18Despite ongoing research in a number of species, the efficiency of embryo production by of sperm fertilization through artificial activation -the parthenote-and will also be introduced. 25Altogether the objective of this review is to have a better understanding on the mechanisms 26 responsible for the resistance to reprogramming; not only because it could improve embryonic 27 development but also as it could benefit therapeutic reprogramming research.

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.

It has been suggested that first embryo cleavage can be related with the embryonic–abembryonic axis at blastocyst stage in mice. Thus, cells of the 2-cell embryo might be already biased to form the inner cell mass or trophectoderm. This study was conducted to observe the possible effects of embryo biopsy on cell allocation patterns during embryo preimplantation in two different mouse strains and the effects of these patterns on further development. First, one blastomere of the 2-cell embryo was injected with a lipophilic tracer and cell allocation patterns were observed at blastocyst stage. Blastocysts were classified into orthogonal, deviant or random pattern. For the first experiment, embryos were biopsied at 8-cell stage and total cell counts (TCC) were annotated. Furthermore, non-biopsied blastocysts were transferred into foster mothers. Then, pups and their organs were weighed two weeks after birth. Random pattern was significantly recurrent (≈60%), against orthogonal (<22%) and deviant (<22%) patterns among groups. These patterns were not affected by biopsy procedure. However, TCC on deviant embryos were reduced after biopsy. Moreover, no differences were found between patterns for implantation rates, litter size, live offspring and organ weights (lungs, liver, pancreas and spleen). However, deviant pups presented heavier hearts and orthogonal pups presented lighter kidneys among the group. In conclusion, these results suggest that single blastomere removal does not disturb cell allocation patterns during pre-implantation. Nonetheless, the results suggest that embryos following different cell allocation patterns present different coping mechanisms against in vitro manipulations and further development might be altered.

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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.