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Human Reproduction, Vol. 15, No. 5, 1149-1154, May 2000
© 2000 European Society of Human Reproduction and Embryology

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

Jan Tesarik1,2,3,6, Zsolt P. Nagy4, Carmen Mendoza2,3,5 and Ermanno Greco4

1 Laboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris, France, 2 MAR & Molecular Assisted Reproduction and Genetics, Granada, Spain, 3 CIVTE, Centre of Insemination In Vitro and Embryo Transfer, Sevilla, Spain, 4 Centre for Reproductive Medicine, European Hospital, Rome, Italy and 5 Department of Biochemistry and Molecular Biology, University of Granada Faculty of Sciences, Granada, Spain

Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplasts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. Here we tested a previously described chemical fusion technique and an original mechanical fusion procedure in this application. Enucleated metaphase II oocytes were first agglutinated with karyoplasts originating from other metaphase II oocytes and then induced to fuse with the use of polyethylene glycol or by micromanipulation with an intracytoplasmic sperm injection (ICSI) micropipette. Both techniques yielded a high frequency of fusion and did not cause oocyte activation. Moreover, the reconstructed oocytes were easily activated by subsequent treatment with ionophore A23187 and 6-dimethylaminopurine. These techniques may be used in attempts to alleviate female infertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual future use in human cloning, they would ensure prolonged exposure of transferred nuclei to metaphase promoting factor, which appears to be required for optimal nuclear reprogramming.

Key words: human oocyte/membrane fusion/nuclear transfer/oocyte activation/polyethylene glycol

6 To whom correspondence should be addressed at: Laboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris, France


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