Construction And Histone Acetylation Modifications Of Cashmere Goat-Bovine Interspecies Reconstructed Embryos

Interspecies somatic cell nuclear transfer(iSCNT) is a valuable tool for studying nucleus-cytoplasm interactions,and it provides a possible alternative to clone endangered animals.However,the low cloning efficiency limits the applications of nuclear transfer technology.Somatic cell nuclear transfer(SCNT) is a remarkable process in which the somatic cell can be converted into a totipotent state in the oocyte through an unknown mechanism.Epigenetic reprogramming of the somatic cell genome has been suggested as a key event occurring in the SCNT,and abnormal reprogramming of the epigenetic marks contributing to the inefficiency of somatic cell nuclear transfer.DNA methylation and histone modification are considered to be important in nuclear reprogramming.Compared to asymmetric modification of DNA methylation,histone modification of cloned embryos has been investigated in limited studies.In present study,the cashmere goat-bovine reconstructed embryos were produced by nuclear transfer.The electro-fusion,activation and in vitro culture systems were optimized to improve the development of reconstructed embryos.The acetylation status of lysine residues on core histones H3 and H4 was also investigated in both the interpecies reconstructed embryos and bovine-bovine cloned embryos at 1-cell stage.Finally,trichostatin A(TSA),a histone deacetylase inhibitor,was used to treat recipient oocytes and interspecies reconstructed embryos to investigate the effects of TSA treatment on the development of reconstructed embryos.1.Construction of cashmere goat-bovine interspecies nuclear transfer embryos In this study,the interspecies reconstructed embryos were produced by transferring the fibroblast cells into enucleated bovine oocytes.Adult cashmere goat ear fibroblast cells were isolated and cultured by tissue bulk attachment.Characters of the fibroblast cells in vitro were analysized.Results indicated that the cultured fibroblast cells had normal morphology,proliferation characteristics and chromosome number.After injecting the fibroblast cell into enucleated oocytes by micromanipulation, the electrical fusion and activation conditions were optimized by using different parameters and activation reagents.For fusion treatment,electrical field intensity(180v/mm,190v/mm),pulse time(20~μs,30μs),pulse number(1 time,2 times) and the recovering interval(0.5hr,1.0hr,＞1.0 hr) were studied.After fusion, different activation methods(7%Ethanol + 6-DMAP,A23187+6-DMAP,Ionomycin + 6-DMAP) were used.The results showed that,in our system,electrical fusion should be done less than 0.5hr after nuclear transfer,and the best parameters were as follows:electrical field intensity 190V/mm,pulse time 20μs,pulse number 2 times. For oocyte activation,A23187 + 6-DMAP treatment could obtain high cleavage rates and 8-cell embryos rates.The interspecies reconstructed embryos were cultured in various medium(SOFaa,M199 and CRlaa) and CRlaa-Granulosa coculture system. Results showed that co-culture system supported reconstructed embryos develop to blastocyst stage,while non-blastocysts were achieved when interspecies reconstructed embryos cultured in SOFaa,M199 and CRlaa alone.2.Effects of TSA-treatment on the development of interspecies reconstructed embryosTrichostatin A,a histone deacetylase inhibitor,can increase histone acetylation in donor cells and enhance gene expression.In this study,bovine oocytes were treated with 1ng/mL TSA during in vitro maturation.Comparable cleavage rates and 8-cell embryos rates were observed between TSA-treated and control groups.However, high morula-blastocyst rate from TSA treated oocytes was observed(18.26%vs control 8.62%,P＜0.05).When interspecies cloned embryos were treated by TSA (1ng/mL and 10ng/mL) during chemical activation for 4 hours,there was no significant difference in 8-cell embryos rates and morula-blastocyst rates between treatment and control groups.High TSA concentration(10ng/mL) significantly decreased the cleavage rates,compared to 1ng/mL TSA treatment(66.67%vs 50.60%, P＜0.01).When TSA treatment lasted from 4 to 24 hours,similar cleavage rates and 8-cell embryos rates were obtained,but morula-blastocyst development rates reduced significantly.It is suggested that longer time for TSA-treatment may cause developmental defects.3.Dynamic reprogramming of histone acetylation modification in the first cell cycle of interspecies reconstructed embryosHistone acetylation modification is considered to be important in nuclear reprogramming.In this study,the acetylation status of lysine residues on core histones H3 and H4 was investigated in the first cell cycle of cashmere goat-bovine and bovine-bovine reconstructed embryos.As results showed,in both reconstructed embryos lysine acetylation on core histones(H3K9 and H3K18) were quickly deacetylated following premature chromosome condensation(PCC),and were reacetylated after activation treatment,while only mild deacetylation occurred on H4K8 after PCC.It is suggested that there was no difference in histone acetylation reprogramming between cashmere goat-bovine and bovine-bovine reconstructed embryos,and the bovine oocyte has the ability to reprogram somatic cells of different genus.