Recent advances in embryology have shown that the sister blastomeres of 2-cell mouse and human embryos differ reciprocally in potency. An open question is whether the blastomeres became different as opposed to originating as different. Here we wanted to test two conflicting models: one proposing that each blastomere contains both animal and vegetal materials in balanced proportions because the plane of first cleavage runs close to the animal-vegetal axis of the fertilized oocyte; and the other model proposing that each blastomere contains variable proportions of animal and vegetal materials because the plane of the first cleavage can vary depending on the topology of fertilization. Therefore, we imposed the fertilization site in three distinct regions of mouse oocytes (animal pole, vegetal pole, equator) via intracytoplasmic sperm injection (ICSI). After the first zygotic cleavage, the sister blastomeres were dissociated and subjected to single-cell transcriptome analysis, keeping track of the original pair associations. Non-supervised hierarchical clustering revealed that the frequency of correct pair matches varied with the fertilization site (vegetal pole > animal pole > equator), thereby, confuting the first model of balanced partitioning. However, the inter-blastomere differences had similar signatures of gene ontology across the three groups, thereby, also confuting the competing model of variable partitioning. These conflicting observations could be reconciled if animal and vegetal materials segregated at the first cleavage: an event considered improbable and possibly deleterious in mammals. We tested this occurrence by keeping the fertilized oocytes immobilized from the time of ICSI until the first cleavage. Image analysis revealed that cleavage took place preferentially along the short (ie equatorial) diameter of the oocyte, thereby partitioning the animal and vegetal materials into the 2-cell blastomeres. Our results point to a simple mechanism by which the two sister blastomeres start out as different, rather than becoming different.
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