title: In vitro development of horse oocytes reconstructed with the nuclei of fetal and adult cells.
abstract: This study investigated the basic conditions required for the production of horse embryos by the transfer of the nuclei of fetal and adult fibroblast cells to enucleated oocytes. Cumulus-oocyte complexes were recovered from abattoir ovaries and matured in vitro in groups of 20-30 for 28-30 h in tissue culture medium 199 containing 20% v:v fetal bovine serum in coculture with equine oviduct epithelial cells. Fetal fibroblast cells (FFC) were derived from a 32-day-old Thoroughbred x Pony fetus, and adult skin fibroblast cells (SFC) were obtained from subdermal biopsies recovered from a 4-yr-old female Pony. The rates of fusion between the recipient cytoplasm with either FFC or SFC were significantly greater when the cells were treated with a combination of direct current (DC) pulses and Sendai virus rather than with DC pulses alone (81%-82% vs. 49%-57%, P < 0.05). There were no differences in the rates of nuclear reprogramming between FFC and SFC (88% vs. 84%), but the rate of cleavage of the resulting embryos to the 2-cell stage was higher when FFC were used (53%) than when SFC were used (35%). Blastocysts were obtained from oocytes reconstructed with both types of donor cells and after culture in vitro for 6-7 days, but the overall proportion of blastocysts produced was very low in both cases (FFC, 4%; SFC, 7%). These results demonstrate a very limited potential for in vitro development of horse embryos after nuclear reprogramming following the transfer of nuclei from either fetal or adult fibroblasts into recipient enucleated oocytes.
authors.affiliation: Department of Clinical Veterinary Medicine, Equine Fertility Unit, University of Cambridge, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, United Kingdom.
authors.forename: Xihe
authors.initials: X
authors.lastname: Li
authors.forename: Lee H-A
authors.initials: LH
authors.lastname: Morris
authors.forename: W R
authors.initials: WR
authors.lastname: Allen
mesh_terms: D000818:Animals; D002455:Cell Division / Q000502:physiology; D002459:Cell Fusion; D002467:Cell Nucleus / Q000502:physiology*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000648:ultrastructure; D004558:Electric Stimulation; D005260:Female; D005333:Fetus / Q000502:physiology; D005347:Fibroblasts; D006736:Horses / Q000502:physiology*; D009865:Oocytes / Q000254:growth & development*; D011247:Pregnancy; D029082:Sendai virus / Q000235:genetics; D012867:Skin / Q000166:cytology