Niknam Lakpour
- Clinical Biochemistry Department, Faculty of Medicine, Kermanshah Medical Sciences University, Kermanshah, Iran
Mohammad Hossein Modarresi
- Nanobiotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran
Hadi Kharazi
- Clinical Biochemistry Department, Faculty of Medicine, Kermanshah Medical Sciences University, Kermanshah, Iran
Mohammad Mehdi Akhondi
- Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Asad Veisi Raygani
- Clinical Biochemistry Department, Faculty of Medicine, Kermanshah Medical Sciences University, Kermanshah, Iran
Jamileh Ghasemi
- Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran
Mahshid Hodjat
- Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran
Mohammad Reza Sadeghi Corresponding Author
- Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran

Received: 10/1/2006 Accepted: 10/1/2006 - Publisher : Avicenna Research Institute

Related Articles


Other Format



Introduction: Leukocytes and defective or dead spermatozoa in human semen are a source for the production of reactive oxygen species (ROS) and subsequent injury to intact sperms. Enzymatic and non-enzymatic defensive mechanisms in semen detoxify these compounds. Glutathione peroxidase-4 (GPX-4 or PHGPX) is a major selenoprotein in sperm and it is one of the enzymatic mechanisms that play multiple roles during spermatogenesis. Some of these roles are formation of the mitochondrial capsule, hydro-peroxide detoxification and sperm chromatin condensation. Any decrease in the enzyme activity or content, may create disorders in spermatogenesis and sperm fertilizing ability. Considering defects in the expression of the enzyme gene or presence of mutations which may cause decreases in PHGPX activity or content, this study was carried out to identify a number of important mutations in GPX-4 gene by PCR-RFLP method in Iranian infertile men. Materials & Methods: This study was performed on 128 Iranian men who had been referred to Avesina Infertility Clinic, including 74 infertile men with defective sperm parameters, 18 normozoospermic and 36 fertile subjects as controls. Mean ± SD for sperm parameters were determined. Genomic DNA was extrac-ted using salting out procedure from peripheral blood leukocytes. PCR-RFLP was done by two sets of primers with 237 bp and 148 bp PCR products that were designed for 1A and 4 exons of GPX-4 gene covering nucleotides of +6 (CT), +17 (GA), +1725 (GA) by MwoI, PshAI and SatI enzymes.Results: Digestion of a 237 bp intact PCR product by MwoI generates two fragments (151 bp and 86 bp). When a mutation occurs in the restriction site +6 (CT), the enzyme would not recognize the sequence, therefore 237 bp segment remains undigested. Treatment of 237 bp segment with PshAI generates two fragments (161 bp and 76) in the intact gene but the same enzyme can not digest 237 bp segment when a mutation occurs in the restriction site +17 (GA). Ultimately, digestion of 148 bp intact segment with SatI generates two fragments (108 bp and 40 bp) but when a mutation occurs in the restriction site +1725 (GA), the enzyme will not recognize the sequence; therefore 148 bp segment remains undigested. Enzy-matic digestion evaluations of 237 bp and 148 bp segments in all participants revealed that neither of the examined mutations existed in GPX-4 gene.Conclusion: According to the results of this study, it is determined that the prevalence of these mutations in Iranian infertile men is probably low and it may have no association with the etiology of the disorder affecting sperm parameters. Hence, a study with a larger number of patients is suggested to determine the exact prevalence of these and other mutations of the gene in Iranian infertile men.

Keywords: PHGPX, Sperm, ROS, Polymorphism, Male infertility, Selenoproteins

To cite this article:


  1. Sharma RK, Agarwal A. Role of reactive oxygen species in male infertility. Urology. 1996;48(6):835-50   [PubMed]
  2. Hull MG, Glazener CM, Kelly NJ, Conway DI, Foster PA, Hinton RA Coulson C, Lambert PA, Watt EM, Desai KM. Population study of causes, treatment, and outcome of infertility. Br Med J. 1985;291:1693-7   [PubMed]
  3. Jones Mann T, Sherins R. Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal effect of acid peroxides and protective action of semi-nal plasma. Fertil Steril. 1979;31:531-7   [PubMed]
  4. Aitken RJ. The Amoroso Lecture. The human sperma-tozoon-a cell in crisis?J Reprod Fertil.1999;115(1):1-7   [PubMed]
  5. Sharma RK, Garwal AA. Reactive oxygen species and male infertility. Urology. 1996;48:835-50   [PubMed]
  6. Aitken RJ. Molecular mechanisms regulating human sperm function. Mol Hum Reprod. 1997;3(3):169-73   [PubMed]
  7. Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human sperma-tozoa. Superoxide dismutase as major enzyme protect-tant against oxygen toxicity. J Androl. 1987;8(5):338-48   [PubMed]
  8. Aitken RJ, Clarkson JS, Fishel S. Generation of reac-tive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod. 1989;41(1):183-97   [PubMed]
  9. Alvarez JG, Storey BT. Spontaneous lipid peroxidation in rabbit epididymal spermatozoa: its effect on sperm motility. Biol Reprod. 1982;27(5):1102-8   [PubMed]
  10. Aitken RJ, Buckingham D, West K, Wu FC, Zikopou-los K, Richardson DW. Differential contribution of leucocytes and spermatozoa to the generation of reac-tive oxygen species in the ejaculates of oligo-zoospermic patients and fertile donors. J Reprod Fertil. 1992;94(2):451-62   [PubMed]
  11. de Lamirande E, Gagnon C. Impact of reactive oxygen species on spermatozoa: a balancing act between bene-ficial and detrimental effects. Hum Reprod. 1995;10 Suppl 1:15-21   [PubMed]
  12. Alkan I, Simsek F, Haklar G, Kervancioglu E, Ozveri H, Yalcin S, et al. Reactive oxygen species production by the spermatozoa of patients with idiopathic inferti-lity: relationship to seminal plasma antioxidants. J Urol. 1997;157(1):140-3   [PubMed]
  13. Lewis SE, Boyle PM, McKinney KA, Young IS, Thompson W. Total antioxidant capacity of seminal plasma is different in fertile and infertile men. Fertil Steril. 1995;64(4):868-70   [PubMed]
  14. Fraga CG, Motchnik PA, Shigenaga MK, Helbock HJ, Jacob RA, Ames BN. Ascorbic acid protects against endogenous oxidative DNA damage in human sperm. Proc Natl Acad Sci U S A. 1991;88(24):11003-6   [PubMed]
  15. Zini A, de Lamirande E, Gagnon C. Reactive oxygen species in semen of infertile patients: levels of super-oxide dismutase- and catalase-like activities in seminal plasma and spermatozoa. Int J Androl. 1993;16(3):183-8   [PubMed]
  16. Peeker R, Abramsson L, Marklund SL. Superoxide dismutase isoenzymes in human seminal plasma and spermatozoa. Mol Hum Reprod. 1997;3(12):1061-6   [PubMed]
  17. Tramer F, Rocco F, Micali F, Sandri G, Panfili E. Antioxidant systems in rat epididymal spermatozoa. Biol Reprod. 1998;59(4):753-8   [PubMed]
  18. Alvarez JG, Storey BT. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete res. 1989;23(1):77-90   [PubMed]
  19. Brigelius-Flohe R. Tissue-specific functions of indivi-dual glutathione peroxidases. Free Rad Biol Med. 1999;27(9-10):951-65   [PubMed]
  20. Kuhn H, Borchert A. Regulation of enzymatic lipid pe-roxidation: the interplay of peroxidizing and peroxide reducing enzymes. Free Rad Biol Med. 2002;33(2): 154-72   [PubMed]
  21. Hall L, Williams K, Perry AC, Frayne J, Jury JA. The majority of human glutathione peroxidase type 5 (GPX5) transcripts are incorrectly spliced: implications for the role of GPX5 in the male reproductive tract. Biochem J. 1998;333 ( Pt 1):5-9   [PubMed]
  22. Fujii T, Endo T, Fujii J, Taniguchi N. Differential expression of glutathione reductase and cytosolic gluta-thione peroxidase, GPX1, in developing rat lungs and kidneys. Free Rad Res. 2002;36(10):1041-9   [PubMed]
  23. Fujii J, Iuchi Y, Matsuki S, Ishii T. Cooperative func-tion of antioxidant and redox systems against oxidative stress in male reproductive tissues. Asian J Androl. 2003;5(3):231-42   [PubMed]
  24. Ursini F, Maiorino M, Valente M, Ferri L, Gregolin C. Purification from pig liver of a protein which pro-tects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase active-ty on phosphatidylcholine hydroperoxides Biochimica et Biophysica Acta. 1982;710:197-211   [PubMed]
  25. Ho YS, Magnenat JL, Bronson RT, Cao J, Gargano M, Sugawara M, et al. Mice deficient in cellular gluta-thione peroxidase develop normally and show no increased sensitivity to hyperoxia. J Biol Chem. 1997; 272(26):16644-51   [PubMed]
  26. Ursini F, Heim S, Kiess M, Maiorino M, Roveri A, Wissing J, et al. Dual function of the selenoprotein PHGPx during sperm maturation. Science. 1999;285 (5432):1393-6   [PubMed]
  27. Foresta C, Flohe L, Garolla A, Roveri A, Ursini F, Maiorino M. Male fertility is linked to the selenopro-tein phospholipid hydroperoxide glutathione peroxi-dase. Biol Reprod. 2002;67(3):967-71   [PubMed]
  28. Maiorino M, Coassin M, Roveri A, Ursini F. Micro-somal lipid peroxidation: effect of vitamin E and its functional interactions with phospholipid hydro-peroxide glutathione peroxidase. Lipids. 1989;24:721-6   [PubMed]
  29. Roveri A, Casasco A, Maiorino M, Dalan P, Calligaro A, Ursini F. Phospholipid hydroperoxide glutathione peroxidase of rat testis. Gonadotropin dependence and immunocytochemical identification. J Biol Chem.1992; 267(9):6142-6   [PubMed]
  30. Pushpa-Rekha TR, Burdsall AL, Oleksa LM, Chisolm GM, Driscoll DM. Rat phospholipid-hydroperoxide glutathione peroxidase. cDNA cloning and identify-cation of multiple transcription and translation start sites. J Biol Chem. 1995;270(45):26993-9   [PubMed]
  31. Pfeifer H, Conrad M, Roethlein D, Kyriakopoulos A, Brielmeier M, Bornkamm GW, et al. Identification of a specific sperm nuclei selenoenzyme necessary for protamine thiol cross-linking during sperm maturation. Faseb J. 2001;15(7):1236-8   [PubMed]
  32. Maiorino MWJ, Brigelius-Flohe´ R, Calabrese F, Roveri A,, Steinert P UF, Flohe´ L. Testosterone mediates expression of the selenoprotein PHGPx by induction of spermatogenesis and not by direct trans-criptional gene activation Faseb J. 1998;12:1359-70   [PubMed]
  33. Nam SY, Fujisawa M, Kim JS, Kurohmaru M, Haya-shi Y. Expression pattern of phospholipid hydro-peroxide glutathione peroxidase messenger ribonucleic acid in mouse testis. Biol Reprod. 1998;58(5):1272-6   [PubMed]
  34. Bauche F, Fouchard MH, Jegou B. Antioxidant system in rat testicular cells. FEBS Lett. 1994;349(3):392-6   [PubMed]
  35. Shalgi R, Seligman J, Kosower NS. Dynamics of the thiol status of rat spermatozoa during maturation: ana-lysis with the fluorescent labeling agent monobro-mobimane. Biol Reprod. 1989;40(5):1037-45   [PubMed]
  36. Godeas C, Tramer F, Micali F, Roveri A, Maiorino M, Nisii C, et al. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) in rat testis nuclei is bound to chromatin. Biochem Mol Med. 1996;59(2):118-24   [PubMed]
  37. Imai H, Suzuki K, Ishizaka K, Ichinose S, Oshima H, Okayasu I, et al. Failure of the expression of phos-pholipid hydroperoxide glutathione peroxidase in the spermatozoa of human infertile males. Biol Reprod. 2001;64(2):674-83   [PubMed]
  38. Kelner MJ, Montoya MA. Structural organization of the human selenium-dependent phospholipid hydro-peroxide glutathione peroxidase gene (GPX4): chro-mosomal localization to 19p13.3. Biochem Biophysic Res Communicat. 1998;249(1):53-5   [PubMed]
  39. Maiorino M, Bosello V, Ursini F, Foresta C, Garolla A, Scapin M, et al. Genetic variations of gpx-4 and male infertility in humans. Biol Reprod. 2003;68(4): 1134-41   [PubMed]
  40. Diaconu M, Tangat Y, Bohm D, Kuhn H, Michelmann HW, Schreiber G, et al. Failure of phospholipid hydro-peroxide glutathione peroxidase expression in oligo-asthenozoospermia and mutations in the PHGPx gene. Andrologia. 2006;38(4):152-7   [PubMed]
  41. Organisation WH. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th Edition. Cambridge, United Kingdom: University Press. 1999
  42. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucle-ated cells. Nucleic Acids Res. 1988;16(3):1215   [PubMed]
  43. Maiorino M, Aumann KD, Brigelius-Flohe R, Doria D, van den Heuvel J, McCarthy J, et al. Probing the presumed catalytic triad of selenium-containing peroxi-dases by mutational analysis of phospholipid hydro-peroxide glutathione peroxidase (PHGPx). Biol Chem Hoppe Seyler. 1995;376(11):651-60   [PubMed]
  44. Gailer J, George GN, Pickering IJ, Madden S, Prince RC, Yu EY, et al. Structural basis of the antagonism between inorganic mercury and selenium in mammals. Chem Res Toxicol. 2000;13(11):1135-42   [PubMed]
  45. Rovio AT, Marchington DR, Donat S, Schuppe HC, Abel J, Fritsche E, et al. Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility. Nat Genet. 2001;29(3):261-2   [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.