ABSTRACT
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.
Subject(s)
Embryonic Development , Homeodomain Proteins/metabolism , Mammals , Oocytes/metabolism , Oogenesis , Animals , Blastocyst/metabolism , Cattle , Cell Lineage/genetics , Embryonic Development/genetics , Endoderm/metabolism , Fetus , Fluorescent Antibody Technique , Gastrulation/genetics , Gene Expression , Gene Knockout Techniques , Homeodomain Proteins/genetics , Humans , Immunohistochemistry , Mice , Oogenesis/genetics , Protein Transport , Zygote/metabolismABSTRACT
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.
Subject(s)
Embryo, Mammalian/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mammals/genetics , Mammals/metabolism , Oocytes/metabolism , Animals , Embryonic Development/genetics , Fluorescent Antibody Technique , Gene Expression Regulation, Developmental , Polymerase Chain ReactionABSTRACT
HOX proteins are transcription factors that play a major role in patterning the body axis of vertebrates from the gastrulation stage. While nothing has been reported so far about their roles at earlier stages, there is evidence that some HOX genes are expressed before gastrulation. The objective of this work was to study the pattern of expression of several HOX genes during oocyte maturation and early embryonic development up to the blastocyst stage. Using nested PCR, HOXD1, HOXA3, HOXD4, HOXB7, HOXB9, and HOXC9 transcripts were detected in bovine oocytes and early embryos at various frequencies depending on the stage of development. Quantitative PCR was performed on bovine oocytes and early embryos: relative expression of HOXD1, HOXA3, and HOXC9 decreased sharply after the 5-8 cell stage. HOXB9 relative expression increased between the oocyte and the morula stage. All transcripts seemed to be of maternal origin before the maternal to embryonic transition, as demonstrated by blocking transcription with α-amanitin. Reverse transcription was performed with either hexamers or oligo-dT, allowing for the determination that HOXC9 transcripts were slightly deadenylated during oocyte maturation; HOXD1, HOXA3, and HOXB9 transcripts were not, indicating that they could be translated. Hoxd1, Hoxa3, Hoxb9, and Hoxc9 expression was also detected in mouse oocytes and early embryos. A similar pattern of expression was found in the two species. In conclusion, mammalian HOX genes might be implicated in the control of oocyte maturation, the maternal-to-embryonic transition or the first steps of embryo differentiation.
