title: Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Reproductive Endocrinology and Infertility, Mayo Clinic, 200St SW, Rochester, MN, 55905, USA.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Meuter
authors.forename: Lisa-Marlen
authors.initials: LM
authors.lastname: Rogmann
authors.forename: Boris J
authors.initials: BJ
authors.lastname: Winterhoff
authors.forename: Tamar
authors.initials: T
authors.lastname: Tchkonia
authors.forename: James L
authors.initials: JL
authors.lastname: Kirkland
authors.forename: Dean E
authors.initials: DE
authors.lastname: Morbeck
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism*; D001755:Blastocyst / Q000378:metabolism*; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology; D006657:Histones / Q000378:metabolism; D066298:In Vitro Techniques / Q000379:methods; D015850:Interleukin-6 / Q000276:immunology; D051379:Mice; D010100:Oxygen / Q000378:metabolism*; D012333:RNA, Messenger; D017382:Reactive Oxygen Species / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism; D054462:p21-Activated Kinases / Q000378:metabolism
chemical_list: D000975:Antioxidants; D015415:Biomarkers; D019941:Cyclin-Dependent Kinase Inhibitor p16; D006657:Histones; D015850:Interleukin-6; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C475961:gamma-H2AX protein, mouse; D054462:p21-Activated Kinases; D001616:beta-Galactosidase; D010100:Oxygen
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