The success of cloned animal “Dolly Sheep” demonstrated the somatic cell nuclear transfer (SCNT) technique holds huge potentials for mammalian asexual reproduction. However, the extremely poor development of SCNT embryos indicates their molecular mechanism remain largely unexplored. Deciphering the spatiotemporal patterns of gene expression in SCNT embryos is a crucial step toward understanding the mechanisms associated with nuclear reprogramming. In this study, a valuable transcriptome recourse of SCNT embryos was firstly established, which derived from different inter-/intra donor cells. The gene co-expression analysis identified 26 cell-specific modules, and a series of regulatory pathways related to reprogramming barriers were further enriched. Compared to the intra-SCNT embryos, the inter-SCNT embryos underwent only complete partially reprogramming. As master genome trigger genes, the transcripts related to TFIID subunit, RNA polymerase and mediators were incomplete activated in inter-SCNT embryos. The inter-SCNT embryos only wasted the stored maternal mRNA of master regulators, but failed to activate their self-sustained pathway of RNA polymerases. The KDM family of epigenetic regulator also seriously delayed in inter-SCNT embryo reprogramming process. Our study provided new insight into understanding of the mechanisms of nuclear reprogramming.

Cloned animals generated by somatic cell nuclear transfer (SCNT) have been reported for many years; however, SCNT is extremely inefficient, and zygotic genome activation (ZGA) is required for SCNT‐mediated somatic cell reprogramming. To identify candidate factors that facilitate ZGA in SCNT‐mediated reprogramming, we performed siRNA‐repressor and mRNA‐inducer screenings, which reveal Dux, Dppa2, and Dppa4 as key factors enhancing ZGA in SCNT. We show that direct injection of ZGA inducers has no significant effect on SCNT blastocyst formation; however, following the establishment of an inducible Dux transgenic mouse model, we demonstrate that transient overexpression of Dux not only improves SCNT efficiency but also increases that of chemically induced pluripotent stem cell reprogramming. Moreover, transcriptome profiling reveals that Dux‐treated SCNT embryos are similar to fertilized embryos. Furthermore, transient overexpression of Dux combined with inactivation of DNA methyltransferases (Dnmts) further promotes the full embryonic development of SCNT‐derived animals. These findings enhance our understanding of ZGA‐regulator function in somatic reprogramming.

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N6‐methyladenosine (m6A) methylation is the most common and abundant modification on mammalian messenger RNA (mRNA) and regulates the pluripotency of embryonic stem cells (ESCs). Research has shown that melatonin plays a fundamental role in DNA and histone modifications. However, the effect of melatonin on RNA modification is unknown. Here, for the first time, we investigated the effect of melatonin on m6A modifications in long‐term‐cultured ESCs. Pluripotency studies indicated that 10 μmol/L melatonin sufficiently maintained ESCs with stemness features over 45 passages (more than 90 days). Notably, treatment of ESCs with melatonin led to a significant decrease in the nuclear presence of m6A methyltransferase complex and decreased global m6A modification. Depletion of melatonin receptor 1 (MT1) by CRISPR/Cas9 significantly reduced the effects of melatonin on ESC pluripotency and m6A modification. Methylated RNA immunoprecipitation sequencing (MeRIP‐seq) revealed that melatonin promotes stabilization of core pluripotency factors, such as Nanog, Sox2, Klf4, and c‐Myc, by preventing m6A‐dependent mRNA decay. Using cell signaling pathway profiling systems, melatonin was shown to regulate m6A modification predominantly through the MT1‐JAK2/STAT3‐Zfp217 signal axis. This study reveals a new dimension regarding melatonin regulation of gene expression at the RNA level.

Bovine embryonic stem cells (bESCs) have not been successfully established yet. One reason could be that CDX2, as the trophectoderm regulator, expresses in bovine inner cell mass (ICM), which probably becomes a technical barrier for maintaining the pluripotency of bESCs in vitro. We hypothesized that CDX2 knockdown (CDX2-KD) could remove such negative effort, which will be helpful for capturing complete and permanent capacity of pluripotency. Expression and localization of pluripotent genes were not affected in CDX2-KD blastocysts. The CDX2-KD bESCs grew into monolayers on feeder layer. Pluripotent genes expressed at an improved levels and lasted longer time in CDX2-KD bESCs, along with down-regulation of DNA methylation on promoters of both OCT4 and SOX2. The cystic structure typical for trophoblast cells did not show during culturing CDX2-KD bESCs. CDX2-KD bESC-derived Embryoid bodies showed with compact morphology and with the improved levels of differentiations in three germ layers. CDX2-KD bESCs still carried the capacity of forming teratomas with three germ layers after long-term culture. In summary, CDX2 in bovine ICM was inducer of trophoblast lineage with negative effect on maintenance of pluripotency of bESCs. Precise regulation CDX2 expression to switch on/off will be studied next for application on establishment of bESCs.