The objective of the first series of experiments was to assess biochemical changes during in vitro maturation of oocytes collected from ovaries of adult cattle and calves (<6 mo old). Activity and/or concentrations of maturation-promoting factor, mitogen-activated protein kinase, and inositol 1,4,5-trisphosphate receptor were determined and they were significantly lower in calf oocytes. Developmental competence of parthenogenic embryos was studied after reciprocal transfer of metaphase II chromosomes between cow and calf oocytes and transfer of cumulus cells into cow and calf ooplasts. Development was higher in embryos originating from adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation. We hypothesized that the germinal vesicle (GV) stage oocyte was capable of inducing a meiotic like division of the somatic cell nucleus. To test this hypothesis, cumulus cells in G0, G1 or metaphase (M) were transferred to GV stage bovine oocytes. Denuded GV oocytes were fused by an electrical pulse and matured in vitro. Chromosome segregation was assessed after staining with Hoechst 33342. All cell stages had similar fusion and survival rates and were capable of undergoing a meiotic-like division on subsequent maturation. Human and mouse fibroblasts and bovine cumulus cells in G1 were transferred to GV or prometaphase I (PM I) stage bovine oocytes. Human and mouse fibroblasts were also transferred to PM 1 mouse oocytes. Meiotic maturation was assed by staining DNA with Hoechst 333342 and chromosome segregation by FISH analysis for bovine chromosomes 6 and 13 and for human chromosomes X and 18. Fusion and survival rates were better when PM I oocytes were used as recipients. Somatic cells were capable of undergoing a meiotic-like division. FISH analysis for reconstructed bovine oocytes revealed just 1 chromosome 6 and 13 per nuclei in 3 of 6 oocytes. After inter-specific nuclear transfer, chromosomes never completed meiotic segregation. These results support the hypothesis that haploidization of somatic cells can be induced after homologous transfer of nuclei into immature oocytes and is influenced by stage of oocyte development.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-3895 |
| Date | 01 January 2004 |
| Creators | Salamone, Daniel F |
| 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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