The Analysis Of Aberrant Reprogramming During Somatic Cloning In Cattle And Studies Of Improving Reprogramming Efficiency

Somatic cloning, also known as somatic cell nuclear transfer (SCNT), is apromising technology with potential applications in therapeutic cloning, producingvaluable recombinant proteins, breeding disease-resistant transgenic livestock, speciespreservation, livestock propagation, man xenotransplantation, and disease models.The cloning efficiency, however, remains low, which is a significant barrier to thewidespread use of such a promising technology. It is generally believed that the lowcloning efficiency is mostly attributed to aberrant nuclear reprogramming of the donorcell by oocytes. In the present study, the pathogenic mechanisms of developmentalabnormity in SCNT cattle were analyzed, and then how to improve the somaticcloning efficiency through enhancing nuclear reprogramming was analyzed.1. The expression and DNA methylation status of imprinted genes in various tissuesfrom deceased cloned calves were analyzed using Quantitative RT-PCR (qRT-PCR),Bisulfite Sequencing PCR (BSP) and Combined Bisulfite Restriction Analysis(COBRA). The results showed that fetal weigh, placental weight and meanplacentome weight were larger, but the number of placentomes was smaller in thedeceased calves with Large Offspring Syndrome (LOS group) compared with thecontrol group. The XIST, PEG3and IGF2were significantly over-expressed inmultiple tissues (e.g. placenta) from the LOS group, whereas expression of H19andIGF2R was significantly reduced in multiple tissues (e.g. placenta) from the LOSgroup compared with controls. In multiple tissues (e.g. placenta) from the LOS group,H19DMR was significantly hypermethylated, but XIST DMR and IGF2R ICR weresignificantly hypomethylated compared with controls. We inferred that abnormalexpression of the imprinted genes in deceased calves could be associated with alteredDNA methylation levels at the DMRs of these imprinted genes. These alterationscould result from aberrant epigenetic nuclear reprogramming during SCNT and maydisrupt normal developmental regulation of placenta and fetus, which may result indevelopmental insufficiencies and ultimately fetal or perinatal death in cloned transgenic calves.2. The pathogenic mechanism of developmental abnormity and death in SCNTcattle was analyzed in placenta from deceased cloned calves (SCNT group) andnormally produced female calves (control group) using Solexa sequencing.(1) Gene expression difference analysis between SCNT and control sample usingRNA-seq (Q) HiSeq high-throughput sequencing revealed664up-regulated and1948down-regulated genes in SCNT group compared with control group. To investigatethe function distribution of differentially expressed genes (DEGs), we performed theGene Ontology (GO) enrichment analysis for the DEGs. DEGs were enriched in thefunctions of catalytic activity. DEGs were enriched in the cellular component of MHCprotein complex and cell surface. DEGs were enriched in the biological process of13GO term, most of them were related with immune system, for instance, immunesystem process, antigen processing and presentation of peptide antigen, immuneresponse, positive regulation of immune system process, and activation of immuneresponse, etc. Pathway enrichment analysis of DEGs showed that25Pathway weresignificantly enriched pathway, and most of them were related with immune system,for instance, Graft-versus-host disease, Allograft rejection, Antigen processing andpresentation, NOD and Toll-like receptor signaling pathway, Leukocytetransendothelial migration, and Autoimmune thyroid disease, etc.(2) The microRNA information was analyzed by small RNA-seq in SCNT andcontrol placentas.328and344unique known bovine microRNAs were identified inSCNT and control placentas, respectively.169unique predicted novel bovinemicroRNAs were found in bovine placentas.135unique known microRNAs weredifferentially expressed between two samples,18microRNAs were up-regulated and117microRNAs were down-regulated in SCNT placentas compared with controls.49unique novel microRNAs were differentially expressed between two samples,35microRNAs were up-regulated and14microRNAs were down-regulated in SCNTplacentas compared with controls.(3) The genome-wide patterns of DNA methylation were analyzed by MeDIP-seqin SCNT and control placentas. The genome-wide map of DNA methylation inruminants (cattle in this study) was, for the fist time, reported in the present study.Generally, cattle display analogous methylation pattern with that of other animals andplants. DNA methylation is enriched in the gene body regions (especially introns) andthe repetitive sequences, and depleted in the transcription start site (TSS) and the transcription termination site (TTS). Most of the CpG islands in the cattle genomewere kept in unmethylated state. There were many differentially methylated genesbetween SCNT and control placentas.37,191CpG islands were found in bovineplacentas.7,482and5,461methylated CpG islands were found in control and SCNTplacentas, respectively. The DNA methylation level of promoters was negativelycorrelated with the gene expression level, indicating its suppressive role in regulatinggene transcription.3. Aberrant reprogramming was found in bovine cloned embryos. The XIST, OCT4,SOX2, NANOG, Rex1, and Fgf4were significantly hypomethylated in SCNTblastocysts compared with IVF blastocysts. The XIST was significantlyover-expressed in SCNT blastocysts compared with IVF blastocysts. The expressionof H3K27me3was aberrant in SCNT blastocysts compared with IVF blastocysts.4. Global gene expression profilings were analyzed in SCNT blastocysts from celllines with high cloning efficiency (high efficiency group), SCNT blastocysts from celllines with low cloning efficiency (low efficiency group), parthenogenetic blastocysts(parthenogenesis group), and IVF blastocysts (IVF grpoup). TCF7, DGCR8, SENP7,DNMT3B, Vimentin, IFI6, EFNA2, F5, IRAK1, PRICKLE1, TNFRSF1A, ARG2,NEK6, and other genes were abnormally expressed in low efficiency group, whichnormally expressed in high efficiency group, compared with the IVF grpoup. Therewere240differentially expressed genes between parthenogenesis group and IVFgrpoup. Expression of PEG3and SNRPN, the paternally expressed imprinted genes,were significantly reduced in parthenogenetic blastocysts compared with the IVFblastocysts.5. The differential bovine oocytes with different nuclear reprogramming capacitywere selected by Brilliant cresyl blue (BCB) staining. The results showed that oocytesof various growth stage selected by BCB staining yield different nuclearreprogramming capacity. The cloned embryos developed from oocytes of finishedgrowth phase (BCB+) showed higher cleavage rate, blastocyst rate, pregnancy rateand full term development rate than these developed from growing oocytes (BCB).Oocytes of finished growth phase support higher developmental competence ofbovine cloned embryos in vitro and in vivo. The global acetylation levels of H3K9and H3K18and the global methylation levels of H3K4in BCB+embryos at thetwo-cell stage were higher than in BCB embryos. BCB+embryos generated moretotal cells, trophectoderm (TE) cells, and inner cell mass (ICM) cells, and fewer apoptotic cells than BCB embryos. The gene expressions in BCB+and BCB blastocysts were also different. These results strongly suggest that BCB+oocytes(finished growth phase) have a higher nuclear reprogramming capacity, and that BCBstaining can be used to select developmentally competent oocytes for nuclear transfer.6. The effects of Oxamflatin, a novel HDACi, on the nuclear reprogramming anddevelopment of bovine SCNT embryos in vitro were analyzed. The results showedthat Oxamflatin treatment (the SCNT embryos treated with1μM Oxamflatinpost-ionomycin for12h) increased the global acetylation levels of H3K9and H3K18and the global methylation levels of H3K4, but decreased the the global methylationlevels of H3K9, reduced the DNA methylation level of satellite I in SCNT blastocysts,increased total and inner cell mass (ICM) cell numbers and the ratio ofICM:trophectoderm (TE) cells, reduced the rate of apoptosis in SCNT blastocysts,modified gene expression and significantly enhanced the development of bovineSCNT embryos in vitro. The study showed that Oxamflatin modifies epigenetic statusand gene expression, increases blastocyst quality, and subsequently enhances thenuclear reprogramming and developmental potential of SCNT embryos.7. The SCNT embryos derived from cell lines of normal expression group (TCF7,DGCR8, SENP7, DNMT3B, Vimentin, IFI6, EFNA2, F5, IRAK1, PRICKLE1,TNFRSF1A, ARG2, NEK6, OCT4, NANOG, SOX2, CDX2, XIST, and IGF2R werenormally expressed in SCNT blastocysts derived from these cell lines compare withIVF blastocysts) showed higher blastocyst rate, pregnancy rate and full termdevelopment rate than SCNT embryos derived from cell lines of two abnormalexpression groups (abnormal expression group1: low efficiency group in4.above-mentioned, TCF7, DGCR8, SENP7, DNMT3B, Vimentin, IFI6, EFNA2, F5,IRAK1, PRICKLE1, TNFRSF1A, ARG2, NEK6, and other genes were abnormallyexpressed in SCNT blastocysts derived from these cell lines compare with IVFblastocysts; abnormal expression group2: OCT4, NANOG, SOX2, CDX2, XIST andIGF2R were abnormally expressed in SCNT blastocysts derived from these cell linescompare with IVF blastocysts). Expression profiling analysis of TCF7, DGCR8,SENP7, DNMT3B, Vimentin, IFI6, EFNA2, F5, IRAK1, PRICKLE1, TNFRSF1A,ARG2, NEK6, OCT4, NANOG, SOX2, CDX2, XIST, and IGF2R in cloned embryoscould be as a method to select good donor cell lines.Aberrant reprogramming, including alterations in gene expressions, DNAmethylation status of genes, histone modifications, and microRNAs in cloned embryos or cloned calves, was found during bovine somatic cloning, which maysubsequently result in developmental insufficiencies and ultimately fetal or perinataldeath in cloned calves. The reprogramming efficiency and even somatic cloningefficiency could be enhanced by selecting good donor cells, selecting oocytes withhigher nuclear reprogramming capacity and the treatments of cloned embryos withepigenetic drugs.