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Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

ABSTRACT: Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.

SUBMITTER: Galan A

PROVIDER: S-EPMC2964308 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

Galán Amparo A Montaner David D Póo M Eugenia ME Valbuena Diana D Ruiz Verónica V Aguilar Cristóbal C Dopazo Joaquín J Simón Carlos C

PloS one 20101026 10

Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyze ...[more]

PMID: 21049019

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Project description:Blastomere cytofragmentation in mammalian embryos poses a significant problem in applied and clinical embryology. Mouse two-cell-stage embryos display strain-dependent differences in the rate of cytofragmentation, with a high rate observed in C3H/HeJ embryos and a lower rate observed in C57BL/6 embryos. The maternally inherited genome exerts the strongest effect on the process, with lesser effects mediated by the paternally inherited genome and the ooplasm. The effect of the maternal genome is transcription dependent and independent of the mitochondrial strain of origin. To identify molecular mechanisms that underlie cytofragmentation, we evaluated transcriptional activities of embryos possessing maternal pronuclei (mPN) of different origins. The mPN from C57BL/6 and C3H/HeJ strains directed specific transcription at the two-cell stage of mRNAs corresponding to 935 and 864 Affymetrix probe set IDs, respectively. Comparing transcriptomes of two-cell-stage embryos with different mPN revealed 64 transcribed genes with differential expression (1.4-fold or greater). Some of these genes occupy molecular pathways that may regulate cytofragmentation via a combination of effects related to apoptosis and effects on the cytoskeleton. These results implicate specific molecular mechanisms that may regulate cytofragmentation in early mammalian embryos. The most striking effect of mPN strain of origin on gene expression was on adenylate cyclase 2 (Adcy2). Treatment with dibutyryl cAMP (dbcAMP) elicits a high rate and severe form of cytofragmentation, and the effective dbcAMP concentration varies with maternal genotype. An activator of exchange proteins directly activated by cAMP (EPACs, or RAPGEF 3 and 4) 8-pCPT-2'-O-methyl-cAMP, elicits a high level of fragmentation while the PKA-specific activator N6-benzoyl-cAMP does not. Inhibition of A kinase anchor protein activities with st-Ht31 induces fragmentation. Inhibition of phosphatidylinositol 3-kinase signaling also induces fragmentation. These results reveal novel mechanisms by which maternal genotype affects cytofragmentation, including a system of opposing signaling pathways that most likely operate by controlling cytoskeletal function.

Dataset Information

Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

Publications

Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

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