Abnormal Epigenetic Modifications in Surviving Somatic Cell Cloned Cattle Associated With Donor Cell Type

Background Studies have shown that the efficiency of somatic cell nuclear transfer (SCNT) is related to the type of donor cell used. Previous studies have shown that fetal oviduct epithelial cells (FOVs) exhibit a higher blastocyst formation rate than fetal fibroblasts (FFBs), but they are associated with lower pregnancy, calving, and full-term rates after implantation. The reason for this difference is unclear. Result In this study, we performed the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), RNA-seq, and 5-hydroxymethylcytidine (5hmC) and 5-methylcytosine (5mC) DNA methylation sequencing methods across the whole genome to analyze the epigenetic differences between cattle cloned using FFBs or FOVs as donor nuclei. The results showed that chromatin openness, gene expression levels, and 5hmC contents were greater in cloned cattle derived from FOV donor nuclei than in those derived from FFBs. ATAC-seq and RNA-seq analyses of cloned bovine ear tissues derived from the same source of donor nuclei showed an obvious clustering tendency. In this study, we also found that the 5hmC content of surviving cloned cattle derived from FFBs was greater than 4‰, whereas it was less than 2‰ in dead cloned cattle. Conclusion We found that there were abnormalities in specific epigenetic modifications and gene expression in living somatic cell cloned cattle derived from different donor nuclei. Although cloned cattle undergo somatic reprogramming and differentiation, they retain the epigenetic imprints of their donor nuclei, and this somatic imprinting may affect the development rate of cloned blastocysts as well as the birth rate and development status of cloned fetuses after implantation.

Transcriptome Analyses Reveal Effects of Vitamin C-Treated Donor Cells on Cloned Bovine Embryo Development

Somatic cell nuclear transfer (SCNT) is a very powerful technique used to produce genetically identical or modified animals. However, the cloning efficiency in mammals remains low. In this study, we aimed to explore the effects of vitamin C (Vc)-treated donor cells on cloned embryos. As a result, Vc treatment relaxed the chromatin of donor cells and improved cloned embryo development. RNA sequencing was adopted to investigate the changes in the transcriptional profiles in early embryos. We found that Vc treatment increased the expression of genes involved in the cell–substrate adherens junction. Gene ontology (GO) analysis revealed that Vc treatment facilitated the activation of autophagy, which was deficient in cloned two-cell embryos. Rapamycin, an effective autophagy activator, increased the formation of cloned blastocysts (36.0% vs. 25.6%, p < 0.05). Abnormal expression of some coding genes and long non-coding RNAs in cloned embryos was restored by Vc treatment, including the zinc-finger protein 641 (ZNF641). ZNF641 compensation by means of mRNA microinjection improved the developmental potential of cloned embryos. Moreover, Vc treatment rescued some deficient RNA-editing sites in cloned two-cell embryos. Collectively, Vc-treated donor cells improved the development of the cloned embryo by affecting embryonic transcription. This study provided useful resources for future work to promote the reprogramming process in SCNT embryos.

Handmade cloned bovine embryos, parthenogenesis and in vitro fertilization: a comparison

<p>The handmade cloning and parthenogenesis as a control leads to the possibility of studying biological processes such as cell reprogramming, epigenetics, embryo genome activation and pre- and post-natal development. The objective of this study was to standardize the process the handmade cloning (HCM) and to compare the resultant embryo production with parthenogenesis and in vitro fertilization (IVF). The primary fibroblast culture was established from explants of ear, the manual cloning technique was standardized with this fibroblast and 10 processes to cloning, parthenogenesis and fertilization in vitro were performed, and the compared production and embryo quality was performed by an ANOVA. An in vitro culture of fibroblast cells was established with optimal characteristics, allowing the genetic material to be used in the process of cloning. The parthenote group without zona pellucida (ZP) exhibited a higher cleavage rate compared with the other groups of parthenotes with ZP and IVF embryo (p&lt;0.05). The number of blastomeres was greater in the IVF (109.81±11.70) compared with the parthenote with ZP (73.73±7.09), without ZP (78.16±7.65) and clone (CL, 77.5±8.23) groups. Embryo production in the CL, ZP, without ZP and IVF groups was not significantly different (29.7, 37.6, 33.8 and 35.2% respectively). The HCM technique was successfully standardized in the laboratory. The resultant embryo production was similar between hand-made cloned embryos, parthenogenesis and in vitro fertilization. The findings of this work give rise to different routes for studying embryology and contribute to the optimization of this technique for commercial purposes.</p>