Melatonin Protects Mouse Type A Spermatogonial Stem Cells against Oxidative Stress via The Mitochondrial Thioredoxin System

Document Type : Original Article

Authors

1 Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran

2 Medical Biology Research Centre, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran

3 Department of Biostatistics, School of Health, Kermanshah University of Medical Sciences, Kermanshah, Iran

4 Medical Technology Research Centre, Institute of Health Technology, Kermanshah University of Medical Sciences, Kermanshah, Iran

Abstract

Objective: Mitochondrial oxidative stress is an important factor in infertility. The mitochondrial thioredoxin system
plays an important role in this condition. N-acetyl-5-methoxy tryptamine (melatonin) plays a role in reducing oxidative
stress and apoptosis in spermatogonial stem cells (SSCs). In this study, we explore the probable protective effects of
melatonin on the mitochondrial thioredoxin system [thioredoxin 2 (Trx2)/Txnip] in SSCs under oxidative stress.
Materials and Methods: In this experimental study, SSCs were co-cultured two-dimensionally (2D) with Sertoli cells
in DMEM culture medium that contained 10% fetal bovine serum (FBS), 1% antibiotics, and 10 ng/ml glial cell-derived
neurotrophic factor (GDNF) for 30 days. The cultured cells were subsequently divided into four groups: control; melatonin
(250 μM, 24 hours); melatonin (250 μM, 24 hours)+hydrogen peroxide (H2O2, 50 μM, 24 hours); and H2O2 (50 μM, 24
hours). Intracellular reactive oxygen species (ROS) production was determined by flow cytometry. Malondialdehyde
(MDA) levels were measured by Fluorometry. The expressions of apoptotic and antioxidant genes and nuclear factor
erythroid 2-related factor 2 (Nrf2), Trx2, and nicotinamide nucleotide transhydrogenase (NNT) proteins were determined
by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Adenosine triphosphate (ATP) levels
were measured by fluorometry.
Results: Melatonin reduced H2O2-induced ROS levels and apoptosis in the SSCs. Melatonin also increased mRNA
expression of Nrf2, Trx2, NNT, Sirtuin 3 (Sirt3), and decreased mRNA expression of Txnip, and increased protein
expressions of Nrf2, Trx2, NNT thereby increasing activity of the mitochondrial thioredoxin system. In addition, melatonin
increased ATP levels.
Conclusion: Melatonin increased Trx2 expression through the Nrf2 pathway. This study suggests that melatonin may
protect SSCs from oxidative stress in diseases related to infertility.

Keywords

Main Subjects

References

  1. Rajender S, Rahul P, Mahdi AA. Mitochondria, spermatogenesis and male infertility. Mitochondrion. 2010; 10(5): 419-428.
  2. Orrenius S, Gogvadze V, Zhivotovsky B. Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol. 2007; 47: 143-183.
  3. Martín M, Macías M, Escames G, León J, Acuña-Castroviejo D. Melatonin but not vitamins C and E maintains glutathione homeostasis in t-butyl hydroperoxide-induced mitochondrial oxidative stress. FASEB J. 2000; 14(12): 1677-1679.
  4. Barati E, Nikzad H, Karimian M. Oxidative stress and male infertility: current knowledge of pathophysiology and role of antioxidant therapy in disease management. Cell Mol Life Sci. 2020; 77(1): 93-113.
  5. Asadi N, Bahmani M, Kheradmand A, Rafieian-Kopaei M. The impact of oxidative stress on testicular function and the role of antioxidants in improving it: a review. J Clin Diagn Res. 2017; 11(5): IE01-IE05.
  6. Shaban S, El-Husseny MWA, Abushouk AI, Salem AMA, Mamdouh M, Abdel-Daim MM. Effects of antioxidant supplements on the survival and differentiation of stem cells. Oxid Med Cell Longev. 2017; 2017: 5032102.
  7. Aponte PM, van Bragt MP, de Rooij DG, van Pelt AM. Spermatogonial stem cells: characteristics and experimental possibilities. APMIS. 2005; 113(11-12): 727-742.
  8. Caroppo E, Dattilo M. Sperm redox biology challenges the role of antioxidants as a treatment for male factor infertility. F&S Reviews. 2022; 3(1): 90-104.
  9. Sadeghi N, Boissonneault G, Tavalaee M, Nasr-Esfahani MH. Oxidative versus reductive stress: a delicate balance for sperm integrity. Syst Biol Reprod Med. 2023; 69(1): 20-31.
  10. Gholami M, Saki G, Hemadi M, Khodadadi A, Mohammadi-Asl J. Melatonin improves spermatogonial stem cells transplantation efficiency in azoospermic mice. Iran J Basic Med Sci. 2014; 17(2): 93-99.
  11. Reiter RJ, Tan DX, Rosales-Corral S, Galano A, Jou MJ, Acuna- Castroviejo D. Melatonin mitigates mitochondrial meltdown: interactions with SIRT3. Int J Mol Sci. 2018; 19(8): 2439.
  12. Gholami M, Saki G, Hemadi M, Khodadadi A, Mohamma-di-Asl J. Effect of melatonin on the expression of apoptotic genes in vitrifiedthawed spermatogonia stem cells type A of 6-day-old mice. Iran J Basic Med Sci. 2013; 16(8): 906-909.
  13. Gholami MR, Saki G, Hemadi M, Khodadadi A, Mohammadi-Asl J. Supplementation vitrified-thawed media with melatonin do not protecting immature mouse testicular tissue from vitrified-thawed induced injury. Asian J Anim Vet Adv. 2012; 7(10): 940-949.
  14. Guo Y, Sun J, Li T, Zhang Q, Bu S, Wang Q, et al. Melatonin ameliorates restraint stress-induced oxidative stress and apoptosis in testicular cells via NF-κB/iNOS and Nrf2/ HO-1 signaling pathway. Sci Rep. 2017; 7(1): 9599.
  15. Zhang K, Lv Z, Jia X, Huang D. Melatonin prevents testicular damage in hyperlipidaemic mice. Andrologia. 2012; 44(4): 230-236.
  16. Lee KM, Lee IC, Kim SH, Moon C, Park SH, Shin DH, et al. Melatonin attenuates doxorubicin-induced testicular toxicity in rats. Andrologia. 2012; 44 Suppl 1: 796-803.
  17. Munro D, Banh S, Sotiri E, Tamanna N, Treberg JR. The thioredoxin and glutathione-dependent H2O2 consumption pathways in muscle mitochondria: involvement in H2O2 metabolism and consequence to H2O2 efflux assays. Free Radic Biol Med. 2016; 96: 334-346.
  18. Roucher-Boulez F, Mallet-Motak D, Samara-Boustani D, Jilani H, Ladjouze A, Souchon PF, et al. NNT mutations: a cause of primary adrenal insufficiency, oxidative stress and extra-adrenal defects. Eur J Endocrinol. 2016; 175(1): 73-84.
  19. Ahmadi Z, Ashrafizadeh M. Melatonin as a potential modulator of Nrf2. Fundam Clin Pharmacol. 2020; 34(1): 11-19.
  20. Morris G, Walker AJ, Walder K, Berk M, Marx W, Carvalho AF, et al. Increasing Nrf2 activity as a treatment approach in neuropsychiatry. Mol Neurobiol. 2021; 58(5): 2158-2182.
  21. Yoshikawa T, Naito Y. What is oxidative stress? JMAJ. 2002; 45(7): 271-276.
  22. Veisi M, Mansouri K, Assadollahi V, Jalili C, Pirnia A, Salahshoor MR, et al. Evaluation of co-cultured spermatogonial stem cells encapsulated in alginate hydrogel with Sertoli cells and their transplantation into azoospermic mice. Zygote. 2022; 30(3): 344-351.
  23. Karimi E, Abbasi S, Abbasi N. Thymol polymeric nanoparticle synthesis and its effects on the toxicity of high glucose on OEC cells: involvement of growth factors and integrin-linked kinase. Drug Des Devel Ther. 2019; 13: 2513-2532.
  24. Rhee SG, Chang TS, Jeong W, Kang D. Methods for detection and measurement of hydrogen peroxide inside and outside of cells. Mol Cells. 2010; 29(6): 539-549.
  25. Basir HRG, Karbasi A, Ravan AP, Abbasalipourkabir R, Bahmani M. Is human umbilical cord mesenchymal stem cell-derived conditioned medium effective against oxidative and inflammatory status in CCl4-induced acute liver injury? Life Sci. 2022; 305: 120730.
  26. Florido J, Martinez-Ruiz L, Rodriguez-Santana C, López-Rodríguez A, Hidalgo-Gutiérrez A, Cottet-Rousselle C, et al. Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport. J Pineal Res. 2022; 73(3): e12824.
  27. Lorestani S, Hashemy SI, Mojarad M, Keyvanloo Shahrestanaki M, Bahari A, Asadi M, et al. Increased glutathione reductase expression and activity in colorectal cancer tissue samples: an investigational study in Mashhad, Iran. Middle East J Cancer. 2018; 9(2): 99-104.
  28. Rashidi Z, Aleyasin A, Eslami M, Nekoonam S, Zendedel A, Bahramrezaie M, et al. Quercetin protects human granulosa cells against oxidative stress via thioredoxin system. Reprod Biol. 2019; 19(3): 245-254.
  29. Li B, He X, Zhuang M, Niu B, Wu C, Mu H, et al. Melatonin ameliorates busulfan-induced spermatogonial stem cell oxidative apoptosis in mouse testes. Antioxid Redox Signal. 2018; 28(5): 385-400.
  30. Anjum S, Rahman S, Kaur M, Ahmad F, Rashid H, Ansari RA, et al. Melatonin ameliorates bisphenol A-induced biochemical toxicity in testicular mitochondria of mouse. Food Chem Toxicol. 2011; 49(11): 2849-2854.
  31. Jou MJ, Peng TI, Yu PZ, Jou SB, Reiter RJ, Chen JY, et al. Melatonin protects against common deletion of mitochondrial DNAaugmented mitochondrial oxidative stress and apoptosis. J Pineal Res. 2007; 43(4): 389-403.
  32. Xu D, Liu L, Zhao Y, Yang L, Cheng J, Hua R, et al. Melatonin protects mouse testes from palmitic acid-induced lipotoxicity by attenuating oxidative stress and DNA damage in a SIRT1-dependent manner. J Pineal Res. 2020; 69(4): e12690.
  33. He X, Ma Q. Redox regulation by nuclear factor erythroid 2-related factor 2: gatekeeping for the basal and diabetes-induced expression of thioredoxin-interacting protein. Mol Pharmacol. 2012; 82(5): 887-897.
  34. Zhou J, Zhang S, Zhao X, Wei T. Melatonin impairs NADPH oxidase assembly and decreases superoxide anion production in microglia exposed to amyloid-beta1-42. J Pineal Res. 2008; 45(2): 157-165.
  35. Yu L, Fan C, Li Z, Zhang J, Xue X, Xu Y, et al. Melatonin rescues cardiac thioredoxin system during ischemia-reperfusion injury in acute hyperglycemic state by restoring Notch1/Hes1/Akt signaling in a membrane receptor-dependent manner. J Pineal Res. 2017; 62(1).
  36. Gameiro PA, Laviolette LA, Kelleher JK, Iliopoulos O, Stephanopoulos G. Cofactor balance by nicotinamide nucleotide transhydrogenase (NNT) coordinates reductive carboxylation and glucose catabolism in the tricarboxylic acid (TCA) cycle. J Biol Chem. 2013; 288(18): 12967-12977.
  37. Yin F, Sancheti H, Cadenas E. Silencing of nicotinamide nucleotide transhydrogenase impairs cellular redox homeostasis and energy metabolism in PC12 cells. Biochim Biophys Acta. 2012; 1817(3): 401-409.
  38. Zhai M, Li B, Duan W, Jing L, Zhang B, Zhang M, et al. Melatonin ameliorates myocardial ischemia reperfusion injury through SIRT3-dependent regulation of oxidative stress and apoptosis. J Pineal Res. 2017; 63(2).
  39. Jiang L, Shestov AA, Swain P, Yang C, Parker SJ, Wang QA, et al. Reductive carboxylation supports redox homeostasis during anchorage- independent growth. Nature. 2016; 532(7598): 255-258.
  40. Rao KNS, Shen X, Pardue S, Krzywanski DM. Nicotinamide nucleotide transhydrogenase (NNT) regulates mitochondrial ROS and endothelial dysfunction in response to angiotensin II. Redox Biol. 2020; 36: 101650.
Cell Journal (Yakhteh)
Volume 25, Issue 11 - Serial Number 118
November 2023
Pages 741-752

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