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Influence of Insulin-Like Growth Factor-I on Cytoplasmic Maturation of Horse Oocytes In Vitro and Organization of the First Cell Cycle Following Nuclear Transfer and Parthenogenesis¹

Department of Clinical Veterinary Medicine,³ University of Cambridge, Equine Fertility Unit, Mertoun Paddocks, Newmarket, Suffolk CB8 9BH, United Kingdom Department of Development and Genetics,⁴ The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom

In vitro maturation of horse oocytes cultured with or without IGF-I supplementation and their first cell cycle organization were studied in reconstructed horse oocytes made by somatic cell nuclear transfer versus intact oocytes stimulated parthenogenetically. The rates of metaphase II oocytes (47% and 45%) and of reconstructed oocytes that developed to the two-cell (27% and 25%) and blastocyst stages (11% and 3%) were not different between the media, with or without IGF-I, respectively. However, significantly more parthenogenetic embryos exhibited two-cell development with IGF-I (P < 0.05). The results also demonstrated that the first cell cycle organization in the reconstructed oocytes involved two different ways of nuclear remodeling. The donor nucleus in the Type I embryo showed normal nuclear remodeling that resulted in normal embryonic development. In the Type II embryos, however, the donor nucleus formed a polyploid nucleus or the embryo fragmented. Addition of IGF-I to the maturation medium significantly increased the rate of normal Type I embryonic development from the reconstructed oocytes (45% vs. 28%, P < 0.05). Maturation-promoting factor (MPF; including cdc2 and cyclin B) and mitogen-activated protein kinase (MAPK; including ERK1 and ERK2) were present at the beginning of culture, just after the oocytes had been harvested from the ovaries. The quantities of cyclin B remained stable no matter how long a period of in vitro culture the oocytes underwent, whereas cdc2 showed a tendency to accumulate in the oocytes toward the end of the 30-h culture period. Addition of IGF-I to the medium may induce a bigger accumulation of MAPK in the cytoplasm of the horse oocyte, especially in the ERK2 component, which might, in turn, increase the chance of the reconstructed oocyte undergoing nuclear remodeling to form a Type I embryo following nuclear transfer.

¹ This study was kindly financed by the Moller Charitable Trust, the Japan Racing Association, and the Thoroughbred Breeders' Association.

² Correspondence: W.R. Allen, TBA Equine Fertility Unit, Mertoun Paddocks, Woodditton Rd., Newmarket, Suffolk CB8 9BH, United Kingdom. FAX: 01638 667207; efu{at}tesco.net

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