Despite the success of animal cloning by somatic cell nuclear transfer (SCNT) in many species, the method is limited by its low efficiency. After zygotic genome activation (ZGA) during mouse development, a large number of endogenous retroviruses (ERVs) are expressed, including the murine endogenous retrovirus‐L (MuERVL/MERVL). In this study, we generate a series of MERVL reporter mouse strains to detect the ZGA event in embryos. We show that the majority of SCNT embryos do not undergo ZGA, and H3K27me3 prevents SCNT reprogramming. Overexpression of the H3K27me3‐specific demethylase KDM6A, but not of KDM6B, improves the efficiency of SCNT. Conversely, knockdown of KDM6B not only facilitates ZGA, but also impedes ectopic Xist expression in SCNT reprogramming. Furthermore, knockdown of KDM6B increases the rate of SCNT‐derived embryonic stem cells from Duchenne muscular dystrophy embryos. These results not only provide insight into the mechanisms underlying failures of SCNT, but also may extend the applications of SCNT.

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.

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.

During the process of embryonic development in mammals, epigenetic modifications must be erased and reconstructed. In particular, the trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcriptional repression and contributes to the maintenance of the pluripotent embryos. In this study, we determined that the global levels of the H3K27me3 marker were elevated in MII oocyte chromatin and decrease to minimal levels at the 8-cell and morula stages. When the blastocyst hatched, H3K27me3 was re-established in the inner cell mass. We also determined that H3K27me3-specific demethylases, UTX and JMJD3, were observed at high transcript and protein levels in mouse preimplantation embryos. In the activated oocytes, when the H3K27me3 disappeared at the 8-cell stage, the UTX (but not JMJD3) protein levels were undetectable. Using RNA interference, we suppressed UTX and JMJD3 gene expression in the embryos and determined that the functions of UTX and JMJD3 were complementary. When JMJD3 levels were decreased by RNA interference, the embryo development rate and quality were improved, but the knockdown of UTX produced the opposite results. Understanding the epigenetic mechanisms controlling preimplantation development is critical to comprehending the basis of embryonic development and to devise methods and approaches to treat infertility.

Chinese Yellow Cattle, an ancient and domesticated breed for draft service, provide unique animal genetic resources with excellent genetic features, including crude feed tolerance, good stress resistance, strong adaptability, and tender meat quality; however, their production performance and meat yield are significantly inferior. Herein, the myostatin gene (MSTN), a negative regulator of skeletal muscle development, was knocked out by CRISPR/Cas9 technology. Eight MSTN gene-edited bull calves (MT) were born, and six of them are well-developed. Compared with the control cattle (WT), the growth trait indexes of MT cattle were generally increased, and the hindquarters especially were significantly improved. The biochemical indexes and the semen characteristics demonstrated that MT bulls were healthy and fertile. Consistent with our conjecture, the wobble and beating of MT bull spermatozoa were significantly higher than that of WT. Nine sperm motility-related proteins and nineteen mitochondrial-related proteins were identified by up-regulation in MT bull spermatozoa using FLQ proteomic technique and act to govern sperm flagellum assembly, organization, and beating and provide sufficient energy for sperm motility. The current study confirmed that the MSTN gene-edited Chinese Yellow cattle have improved growth traits and normal fertility, which can be used for beef cattle production and breeding.