A microsurgical technique for enucleation of multipronuclear human zygotes.

Tripronuclear human zygotes were used to determine the feasibility of an enucleation procedure without the use of cytoskeleton inhibitors to obtain intact cytoplasts for nuclear transfer research. Of 61 tripronuclear human zygotes manipulated, 100% of the zygotes survived after removal of one pronucleus and 90.1% after complete enucleation, proving the reliability of the proposed microsurgical technique. Morphological changes were observed in the resulting cytoplasts during extended culture. At 36 h post-insemination, 87.5% of cultured cytoplasts showed morphological changes, which included cleavage into two 'blastomeres', three 'blastomeres' with various degrees of fragmentation, one cell with fragments or gross fragmentation. Comparison with the diploid human zygotes from the same harvests, which had undergone the first cleavage division by this time, showed similar timing in cleavage suggesting autonomous cytoplasmic activity in human zygotes.