Aberrant Expression of TET2 Accounts for DNA Hypomethylation in Varicocele

Document Type : Original Article

Authors

1 ACECR Institute of Higher Education, Isfahan Branch, Isfahan, Iran

2 Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

3 Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

Abstract

Objective: Epigenetic modifications such as DNA methylation play a key role in male infertility etiology. This study aimed
to explore the global DNA methylation status in testicular spermatogenic cells of varicocele-induced rats and consider
their semen quality, with a focus on key epigenetic marks, namely 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine
(5-hmC), as well as the mRNA and proteins of ten-eleven translocation (TET) methylcytosine dioxygenases 1-3.
Materials and Methods: In this experimental study, 24 mature male Wistar rats (8 in each group) were assigned
amongst the control, sham, and varicocele groups. Sperm quality was assessed, and DNA methylation patterns of
testicular spermatogenic cells were investigated using reverse transcription-polymerase chain reaction (RT-PCR),
western blot, and immunofluorescence techniques.
Results: Sperm parameters, chromatin and DNA integrity were significantly lower, and sperm lipid peroxidation significantly
increased in varicocele-induced rats in comparison with control rats. During spermatogenesis in rat testis, 5-mC and 5-hmC
epigenetic marks, and TET1-3 mRNA and proteins were expressed. In contrast to the 5-mC fluorescent signal which was
presented in all testicular cells, the 5-hmC fluorescent signal was presented exclusively in spermatogonia and a few spermatids.
In varicocele-induced rats, the 5-mC signal decreased in all cells within the tubules, whereas a strong signal of 5-hmC was
detected in seminiferous tubules compared to the control group. As well, the levels of TET2 mRNA and protein expression
were significantly upregulated in varicocele-induced rats in comparison with the control group. Also, our results showed that
the varicocele-induced animals exhibited strong fluorescent signals of TET1-3 in testicular cells, whereas weak fluorescent
signals were identified in the seminiferous tubules of the control animals.
Conclusion: Consequently, we showed TET2 upregulation and the 5-hmC gain at testicular levels are associated with
varicocele and sperm quality decline, and therefore they can be exploited as potential biomarkers of spermatogenesis.

Keywords

Main Subjects

References

  1. Santana VP, James ER, Miranda-Furtado CL, Souza MF, Pompeu CP, Esteves SC, et al. Differential DNA methylation pattern and sperm quality in men with varicocele. Fertil Steril. 2020; 114(4): 770-778.
  2. Santana VP, Miranda-Furtado CL, de Oliveira-Gennaro FG, Dos Reis RM. Genetics and epigenetics of varicocele pathophysiology: an overview. J Assist Reprod Genet. 2017; 34(7): 839-847.
  3. Sadeghi N, Erfani-Majd N, Tavalaee M, Tabandeh MR, Drevet JR, Nasr-Esfahani MH. Signs of ROS-associated autophagy in testis and sperm in a rat model of varicocele. Oxid Med Cell Longev. 2020; 2020: 5140383.
  4. Zhang QF, Wang S, Zhang H, Liu QL, Wei Y, Deng W, et al. Effects of alpha-lipoic acid on sperm quality in patients with varicocelerelated male infertility: study protocol for a randomized controlled clinical trial. Trials. 2022; 23(1): 1002.
  5. Lorian K, Kadkhodaee M, Kianian F, Abdi A, Ranjbaran M, Ashabi G, et al. Long-term NaHS administration reduces oxidative stress and apoptosis in a rat model of left-side varicocele. Andrologia. 2020; 52(2): e13496.
  6. Razi M, Tavalaee M, Sarrafzadeh-Rezaei F, Moazamian A, Gharagozloo P, Drevet JR, et al. Varicocoele and oxidative stress: New perspectives from animal and human studies. Andrology. 2021; 9(2): 546-558
  7. Rotondo JC, Lanzillotti C, Mazziotta C, Tognon M, Martini F. Epigenetics of male infertility: the role of DNA methylation. Front Cell Dev Biol. 2021; 9: 689624.
  8. Onodera A, González-Avalos E, Lio CJ, Georges RO, Bellacosa A, Nakayama T, et al. Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells. Genome Biol. 2021; 22(1): 186.
  9. Li J, Xu J, Yang T, Chen J, Li F, Shen B, et al. Genome-wide methylation analyses of human sperm unravel novel differentially methylated regions in asthenozoospermia. Epigenomics. 2022; 14(16): 951-964.
  10. Tavalaee M, Bahreinian M, Barekat F, Abbasi H, Nasr-Esfahani MH. Effect of varicocelectomy on sperm functional characteristics and DNA methylation. Andrologia. 2015; 47(8): 904-909.
  11. Bahreinian M, Tavalaee M, Abbasi H, Kiani-Esfahani A, Shiravi AH, Nasr-Esfahani MH. DNA hypomethylation predisposes sperm to DNA damage in individuals with varicocele. Syst Biol Reprod Med. 2015; 61(4): 179-186.
  12. Rashidi M, Tavalaee M, Abbasi H, Nomikos M, Nasr-Esfahani MH. Increased de novo DNA methylation enzymes in sperm of individuals with varicocele. Cell J. 2021; 23(4): 389-396.
  13. Santana VP, Miranda-Furtado CL, Pedroso DCC, Eiras MC, Vasconcelos MAC, Ramos ES, et al. The relationship among sperm global DNA methylation, telomere length, and DNA fragmentation in varicocele: a cross-sectional study of 20 cases. Syst Biol Reprod Med. 2019; 65(2): 95-104.
  14. NRC. Guide for the care and use of laboratory animals. 8th ed. Washington (DC): National Academies Press (US); 2011.
  15. Turner TT. The study of varicocele through the use of animal models. Hum Reprod Update. 2001; 7(1): 78-84.
  16. Aitken RJ, Wingate JK, De Iuliis GN, McLaughlin EA. Analysis of lipid peroxidation in human spermatozoa using BODIPY C11. Mol Hum Reprod. 2007; 13(4): 203-211.
  17. Rahmani M, Tavalaee M, Hosseini M, Eskandari A, Shaygannia E, Sadeghi N, et al. Deferasirox, an iron-chelating agent, improves testicular morphometric and sperm functional parameters in a rat model of varicocele. Oxid Med Cell Longev. 2021; 2021: 6698482.
  18. Ni K, Dansranjavin T, Rogenhofer N, Oeztuerk N, Deuker J, Bergmann M, et al. TET enzymes are successively expressed during human spermatogenesis and their expression level is pivotal for male fertility. Hum Reprod. 2016; 31(7): 1411-1424.
  19. Salmani S, Razi M, Sarrafzadeh-Rezaei F, Mahmoudian A. Testosterone amplifies HSP70-2a, HSP90 and PCNA expression in experimental varicocele condition: Implication for DNA fragmentation. Reprod Biol. 2020; 20(3): 384-395.
  20. Wang K, Gao Y, Wang C, Liang M, Liao Y, Hu K. Role of oxidative stress in varicocele. Front Genet. 2022; 13: 850114.
  21. Majzoub A, Cho CL, Agarwal A, Esteves SC. Oxidative stress and varicocele pathophysiology series. In: Esteves Sc, Cho CL, MajzoubA Agarwal A, editors. Varicocele and male infertility: a complete guide. Cham: Springer International Publishing; 2019; 55-71.
  22. Gao Y, Zhao Y, Zhang H, Zhang P, Liu J, Feng Y, et al. Pubertal exposure to low doses of zearalenone disrupting spermatogenesis through ERα related genetic and epigenetic pathways. Toxicol Lett. 2019; 315: 31-38.
  23. Shaygannia E, Nasr-Esfahani MH, Sotoodehnejadnematalahi F, Parivar K. Is ferroptosis involved in ROS-induced testicular lesions in a varicocele rat model? Basic Clin Androl. 2021; 31(1): 10.
  24. Hassanin AM, Ahmed HH, Kaddah AN. A global view of the pathophysiology of varicocele. Andrology. 2018; 6(5): 654-661.
  25. Zhang Q, Zhang F, Gao HH, Zhang JM. Effects of varicocele on DNA methylation pattern of H19 and Snrpn gene in spermatozoa and behavioural characteristics of adult rat offspring. Andrologia. 2017; 49(1).
  26. Godschalk RWL, Janssen MCM, Vanhees K, van Doorn-Khosrovani SBVW, van Schooten FJ. Maternal exposure to genistein duringpregnancy and oxidative DNA damage in testes of male mouse offspring. Front Nutr. 2022; 9: 904368.
  27. Bruno S, Williams RJ, Del Vecchio D. Epigenetic cell memory: the gene’s inner chromatin modification circuit. PLoS Comput Biol. 2022; 18(4): e1009961.
  28. Efimova OA, Pendina AA, Tikhonov AV, Parfenyev SE, Mekina ID, Komarova EM, et al. Genome-wide 5-hydroxymethylcytosine patterns in human spermatogenesis are associated with semen quality. Oncotarget. 2017; 8(51): 88294-88307.
  29. Nettersheim D, Heukamp LC, Fronhoffs F, Grewe MJ, Haas N, Waha A, et al. Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development. PLoS One. 2013; 8(12): e82881.
  30. Gan H, Wen L, Liao S, Lin X, Ma T, Liu J, et al. Dynamics of 5-hydroxymethylcytosine during mouse spermatogenesis. Nat Commun. 2013; 4: 1995.
  31. Olszewska M, Kordyl O, Kamieniczna M, Fraczek M, Jędrzejczak P, Kurpisz M. Global 5mC and 5hmC DNA levels in human sperm subpopulations with differentially protaminated chromatin in normo- and oligoasthenozoospermic males. Int J Mol Sci. 2022; 23(9): 4516.
  32. Montjean D, Zini A, Ravel C, Belloc S, Dalleac A, Copin H, et al. Sperm global DNA methylation level: association with semen parameters and genome integrity. Andrology. 2015; 3(2): 235-240.
  33. Hernández-Cruz EY, Arancibia-Hernández YL, Loyola-Mondragón DY, Pedraza-Chaverri J. Oxidative stress and its role in Cd-induced epigenetic modifications: use of antioxidants as a possible preventive strategy. Oxygen. 2022; 2(2): 177-210.
  34. Madugundu GS, Cadet J, Wagner JR. Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA. Nucleic Acids Res. 2014; 42(11): 7450-7460.
  35. Ko M, An J, Bandukwala HS, Chavez L, Aijö T, Pastor WA, et al. Modulation of TET2 expression and 5-methylcytosine oxidation by the CXXC domain protein IDAX. Nature. 2013; 497(7447): 122-126.
  36. Jin C, Lu Y, Jelinek J, Liang S, Estecio MR, Barton MC, et al. TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells. Nucleic Acids Res. 2014; 42(11): 6956-6971.
  37. Santiago M, Antunes C, Guedes M, Iacovino M, Kyba M, Reik W, et al. Tet3 regulates cellular identity and DNA methylation in neural progenitor cells. Cell Mol Life Sci. 2020; 77(14): 2871-2883.
  38. van der Wijst MG, Venkiteswaran M, Chen H, Xu GL, Plösch T, Rots MG. Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase. Epigenetics. 2015; 10(8): 671-676.
  39. Li Y, Zhou T, Su YF, Hu ZY, Wei JJ, Wang W, et al. Prokineticin 2 overexpression induces spermatocyte apoptosis in varicocele in rats. Asian J Androl. 2020; 22(5): 500-506.
  40. Aitken RJ, Gibb Z, Baker MA, Drevet J, Gharagozloo P. Causes and consequences of oxidative stress in spermatozoa. Reprod Fertil Dev. 2016; 28(1-2): 1-10.

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