title: P66Shc, a key regulator of metabolism and mitochondrial ROS production, is dysregulated by mouse embryo culture.
abstract: Do high oxygen tension and high glucose concentrations dysregulate p66Shc (Src homologous-collagen homologue adaptor protein) expression during mouse preimplantation embryo culture?
  
  
  
  Compared with mouse blastocysts in vivo, P66Shc mRNA and protein levels in blastocysts maintained in vitro increased under high oxygen tension (21%), but not high glucose concentration.
  
  
  
  Growth in culture adversely impacts preimplantation embryo development and alters the expression levels of the oxidative stress adaptor protein p66Shc, but it is not known if p66Shc expression is linked to metabolic changes observed in cultured embryos.
  
  
  
  We used a standard wild-type CD1 mouse model of preimplantation embryo development and embryo culture with different atmospheric oxygen tension and glucose media concentrations. Changes to p66Shc expression in mouse blastocysts were measured using quantitative RT-PCR, immunoblotting and immunofluorescence followed by confocal microscopy. Changes to oxidative phosphorylation metabolism were measured by total ATP content and superoxide production. Statistical analyses were performed on a minimum of three experimental replicates using Students' t-test or one-way ANOVA.
  
  
  
  P66Shc is basally expressed during in vivo mouse preimplantation development. Within in vivo blastocysts, p66Shc is primarily localized to the cell periphery of the trophectoderm. Blastocysts cultured under atmospheric oxygen levels have significantly increased p66Shc mRNA transcript and protein abundances compared to in vivo controls (P < 0.05). However, the ratio of phosphorylated serine 36 (S36) p66Shc to total p66Shc decreased in culture regardless of O2 atmosphere used, supporting a shift in the mitochondrial fraction of p66Shc. Total p66Shc localized to the cell periphery of the blastocyst trophectoderm and phosphorylated S36 p66Shc displayed nuclear and cytoplasmic immunoreactivity, suggesting distinct compartmentalization of phosphorylated S36 p66Shc and the remaining p66Shc fraction. Glucose concentration in the culture medium did not significantly change p66Shc mRNA or protein abundance or its localization. Blastocysts cultured under low or high oxygen conditions exhibited significantly decreased cellular ATP and increased superoxide production compared to in vivo derived embryos (P < 0.05).
  
  
  
  This study associates embryonic p66Shc expression levels with metabolic abnormalities but does not directly implicate p66Shc in metabolic changes. Additionally, we used one formulation of embryo culture medium that differs from that used in other mouse model studies and from clinical media used to support human blastocyst development. Our findings may, therefore, be limited to this media, or may be a species-specific phenomenon.
  
  
  
  This is the first study to show distinct immunolocalization of p66Shc to the trophectoderm of mouse blastocysts and that its levels are abnormally increased in embryos exposed to culture conditions. Changes in p66Shc expression and/or localization could possibly serve as a molecular marker of embryo viability for clinical applications. The outcomes provide insight into the potential metabolic role of p66Shc. Metabolic anomalies are induced even under the current optimal culture conditions, which could negatively impact trophectoderm and placental development.
  
  
  
  Not applicable.
  
  
  
  Canadian Institutes of Health Research (CIHR) operating funds, Ontario Graduate Scholarship (OGS). There are no competing interests.
authors.affiliation: Departments of Physiology and Pharmacology, The University of Western Ontario, Canada.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Edwards
authors.affiliation: Departments of Physiology and Pharmacology, The University of Western Ontario, Canada Obstetrics and Gynaecology, Schulich School of Medicine & Dentistry, The University of Western Ontario, Canada The Children's Health Research Institute (CHRI), Lawson Health Research Institute, London, Ontario, Canada N6A 5C1.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Watson
authors.affiliation: Departments of Physiology and Pharmacology, The University of Western Ontario, Canada Obstetrics and Gynaecology, Schulich School of Medicine & Dentistry, The University of Western Ontario, Canada The Children's Health Research Institute (CHRI), Lawson Health Research Institute, London, Ontario, Canada N6A 5C1 dean.betts@schulich.uwo.ca.
authors.forename: Dean H
authors.initials: DH
authors.lastname: Betts
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D004195:Disease Models, Animal; D046149:Embryo Culture Techniques; D047108:Embryonic Development / Q000235:genetics / Q000502:physiology; D005260:Female; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000494:pharmacology; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D005947:Glucose
keywords: blastocyst; embryo culture; metabolism; mitochondria; p66Shc; preimplantation embryo; reactive oxygen species, stress adaptor