Cell-cycle phase effect on the survival of mouse embryos after nuclear transfer

The aim of this project was to investigate how the cell-cycle of both nucleardonor 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 101iM nocodazole or culture for four hours in 0.1 pg/mi colcemid. Secondly, treatment with 1 pg/mI of aphidicolin of embryos previously synchronized in mitosis was able to synchronize the blastomeres at the GuS border without any apparent effect upon development to blastocyst. A method of parthenogenetic activation of recently ovulated and preovulatoiy 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 (nocodazole) 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. After mapping cell-cycle events in activated oocytes, and 4-cell and 8-cell stage mouse embryos, nuclear donor cell-cycle effect and its interaction with cytoplast cellcycle were studied. Mouse 4-cell stage were used as donors of nuclei in Gi-phase, early S-phase and late S-G2 phase. The recipients were enucleated cytoplast either non-activated (meiosis II) or preactivated 6-8hr (early S) or 12-16 hr (late S) before