Use of trichostatin A alters the expression of HDAC3 and KAT2 and improves in vitro development of bovine embryos cloned using less methylated mesenchymal stem cells
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The aim of this work was to investigate the methylation and hydroxymethylation status of mesenchymal stem cells (MSC) from amniotic fluid (MSC-AF), adipose tissue (MSC-AT) and fibroblasts (FIB-control) and to verify the effect of trichostatin A (TSA) on gene expression and development of cloned bovine embryos produced using these cells. Characterization of MSC from two animals (BOV1 and BOV2) was performed by flow cytometry, immunophenotyping and analysis of cellular differentiation genes expression. The cells were used in the nuclear transfer in the absence or presence of 50 nM TSA for 20 hr in embryo culture. Expression of HDAC1, HDAC3 and KAT2A genes was measured in embryos by qRT-PCR. Methylation results showed difference between animals, with MSC from BOV2 demonstrating lower methylation rate than BOV1. Meanwhile, MSC-AF were less hydroxymethylated for both animals. MSC-AF from BOV2 produced 44.92 ± 8.88% of blastocysts when embryos were exposed to TSA and similar to embryo rate of MSC-AT also treated with TSA (37.96 ± 15.80%). However, when methylation was lower in FIB compared to MSC, as found in BOV1, the use of TSA was not sufficient to increase embryo production. MSC-AF embryos expressed less HDAC3 when treated with TSA, and expression of KAT2A was higher in embryos produced with all MSC and treated with TSA than embryos produced with FIB. The use of MSC less methylated and more hydroxymethylated in combination with embryo incubation with TSA can induce lower expression of HDAC3 and higher expression of KAT2A in the embryos and consequently improve bovine embryo production.