Effect Of Nuclear-Cytoplasmic Interaction On Donor Nuclei Reprogramming

Somatic cell nuclear transfer (SCNT) has wide applications in agriculture and biomedical research. Yet low efficiency of cloning remains the major obstacle to the development and widespread use of this technology. SCNT includes complicated protocols; therefore many variables will have great effects on nuclear transfer efficiency. Intensive investigation has been performed on the nuclear transfer procedures, which includes but is not limited to enucleation, fusion, activation, and in vitro culture system of embryos. However, in vitro development rates of cloned embryos vary extensively and cloned fetus often fails to develop to term. Besides improvement of the SCNT procedures, thus, attention should be paid to the source, quality and epigenetic state of initial biological materials for producing cloned embryos. Therefore, investigation on donor cells, oocytes and nuclear-cytoplasmic interaction was performed in the present study.In the present study, we first optimized the in vitro maturation system of bovine oocytes. Bovine oocytes were exposed to four different hormonal regimes in TCM-199 medium. No significant difference was observed in maturation rates in four groups. Human menopausal gonadotropin (HMG) provided steady maturation results in replicates. Maturation of oocytes was promoted by supplementation with 17β-estradiol to some extent. Combination of HMG and E2 gave rise to steady and efficient mature results. Then we studied the effect of epidermal growth factor (EGF) present in the above defined medium on bovine oocyte maturation, in vitro development and quality of parthenogenetic embryos. The results showed that the presence of EGF at 30 ng/ml concentration significantly increased maturation rate and blastocyst rate and reduced apoptotic cells in parthenogenetic blastocysts. Based on the above results, the final oocyte maturation (OM) solution were TCM-199 supplemented with 0.075 IU/ml HMG,1μg/ml E2,30 ng/ml EGF,10%(v/v)FBS.Then the present work investigated changes to G0/G1 phase of donor cells treated with serum starvation or contact inhibition on in vitro development and quality of nuclear transfer embryos. No significant difference was observed in fused oocyte rate, cleavage rate and blastocyst rate between serum starvation and contact inhibition. However, more apoptotic cells were detected in blastocysts derived from donor cells treated with serum starvation. The passages of donor cells had an adverse effect on nuclear transfer. The 2nd to 5th passages of cell line were better than the other passages. Especially, repetitious passage impaired embryo competence and increased apoptotic rate in nuclear transfer blastocysts. Therefore, in this study, the 2nd to 5th passages of cell line were utilized as nuclei donors.The present study investigated the effects of donor cells pretreated with oocyte extracts on in vitro development of cloned embryos. Bovine fibroblasts were exposed to immature, mature and parthenogenetic oocyte extracts respectively before nuclear transfer. The detectable expression of totipotent genes and global deacetylation in the treated cells showed that extracts could reprogram fibroblasts. Although all three groups of extracts exhibited reprogramming capacity, embryo development was not compliant with reprogramming effect. Improved quality and development of blastocysts were observed only in the mature extract treated group. We demonstrated that pretreatment of donor cells with mature oocyte extract improved in vitro development of cloned embryos. The present study demonstrated that it was synchronization between the epigenetic state of donor nuclei and recipient cytoplasm that played a more important role in nuclear-cytoplasmic interaction. Our results suggested that, rather than reprogramming donor nuclei to a lower differentiation state, reprogramming donor nuclei to a state synchronised with recipient cytoplasm before nuclear transfer would be beneficial for the development of cloned embryos.The present study utilized mouse and bovine, huge differences in gene expression patterns and developmental program existed between the two species, to investigate the modulation of donor nuclei and cytoplasm on early embryogenesis. Mouse-bovine interspecies nuclear transfer embryos were constructed. The timing of in vitro development and morphology of heterogenous embryos and expression patterns of zygotic activated genes in heterogenous embryos were assessed. The results showed that the development of interspecies cloned embryos was modulated by bovine ooplasm before zygotic gene activation (ZGA). At this time, heterogenous embryos presented bovine phenotype in the timing and morphology. ZGA of mouse nuclei were controlled by bovine ooplasm and would be activated until 8-cell embryo, furthermore, this activation was incompletely. Retardant and incomplete activation of zygotic genes on mouse nuclei might result in 8-cell block of mouse-bovine interspecies nuclear transfer embryos.Finally, the present study investigated the differential detrimental effects of heat shock on the nuclear or cytoplasmic component of bovine embryos during the first cell cycle. The parthenogenetic embryos were incubated at 38.5 or 41℃for 7 h, respectively; then the nuclei of embryos cultured at 41℃were transferred into enucleated 38.5℃cytoplasts to construct heat shock nuclei-normal cytoplasm embryos (HN embryos) and 38.5℃nuclei were transferred into 41℃cytoplasts to construct heat shock cytoplasm-normal nuclei embryos (HC embryos). In vitro culture showed that blastocyst formation rate for HC embryos decreased significantly compared to control parthenogenetic embryos and HN embryos. Incomplete activation of zygotic genes was observed in nuclear exchange embryos, especially in HC embryos. It suggested that heat shock might disrupt some cytoplasmic factors required for zygotic gene activation. Heat shock induced apoptosis was observed in both HN and HC blastocysts and the incidence of apoptosis in HC blastocysts was significantly higher than that in HN embryos. These results suggested that, although heat shock injuries existed in both nuclear and cytoplasmic components; cytoplasmic component is more susceptible to heat shock than nuclear component.