Porcine SCNT Embryo Development And Its Early Gene Expression Influenced By Oocyte Nucleus

At present, the mammalian somatic cell nuclear transfer reprogramming efficiency is still very low, as the primary animal model for therapeutic cloning, pig nuclear transfer embtyonic stem cell lines is still not established. The technical bottleneck is that somatic cell nuclear reprogramming is not fully at all. The oocyte nucleus as maternal genetic material has huge influence on embryonic development and even somatic cell reprogramming. Therefore, on the background of pig nuclear transfer system, to detect intact oocyte SCNT embryo's development in vitro, combining with high throughput sequencing technology, research the specific gene expression patterns in the efficient reprogramming system, and lay the foundation of SCNT embryo reprogramming mechanism in nuclear transfer embryo as well as the further research of functional genes, hope to find out the key point to improve the efficiency of somatic cell reprogramming. The main contents of this research are as follows:1. Pig egg's influence on nuclear transfer embryo development and the efficiency of somatic cell reprogrammingBuilding pig intact oocyte SCNT embryo, observe its development potential. We totally injected 539 porcine cumulus cells into the intact oocytes, at the same time, we injected 461 porcine cumulus cells into enucleated oocytes. After fusion and activation, we observed 260 polyploid reconstructed embryos develop into blastocysts, and then only 93 enucleated oocyte SCNT embryos reached the blastocyst stage. This result indicated that the cleavage rate(81.15% vs 91.05%) and blastocyst rate (20.15% vs 48.29%) of intact oocyte SCNT reconstructed embryos had a significantly increase, especially the blastocyst rate had improved more than two times.2. Identify chromosome number of the donor cell, enucleated oocyte SCNT embryo, and intact oocyte SCNT embryoWe identified the chromosome number of somatic cells for transplantation, enucleated oocyte SCNT reconstructed embryos and intact oocyte SCNT reconstructed embryos respectively, to ensure the chromosome number are all in line with expectations (38/38/57, the chromosome number of normal pig is 38). Some intact oocyte SCNT reconstructed embryos shows the characteristic of tetraploid (76), we speculated that when the reconstructed embryos were electric-activated, the sister chromatid of oocytes separated, but the second polar body were not extruded.3. The differences of morphology and cell number of blastocyst between intact oocyte SCNT reconstructed embryo and enucleated oocyte SCNT reconstructed embryo, and parthenogenetic embryoWe made a statistics of two kinds of reconstructed blastocysts'total cell number, and took the parthenogenetic blastocysts as control. We found that the average cell numbers of enucleated oocyte SCNT blastocysts are the least (43.30), parthenogenetic blastocysts are at most (58.60), and intact oocyte SCNT blastocysts are between them (56.95). In morphology, the parthenogenetic blastocysts have a larger diameter and a lighter color, the enucleated oocyte SCNT blastocysts and intact oocyte SCNT blastocysts have a smaller diameter and darker color. Moreover, most of the intact oocyte SCNT blastocysts are hatched blastocysts and have an "8" shape. But a majority of the enucleated oocyte SCNT blastocysts are expanded blastocysts, not hatched.4. The influence of Mechanical damage to nuclear transfer embryonic developmentWe through the method that making small holes in the oocyte, simulate the mechanical damage in the process of nuclear transfer, to verify whether the inevitable mechanical damage is the principal factor to affect embryonic development. The results show that mechanical damage can result in decrease of cleavage rate and blastocyst rate of parthenogenetic embryos (cleavage rate: 93.136% vs 95.032%, P?0.36; blastocyst rate:56.14% vs 64.42%, P?0.014), and blastula cells were reduced (52.65 vs 55.40). The exclusion of mechanical damage still do not constitute all the factors of a significantly enhanced viability of intact oocyte SCNT embryos.5. The influence of chromosome doubling on the effect of embryonic development and whether the oocyte nucleus could be replaced by another somatic cell nucleusWe replace the oocyte nucleus with somatic cell nucleus in the intact oocyte SCNT mbryo, and inject another somatic cell to build polyploid embryo, to confirm whether intact oocyte SCNT embryo development advantage is only caused by chromosome doubling and whether oocyte nucleus can be replaced by any other somatic cell nucleus, the blastocyst rate of polyploid embryos after replacing was only 10.5%, far below polyploid embryos constructed with maternal nucleus and somatic cell nucleus (50.6%). These data indicates that in the intact polyploid embryo model, oocyte nucleus plays an extremely important role in the molecular level and is irreplaceable.6. The relationship of ovary type to oocyte developmental potentialWe respectively collect the ovarian with or without red body,to detect their maturing rate and blastocyst rate, found that the red body ovarian eggs with high development efficiency, the oocyte mature rate (75.77%vs 68.97%, P>0.05)? cleavage rate(95.33%vs 94.00%, P>0.05)? blastocyst rate(68.67%vs 59.00, P>0.05).Then we can choose the ovaries with red body for oocytes, and then build nuclear transfer embryos, for subsequent gene expression profiles.7. Build the gene expression profile of intact oocyte SCNT embryo and enucleated oocyte SCNT embryoIn order to explore the molecular mechanism of oocyte nucleus'role in somatic cell reprogramming, and understand the specific gene expression patterns of two kinds of nuclear transfer embryos, we collected enucleated oocyte SCNT embryos and intact oocyte SCNT embryos respectively, we choose the developmental block stage (4cell) and the first cleavage stage (2cell) to proceed single cell gene expression spectrum sequencing (single-cell RNA sequencing).8. Differentially expressed gene screening and analysisTwo kinds of embryos have big differences in transcriptional level. In 2cell stage, compared with enucleated oocyte SCNT embryo, intact oocyte SCNT embryo has 1738 genes up-regulated,728 genes down-regulated (?log2Ratio??5). In 4cell stage, compared with enucleated oocyte SCNT embryo, intact oocyte SCNT embryo has 2941 genes appear up-regulated,1682 genes down-regulated (?log2Ratio??5). WEGO analysis of differently expressed genes shows that the deepest enrichment degrees of gene cluster are focus on binding regulation, catalytic activity and molecular transduction activity, respectively. Other genes appeared up-regulation in intact SCNT embryo are most involved in various metabolic processes. Finally, we found the Ribosome and Oxidative phosphorylation pathway show down-regulate in enucleated oocyte SCNT embryo and up-regulate in intact oocyte SCNT embryo. And then the signal path of protein processing in endoplasmic reticulum had an obvious enrichment in the differently expressed genes KEGG pathway results at 4cell stage. Endoplasmic reticulum closely near to the nucleus structure. In the process of enucleation, the endoplasmic reticulum may be also removed. Thus we speculate that the deficiency of protein processing in endoplasmic reticulum, ribosome and oxidative phosphorylation may be the primary causes why enucleated oocyte SCNT embryo shows the development of disadvantage compared with intact oocyte SCNT embryo.9. Expression pattern analysis of transcription activity genes expressed in nucleus part267 transcriptional activity related genes in DEGs (differentially expressed genes) were identified expressed in nuclear part. Most of these genes show faint expression in enucleated oocyte SCNT embryo, but have a significantly up-regulate in intact oocyte SCNT embryo.10. Gene expression quantity verification by QRT-PCRWe randomly selected six high expression genes:DNMT1, FTL, POLR1D, RPS3, RPS20 and BUB3 for QRT-PCR detection respectively. Then make a comparison between test results and RNA-seq results, found that 6 genes expression trend are basically identical under two kinds of detection methods.11. Endoplasmic reticulum fluorescent tracer testWe stained the M ? matured oocytes and enucleated oocytes with endoplasmic reticulum red fluorescent probe ER-Tracker, to compare the endoplasmic reticulum structure in two cells and identify whether endoplasmic reticulum was removed in the process of enucleation.