Dissecting the First Transcriptional Divergence During Human Embryonic Development

The trophoblast cell lineage is specified early at the blastocyst stage, leading to the emergence of the trophectoderm and the pluripotent cells of the inner cell mass. Using a double mRNA amplification technique and a comparison with transcriptome data on pluripotent stem cells, placenta, germinal and adult tissues, we report here some essential molecular features of the human mural trophectoderm. In addition to genes known for their role in placenta (CGA, PGF, ALPPL2 and ABCG2), human trophectoderm also strongly expressed Laminins, such as LAMA1, and the GAGE Cancer/Testis genes. The very high level of ABCG2 expression in trophectoderm, 7.9-fold higher than in placenta, suggests a major role of this gene in shielding the very early embryo from xenobiotics. Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression. A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta. The induction of this network could be recapitulated in an in vitro trophoblast differentiation model

Transcriptome Analysis during Human Trophectoderm Specification Suggests New Roles of Metabolic and Epigenetic Genes

In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts

Slow freezing and vitrification differentially modify the gene expression profile of human metaphase II oocytes.

International audienceBACKGROUND: Cryopreservation is now considered as an efficient way to store human oocytes to preserve fertility. However, little is known about the effects of this technology on oocyte gene expression. The aim of this study was to examine the effect of the two cryopreservation procedures, slow freezing and vitrification, on the gene expression profile of human metaphase II (MII) oocytes. METHODS: Unfertilized MII oocytes following ICSI failure were cryopreserved either by slow freezing or by the Cryotip method for vitrification. After thawing, total RNA was extracted and analyzed using Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays. The gene expression profiles and associated biological pathways in slowly frozen/thawed and vitrified MII oocytes were determined and compared with those of non-cryopreserved MII oocytes used as controls. RESULTS: Both cryopreservation procedures negatively affected the gene expression profile of human MII oocytes in comparison with controls. However, slowly frozen and vitrified MI oocytes displayed specific gene expression signatures. Slow freezing was associated with down-regulation of genes involved in chromosomal structure maintenance (KIF2C and KIF3A) and cell cycle regulation (CHEK2 and CDKN1B) that may lead to a reduction in the oocyte developmental competence. In vitrified oocytes, many genes of the ubiquitination pathway were down-regulated, including members of the ubiquitin-specific peptidase family and subunits of the 26S proteasome. Such inhibition of the degradation machinery might stabilize the maternal protein content that is necessary for oocyte developmental competence. CONCLUSIONS: The low pregnancy rates commonly observed when using human MII oocytes after slow freezing-thawing may be explained by the alterations of the oocyte gene expression profile

List of the 100 genes with the highest fold change in day 3 human embryos in comparison to TE samples.

<p>List of the 100 genes with the highest fold change in day 3 human embryos in comparison to TE samples.</p

Gene Ontology (GO) annotations of the day 3 embryo and TE molecular signatures.

<p>We compared the GO annotations of genes specifically over-expressed in day 3 embryos and in TE cells by using the Babelomics web tool (<a href="http://babelomics.bioinfo.cipf.es/" target="_blank">http://babelomics.bioinfo.cipf.es/</a>). Histograms show the percentage of genes with a specific GO annotation in day 3 embryos (white) or in TE samples (black). Only GO categories which differed significantly (<i>p</i> value <0.01) between the two groups are shown.</p

List of the 100 genes with the highest fold change in TE samples in comparison to day 3 embryos.

<p>List of the 100 genes with the highest fold change in TE samples in comparison to day 3 embryos.</p

Up-regulated genes that are related to the <i>NANOG</i> pathway, or to metabolic and epigenetic functions in day 3 human embryos and TE samples.

<p>(A) The interaction network was generated with the Ingenuity software and shows that many genes from the <i>NANOG</i> pathway are up-regulated (red) in day 3 embryos. (B) Top-ranked functional networks in which are involved transcription factors (<i>GATA2</i> and <i>GATA3</i>), or genes that regulate steroidogenesis (including <i>HSD1B3</i>), DNA repair (<i>TDG</i> and <i>BRCA1</i>) or epigenetic modifications (including <i>DNMT3L)</i> and that are up-regulated in TE samples. The color intensity indicates their degree of up-regulation. Uncolored genes were identified as not differentially expressed by our analysis, but were, nevertheless, integrated into the computationally generated networks on the basis of the evidence stored in the IPA knowledge memory indicating a relevance to this network. In each network, nodes indicate genes, a plain line indicates direct interaction, a dashed line indicates indirect interaction; a line without arrowhead indicates binding only; a line with an arrowhead indicates “acts on”.</p

Day 3 embryo and TE molecular signatures:

<p>Heat map of the molecular signatures in six day 3 embryos and five TE samples. Each horizontal line represents a gene and each column represents a single sample. The color intensity indicates the level of gene expression (red for up-regulation and blue for down-regulation) “see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039306#pone.0039306.s006" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039306#pone.0039306.s007" target="_blank">S2</a>”.</p

Differential expression of apoptosis and stemness-related genes in day 3 embryos and TE samples.

<p>(A) Histograms show the microarray signal values for apoptosis-related genes in day 3 embryos (black) and TE samples (white). (B) The mean expression level of 48 stemness genes in six day 3 embryos and five TE samples was plotted on a logarithmic scale in a radar graph. Asterisks indicate a statistically significant difference (<i>P</i><0.05) between TE and day 3 embryos (Mann-Whitney test).</p