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

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

Lessly P Sepulveda-Rincon et al. Biol Reprod.

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.

Keywords: H3 arginine methylation; blastocyst; embryo biopsy; patterning; preimplantation development; time-lapse microscopy.

Figures

FIG. 1
FIG. 1
Cell-allocation patterns at the blastocyst stage. Examples of bovine (A) and mouse (B) blastocysts observed after DiI labeling (Z-projections of the Apotome images) with drawings of the boundary line between the labeled/unlabeled cells (green) and the blastocoel cavity floor (blue). According to the angle between these two lines (≤30° or >30°), blastocysts were scored as orthogonal or deviant, respectively. When the labeled and nonlabeled cells were intermingled, making it impossible to draw a boundary line between them, blastocysts were scored as random. Bar = 50 μm for bovine embryos and 20 μm for mouse embryos. The proportion of the different cell allocation patterns observed at the blastocyst stage after DiI labeling, in bovine (A') and mouse (B'). Expressed values represent the mean percent ± SEM of each pattern among repetitions of the experiment. The total number of blastocysts scored is indicated below each column. For each graph, values with different superscripts (a, b) indicate significantly different values (P < 0.05; Bonferroni post hoc test).
FIG. 2
FIG. 2
Sequential images of Day 3/Day 4 bovine embryos labeled with DiI and Ras-GFP. Examples of the separated and the intermingled pattern observed in bovine embryos labeled at the 2-cell stage with the lipophilic tracer DiI in one blastomere and with Ras-eGFP in the other one. The selected images correspond to three-dimensional reconstructions obtained with the Zeiss Zen software from the time-lapse movies (Supplemental Movies S1 and S2), with a 4-h interval. Bars = 50 μm.
FIG. 3
FIG. 3
Sequential images of labeled bovine embryos up to the blastocyst stage. Bovine embryos with the DiI/Ras-GFP double labeling were observed by time-lapse microscopy from Day 3 up to the blastocyst stage. Examples (three-dimensional reconstructions) shown here correspond to images taken every 4 h between Day 3 and 4 (A, B) and then every 12 h (A', B'). Whereas the bovine embryo with an intermingled pattern (A) became a random blastocyst (A'), the separated one (B) became a deviant blastocyst (B'). Bars = 50 μm.
FIG. 4
FIG. 4
Scoring of the bovine blastocyst according to their size. Bovine blastocysts were classified into two groups: stage 6 blastocyst and stage 7–8 expanded/hatched blastocyst and were then analyzed for their DiI allocation. Examples of each type of blastocyst (bars = 50 μm) are shown on top of the graph that combines both parameters. The total number of blastocysts scored is indicated below each column.
FIG. 5
FIG. 5
Immunodetection of SOX2. Immunodetection of Sox2 (green) and labeling of DNA by DAPI (blue) was performed in each group of bovine blastocysts (bar = 50 μm). The total number of DAPI- and SOX2-positive cells was determined on z-projections using the cell counter plugin of ImageJ software.
FIG. 6
FIG. 6
Immunodetection of histone H3 arginine methylation. Immunodetection of H3R2me2 (green) and labeling of DNA (red) was performed in bovine (A) and mouse (B) 4-cell stage embryos (bar = 10 μm). Quantification was then performed with ImageJ software as described in Materials and Methods. Each bar represents the relative H3R2me2 fluorescence level of one of the four blastomeres either in bovine (blue bars, n = 35) or mouse (gray bars, n = 15) embryos. The results for the two species are not statistically different (P > 0.05), however there is significant differences between blastomeres (P = 0.03).

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