221 HOXB9 IS DIFFERENTIALLY EXPRESSED IN THE TWO FIRST CELL LINES OF THE MAMMALIAN EMBRYO

Abstract

Hox proteins are transcription factors known to be essential for embryo patterning. The detection of some Hox transcripts in oocytes and early embryos suggests that they could play a role before gastrulation. We previously demonstrated Hoxb9 expression in oocytes and from the zygote to the blastocyst stage in the mouse and the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436). The protein is present at all stages and in all cells with a strong nuclear staining in both species. The objective of this study was to perform an in-depth study at the blastocyst stage to compare the level of the nuclear protein between the inner cell mass (ICM) and the trophectoderm (TE) from the early to the expanded blastocyst stage. In vitro produced bovine blastocysts were collected at Day 6, Day 7.5, and Day 8 post-insemination. Hoxb9 proteins were detected by whole-mount immunofluorescence. TE nuclei were strongly stained at all stages while from D6 but especially from D7.5, the level of HOXB9 seemed to decrease in ICM nuclei with an increasing heterogeneity of staining between ICM nuclei. A light and apparently stable staining was also observed in the cytoplasm. Confocal images were quantified (Nis-element 3.1, Nikon). For each cell of TE or ICM, the ratio between the mean intensity of the nucleus and the mean intensity of the corresponding total cytoplasm was calculated. Whatever the stages, TE ratios were significantly (Mann–Whitney test; P < 0.0001) higher than ICM ratios, suggesting that HOXB9 is present in higher amounts in TE than in ICM cells. This observation could be correlated with the reduced HOXB9 relative expression observed in blastocysts. Moreover, the proportion of blastocysts showing a reduction of HOXB9 staining in at least one nucleus significantly increased from Day 6 to Day 7.5 blastocysts and Day 8 blastocysts (from 26% to 74% or 85%, chi-squared test; P < 0.001). Mouse zygotes, collected from superovulated mice, were cultured in vitro and embryos were collected 72 h, 80 h, 92 h and 100 h post-hCG injection. A similar nuclear staining was observed in all cells until 80 h post-hCG injection, while heterogeneity of staining appeared in ICM cells 92 h post-hCG, but especially in 100 h post-hCG embryos. The quantitative study was performed only on this latest stage and confirmed the stronger staining in TE than in ICM nuclei (Mann–Whitney test; P < 0.0001) observed in the bovine. At this stage, 82% of blastocysts presented a reduced Hoxb9 staining in some or all ICM nuclei. In conclusion, Hoxb9 protein is detected in all blastocyst nuclei both in the mouse and in the bovine. However, the protein seems globally less abundant in the ICM than in the TE cells. Moreover, the percentage of bovine blastocysts showing a reduction in HOXB9 staining intensity in ICM nuclei increases with blastocyst expansion. These results suggest an involvement of Hoxb9 in cell lineage differentiation in mammals.

C. S. holds a FRIA PhD grant from the FRS-FNRS (Belgium). This study is supported by the FRS-FNRS and by an Action de Recherche Concertée.