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. This model of totipotency was inferred from a minority of cases in which blastomeres produced monozygotic twins in mice. Was this due to experimental limitation or biological constraint? Here we removed experimental obstacles and achieved reliable quantification of the prevalence of dual totipotency among mouse two-cell stage blastomeres. We separated the blastomeres of 1,252 two-cell embryos, preserving 1,210 of the pairs. Two classes of monozygotic twins became apparent at the blastocyst stage: 27% formed a functional epiblast in both members (concordant), and 73% did so in only one member of the pair (discordant) – a partition that proved insensitive to oocyte quality, sperm-entry point, culture environment and pattern of cleavage. In intact two-cell embryos, the ability of sister blastomeres to generate epiblast was also skewed. Class discovery clustering of the individual blastomeres’ and blastocysts’ transcriptomes points to an innate origin of concordance and discordance rather than developmental acquisition. Our data place constraints on the commonly accepted idea that totipotency is allocated equally between the two-cell stage blastomeres in mice.

Early mouse embryos have an atypical translational machinery that consists of cytoplasmic lattices and is poorly competent for translation. Hence, the impact of transcriptomic changes on the operational level of proteins is predicted to be relatively modest. To investigate this, we performed liquid chromatography–tandem mass spectrometry and mRNA sequencing at seven developmental stages, from the mature oocyte to the blastocyst, and independently validated our data by immunofluorescence and qPCR. We detected and quantified 6,550 proteins and 20,535 protein-coding transcripts. In contrast to the transcriptome – where changes occur early, mostly at the 2-cell stage – our data indicate that the most substantial changes in the proteome take place towards later stages, between the morula and blastocyst. We also found little to no concordance between the changes in protein and transcript levels, especially for early stages, but observed that the concordance increased towards the morula and blastocyst, as did the number of free ribosomes. These results are consistent with the cytoplasmic lattice-to-free ribosome transition being a key mediator of developmental regulation. Finally, we show how these data can be used to appraise the strengths and limitations of mRNA-based studies of pre-implantation development and expand on the list of known developmental markers.

BackgroundWhile DNA and RNA methods are routine to disrupt the expression of specific genes, complete understanding of developmental processes requires also protein methods, because: oocytes and early embryos accumulate proteins and these are not directly affected by DNA and RNA methods. When proteins in the oocyte encounter a specific antibody and the TRIpartite Motiv-containing 21 (TRIM21) ubiquitin-protein ligase, they can be committed to degradation in the proteasome, producing a transient functional knock-out that reveals the role of the protein. However, there are doubts about whether this targeted proteolysis could be successfully used to study mammalian development, because duration of the transient effect is unknown, and also because amounts of reagents delivered must be adequate in relation to the amount of target protein, which is unknown, too.ResultsWe show that the mouse egg contains up to 1E-02 picomoles/protein, as estimated by mass spectrometry using the intensity-based absolute quantification (iBAQ) algorithm. However, the egg can only accommodate ≈1E-04 picomoles of antibody or TRIM21 without incurring toxic effects. Within this framework, we demonstrate that TRIM21-mediated protein depletion efficiently disrupts the embryonic process of trophectoderm formation, which critically depends on the TEA domain family member 4 (Tead4) gene. TEAD4 depletion starting at the 1-cell stage lasts for 3 days prior to a return of gene and protein expression to baseline. This time period is long enough to result in a phenotype entirely consistent with that of the published null mutation and RNA interference studies: significant underexpression of trophectodermal genes Cdx2 and Gata3 and strongly impaired ability of embryos to cavitate and implant in the uterus. Omics data are available via ProteomeXchange (PXD012613) and GEO (GSE124844).ConclusionsTRIM21-mediated protein depletion can be an effective means to disrupt gene function in mouse development, provided the target gene is chosen carefully and the method is tuned accurately. The knowledge gathered in this study provides the basic know-how (prerequisites, requirements, limitations) to expedite the protein depletion of other genes besides Tead4.

The mammalian zygote is a totipotent cell that generates all the cells of a new organism through embryonic development. However, if one asks about the totipotency of blastomeres after one or two zygotic divisions, opinions differ. As it is impossible to determine the individual developmental potency of early blastomeres in an intact embryo, experiments of blastomere isolation were conducted in various species, showing that two-cell blastomeres could give rise to a new organism when sister cells were separated. A mainstream interpretation was that each of the sister mammalian blastomeres was equally totipotent. However, reevaluation of those experiments raised some doubts about the real prevalence of cases in which this interpretation could truly be validated. We compiled experiments that tested the individual developmental potency of early mammalian blastomeres in a cell-autonomous way (i.e. excluding nuclear transfer and chimera production). We then confronted the developmental abilities with reported molecular differences between sister blastomeres. The reevaluated observations were at odds with the mainstream view: A viable two-cell embryo can already include one non-totipotent blastomere. We were, thus, led to propose a revised model for totipotency continuity based on the construction of the zygote as a mosaic, which accounts for differential inheritance of totipotency-relevant components between sister blastomeres. This takes place with no preordained mechanisms that would ensure a reproducible partition. This model, which is compatible with the body of data on regulative properties of mammalian early embryos, aims at tempering the rigid interpretation that discounted maternal constraints on totipotency.Reproduction (2019) 158 R49-R65

It is widely held that the first two blastomeres of mammalian embryos are equally totipotent and that this totipotency belongs to the group of regulative properties. However, this interpretation neglects an important aspect: evidence only came from successful monozygotic twins which can speak only for those pairs of half-embryos that are able to regulate in the first place. Are the frequently occurring incomplete pairs simply an artefact, or do they represent a real difference, be it in the imperfect blastomere’s ability to regulate growth or in the distribution of any compound X that constrains regulation? Using the model system of mouse embryos bisected at the 2-cell stage after fertilization, we present evidence that the interblastomere differences evade regulation by external factors and are already latent in oocytes. Specifically, an interblastomere imbalance of epiblast production persists under the most diverse culture conditions and applies to the same extent in parthenogenetic counterparts. As a result, cases in which twin blastocysts continued to develop in only one member account for 65 and 57% of zygotic and parthenogenetic pairs, respectively. The interblastomere imbalance is related to the subcellular distribution of gene products, as documented for the epiblast-related gene Cops3, using mRNA FISH in super-resolution mode confocal microscopy. Blastomere patterns of Cops3 mRNA distribution are α-amanitin-resistant. Thus, the imbalance originates not from de novo transcription, but from influences which are effective before fertilisation. These data expose previously unrecognized limits of regulative capacities of 2-cell stage blastomeres and point to aspects of cytoplasmic organization of the mouse oocyte that segregate unequally to blastomeres during cleavage.