Effect Of ACY-1215 On The Development Of Early Bovine Somatic Cell Nuclear Transfer Embryos

Somatic cell nuclear transfer(SCNT)has been changing so fast.The current research is to clarify the related epigenetic abnormalities associated with SCNT and how to correct abnormalities.The ultimate goal is to increase the birth rate.the current research has made it clear that most of the nuclear epigenetic modifications can be erased by oocytes reprogramming,but a few genomic imprints,especially those deriving from donor cells,are difficult to be successfully erased,which is the key reason for the abnormal development of cloned embryos.So how to correct the imprinting by regulating the level of DNA methylation and histone modification may be an important direction to improve the reprogramming of somatic cell nuclear transfer embryos.As a histone deacetylase,HDAC6 has the following functions:(1)It regulates signal transduction by shuttling between cytoplasm and nucleus,due to the two functional catalytic domains;(2)It take part in chromatin structure and modification gene transcription;(3)It also supervises the acetylation level of α-tubulin in the cytoskeleton.However,it is not clear whether HDAC6 has an effect on the reprogramming of nuclear transfer embryos.ACY-1215 is an effective inhibitor of HDAC6 selection.It has been widely used in cancer and neurodegenerative diseases,and regulates mitosis and epigenetic modification.Therefore,in this study,ACY-1215 was adopted to treat cloned embryos,with the purpose of traversing its consequence on the development of bovine SCNT embryo.The end results are shown below:1.In SCNT and IVF embryos,patterns of dynamic changes of acetylated tubulin at different stages prior were tested.By SCNT,IVF and immunofluorescence experiments,ac α-tubulin was significantly higher in the IVF group than SCNT(p < 0.05)2.In qPCR and immunofluorescence experiments,the presence of 20 n M of drug inhibits HDAC6 by different concentration of ACY-1215.The α-tubulin level of the SCNT group was closer to that of the IVF group(p > 0.05),and the proportion of spindle disorders in the SCNT group was reduced(p < 0.05).3.The presence of ACY-1215 delayed the average first cleavage of SCNT embryos(p <0.05),but had no effect on the cleavage rate(p > 0.05).And the blastocyst-related quality evaluation index and the formation rate of blastocyst is improved(p < 0.05)4.By testing the dynamic changes of H3K9 ac and H4K16 ac in different groups,it was found that H3K9 ac and H4K16 ac in the control group were significantly lower than the IVF group at the same period(p < 0.05).However,H3K9 ac and H4K16 ac in the ACY-1215 group were significantly higher than those in the control group(p < 0.05).In some periods,H3K9 ac and H4K16 ac in the ACY-1215 group were significantly higher than IVF group(p < 0.05)It shows that the reprogramming of nuclear transfer embryos has been improved to a certain extent.The results show that ACY-1215 improves the level of tubulin acetylation before the first cleavage of somatic cell nuclear transfer embryos.The cleavage time and later development is improved by ACY-1215.It repairs the abnormal appearance of somatic cell nuclear transfer embryos,making it closer to the in vitro fertilization group.All in all,our discovery provide a technical reference for somatic cell nuclear transfer technology and has certain application prospects.