Transcriptome of the Inner Cell Mass and Trophectoderm in Bovine Blastocyst as Determined by Next Generation Sequencing.

Abstract

The first distinct differentiation in mammals occurs at the blastocyst stage. Here we determined global gene expressions patterns in the inner cell mass (ICM) and trophectoderm (TE) isolated from bovine blastocysts using next generation sequencing and evaluated how CSF2, which increases embryo competence to complete development to term, alters gene expression. Bovine embryos were produced in vitro and were treated with 0 or 10 ng/ml CSF2 from Day 6 to 8 after insemination. Blastocysts were harvested at Day 8, ICM and TE isolated, and cDNA sequenced using SOLiD 4.0 (n=3 separate pools of cDNA per treatment-cell type). A total of 870 genes were differentially expressed between ICM and TE (411 upregulated in ICM and 459 upregulated in TE). CSF2 changed expression of 25 genes in ICM and 23 in TE. Numerous genes not considered as ICM or TE markers in the mouse or human were identified. In addition, genes characteristic of ICM (NANOG, SOX2, and STAT3) and TE (ATP1B3, ELF5, and KRT18) in mouse and human showed similar patterns in bovine. Upregulated genes in ICM were consistent with maintenance of pluripotency and activation of cell proliferation. Enriched gene ontology (GO) terms included regulation of transcription, transcription regulator activity and Jak-STAT signaling pathway, pathways in cancer and chemokine signaling pathway. The last term indicates possible involvement of cytokines for communication with TE or regulation of ICM. Upregulated genes in TE were consistent with increased metabolic activity as well as interactions with the maternal environment with respect to endo- and exocytosis (GO terms included vesicle-mediated transport and lysosomes), secretion of signaling molecules (PAG2 and IFNT; steroidogenesis GO terms), and changes in cellular architecture (GO terms included actin cytoskeleton and cell projection). The percent of genes overexpressed in ICM that were classified as CpG positive was lower (P<0.015) than for genes overexpressed in TE. In conclusion, the transcriptome of the ICM and TE reflect their developmental fate and spatial orientation in the embryo, with overexpression of genes involved in pluripotency and cell proliferation in ICM and overexpression of genes required for interactions with the maternal system and for cellular rearrangement in the TE.