Development of early embryonic stages before activation of the embryonic genome depends on sufficiently stored products of the maternal genome, adequate recruitment and degradation of mRNAs, as well as activation, deactivation, and relocation of proteins. By application of an isobaric tagging for relative and absolute quantification (iTRAQ)-based approach, the proteomes of bovine embryos at the zygote and 2-cell and 4-cell stage with MII oocytes as a reference were quantitatively analyzed. Of 1072 quantified proteins, 87 differed significantly in abundance between the four stages. The proteomes of 2-cell and 4-cell embryos differed most from the reference MII oocyte, and a considerable fraction of proteins continuously increased in abundance during the stages analyzed, despite a strongly attenuated rate of translation reported for this period. Bioinformatic analysis revealed particularly interesting proteins involved in the p53 pathway, lipid metabolism, and mitosis. Verification of iTRAQ results by targeted SRM (selected reaction monitoring) analysis revealed excellent agreement for all five proteins analyzed. By principal component analysis, SRM quantifications comprising a panel of only five proteins were shown to discriminate between all four developmental stages analyzed here. For future experiments, an expanded SRM protein panel will provide the potential to detect developmental disturbances with high sensitivity and enable first insights into the underlying molecular pathways.
STUDY QUESTIONIs 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)?SUMMARY ANSWERPostovulatory 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.WHAT IS KNOWN ALREADYOocyte 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.STUDY DESIGN, SIZE, DURATIONGerminal 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).MATERIAL AND METHODSGV 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).MAIN RESULTS AND ROLE OF CHANCEThe significant reduction in abundance of pr...
During mammalian embryo development, the zygote undergoes embryonic cleavage in the oviduct and reaches the uterus at the morula stage, when compaction and early lineage specification take place. To increase knowledge about the associated changes of the embryonic protein repertoire, we performed a comprehensive proteomic analysis of in vitro produced bovine morulae and blastocysts (six biological replicates), using an iTRAQ-based approach. A total of 560 proteins were identified of which 502 were quantified. The abundance of 140 proteins was significantly different between morulae and blastocysts, among them nucleophosmin (NPM1), eukaryotic translation initiation factor 5A-1 (EIF5A), receptor of activated protein kinase C 1 (GNB2L1/RACK1), and annexin A6 (ANXA6) with increased, and glutathione S-transferase mu 3 (GSTM3), peroxiredoxin 2 (PRDX2), and aldo-keto reductase family 1 member B1 (AKR1B1) with decreased abundance in blastocysts. Seventy-three percent of abundance altered proteins increased, reflecting an increase of translation activity in this period. This is further supported by an increase in the abundance of proteins involved in the translation machinery and the synthesis of ATP. Additionally, a complementary 2D saturation DIGE analysis led to the detection of protein isoforms, e.g. of GSTM3 and PRDX2, relevant for this period of mammalian development, and exemplarily verified the results of the iTRAQ approach. In summary, our systematic differential proteome analysis of bovine morulae and blastocysts revealed new molecular correlates of early lineage specification and differentiation events during bovine embryogenesis.
Embryo-maternal communication, as well as recognition and establishment of pregnancy, is predominantly controlled by secretion of hormones and proteins. Furthermore, uterine fluid serves as a histotroph and contains factors essential for embryo development and elongation, making it a meaningful source for proteomic approaches of early embryonic development. The studies included in this review address the analysis of uterine fluid and endometrium during the oestrous cycle, early pregnancy, maternal effects on embryo development and embryo-induced alterations to the maternal environment. Clear differences between the proteome of uterine fluid and blood plasma unravelled the active contribution of the endometrium to the composition of uterine luminal fluid. Proteins were identified which may contribute either to growth enhancement of blastocysts or influence secretion of "Interferon tau". Several studies suggested important roles of intrauterine factors including "Tissue inhibitor of metalloproteinases 2" for pregnancy establishment and "Acyl-CoA-binding protein" for the embryo prior to implantation. A meta-analysis of quantitative proteome studies revealed functional clusters including the terms "degradation of extracellular matrix", "platelet degranulation" and "MHC class II antigen presentation". Proteomic studies targeting oocytes and early embryos provided evidence for the function of "Cyclin B" in mitotic resumption, for "14-3-3 epsilon" to activate "maturation promotion factor" for promotion of mitosis during in vitro maturation, or for the inhibition of proteasomes during the late phase of maturation. Analysis of blastocyst derived trophectoderm cell lines suggested effects of annexin down-regulation on the failure rate of nuclear transfer pregnancies. Finally, absolute quantification of "Y-box-binding protein 2" and "Insulin-like growth factor 2 mRNA-binding protein 3" during early embryogenesis by "selected reaction monitoring" demonstrated the dynamism of proteome alterations during the first embryonic cell divisions.
Development of early embryonic stages before activation of the embryonic genome depends on sufficiently stored products of the maternal genome and adequate activation, deactivation, and relocation of proteins. To establish protein function, several posttranslational events (e.g. proteolytic activation, phosphorylation, or secretion) are frequently essential and thereby prevent prediction of protein abundance from transcript abundance. Consequently, proteomic studies are indispensable to characterise the molecular processes governing early embryonic development and to establish corresponding regulatory networks. Here, we present a quantitative proteome analysis of bovine zygotes and embryos at the 2-cell and 4-cell stage. Cumulus-oocyte complexes (COC) were prepared from bovine ovaries obtained from a local abattoir and selected for a compact layer of cumulus cells. In vitro maturation, fertilization, and embryo production were performed according to standard procedures. For quantitative isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, protein from batches of 50 MII oocytes (serving as a reference), zygotes, 2-cell and 4-cell stage embryos, respectively, was extracted. Quantitative proteome analysis of iTRAQ-labelled tryptic peptides was performed on an Orbitrap XL instrument (Thermo Fisher, Waltham, MA, USA) coupled to an Eksigent nano-liquid chromatography system (AB Sciex, Framingham, MA, USA). The tandem MS data were analysed by MASCOT and filtered for a false discovery rate (FDR) of <1%. Quantification of iTRAQ signals was accomplished with the Q+ module of the Scaffold software (Proteome Software Inc., Portland, OR, USA). t-Tests, ANOVA and principal component analysis (PCA) analysis were performed using R (R Core Development Team, Vienna, Austria). From 4 biological replicates, 1072 proteins were identified and quantified. Eighty-seven differed significantly in abundance between the 4 stages (log2 fold change ≥ |0.6|, P ≤ 0.05). The proteomes of 2-cell and 4-cell embryos differed most from the reference MII oocyte, and a considerable fraction of proteins continuously increases in abundance during the stages analysed. Bioinformatic analysis of abundance altered proteins provided evidence that the proteins RPS14 and HNRNPK involved in the p53 pathway play a major role during early development, as well as proteins of the lipid metabolism, in particular APOA1. Furthermore, a group of proteins (e.g. SPTBN1, PPP1CC, RABGAP1, STMN1, and WEE2) is engaged in mitosis. In addition, we detected relevant differences between transcript and protein abundance levels; for example, for WEE2. In conclusion, this study identified and quantified numerous proteins important for early embryogenesis so far not described in the mammalian system, and contributed protein profiles for key players previously described. Our results highlight the importance of innovative proteomic tools and workflows to complement transcriptome data of early embryogenesis.
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