The Mechanisms Of Telomere Reprogramming In The Cloned Mouse,Bovine And Ovine Embryos

Somatic cell nuclear transfer（SCNT）is the sole reproductive biotechnology that endows the differentiated cell genome with totipotency or pluripotency via a series of sequential molecular reprogramming.Therefore,the SCNT technique holds considerable promise for farm animal breeding and regenerative medicine.Telomeres are formed from a unique DNA nucleotide sequence（TTAGGG repeats,also termed a“G-tail”）and associated protective proteins,capping and protecting the ends of eukaryotic chromosomes from end-to-end fusion,degradation,and recombination.Telomeres are formed from a unique DNA nucleotide sequence（TTAGGG repeats,also termed a“G-tail”）and associated protective proteins,capping and protecting the ends of eukaryotic chromosomes from end-to-end fusion,degradation,and recombination.Although there has been some research on telomere elongation in induced pluripotent stem cells,insufficient data are available regarding the mechanism of telomere elongation during SCNT reprogramming.In order to explore the role of telomere reprogramming in SCNT embryos,we designed a series of experiments:First,the dynamic changes of telomere length and its effect on embryonic development in both mouse SCNT and intracytoplasmic sperm injection（ICSI）pre-implantation embryos were detected,and the results were verified in bovine and ovine SCNT embryos,respectively.Secondly,small molecular compounds were screened to find a way to promote telomere elongation of SCNT embryos.Finally,SCNT embryos treated with small molecule compound were transplanted to pseudo-pregnant recipients to detect the effect of small molecule compound on birth efficiency of cloned mice.The main results are as follows:1.The telomere elongation mainly occurred during zygotic genome activation（ZGA）stage in both SCNT and ICSI embryos,but the length of telomeres in SCNT embryos was significantly shorter than that in the ICSI embryos.Detections of embryo biopsy and telomere length showed that the length of telomeres in SCNT embryos dominated determined the quality of embryo development.2.Additions of a series small molecule compounds of trichostatin A（TSA）,azacytidine（Aza）,melatonin,vitamin C（Vc）,sodium butyrate（NaB）,and mirdametinib（PD0325901）to the culture medium,and the hypoxia（5%O₂）culture environment,indicated that the relative lengths of telomeres in SCNT embryos were significantly extended when the TSA,melatonin,NaB,PD0325901 or 5%O₂ were used.Furthermore,the effect of melatonin on telomere elongation was the most significant.Examination by qPCR and Q-FISH showed that the telomere length in melatonin-treated SCNT embryos has been extended 2-fold higher than the control embryos,the blastocyst development of SCNT embryos was also improved from10.89%to 58.12%.3.Single cell RNA-seq test indicated that the transcriptome of melatonin-treated SCNT embryos was close to that of ICSI embryos,and melatonin obviously attenuated the ZGA defect in SCNT embryos.By analyzing the differentially expressed genes（DEGs）,we found that melatonin activated the expressions of telomere related genes Zscan4 and Tcstv3 in SCNT embryos.4.In order to further verify the role of melatonin in SCNT embryos,the Zscan4transcription expression vector was constructed,and the Zscan4-mRNA was directly injected into SCNT embryos.The result showed that the telomere lengths in SCNT embryos were significantly extended.However,the blastocyst development of the injected SCNT embryos did not improved in comparison with the melatonin treatment group.The reason for that is probably in addition to promote telomere elongation,melatonin also reduces damages from the reactive oxygen species（ROS）to embryos.5.Results from immunofluorescence and western blot showed that melatonin inhibited heterochromatic epigenetic modifications related to gene silencing including DNA methylation and H3K9me3,while histone H3K27me3 modification remained unchanged.The expression of DNA demethylase Tet3 was significantly up-regulated in the melatonin treatment group.Moreover,similar results that melatonin improved telomere elongation and embryo development were obtained in the cloned bovine and ovine embryos.6.Treatment of the cloned embryos with melatonin significantly increased the birth rate of tail-tip fibroblasts cell-derived cloned mice from 0.3%to 0.8%.In addition,we also found that injection of melatonin into pseudo-pregnant recipients synergistically enhanced the production of cloned animals（0.3%vs.1.5%）,and increased the birth rate of cumulus cell-derived cloned mice by 7-folds（7.1%vs.1.0%）.In summary,this study successfully revealed the process and mechanism of telomere reprogramming in the SCNT embryos,and found that melatonin virtually promoted telomere elongation,repaired abnormal epigenetic modifications and ROS level during reprogramming,and eventually increased the birth rate of cloned mice.