Dataset Information

Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine.

ABSTRACT: Accumulating evidence indicates the absence of paternally derived miRNAs, piwiRNAs, and proteins may be one important factor contributing to developmental failure in somatic cell cloned embryos. In the present study, we found microRNA-449b (miR-449b) was highly expressed in sperm. Target gene predictions and experimental verification indicate that several embryonic development-related genes, including CDK6, c-MYC, HDAC1 and BCL-2, are targets of miR-449b. We therefore investigated the role of miR-449b using somatic cell nuclear transfer (SCNT) embryo model. Bovine fetal fibroblasts, expressing miR-449b through a doxycycline (dox) induced expression system were used as nuclear donor cells for SCNT. The results showed that miR-449b expression in SCNT embryos significantly enhanced the cleavage rate at 48?h after activation and the levels of H3K9 acetylation at the 2-cell to 8-cell stages, meanwhile, significantly decreased the apoptosis index of blastocysts. In addition, we verified miR-449b could regulate the expression levels of CDK6, c-MYC, HDAC1 and BCL-2. In conclusion, the present study shows that miR-449b expression improves the first cleavage division, epigenetic reprogramming and apoptotic status of bovine preimplantation cloned embryos.

SUBMITTER: Wang M

PROVIDER: S-EPMC5645405 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine.

Wang Mengyun M Gao Yang Y Qu Pengxiang P Qing Suzhu S Qiao Fang F Zhang Yong Y Mager Jesse J Wang Yongsheng Y

Scientific reports 20171017 1

Accumulating evidence indicates the absence of paternally derived miRNAs, piwiRNAs, and proteins may be one important factor contributing to developmental failure in somatic cell cloned embryos. In the present study, we found microRNA-449b (miR-449b) was highly expressed in sperm. Target gene predictions and experimental verification indicate that several embryonic development-related genes, including CDK6, c-MYC, HDAC1 and BCL-2, are targets of miR-449b. We therefore investigated the role of mi ...[more]

PMID: 29042680

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Project description:BackgroundLong non-coding RNAs (lncRNAs), a type of epigenetic regulator, are thought to play important roles in embryonic development in mice, and several developmental defects are associated with epigenetic modification disorders. The most dramatic epigenetic reprogramming event occurs during somatic cell nuclear transfer (SCNT) when the expression profile of a differentiated cell is abolished, and a newly embryo-specific expression profile is established. However, the molecular mechanism underlying somatic reprogramming remains unclear, and the dynamics and functions of lncRNAs in this process have not yet been illustrated, resulting in inefficient reprogramming.ResultsIn this study, 63 single-cell RNA-seq libraries were first generated and sequenced. A total of 7009 mouse polyadenylation lncRNAs (including 5204 novel lncRNAs) were obtained, and a comprehensive analysis of in vivo and SCNT mouse pre-implantation embryo lncRNAs was further performed based on our single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs were expressed in a developmental stage-specific manner during mouse early-stage embryonic development, whereas a more temporal and spatially specific expression pattern was identified in mouse SCNT embryos with changes in the state of chromatin during somatic cell reprogramming, leading to incomplete zygotic genome activation, oocyte to embryo transition and 2-cell to 4-cell transition. No obvious differences between other stages and mouse NTC or NTM embryos at the same stage were observed. Gene oncology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and weighted gene co-expression network analysis (WGCNA) of lncRNAs and their association with known protein-coding genes suggested that several lncRNAs and their associated with known protein-coding genes might be involved in mouse embryonic development and cell reprogramming.ConclusionsThis is a novel report on the expression landscapes of lncRNAs of mouse NT embryos by scRNA-seq analysis. This study will provide insight into the molecular mechanism underlying the involvement of lncRNAs in mouse pre-implantation embryonic development and epigenetic reprogramming in mammalian species after SCNT-based cloning.

Project description:Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

Dataset Information

Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine.

Publications

Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine.

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