Published On: Tue, Apr 23rd, 2019

Characterization and comparative analyses of transcriptomes of cloned and in vivo fertilized porcine pre-implantation embryos [RESEARCH ARTICLE]


Multi-omics sequencing of various species has developed rapidly, and several recent studies have reported the gene expression dynamics of human and mouse pre-implantation embryos by RNA-Seq, which has important implications for the study of embryonic development mechanisms (Petropoulos et al., 2016; Stirparo et al., 2018; Xue et al., 2013; Yan et al., 2013). However, in the study of porcine embryo development, there are very few reports highlighting the whole genome gene expression regulatory mechanism of the pre-implantation of cloned embryos or normal embryos. Here, we utilized RNA-Seq to systematically characterize the transcriptome profiles of porcine matured oocytes developing into the blastocyst stage. The developmental efficiency of cloned embryos may differ from that of in vitro culture conditions, but to avoid any additional stress on cloned embryos due to surgical transplantation and embryo flushing from the porcine uterus, we cultured the cloned embryos in vitro and rigorously selected embryos with better morphology, no fragmentation and homogeneous blastomeres for the construction of sequencing libraries (Cagnone and Sirard, 2016; Østrup et al., 2013). Quality control after cDNA amplification during the construction of all the sequencing libraries showed normal peaks and no RNA degradation. Consequently, our cloned embryo samples were suitable for studying the typical characteristics and defects in gene expression in the cloned embryos at each specific cleavage period. In this study, in vivo fertilized embryos were used as a control group. Utilizing fertilized embryos offers advantages, as the development efficiency of in vivo derived porcine embryos is higher compared to in vitro fertilized embryos, which are commonly used as controls in multiple publications (Bauer et al., 2010; Lee et al., 2018). In pigs, the efficiency of in vitro fertilization is very low because of several problems, such as incomplete maturation of oocytes after in vitro maturation, a high incidence of polyspermy and poor quality of blastocysts (Grupen, 2014). Additionally, although it is technically feasible to perform RNA-Seq on a single embryo or even a single cell, for this experiment, we decided to prepare each sample with about ten oocytes or embryos. Gene expression patterns between the individual embryos may exhibit individual differences, and some embryos may have cytogenetic abnormalities, such as gene mutations, which may directly affect their gene expression profiles. Transcriptome analysis of a single embryo or of a single blastomere may reflect cellular abnormalities within that particular embryo. Without many biological duplicates, using multiple mixed embryos is more suitable for the analysis of transcriptome characteristics at a particular developmental stage (Graf et al., 2014). Since the sample preparation had to be completed in a very short time, we did not conduct gender identification for IV-D embryos. All male and female embryos of the same cleavage period collected from a pregnant sow were mixed into a single sample for sequencing. However, it is important to note that the NT-LW and NT-D embryos collected for RNA-Seq were all male. Gender differences between the three groups of embryos may also result in differences in gene expression, especially for genes located on the sex chromosomes. Based on the analysis of our transcriptomic data of single 4-cell embryos (unpublished data), the sex of in vivo fertilized 4-cell embryos mainly influenced gene expression levels on the sex chromosomes, but other autosomal genes were not affected. Therefore, we only considered the influence of gender on sex chromosomes. The results described above indicate that the RNA integrity of our embryonic samples was high, resulting in good quality in the sequencing libraries. The mapping rate of all sequenced clean reads to the reference genome was more than 90%, and the average number of genes detected more than 10,000 (with FPKM value1). These results are comparable with other single-cell RNA-Seq methods reported in previous studies (Biase et al., 2014; Wang et al., 2017b), indicating that the transcriptome sequencing method used in this experiment is likely valid, the reliability and accuracy of the transcriptome data are high and this analysis has the potential to provide meaningful reference data for future research.

After the donor cells were transferred to the recipient oocyte, the cloned embryos underwent nuclear silencing of gene expression, epigenetic memory erasure in somatic cells, embryonic genome activation and reestablishment of new epigenetic modifications (Saitou et al., 2012; Xu and Xie, 2018). Donor cells determine the correctness and completeness of the nuclear reprogramming process, which is one of the most critical factors affecting cloning efficiency. Many previous studies have shown that donor cell type, differentiation, passage number and epigenetic modification state can all influence cloning efficiency (Bonk et al., 2007; Hirasawa et al., 2013; Zhai et al., 2018). Using several years of statistical analyses of pig cloning experimental data, our research team discovered that the cloning efficiency of Laiwu pigs was significantly higher compared to that of Duroc pigs (Li et al., 2013c). Laiwu pigs are one of the most prolific pig breeds in China. Duroc sires imported from the United States are utilized most frequently as a terminal/paternal sire in the three-breed terminal cross-breeding program. Duroc boars are the predominant terminal sires worldwide, and they provide 100% heterosis when mated to Yorkshire×Landrace F1 females. According to the results of the preliminary experiments, the blastocyst development efficiency of the Laiwu and Duroc cloned 1-cell embryos collected in our sequencing experiment reached 30% and 20%, respectively, indicating that a significant difference exists between the cloning efficiency of the two donor cell lines. In this study, we compared the differences in gene expression patterns of NT-LW and NT-D embryos. We sought to identify the intrinsic molecular mechanisms that underlie differences in cloning efficiency between these two different breeds of pigs. Our analysis indicated that the number of genes that failed to be appropriately activated in the 4-cell NT-LW embryos was significantly lower compared to the NT-D embryos. Another important feature of the NT-LW embryos is that the pattern of pluripotent gene expression changes from the cloned 1-cell embryo to the blastocyst stage is more similar to a normally fertilized embryo. In particular, the variation tendency of MYC, NANOG, KLF4, BMP4 and POU5F1 in the early development of the NT-LW embryos and normal embryos was consistent while the dysregulation of pluripotent genes in the NT-D embryos was more variable. These pluripotent genes are key candidates that can drive the development of early embryos, which can likely explain the higher developmental potential of NT-LW embryos.

In the present study, we obtained the dynamic transcriptional profiles of porcine cloned and in vivo fertilized embryos. Our data provide comprehensive insight into the aberrant gene expression patterns in porcine cloned embryos produced via SCNT. The profiling of DEGs, identified pathways and key TFs will facilitate in-depth understanding the molecular mechanisms underlying the failure of SCNT. The dysregulated critical genes observed in cloned embryos could serve as potential candidate genes for the embryonic competence gene markers selection and verification. We identified many previously unknown genes abnormally expressed in cloned porcine embryos at seven cleavage stages. Dozens of TFs were found to be abnormally expressed in cloned porcine embryos at specific stages, of which several TFs (MYC, TP53, TET1, MYCN, SUV39H2, KDM4A, KDM4D, KDM5B) have been reported to play essential roles in mouse and human early cloned embryo development, but are not well understood in pigs (Liu et al., 2016; Matoba et al., 2014; Matoba and Zhang, 2018). We have found the top significant pathways associated with the DEGs in cloned and normal embryos to include oxidative phosphorylation, mitochondrial dysfunction, protein ubiquitination and cholesterol biosynthesis. In addition, we focused on genes related to histone lysine methylation, which is a hot topic in the study of improving mouse SCNT efficiency in recent years. For this reason, we provide evidence that H3K4 and H3K9 methylation contribute to resistance to transcriptional reprogramming in cloned porcine embryos.

In summary, for the first time, we reported the whole genome transcriptomes of the three sources of porcine pre-implantation embryos, including Laiwu and Duroc cloned embryos and in vivo fertilized Duroc embryos. Our data not only provided a valuable resource for dissection of gene regulatory mechanisms underlying the development of porcine embryos, but also identified novel molecular defects and transcriptome changes during SCNT reprogramming. Further work based on these results might significantly increase the efficiency of pig SCNT technology.

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