Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance

Abstract

BackgroundWhile initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3’ tag digital gene expression (3’DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance.ResultsUsing 3’DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT.ConclusionsChanges in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage.

The global effect of heat on gene expression in cultured bovine mammary epithelial cells

“…In all HSPs, the most pronounced stress-related changes and involvement in different cell functions have been demonstrated for HSP70 (Grover 2002;Jego et al 2010;Sirotkin and Bauer 2011). The bovine two-cell embryo is capable of increasing transcription of HSPA1A and synthesis of HSP70 in response to heat shock (Sakatani et al 2013). The mRNA and protein expression of HSP70 increased after 42°C heat treatment in cultured bovine mammary epithelial cells (Han et al 2014).…”

Developmental competence and expression profile of genes in buffalo (Bubalus bubalis) oocytes and embryos collected under different environmental stress

“…Transcription levels of GAPDH vary among cell types (Barber et al 2005), developmental stages (Robert et al 2002) and experimental conditions (Ito et al 1996;Zhong and Simons 1999).These findings underline the priority of reference gene validation for each specific developmental stage and for each season. This report also raises concern as GAPDH has been generally used as an internal control gene in studies involving the effect of seasons on oocyte and embryo gene expression in cattle and buffaloes (Sakatani et al 2013;Yadav et al 2013). …”

The embryonic stress response to in vitro culture: insight from genomic analysis

“…These regulators consist of not only chaperone proteins (Edwards & Hansen 1996) as well as apoptosis-related proteins such as Bax, Bid and Caspase-3 (Yadav et al 2013) but also proteins associated with developmental competence genes, such as PLAC8 and CDX2 (Silva et al 2013). A transcriptional dysregulation subject to wnt signalling in conjunction with heparin sulphate proteoglycans also may result from HS exposure (Sakatani et al 2013).…”

“…The early stages of bovine pre-EGA embryo development are particularly sensitive to heat shock; this is associated with major changes in the transcriptomic profile at the morula stage (Sakatani et al 2013). Responses to heat shock include the production of homeostatic regulators involved in the unfolding response via the ubiquitination pathway and the glutathione scavenging response to oxidative stress (Arechiga et al 1995).…”