| Porcine somatic nuclear transfer (SCNT)holds tremendous value in animal husbandry, medicine and biology basic research, asdemonstrated in conservation of endangered elite genetics, multiplication of excellent breeds, production of donor organs for xenotransplantation, making human disease models, quality control in humanasssisted reproductive technologies, production of high value recombinant proteins, and acting as ideal research materials for reproduction and development. However, poorer efficiency caused by incomplete epigenetic reprogramming and other factors hinderswider application of porcine SCNT. Epigenetic eventsincludeDNA methylation/demethylationand histone modifications (methylation, acetylation, glycosylation, phosphorylation, ubiquitination and SUMO etc). Treatment of nuclear donor cells with appropriate epigenetic modifiers can promote cloning efficiency. EPZ004777 (EPZ), a specific inhibitor of DOT1L (a methyltransferase of H3K79), can significantly improve the generation and quality of mouse induced pluripotent stem cells, suggesting that H3K79 dimethylation (H3K79me2) is involved in regulation of cells’pluripotency. To date, however, if H3K79me2 regulates development competency of animal cloned embryos is unclear. Thus, we aimed to examinethe dynamic changes of H3K79me2 inpreimplantation cloned embryos of pigs, andto explore effect of EPZ treatment of embryos on in vitrodevelopment fate in order to lay foundation for future revealing of H3K79me2’s role and mechanisms in contolling cell’s pluripotency.Experiment 1 was designed to reveal the reprogramming pattern of H3K79me2 in pig preimplantation embryos.During the preimplantation development of porcine in vitro fertilization (IVF) embryos, H3K79me2 signal of porcine SCNT embryos at different stages was analyzed by immunofluorescence staining. We found that, in IVF and SCNT embryos, H3K79me2 signal was retained in high level in starting stage, and disappeared in pronuclear followed by reappearing in morula stage and further enhanced in blastocyst stage. From the stage of EXPB to HB, H3K79me2 signal did not change obviously in IVF embryos, while it increased continuously in SCNT embryos. Moreover, from the stage of pronuclear (16hpa NTE and 18hpi IVFE) to 8-cell, H3K79me2 signals were decreased to lowest level in both NTE and IVFE, while it was higher in IVFE than that in NTE after starting and morula stages. These results indicated the abnormal reprogramming of H3K79me2 during preimplantation d development of porcine SCNT embryos.Experiment 2 wasto investigate effects of EPZ on prepimplantation development of porcine clonedembryos in vitro. SCNT embryos were treated with PZM-3 medium including 0.5nM,5nM,50nM EPZ and 1‰ DMSO (v/v, control group) for 24h, respectively. Then they were transferred into fresh PZM-3 without EPZ. We found that there was no significant difference for cleavage rate among groups48hafter treatment, while the blastocyst rate of 0.5nM EPZ group was obviously higher than that of control group (28.97±2.65% vs.17.13±2.69%). No obvious difference was observed for the total cell number of blastocyst among groups. We further treated the SCNT embryos with 0.5nM EPZ for Oh (control group),12h,24h and 36h, respectively. No significant difference was found for cleavage rate among groups48hafteronset of treatment, while the blastocyst rate of 12h and 24h group both was significant higher than that of control and 36h group (28.56±3.51%,28.34±3.00% vs.16.32±1.93%,17.93±0.64%). Except for the remarkable decrease of 36h treatment group, no obvious difference was observed for the total cell number of blastocyst among other three group. These results suggested that, treatment of porcine SCNT embryos with 0.5nM EPZ for 12-24h, could improve their development during early stage, while higher concentration or longer term treatment of EPZ impair the development of porcine SCNT embryos.Experiment 3 aimsto estimate whether the EPZ favored the in vitro development of porcine SCNT embryos by regulating H3K79me2 reprogramming.Porcine SCNT embryos were treated with 0.5nM EPZ, and then the H3K79me2 signal and expression of DOT1L atdifferent development stages was analyzed. We found that, H3K79me2 signal in control group (without EPZ treatment) was decreased slowly from the time of electric stimulation to 4hpa, and it disappeared in 8hpa stage. In EPZ treatment group, H3K79me2 signal started decreasing from 2hpa, and disappeared in 8hpa stage. The mRNA level of DOT1L in EPZ treatment group was lower than that in control group, while the difference was not significant. These results revealed that EPZ repressed the expression of DOT1L partially down-regulated the level of H3K79me2, and which thereby improvedthe reprogramming of H3K79, which contribute to the preimplantation development of porcine SCNT embryos.Experiment 4 was to further reveal the mechanism of EPZ improving the in vitro development of porcine SCNT embryos.We firstly evaluated the H3K79me2 level in EPZ treatment blastocyst in different stage (EB, EXPB and HB). The pattern of H3K79me2 expression in EPZ treatment SCNT blastocyst was similar with that in non-EPZ treatment SCNT blastocyst. Although the H3K79me2 level in EPZ treatment SCNT blastocyst was a little bit higher than that in non-EPZ treatment SCNT blastocyst, it was lower than that in IVF blastocyst. Then we analyzed the gene expression in EPZ treatment (12h and 24h) and non-EPZ treatment SCNT blastocyst (control group), and we found that OCT4, CDX2 and GATA4 were significantly up-regulated in EPZ treatment group, andSox2 was higher in 12h EPZ treatment group than that in control group, while it is similar between 24h EPZ treatment group and control group. Lin28 was higher in 24h EPZ treatment group than that in control group, while it is indistinguishable between 12h EPZ treatment group and control group. There was no obvious difference for CDX2 and GATA4 expression between 12h EPZ treatment group and 24h EPZ treatment group. mRNA level of SOX2 and LIN28 in12h EPZ treatment was higher than that in 24h EPZ treatment, while the expression level of OCT4 in 24h EPZ treatment was higher than that in 12h EPZ treatment.Altogether, to the best of our knowledge, we are the firstwho revealed the H3K79me2 reprogramming in SCNT and IVF embryos during preimplantation development of porcine early embryos. We also demonstrated that treatment of porcine SCNT embryos with 0.5nM EPZ for 12h or 24h could modulate reprogramming of H3K79me2, and thereby improvethe preimplantation development of porcine SCNT embryos. H3K79me2 also involved in the fate of porcine SCNT embryos during early stage. Thus, we enriched the culture program of porcine SCNT embryos, and accelerated the application of SCNT technology, while further study was needed to investigate the effect of EPZ on the in vivo development of porcine SCNT embryos. |
PDF Full Text Request