BackgroundWe 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.ResultsThe 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.ConclusionsTogether, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals.
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.
Several studies have demonstrated differences in developmental rates and metabolism between bovine female and male embryos after IVF. Such differences seem related to the activity of both X chromosomes in female embryos up to the blastocyst stage and can lead to a shift in sex ratio. Developmental differences between male and female embryos are influenced by culture conditions. The objective of this study was to evaluate developmental and apoptotic rates of male and female bovine embryos in 2 SOF-based culture media, one with 5% FCS and the other with 4 mg mL–1 of BSA. Sex-sorted semen of one bull was used to produce cohorts of embryos of the desired sex. In preliminary experiments, IVF procedures were adapted to the use of sexed semen, and the purity of the sexed semen was verified through embryo sexing. The levels of apoptosis were assessed in Day-7 blastocysts using 2 techniques on the same embryos: TUNEL and detection of cleaved caspase-3 by immunostaining (caspase staining). Analysis by confocal microscopy and subsequent 3-D reconstruction allowed a precise cell count. A higher blastocyst rate on cleaved embryos was observed at Day 8 post-insemination for male than for female embryos in both media (BSA: male: 36.7 ± 4.0%, female: 28.6 ± 3.7%; FCS: male: 41.7 ± 2.9%, female: 31.7 ± 4.7%; ANOVA 3, P = 0.01). No significant difference in cell number was observed between male and female blastocysts (BSA: male: 188 ± 9, female: 170 ± 9; FCS: male: 186 ± 6, female: 177 ± 7; ANOVA 3, P = 0.14). In both media a higher proportion of cells showing caspase staining was observed in female than in male embryos (BSA: male: 7.3 ± 1.3%, female: 9.4 ± 2.1%; FCS: male: 9.2 ± 0.6%, female: 14.2 ± 1%; ANOVA 3, P = 0.01), whereas the proportion of stained cells was higher in FCS than in BSA medium whatever the sex (ANOVA 3, P = 0.02). The same tendency, although not significant, was obtained for the proportion of cells showing TUNEL staining with higher values in female than in male embryos (BSA: male: 9.3±2.1%, female: 10.5 ± 2.6%; FCS: male: 13.1 ± 0.9%, female: 16.5 ± 1.1%; ANOVA 3, P = 0.07) and higher values in FCS than in BSA medium whatever the sex (ANOVA 3, P = 0.05). A tendency for a higher proportion of double-stained cells (TUNEL and caspase-positive) was also observed in female embryos whatever the medium (BSA: male: 3.8 ± 1.1%, female: 5 ± 1.6%; SOF: male: 4.8 ± 0.5%, female: 7.0 ± 0.7%; ANOVA 3, P = 0.08). Intriguingly, only about half of the stained cells showed the double staining (TUNEL and caspase). This could be explained by the fact that caspase-3 activation can appear before DNA fragmentation during the apoptosis process and caspase staining disappear when TUNEL staining is still visible. But both caspase-3 activation and DNA fragmentation can also occur independently of apoptosis. In conclusion, male embryos seem to show a higher developmental rate in both media and could be less affected by apoptosis than female ones, particularly when cultured with FCS. Those experiments have to be repeated with the sexed sperm of another bull to draw final conclusions.
Hox genes encode for homeodomain transcription factors well known to be involved in developmental control after gastrulation. However, the expression of some of these genes has been detected during oocyte maturation and early embryo development. An interesting expression profile has been obtained for HOXB9 in the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436): its relative expression increases between the immature oocyte and the zygote, further increases at the 5- to 8-cell stage to peak at the morula stage before decreasing at the blastocyst stage. The main objective of this work is to establish the HOXB9 protein profile from the immature oocyte to the blastocyst in the bovine. Bovine embryos were produced in vitro from immature oocytes obtained from slaughterhouse ovaries. Embryos were collected at the following stages: immature oocyte, mature oocyte, zygote (18 h post-insemination, hpi), 2-cell (26 hpi), 5 to 8 cell (48 hpi), 9 to 16 cell (96 hpi), morula (120 hpi), and blastocyst (180 hpi). The presence and distribution of HOXB9 proteins were detected by whole-mount immunofluorescence followed by confocal microscopy using an anti-human HOXB9 polyclonal antibody directed against a sequence showing 100% homology with the bovine protein. Its specificity to the bovine protein was controlled by Western blot on total protein extract from the bovine uterus and revealed, among a few bands of weak intensities, 2 bands of high intensity corresponding to the expected size. Oocytes or embryos were fixed and incubated overnight with rabbit anti-HOXB9 (Sigma, St. Louis, MO, USA) and mouse anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA) primary antibodies and then for 1 h with goat anti-rabbit Alexafluor 555 conjugated (Cell Signaling Technology, Beverly, MA, USA) and goat anti-mouse FITC-conjugated (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibodies. Embryos were then mounted in Vectashield containing DAPI. HOXB9 is detected from the immature oocyte to the blastocyst stage. At the immature oocyte stage, it is mainly localised in the germinal vesicle with a weak signal in the cytoplasm. At the mature oocyte stage, HOXB9 labelling is present in the cytoplasm. At the zygote stage, a stronger immunoreactivity is observed in the pronuclei than in the cytoplasm. From the 2-cell stage to the morula stage, the presence of HOXB9 is also more important in the nuclei than in the cytoplasm. HOXB9 is also observed at the blastocyst stage where it is localised in the nuclei of the trophectoderm cells, whereas an inconstant or weaker labelling is observed in the inner cell mass cells. In conclusion, we have shown for the first time the presence of the HOXB9 protein throughout early bovine embryo development. The results obtained suggest the presence of the maternal HOXB9 protein because it is already detected before the maternal to embryonic transition that occurs during the fourth cell cycle in the bovine. Finally, the pattern obtained at the blastocyst stage suggests a differential role of HOXB9 in the inner cell mass and trophectoderm cells. C. Sauvegarde holds a FRIA PhD grant from the Fonds National de la Recherche Scientifique (Belgium).
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