Submitted to: Journal of Molecular Reproduction and Development
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/4/2004
Publication Date: 10/1/2004
Citation: Talbot, N.C., Caperna, T.J., Powell, A.M., Garrett, W.M., Ealy, A.D. 2004. Isolation and characterization of a bovine trophectoderm cell line derived from a parthenogenetic blastocyst. Molecular Reproduction and Development. 69(2):164-173. Interpretive Summary: The work describes the isolation, development, and characterization of a bovine trophectoderm cell line (designated BPT-1) that was derived from a parthenogenetic in vitro produced bovine embryo, i.e., an embryo produced without sperm having fertilizing the egg. Therefore, the cell's of the cell line only contain genes from the egg's nucleus (maternal genetic complement) and do not contain genes from a sperm (paternal genetic complement). The trophectoderm is the first specialized tissue of the early embryo to form. Although the trophectoderm does not contribute to the embryo itself, i.e., is an extraembryonic tissue, it plays vitally important roles in pre-implantation and post-implantation development. Most notably in the bovine embryo's early stage it signals to the cow that she is pregnant. Later on in development, it is one of the primary extraembryonic tissues that forms the placenta, thus, insuring that nutrients flow from the mother to the developing fetus. Fetuses made by cloning often have trouble with forming the placenta and so often abort for that reason. Parthenogenetic fetuses always have poor formation of the placenta and always abort. Therefore, the BPT-1 cell line provides an in vitro model of parthenogenote trophectoderm whose biological characteristics can be compared to trophectoderm cell lines derived from bovine embryos produced by normal fertilization or cloning. Such comparitive studies may lead to an understanding as to why cloned animals so often fail to form healthy placentas. This may help in the efforts to improve the efficiency of animal cloning which is important because, via cloning, it may be possible to rapidly improve various traits of cattle.
Technical Abstract: A bovine trophectoderm cell line was established from a parthenogenetic in vitro-produced blastocyst. To initiate the cell line, 8-day parthenogenetic blastocysts were attached to a feeder layer of STO fibroblasts and primary outgrowths occurred that consisted of trophectoderm, endoderm, and very occasionally epiblast tissue. Any endoderm and epiblast outgrowths were removed from the primary cultures within the first ten days of culture by dissection. One of the primary trophectoderm cell cultures was chosen for further propagation and was passaged by physical dissociation and replating on STO feeder cells. The cell culture, designated BPT-1, was maintained in T25 flasks and passaged at a 1:3 split ratio for the first 15 passages approximately once every 2 wk. Thereafter, the cell culture was passaged at 1:10 - 1:40 split ratios. Transmission electron microscopic examination showed the cells to be a polarized epithelium with apical microvilli, a thin basal lamina, and lateral junctions consisting of tight junctions and desmosomes. Lipid vacuoles and digestive vacuoles were also prominent features of the BPT-1 cells. Metaphase spread analysis at passage 59 indicated a near diploid cell population (2n = 60) with a mode and median of 60 and a mean of 64. BPT-1 cells secreted interferon-tau into the medium as measure by anti-viral assay and Western blot analysis. The cell line provides an in vitro model of parthenogenote trophectoderm whose biological characteristics can be compared to trophectoderm cell lines derived from bovine embryos produced by normal fertilization or nuclear transfer.