Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals.

BACKGROUND: We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed. RESULTS: The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells. CONCLUSIONS: Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals.

Female bovine blastocysts are more prone to apoptosis than male ones.

Female and male embryos show differences in gene expression and metabolism from the onset of their genome. Those differences are affected by environmental factors. The objective of the study was to compare the apoptotic rates of in vitro-produced female and male bovine blastocysts cultured in different conditions. Day 7 blastocysts obtained after IVF with sex-sorted semen and culture in two synthetic oviductal fluid-based media (containing fetal calf serum [FCS] or BSA, insulin, transferrin, and selenium) were simultaneously evaluated for two markers of apoptosis after 3D reconstruction from confocal images: active caspase 3 by immunofluorescence and DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling. Higher levels of apoptotic cells were observed in female embryos whatever the culture condition but with a more pronounced difference in FCS medium. This result was confirmed using the unsexed semen of two bulls. The sex effect on apoptosis was detected in both the inner cell mass and the trophectoderm but was dependent on the embryonic size. In conclusion, this study reported that female bovine blastocysts are more prone to apoptosis than male ones but that culture in FCS exacerbates the differences in apoptosis between sexes, especially in small blastocysts.

How to study HOX gene expression and function in mammalian oocytes and early embryos.

Mammalian oocytes and early embryos have unique characteristics and can only be obtained in small amounts. As a consequence, the techniques to be used to characterize gene expression and function have to be adapted. It is also important to keep in mind that differences exist between mammalian species. Here we describe a set of techniques useful to analyze gene expression in oocytes and early bovine embryos, including techniques to quantify maternal and embryonic transcripts by RT-qPCR, to evaluate the translation potential of maternal transcripts, to knock down HOX transcripts by injection of siRNA, and to localize HOX proteins by whole-mount immunofluorescence.