Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisit
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RESEARCH
Open Access
Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance Miki Sakatani1, Luciano Bonilla2,6, Kyle B Dobbs2, Jeremy Block2,3, Manabu Ozawa2,4, Savita Shanker5, JiQiang Yao5 and Peter J Hansen2*
Abstract Background: While 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. Results: Using 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 8.0. A total of three replicates of 50 morulae each that met this criterion were obtained for each treatment. RNA amplification and 3’-DGE sequencing
Total RNA was processed for cDNA synthesis using the Ovation 3’-DGE system (NuGEN Technologies, Inc., San Carlos, CA, USA). The 3’-DGE libraries were constructed from the resulting double stranded cDNA using the TruSeq™ DNA library preparation kit according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). In brief, 500 ng cDNA was sheared and pooled with 500 ng of un-sheared cDNA and end-repaired by enzymatic polishing with T4 DNA polymerase and E.coli DNA polymerase 1 Klenow fragment. A single ‘A’ base was added (A tailing) to the 3’end of the repaired fragments. Illumina pair-end adaptors, essentially consisting of the sequencing primer-annealing sequences, were then ligated to the A-tailed fragments via a 3’ thymine overhang followed by purification using Agentcourt AmpureXP beads (Beckman Coulter, Inc.). The purified ligated DNA was subjected to 11 cycles of PCR amplification to enrich the adaptor modified cDNA libraries using primers complementary to
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