title: Use of a chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos.
abstract: The mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (+/-SEM) total cell numbers were 72.2+/-3.1 and 93.1+/-5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25+/-9.9, 166.14+/-9.9 and 105+/-6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p < 0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.
authors.affiliation: Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Canada.
authors.forename: R
authors.initials: R
authors.lastname: de la Fuente
authors.forename: W A
authors.initials: WA
authors.lastname: King
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002452:Cell Count; D002463:Cell Membrane Permeability; D004396:Coloring Agents; D004622:Embryo, Mammalian / Q000166:cytology; D005456:Fluorescent Dyes; D007211:Indoles; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D011419:Propidium; D013194:Staining and Labeling / Q000379:methods*; D013552:Swine; D013696:Temperature; D014327:Trophoblasts / Q000166:cytology*
chemical_list: D004396:Coloring Agents; D005456:Fluorescent Dyes; D007211:Indoles; D007476:Ionophores; D011419:Propidium; D000001:Calcimycin; C007293:DAPI; D002118:Calcium