Aberrant epigenetic reprogramming often results in developmental defects in somatic cell nuclear transfer (SCNT) embryos during embryonic genome activation (EGA). Bovine eight-cell SCNT embryos exhibit global hypermethylation of histone H3 lysine 9 tri- and di-methylation (H3K9me3/2), but the intrinsic reason for this remains elusive. Here, we provide evidence that two H3K9 demethylase genes, lysine-specific demethylase 4D () and 4E (), are related to active H3K9me3/2 demethylation in fertilized (IVF) embryos and are deficiently expressed in cloned embryos at the time of EGA. Moreover, KDM4E plays a more crucial role in IVF and SCNT embryonic development, and overexpression of KDM4E can restore the global transcriptome, improve blastocyst formation and increase the cloning efficiency of SCNT embryos. Our results thereby indicate that KDM4E can function as a crucial epigenetic regulator of EGA and as an internal defective factor responsible for persistent H3K9me3/2 barriers to SCNT-mediated reprogramming. Furthermore, we show that interactions between RNA and KDM4E are essential for H3K9 demethylation during EGA. These observations advance the understanding of incomplete nuclear reprogramming and are of great importance for transgenic cattle procreation.

BackgroundDNA methylation is an important epigenetic modification that is essential for epigenetic gene regulation in development and disease. To date, the genome-wide DNA methylation maps of many organisms have been reported, but the methylation pattern of cattle remains unknown.ResultsWe showed the genome-wide DNA methylation map in placental tissues using methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq). In cattle, the methylation levels in the gene body are relatively high, whereas the promoter remains hypomethylated. We obtained thousands of highly methylated regions (HMRs), methylated CpG islands, and methylated genes from bovine placenta. DNA methylation levels around the transcription start sites of genes are negatively correlated with the gene expression level. However, the relationship between gene-body DNA methylation and gene expression is non-monotonic. Moderately expressed genes generally have the highest levels of gene-body DNA methylation, whereas the highly, and lowly expressed genes, as well as silent genes, show moderate DNA methylation levels. Genes with the highest expression show the lowest DNA methylation levels.ConclusionsWe have generated the genome-wide mapping of DNA methylation in cattle for the first time, and our results can be used for future studies on epigenetic gene regulation in cattle. This study contributes to the knowledge on epigenetics in cattle.

Somatic cell nuclear transfer (SCNT) is a promising technology, but its application is hampered by its low efficiency. Hence, the majority of SCNT embryos fail to develop to term. In this study, the antioxidant melatonin reduced apoptosis and reactive oxygen species (ROS) in bovine SCNT embryos. It also increased cell number, inner cell mass (ICM) cell numbers, and the ratio of ICM to total cells while improving the development of bovine SCNT embryos in vitro and in vivo. Gene expression analysis showed that melatonin suppressed the expression of the pro-apoptotic genes p53 and Bax and stimulated the expression of the antioxidant genes SOD1 and Gpx4, the anti-apoptotic gene BCL2L1, and the pluripotency-related gene SOX2 in SCNT blastocysts. We also analyzed the epigenetic modifications in bovine in vitro fertilization, melatonin-treated, and untreated SCNT embryos. The global H3K9ac levels of melatonin-treated SCNT embryos at the four-cell stage were higher than those of the untreated SCNT embryos. We conclude that exogenous melatonin affects the expression of genes related to apoptosis, antioxidant function, and development. Moreover, melatonin reduced apoptosis and ROS in bovine SCNT embryos and enhanced blastocyst quality, thereby ultimately improving bovine cloning efficiency.

microRNAs (miRNAs) are small non-coding RNAs that participates in the regulation of many physiological pathways, but a role for spermatozoon-delivered miRNAs in fertilization and embryonic development remains controversial. A library of miRNAs in bovine sperm was constructed using Illumina high-throughput sequencing technology, along with the predication and the pathway analysis of target genes. miRNAs in mammalian spermatozoon were systematically investigated, and a protocol for RNA isolation from the cauda region of an epididymal biopsy was established. Unique sequences that were 18-26 nucleotides in length were mapped to specific precursors in miRBase 20.0 using BLAST. A total of 951 known miRNAs and 8 novel, highly expressed miRNA candidates were identified. The search for endogenous sperm miRNAs will contribute to a preliminary database for functional and molecular mechanistic studies in embryonic development and sperm epigenetic programming.