High homology between a trophoblastic protein (trophoblastin) isolated from ovine embryo and α-interferons

AbstractOvine trophoblastic protein B (oTPB), an embryonic protein, is a 20 kDa secretory protein which is synthesized by the ovine conceptus from days 12 to 22 of pregnancy. oTPB was purified by HPLC using ion-exchange chromatography on a DEAE column and was subsequently chromatographed on a reversed-phase column. Automated Edman degradation was then used to determine the N-terminal amino acid sequence up to 45 residues. The sequence data reveal a significant homology between oTPB and bovine interferons α of class II: 64% of the amino acids are identical and 75% are homologous. A highly conserved region including residues 23–44 exhibits 82% homology. Identity between oTPB and either HuIFN-α.9 or MuIFNα. 1 is 55%. These alignments between oTPB and IFNs occur at the N-terminus of the mature proteins and proceed without deletion. These results suggest that oTPB is an embryonic interferon

Paracrine effects of embryo-derived FGF4 and BMP4 during pig trophoblast elongation

The crosstalk between the epiblast and the trophoblast is critical in supporting the early stages of conceptus development. FGF4 and BMP4 are inductive signals that participate in the communication between the epiblast and the extraembryonic ectoderm (ExE) of the developing mouse embryo. Importantly, however, it is unknown whether a similar crosstalk operates in species that lack a discernible ExE and develop a mammotypical embryonic disc (ED). Here we investigated the crosstalk between the epiblast and the trophectoderm (TE) during pig embryo elongation. FGF4 ligand and FGFR2 were detected primarily on the plasma membrane of TE cells of peri-elongation embryos. The binding of this growth factor to its receptor triggered a signal transduction response evidenced by an increase in phosphorylated MAPK/ERK. Particular enrichment was detected in the periphery of the ED in early ovoid embryos, indicating that active FGF signalling was operating during this stage. Gene expression analysis shows that CDX2 and ELF5, two genes expressed in the mouse ExE, are only co-expressed in the Rauber's layer, but not in the pig mural TE. Interestingly, these genes were detected in the nascent mesoderm of early gastrulating embryos. Analysis of BMP4 expression by in situ hybridisation shows that this growth factor is produced by nascent mesoderm cells. A functional test in differentiating epiblast shows that CDX2 and ELF5 are activated in response to BMP4. Furthermore, the effects of BMP4 were also demonstrated in the neighbouring TE cells, as demonstrated by an increase in phosphorylated SMAD1/5/8. These results show that BMP4 produced in the extraembryonic mesoderm is directly influencing the SMAD response in the TE of elongating embryos. These results demonstrate that paracrine signals from the embryo, represented by FGF4 and BMP4, induce a response in the TE prior to the extensive elongation. The study also confirms that expression of CDX2 and ELF5 is not conserved in the mural TE, indicating that although the signals that coordinate conceptus growth are similar between rodents and pigs, the gene regulatory network of the trophoblast lineage is not conserved in these species

Temporal and Spatial Expression of Muc1 During Implantation in Sows

Recent evidence points to an important role for Muc1 in embryo implantation. In this study, Real-time PCR and immunohistochemistry were used to study mRNA and protein levels at, and between, the attachment sites of the endometrium of Day 13, 18 and 24 pregnant sows. The results indicate that Muc1 mRNA expression was higher between attachment sites than at attachment sites during implantation and this effect was significant on Day 13 (P < 0.01) and 24 (P < 0.01). Intense Muc1 immunostaining was observed in luminal epithelium and stroma and the staining between attachment sites was stronger than at attachment sites on Days 13 and 18. Collectively, these results suggest the crucial role of Muc1 in successful implantation and embryo survival

Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way

Place de la microscopie dans l'etude des conditions de l'implantation chez les ruminants

National audienc

Le developpement embryonnaire normal avant l'implantation

150 ref.National audienc

Blastocyst development and implantation

chap. 20International audienc

Developpement et implantation du blastocyste

chap. 20National audienc

Organisation cellulaire des cellules trophoblastiques dans l'élongation du conceptus ovin

International audienceWe present a computational approach to analyze the cellular organization during the elongation process of the ovine conceptus. First, we selected a set of mathematical descriptors to quantify cell geometry and cell neighborhood within the external epithelial layer of the conceptus: the trophoblast. Second, we established a hybrid image segmentation framework, and analyzed the extracted features with statistical tools to describe and compare the spatio-temporal dynamics of cellular organization within this epithelium. The main results indicated that the average geometry and neighborhood of the trophoblast cells are relatively stable from a sampling position to another and from a stage of development to another. Further, their elongation axes are randomly distributed. The cellular organization fits the Poisson's process. Moreover, no clustering structures or grid-like regular point patterns are found inside the studied cell population. This suggests that the trophoblast elongation observed in ruminants is not due to the geometrical change of cell shape but might be the consequence of cell addition associated with peculiar plans of cell division or intercalation.L'objectif de cette étude est une approche statistique de l'organisation cellulaire au cours de l'élongation du conceptus ovin. Nous avons sélectionné un ensemble de descripteurs mathématiques pour quantifier la géométrie et la topologie des cellules ainsi que des outils de la statistique spatiale permettant d'évaluer la dynamique spatio-temporelle d'organisation des cellules au niveau d'un tissu. Les analyses montrent une relative stabilité de la forme et de la topologie des cellules trophoblastiques au cours de l'élongation du conceptus. Aucune orientation particulière des cellules n'a pu être démontrée. L'organisation spatiale des cellules correspond bien à un processus de Poisson et aucune forme d'agrégation ou de dispersion régulière des cellules n'a été observée. Ceci suggère que l'élongation du conceptus observée chez les ruminants n'est pas due au changement des formes mais pourrait être la conséquence d'adjonction de cellules selon des plans de divisions particuliers ou des patrons coordonnés d'intercalation des cellules