Evaluation of the NOX5 protein expression and oxidative stress in sperm from asthenozoospermic men compared to normozoospermic men
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NADPH oxidase 5 (NOX5), the main isoform of NOX in spermatozoa, has been recognized as the main active generators of reactive oxygen species (ROS), including superoxide anion (O 2 −. ) and hydrogen peroxide (H2O2). ROS have been shown to play important roles in many physiological and pathological conditions in spermatozoa. The present study aims to investigate the alterations of NOX5 protein expression and oxidative stress (OS) status in asthenozoospermic men compared to normozoospermic men.
Semen samples were collected from 25 asthenozoospermic men and 28 normozoospermic men. In this study, NOX5 protein expression was evaluated by Western blotting. An OS status was evaluated by measuring of ROS (O 2 −. and H2O2), DNA damage and plasma membrane integrity in spermatozoa.
The protein expression of NOX5 (p < 0.0001) was remarkably higher in asthenozoospermic men in comparison to normozoospermic men. In addition, the percentages of intracellular O 2 −. (p < 0.0001), H2O2 (p < 0.0001) in viable spermatozoa, apoptotic sperm cells with altered plasma membrane (p < 0.001) and DNA damage (p = 0.001) were significantly increased in asthenozoospermic men compared to normozoospermic men.
The present study provides evidence that the overexpression of NOX5 protein may induce excessive ROS production and oxidative stress damages to DNA and plasma membrane integrity in asthenozoospermic men.
KeywordsNADPH oxidase 5 Male infertility Oxidative stress Reactive oxygen species
Reactive oxygen species
Sperm chromatin dispersion
Sperm DNA fragmentation
World Health Organization
2′, 7′-dichlorodihydrofluorescin diacetate
We thank Mehdi Baratchian and Fatima Zahraa Fouani for grammatical editing of the manuscript. The authors would like to thank Omid Clinic particularly Dr. Saeidi for providing samples.
AV performed experiments and wrote the manuscript. HT, MD and MRS, contributed equally to this work by helping to design experiments. SA and NL supplied semen samples. MK analyzed data. All authors read and approved the final version of the manuscript.
This work was supported by the Tehran University of Medical Sciences, Tehran, Iran (Grant numbers 21660 and 30756).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
All procedures performed in this study were in accordance with the ethical standards of the ethical committee (no.Ir.tums.rec.1395.2706) of Tehran University of Medical Sciences (Tehran, Iran).
All participants were informed of the study, and a written consent was obtained from them.
Consent for publication
All authors approve the manuscript for publication.
- 1.Chen F, Wang Y, Barman S Jr, Fulton D (2015) Enzymatic regulation and functional relevance of NOX5. Curr Pharm Des 21(41):5999–6008Google Scholar
- 4.Richer SC, Ford WC (2001) A critical investigation of NADPH oxidase activity in human spermatozoa. Mol Hum Reprod 7(3):237–244Google Scholar
- 5.Alvarez JG, Touchstone JC, Blasco L, Storey BT (1987) Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl 8(5):338–348Google Scholar
- 9.Petrushanko IY, Lobachev VM, Kononikhin AS, Makarov AA, Devred F, Kovacic H, Kubatiev AA, Tsvetkov PO (2016) Oxidation of Са2 + -binding domain of NADPH oxidase 5 (NOX5): toward understanding the mechanism of inactivation of NOX5 by ROS. PLoS One 11(7):e0158726Google Scholar
- 10.Ghani E, Keshtgar S, Habibagahi M, Ghannadi A, Kazeroni M (2013) Expression of NOX5 in human teratozoospermia compared to normozoospermia. Andrologia 45(5):351–356Google Scholar
- 13.Wright C, Milne S, Leeson H (2014) Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod Biomed Online 28(6):684–703Google Scholar
- 14.Mohammadi F, Varanloo N, Nasrabadi MH, Vatannejad A, Amjadi FS, Masroor MJ, Bajelan L, Mehdizadeh M, Aflatoonian R, Zandieh Z (2019) Supplementation of sperm freezing medium with myoinositol improve human sperm parameters and protects it against DNA fragmentation and apoptosis. Cell Tissue Bank. https://doi.org/10.1007/s10561-018-9731-0 Google Scholar
- 15.Brar SS, Corbin Z, Kennedy TP, Hemendinger R, Thornton L, Bommarius B, Arnold RS, Whorton AR, Sturrock AB, Huecksteadt TP, Quinn MT, Krenitsky K, Ardie KG, Lambeth JD, Hoidal JR (2003) NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells. Am J Physiol Cell Physiol 285(2):C353–C369. https://doi.org/10.1152/ajpcell.00525.2002 Google Scholar
- 16.Guzik TJ, Chen W, Gongora MC, Guzik B, Lob HE, Mangalat D, Hoch N, Dikalov S, Rudzinski P, Kapelak B (2008) Calcium-dependent NOX5 nicotinamide adenine dinucleotide phosphate oxidase contributes to vascular oxidative stress in human coronary artery disease. J Am Coll Cardiol 52(22):1803–1809Google Scholar
- 17.Aitken RJ, Ryan AL, Curry BJ, Baker MA (2003) Multiple forms of redox activity in populations of human spermatozoa. Mol Hum Reprod 9(11):645–661Google Scholar
- 18.El Jamali A, Valente AJ, Lechleiter JD, Gamez MJ, Pearson DW, Nauseef WM, Clark RA (2008) Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl. Free Radic Biol Med 44(5):868–881. https://doi.org/10.1016/j.freeradbiomed.2007.11.020 Google Scholar
- 19.World Health Organization (2010) WHO laboratory manual for the examination and processing of human semen, 5th edn. WHO Press, GenevaGoogle Scholar
- 20.Wang G, Guo Y, Zhou T, Shi X, Yu J, Yang Y, Wu Y, Wang J, Liu M, Chen X, Tu W, Zeng Y, Jiang M, Li S, Zhang P, Zhou Q, Zheng B, Yu C, Zhou Z, Guo X, Sha J (2012) In-depth proteomic analysis of the human sperm reveals complex protein compositions. J Proteom 79:114–122. https://doi.org/10.1016/j.jprot.2012.12.008 Google Scholar
- 22.Mahfouz RZ, du Plessis SS, Aziz N, Sharma R, Sabanegh E, Agarwal A (2008) Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress. Fertil Steril 93(3):814–821. https://doi.org/10.1016/j.fertnstert.2008.10.068 Google Scholar
- 23.Ghorbani M, Vatannejad A, Khodadadi I, Amiri I, Tavilani H (2016) Protective effects of glutathione supplementation against oxidative stress during cryopreservation of human spermatozoa. Cryo Lett 37(1):34–40Google Scholar
- 25.Choucair FB, Rachkidi EG, Raad GC, Saliba EM, Zeidan NS, Jounblat RA, Jaoude IFA, Hazzouri MM (2016) High level of DNA fragmentation in sperm of Lebanese infertile men using sperm chromatin dispersion test. Middle East Fertil Soc J 21(4):269–276Google Scholar
- 27.Mahfouz R, Sharma R, Thiyagarajan A, Kale V, Gupta S, Sabanegh E, Agarwal A (2010) Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species. Fertil Steril 94(6):2141–2146Google Scholar
- 29.Alvarez JG, Aitken RJ (2012) Lipid peroxidation in human spermatozoa. In: Agarwal AAR, Alvarez JG (eds) Studies on men’s health and fertility. Humana Press, New York, pp 119–130Google Scholar
- 31.Aitken RJ, Baker MA, Nixon B (2017) Are sperm capacitation and apoptosis the opposite ends of a continuum driven by oxidative stress? Asian J Androl 17(4):633Google Scholar
- 32.Tavilani H, Vatannejad A, Akbarzadeh M, Atabakhash M, Khosropour S, Mohaghgeghi A (2014) Correlation between lipid profile of sperm cells and seminal plasma with lipid profile of serum in infertile men. Avicenna J Med Biochem 2(1):e19607Google Scholar
- 33.Vicari E, La Vignera S, Castiglione R, Calogero AE (2006) Sperm parameter abnormalities, low seminal fructose and reactive oxygen species overproduction do not discriminate patients with unilateral or bilateral post-infectious inflammatory prostato-vesiculo-epididymitis. J Endocrinol Invest 29(1):18Google Scholar
- 34.Condorelli RA, Calogero AE, Vicari E, Duca Y, Favilla V, Morgia G, Cimino S, Di Mauro M, La Vignera S (2013) Prevalence of male accessory gland inflammations/infections in patients with Type 2 diabetes mellitus. J Endocrinol Invest 36(9):770–774Google Scholar
- 35.Condorelli RA, Russo GI, Calogero AE, Morgia G, La Vignera S (2017) Chronic prostatitis and its detrimental impact on sperm parameters: a systematic review and meta-analysis. J Endocrinol Invest 40(11):1209–1218Google Scholar
- 36.Vignera S, Vicari E, Condorelli R, Dâ€™Agata R, Calogero AE (2011) Hypertrophic-congestive and fibro-sclerotic ultrasound variants of male accessory gland infection have different sperm output. J Endocrinol Invest 10(34):e330–e335Google Scholar
- 37.La Vignera S, Condorelli R, Dâ€™Agata R, Vicari E, Calogero AE (2012) Semen alterations and flow-citometry evaluation in patients with male accessory gland infections. J Endocrinol Invest 35(2):219–223Google Scholar
- 38.Kamiguti AS, Serrander L, Lin K, Harris RJ, Cawley JC, Allsup DJ, Slupsky JR, Krause KH, Zuzel M (2005) Expression and activity of NOX5 in the circulating malignant B cells of hairy cell leukemia. J Immunol 175(12):8424–8430Google Scholar
- 40.Serrander L, Jaquet V, Bedard K, Plastre O, Hartley O, Arnaudeau S, Demaurex N, Schlegel W, Krause KH (2007) NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation. Biochimie 89(9):1159–1167. https://doi.org/10.1016/j.biochi.2007.05.004 Google Scholar
- 41.Miraglia E, Lussiana C, Viarisio D, Racca C, Cipriani A, Gazzano E, Bosia A, Revelli A, Ghigo D (2010) The pentose phosphate pathway plays an essential role in supporting human sperm capacitation. Fertil Steril 93(7):2437–2440Google Scholar
- 42.Vernet P, Fulton N, Wallace C, Aitken RJ (2001) Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa. Biol Reprod 65(4):1102–1113Google Scholar
- 43.Jamaludin N, Razak SSA, Jaffar FHF, Osman K, Ibrahim SF (2017) The effect of Smartphoneâ€™s radiation frequency and exposure duration on NADPH oxidase 5 (NOX5) level in sperm parameters. Sains Malays 46(9):1597–1602Google Scholar
- 44.Amaral A, Lourenço B, Marques M, Ramalho-Santos J (2013) Mitochondria functionality and sperm quality. Reproduction 146(5):R163–R174Google Scholar
- 45.Davila MP, Muñoz PM, Tapia JA, Ferrusola CO, Peña FJ (2015) Inhibition of mitochondrial complex I leads to decreased motility and membrane integrity related to increased hydrogen peroxide and reduced ATP production, while the inhibition of glycolysis has less impact on sperm motility. PLoS One 10(9):e0138777Google Scholar
- 46.Weng S-L, Taylor SL, Morshedi M, Schuffner A, Duran EH, Beebe S, Oehninger S (2002) Caspase activity and apoptotic markers in ejaculated human sperm. Mol Hum Reprod 8(11):984–991Google Scholar
- 47.Wei X, Li Q, Han Z, Lin D, Yu P (2015) Differences in caspase-8 and-9 activity and sperm motility in infertile males of Li nationality in China. Int J Clin Exp Med 8(3):4721Google Scholar
- 48.Moradi MN, Karimi J, Khodadadi I, Amiri I, Karami M, Saidijam M, Vatannejad A, Tavilani H (2018) Evaluation of the p53 and Thioredoxin reductase in sperm from asthenozoospermic males in comparison to normozoospermic males. Free Radic Biol Med. https://doi.org/10.1016/j.freeradbiomed.2017.12.038 Google Scholar
- 49.Shabani Nashtaei M, Amidi F, Gilani S, Aleyasin A, Bakhshalizadeh S, Naji M, Nekoonam S (2016) Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5′ AMP-activated protein kinase activation. Andrology 5(2):313–326Google Scholar
- 50.Najafi A, Asadi E, Moawad AR, Mikaeili S, Amidi F, Adutwum E, Safa M, Sobhani AG (2016) Supplementation of freezing and thawing media with brain-derived neurotrophic factor protects human sperm from freeze–thaw-induced damage. Fertil Steril 106(7):1658–1665Google Scholar
- 51.Sp Carnesecchi, Rougemont AL, Doroshow JH, Nagy M, Mouche S, Gumy-Pause F, Szanto I (2015) The NADPH oxidase NOX5 protects against apoptosis in ALK-positive anaplastic large-cell lymphoma cell lines. Free Radical Biol Med 84:22–29Google Scholar
- 52.Weyemi U, Redon CE, Aziz T, Choudhuri R, Maeda D, Parekh PR, Bonner MY, Arbiser JL, Bonner WM (2015) Inactivation of NADPH oxidases NOX4 and NOX5 protects human primary fibroblasts from ionizing radiation-induced DNA damage. Radiat Res 183(3):262–270Google Scholar
- 54.Agarwal A, Tvrda E, Sharma R (2014) Relationship amongst teratozoospermia, seminal oxidative stress and male infertility. Reprod Biol Endocrinol 12:1–8Google Scholar
- 55.Condorelli RA, Calogero AE, Mongioi L, Vicari E, Russo GI, Lanzafame F, La Vignera S (2016) Varicocele and concomitant dilation of the periprostatic venous plexus: effects on semen viscosity sperm parameters. J Endocrinol Invest 39(5):543–547Google Scholar
- 56.Kawahara T, Jackson HM, Smith SME, Simpson PD, Lambeth JD (2011) Nox5 forms a functional oligomer mediated by self-association of its dehydrogenase domain. Biochemistry 50(12):2013–2025Google Scholar
- 57.Pandey D, Fulton DJR (2011) Molecular regulation of NADPH oxidase 5 via the MAPK pathway. Am J Physiol-Heart Circul Physiol 300(4):H1336–H1344Google Scholar