Variable partition of non-homogeneous ooplasm sets the stage for divergent potency of 2-cell stage blastomeres

Following fertilization in mice and humans, the first two blastomeres are not equivalent, but one produces more epiblast than the other (imbalance); therefore, they do not feature equal totipotency. Research into the causes has overlooked that the epiblast imbalance is preceded by a fertilization imbalance, since in nature, the spermatozoon fertilizes the oocyte preferentially in the animal hemisphere near the animal-vegetal midline (equator). We conceived a hypothesis that the two imbalances are linked to each other, and broke it down into testable predictions. If the two imbalances were interdependent, then changing the site of sperm entry into the oocyte should change the extent of the epiblast imbalance. Thus, we evened out the fertilization imbalance, using ICSI to fertilize mouse oocytes also in the vegetal hemisphere and the equator. Resultant embryos were split at the 2-cell stage, and the twin blastocysts originating from the sister blastomeres were analyzed. Against the similarity in mRNA levels of epiblast genes, twin blastocysts differed in epiblast function, as measured by NANOG protein expression and derivation of embryonic stem cells, and the epiblast imbalance was greater after oocyte fertilization at the equator. There is no simple way to explain the positional effect other than through differences in the molecular composition of the ooplasm, which, moreover, should also be apportioned variably at the first zygotic division. We tested these predictions by measuring the orientation of the first zygotic division regarding the ICSI site, and the composition of bisected oocytes' hemispheres using half-cell proteomics. Since we found that the hemispheres have different compositions depending on the bisection axis, and the angle of the first division is variable, we propose that the variable partition of non-homogeneous ooplasm sets the stage for the epiblast imbalance. These results revive the role of the oocyte's molecular architecture on embryogenesis in a mammalian species hitherto considered mostly regulative in development.