A microsurgical technique for enucleation of multipronuclear human zygotes

Tripronuclear human zygotes were used to determine the feasibility of an en ucleation procedure without the use of cytoskeleton inhibitors to obtain in tact cytoplasts for nuclear transfer research. Of 61 tripronuclear human zy gotes manipulated, 100% of the zygotes survived after removal of one pronuc leus and 90.1% after complete enucleation, proving the reliability of the p roposed microsurgical technique. Morphological changes were observed in the resulting cytoplasts during extended culture. At 36 h post-insemination, 8 7.5% of cultured cytoplasts showed morphological changes, which included cl eavage into two 'blastomeres', three 'blastomeres' with various degrees of fragmentation, one cell with fragments or gross fragmentation. Comparison w ith the diploid human zygotes from the same harvests, which had undergone t he first cleavage division by this time, showed similar timing in cleavage suggesting autonomous cytoplasmic activity in human zygotes.