We propose to investigate nucleo-cytoplasmic interactions which take place during normal development and to determine the mechanism by which these interactions are controlled. The role of various components of the mammalian zygote (male genome, female genome, egg cytoplasm) in development will be analyzed using recently developed nuclear transfer techniques. Our first major objective will be to determine the reasons for developmental failure of biparental androgenones (embryos with two male pronuclei) and biparental gynogenones (two female pronuclei). Development of chimeric embryos produced by aggregation of gynogenetic or androgenetic and normal embryos will be monitored in order to determine whether embryo- or cell-lethality is ultimately responsible for death of isoparental embryos. Haploid nuclei obtained from successive stages of male gametogenesis will be transferred into zygotes from which male nuclei were removed. These experiments will address the timing of appearance of functional differences between the male and the female genome. The second major objective will be to analyze development of enucleated zygotes into which nuclei from older developmental stages are transferred. We will determine if repeated nuclear transfer will improve the development of such embryos. The use of cytoplasm from older embryos (4- and 8-cell stage) as the nuclear recipient will be explored. Using 2D gel analysis we will monitor the changes in genome expression following nuclear transfer. Nuclear morphology, changes in nuclear proteins (lamins) and the structure of the mitotic apparatus in these nuclear transfer embryos will be investigated.
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