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Joint Analysis of Genome-wide DNA Methylation and Transcription Sequencing Identifies the Role of BAX Gene in Heat Stress–Induced-Sertoli Cells Apoptosis

  • Male Reproduction: Original Article
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Abstract

The problem of male infertility is a global health crisis and poses a serious threat to the well-being of families. Under heat stress (HS), the reduction of Sertoli cells (SCs) inhibits energy transport and nutrient supply to germ cells, leading to spermatogenesis failure. DNA methylation of genes is a central epigenetic regulatory mechanism in mammalian reproduction. However, it remains unclear how DNA methylation regulates gene expression in heat-stressed SCs. In this study, we investigated whether the decrease in SC levels during HS could be related to epigenetic DNA modifications. The cells exposed to HS showed changes in differential methylation cytosines and regions (DMCs/DMRs) and differential expression genes (DEGs), but not in global DNA methylations. One of the most important biological processes affected by HS is cell apoptosis induced by the intrinsic apoptotic signaling pathway (GO: 2,001,244, P < 0.05) by enrichment in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The joint analysis showed that several gene expressions in RNA-seq and WGBS overlapped and the shortlisted genes BAX, HSPH1, HSF1B, and BAG were strongly correlated with stress response and apoptosis. Methylation-specific PCR (MSP) and flow cytometry (FCM) analyzes showed that reduced promoter methylation and enhanced gene expression of BAX with a consequence of apoptosis. The activity of BAX, as well as an increase in its expression, is likely to result in a reduction of SCs population which could further impair ATP supply and adversely affect membrane integrity. These findings provide novel insights into the molecular mechanisms through which stressors cause male reproductive dysfunction and a new molecular etiology of male infertility.

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Abbreviations

WGBS:

Whole genomic DNA bisulfite methylation sequencing

bp:

Base pair

RNA-seq:

RNA transcription sequencing

DMC:

Differentially methylated cytosine

DMR:

Differentially methylated region

DNMT:

DNA methyltransferases

DEGs:

Differential expression genes

SCs:

Sertoli cells

References

  1. Wang X, Kadarmideen HN. An epigenome-wide DNA methylation map of testis in pigs for study of complex traits. Front Genet. 2019;10:405. https://doi.org/10.3389/fgene.2019.00405

  2. Berger SL, Kouzarides T, Shiekhattar R, Shilatifard A. An operational definition of epigenetics. Gene Dev. 2009;23(7):781–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Noyer-Weidner M, Trautner TA. Methylation of DNA in Prokaryotes Exs. 1993;64:39–108.

    Article  CAS  PubMed  Google Scholar 

  4. Fisher O, Siman-Tov R, Ankri S. Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica. Nucleic Acids Res. 2004;32(1):287–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Anway MD, Memon MA, Uzumcu M, Skinner MK. Transgenerational effect of the endocrine disruptor vinclozolin on male spermatogenesis. J Androl. 2006;27(6):868–79.

    Article  CAS  PubMed  Google Scholar 

  6. Anway MD. Epigenetic transgenerational actions of endocrine disruptors and male fertility (June, pg 1466, 2005). Sci. 2010;328(5979):690–690.

    Google Scholar 

  7. Rahman MB, Schellander K, Luceno NL, Van Soom A. Heat stress responses in spermatozoa: mechanisms and consequences for cattle fertility. Theriogenol. 2018;113:102–12.

    Article  CAS  Google Scholar 

  8. Sadler-Riggleman I, Klukovich R, Nilsson E, Beck D, Xie YM, Yan W, Skinner MK. Epigenetic transgenerational inheritance of testis pathology and Sertoli cell epimutations: generational origins of male infertility. Environ Epigen. 2019;5(3). https://doi.org/10.1093/eep/dvz013

  9. Rebourcet D, O’Shaughnessy PJ, Pitetti JL, Monteiro A, O’Hara L, Milne L, Tsai YT, Cruickshanks L, Riethmacher D, Guillou F, et al. Sertoli cells control peritubular myoid cell fate and support adult Leydig cell development in the prepubertal testis. Development. 2014;141(10):2139–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rebourcet D, Darbey A, Monteiro A, Soffientini U, Tsai YT, Handel I, Pitetti JL, Nef S, Smith LB, O’Shaughnessy PJ. Sertoli cell number defines and predicts germ and Leydig cell population sizes in the adult mouse testis. Endocrinol. 2017;158(9):2955–69.

    Article  CAS  Google Scholar 

  11. Yokonishi T, McKey J, Ide S, Capel B. Sertoli cell ablation and replacement of the spermatogonial niche in mouse. Nat Commun. 2020;11(1). https://doi.org/10.1038/s41467-019-13879-8

  12. Ross JW, Hale BJ, Gabler NK, Rhoads RP, Keating AF, Baumgard LH. Physiological consequences of heat stress in pigs. Anim Prod Sci. 2015;55(11–12):1381–90.

    Article  CAS  Google Scholar 

  13. Fujisawa M, Yamazaki T, Dobashi M, Okada H, Kamidono S. Sertoli cell number in testes of azoospermic men: trends in biopsy specimens. Arch Androl. 2001;47(2):103–6.

    Article  CAS  PubMed  Google Scholar 

  14. Johnston H, Baker PJ, Abel M, Charlton HM, Jackson G, Fleming L, Kumar TR, O’Shaughnessy PJ. Regulation of Sertoli cell number and activity by follicle-stimulating hormone and androgen during postnatal development in the mouse. Endocrinol. 2004;145(1):318–29.

    Article  CAS  Google Scholar 

  15. Aitken RJ, Koppers AJ. Apoptosis and DNA damage in human spermatozoa. Asian J Androl. 2011;13(1):36–42.

    Article  CAS  PubMed  Google Scholar 

  16. Houston BJ, Nixon B, Martin JH, De Iuliis GN, Trigg NA, Bromfield EG, McEwan KE, Aitken RJ. Heat exposure induces oxidative stress and DNA damage in the male germ line. Biol Reprod. 2018;98(4):593–606.

    Article  PubMed  Google Scholar 

  17. Hengartner MO. The biochemistry of apoptosis. Nature. 2000;407(6805):770–6.

    Article  CAS  PubMed  Google Scholar 

  18. Blatt NB, Glick GD. Signaling pathways and effector mechanisms pre-programmed cell death. Bioorgan Med Chem. 2001;9(6):1371–84.

    Article  CAS  Google Scholar 

  19. Said TM, Paasch U, Glander HJ, Agarwal A. Role of caspases in male infertility. Hum Reprod Update. 2004;10(1):39–51.

    Article  CAS  PubMed  Google Scholar 

  20. Hu Y, Deng J, Tian K, Yang WR, Luo NJ, Lian Y, Gan L, Tang XY, Luo HY, Zhang JJ, et al. MiR-8-3p regulates hyperthermia-induced lactate secretion by targeting PPP2R5B in boar Sertoli cells. Mol Reprod Dev. 2019;86(11):1720–30.

    Article  CAS  PubMed  Google Scholar 

  21. Qiao B, Huang J, Mei Z, Lam AK, Zhao J, Ying L. Analysis of immune microenvironment by multiplex immunohistochemistry staining in different oral diseases and oral squamous cell carcinoma. Front Oncol. 2020;10: 555757.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Cohen GM, Sun XM, Fearnhead H, Macfarlane M, Brown DG, Snowden RT, Dinsdale D. Formation of Large molecular-weight fragments of DNA is a key committed step of apoptosis in thymocytes. J Immunol. 1994;153(2):507–16.

    Article  CAS  PubMed  Google Scholar 

  23. Zinkel S, Gross A, Yang E. BCL2 family in DNA damage and cell cycle control. Cell Death Differ. 2006;13(8):1351–9.

    Article  CAS  PubMed  Google Scholar 

  24. Guerrero-Bosagna C, Savenkova M, Haque MM, Nilsson E, Skinner MK. Environmentally induced epigenetic transgenerational inheritance of altered Sertoli cell transcriptome and epigenome: molecular etiology of male infertility. PLoS ONE 2013;8(3). https://doi.org/10.1371/journal.pone.0059922

  25. Sharpe RM, Skakkebaek NE. Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects. Fertil Steril. 2008;89(2 Suppl):e33-38.

    Article  PubMed  Google Scholar 

  26. Skinner MK, Griswold MD. Sertoli cells synthesize and secrete transferrin-like protein. J Biol Chem. 1980;255(20):9523–5.

    Article  CAS  PubMed  Google Scholar 

  27. Salilew-Wondim D, Saeed-Zidane M, Hoelker M, Gebremedhn S, Poirier M, Pandey HO, Tholen E, Neuhoff C, Held E, Besenfelder U et al. Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation. BMC Genom. 2018;19. https://doi.org/10.1186/s12864-018-4826-3

  28. Deshmukh RS, Ostrup O, Ostrup E, Vejlsted M, Niemann H, Lucas-Hahn A, Petersen B, Li JA, Callesen H, Hyttel P. DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer. Epigenetics-Us. 2011;6(2):177–87.

    Article  CAS  Google Scholar 

  29. Patil V, Ward RL, Hesson LB. The evidence for functional non-CpG methylation in mammalian cells. Epigenetics-Us. 2014;9(6):823–8.

    Article  Google Scholar 

  30. Li L, Tan HP, Gu ZT, Liu ZF, Geng Y, Liu YS, Tong HS, Tang YQ, Qiu JM, Su L. Heat stress induces apoptosis through a Ca2+-mediated mitochondrial apoptotic pathway in human umbilical vein endothelial cells. PLoS ONE 2014;9(12). https://doi.org/10.1371/journal.pone.0111083

  31. Hawes NA, Tremblay LA, Pochon X, Dunphy B, Fidler AE, Smith KF. Effects of temperature and salinity stress on DNA methylation in a highly invasive marine invertebrate, the colonial ascidian Didemnum vexillum. PeerJ. 2018;6: e5003.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Cui Y, Liu B, Xie J, Xu P, Habte-Tsion HM, Zhang Y. Effect of heat stress and recovery on viability, oxidative damage, and heat shock protein expression in hepatic cells of grass carp (Ctenopharyngodon idellus). Fish Physiol Biochem. 2014;40(3):721–9.

    Article  CAS  PubMed  Google Scholar 

  33. Sajjanar B, Siengdee P, Trakooljul N, Liu X, Kalbe C, Wimmers K, Ponsuksili S. Cross-talk between energy metabolism and epigenetics during temperature stress response in C2C12 myoblasts. Int J Hyperther. 2019;36(1):776–84.

    Article  Google Scholar 

  34. Hao Y, Cui Y, Gu X. Genome-wide DNA methylation profiles changes associated with constant heat stress in pigs as measured by bisulfite sequencing. Sci Rep. 2016;6:27507.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Clark SJ, Harrison J, Frommer M. CpNpG methylation in mammalian cells. Nat Genet. 1995;10(1):20–7.

    Article  CAS  PubMed  Google Scholar 

  36. Aranyi T, Faucheux BA, Khalfallah O, Vodjdani G, Biguet NF, Mallet J, Meloni R. The tissue-specific methylation of the human Tyrosine Hydroxylase gene reveals new regulatory elements in the first exon. J Neurochem. 2005;94(1):129–39.

    Article  CAS  PubMed  Google Scholar 

  37. Almamun M, Levinson BT, Gater ST, Schnabel RD, Arthur GL, Davis JW, Taylor KH. Genome-wide DNA methylation analysis in precursor B-cells. Epigenetics-Us. 2014;9(12):1588–95.

    Article  Google Scholar 

  38. Glastad KM, Hunt BG, Goodisman MAD. DNA methylation and chromatin organization in insects: insights from the ant Camponotus floridanus. Genome Biol Evol. 2015;7(4):931–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S, Rebhan M, Schubeler D. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet. 2007;39(4):457–66.

    Article  CAS  PubMed  Google Scholar 

  40. Chen X, Shen LH, Gui LX, Yang F, Li J, Cao SZ, Zuo ZC, Ma XP, Deng JL, Ren ZH, et al. Genome-wide DNA methylation profile of prepubertal porcine testis. Reprod Fertil Dev. 2018;30(2):349–58.

    Article  CAS  PubMed  Google Scholar 

  41. Gump JM, Thorburn A. Autophagy and apoptosis: what is the connection? Trends Cell Biol. 2011;21(7):387–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Bao ZQ, Liao TT, Yang WR, Wang Y, Luo HY, Wang XZ. Heat stress-induced autophagy promotes lactate secretion in cultured immature boar Sertoli cells by inhibiting apoptosis and driving SLC2A3, LDHA, and SLC16A1 expression. Theriogenol. 2017;87:339–48.

    Article  CAS  Google Scholar 

  43. Fan XT, Hou TT, Zhang S, Guan YJ, Jia J, Wang ZZ. The cellular responses of autophagy, apoptosis, and 5-methylcytosine level in zebrafish cells upon nutrient deprivation stress. Chemosphere. 2020;241. https://doi.org/10.1016/j.chemosphere.2019.124989

  44. Hu Y, Hu H, Yin L, Wang L, Luo K, Luo N. Arachidonic acid impairs the function of the blood-testis barrier via triggering mitochondrial complex-ROS-P38 MAPK axis in hyperthermal Sertoli cells. Ecotoxicol Environ Saf. 2023;252: 114598.

    Article  CAS  PubMed  Google Scholar 

  45. Thorburn A. Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis. 2008;13(1):1–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Khan A, Dou JH, Wang YC, Jiang XL, Khan MZ, Luo HP, Usman T, Zhu HB. Evaluation of heat stress effects on cellular and transcriptional adaptation of bovine granulosa cells. J Anim Sci Biotechno. 2020;11:1–20. https://doi.org/10.1186/s40104-019-0408-8

  47. Charmandari E, Tsigos C, Chrousos G. Endocrinology of the stress response. Annu Rev Physiol. 2005;67:259–84.

    Article  CAS  PubMed  Google Scholar 

  48. Tsujimoto Y. Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria? Genes to cells:Devoted to Mol Cell Mech. 1998;3(11):697–707.

    Article  CAS  Google Scholar 

  49. Garrido C, Galluzzi L, Brunet M, Puig PE, Didelot C, Kroemer G. Mechanisms of cytochrome c release from mitochondria. Cell Death Differ. 2006;13(9):1423–33.

    Article  CAS  PubMed  Google Scholar 

  50. Russell LD, Chiarini-Garcia H, Korsmeyer SJ, Knudson CM. Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis. Biol Reprod. 2002;66(4):950–8.

    Article  CAS  PubMed  Google Scholar 

  51. Alipour M, Zargar SJ, Safarian S, Fouladdel S, Azizi E, Jafargholizadeh N. The study of DNA methylation of bax gene promoter in breast and colorectal carcinoma cell lines. Iranian J Cancer Prevent. 2013;6(2):59–64.

    CAS  Google Scholar 

  52. Ahani-Nahayati M, Solali S, Shams Asenjan K, Movassaghpour Akbari AA, Talebi M, Zadi Heydarabad M, Baharaghdam S, Farshdousti HM. Promoter methylation status of survival-related genes in MOLT- 4 cells co-cultured with bone marrow mesenchymal stem cells under hypoxic conditions. Cell J. 2018;20(2):188–94.

    PubMed  PubMed Central  Google Scholar 

  53. Wang X, Li B. DNMT1 regulates human endometrial carcinoma cell proliferation. Onco Targets Ther. 2017;10:1865–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Maor GL, Yearim A, Ast G. The alternative role of DNA methylation in splicing regulation. Trends Genet. 2015;31(5):274–80.

    Article  Google Scholar 

  55. Skinner MK, Ben Maamar M, Sadler-Riggleman I, Beck D, Nilsson E, McBirney M, Klukovich R, Xie YM, Tang C, Yan W. Alterations in sperm DNA methylation, non-coding RNA and histone retention associate with DDT-induced epigenetic transgenerational inheritance of disease. Epigenet Chromatin. 2018;11. https://doi.org/10.1186/s13072-018-0178-0

  56. Tong DD, Zhang J, Wang XF, Li Q, Liu LY, Yang J, Guo B, Ni L, Zhao LY, Huang C. MeCP2 facilitates breast cancer growth via promoting ubiquitination-mediated P53 degradation by inhibiting RPL5/RPL11 transcription. Oncogenesis 2020;9(5). https://doi.org/10.1038/s41389-020-0239-7

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Acknowledgements

We thank the sponsors of the Funding for their support.

Funding

This work was supported by grants from the Science and Technology Cooperation Project of Zunyi City (NO. HZ Zi (2022) 294 and HZ Zi (2022) 345) and Research project of Science and Technology Department of Guizhou Province (Qian Ke He basic-ZK[2023]-547) as well as Youth Program of Guizhou Provincial Department of Education (Qian Jiao Ji [2022]227). The funders had no role in experiment design and preparation of the manuscript.

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Authors and Affiliations

  1. Department of Reproductive Medicine, Department of Obstetrics and Gynecology, Affiliated Hospital of Zunyi Medical University, Affiliated Hospital of Zunyi Medical University, Zunyi, 563000, China

    Yu Hu, QingHan Li, ZhengLi Qian,  BeiXiao & KeYan Luo

  2. Department of Preclinical Medicine, Zunyi Medical University, Zunyi, 563000, China

    NanJian Luo

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  1. Yu Hu
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  2. QingHan Li
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  3. ZhengLi Qian
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  4. BeiXiao
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NJL conceived the project, YH designed the experiments and wrote the manuscript, QHL analyzed the data. ZLQ, BX, and KYL provided advice on manuscript. KYL and NJL are co-corresponding authors.

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Correspondence to KeYan Luo or NanJian Luo.

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The study was approved by Zunyi Medical University Animal Ethics Committee. The experimental procedures, including animal welfare, were in strict accordance with the Guidelines on Ethical Treatment of Experimental Animals (SYXK(QIAN)2014–003), which issued by GuiZhou Municipal People’s Government.

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Hu, Y., Li, Q., Qian, Z. et al. Joint Analysis of Genome-wide DNA Methylation and Transcription Sequencing Identifies the Role of BAX Gene in Heat Stress–Induced-Sertoli Cells Apoptosis. Reprod. Sci. (2024). https://doi.org/10.1007/s43032-023-01430-6

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