Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes

Most current studies of nuclear transfer in mammalian oocytes have used ele ctrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplas ts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. H ere we tested a previously described chemical fusion technique and an origi nal mechanical fusion procedure in this application. Enucleated metaphase I I oocytes were first agglutinated with karyoplasts originating from other m etaphase II oocytes and then induced to fuse with the use of polyethylene g lycol or by micromanipulation with an intracytoplasmic sperm injection (ICS I) micropipette, Both techniques yielded a high frequency of fusion and did not cause oocyte activation. Moreover, the reconstructed oocytes were easi ly activated by subsequent treatment with ionophore A23187 and 6-dimethylam inopurine. These techniques may be used in attempts to alleviate female inf ertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual futu re use in human cloning, they would ensure prolonged exposure of transferre d nuclei to metaphase promoting factor, which appears to be required for op timal nuclear reprogramming.