Study On Nuclear Reprogramming Of Somatic Cell Nuclear Cloned Bovine Embryos

Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Successful SCNT requires erasing the inherent differentiation memory or program, present in differentiated donor nuclei and reprogram dedifferentiated state of nucleus to development-specific totipotent pattern like the normal embryos. All these changes refer to remodeling. Differentiated donor cells were difficult to accomplish such a lot of work in a short period, so it was one of the reasons of low SCNT efficiency. In this study, we used confocal microscopy to investigate the distribution pattern of Ca²⁺ and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization (IVF), parthenogenetic activation (PA) and SCNT embryo development. To investigate the effect of MG-132 on the nuclear remodeling of donor cells after transferring into bovine oocytes.1. The establishment of an ear marginal fibroblast cell from Nanyang bovineAn ear marginal fibroblast cell was established from Nanyang bovine using a primary explant technique and cell cryopreservation biotechniques. Cell morpHology, dynamic growth and contamination were tested, and the karyotype, levels of isoenzymes of lactic dehydrogenase and malic dehydrogenase were analyzed.2. Ca²⁺ and its dynamic changes in the processes of bovine oocytes maturation, in IVF, PA and SCNT embryo development.In this study, we used confocal microscopy to investigate the distribution pattern of Ca²⁺ and its dynamic changes in the processes of bovine oocytes maturation, IVF, PA and SCNT embryo development. During the germinal vesicle (GV) and GV breakdown stage, Ca²⁺ was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca²⁺ was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca²⁺ was present throughout the blastomere. In PA embryos, Ca²⁺ was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell pHase and the morula. At 6 h after activation, there was almost no distribution of Ca²⁺ in the SCNT embryos. However, Ca²⁺ was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca²⁺ showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca²⁺ location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate.3. The effect of MG-132 on the nuclear remodelingTo investigate the effect of MG-132 on the nuclear remodeling of donor cells after transferring into bovine oocytes. We studied the role of MG-132 by examination the developmental potential of reconstructed embryos that got from nuclear transfer. The entire process of nuclear transfer was acting by MG-132, we verify the results by immunofluorescence. The reconstructed embryos 4 h after fusion showed strong lamin staining of the control group. The group of adding MG-132 at 4h after fusion there showed patchy nuclear membrance attaining and significant chromatin condensation. The results indicate that adding of 5μmol/mL MG-132 in SCNT might promote the reprogramming of reconstructed embryos. Then 5μmol/mL MG-132 was addedn in nuclear transfer operation. We compared the expression of lamin A/C in PA,IVF and SCNT embryos. The results indicate that lamin A/C is an indicator of erroneous reprogramming. Lamin A/C signal was negative in early reconstructed embryos, expressed in 8-cell stage. Lamin A/C was expressed in tropHectodermal cells of blastocysts, but signal was diminished in ICM. This is same to IVF embryos of lamin A/C expression. It proved that adding of MG-132 can improve reprogramming. In SCNT embryos, no lamin A/C was expressed in 4-cell stage. It began to express in 8-cell stag; Lamin A/C was expressed in 16-cell stage, morula stage and blastocysts. Signal was expressed in trophectodermal cells and ICM of blastocysts. But in IVF embryos, Lamin A/C was expressed in trophectodermal cells of blastocysts, but signal was diminished in ICM. It need further study to define the different expression in blastocysts effecton the embryonic development.4. The expressiong of Oct-4 in IVF and SCNT embryosOct-4 was almost no expressed in 2-4 cell phase, and expressed from 8-16 cell stage. Strong oct-4 signal was expressed in morula and blastocysts. The expression of oct-4 in hatching blastocyst stage was same to hatching blastocyst stage, but it was not decreased in trophoblast cells. The initial expression of SCNT oocytes was same to IVF, but the expression was not decreased in trophoblast cells in IVF oocytes. If this different expression will affect the development of post-implantation embryo, it remains to be study. This rearcher was investigated the distribution pattern of Ca2+ and its dynamic changes in the processes of bovine oocytes maturation, IVF, PA and SCNT embryos development, further research is expected to improve the SCNT efficiency. The research of lamin A/C expression showed that MG132 could be used for nuclear transfer process, and could improve the reprogramming of reconstructed embryos, and the expression of Oct-4 was same to IVF embryos.