Optimizing Acridine Orange Staining for Assessment of Protective Effects of Saffron and Vitamin E on Rat Sperm DNA Structure

Document Type : Original Article(s)

Authors

1 Professor, Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 PhD student, Department of Tissue Engineering, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

3 Associate Professor, Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: In recent decades, relation between reactive oxygen species (ROS) concentration and semen quality was noted. Traditionally, saffron has been not only considered as a food additive but also as a medicinal herb, which has good antioxidant properties. Vitamin E is considered as a endogenous and supplementary antioxidant which can protects the cell. The aim of this study was to evaluate the protection influence of saffron and vitamin E on sperm DNA against acid.Methods: Thirty adult male Wistar rats randomly divided into 3 equal groups of saffron, vitamin E and controlwhich received saffron (100 mg/kg/day), vitamin E (100 mg/kg/day) and distilled water (0.5 cc/day), respectively. After 60 days, cauda epididymis dissected and sperm were used for analysis of sperm chromatin susceptibility to acid denaturation via acridine orange (AO) staining. Acridine orange staining was carried out with or without acid detergent incubation.Findings: Both, saffron and vitamin E, decreased sperm DNA damage against acid (P < 0.001).Conclusion: Saffron and vitamin E protect DNA against denaturation, probably because of their powerful antioxidant properties.

Keywords

References

  1. Geva E, Lessing JB, Lerner-Geva L, Amit A. Free radicals, antioxidants and human spermatozoa: clinical implications. Hum Reprod 1998; 13(6): 1422-4.
  2. Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol 2005; 3: 28.
  3. Giudice LC. Infertility and the environment: the medical context. Semin Reprod Med 2006; 24(3): 129-33.
  4. Balhorn R. The protamine family of sperm nuclear proteins. Genome Biol 2007; 8(9): 227.
  5. Sadeghi MR, Lakpour N, Heidari-Vala H, Hodjat M, Amirjannati N, Hossaini JH, et al. Relationship between sperm chromatin status and ICSI outcome in men with obstructive azoospermia and unexplained infertile normozoospermia. Rom J Morphol Embryol 2011; 52(2): 645-51.
  6. Hanukoglu I. Antioxidant protective mechanisms against reactive oxygen species (ROS) generated by mitochondrial P450 systems in steroidogenic cells. Drug Metab Rev 2006; 38(1-2): 171-96.
  7. Turner TT, Lysiak JJ. Oxidative stress: a common factor in testicular dysfunction. J Androl 2008; 29(5): 488-98.
  8. Nasr-Esfahani MH, Razavi S, Mardani M. Relation between different human sperm nuclear maturity tests and in vitro fertilization. J Assist Reprod Genet 2001; 18(4): 219-25.
  9. Hoshi K, Katayose H, Yanagida K, Kimura Y, Sato A. The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm. Fertil Steril 1996; 66(4): 634-9.
  10. Halliwell B. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 1994; 344(8924): 721-4.
  11. Bansal AK, Bilaspuri GS. Impacts of oxidative stress and antioxidants on semen functions. Vet Med Int 2010; 2010.
  12. Abdullaev, F. Biological properties and medicinal use of Saffron (Crocus sativus l.). Acta Hort (ISHS) 2007; 739: 339-45.
  13. Khayatnouri M, Safavi SE, Safarmashaei S, Babazadeh D, Mikailpourardabili B. The effect of Saffron orally administration on spermatogenesis index in rat. Advances in Environmental Biology 2011; 5(7): 1514-21.
  14. Heidary M, Vahhabi S, Reza NJ, Delfan B, Birjandi M, Kaviani H, et al. Effect of saffron on semen parameters of infertile men. Urol J 2008; 5(4): 255-9.
  15. Vega SG, Guzman P, Garcia L, Espinosa J, Cortinas de NC. Sperm shape abnormality and urine mutagenicity in mice treated with niclosamide. Mutat Res 1988; 204(2): 269-76.
  16. Linder RE, Klinefelter GR, Strader LF, Narotsky MG, Suarez JD, Roberts NL, et al. Dibromoacetic acid affects reproductive competence and sperm quality in the male rat. Fundam Appl Toxicol 1995; 28(1): 9-17.
  17. Jasko DJ. Evaluation of stallion semen. Vet Clin North Am Equine Pract 1992; 8(1): 129-48.
  18. Linder RE, Strader LF, Slott VL, Suarez JD. Endpoints of spermatotoxicity in the rat after short duration exposures to fourteen reproductive toxicants. Reprod Toxicol 1992; 6(6): 491-505.
  19. Neild D, Chaves G, Flores M, Mora N, Beconi M, Aguero A. Hypoosmotic test in equine spermatozoa. Theriogenology 1999; 51(4): 721-7.
  20. Amann R, Graham J. Spermatozoal function. In: McKinnon A, Voss JL, editors. Equine reproduction. Philadelphia, PA: Lea and Febiger; 1993. p. 715- 45.
  21. Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool 1999; 284(6): 696-704.
  22. Wyrobek AJ, Bruce WR. Chemical induction of sperm abnormalities in mice. Proc Natl Acad Sci U S A 1975; 72(11): 4425-9.
  23. Sailer BL, Jost LK, Evenson DP. Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J Androl 1995; 16(1): 80-7.
  24. Bochenek M, Smorag Z, Pilch J. Sperm chromatin structure assay of bulls qualified for artificial insemination. Theriogenology 2001; 56(4): 557-67.
  25. Lopes S, Jurisicova A, Casper RF. Gamete-specific DNA fragmentation in unfertilized human oocytes after intracytoplasmic sperm injection. Hum Reprod 1998; 13(3): 703-8.
  26. Rybar R, Faldikova L, Faldyna M, Machatkova M, Rubes J. Bull and boar sperm DNA integrity evaluated by sperm chromatin structure assay in the Czech Republic. Veterinarni Medicina 2004; 49(1): 1-8.
  27. Mahi CA, Yanagimachi R. Induction of nuclear decondensation of mammalian spermatozoa in vitro. J Reprod Fertil 1975; 44(2): 293-6.
  28. Bucak MN, Sariozkan S, Tuncer PrB, Sakin F, Atessahin A, Kulaksiz R, et al. The effect of antioxidants on post-thawed Angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Ruminant Research 2010; 89(1): 24-30.
  29. Agarwal A, Makker K, Sharma R. Clinical relevance of oxidative stress in male factor infertility: an update. Am J Reprod Immunol 2008; 59(1): 2-11.
  30. Sigman M, Jarow JP. Male infertility. In: Wein AJ, editor. Campbell-Walsh urology. 9th ed. Philadelphia, PA: Sanders Elsevier; 2007. p. 609-53.
  31. Razavi SH, Nasr-Esfahani MH, Deemeh MR, Shayesteh M, Tavalaee M. Evaluation of zeta and HA-binding methods for selection of spermatozoa with normal morphology, protamine content and DNA integrity. Andrologia 2010; 42(1): 13-9.
  32. Nasr-Esfahani MH, Salehi M, Razavi S, Anjomshoa M, Rozbahani S, Moulavi F, et al. Effect of sperm DNA damage and sperm protamine deficiency on fertilization and embryo development post-ICSI. Reprod Biomed Online 2005; 11(2): 198-205.
  33. Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction 2001; 122(4): 497-506.
  34. Suleiman SA, Ali ME, Zaki ZM, el-Malik EM, Nasr MA. Lipid peroxidation and human sperm motility: protective role of vitamin E. J Androl 1996; 17(5): 530-7.
  35. Hosseinzadeh H, Ziaee T, Sadeghi A. The effect of saffron, Crocus sativus stigma, extract and its constituents, safranal and crocin on sexual behaviors in normal male rats. Phytomedicine 2008; 15(6-7): 491-5.
  36. Uzunhisarcikli M, Kalender Y, Dirican K, Kalender S, Ogutcu A, Buyukkomurcu F. Acute, subacute and subchronic administration of methyl parathion-induced testicular damage in male rats and protective role of vitamins C and E. Pesticide Biochemistry and Physiology 2007; 87(2): 115-22.
  37. Bansal A, Bilaspuri G. Antioxidant effect of vitamin E on motility, viability and lipid peroxidation of cattle spermatozoa under oxidative stress. Animal Science Papers and Reports 2009; 27(1): 5-14.
  38. Kumar V, Bhat Z, Kumar D, Khan N, Chashoo I, Shah M. Pharmacological profile of crocus sativus-a comprehe sive review. Pharmacologyonline 2011; 3: 799-811.
  39. Dominguez-Rebolledo AE, Fernandez-Santos MR, Bisbal A, Ros-Santaella JL, Ramon M, Carmona M, et al. Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments. Reprod Fertil Dev 2010; 22(5): 856-70.
  40. Safarinejad MR, Shafiei N, Safarinejad S. A prospective double-blind randomized placebo-controlled study of the effect of saffron (Crocus sativus Linn.) on semen parameters and seminal plasma antioxidant capacity in infertile men with idiopathic oligoasthenoteratozoospermia. Phytother Res 2011; 25(4): 508-16.
  41. Paradiso GG, Gravina GL, Angelozzi G, Sacchetti A, Innominato PF, Pace G, et al. May antioxidant therapy improve sperm parameters of men with persistent oligospermia after retrograde embolization for varicocele? World J Urol 2008; 26(1): 97-102.
  42. Keskes-Ammar L, Feki-Chakroun N, Rebai T, Sahnoun Z, Ghozzi H, Hammami S, et al. Sperm oxidative stress and the effect of an oral vitamin E and selenium supplement on semen quality in infertile men. Arch Androl 2003; 49(2): 83-94.
  43. Momeni HR, Soleimani Mehranjani M, Abnosi MH, Mahmoodi M. Effects of vitamin E on sperm parameters and reproductive hormones in developing rats treated with para-nonylphenol. Iran J Reprod Med 2009; 7(3): 111-6.
  44. Premkumar K, Thirunavukkarasu C, Abraham SK, Santhiya ST, Ramesh A. Protective effect of saffron (Crocus sativus L.) aqueous extract against genetic damage induced by anti-tumor agents in mice. Hum Exp Toxicol 2006; 25(2): 79-84.
  45. Hosseinzadeh H, Abootorabi A, Sadeghnia HR. Protective effect of Crocus sativus stigma extract and crocin (trans-crocin 4) on methyl methanesulfonate-induced DNA damage in mice organs. DNA Cell Biol 2008; 27(12): 657-64.
  46. Greco E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Tesarik J. Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J Androl 2005; 26(3): 349-53.
  47. Tunc O, Thompson J, Tremellen K. Improvement in sperm DNA quality using an oral antioxidant therapy. Reprod Biomed Online 2009; 18(6): 761-8.
  48. Menezo YJ, Hazout A, Panteix G, Robert F, Rollet J, Cohen-Bacrie P, et al. Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. Reprod Biomed Online 2007; 14(4): 418-21.
  49. Piomboni P, Gambera L, Serafini F, Campanella G, Morgante G, De Leo V. Sperm quality improvement after natural anti-oxidant treatment of asthenoteratospermic men with leukocytospermia. Asian J Androl 2008; 10(2): 201-6.
Journal of Isfahan Medical School
Volume 32, Issue 293 - Serial Number 293
May and June 2014
Pages 1061-1072

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History

  • Receive Date: 26 April 2014
  • Revise Date:
  • Accept Date: 06 April 2022

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