DOI QR코드

DOI QR Code

p66Shc in sheep preimplantation embryos: Expression and regulation of oxidative stress through the manganese superoxide dismutase-reactive oxygen species metabolic pathway

  • Tong Zhang (School of Medicine, Shanxi Datong University) ;
  • Jiaxin Zhang (Key Laboratory of Animal Genetics, Breeding and Reproduction, Inner Mongolia Autonomous Region) ;
  • Ruilan Li (School of Medicine, Shanxi Datong University)
  • Received : 2022.10.20
  • Accepted : 2023.02.23
  • Published : 2023.07.01

Abstract

Objective: p66Shc, a 66 kDa protein isoform encoded by the proto-oncogene SHC, is an essential intracellular redox homeostasis regulatory enzyme that is involved in the regulation of cellular oxidative stress, apoptosis induction and the occurrence of multiple age-related diseases. This study investigated the expression profile and functional characteristics of p66Shc during preimplantation embryo development in sheep. Methods: The expression pattern of p66Shc during preimplantation embryo development in sheep at the mRNA and protein levels were studied by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The effect of p66Shc knockdown on the developmental potential were evaluated by cleavage rate, morula rate and blastocyst rate. The effect of p66Shc deficiency on reactive oxygen species (ROS) production, DNA oxidative damage and the expression of antioxidant enzymes (e.g., catalase and manganese superoxide dismutase [MnSOD]) were also investigated by immunofluorescence staining. Results: Our results showed that p66Shc mRNA and protein were expressed in all stages of sheep early embryos and that p66Shc mRNA was significantly downregulated in the 4-to 8-cell stage (p<0.05) and significantly upregulated in the morula and blastocyst stages after embryonic genome activation (EGA) (p<0.05). Immunofluorescence staining showed that the p66Shc protein was mainly located in the peripheral region of the blastomere cytoplasm at different stages of preimplantation embryonic development. Notably, serine (Ser36)-phosphorylated p66Shc localized only in the cytoplasm during the 2- to 8-cell stage prior to EGA, while phosphorylated (Ser36) p66Shc localized not only in the cytoplasm but also predominantly in the nucleus after EGA. RNAi-mediated silencing of p66Shc via microinjection of p66Shc siRNA into sheep zygotes resulted in significant decreases in p66Shc mRNA and protein levels (p<0.05). Knockdown of p66Shc resulted in significant declines in the levels of intracellular ROS (p<0.05) and the DNA damage marker 8-hydroxy2'-deoxyguanosine (p<0.05), markedly increased MnSOD levels (p<0.05) and resulted in a tendency to develop to the morula stage. Conclusion: These results indicate that p66Shc is involved in the metabolic regulation of ROS production and DNA oxidative damage during sheep early embryonic development.

Keywords

Acknowledgement

This work was financially supported by the National Natural Science Foundation of China (31460598), Youth Science and Technology Research Fund of the Basic Research Program of Shanxi Province (20210302124293), and Applied Basic Research Project of Datong (2022065).

References

  1. Paramio MT, Izquierdo D. Recent advances in in vitro embryo production in small ruminants. Theriogenology 2016;86:152-9. https://doi.org/10.1016/j.theriogenology.2016.04.027
  2. Falchi L, Ledda S, Zedda MT. Embryo biotechnologies in sheep: Achievements and new improvements. Reprod Domest Anim 2022;57:22-33. https://doi.org/10.1111/rda.14127
  3. Hardy MLM, Day ML, Morris MB. Redox regulation and oxidative stress in mammalian oocytes and embryos developed in vivo and in vitro. Int J Environ Res Public Health 2021;18:11374. https://doi.org/10.3390/ijerph182111374
  4. Sen U, Kuran M. Low incubation temperature successfully supports the in vitro bovine oocyte maturation and subsequent development of embryos. Asian-Australas J Anim Sci 2018;31:827-34. https://doi.org/10.5713/ajas.17.0569
  5. Soto-Heras S, Paramio MT. Impact of oxidative stress on oocyte competence for in vitro embryo production programs. Res Vet Sci 2020;132:342-50. https://doi.org/10.1016/j.rvsc.2020.07.013
  6. Pintus E, Ros-Santaella JL. Impact of oxidative stress on male reproduction in domestic and wild animals. Antioxidants 2021;10:1154. https://doi.org/10.3390/antiox10071154
  7. Betts DH, Madan P. Permanent embryo arrest: molecular and cellular concepts. Mol Hum Reprod 2008;14:445-53. https://doi.org/10.1093/molehr/gan035
  8. Takahashi M. Oxidative stress and redox regulation on in vitro development of mammalian embryos. J Reprod Dev 2012;58:1-9. https://doi.org/10.1262/jrd.11-138N
  9. Bhat SS, Anand D, Khanday FA. p66Shc as a switch in bringing about contrasting responses in cell growth: implications on cell proliferation and apoptosis. Mol Cancer 2015;14:76. https://doi.org/10.1186/s12943-015-0354-9
  10. Mir HA, Ali R, Mushtaq U, Khanday FA. Structure-functional implications of longevity protein p66Shc in health and disease. Ageing Res Rev 2020;63:101139. https://doi.org/10.1016/j.arr.2020.101139
  11. Sirard MA. Factors affecting oocyte and embryo transcriptomes. Reprod Domest Anim 2012;47:148-55. https://doi.org/10.1111/j.1439-0531.2012.02069.x
  12. Graf A, Krebs S, Zakhartchenko V, Schwalb B, Blum H, Wolf E. Fine mapping of genome activation in bovine embryos by RNA sequencing. Proc Natl Acad Sci USA 2014;111:4139-44. https://doi.org/10.1073/pnas.1321569111
  13. Ren K, Li X, Yan J, et al. Knockdown of p66Shc by siRNA injection rescues arsenite-induced developmental retardation in mouse preimplantation embryos. Reprod Toxicol 2014;43:8-18. https://doi.org/10.1016/j.reprotox.2013.10.008
  14. Betts DH, Bain NT, Madan P. The p66(Shc) adaptor protein controls oxidative stress response in early bovine embryos. PLoS One 2014;9:e86978. https://doi.org/10.1371/journal.pone.0086978
  15. Zhang T, Li R, Fan X, et al. Expression of p66Shc and its relationship with cytoplasmic redox homeostasis in sheep oocytes. Scientia Agricultura Sinica 2019;52:2183-92.
  16. Zhang T, Zhao X, Hai R, Li R, Zhang W, Zhang J. p66Shc is associated with hydrogen peroxide-induced oxidative stress in preimplantation sheep embryos. Mol Reprod Dev 2019;86:342-50. https://doi.org/10.1002/mrd.23110
  17. Zhang T, Fan X, Li R, Zhang C, Zhang J. Effects of pre-incubation with C-type natriuretic peptide on nuclear maturation, mitochondrial behavior, and developmental competence of sheep oocytes. Biochem Biophys Res Commun 2018;497:200-6. https://doi.org/10.1016/j.bbrc.2018.02.054
  18. Migliaccio E, Giorgio M, Mele S, et al. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 1999;402:309-13. https://doi.org/10.1038/46311
  19. Xiao Y, Xia J, Cheng J, et al. Inhibition of S-adenosylhomocysteine hydrolase induces endothelial dysfunction via epigenetic regulation of p66shc-mediated oxidative stress pathway. Circulation 2019;139:2260-77. https://doi.org/10.1161/CIRCULATIONAHA.118.036336
  20. Boengler K, Bornbaum J, Schluter KD, Schulz R. P66shc and its role in ischemic cardiovascular diseases. Basic Res Cardiol 2019;114:29. https://doi.org/10.1007/s00395-019-0738-x
  21. Gao L, Shan W, Zeng W, et al. Carnosic acid alleviates chronic alcoholic liver injury by regulating the SIRT1/ChREBP and SIRT1/p66shc pathways in rats. Mol Nutr Food Res 2016;60:1902-11. https://doi.org/10.1002/mnfr.201500878
  22. Zhu J, Moawad AR, Wang CY, Li HF, Ren JY, Dai YF. Advances in in vitro production of sheep embryos. Int J Vet Sci Med 2018;6:S15-26. https://doi.org/10.1016/j.ijvsm.2018.02.003
  23. Mondal S, Mor A, Reddy IJ, Nandi S, Gupta PSP, Mishra A. In vitro embryo production in sheep. In: Herrick, J, editor. Comparative embryo culture. Methods in molecular biology. New York, NY, USA: Humana; 2019. p. 131-40. https://doi.org/10.1007/978-1-4939-9566-0_9
  24. Belli M, Zhang L, Liu X, et al. Oxygen concentration alters mitochondrial structure and function in in vitro fertilized preimplantation mouse embryos. Hum Reprod 2019;34:601-11. https://doi.org/10.1093/humrep/dez011
  25. Lin J, Wang L. Oxidative stress in oocytes and embryo development: Implications for in vitro systems. Antioxid Redox Signal 2021;34:1394-406. https://doi.org/10.1089/ars.2020.8209
  26. Haslem L, Hays JM, Hays FA. p66Shc in cardiovascular pathology. Cells 2022;11:1855. https://doi.org/10.3390/cells11111855
  27. Edwards NA, Watson AJ, Betts DH. Knockdown of p66Shc alters lineage-associated transcription factor expression in mouse blastocysts. Stem Cells Dev 2018;27:1479-93. https://doi.org/10.1089/scd.2018.0131
  28. Trinei M, Migliaccio E, Bernardi P, Paolucci F, Pelicci P, Giorgio M. p66Shc, mitochondria, and the generation of reactive oxygen species. Methods Enzymol 2013;528:99-110. https://doi.org/10.1016/B978-0-12-405881-1.00006-9
  29. Wianny F, Zernicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol 2000;2:70-5. https://doi.org/10.1038/35000016
  30. Sakurai N, Fujii T, Hashizume T, Sawai K. Effects of downregulating oct-4 transcript by RNA interference on early development of porcine embryos. J Reprod Dev 2013;59:353-60. https://doi.org/10.1262/jrd.2013-003
  31. Gertz M, Steegborn C. The Lifespan-regulator p66Shc in mitochondria: redox enzyme or redox sensor? Antioxid Redox Signal 2010;13:1417-28. https://doi.org/10.1089/ars.2010.3147
  32. Yang SK, Xiao L, Li J, Liu F, Sun L. Oxidative stress, a common molecular pathway for kidney disease: role of the redox enzyme p66Shc. Ren Fail 2014;36:313-20. https://doi.org/10.3109/0886022X.2013.846867
  33. Turco MY, Matsukawa K, Czernik M, et al. High levels of anandamide, an endogenous cannabinoid, block the growth of sheep preimplantation embryos by inducing apoptosis and reversible arrest of cell proliferation. Hum Reprod 2008;23:2331-8. https://doi.org/10.1093/humrep/den258
  34. Liang S, Jin YX, Yuan B, Zhang JB, Kim NH. Melatonin enhances the developmental competence of porcine somatic cell nuclear transfer embryos by preventing DNA damage induced by oxidative stress. Sci Rep 2017;7:11114. https://doi.org/10.1038/s41598-017-11161-9
  35. Remiao MH, Lucas CG, Domingues WB, et al. Melatonin delivery by nanocapsules during in vitro bovine oocyte maturation decreased the reactive oxygen species of oocytes and embryos. Reprod Toxicol 2016;63:70-81. https://doi.org/10.1016/j.reprotox.2016.05.016
  36. Semenova NV, Madaeva IM, Brichagina AS, Kolesnikov SI, Kolesnikova LI. 8-Hydroxy-2'-deoxyguanosine as an oxidative stress marker in insomnia. Bull Exp Biol Med 2021;171:384-7. https://doi.org/10.1007/s10517-021-05233-0
  37. Deluao JC, Winstanley Y, Robker RL, Pacella Ince L, Gonzalez M, McPherson NO. Reactive oxygen species in the mammalian pre-implantation embryo. Reproduction 2022;164:F95-108. https://doi.org/10.1530/REP-22-0121