Cell and Tissue Research

, Volume 375, Issue 2, pp 543–558 | Cite as

The possible protective role of zinc oxide nanoparticles (ZnONPs) on testicular and epididymal structure and sperm parameters in nicotine-treated adult rats (a histological and biochemical study)

  • Dalia A. MohamedEmail author
  • Shaimaa A. Abdelrahman
Regular Article


Exposure to nicotine in smoking contributes to most unexplained male infertility but the mechanisms remain to be fully elucidated. Zinc (Zn) is an essential trace element in normal growth, development and reproduction. Zinc oxide nanoparticles (ZnONPs) are well-known antioxidants. Therefore, this work was designed to investigate the potential ability of ZnONPs to protect testis and epididymis in nicotine-treated rats. Forty adult male Wistar albino rats were divided into control group and two experimental groups (treated and supplemented rats). In the treated group, rats received nicotine at a dose of 1 mg/kg/day orally for 30 days. Rats in the supplemented group received ZnONPs (10 mg/kg/day) with nicotine (1 mg/kg/day), orally for the same period. Testicular and epididymal sections were stained with H&E to assess the histological changes. Negrosin-eosin staining of epididymal sperms was performed to assess their viability and morphological changes. Serum testosterone, FSH and LH levels were assessed. Also, oxidative stress parameters and semiquantitative real-time PCR for steroidogenic enzymes were measured. Morphometric studies of both organs were statistically analyzed. Mild to severe testicular and epididymal structural changes together with sperm morphological abnormalities were detected in nicotine-treated rats. Biochemical results also showed a decrease in all measured parameters except for an increased malondialdehyde (MDA) level that meant deterioration of their reproductive function. On the other hand, ZnONP supplementation in the last group showed an obvious improvement in all investigated parameters.


Zinc oxide nanoparticles Nicotine Testis Epididymis Rats 





Zinc oxide nanoparticles


World Health Organization


Steroidogenic acute regulatory protein


Semiquantitative reverse transcriptase-PCR


Follicle-stimulating hormone


Luteinizing hormone


Enzyme-linked immune sorbent assay


Coefficient of variation


Phosphate-buffered saline


Ethylenediaminetetraacetic acid


Thiobarbituric acid reactive substances




Superoxide dismutase




Round per minute


Total sperm abnormalities


Outer mitochondrial membrane


Seminiferous tubules


Nitroblue tetrazolium to measure neutrophil oxidative burst




Glutathione peroxidase


Body weight


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abdul-Ghani AS (2014) Studies on cigarette smoke induced oxidative DNA damage and reduced spermatogenesis in rats. J Environ Biol 35:943–947Google Scholar
  2. Acharya UR, Mishra M, Patro J, Panda MK (2008) Effect of vitamin C and E on spermatogenesis in mice exposed to cadmium. Reprod Toxicol 25(1):84–88Google Scholar
  3. Afifi M, Almaghrabi OA, Kadasa NM (2015) Ameliorative effect of zinc oxide nanoparticles on antioxidants and sperm characteristics in streptozotocin-induced diabetic rat testes. BioMed Res Int 19:1–6Google Scholar
  4. Akingebemi BT, Sottas CM, Koulova A, Klinefelter GR, Hardy MP (2004) Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells. Endocrinology 145:592–603Google Scholar
  5. Al-Ani NK, Al-Kawaz U, Saeed BT (2015) Protective influence of zinc on reproductive parameters in male rat treated with cadmium. Am J Med Med Sci 5(2):73–81Google Scholar
  6. Arrighi S, Bosil G, Groppetti D, Cremonesi F (2010) Morpho- and histometric evaluations on the testis and epididymis in buffalo bulls during the different reproductive seasons. Open Anat J 2, 29–33.Google Scholar
  7. Babaei H, Azaril O, Kheirandish R, Abshenas J, Mohammadi N (2010) Zinc therapy improves deleterious effects of experimental unilateral cryptorchidism: histopathological evaluation of testes. IJVS 5(1, 2):77–88Google Scholar
  8. Bancroft J, Layton C (2013) Hematoxylin and eosin. In: Suvarna SK, Layton C, Bancroft JD (eds) Theory and practice of histological techniques, Ch. 10 and 11, 7th edn. Churchill Livingstone of Elsevier, Philadelphia, pp. 172–214.Google Scholar
  9. Bedwal S, Prasad S, Nair N, Saini MR, Bedwal RS (2009) Catalase in testes and epididymidis of Wistar rats fed zinc deficient diet. Indian J Pharmac Sci 71(1):55–58Google Scholar
  10. Bellinger LL, Wellman PJ, Harris R, Kelso EW, Kramer PR (2010) The effects of chronic nicotine on meal patterns, food intake, metabolism and body weight of male rats. Pharmacol Biochem Behav 95:92–99Google Scholar
  11. Berchtold MW (1989) A simple method for direct cloning and sequencing cDNA by the use of a sinle specific oligonucleotide and oligo (dT) in a polymerase chain reaction (PCR). Nucleic Acid Res 17(11):453Google Scholar
  12. Bin Dohaish E, Ali A, Melebary S (2008) Histological changes in the testes of two strains of mice: effect of ecological factors. Saudi J Biol Sci 15(2):279–287Google Scholar
  13. Bishop C, Parker GC, Coscina DV (2002) Nicotine and its withdrawal after feeding induced by paraventricular hypothalamic injections of neuropeptide Y in Sprague- Dawley rats. Psychopharmacology 162(3):265–272Google Scholar
  14. Bose M, Debnath D, Chen Y, Bose HS (2007) Folding, activity and import of steroidogenic acute regulatory protein into mitochondria changed by nicotine exposure. J Mol Endocrinol 39(1):67–79Google Scholar
  15. Burns LH (2007) Psychiatric aspects of infertility and infertility treatments. Psychiatr Clin North Am 30:689–716Google Scholar
  16. Cao Y, Li YS, Li ZJ, Wang F, Li CM (2015) Dietary zinc may attenuate heat-induced testicular oxidative stress in mice via up-regulation of Cu-Zn SOD. Genet Mol Res 14(4):16,616–16,626Google Scholar
  17. Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J 326:1–16Google Scholar
  18. Dai JB, Wang ZX, Qiao ZD (2015) The hazardous effects of tobacco smoking on male fertility. Asian J Androl 17:954–960Google Scholar
  19. Duru FI, Yama OE, Noronha CC, Okanlawon AO (2011) Alterations in morphometry and malondialdehyde levels in adult Sprague-Dawley rat testes in three obstructive vasectomy models: effect of melatonin. Asian J Pharm Clin Res 4(2):27–30Google Scholar
  20. Elshal MF, El-Sayed IH, Elsaied MA, El-Masry SA, Kumosani TA (2009) Sperm head defects and disturbances in spermatozoal chromatin and DNA integrities in idiopathic infertile subjects: association with cigarette smoking. Clin Biochem 42:589–594Google Scholar
  21. Emadi L, Azari O, Gholipour H, Saeed M (2012) Effect of vitamin C on epididymal spermquality in the rat experimentally induced unilateral cryptorchidism. IJVS 7(1, 2):63–74Google Scholar
  22. Ezzatabadipour M, Azizollahi S, Sarvazad A, Mirkahnooj Z, Mahdinia Z, Nematollahi-Mahani SN (2012) Effects of concurrent chronic administration of alcohol and nicotine on rat sperm parameters. Andrologia 44(5):330–336Google Scholar
  23. Hirst SM, Krakatoa A, Singh S et al (2013) Bio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in mice. Environ Toxicol 28(2):107–118Google Scholar
  24. Hsieh CH (2007) Spherical zinc oxide nano particles from zinc acetate in the precipitation method. J Chin Chem Soc 54(1):31–34Google Scholar
  25. Imran A, Muhammad S, Khalid FY (2003) Study of the effects of lead poisoning on the testes in albino rats. Pak J Med Res 42(3):97–101Google Scholar
  26. Jalili C, Khani F, Salahshoor MR, Roshankhah S (2014) Protective effect of curcumin against nicotine-induced damage on reproductive parameters in male mice. Int J Morphol 32(3):844–849Google Scholar
  27. Jana K, Samanta PK, De DK (2010) Nicotine diminishes testicular gametogenesis, steroidogenesis, and steroidogenic acute regulatory protein expression in adult albino rats: possible influence on pituitary gonadotropins and alteration of testicular antioxidant status. Toxicol Sci 116:647–659Google Scholar
  28. Kapawa A, Giannakos D, Tsoukanelis K, Kanakas N, Kalogiannis D, Agapitos E, Loutradis D, Miyagawa I, Sofikitis N (2004) Effects of paternal cigarette smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation in rats. Andrologia 36:57–68Google Scholar
  29. Kara A, Unal D, Simsek N, Yucel A, Yucel N, Selli J (2014) Ultra- structural changes and apoptotic activity in cerebellum of post- menopausal-diabetic rats: a histochemical and ultra-structural study. J Gynecol Endocrinol 30(3):226–231Google Scholar
  30. Klein LC, Corwin EJ, Ceballos RM (2004) Leptin, hunger, and body weight: influence of gender, tobacco smoking, and smoking abstinence. Addic Behav 29:921–927Google Scholar
  31. Knobil E (1980) The neuroendocrine control of the menstrual cycle. Rec Prog Horm Res 36:52–88Google Scholar
  32. La Maestra S, De Flora S, Micale RT (2015) Effect of cigarette smoke on DNA damage, oxidative stress, and morphological alterations in mouse testis and spermatozoa. Int J Hyg Environ Health 218:117–122Google Scholar
  33. Linder RE, Klinefelter GR, Strader LF, Narotsky MG, Suarez JD, Roberts NL, Perreault SD (1995) Dibromoacetic acid affects reproductive competence and sperm quality in the male rat. Fund Appl Toxicol 2825:9–17Google Scholar
  34. Liu L, Wang JF, Fan J, Rao YS, Liu F, Yan YE, Wang H (2016) Nicotine suppressed fetal adrenal star expression via YY1 mediated-histone deacetylation modification mechanism. Int J Mol Sci 17(9):1477Google Scholar
  35. Maruyama Y, Aoki N, Suzuki Y, Ohno Y, Imamura M, Saika T, Sinohara H, Yamamoto T (1987) Sex-steroid-binding plasma protein (SBP), testosterone, oestradiol and dehydroepiandrosterone Reprod, (DHEA) in prepuberty and puberty. Acta Endocrinol. 114:60–67Google Scholar
  36. Mathur PP, D’Cruz SC (2011) The effect of environmental contaminants on testicular function. Asian J Androl 13:585–591 (review)Google Scholar
  37. Miranda-Spooner M, Paccola CC, Neves FM, Oliva SU, Miraglia SM (2016) Late reproductive analysis in rat male offspring exposed to nicotine during pregnancy and lactation. Andrology 4(2):218–231Google Scholar
  38. Mohammadi T, Hoveizi E, Khajehpour L, Jelodar Z (2017) Protective effects of zinc oxide nanoparticles on testis histological structure in cyclophosphamide treated adult mice. J Mazandaran Univ Med Sci 26(144):19–27Google Scholar
  39. Mostafa T (2010) Cigarette smoking and male infertility. Rev J Adv Res 1:179–186Google Scholar
  40. Nair SV, Rajamohan T (2014) The role of coconut water on nicotine-induced reproductive dysfunction in experimental male rat model. Food Nutr Sci 5:1121–1130Google Scholar
  41. Narayana K, D’Souza UJ, Rao KP (2002) Ribavirin induced sperm shape abnormalities in Wistar rat. Mut Res 513:193–196Google Scholar
  42. Nerín I, Beamonte A, Gargallo P, Jiménez-Muro A, Marqueta A (2007) Weight gain and anxiety levels in recent ex-smokers. Arch Bronconeumol 43:9–15Google Scholar
  43. Nolte T, Harleman JH, Jahn W (1995) Histopathology of chemically induced testicular atrophy in rats. Exp Toxic Pathol 47:267–286Google Scholar
  44. Oyeyemi WA, Shittu ST, Kolawole TA, Ubanecheand P, Akinola AO (2015) Protective effect of vitamin E on nicotine induced reproductive toxicity in male rats. Nigerian J Basic Appl Sci 23(1):7–13Google Scholar
  45. Oyeyipo IP, Raji Y, Emikpe BO, Bolarinwa AF (2010) Effects of oral administration of nicotine on organ weight, serum testosterone level and testicular histology in adult male rats. Nig J Physiol Sci 25:81–86Google Scholar
  46. Oyeyipo IP, Raji Y, Emikpe BO, Bolarinwa AF (2011) Effects of nicotine on sperm characteristics and fertility profile in adult male rats: a possible role of cessation. J Reproductive Infertility 12:201–207.Google Scholar
  47. Practice Committee of the American Society for Reproductive Medicine [PCASRM] (2006) Smoking and infertility. Fertil Steril 86:172–177Google Scholar
  48. Reddy S, Londonkar R, Reddy S, Patil SB (1998) Testicular changes due to graded doses of nicotine in albino mice. Indian J Physiol Pharmacol 42:276–280Google Scholar
  49. Ribeiro AG, Ribeiro-paes GT, Stessuk T, Kozma RL, Isabel Camargo IC, Biosci J (2016) Histomorphometrical evaluation in testis and epididymis of rats with pulmonary emphysema experimentally induced by prolonged exposure to cigarette smoke. Biosci J Uberlandia 32(4):1092–1102Google Scholar
  50. Sankako MK, Garcia PC, Piffer RC, Dallaqua B, Damasceno DC, Oduvaldo CM (2012) Pereira1 Possible mechanism by which zinc protects the testicular function of rats exposed to cigarette smoke. Pharmacol Rep 64:1537–1546Google Scholar
  51. Torabi F, Shafaroudi MM, Rezaei N (2017) Combined protective effect of zinc oxide nanoparticles and melatonin on cyclophosphamide-induced toxicity in testicular histology and sperm parameters in adult Wistar rats. Int J Reprod BioMed 15(7):403–412Google Scholar
  52. Vaseem M, Umar A, Hahn YB (2010) ZnO nanoparticles: growth, properties, and applications of copper oxide and nickel oxide/hydroxide nanostructures. Chapter 2 in book: Metal Oxide Nanostructures and Their Applications. American Scientific Publishers, pp 1–39Google Scholar
  53. Wakabayashi K (1977) Heterogeneity of rat luteinizing hormone revealed by radioimmunoassy and electrofocusing studies. Endocrinol Japon 24:473–485Google Scholar
  54. WHO (2010) laboratory manual for the examination and processing of human semen. 5th edn. Sexual and reproductive health (Updated July 2013).
  55. Wu YL, Tok AI, Boey FY, Zeng XT, Zhang XH (2007) Surface modification of ZnO nanocrystals. Appl Surf Sci 253:5473–5479Google Scholar
  56. Zhao Y, Tan Y, Dai J, Li B, Guo L, Cui J, Wang G, Shi X, Zhang X, Mellen N, Li W (2011) Exacerbation of diabetes-induced testicular apoptosis by zinc deficiency is most likely associated with oxidative stress, p38 MAPK activation, and p53 activation in mice. Toxicol Lett 200(1):100–106Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Histology and Cell Biology, Faculty of MedicineZagazig UniversityZagazigEgypt

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