Pluripotency genes are implicated in mouse embryonic genome activation (EGA) and pluripotent lineage specification. Moreover, their expression levels have been correlated with embryonic term development. In bovine, however, little information is available about dynamics of pluripotency genes during these processes. In this study, we charted quantitative and/or qualitative spatio-temporal expression patterns of transcripts and proteins of pluripotency genes (OCT4, SOX2 and NANOG) and mRNA levels of some of their downstream targets in bovine oocytes and early embryos. Furthermore, to correlate expression patterns of these genes with term developmental potential, we used cloned embryos, having similar in vitro but different full term development rates. Our findings affirm: firstly, the core triad of pluripotency genes is probably not implicated in bovine EGA since their proteins were not detected during pre-EGA phase, despite the transcripts for OCT4 and SOX2 were present. Secondly, an earlier ICM specification of transcripts and proteins of SOX2 and NANOG makes them pertinent candidates of bovine pluripotent lineage specification than OCT4. Thirdly, embryos with low term development potential have higher transcription rates; nevertheless, precarious balance between pluripotency genes is maintained. This balance presages normal in vitro development but, probably higher transcription rate disturbs it at later stage that abrogates term development.

The first lineage specification during mammalian embryo development can be visually distinguished at the blastocyst stage. Two cell lineages are observed on the embryonic-abembryonic axis of the blastocyst: the inner cell mass and the trophectoderm. The timing and mechanisms driving this process are still not fully understood. In mouse embryos, cells seem prepatterned to become certain cell lineage because the first cleavage plane has been related with further embryonic-abembryonic axis at the blastocyst stage. Nevertheless, this possibility has been very debatable. Our objective was to determine whether this would be the case in another mammalian species, the bovine. To achieve this, cells of in vitro produced bovine embryos were traced from the 2-cell stage to the blastocyst stage. Blastocysts were then classified according to the allocation of the labeled cells in the embryonic and/or abembryonic part of the blastocyst. Surprisingly, we found that there is a significant percentage of the embryos (∼60%) with labeled and nonlabeled cells randomly distributed and intermingled. Using time-lapse microscopy, we have identified the emergence of this random pattern at the third to fourth cell cycle, when cells started to intermingle. Even though no differences were found on morphokinetics among different embryos, these random blastocysts and those with labeled cells separated by the embryonic-abembryonic axis (deviant pattern) are significantly bigger; moreover deviant embryos have a significantly higher number of cells. Interestingly, we observed that daughter cells allocation at the blastocyst stage is not affected by biopsies performed at an earlier stage.

Rabbit embryo-fetal fluid (EFF) contains regulatory factors of cell proliferation which increase the duration of the cell cycle, induce a quiescent status in some cells and lead up to cell death in others. The objective of this study was to demonstrate which of the two processes, namely necrosis or apoptosis, was responsible for the cell death. Inhibitors of protein synthesis, and nuclease and phospholipase A2 activities did not restore the viability of the cells treated with EFF. Using a combination of DNA labelling and extraction, it was possible to show that a large proportion of DNA was fragmented in the cells released in the supernatant while only a very small portion of DNA was fragmented in the monolayer cells. EFF did not induce fragmentation of DNA into nucleosome-sized subunits as analysed using polyacrylamide gel electrophoresis. Nevertheless, using cytofluorometric analysis, it was possible to demonstrate that 50% of the cells released in the supernatant contained a lower quantity of DNA per cell than in the control cells. This was also observed with EFF-treated monolayer cells but not in the control monolayer cells. The reduction of the DNA content per monolayer cell became significant at 48 h of treatment with EFF. Electron microscopic analysis did not reveal blebbing of the cells. However, depletion of glycogen, condensation of mitochondria and increasing number of lysosomes and residual bodies were observed upon treatment with EFF. From these experiments we conclude that the DU-145 cells treated with EFF do not die by apoptosis, but rather seem to die by necrosis.

The first lineage specification occurs during pre-implantation mammalian development. At the blastocyst stage, 2 cell lineages can be distinguished: the inner cell mass (ICM) and the trophectoderm (TE). The exact timing when embryo cells are skewed to these lineages is not clearly determined in mammalian species. In murine embryos, it has been suggested that the first cleavage plane might be related to the embryonic-abembryonic (Em-Ab) axis at blastocyst stage. Thus, the daughter cells of the 2-cell embryo might already be predisposed to a specific cell lineage further on development. The objective of the present study was to observe how the first cleavage in bovine embryos may be related to cell lineage allocation at the blastocyst stage, using a noninvasive tracing approach. Bovine oocytes were harvested, in vitro matured, and fertilised. At the 2-cell stage, embryos were injected in one blastomere with the membrane tracer DiI. At the blastocyst stage, embryos (n = 346) were classified as orthogonal when the Em-Ab axis was orthogonally divided by the borderline between labelled and non-labelled cells; as deviant if the borderline was overlapping the Em-Ab axis; and as random when the labelled and non-labelled cells were randomly distributed. Total cell count (TCC) and the ICM/TE ratio was allowed by DNA staining with 4′,6-diamidino-2-phenylindole (DAPI) and by immunostaining of the ICM with Sox2 antibody. Analysis of variance was performed by one-way ANOVA employing IBM SPSS v21 (SPSS Inc., Chicago, IL, USA) to determine any difference between the cell lineage allocation patterns, TCC, and the ICM/TE ratio. P-values = 0.05 were considered significant. All values are reported as mean ± standard error of mean. Within 40 repetitions, the blastocyst classification was as follows: orthogonal 14.9% (±2.32, n = 56), deviant 22.2% (±2.58, n = 80), and random 62.9% (±2.64, n = 210). A significant difference was found in the incidence between the random group against the orthogonal and deviant, but not between the latter two. Regarding TCC, a significant difference was observed only between the orthogonal (99.6 ± 11.7 cells, n = 15) and deviant (135 ± 7.3 cells, n = 25) groups, but not with random embryos (116 ± 5.5 cells, n = 42). Finally, no significant difference was found among the groups concerning the ICM/TE ratio (0.43 ± 0.07 for orthogonal, n = 7; 0.54 ± 0.06 for deviant, n = 14; and 0.40 ± 0.03 for random embryos, n = 26). In conclusion, bovine embryos present a marked tendency for a random distribution of the daughter cells derived from the 2-cell blastomeres. However, around 37% of the blastocysts present a patterned cell division, where the daughter cells remain together through pre-implantation development. The effect of these cell lineage allocation patterns on implantation and further embryo development needs to be addressed.The authors acknowledge Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73) and CONACyT Mexico for funding.