. Thirty-five days after the treatment, the mice testes were dissected and prepared for apoptosis study by trans-mission electron microscopy (TEM) and TUNEL assays. The results were analyzed by ANOVA test using SPSS software and a p<0.05 was regarded statistically significant. results: a single dose of busulfan induced ultrastructural features of apoptosis in all the experi-mental groups such as: chromatin marginalization of germ cell nuclei, especially in spermatogo-nia, deformation of nuclei, separation of germ cells and presence of large spaces between adjacent cells, cell shrinkage, occasional presence of vacuoles in germ cells and apoptotic bodies in sertoli cells. ultrastructural features of apoptosis were more frequent in spermatogonia and primary sper-matocytes. apoptosis was observed less frequently in the control group (2.77±0.57%), particularly in primary spermatocytes. however, the number of apoptotic cells in 10mg g and 20mg g busulfan treated groups were (58.23±7.32% and 54.09±6.23%) significantly higher than the number of apoptotic cells in 40mg g treated group (28.16±5.67%), (p<0.001). conclusion: administration of a single dose of busulfan in various doses of 10mg g, 20mg g and 40mg g, induces apoptosis in male germ cells, especially in spermatogonia and primary sper-matocytes. degree of apoptosis in male germ cells in busulfan treated groups is not dose depen-dent. fewer number of apoptotic cells in 40mg g busulfan treated group may be due to more declines in the number of germ cells in comparison to 10 and 20mg g busulfan treated groups.< pan>

Keywords: Busulfan, Chemotherapy, Germ cells, Apoptosis, Spermatogenesis, Azoospermia, Sertoli cell, Testis

To cite this article:


  1. Billig H, Chun SY, Eisenhauer K, J.W. Hsueh A. Gonadal cell apoptosis :hormone regulated cell demise. Hum Reprod Update.1996;2(2):103-17.   [PubMed]
  2. Print CG, Loveland KL. Germ cell suicide: new insights into apoptosis during spermatogenesis. Bio-essay. 2000;22:423-30.   [PubMed]
  3. Ohta H, Aizawa SH, Nishimune Y.Functional analysis of the p53 gene in apoptosis induced by heat stress or loss of stem cell factor signaling in mouse male germ cells. Biol Reprod. 2003;68:2249-54.   [PubMed]
  4. Shuttlesworth GA, de Rooij DG, Huhtaniemi I, Reiss-mann T, Russell LD, Shetty G, et al. Enhancement of A Spermatogonial proliferation and differentiation in irradiated rats by GnRH antagonist administration. Endocrinology. 2000;141:37-49.   [PubMed]
  5. Tohda A, Matsumiya K, Tadokoro Y, Yomogida K, Miyagawa Y, DohmaeK, et al. Testosterone suppresses spermatogenesis in juvenile spermatogonial depletion (jsd) mice. Biol Reprod. 2001;65:532-7.   [PubMed]
  6. Omezzine A, Chater S, Mauduit C,Florin A, Tabone E, Chuzel F, et al. Long-term apoptotic cell death process with increased expression and activation of caspase 3 and-6 in adult rat germ cells exposed in utero to fluta-mide. Endocrinology.2003;144(2):648-61.   [PubMed]
  7. Roberts R. Apoptosis in toxicology. 2nd ed. Taylor & Francies, London. 2000;p 130-204.
  8. Anderson PhO, Knoben JE, Troutman WG. Handbook of clinical data. 10th Edition. Mc Graw Hill, New york. 2002;p:136-40.
  9. Choi YJ, Ok DW, Kwon DN, Chung JI, Kim HC, Yeo SM, et al. Murine male germ cell apoptosis induced by busulfan treatment correleates with loss of c-kit-expression in a Fas/FasL-and P53-independent manner. FEBS Lett. 2004;575(1-3):41-51.   [PubMed]
  10. Van Keulen CJ, de Rooij DG. Spermatogenetic clones developing from repopulating stem cells surviving a high dose of an alkylating agent. Cell Tissue Kinet. 1975;8(6):543-51.   [PubMed]
  11. Bishop JB, Wassom JS. Toxicological review of busulfan (Myleran). Mutat Res. 1986;168(1):15-45.   [PubMed]
  12. Bucci LR, Meistrich ML. Effects of busulfan on mu-rine spermatogenesis: cytotoxicity, sterility, sperm ab- normalities, and dominant lethal mutations. Mutat Res. 1987;176(2):259-68.   [PubMed]
  13. Janes GF, Pomerantz DK. The effect of prenatal treat-ment with busulfan on in vitro androgen production by testes from rats of various ages. Can J Physiol Pharma-col. 1985;63(9):1155-58.   [PubMed]
  14. Boujrad N, Hochereau-de Rivers MT, 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. 1995; 27(4):223-8.   [PubMed]
  15. Grigg AP, Mclachlan R, Zaja J, Szer J. Reproductive status in long term bone marrow transplant survivors receiving busulfan- cychlophosphamide. Bone Marrow Transplant. 2000;26(10):1086-95.   [PubMed]
  16. Afify Z, Shaw PJ, Clavano-Harding A, Cowell CT. Growth and endocrine function in children with acute myeloid leukemia after bone marrow transplantation usingbusulfan/ cychlophosphamide. Bone Marrow Transplant. 2000;25(10):1087-92.   [PubMed]
  17. Veccino P, Uranga J, Arechaga J. Suppression of sper-matogenesis for cell transplantation in adult mice. Protoplasma. 2001;217(4):191-8.   [PubMed]
  18. Honaramooz A, Behboodi E, Hausler CL, Blash S, Ayres S, et al. Depletion of endogenous germ cells in male pigs and goats in preparation for germ cell trans-plantation. J Androl .2005;26(6):698-705.   [PubMed]
  19. Boujrad N, Hochereau-de Rivers MT, 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. 1995; 27(4):223-8.   [PubMed]
  20. Howell SJ, Shalet SM. Testicular function following chemotherapy. Hum Reprod Update. 2001;7(4):363-9.   [PubMed]
  21. Mohammad-Ghasemi F, Soleimanirad J, Ghanbari AA. A morphologic and morphometric study of adult mouse testis following different doses of busulfan administration. J Reprod Infertil. 2006;7(1):25-36.
  22. Udagawa K, Ogawa T, Watanabe T, Yumura Y, Take-da M, Hosaka M. GnRH analog, leuprorelin acetate, promotes regeneration of rat spermatogenesis after severe chemical damage. Int J Urol. 2001;8:615-22.   [PubMed]
  23. Labat-Moleur F, Guillermet C, Lorimier P, Robert C, Lantuejoul S, Brambilla E, Negoescu A. TUNEL apop-totic cell detection in tissue sections: critical evaluation and improvement. J Histochem Cytochem. 1998;46(3): 327-34.   [PubMed]
  24. Sawhney P, Gimmona J, Meistrich ML, Richburg JH. Cisplatin-induced long-term failure of spermatogenesis in adult c57/BL/6J Mice. J Androl. 2005;26(1):136-45.   [PubMed]
  25. Swada H, Esaki M. Electron microscopic observation of 137Cs-irradiated rat testis: Production of basal lami-nae for germ cells, despite their absence. J Electron Microsc. 2003;52(4):391-7.   [PubMed]
  26. Newton SC, Blaschuk OW, Millette CF. N-cadherin mediates Sertoli cell-spermatogenic cell adhesion. Dev Dyn. 1993;197(1):1-13.   [PubMed]
  27. Day ML, Zhao X, Vallorosi CJ, Putzi M, Powell CT, Lin C, et al. E-cadherin mediates aggregation depen-dent survival of prostate and mammary epithelial cells through the retinoblastoma cell cycle control pathway. J Biol Chem. 1999;274(14):9656-64.   [PubMed]
  28. Allan DJ, Gobe GC, Harmon BV. Sertoli cell death by apoptosis in the immature rat testis following x-irra-diation. Scaning Microsc. 1998;2(1):503-12.   [PubMed]
  29. Ateeq B, Farah MA, Ahmad W. Evidence of apoptotic effects of 2,4-D and butachlor on walking catfish, clarios batrachus, by transmission electron microscopy and DNA degradation studies. Life Science. 2006;78 (9):977-86.   [PubMed]
  30. Andrew W, Vicki D, Berton F, Hill D, Mansow S. Apoptosis and cell differentiation. 2nd Edition. Boeh-ringer Mannheim. 1998;p:116-20.
  31. Francavilla S, D'Abrizio P, Cordeschi G, Pelliccione F, Necozione S, Ulisse S,et al. Fas expression corre-lates with human germ cell degeneration in meiotic and post-meiotic arrest of spermatogenesis. Mol Hum Reprod. 2002;8(3):213-20.   [PubMed]
  32. Print CG, Loveland KL. Germ cell suicide: new insights into apoptosis during spermatogenesis. 2000; Bioessays. 22(5):423-30.   [PubMed]
  33. Shiratsuchi A, Kawasaki Y, Ikemoto M, Arai H, Nakanishi Y. Role of class B scavenger receptor type I in phagocytosis of apoptotic rats spermatogenic cells by sertoli cells. J Biol Chem. 1999;274(9):5901-08.   [PubMed]
  34. Tesarik J, Greco F, Cohen-Bacrie P, Mendoza C. Germ cell apoptosis in men with complete and income-plete spermiogenesis failure. Mol Hum Reprod. 1998;4 (8):757-62.   [PubMed]
  35. Kopcky M, Semecky V, Nachtigal P. Vimentin ex-pression during altered spermatogenesis in rats. Acta Histochem. 2005;107(4):279-89.   [PubMed]
  36. Brinkworth MH, Nieschlag E. Association of cychlo-phosphamide-induced male mediated, foetal abnor-malities with reduced paternal germ cell apoptosis. Mutat Res. 2000;447:149-54.   [PubMed]
  37. Sawhney P, Gimmona J,Meistrich ML, Richburg JH. Cisplatin induced long-term failure of spermatogenesis in adult C57 /BL/6J mice. J Androl. 2005;26(1):136-45.   [PubMed]
  38. Zhang X, Yamamoto N, Soramoto S, Takenaka I. Cis-platin-Induced germ cell apoptosis in mouse testes. Arch Androl. 2001;46(1):43-9.   [PubMed]
  39. Cai L, Hales BF, Robaire B. Induction of apoptosis in the germ cells of adult male rats after exposure to cyclophosphamide. Biol Reproduction. 1997;56(6): 1490-7.   [PubMed]
  40. Krishnamurty H, Weinbauer GF, Aslam H, Yeung CH. Quantification of apoptotic testicular germ cells in normal and Methoxyacetic acid-treated mice as deter-mined by flow cytometry. J Androl. 1998;19(6): 710-7.   [PubMed]
  41. Udagawa K, Takeda M, Hosaka M, Kubota Y, Ogawa T. Recovery of spermatogenesis by high dose gonado-tropin-releasing hormone analoge treatment in rat cryptorchid testis after orchiopexy. J Urol. 2002;168 (3):1279-83.   [PubMed]


Home | About Us | Current Issue | Past Issues | Submit a Manuscript | Instructions for Authors | Subscribe | Search | Contact Us

"Journal of Reproduction & Infertility" is owned, published, and managed by Avicenna Research Institute .
Creative Commons License

This work is licensed under a Creative Commons Attribution –NonCommercial 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.

Journal of Reproductoin and Infertility (JRI) is a member of COMMITTEE ON PUBLICATION ETHICS . Verify here .

©2024 - eISSN : 2251-676X, ISSN : 2228-5482, For any comments and questions please contact us.