title: Ploidy of bovine nuclear transfer blastocysts reconstructed using in vitro produced blastomere donors.
abstract: The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro development. Because little is known about ploidy errors in nuclear transfer embryos, this was examined using embryos reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6-dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blastomeres from day 4-5 in vitro produced donors. The embryos were cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 +/- 20.2 (mean % +/- SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8%) were potentially abnormal because in these, 1.1%, 1.4%, 5.3%, 7.5%, 26.3%, 30.4%, and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors comprised mainly triploid (8.2 +/- 10.3 [0-26.3]: % +/- SD [range]) and tetraploid (10.6 +/- 19.9 [0-54.9]) nuclei with other ploidy combinations accounting for only 0.9 +/- 2.1 [0-2.1]% of deviant nuclei. The proportion of completely normal nuclear transfer embryos was no less than those produced by in vitro fertilization but the distribution of chromosome abnormalities was different (p = 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chromosome abnormalities to embryonic loss in the remaining embryos deserves further investigation.
authors.affiliation: Section for Reproductive Biology, Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, 8830 Tjele, Denmark. Paul.Booth@agrsci.dk
authors.forename: P J
authors.initials: PJ
authors.lastname: Booth
authors.forename: D
authors.initials: D
authors.lastname: Viuff
authors.forename: P D
authors.initials: PD
authors.lastname: Thomsen
authors.forename: P
authors.initials: P
authors.lastname: Holm
authors.forename: T
authors.initials: T
authors.lastname: Greve
authors.forename: H
authors.initials: H
authors.lastname: Callesen
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D001757:Blastomeres / Q000648:ultrastructure*; D000001:Calcimycin / Q000494:pharmacology; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D002869:Chromosome Aberrations; D002875:Chromosomes / Q000648:ultrastructure; D019976:Cloning, Organism / Q000379:methods*; D046149:Embryo Culture Techniques / Q000379:methods*; D044127:Epigenesis, Genetic; D005307:Fertilization in Vitro; D017404:In Situ Hybridization, Fluorescence; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000378:metabolism; D011003:Ploidies; D013997:Time Factors
chemical_list: D000001:Calcimycin; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine