Optimization of techniques required for somatic cell nuclear transfer using a mouse model

Introduction: Somatic cell nuclear transfer (SCNT) is the removal (enucleation) of chromosomes from a metaphase II oocyte, followed by the transfer and fusion of a donor somatic cell to the enucleated oocyte (cytoplast). The reconstructed oocyte is then activated to induce embryonic development. One of the main purposes of creating SCNT blastocysts is the derivation of embryonic stem cells (ESCs) for therapeutic cloning. The consensus for the low SCNT blastulation rate (12-15% in mice and 10% in humans) is epigenetic reprogramming failure of the donor somatic cell nucleus by the oocyte. The optimization and technical efficiency of SCNT was investigated in this study. Methods: Female B6D2F1 mice were used as oocyte and cumulus cell (somatic cell) donors. In study 1, non-invasive spindle imaging by Hoffman modulation contrast microscopy was used to identify the spindle within 484 oocytes. Once located, the spindles were enucleated. Successful enucleation was confirmed through artificial activation (317 cytoplasts) using calcium ionophores and kinase or protein synthesis inhibitors that caused fragmentation of the cytoplasts; and Hoechst DNA staining (167 cytoplasts) to microscopically confirm the absence of chromosomes. Appropriate controls for the techniques were included. In study 2, enucleation of 564 oocytes was followed by the transfer a single cumulus cell exposed to a membrane fusogen into close contact with the oolemma of the cytoplast. Post fusion, the reconstructed oocytes were artificially activated and subsequently cultured to the blastocyst stage, with the addition of a histone deacetylase inhibitor to aid epigenetic reprogramming. The reconstructed oocytes (80-90%) were expected to survive nuclear transfer; with 70-80% surviving activation, 60-70% pseudo-pronucleus formation; 50-60% cleavage to the 2-cell stage after 24 hours of nuclear transfer; and 30-50% development to the morula/blastocyst stage 72-96 hours post nuclear transfer. Results: The first enucleation confirmation technique was the analysis of cytoplast fragmentation between 16-18 hours after activation, which indicated that 85% of cytoplasts were effectively enucleated. A cohort of non-enucleated control oocytes confirmed the efficiency of the activation protocol by showing a pseudo-pronucleus formation rate of 95.4%. The second confirmation technique revealed an enucleation efficiency of 97.5%, which was confirmed by the absence of chromosomes in stained cytoplasts. The staining protocol was verified using a group of non-enucleated control oocytes, resulting in 100% of the oocytes presenting with stained and visible chromosomes. In study 2, enucleation was performed with a survival rate of 99.1%. The cytoplasts then underwent cumulus cell nuclear transfer with 100% survival. Subsequently, a fusion rate of 72.3% and an activation rate of 81.7% was achieved in this study. Blastocyst formation by SCNT was significantly lower than that of the control group (5.4% vs. 55.1%), and more poor-quality blastocysts were produced by SCNT (63.6%). Therefore, according to statistical analyses, the chance of forming a blastocyst by SCNT in this project was 0.041-fold that of the control group. Discussion: Results revealed that the rates of enucleation and nuclear transfer survival, fusion efficiency, activation survival, pseudo-pronucleus formation, cell division, compaction and morula development of the SCNT embryos were as good as those reported. However, the formation of SCNT blastocysts was below the published average, which may be a consequence of epigenetic reprogramming failure. Experimental adaptations such as medium supplementation or nuclear reprogramming strategies can be applied to improve epigenetic reprogramming by SCNT, which could be evidenced by a greater number of embryos progressing to good-quality blastocysts. In humans, use of SCNT as a tool to generate specific ESCs from the somatic cells of an individual could ultimately lead to the analysis of disease mechanisms, as well as improve the efficiency of cell-based therapies with a negligible risk of immune rejection in the treatment of degenerative diseases. SCNT is a cutting-edge technique that can offer innovative clinical applications in the field of assisted reproduction such as preventing the transmission of mitochondrial DNA diseases from mother to child, as well as the treatment of ooplasm pathologies.Dissertation (MSc)--University of Pretoria, 2018.Obstetrics and GynaecologyMScUnrestricte