A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis

Abstract

BackgroundGenes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed.ResultsBetween morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated.ConclusionThe data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1448-x) contains supplementary material, which is available to authorized users.

Single-cell gene expression of the bovine blastocyst

“…2017) and is more highly expressed in the ICM than in the TE at Day 7–9 (Ozawa et al . 2012; Brinkhof et al . 2015; Hosseini et al .…”

OCT4/POU5F1 is required for NANOG expression in bovine blastocysts

“…1E). To examine if genes specific to EPI, HB, or TE were affected by loss of OCT4, we compared the DATs in OCT4 KO embryos to sets of genes reported to be lineage specific in mouse, human, or bovine embryos (4,6,17,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Only genes with different transcript abundance in both OCT4KO tm1 vs. IVP Ctrl and OCT4KO tm1 vs. NT Ctrl, but not in NT Ctrl vs. IVP Ctrl, were considered to be affected by the loss of OCT4.…”

Initiation of X Chromosome Inactivation during Bovine Embryo Development

“…Inner cell mass (ICM) and trophectoderm (TE) cells were separated and collected from day 9 blastocysts using tungsten needles, as described previously [22]. Isolated ICMs and TE cells were immediately placed in groups (range [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] in RNase free tubes on ice containing 100 µL RLT buffer (Qiagen, Venlo, The Netherlands) and then stored at −80 • C until RNA isolation.…”

“…To isolate the COCs, sediment from the fluid was transferred to Petri dishes and COCs were identified using a stereomicroscope. The COCs were then matured by 23 h incubation at 39 • C in a humidified atmosphere of 5% CO 2 in air, as described previously [22]. Next, the matured oocytes were…”