| 5-aza-2’-deoxycytidine (5-aza-dc) and trichostatin A (TSA) are two major inhibitors for epigenetic reprogramming. Treating donor cells or somatic cell nuclear transfer embryos with 5-aza-dc and TSA can inhance developmental capacities of SCNT embryos through alteration of epigenetic marks including DNA methylation, histone acetylation, chromatin re-organization and rehabilitation of differentiated donor cells back to a totipotent stage, and can improve the efficiency of SCNT. Previously, TSA at low concerntration was reported that it could increase efficiencies of parthenogenetic development. However, to date, the effect of these inhibitors on androgenetic embryos have not been reported. To improve in vitro development of androgentic embryos, different attributes like concentration of either of the inhibitor, time and stage of embryo were evaluated. Moreover, the probable mechanism of TSA for bovine androgenetic embryo development by detecting the acetylation levels of H3 and genes relative expression levels related for epigenetic modification was also studied:In this research, we utilized TSA and 5-aza-dc to treat bovine androgenetic embryos, and then observed the effect of in vitro development of these androgenetic embryos. Different parameters of the study were:concentration of either of the inhibitor (TSA: Onmol/L,5nmol/L,10nmol/L,15nmol/L,20nmol/L,50nmol/L; 5-aza-dc: 0nmol/L, 0.1nmol/L, 1nmol/L, 10nmol/L), treatment times (TSA:Oh, 10h,15h,20h,25h; 5-aza-dc: Oh,15h,24h,48h) and different stages of embryo (control Onmol/L, recover stage, activation stage, culture stage).To measure the level of histone acetylation in 2-5 days embryos cultured with or without TSA, another experiment was designed. Four groups, two control groups (androgenetic embryo with Onmol/L TSA and parthenogenetic embryo with Onmol/L TSA) and two treatment groups (5nmol/L,20nmol/L) constituted this experiment. To measure the relative expression of genes related for epigenetic modification, we designed another experiment. Control group(Onmol/L) and two treatment groups(5nmol/L,20nmol/L) constituted this experiment with treating 15 hours during culture stage. 1. Effects of TSA treatment on the development of bovine preimplantation androgenetic embryosThe rates of cleavage,16-cell embryos and morula were higher in the groups treated for 5nmol/L,10nmol/L,15nmol/L,20nmol/L compared with the groups treated for Onmol/L,50nmol/L. But there were significantly higher (P<0.05) in the groups treated for 5nmol/L,10nmol/L compared with the groups treated for Onmol/L,50nmol/L. The rates of blastocyst formation were significantly higher (P<0.05) in the groups treated for 5nmol/L,10nmol/L compared with control group and there was no difference between other groups.For comparison of the exposure times of TSA treatment, androgenetic embryos were cultured in SOF supplemented with 10nmol/L of TSA for 0,10,15,20,25h from embryo culture stage. Treatment with TSA for 15h,20h increased the percentage cleavage (P<0.05) compared to the control groups and there was no difference with other two groups treated with TSA for 10h,25h. The rates of 16-cell embryos were higher (P<0.05) in the groups treated 10h,15h,20h,25h compared to control group. The percentage of blastocyst increased in the groups treated 15h,20h,25h than control group and the group treated 15h was significant different (9.4% vs.1.5% P<0.05) than control group.In relation to stage of embryo, there was no difference between different groups with the rates of cleavage,16-cell embryo and morula. The rates of blastocyst were higher in group treated TSA in culture stage compared to other groups (9.7% vs.0.0%P<0.05)2. Effects of 5-aza-dc treatment on the development of bovine preimplantation androgenetic embryosFor 5-aza-dc treatment, the rates of different embryos in all treatment groups were higher than control group, but there was no difference (P>0.05). The percentage of cleavage in 1nmol/L treatment group was significant higher compared to control group, and the rate of blastocyst reached to 7.6%, and was higher than other groups. These results indicated that treatment with lnmol/L 5-aza-dc was the best than other different concentration for bovine androgenetic embryo development.The rates of cleavage,16-cell embryo and morula was not significant in the groups treated 15h,24h,48h. There was significant difference between group treated with 24h and control group, but there was no difference in the groups treated 15h,48h and control group. The percentage of blastocyst in the group treated 24h was 9.0% and were higher than other groups. When androgenetic embryo treated with 48h by 5-aza-dc, the rates of blastocyst was 3.3% and there was no difference with other groups.3. Effects of TSA treatment on the histone acetylation and relative expression of genes related with epigenetic modificationIn another experiment the effect of TSA on histone H3 acetylation was evaluated. There was no difference between the groups treated 5nmol/L,20nmol/L and Onmol/L during different embryo stages. But the average intensity of AcH3 level of androgenetic embryos was lower than parthenogenetic embryos with the same stage. The histone acetylation of androgenetic embryos with the same treatment had difference between different stages.The relative expression of HDAC1 gene was significant difference between three groups and the expression of TSA treatment group was significant lower than control. Like HDAC1 gene, the expression of HDAC2 gene significantly decreased in treatment groups than control, but there was no difference between the groups treated with 5nmol/L and 20nmol/L TSA. Genes related with DNA methylation, such as DNMT3a, DNMT3b, DNMT1 had a high expression in control group and the expression gradually decreased with a high TSA treatment. Not like other DNA methylation genes, the expression of DNMT3b gene was not significant different in treated group with 5nmol/L TSA compared to control. The expression of HAT1 gene was higher in control group than TSA treatment groups. |
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