title: Aggregating embryonic but not somatic nuclear transfer embryos increases cloning efficiency in cattle.
abstract: Our objectives were to compare the cellular and molecular effects of aggregating bovine embryonic vs. somatic cell nuclear transfer (ECNT vs. SCNT) embryos and to determine whether aggregation can improve cattle cloning efficiency. We reconstructed cloned embryos from: 1) morula-derived blastomeres, 2) six adult male ear skin fibroblast lines, 3) one fetal female lung fibroblast line (BFF), and 4) two transgenic clonal strains derived from BFF. Embryos were cultured either singularly (1X) or as aggregates of three (3X). In vitro-fertilized (IVF) 1X and 3X embryos served as controls. After aggregation, the in vitro development of ECNT but not that of SCNT or IVF embryos was strongly compromised. The inner cell mass (ICM), total cell (TC) numbers, and ICM:TC ratios significantly increased for all the aggregates. The relative concentration of the key embryonic transcript POU5F1 (or OCT4) did not correlate with these increases, remaining unchanged in the ECNT and IVF aggregates and decreasing significantly in the SCNT aggregates. Overall, the IVF and 3X ECNT but not the 1X ECNT embryos had significantly higher relative POU5F1 levels than the SCNT embryos. High POU5F1 levels correlated with high in vivo survival, while no such correlation was noted for the ICM:TC ratios. Development to weaning was more than doubled in the ECNT aggregates (10/51 or 20% vs. 7/85 or 8% for 3X vs. 1X, respectively; P < 0.05). In contrast, the SCNT and IVF controls showed no improvement in survival. These data reveal striking biological differences between embryonic and somatic clones in response to aggregation.
authors.affiliation: AgResearch, Ruakura Research Centre, Reproductive Technologies, Private Bag 3123, Hamilton, New Zealand.
authors.forename: Pavla M
authors.initials: PM
authors.lastname: Misica-Turner
authors.forename: Fleur C
authors.initials: FC
authors.lastname: Oback
authors.forename: Michael
authors.initials: M
authors.lastname: Eichenlaub
authors.forename: David N
authors.initials: DN
authors.lastname: Wells
authors.forename: Björn
authors.initials: B
authors.lastname: Oback
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000831:Animals, Newborn / Q000254:growth & development; D001755:Blastocyst; D053624:Blastocyst Inner Cell Mass; D002417:Cattle / Q000196:embryology*; D002478:Cells, Cultured; D019976:Cloning, Organism; D004622:Embryo, Mammalian / Q000378:metabolism; D047108:Embryonic Development; D005260:Female; D005307:Fertilization in Vitro; D066298:In Vitro Techniques; D008297:Male; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D027724:Reproductive Techniques, Assisted; D016019:Survival Analysis; D014327:Trophoblasts / Q000166:cytology; D014886:Weaning
chemical_list: D050814:Octamer Transcription Factor-3