m‐carboxycinnamic acid bishydroxamide improves developmental competence, reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos

Abstract

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m-carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand-made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency-related genes OCT-4 and NANOG, and anti-apoptotic gene BCL-XL, and decreased (p < .05) that of pro-apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis-related genes p53 and CASPASE3 and epigenetics-related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.

RNA sequencing and transcriptome analysis of buffalo (Bubalus bubalis) blastocysts produced by somatic cell nuclear transfer and in vitro fertilization

“…We found that a majority of the epigenetic regulators, such as DNA methyltransferases (DNMTs), histone deacetylases (HDACs), and histone methyltransferases were upregulated in NT compared with IVF blastocysts which agrees with a previous report in bovine (Min et al, 2015). We have attempted correcting these critical nuclear reprogramming-related errors by treating NT embryos with epigenetic modifiers such as scriptaid (Panda et al, 2012), trichostatin A Selokar et al, 2016), valproic acid , and m-carboxycinnamic acid bishydroxymide (Agrawal et al, 2018a(Agrawal et al, , 2018b, which are HDAC inhibitors, to reduce histone acetylation, and 5-aza-2ʹ-deoxycytidine, a DNMT inhibitor, to reduce DNA methylation .…”

“…We found that a majority of the epigenetic regulators, such as DNA methyltransferases (DNMTs), histone deacetylases (HDACs), and histone methyltransferases were upregulated in NT compared with IVF blastocysts which agrees with a previous report in bovine (Min et al, ). We have attempted correcting these critical nuclear reprogramming‐related errors by treating NT embryos with epigenetic modifiers such as scriptaid (Panda et al, ), trichostatin A (Saini et al, , ; Selokar et al, ), valproic acid (Selokar et al, ), and m‐carboxycinnamic acid bishydroxymide (Agrawal et al, , ), which are HDAC inhibitors, to reduce histone acetylation, and 5‐aza‐2′‐deoxycytidine, a DNMT inhibitor, to reduce DNA methylation (Saini et al, , ). Although treatments with these epigenetic modifiers resulted in significant improvements in blastocyst rate and embryo quality, as indicated by higher total cell number and lower level of apoptosis, whether these beneficial effects will be translated to improvements in live birth rate is not known.…”

Approaches used to improve epigenetic reprogramming in buffalo cloned embryos

“…Different approaches have been used to correct the epigenetic reprogramming in cloned embryos such as treatment of somatic cells, oocytes and fused embryos with epigenetic modifiers or with oocytes or stem cells extract, overexpression or suppression of important regulatory genes in somatic cells or one cell stage cloned embryos ( Figure ). Buffalo somatic cells and/or fused embryos were treated with different epigenetic modulating agents, such as trichostatin A (TSA), 5-aza-2’-deoxycytidine (5-aza-dC), valproic acid (VPA) and m-carboxycinnamic acid bishydroxamide (CBHA) 14 16 17 18 . These studies showed that treatment of buffalo donor cells and/or one cell stage fused embryos or both with epigenetic modulators, alone or in combination, resulted in higher blastocyst production rates and lower level of apoptosis in generated cloned blastocysts 14 15 .…”

“…This study demonstrated that treatment of donor cells with VPA did not improve the blastocyst production rate. Agrawal et al 17 reported that 10 μM of CBHA could be used to improve the blastocyst rates and quality of cloned embryos.…”

Asian water buffalo: domestication, history and genetics

“…Until now genetic studies have mainly addressed genetic diversity (Barker et al ; Lei et al ; Kumar et al ; Zhang et al ; Colli et al ) and identification of genes and molecular markers that are associated with desirable traits (de Camargo et al ; Yusnizar et al ; Iannaccone et al ; Liu et al ), but to date only a few epigenetic studies have been carried out on water buffalo (Verma et al ; Agrawal et al ; Sharma et al ).…”