Random Allocation of Blastomere Descendants to the Trophectoderm and ICM of the Bovine Blastocyst

Abstract

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.

Embryo cell allocation patterns are not altered by biopsy but can be linked with further development

“…A total of 287 B6CBAF1 × B6CBAF1 blastocysts in the control group and 214 blastocysts in the biopsied group were successfully classified according to their cell allocation pattern as previously reported on ( Sepulveda-Rincon et al 2016 ) ( Fig. 1 ).…”

“…As previously reported ( Sepulveda-Rincon et al 2016 ), the lipophilic tracer CM-DiI (Chloromethyl DiI, Molecular probes) was dissolved in olive oil at a final concentration of 2 mg/mL. Prior to labelling, the FemtoTip II (Eppendorf, Hamburg, Germany) was backfilled with the dye.…”

“…Blastocysts were scored for the different pattern categories as described previously ( Sepulveda-Rincon et al 2016 ). Briefly, blastocysts were placed in 5 µL EmbryoMax Hepes (Merck Millipore) medium drops on 60 mm petri dish and on a Leica DMI3000 B inverted microscope, coupled with red fluorescent filters corresponding to maximum excitation/emission wavelengths of 553 nm and 570 nm respectively.…”

“…It is still unclear if mammalian embryos are pre-patterned or the presence of stochastic development is just a reflex of the great plasticity of mammalian embryos. Research performed on cell allocation patterns suggest that it might be a common characteristic during pre-implantation embryo development of different mammalian species ( Park et al 2009 , Hosseini et al 2016 , Sepulveda-Rincon et al 2016 ). However, there is a lack of evidence on the mechanism(s) leading to these cell allocation patterns.…”

Totipotency continuity from zygote to early blastomeres: a model under revision

“…In addition to mice, four-cell stage molecular diversification was also studied in other mammals. When chromatin modifications were examined in four-cell bovine (H3R2me2) and rabbit (acetylated histone H4K5) embryos, significant interblastomere differences were noticed, comparable with those seen in the mouse (Chen et al 2012, Sepulveda-Rincon et al 2016. In human embryos, Chavez and colleagues noticed marked differences in chromatin modifications (H3-S28P and H4-R3me2) as early as the four-cell stage (Chavez et al 2014).…”