The aim of the project was to optimize cell cycle co-ordination in murine embryos reconstructed by nuclear transfer. The project also involved studies of oocyte activation, cell cycle length and synchronization, evaluation of MPF, and DNA replication in early embryos. Embryos were successfully arrested at mitosis by culturing them up to 16 hours with 2.5 mM 6-dimethylaminopurine (DMAP). A comparative study of different concentrations of 6-DMAP and 10 μM nocodazole for holding the embryos at mitosis for 12 hours showed a greater effectiveness of nocodazole. In most of the experiments 25 mM strontium chloride was used as the method for the artificial activation of cytoplasts and reconstituted embryos. Later on, a new method for parthenogenetic activation of metaphase II oocytes or recipient cytoplasts was established. The effect of meiosis/mitosis/maturation promotion factor (MPF) on the development of embryos reconstructed at different times in relation to activation was investigated and its level was studied by looking at nuclear envelope breakdown (NEBD) and nuclear envelope breakdown and premature chromatin condensation. After determining the timing of G1 duration and S-phase initiation of 3<SUP>rd</SUP> and 4<SUP>th</SUP> cell cycle, nuclear transfer experiments were performed to investigate the effect of nuclear donor cell cycle stage and its interaction with recipient cell cycle stage. The studies confirm the importance of cell cycle co-ordination in the development of reconstructed embryos. By appropriate co-ordination of cell cycle stages it has been possible for the first time to obtain similar development with 4-cell blastomeres with donor cell nuclei at any stage of the cell cycle. Moreover, the use of strontium chloride + 6-DMAP activated cytoplasts with 4-cell donor nuclei from different cell cycle can produce embryos that will develop with a higher frequency to morula/blastocyst than strontium chloride alone.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:640372 |
| Date | January 1998 |
| Creators | Ali, Safdar |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/16955 |
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