During mammalian pre-implantation embryonic development dramatic and orchestrated changes occur in gene transcription. The identification of the complete changes has not been possible until the development of the Next Generation Sequencing Technology. Here we report comprehensive transcriptome dynamics of single matured bovine oocytes and pre-implantation embryos developed in vivo. Surprisingly, more than half of the estimated 22,000 bovine genes, 11,488 to 12,729 involved in more than 100 pathways, is expressed in oocytes and early embryos. Despite the similarity in the total numbers of genes expressed across stages, the nature of the expressed genes is dramatically different. A total of 2,845 genes were differentially expressed among different stages, of which the largest change was observed between the 4- and 8-cell stages, demonstrating that the bovine embryonic genome is activated at this transition. Additionally, 774 genes were identified as only expressed/highly enriched in particular stages of development, suggesting their stage-specific roles in embryogenesis. Using weighted gene co-expression network analysis, we found 12 stage-specific modules of co-expressed genes that can be used to represent the corresponding stage of development. Furthermore, we identified conserved key members (or hub genes) of the bovine expressed gene networks. Their vast association with other embryonic genes suggests that they may have important regulatory roles in embryo development; yet, the majority of the hub genes are relatively unknown/under-studied in embryos. We also conducted the first comparison of embryonic expression profiles across three mammalian species, human, mouse and bovine, for which RNA-seq data are available. We found that the three species share more maternally deposited genes than embryonic genome activated genes. More importantly, there are more similarities in embryonic transcriptomes between bovine and humans than between humans and mice, demonstrating that bovine embryos are better models for human embryonic development. This study provides a comprehensive examination of gene activities in bovine embryos and identified little-known potential master regulators of pre-implantation development.

Although leukemia inhibitory factor (LIF) maintains the ground state pluripotency of mouse embryonic stem cells and induced pluripotent stem cells (iPSCs) by activating the Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) pathway, the mechanism remained unclear. Stat3 has only been shown to promote complete reprogramming of epiblast and neural stem cells and partially reprogrammed cells (pre-iPSCs). We investigated if and how Jak/Stat3 activation promotes reprogramming of terminally differentiated mouse embryonic fibroblasts (MEFs). We demonstrated that activated Stat3 not only promotes but also is essential for the pluripotency establishment of MEFs during reprogramming. We further demonstrated that during this process, inhibiting Jak/Stat3 activity blocks demethylation of Oct4 and Nanog regulatory elements in induced cells, which are marked by suppressed endogenous pluripotent gene expression. These are correlated with the significant upregulation of DNA methyltransferase (Dnmt) 1 and histone deacetylases (HDACs) expression as well as the increased expression of lysine-specific histone demethylase 2 and methyl CpG binding protein 2. Inhibiting Jak/Stat3 also blocks the expression of Dnmt3L, which is correlated with the failure of retroviral transgene silencing. Furthermore, Dnmt or HDAC inhibitor but not overexpression of Nanog significantly rescues the reprogramming arrested by Jak/Stat3 inhibition or LIF deprivation. Finally, we demonstrated that LIF/Stat3 signal also represents the prerequisite for complete reprogramming of pre-iPSCs. We conclude that Jak/Stat3 activity plays a fundamental role to promote pluripotency establishment at the epigenetic level, by facilitating DNA demethylation/de novo methylation, and open-chromatin formation during late-stage reprogramming. STEM CELLS 2012;30:2645–2656

DNA methylation is an important epigenetic modification that undergoes dynamic changes in mammalian embryogenesis, during which both parental genomes are reprogrammed. Despite the many immunostaining studies that have assessed global methylation, the gene-specific DNA methylation patterns in bovine preimplantation embryos are unknown. Using reduced representation bisulfite sequencing, we determined genome-scale DNA methylation of bovine sperm and individual in vivo developed oocytes and preimplantation embryos. We show that (1) the major wave of genome-wide demethylation was completed by the 8-cell stage; (2) promoter methylation was significantly and inversely correlated with gene expression at the 8-cell and blastocyst stages; (3) sperm and oocytes have numerous differentially methylated regions (DMRs)—DMRs specific for sperm were strongly enriched in long terminal repeats and rapidly lost methylation in embryos; while the oocyte-specific DMRs were more frequently localized in exons and CpG islands (CGIs) and demethylated gradually across cleavage stages; (4) DMRs were also found between in vivo and in vitro matured oocytes; and (5) differential methylation between bovine gametes was confirmed in some but not all known imprinted genes. Our data provide insights into the complex epigenetic reprogramming of bovine early embryos, which serve as an important model for human preimplantation development.

DNA methylation undergoes drastic fluctuation during early mammalian embryogenesis. The dynamics of global DNA methylation in bovine embryos, however, have mostly been studied by immunostaining. We adopted the whole genome bisulfite sequencing (WGBS) method to characterize stage-specific genome-wide DNA methylation in bovine sperm, immature oocytes, oocytes matured in vivo and in vitro, as well as in vivo developed single embryos at the 2-, 4-, 8-, and 16-cell stages. We found that the major wave of genome-wide DNA demethylation was complete by the 8-cell stage when de novo methylation became prominent. Sperm and oocytes were differentially methylated in numerous regions (DMRs), which were primarily intergenic, suggesting that these non-coding regions may play important roles in gamete specification. DMRs were also identified between in vivo and in vitro matured oocytes, suggesting environmental effects on epigenetic modifications. In addition, virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, by using RNA-seq data generated from embryos at the same developmental stages, we revealed a weak inverse correlation between gene expression and promoter methylation. This comprehensive analysis provides insight into the critical features of the bovine embryo methylome, and serves as an important reference for embryos produced in vitro, such as by in vitro fertilization and cloning. Lastly, these data can also provide a model for the epigenetic dynamics in human early embryos.