title: Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes.
abstract: OBJECTIVE
  Is the postovulatory aging-dependent differential decrease of mRNAs and polyadenylation of mRNAs coded by maternal effect genes associated with altered abundance and distribution of maternal effect and RNA-binding proteins (MSY2)?
  
  
  CONCLUSIONS
  Postovulatory aging results in differential reduction in abundance of maternal effect proteins, loss of RNA-binding proteins from specific cytoplasmic domains and critical alterations of pericentromeric proteins without globally affecting protein abundance.
  
  
  BACKGROUND
  Oocyte postovulatory aging is associated with differential alteration in polyadenylation and reduction in abundance of mRNAs coded by selected maternal effect genes. RNA-binding and -processing proteins are involved in storage, polyadenylation and degradation of mRNAs thus regulating stage-specific recruitment of maternal mRNAs, while chromosomal proteins that are stage-specifically expressed at pericentromeres, contribute to control of chromosome segregation and regulation of gene expression in the zygote.
  
  
  METHODS
  Germinal vesicle (GV) and metaphase II (MII) oocytes from sexually mature C57B1/6J female mice were investigated. Denuded in vivo or in vitro matured MII oocytes were postovulatory aged and analyzed by semiquantitative confocal microscopy for abundance and localization of polyadenylated RNAs, proteins of maternal effect genes (transcription activator BRG1 also known as ATP-dependent helicase SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) and NOD-like receptor family pyrin domain containing 5 (NLRP5) also known as MATER), RNA-binding proteins (MSY2 also known as germ cell-specific Y-box-binding protein, YBX2), and post-transcriptionally modified histones (trimethylated histone H3K9 and acetylated histone H4K12), as well as pericentromeric ATRX (alpha thalassemia/mental retardation syndrome X-linked, also termed ATP-dependent helicase ATRX or X-linked nuclear protein (XNP)). For proteome analysis five replicates of 30 mouse oocytes were analyzed by selected reaction monitoring (SRM).
  
  
  METHODS
  GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)20 nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE).
  
  
  RESULTS
  The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and abundance of acetylated histone H4K12, and decreased pericentromeric trimethylated histone H3K9 (all P < 0.001). Spreading revealed a highly significant increase in pericentromeric ATRX (P < 0.001) upon ageing. Thus, the significantly reduced abundance of MSY2 protein, especially at the SCRD and the spindle may disturb the spatial control and timely recruitment, deadenylation and degradation of developmentally important RNAs. An autonomous program of degradation appears to exist which transiently and specifically induces the loss and displacement of transcripts and specific maternal proteins independent of fertilization in aging oocytes and thereby can critically affect chromosome segregation and gene expression in the embryo after fertilization.
  
  
  CONCLUSIONS
  We used the mouse oocyte to study processes associated with postovulatory aging, which may not entirely reflect processes in aging human oocytes. However, increases in spindle abnormalities, unaligned chromosomes and H4K12 acetylated histones, as well as in mRNA abundance and polyadenylation have been observed also in aged human oocytes suggesting conserved processes in aging.
  
  
  CONCLUSIONS
  Postovulatory aging precociously induces alterations in expression and epigenetic modifications of chromatin by ATRX and in histone pattern in MII oocytes that normally occur after fertilization, possibly contributing to disturbances in the oocyte-to-embryo transition (OET) and the zygotic gene activation (ZGA). These observations in mouse oocytes are also relevant to explain disturbances and reduced developmental potential of aged human oocytes and caution to prevent oocyte aging in vivo and in vitro.
  
  
  BACKGROUND
  The study has been supported by the German Research Foundation (DFG) (EI 199/7-1 | GR 1138/12-1 | HO 949/21-1 and FOR 1041). There is no competing interest.
authors.affiliation: Institute of Gene Technology/Microbiology, University of Bielefeld, Bielefeld, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Trapphoff
authors.affiliation: Institute of Gene Technology/Microbiology, University of Bielefeld, Bielefeld, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Heiligentag
authors.affiliation: Institute of Anatomy, University Hospital, University Duisburg-Essen, Essen, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Dankert
authors.affiliation: Institute of Human Genetics, University Hospital, University Duisburg-Essen, Essen, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Demond
authors.affiliation: Laboratory for Functional Genome Analysis, Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Deutsch
authors.affiliation: Laboratory for Functional Genome Analysis, Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Fröhlich
authors.affiliation: Laboratory for Functional Genome Analysis, Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany.
authors.forename: G J
authors.initials: GJ
authors.lastname: Arnold
authors.affiliation: Institute of Anatomy, University Hospital, University Duisburg-Essen, Essen, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Grümmer
authors.affiliation: Institute of Human Genetics, University Hospital, University Duisburg-Essen, Essen, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Horsthemke
authors.affiliation: Institute of Gene Technology/Microbiology, University of Bielefeld, Bielefeld, Germany eiri@uni-bielefeld.de.
authors.forename: U
authors.initials: U
authors.lastname: Eichenlaub-Ritter
mesh_terms: D000818:Animals; D000941:Antigens / Q000378:metabolism*; D016922:Cellular Senescence / Q000502:physiology*; D002503:Centromere / Q000378:metabolism*; D004527:Egg Proteins / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D051379:Mice; D009865:Oocytes / Q000378:metabolism*; D010060:Ovulation / Q000378:metabolism*; D020543:Proteome; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*
chemical_list: D000941:Antigens; D004527:Egg Proteins; C416503:Nalp5 protein, mouse; D020543:Proteome; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C116047:Ybx2 protein, mouse
keywords: RNA dynamics; aging; centromere; gene expression; histone pattern; maternal effect genes; oocyte; spindle chromosome complex