Abstract
Imprinting disorder during somatic cell nuclear transfer usually leads to the abnormality of cloned animals and low cloning efficiency. However, little is known about the role of donor cell imprinting in the development of cloned embryos. Here, we demonstrated that the imprinting (H19/Igf2) in porcine fetus fibroblasts derived from the morphologically abnormal cloned fetuses (the abnormal imprinting group) was more hypomethylated, and accordingly, significantly higher H19 transcription and lower Igf2 expression occurred in comparison with those in fibroblasts derived from morphologically normal cloned fetuses (the normal imprinting group) or donor fetus fibroblasts (the control group). When these fibroblasts were used as donor cells, the abnormal imprinting group displayed an even lower imprinting methylation level, in correspondence to the significantly downregulated expression of Dnmt1, Dnmt3a and Zfp57, and a markedly reduced blastocyst rate, while the normal imprinting group took on the similar patterns of imprinting, gene expression and embryo development to the control group. When 5-aza-dC was applied to reduce the fibroblasts imprinting methylation level in the normal imprinting group, cloned embryos displayed the more severely impaired imprinting and significantly lower blastocyst rate. While the upregulated H19 transcription in the abnormal imprinting group was knocked down, the imprinting statuses were partly rescued, and the cleavage and blastocyst rates significantly increased in cloned embryos. In all, donor cell imprinting disorder reduced the developmental efficiency of cloned embryos. This work provides a new insight into understanding the molecular mechanism of donor cells regulating the cloned embryo development.
Highlights
Somatic cell nuclear transfer (SCNT) has achieved in many species, owning a broad application prospect in the basic research, agriculture, biomedicine, etc [1]
Our results demonstrated that when fibroblasts derived from the morphologically abnormal cloned fetus with the hypomethylated H19/Igf2 imprinting were used as donor cells, cloned embryos displayed a markedly reduced development and an even more severely impaired H19/Igf2 imprinting, and downregulation of H19/Igf2 methylation level in normal imprinting porcine fetal fibroblasts (PFFs) by 5-azadC resulted in the significantly lower blastocyst rate
The methylation statuses and transcription of H19/Igf2 and the expression of genes related to the imprinting methylation maintenance were further detected in PFFs derived from the morphologically abnormal and normal cloned fetuses (Figure 1 and Supplementary Figures A1, A2, A3, A4)
Summary
Somatic cell nuclear transfer (SCNT) has achieved in many species, owning a broad application prospect in the basic research, agriculture, biomedicine, etc [1]. The overall cloning efficiency remains low, and the developmental abnormalities frequently occur, limiting the wide application of cloning technology [2, 3]. It is generally believed that the developmental abnormalities of cloned animals and low cloning efficiency are largely due to the imprinting disorder [4]. Imprinting is an epigenetic regulatory mechanism to ensure a monoallelic parental-specific expression pattern and the normal growth and development of embryos [5]. The imprinting disorder would alter the expression patterns of imprinted genes, resulting in the poor embryo www.impactjournals.com/oncotarget development. Imprinting has been considered as an excellent model to evaluate the developmental efficiency of cloned embryos
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