Transcriptomic signature in bovine blastocysts resulting from oxidative stress exposure at the time of embryonic genome activation
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ABSTRACT:
ORGANISM(S): Bos taurus
PROVIDER: GSE42281 | GEO | 2012/11/14
SECONDARY ACCESSION(S): PRJNA179521
REPOSITORIES: GEO
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Project description:Organophosphate (OP) compounds are used as insecticides, acaricides, and chemical agents and share a common neurotoxic mechanism of action. The biochemical alterations leading to many of the deleterious effects have been studied in neuronal cell lines, however, non-neuronal toxic effects of OPs are far less well characterized in vitro, and specifically in cell lines representing oral routes of exposure. To address this void, the human salivary gland (HSG) cell line, representing likely interactions in the oral cavity, was exposed to the representative OP paraoxon (PX; O,O-diethyl-p-nitrophenoxy phosphate) over a range of concentrations (0.01-100 μM) and analyzed for cytotoxicity. PX induced cytotoxicity in HSG cells at most of the exposure concentrations as revealed by MTT assay, however, the release of LDH only occurred at the highest concentration of PX tested (100 μM) at 48 h. Slight increases in cellular ATP levels were measured in PX-exposed (10 μM) HSG cells at 24 h. Exposing HSG cells to 10 μM PX also led to an increase in DNA fragmentation prior to loss of cellular membrane integrity implicating reactive oxygen species (ROS) as a trigger of toxicity. The ROS genes gss, gstm2, gstt2 and sod2 were upregulated, and the presence of superoxide following 10 μM PX exposure was determined via dihydroethidium fluorescence studies further implicating PX-induced oxidative stress in HSG cells.
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Project description:BackgroundAberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis.ResultsThe 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes.ConclusionIn vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.
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