Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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The requirement of leukemia inhibitory factor or epidermal growth factor for pre-implantation embryogenesis

ABSTRACT: In this study, we further verified the role of LIF by looking at gene expressions in morula stage embryos through DNA microarray. Our results showed that LIF knockdown affected 373 gene expressions, and after LIF supplementation we found that the epidermal growth factor (EGF) is most affected by LIF expression. In order to observe the correlation between LIF and EGF, the LIF knockdown embryos were supplemented with various growth factors including LIF, EGF, GM-CSF, TGF, and IGF II. Only LIF and EGF caused the rate of blastocyst development to recover from 52% of control significantly to 83% and 93%, respectively, and all of the parameters, such as the diameter of blastocysts, the numbers of blastomeres, and cells in ICM and TE, to mostly be restored. Moreover, EGF knockdown also impaired blastocyst development which was reversed by LIF or EGF supplementation. These data suggest that the two growth factors EGF and LIF can be compensatory to each other during early embryonic development, and one of them is necessary for sustaining the normal development of preimplantation embryos. Two-condition experiment, mouse embryos vs. LIF-deficiency mouse embryos at morula stage

ORGANISM(S): Mus musculus

SUBMITTER: Jer-Yuh Liu

PROVIDER: E-GEOD-71736 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Despite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro is still lower than their in vivo counterparts. To bring knowledge to answer this question, we used a mass spectrometry (MS) approach to compare the protein content of bovine embryos that were conceived in vivo or produced in vitro. A total of 38 pools of grade-1 quality bovine embryos at the 4-6 cell, 8-12 cell, morula, compact morula and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) according to embryo origin were evidenced, including 463 proteins more abundant in vivo than in vitro across development, and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity while those overabundant after morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other proteins previously shown to interact with early embryos in vitro were among the most overabundant proteins after in vivo development. In conclusion, the maternal environment led to faster degradation of mitochondrial proteins at early embryo developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation and a global upregulation of carbohydrate and small molecule metabolic pathways compared to in vitro produced embryos. Furthermore, our data confirm that embryos developed in vivo uptake large amounts of oviduct fluid-derived proteins as soon as the 4-6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help designing better in vitro culture systems.

Project description:The transition between morula and blastocyst stage during preimplantation development represents the first differentiation event of embryogenesis. Morula cells undergo the first cellular specialization and produce two well-defined populations of cells, the trophoblast and the inner cell mass (ICM). Embryonic stem cells (ESCs) with unlimited self-renewal capacity are believed to represent the in vitro counterpart of the ICM. Both mouse and rat ESCs can be derived from the ICM cells, but their in vitro stability differs. In this study we performed a microarray analysis in which we compared the transcriptome of mouse and rat morula, blastocyst, and ICM. This cross-species comparison represents a good model for understanding the differences in derivation and cultivation of ESCs observed in the two species. In order to identify alternative regulation of important molecular mechanisms the investigation of differential gene expression between the two species was extended at the level of signaling pathways, gene families, and single selected genes of interest. Some of the genes differentially expressed between the two species are already known to be important factors in the maintenance of pluripotency in ESCs, like for example Sox2 or Stat3, or play a role in reprogramming somatic cells to pluripotency like c-Myc, Klf4 and p53 and therefore represent interesting candidates to further analyze in vitro in the rat ESCs. This is the first study investigating the gene expression changes during the transition from morula to blastocyst in the rat preimplantation development. Our data show that in the pluripotent pool of cells of the rat and mouse preimplantation embryo substantial differential regulation of genes is present, which might explain the difficulties observed for the derivation and culture of rat ESCs using mouse conditions Casanova EA, Okoniewski MJ, Cinelli P. Cross-Species Genome Wide Expression Analysis during Pluripotent Cell Determination in Mouse and Rat Preimplantation Embryos.PLoS One. 2012;7(10):e47107. doi: 10.1371/journal.pone.0047107. Epub 2012 Oct 15. We collected morula and blastocysts stage embryos from mouse and rat and, by immunosurgery, we isolated the ICM cells from the blastocysts. All the embryos and ICMs were pooled into two groups for every developmental stage. Pooling of embryos for RNA extraction in this study was chosen mainly because of the low amounts of RNA that can be isolated from preimplantation embryos, and in addition because of the heterogeneity of the cell populations present in the embryos. For the analysis we pooled a large number (n >100) of the independent isolated embryos to achieve a sufficient accuracy of biological pooling . Due to the difficulties to isolate a larger number of embryos from mice and rats, we performed the microarray study by using two replicate samples per developmental stage.

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The requirement of leukemia inhibitory factor or epidermal growth factor for pre-implantation embryogenesis

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