Studies Of Somatic Cell Nuclear Transfer And Mitochondrial Fate

Interspecies somatic cell nuclear transfer (iSCNT) breachs the rule of "reproductive isomatic" among animal species and involves in the interactions of species in molecular, cellular and physilogical level. It is a new approach to research the mechanism of nucleus-cytoplasmic interaction,and rescue those highly endangered species. In addition, it may be a hopeful approach to revive those extinct species in the world. However, the heteroplasmy of mtDNA in iSCNT embryos caused by whole cell electro fusion during iSCNT is an important issue. In order to improve the efficiency of iSCNT, in this dissertation, studies have investigated mitochondrial fate in iSCNT embryos.Experiment 1: Preparation of bovine and ovine specific primers. The soft primer 5.0 was used to designed two sets of species-specific primers. MtDNA used as PCR template after lysising oocytes. PCR products were integrated into pMD18-T vectors and cloned in DH5-a. Seven 10-fold dilutions for bovine and six 10-fold dilutions for ovine were made in order to set up a standard curve for PCR quantification. The results indicated DNA standards were well proportional and the standard curves were generated from seven or six points spanning the expected unknown values.Experiment 2: Effects of copy numbers of bovine oocyte mtDNA on the quality of oocytes and subsequent embryos. We classified cumulus oocyte complexes(COCs) as good (G) and poor (P) quality oocyte based on cytoplasmic appearance and cumulus characteristics, and assessed mtDNA copy numbers in the G and P oocytes with real-time PCR. The results revealed that there was a significant difference in the mtDNA copy numbers between the G-oocyte (n=30) and P-oocyte (n=30). In the G oocyte group, the mean mtDNA copy number for cleaved embryos (n=30) was significantly higher than uncleaved embryos (n=18). Similarly, for P oocytes, the mean mtDNA copy numbers for cleaved embryos (n=30) was significantly different compared to uncleaved embryos (n=24). Therefore, G oocytes contant more mtDNA than that of P oocytes. In addition, there were higher cleavage rate, blastocyst formation rate and blastocyst cell numbers in G oocytes than those of P oocytes.Experiment 3: Effects of granulosa cell mitochondria transfer on the early development of bovine embryos in vitro. The mitochondria were isolate by fractionation and injected into the parthenogenetic embryos and ICSI embryos. The results showed a significant difference in mtDNA copy number between G (361 113±147 114) and P (198 293±174 178) oocytes (P<0.01). The rates of morula, blastocyst and hatched blastocysts derived from P-oocytes+mitochondria were similar to those of G-oocytes, but significantly higher than P-oocytes without exogenous mitochondria in both the 1CSI and parthenogcnetic activation experiments. We found no difference in blastomere numbers between G-oocytes and P-oocytes+mitochondria in either experiment, but blastomere numbers in these two groups were significantly higher than in P-oocyte groups without exogenous mitochondria. These data suggest that mtDNA content is very important for early embryo development. Furthermore, the transfer of mitochondria from the same breed may improve embryo quality during preimplantation development.Experiment 4: The fate of donor mitochondria following somatic cell nuclear transfer. Granulosa cells, fetal fibroblasts and adult fibroblasts labeled with MitoTracker Green fluorochrome were injected into enucleated bovine oocytes and cultured in vitro. The results indicated: l)MitoTracker labeling on donor cells did not have a detrimental effect on the development of reconstructed embryos following nuclear transfer; 2)There were no difference in embryonic development potential between three types of reconstructed embryos; 3)Prior to the 16-cell stage, the rates of embryo with fluorescence between different reconstructed embryos were not different. While following the 16-cell stage, the rate of reconstructed embryos with fluorescence derived from granulosa cell was lower than that of other two types of reconstaicted embryos. Therefore, the development potential of reconstructed embryos is not related with the type of donor cell, and the efficiency of mitochondria fusion in reconstructed embryo derived from granulose cell is better than in those derived from fetal and adult fibroblasts.Experiment 5: Reconstruction of interspecies somatic cell nuclear transfer embryos. We generated bovine- ovine reconstructed embryos via iSCNT using bovine oocytes as recipient cytoplasm and ovine fetal fibroblast as donor cells. Chromosome composition, the total cell number of blastocyst and embryonic morphology were analyzed. The results indicated the following: 1) cell nuclei of ovine fetal fibroblasts can dedifferentiate in enucleated bovine ooplasm, and the reconstructed embryos can develop to blastocysts; 2) 66% of iSCNT embryos had the same number of chromosome as that of donor cell, and the total cell number of iSCNT blastocysts was comparable to that of sheep parthenogenetic blastocysts. Experiment 6: Mitochondria segregation in blastomeres derived from bovine-ovine reconstructed. mtDNA copy numbers both from donor cell and recipient cytoplasm were assessed by real-time PCR in individual blastocysts and blastomeres from 1 -cell to 16-cell stage reconstructed embryos. The results indicated the following: 1) RT-PCR analysis in individual blastomeres revealed that the ratio of donor cell mtDNA : recipient cytoplasm mtDNA remained constant (1%) from the 1-cell to 8-cell stage. However, the ratio decreased from 0.6% at the 16-cell stage to 0.1% at the blastocyst stage; 4) Both donor cell- and recipient cytoplasm-derived mitochondria distributed unequally in blastomeres with progression of cell mitotic division. Considerable unequal mitochondrial segregation occurred between blastomeres from the same iSCNT embryos.