The aim of this project was to investigate how the cell-cycle of both nuclear-donor and recipient donor affects the development of embryos reconstituted by nuclear transfer. Two methods of synchronising embryos were established. Embryos could be held in mitosis by culture for nine hours in 10μM nocodazole or culture for four hours in 0.1 μg/ml colcemid. Secondly, treatment with 1 μg/ml of aphidicolin of embryos previously synchronized in mitosis was able to synchronize the blastomeres at the G1/S border without any apparent effect upon development to blastocyst. A method of parthenogenetic activation of recently ovulated and preovulatory oocytes was established, involving the culture of the oocytes for 60 minutes in 25 mM strontium in a calcium magnesium free M16 medium. Spontaneous activation was almost non existent in oocytes recovered from the ovary or in those recovered very early after ovulation (14 hours after hCG). It was possible now to conduct experiments on nuclear transfer in which the timing of activation was strictly controlled. To study the effect of variations in the time of fusion in relation to activation, late 2-cell stage nuclei were fused six hours before, at the same time or 12 h after activation. After activation of enucleated oocytes, the cytoplast fragmented. This phenomenon appears to be mediated by microtubules, since culture of activated cytoplast with inhibitors of microtubules polymerisation (nocadazole) inhibited fragmentation. A "nuclear transfer" induced activation was observed, although the cytoplast donor oocytes were not activated by the procedures involved in the recovery or the enucleation methods. A greater proportion of reconstituted embryos developed to blastocyst when nuclei were transferred to preactivated cytoplasts. These results confirm the importance of controlling cell cycle during nuclear transfer and emphasise the advantage of transferring nuclei in G1-phase. In addition, they suggest that development may be achieved from later stages, provided that the nuclei are transferred into appropriate recipients.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:660246 |
| Date | January 1995 |
| Creators | Otaegui, Pedro Jose |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15565 |
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