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Characterization and comparative analyses of transcriptomes of cloned and in vivo fertilized porcine pre-implantation embryos.

ABSTRACT: Somatic cell nuclear transfer (SCNT) is the only method known to rapidly reprogram differentiated cells into totipotent embryos. Most cloned embryos become arrested before implantation and the details of the underlying molecular mechanism remain largely unknown. Dynamic regulation of the transcriptome is a key molecular mechanism driving early embryonic development. Here, we report comprehensive transcriptomic analysis of cloned embryos (from Laiwu and Duroc pigs) and in vivo fertilized embryos (from Duroc pigs) using RNA-sequencing. Comparisons between gene expression patterns were performed according to differentially expressed genes, specific-expressed genes, first-expressed genes, pluripotency genes and pathway enrichment analysis. In addition, we closely analyzed the improperly expressed histone lysine methyltransferases and histone lysine demethylases during cell reprogramming in cloned embryos. In summary, we identified altered gene expression profiles in porcine cloned pre-implantation embryos in comparison to normal in vivo embryos. Our findings provide a substantial framework for further discovery of the epigenetic reprogramming mechanisms in porcine SCNT embryos.

SUBMITTER: He X

PROVIDER: S-EPMC6504007 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Characterization and comparative analyses of transcriptomes of cloned and <i>in vivo</i> fertilized porcine pre-implantation embryos.

He Xiaoyan X Tan Cheng C Li Zicong Z Zhao Chengfa C Shi Junsong J Zhou Rong R Wang Xingwang X Jiang Gelong G Cai Gengyuan G Liu Dewu D Wu Zhenfang Z

Biology open 20190423 4

Somatic cell nuclear transfer (SCNT) is the only method known to rapidly reprogram differentiated cells into totipotent embryos. Most cloned embryos become arrested before implantation and the details of the underlying molecular mechanism remain largely unknown. Dynamic regulation of the transcriptome is a key molecular mechanism driving early embryonic development. Here, we report comprehensive transcriptomic analysis of cloned embryos (from Laiwu and Duroc pigs) and <i>in vivo</i> fertilized e ...[more]

PMID: 30952695

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Project description:BackgroundIntergenic transcription, either failure to terminate at the transcription end site (TES), or transcription initiation at other intergenic regions, is present in cultured cells and enhanced in the presence of stressors such as viral infection. Transcription termination failure has not been characterized in natural biological samples such as pre-implantation embryos which express more than 10,000 genes and undergo drastic changes in DNA methylation.ResultsUsing Automatic Readthrough Transcription Detection (ARTDeco) and data of in vivo developed bovine oocytes and embryos, we found abundant intergenic transcripts that we termed as read-outs (transcribed from 5 to 15 kb after TES) and read-ins (transcribed 1 kb up-stream of reference genes, extending up to 15 kb up-stream). Read-throughs (continued transcription from TES of expressed reference genes, 4-15 kb in length), however, were much fewer. For example, the numbers of read-outs and read-ins ranged from 3,084 to 6,565 or 33.36-66.67% of expressed reference genes at different stages of embryo development. The less copious read-throughs were at an average of 10% and significantly correlated with reference gene expression (P < 0.05). Interestingly, intergenic transcription did not seem to be random because many intergenic transcripts (1,504 read-outs, 1,045 read-ins, and 1,021 read-throughs) were associated with common reference genes across all stages of pre-implantation development. Their expression also seemed to be regulated by developmental stages because many were differentially expressed (log2 fold change ≥ 2, P < 0.05). Additionally, while gradual but un-patterned decreases in DNA methylation densities 10 kb both up- and down-stream of the intergenic transcribed regions were observed, the correlation between intergenic transcription and DNA methylation was insignificant. Finally, transcription factor binding motifs and polyadenylation signals were found in 27.2% and 12.15% of intergenic transcripts, respectively, suggesting considerable novel transcription initiation and RNA processing.ConclusionIn summary, in vivo developed oocytes and pre-implantation embryos express large numbers of intergenic transcripts, which are not related to the overall DNA methylation profiles either up- or down-stream.

Project description:The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization (IVF) were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (> 2-fold vs. controls) than did the extraembryonic tissues (P < 1.0 × 10–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryo transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages, although these alterations were not always statistically significant for all three SCNT groups because of variability. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

Project description:The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.

Dataset Information

Characterization and comparative analyses of transcriptomes of cloned and in vivo fertilized porcine pre-implantation embryos.

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Characterization and comparative analyses of transcriptomes of cloned and <i>in vivo</i> fertilized porcine pre-implantation embryos.

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