Sue Varmuza
Department of Zoology; University of Toronto
The study of genomic imprinting in mammals started with analysis of parthenogenetic embryos. At the phenotypic level, embryos with two maternal genomes and no paternal genome proceed through early development unimpaired, and only begin to fail after implantation. The most recognizable early defect is reduced or non-existent trophoblast, the tissue that gives rise to the placenta. This and other observations led us to propose the Ovarian Time Bomb Hypothesis (OTBH; Varmuza and Mann, TIG 10:188, 1994), which states that genomic imprinting is a gene silencing mechanism adapted to the maternal germline in mammals to protect females from ectopic parthenogenetic trophoblast. In order to develop tools to test this hypothesis, we applied the procedure for establishing Trophoblast Stem cells (TS cells) devised by the Rossant lab to parthenogenetic embryos, and were successful in making four different TS cell lines, three from MI oocyte derived blastocysts and one from MII derived blastocysts. Initial molecular characterization, including microarray analysis, indicates that these cells are indistinguishable from fertilized TS cells, with the single exception of null expression of the paternally expressed gene Snrpn. The only significant difference between parthenogenetic and fertilized TS cells was the frequency with which they could be derived. In our hands, fertilized blastocyst outgrowths produced TS cells at robust rates (5-10 colonies per blastocyst), while parthenogenetic blastocyst outgrowths produced only 4 colonies in 50 outgrowths, 50-100 times less frequently than fertilized embryos. This led us to hypothesize that those few TS cells that arose in parthenogenetic outgrowths were probably a result of very low frequency stochastic loss of imprinting of a gene or genes required for either establishment or maintenance of stem cells, or both. The corollary to this hypothesis posits that the early failure of parthenogenetic embryos, and in particular parthenogenetic trophoblast, is a result of impaired stem cell function.