Effect Of Histone Deacetylase Inhibitors On In Vitro Developmental Competence Of PIG Somatic Cell Nuclear Transfer Embryos

Objective:Since the first successful cloned sheep by somatic cell nuclear transfer (SCNT), many other Species have also been cloned. Such as goat, cattle, cats, rats, mules, horse, and dogs. However, the low success rate of animal cloning by SCNT associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. HDACi, such as LBH589, CUDC-101, PXD101, have been known to enhance the level of histone acetylation, but yet never used in SCNT embryos.In present study, for the first time, attempted to examine the effect of LBH589, CUDC-101and PXD101after the pig somatic cell nuclear transfer embryos and determinate optimal concentration and duration.Method:To selected optimal concentration, the pig SCNT embryos were treated with different concentrantions of three kinds of HDACi (LBH589, CUDC-101and PXD101) for24h, and pig SCNT embryos were treated with optimal concentration for different time. After pig SCNT embryos treated with optimal condition, detecting the level of acetylation. In addition, LBH589, CUDC-101and PXD101treated pig SCNT embryos were transferred into the oviducts of surrogates on the day on which estrus began or1day afterResult:(1) We compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with50nM LBH589for24h showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with5or500nM LBH589(32.4%vs.11.8%,12.1%, and10.0%, respectively, P<0.05). we examined the in vitro developmental competence of nuclear transfer embryos treated with50nM LBH589for various intervals after activation and6-dimethylaminopurine (6-DMAP). Embryos treated for24h had higher rates of blastocyst formation than the other groups. When the acetylation of H4K12was examined in SCNT embryos treated for6h with50nM LBH589by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control.(2) We found that treatment with1μM CUDC-101for24h significantly improved the development of pig SCNT embryos. Compared with the control group, the blastocyst rate was higher (18.5%vs.10.3%, P<0.05). We then investigated the acetylation level of histone H3K9in SCNT embryos treated with CUDC-101were compared only against untreated embryos. The acetylation level of control SCNT embryos was lower than that of CUDC-101-treated embryos at pseudo-pronuclear stages.(3) We compared the in vitro developmental competence of SCNT embryos treated with various concentrations of PXD101for24h. Treatment with0.5μM PXD101significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3%vs.11.5%, P<0.05). We tested the in vitro developmental competence of SCNT embryos treated with0.5μM PXD101for various amounts of times following activation. Treatment for24h significantly improved the development of pig SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7%vs.10.6%, P<0.05). PXD101treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9in embryos at the pseudo-pronuclear and2-cell stages. We were discovered fetuses after pig SCNT embryos treated with LBH589, CUDC-101and PXD101and transferred the into oviducts of surrogates on the day of, or1day after, the onset of estrus.Conclusion:(1) Treatment50nM LBH589for24h significantly improved the in vitro development of pig SCNT embryos (P<0.05).(2) Treated with1uM CUDC-101for24h significantly improved the in vitro development of pig SCNT embryos (P<0.05).(3)0.5uM PXD101treated for24h significantly improved the in vitro development of pig SCNT embryos (P<0.05)...