title: Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication.
abstract: Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P < 0.05), respectively. Epifluorescence microscopy showed that the proportion of blastocysts with eGFP-positive cells in the ICM was higher in the PHA group than in the no-PHA group (40% vs. 16%; P < 0.05). Confocal laser scanning microscopy revealed that the total cell numbers of blastocysts from the PHA group of aggregation chimeras (n = 17; 207.8 ± 67.3 [mean ± standard deviation]) were higher (P < 0.05) than those of embryos without ZP and exposed to PHA (n = 30; 159.6 ± 42.2) and of handling control embryos (n = 19; 176.9 ± 53.3). The same was true for ICM cell counts (56.5 ± 22.0 vs. 37.7 ± 14.2 and 38.7 ± 12.4) and TE cell counts (151.2 ± 58.0 vs. 121.9 ± 37.4 and 138.3 ± 53.0), whereas the ICM/total cell number ratio was not different between the groups. Of the 17 chimeric blastocysts analyzed by confocal laser scanning microscopy, nine had eGFP-positive cells (three of them in the ICM, three in the TE, and three in both lineages). When integration in the ICM occurred, the number of eGFP-positive cells in this compartment was 8.3 ± 2.3 (mean ± standard error of the mean). We conclude that PHA is advantageous for the formation of aggregation chimeras, but the approach tested in the present study with only two donor blastomeres and two host embryos did not result in multiplication of genetically valuable donor embryos.
authors.affiliation: Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Kilian
authors.initials: K
authors.lastname: Simmet
authors.affiliation: Department of Animal Health, Bayern-Genetik GmbH, Poing, Germany.
authors.forename: Myriam
authors.initials: M
authors.lastname: Reichenbach
authors.affiliation: Bavarian State Research Center for Agriculture, Institute of Animal Breeding, Poing, Germany.
authors.forename: Horst-Dieter
authors.initials: HD
authors.lastname: Reichenbach
authors.affiliation: Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians-University, Munich, Germany. Electronic address: ewolf@lmb.uni-muenchen.de.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
mesh_terms: D000818:Animals; D001757:Blastomeres / Q000166:cytology / Q000502:physiology*; D002417:Cattle / Q000196:embryology; D002459:Cell Fusion / Q000662:veterinary; D002678:Chimera / Q000502:physiology*; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian; D047108:Embryonic Development / Q000235:genetics; D005260:Female; D049452:Green Fluorescent Proteins; D010835:Phytohemagglutinins / Q000494:pharmacology*
chemical_list: D010835:Phytohemagglutinins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Aggregation; Bovine; Chimera; Lineage differentiation; Multiplication