Studies On Porcine Somatic Nuclear Transfer And Some Related Problems

Nuclear transfer technique is important not only for the study of nuclear-cytoplasm interaction, but also has extensive applications in human clinics and animal husbandry. it has been proved that porcine somatic cloning is the most difficult among all mammalian species. In this experiment, we studied porcine cloning by transplantation of cumulus cell and cultured granulosa cell nuclei into enucleated in vitro matured oocytes, and some issues related to it, such as oocyte activation, cell fusion and oocyte maturation. The results are as follows:1.Premature chromosome consendesion (PCC) occurred when the cumulus cell nuclei was transferred into enucleated oocyte by directly injection or cell fusion. Similiar changes occurred in nuclei when they were transferred into enucleated oocytes either by direct injection or by cell fusion. But there was a more advanced development when the nuclei were transferred by cell fusion. Nuclear swelling occurred in 48% of the reconstructed embryos 4 hours after electrical fusion, but 10.8 %reconstructed embryos underwent nuclear swelling 4 hours after injection.2.Nuclear transfer embryos reconstructed with cumulus cell nuclei developed into blastocyst in surrogate animals.3.When the porcine-porcine and porcine-mouse nuclear transfer embryos were activated, in the former only one pronucleus was observed in most cases, but in the later two pronuclei were found mostly.4.Matruted oocyte can be effectively activated by lonomycinl6-DMAPor Ionomycin alone and the activation rates reached 84.3 o/o and 88.9 % ,respectively; there was no significant difference in activation rates when oocyte was activated by electronic pulse/ 6-DMAP and Ionomycini6-DMAP methods ,but the cleavage rate of oocytes activated with electronic pulse/6DMAP was significantly higher than those activated by lonomycin/6-DMAP.5.The-fusion rate was improved when mild strength and long duration pulses were applied and was markedly higher than that obtained with pulses of strong strength and short duration.6.Four types GV chromatin configuration were observed with Hocest33342 staining in the porcine oocytes.With the follicular development, the proportion of GV-l oocyte was increased. Qocytes from the pre-antral follicles were all at GV-0 stage. The proportion of GV-3 oocyte was increased with time during in vitro maturation. No GV-3 stage was observed, but GV-2 stage was found in vivo 18-24 hours after hCG injection.7.The different GV chromatin configuration was not synchronized when A and B types of oocytes were pre-cultured in absence of hormone for 12 hours, but the proportion of maturedoocyte in A types with intact polar body was increased.8.Hormone level in maturation medium affected cumulus expansion of COC, but cumulus expansion became less dependent on hormone level with time of culture. Porcine immatured oocytes, granulosa cells from small antral follicles and cumulus cells of immatured oocytes secreted cumulus-expansion enabling factors. Granulosa cells from 3-6mm follicles did not produce CEEF, but there was some CEEF in the follicular fluid of this sized follicles.