| The practical application of in vitro production of embryos has been limited by the lower efficiency and quality, as well as the aberrant development of embryos. These obstacles not only restrict the application of reproductive biotechnology in domestic animals breeding, but also threaten the clinical use of artificial reproductive technology (ART). Many previous studies have profiled the transcriptome of in vitro produced embryos in mouse, pig, and bovine, and found many candidate genes that are likely responsible for the abnormal embryonic development. However, the mechanisms responsible for the aberrant gene expression after in vitro fertilization and culture are still unclear. A wildly accepted theory is abnormal epigenetic modifications, including DNA methylation, miRNA, et cl. mediated the in vitro environment induced aberrant gene expression. In this study, using high-throughput approaches, including RNA-seq, MeDIP-seq, small RNA-seq, we profiled a dynamic and high-dimensional omic data of in vivo and in vitro produced early embryos. Further exploration of these data is based on the integrated analysis, which could provide a detailed regulative network of epigenetic modification and gene expression.Using mice as model, we collected embryonic and extra-embryonic tissues at E3.5, E7.5, and E10.5, and study the effect of in vitro fertilization and culture on the epigenetic modification and gene expression patterns of embryos, as well, we also recorded the phenotypes of in vivo and in vitro produced embryos during sampling process. Our results showed there is not significant difference in implantation rates between in vivo and in vitro produced embryos. However, the rate of normal embryo of in vitro group is relative lower than in vivo group, implying an impaired developmental potential of in vitro embryos. Detailed analysis of embryos at different stages indicated that lethality of in vitro produced embryos occurred mainly before E7.5. In addition, in vitro produced fetus showed a lower body weight at birth, and an increased placental weight, implying that the development of both embryonic and extra-embryonic tissues was compromised in in vitro produced embryos.The dynamic comparison of transcriptome between in vivo and in vitro produced embryos indicated some basic biological processes, such as cell adhesion, ion transport, and energy metabolism, were disturbed consistently during per-implantation period. Interestingly, the differentially expressed genes (DEGs) at three time points were highly associated with the pathology of neuron degenerative diseases (NDs), cardiomyopathies, and cancers. According to "fetal origins of adult disease" hypothesis (Barker hypothesis), and clinical epidemiological analysis of child and adult disease of ART offspring, our data implies that the ART induced aberrant expression of some key genes would predispose offspring to an increased risk of special adult disease. These DEGs are potential candidates for further exploring the mechanism(s) of ART induced health problems.Based on the methylome obtained from MeDIP-seq, we found both in vivo and in vitro produced embryos underwent de novo methylation from blastocysts, which were characterized with a global hypomethylation. Compared with in vivo embryos, many genes displayed a consistently aberrant DNA methylation level at promoter region. Fuctional clustering analysis showed these genes are mainly involved in the NDs and cardiomuscular diseases, which is in agreement with the indications of transcriptomic data.To better understand the epigenetically involved mechanisms responsible for the different gene expression patterns of in vitro embryo, integrated analysis were applied using our transcriptome, methylome and small RNA data. The results showed that gene expression were mainly regulated by microRNA, which is possibly due to the hypomethylation of blastocysts. It is unexpected that the methylation of promoter displayed the highest correlation with gene expression at E3.5. From then on, during the post-implantation period, gene expression were increasingly regulated by the methylation of gene body. During the embryonic development, gene with a high-level abundance showed a relative stable expression pattern. These genes were mainly responsible for the basic functions. With respect to the DEGs between in vivo and in vitro embryos, aberrant methylation modifications contribute largely to the abnormal gene expression patterns, although the embryos (blastocysts) displayed a global hypomethyaltion at this time point. microRNA, accounted for the largest part of the regulation of DEGs, and this effect of microRNA were increasingly enhanced during development. Methylation, could not only influence gene expression directly, but also impact gene expression indirectly through the effect of microRNA, which showed a very complicated processes of epigenetically regulated gene expression. Moreover, NDs are likely to be induced by the aberrant methylation of gene body of some key genes.Our study, for the first time, displayed a high-dimensional and dynamic omic profile of in vivo and in vitro embryos. We found many DEGs and differentially methylated regions (DMRs) that are likely responsible for the aberrant development, and increased healthy problems of in vitro embryos. In addition, we explored the epigenetical mechanisms of gene expression regulation. These database and results provide a comprehensive and detailed reference for studying the underlying mechanisms responsible for many aberrations and dysfunctions of in vitro produced embryos. |
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