Single cell analysis of sister blastomeres of parthenogenetic 2-cell mouse embryos respecting pair associations

Following fertilization in mammals, it is generally accepted that totipotent cells are exclusive to the zygote and to each of the two blastomeres originating from the first mitotic division. We counter that this classic view needs to be revised, because we have presented compelling evidence that the sister blastomeres are both totipotent in only a subset of 2-cell stage mouse embryos (PMID 28811525). Building on our previous findings, we here ask the question if the interblastomere differences depend - at least in part - on the contribution of sperm, since the area of sperm entry is inherited preferentially by one blastomere. To this end, we created sister 2-cell stage blastomeres without sperm entry point, by parthenogenesis. We compare the transcriptomes of the sister blastomeres, to see if the interblastomere mRNA differences observed after fertilization (GSE94050) are still there when the sperm entry point is missing. After parthenogenetic activation and short culture in KSOM(aa) medium, 2-cell embryos were split, and the individual blastomeres were lysed immediately, keeping track of the original pair associations (e.g. blastomere '1a' and blastomere '1b' both derived from same 2-cell embryo number '1'). Transcriptome analysis followed by intra-pair mRNA ratio analysis revealed differences between the embryos of the same pair. These differences were fewer, but not substantially fewer, than the differences observed in the sister blastomeres of fertilized 2-cell embryos (GSE94050). Starting pair association (e.g. blastomere 'a' and 'b' from the same 2-cell embryo) was rigorously observed. Samples were subjected to Affymetrix microarray analysis, performed by Anika Witten c/o Institut fuer Humangenetik, Universitaetsklinikum Muenster.