Somatic cell nuclear transfer (SCNT) is an important technique for life science research. However, most SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we show that abnormal Xi occurs in somatic cell NT blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. H3K27me3 removal by Kdm6a mRNA overexpression could significantly improve preimplantation development of NT embryos, and even reached a 70.2% blastocyst rate of cleaved embryos compared with the 38.5% rate of the control. H3K27me3 levels were significantly reduced in blastomeres from cloned blastocysts after overexpression of Kdm6a. qPCR indicated that rDNA transcription increased in both NT embryos and 293T cells after overexpression of Kdm6a. Our findings demonstrate that overexpression of Kdm6a improved the development of cloned mouse embryos by reducing H3K27me3 and increasing rDNA transcription.

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Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a 2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a 4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a 4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a 4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study.

Methyl-CpG-binding domain proteins (MBPs) connect DNA methylation and histone modification, which are the key changes of somatic cell reprogramming. Methyl-CpG-binding protein 2 (MeCP2) was the first discovered MBP that has been extensively studied in the neurodevelopmental disorder Rett syndrome. However, a role for MeCP2 during cellular reprogramming associated with somatic cell nuclear transfer (SCNT) has not been examined. In this study, we discovered that MeCP2 expression was significantly lower in embryos generated by SCNT compared with those generated by intracytoplasmic sperm injection (ICSI). We genetically modified mouse embryonic fibroblasts (MEFs) to overexpress MeCP2 and serve as donor cells for nuclear transfer (NT) to investigate the effects of MeCP2 on preimplantation development of SCNT embryos. The blastocyst rate (35.71%) of MeCP2 overexpressed embryos (NT(+)) was significantly greater than in nontransgenic embryos (NT(-), 24.29%). Furthermore, immunofluorescence experiments revealed that 5-methylcytosine (5mC) was transferred to 5-hydroxymethylcytosine (5hmC) to a greater extent in NT(+) embryos than in NT(-) embryos. Real-time PCR evaluation of gene expression also showed that embryonic development-associated genes, such as Oct4 and Nanog, were significantly higher in the NT(+) group compared to the NT(-) group. Collectively, these results suggested that MeCP2 facilitated Tet3 activity, enhanced expression of pluripotency-related genes, and eventually improved the development of NT embryos. Finally, we performed chromatin immunoprecipitation to identify direct targets of MeCP2 and constructed a protein interaction network to elucidate several putative MeCP2 targets.

Somatic cell nuclear transfer is an important technique for life science research, but its efficiency is still extremely low, and most genes that are important during early development, such as X chromosome-linked genes, are not appropriately expressed during this process. Poly (ADP-ribose) polymerase (PARP) is an enzyme that transfers ADP ribose clusters to target proteins. PARP family members such as PARP1 participate in cellular signalling pathways through poly (ADP-ribosylation) (PARylation), which ultimately promotes changes in chromatin structure, gene expression, and the localization and activity of proteins that mediate signalling responses. PARP1 is associated with X chromosome inactivation (Xi). Here, we showed that abnormal Xi occurs in somatic cell nuclear transfer (NT) blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. Parp1 expression was higher in female NT blastocysts than that in intracytoplasmic sperm injection (ICSI) embryos but not in male NT blastocysts. After knocking down Parp1 expression, both the pre-rRNA 47S and X-inactivation-specific transcript (Xist) levels increased. Moreover, the expression of genes on the inactivated X chromosome, such as Magea6 and Msn, were also increased in the NT embryos. However, the development of Parp1si NT embryos was impaired, although total RNA sequencing showed that overall gene expression between the Parp1si NT blastocysts and the control was similar. Our findings demonstrate that increases in the expression of several genes on the X chromosome and of rRNA primary products in NT blastocysts with disrupted Parp1 expression are insufficient to rescue the impaired development of female cloned mouse embryos and could even exacerbate the associated developmental deficiencies.