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Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen

  • Embryo Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

We aimed to determine whether embryo culture induces markers of cellular senescence and whether these effects were dependent on culture conditions.

Methods

Murine blastocysts were derived in vitro and in vivo and assessed for 2 primary markers of senescence: senescence-associated β-galactosidase (SA-β-gal) and phosphorylated H2A.X (γ-H2A.X), the latter being a mark of DNA oxidative damage. Expression of senescence-associated genes p21, p16, and interleukin 6 (IL6) were also assessed.

Results

Compared with in vivo–derived blastocysts, in vitro embryos had high levels of SA-β-gal, nuclear γ-H2A.X, and p21 mRNA expression, indicating that a senescence-like phenotype is induced by in vitro culture. To determine the role of culture conditions, we studied the effect of oxygen (5 % vs 20 %) and protein supplementation on senescence markers. Blastocysts in reduced oxygen (5 %) had low levels of both SA-β-gal and γ-H2A.X compared with blastocysts cultured in ambient oxygen. Senescence markers also were reduced in the presence of protein, suggesting that antioxidant properties of protein reduce oxidative DNA damage in vitro.

Conclusion

Elevated SA-β-gal, γ-H2A.X, and p21 suggest that in vitro stress can induce a senescence-like phenotype. Reduced oxygen during embryo culture minimizes these effects, providing further evidence for potential adverse effects of culturing embryos at ambient oxygen concentrations.

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Abbreviations

ANOVA:

Analysis of variance

BSA:

Bovine serum albumin

DAPI:

4’,6-diamidino-2-phenylindole

FVB:

Friend Virus B

γ-H2A.X:

phosphorylated histone H2A.X

HEPES:

N-2-hydroxyethylpiperazine-N’-2-ethane-sulfonate

Ig:

Immunoglobulin

IL6 :

Interleukin 6

IVF:

In vitro fertilization

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

PVA:

Polyvinyl alcohol

ROS:

Reactive oxygen species

SA-β-galactosidase:

Senescence-associated β-galactosidase

References

  1. Thomas MR, Sparks AE, Ryan GL, Van Voorhis BJ. Clinical predictors of human blastocyst formation and pregnancy after extended embryo culture and transfer. Fertil Steril. 2010;94(2):543–8. Epub 2009 May 5.

    Article  PubMed  Google Scholar 

  2. Doherty AS, Mann MR, Tremblay KD, Bartolomei MS, Schultz RM. Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod. 2000;62(6):1526–35.

    Article  PubMed  CAS  Google Scholar 

  3. Rinaudo P, Schultz RM. Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos. Reproduction. 2004;128(3):301–11.

    Article  PubMed  CAS  Google Scholar 

  4. Hardy K. Cell death in the mammalian blastocyst. Mol Hum Reprod. 1997;3(10):919–25.

    Article  PubMed  CAS  Google Scholar 

  5. Jurisicova A, Acton BM. Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development. Reproduction. 2004;128(3):281–91.

    Article  PubMed  CAS  Google Scholar 

  6. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961;25:585–621.

    Article  PubMed  CAS  Google Scholar 

  7. Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol. 2007;8(9):729–40.

  8. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995;92(20):9363–7.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  9. Fragkos M, Jurvansuu J, Beard P. H2AX is required for cell cycle arrest via the p53/p21 pathway. Mol Cell Biol. 2009;29(10):2828–40. Epub 2009 Mar 9.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  10. Passos JF, von Zglinicki T, Kirkwood TB. Mitochondria and ageing: winning and losing in the numbers game. Bioessays. 2007;29(9):908–17.

    Article  PubMed  CAS  Google Scholar 

  11. Frippiat C, Chen QM, Zdanov S, Magalhaes JP, Remacle J, Toussaint O. Subcytotoxic H2O2 stress triggers a release of transforming growth factor-beta 1, which induces biomarkers of cellular senescence of human diploid fibroblasts. J Biol Chem. 2001;276(4):2531–7. Epub 2000 Nov 1.

    Article  PubMed  CAS  Google Scholar 

  12. Passos JF, Saretzki G, von Zglinicki T. DNA damage in telomeres and mitochondria during cellular senescence: is there a connection. Nucleic Acids Res. 2007;35(22):7505–13. Epub 2007 Nov 5.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  13. Favetta LA, St John EJ, King WA, Betts DH. High levels of p66shc and intracellular ROS in permanently arrested early embryos. Free Radic Biol Med. 2007;42(8):1201–10. Epub 2007 Jan 13.

    Article  PubMed  CAS  Google Scholar 

  14. Betts DH, King WA. Genetic regulation of embryo death and senescence. Theriogenology. 2001;55(1):171–91.

    Article  PubMed  CAS  Google Scholar 

  15. Betts DH, Madan P. Permanent embryo arrest: molecular and cellular concepts. Mol Hum Reprod. 2008;14(8):445–53. Epub 2008 May 29.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  16. McLaren A, Bowman P. Genetic effects on the timing of early development in the mouse. J Embryol Exp Morphol. 1973;30(2):491–8.

    PubMed  CAS  Google Scholar 

  17. Lawless C, Wang C, Jurk D, Merz A, Zglinicki TV, Passos JF. Quantitative assessment of markers for cell senescence. Exp Gerontol. 2010;45(10):772–8. Epub 2010 Feb 1.

    Article  PubMed  CAS  Google Scholar 

  18. Willems E, Mateizel I, Kemp C, Cauffman G, Sermon K, Leyns L. Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells. Int J Dev Biol. 2006;50(7):627–35.

    Article  PubMed  CAS  Google Scholar 

  19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 2001;25(4):402–8.

    Article  PubMed  CAS  Google Scholar 

  20. Rodier F, Campisi J. Four faces of cellular senescence. J Cell Biol. 2011;192(4):547–56. Epub 2011 Feb 14.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Takahashi M, Keicho K, Takahashi H, Ogawa H, Schultz RM, Okano A. Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay. Theriogenology. 2000;54(1):137–45.

    Article  PubMed  CAS  Google Scholar 

  22. Guerin P, El Mouatassim S, Menezo Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update. 2001;7(2):175–89.

    Article  PubMed  CAS  Google Scholar 

  23. Kitagawa Y, Suzuki K, Yoneda A, Watanabe T. Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology. 2004;62(7):1186–97.

    Article  PubMed  CAS  Google Scholar 

  24. Van Soom A, Yuan YQ, Peelman LJ, de Matos DG, Dewulf J, Laevens H, et al. Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition. Theriogenology. 2002;57(5):1453–65.

    Article  PubMed  Google Scholar 

  25. Pomar FJ, Teerds KJ, Kidson A, Colenbrander B, Tharasanit T, Aguilar B, et al. Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study. Theriogenology. 2005;63(8):2254–68. Epub 2004 Nov 28.

    Article  PubMed  CAS  Google Scholar 

  26. Takahashi M, Saka N, Takahashi H, Kanai Y, Schultz RM, Okano A. Assessment of DNA damage in individual hamster embryos by comet assay. Mol Reprod Dev. 1999;54(1):1–7.

    Article  PubMed  CAS  Google Scholar 

  27. Jurisicova A, Latham KE, Casper RF, Casper RF, Varmuza SL. Expression and regulation of genes associated with cell death during murine preimplantation embryo development. Mol Reprod Dev. 1998;51(3):243–53.

    Article  PubMed  CAS  Google Scholar 

  28. Lichnovsky V, Kolar Z, Murray P, Hlobilkova A, Cernochova D, Pospisilova E, et al. Differences in p53 and Bcl-2 expression in relation to cell proliferation during the development of human embryos. Mol Pathol. 1998;51(3):131–7.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  29. Frenkel J, Sherman D, Fein A, Schwartz D, Almog N, Kapon A, et al. Accentuated apoptosis in normally developing p53 knockout mouse embryos following genotoxic stress. Oncogene. 1999;18(18):2901–7.

    Article  PubMed  CAS  Google Scholar 

  30. Chandrakanthan V, Chami O, Stojanov T, O’Neill C. Variable expressivity of the tumour suppressor protein TRP53 in cryopreserved human blastocysts. Reprod Biol Endocrinol. 2007;5:39.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Ganeshan L, Li A, O’Neill C. Transformation-related protein 53 expression in the early mouse embryo compromises preimplantation embryonic development by preventing the formation of a proliferating inner cell mass. Biol Reprod. 2010;83(6):958–64. Epub 2010 Aug 25.

    Article  PubMed  CAS  Google Scholar 

  32. Leidenfrost S, Boelhauve M, Reichenbach M, Gungor T, Reichenbach HD, Sinowatz F, et al. Cell arrest and cell death in mammalian preimplantation development: lessons from the bovine model. PLoS ONE. 2011;6(7):e22121. Epub 2011 Jul 21.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  33. Ishii N, Oohira T, Oshima A, Sakuraba H, Endo F, Matsuda I, et al. Clinical and molecular analysis of a Japanese boy with Morquio B disease. Clin Genet. 1995;48(2):103–8.

    Article  PubMed  CAS  Google Scholar 

  34. Severino J, Allen RG, Balin S, Balin A, Cristofalo VJ. Is beta-galactosidase staining a marker of senescence in vitro and in vivo? Exp Cell Res. 2000;257(1):162–71.

    Article  PubMed  CAS  Google Scholar 

  35. Zwerschke W, Mazurek S, Stockl P, Hutter E, Eigenbrodt E, Jansen-Durr P. Metabolic analysis of senescent human fibroblasts reveals a role for AMP in cellular senescence. Biochem J. 2003;376(Pt 2):403–11.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  36. Sitte N, Merker K, Grune T, von Zglinicki T. Lipofuscin accumulation in proliferating fibroblasts in vitro: an indicator of oxidative stress. Exp Gerontol. 2001;36(3):475–86.

    Article  PubMed  CAS  Google Scholar 

  37. Liton PB, Lin Y, Gonzalez P, Epstein DL. Potential role of lysosomal dysfunction in the pathogenesis of primary open angle glaucoma. Autophagy. 2009;5(1):122–4. Epub 2009 Jan 31.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  38. Mah LJ, El-Osta A, Karagiannis TC. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia. 2010;24(4):679–86. Epub 2010 Feb 4.

    Article  PubMed  CAS  Google Scholar 

  39. von Zglinicki T, Saretzki G, Ladhoff J, d’Adda di Fagagna F, Jackson SP. Human cell senescence as a DNA damage response. Mech Ageing Dev. 2005;126(1):111–7.

    Article  Google Scholar 

  40. d’Adda di Fagagna F. Living on a break: cellular senescence as a DNA-damage response. Nat Rev Cancer. 2008;8(7):512–22.

    Article  PubMed  Google Scholar 

  41. Mu XF, Jin XL, Farnham MM, Li Y, O’Neill C. DNA damage-sensing kinases mediate the mouse 2-cell embryo’s response to genotoxic stress. Biol Reprod. 2011;85(3):524–35. Epub 2011 May 18.

    Article  PubMed  CAS  Google Scholar 

  42. Hamatani T, Carter MG, Sharov AA, Ko MS. Dynamics of global gene expression changes during mouse preimplantation development. Dev Cell. 2004;6(1):117–31.

    Article  PubMed  CAS  Google Scholar 

  43. Adiga SK, Toyoshima M, Shimura T, Takeda J, Uematsu N, Niwa O. Delayed and stage specific phosphorylation of H2AX during preimplantation development of gamma-irradiated mouse embryos. Reproduction. 2007;133(2):415–22.

    Article  PubMed  CAS  Google Scholar 

  44. Beausejour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P, et al. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J. 2003;22(16):4212–22.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  45. Sviderskaya EV, Gray-Schopfer VC, Hill SP, Smit NP, Evans-Whipp TJ, Bond J, et al. p16/cyclin-dependent kinase inhibitor 2A deficiency in human melanocyte senescence, apoptosis, and immortalization: possible implications for melanoma progression. J Natl Cancer Inst. 2003;95(10):723–32.

    Article  PubMed  CAS  Google Scholar 

  46. Herbig U, Jobling WA, Chen BP, Chen DJ, Sedivy JM. Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). Mol Cell. 2004;14(4):501–13.

    Article  PubMed  CAS  Google Scholar 

  47. Freedman DA, Folkman J. CDK2 translational down-regulation during endothelial senescence. Exp Cell Res. 2005;307(1):118–30. Epub 2005 Apr 21.

    Article  PubMed  CAS  Google Scholar 

  48. Egashira A, Kano K, Naito K. Preimplantation-embryo-specific cell-cycle regulation is attributable to a low expression of retinoblastoma protein rather than its phosphorylation. J Reprod Dev. 2011;57(4):492–9. Epub Apr 26.

    Article  PubMed  CAS  Google Scholar 

  49. Brown JP, Wei W, Sedivy JM. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science. 1997;277(5327):831–4.

    Article  PubMed  CAS  Google Scholar 

  50. Dumoulin JC, Land JA, Van Montfoort AP, Nelissen EC, Coonen E, Derhaag JG, et al. Effect of in vitro culture of human embryos on birthweight of newborns. Hum Reprod. 2010;25(3):605–12. Epub 2010 Jan 18.

    Article  PubMed  Google Scholar 

  51. Kallen B, Finnstrom O, Lindam A, Nilsson E, Nygren KG, Olausson PO. Blastocyst versus cleavage stage transfer in in vitro fertilization: differences in neonatal outcome? Fertil Steril. 2010;94(5):1680–3. Epub 2010 Feb 4.

    Article  PubMed  Google Scholar 

  52. Catt JW, Henman M. Toxic effects of oxygen on human embryo development. Hum Reprod. 2000;15 Suppl 2:199–206.

    Article  PubMed  Google Scholar 

  53. Orsi NM, Leese HJ. Protection against reactive oxygen species during mouse preimplantation embryo development: role of EDTA, oxygen tension, catalase, superoxide dismutase and pyruvate. Mol Reprod Dev. 2001;59(1):44–53.

    Article  PubMed  CAS  Google Scholar 

  54. Meintjes M, Chantilis SJ, Douglas JD, Rodriguez AJ, Guerami AR, Bookout DM, et al. A controlled randomized trial evaluating the effect of lowered incubator oxygen tension on live births in a predominantly blastocyst transfer program. Hum Reprod. 2009;24(2):300–7. Epub 2008 Oct 16.

    Article  PubMed  Google Scholar 

  55. Kovacic B, Sajko MC, Vlaisavljevic V. A prospective, randomized trial on the effect of atmospheric versus reduced oxygen concentration on the outcome of intracytoplasmic sperm injection cycles. Fertil Steril. 2010;94(2):511–9. Epub 2009 May 5.

    Article  PubMed  Google Scholar 

  56. Bontekoe S, Mantikou E, van Wely M, Seshadri S, Repping S, Mastenbroek S. Low oxygen concentrations for embryo culture in assisted reproductive technologies. Cochrane Database Syst Rev. 2012;7:CD008950.

    PubMed  Google Scholar 

  57. Knijn HM, Gjorret JO, Vos PL, Hendriksen PJ, van der Weijden BC, Maddox-Hyttel P, et al. Consequences of in vivo development and subsequent culture on apoptosis, cell number, and blastocyst formation in bovine embryos. Biol Reprod. 2003;69(4):1371–8. Epub 2003 Jun 25.

    Article  PubMed  CAS  Google Scholar 

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The authors declare that they have no conflict of interest.

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Correspondence to Dean E. Morbeck.

Additional information

Capsule In vitro culture of murine embryos induces cellular and molecular changes that are similar to changes observed in cellular senescence.

Alexandra Meuter and Lisa-Marlen Rogmann contributed equally to the manuscript.

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Meuter, A., Rogmann, LM., Winterhoff, B.J. et al. Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen. J Assist Reprod Genet 31, 1259–1267 (2014). https://doi.org/10.1007/s10815-014-0299-8

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  • DOI: https://doi.org/10.1007/s10815-014-0299-8

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