This study evaluated procedures to improve nuclear transplantation efficiency in rabbit embryos. Recently ovulated oocytes were enucleated and fused at a higher rate than aged oocytes and were efficiently activated by multiple electrical pulses. Repeated stimulations also improved development of reconstituted embryos in vitro. Also, manipulating oocytes in bicarbonate-buffered medium, and cytochalasin B in the post-fusion medium enhanced development in vitro and to term. The profile of nuclear remodelling in reconstituted embryos was characterized and the relationship between chromatin behavior after transfer and embryo development determined. Upon blastomere fusion to non-activated cytoplasm, nuclear remodelling was characterized by premature chromosome condensation (PCC) and nuclear swelling. Fusion to activated cytoplasm prevented PCC and extensive nuclear swelling, but allowed development to blastocysts. The results indicate that remodelling of the donor nucleus was not essential for development to blastocysts; however, it was beneficial. The influence of cell cycle stage of the donor nucleus on development of reconstituted embryos was determined. Use of early cell cycle nuclei resulted in high rate of development to blastocysts, whereas mid and late cell cycle donors led to reduced development. Synchronizing blastomeres in G1 entailed incubation in colcemid to arrest embryos in M phase and in aphidicolin to synchronize embryos at the G1/S boundary. Development to blastocysts was enhanced with G1 nuclei, as opposed to late S. Therefore, extent of development was reduced as donor nuclei progressed in the cell cycle. The effect of donor cell cycle stage on chromatin and spindle morphology in manipulated embryos was determined. After blastomere fusion, an attempt to form a spindle and a metaphase plate occurred in G1, early S and, to some extent, late S transplants. These structures displayed minor and gross abnormalities in early and late S transplants, respectively. G1 and early S transplants contained normal chromosomes, whereas defects occurred in late S transplants. Therefore, PCC in G1 and early S had a minor influence on chromosome constitution of manipulated embryos. PCC in late S, however, affected chromosome conformation and may account for impaired embryo development.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8027 |
| Date | 01 January 1991 |
| Creators | Collas, Philippe |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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