Isabelle Hue and Jean-Paul Renard
Michel Guillomot, Annick Turbe, Isabelle Hue and Jean-Paul Renard
The high rates of embryonic mortalities which follow in vitro production of ruminant embryos have emphasized the need for increased knowledge of early development. It is likely that early failures in embryonic development and placenta formation involve abnormal differentiation of mesoderm. The aim of this study was to investigate the pattern of expression of two T-box genes known to control the gastrulation process, Brachyury and Eomesodermin, by whole-mount in situ hybridization. To allow a more precise comparison of both expression patterns between embryos, we describe a new staging of pre-implanted ovine embryos by gross morphology and histology from pre-gastrulation stages to the beginning of neurulation. In pre-streak embryos primitive mesoderm cells delaminated in between the primitive endoderm and the epiblast. At that stage, no expression of Brachyury or Eomesodermin could be detected in the embryos. Early expression of both T-genes was observed by the early-streak stages in epiblast cells located close to the presumptive posterior pole of the embryos. Later on, during gastrulation both genes followed a pattern of expression similar to the ones described in other mammals. These observations suggest that other genes, which remain to be identified, are responsible for extra-embryonic mesoderm differentiation in ruminant embryos.
Isabelle Hue, Isabelle Dufort, Anaïs Vitorino Carvalho, Denis Laloe, Nathalie Peynot, Severine A Degrelle, Christoph Viebahn and Marc-Andre Sirard
Embryo transfer in cattle is performed with blastocysts produced in vivo or in vitro using defined media. However, outdated systems such as those that use serum and co-culture remain of interest for research purposes. Here, we investigated the effect of additional culture time on in vitro-produced embryos. Specifically, we compared embryos that formed a blastocoel at different times after fertilisation to those that stayed in culture for up to two additional days with respect to their development in vivo after temporary transfer to oestrus-synchronised recipients. A pre-transfer set (D6, D6+1, D6+2, D7, D7+1, D8) was examined using microarray analyses, and correlated with a post-transfer set that included two different days of transfer (D6-T6, D6+2-T8, D7+1-T8, D8-T8). All surviving conceptuses reached primitive-streak stages and filamentous sizes similarly to in vivo (D18) or in vitro controls (D7/T7). The recovery rate differed between D6 and D8 embryos that were immediately transferred (58% vs 25%). With an intermediate survival rate (33%), the D6 embryos with two additional days in culture produced 9 times more IFN-tau at D18 than the D6 embryos that were immediately transferred. At the end of culture, D6 and D6+2 embryos displayed the highest number of gene expression differences. Despite a mortality of 40-60%, no signature was detectable in any of the transferred groups that would account for the embryos’ fates. Initially reputed to be beneficial in producing more blastocysts our culture system of B2 medium plus serum and co-culture generated blastocysts that were distinct from those developed in vivo (D7).
Séverine A Degrelle, Kim-Anh Lê Cao, Yvan Heyman, Robin E Everts, Evelyne Campion, Christophe Richard, Céline Ducroix-Crépy, X Cindy Tian, Harris A Lewin, Jean-Paul Renard, Christèle Robert-Granié and Isabelle Hue
Axis specification in mouse is determined by a sequence of reciprocal interactions between embryonic and extra-embryonic tissues so that a few extra-embryonic genes appear as ‘patterning’ the embryo. Considering these interactions as essential, but lacking in most mammals the genetically driven approaches used in mouse and the corresponding patterning mutants, we examined whether a molecular signature originating from extra-embryonic tissues could relate to the developmental stage of the embryo proper and predict it. To this end, we have profiled bovine extra-embryonic tissues at peri-implantation stages, when gastrulation and early neurulation occur, and analysed the subsequent expression profiles through the use of predictive methods as previously reported for tumour classification. A set of six genes (CALM1, CPA3, CITED1, DLD, HNRNPDL, and TGFB3), half of which had not been previously associated with any extra-embryonic feature, appeared significantly discriminative and mainly dependent on embryonic tissues for its faithful expression. The predictive value of this set of genes for gastrulation and early neurulation stages, as assessed on naive samples, was remarkably high (93%). In silico connected to the bovine orthologues of the mouse patterning genes, this gene set is proposed as a new trait for embryo staging. As such, this will allow saving the bovine embryo proper for molecular or cellular studies. To us, it offers as well new perspectives for developmental phenotyping and modelling of embryonic/extra-embryonic co-differentiation.