Embryonic and extra-embryonic lineages are separated at the blastocyst stage in the mouse at the onset of implantation but well ahead of implantation in most mammals. To provide information on the development of the trophoblast lineage in late-implanting bovine embryos, we combined the use of molecular markers defining embryonic and extra-embryonic lineages in the mouse with a transcriptomic approach dedicated to the early steps of the elongation process, a characteristic feature of blastocyst development in ruminants. In this study, we present molecular evidence for differences between the cow and the mouse in the programming of trophoblast differentiation. This different programming encompasses: (i) the expression of epiblast specifying genes (Oct-4, Nanog) in bovine trophoblast cells at the onset of elongation, (ii) the transcription of proliferation markers in early elongating blastocysts, (iii) the early detection of trophoblast-specific transcripts related to extra-embryonic tissue's differentiation (Hand1, Ets2, IFN-tau) and (iv) the identification of a new transcript (c12) which displays a reciprocal pattern to that of Oct-4 and Nanog genes in the embryonic cells and for which no equivalent has thus far been found in the mouse. Altogether, these results tended to show that early elongation is a critical transition in bovine trophoblast development.

At implantation the endometrium undergoes modifications necessary for its physical interactions with the trophoblast as well as the development of the conceptus. We aim to identify endometrial factors and pathways essential for a successful implantation in the caruncular (C) and the intercaruncular (IC) areas in cattle. Using a 13,257-element bovine oligonucleotide array, we established expression profiles at day 20 of the estrous cycle or pregnancy (implantation), revealing 446 and 1,295 differentially expressed genes (DEG) in C and IC areas, respectively (false discovery rate ϭ 0.08). The impact of the conceptus was higher on the immune response function in C but more prominent on the regulation of metabolism function in IC. The C vs. IC direct comparison revealed 1,177 and 453 DEG in cyclic and pregnant animals respectively (false discovery rate ϭ 0.05), with a major impact of the conceptus on metabolism and cell adhesion. We selected 15 genes including C11ORF34, CXCL12, CXCR4, PLAC8, SCARA5, and NPY and confirmed their differential expression by quantitative RT-PCR. The cellular localization was analyzed by in situ hybridization and, upon pregnancy, showed gene-specific patterns of cell distribution, including a high level of expression in the luminal epithelium for C11ORF34 and MX1. Using primary cultures of bovine endometrial cells, we identified PTN, PLAC8, and CXCL12 as interferon-(IFNT) target genes and MSX1 and CXCR7 as IFNT-regulated genes, whereas C11ORF34 was not an IFNT-regulated gene. Our transcriptomic data provide novel molecular insights accounting for the biological functions related to the C or IC endometrial areas and may contribute to the identification of potential biomarkers for normal and perturbed early pregnancy.transcriptome; interferon-tau; pregnancy; cattle IN MAMMALS, the establishment and maintenance of pregnancy require a subtle and tightly regulated communication between the conceptus (embryo and embryonic annexes) and the maternal environment (85). The success of implantation relies on several essential steps including the adjustment of the uterine environment to support the development of the conceptus and the profound remodeling of the endometrium structure necessary for the apposition, adhesion, and invasion phases (36). In contrast to human and rodents, the invasion of the maternal tissue by the fetal tissue is very limited in ruminants (71) and leads to a synepitheliochorial placentation (86). Since the trophoblast appears to be intrinsically invasive in mammals (11), apposition, adhesion, and invasion processes are thought to be controlled by the endometrium (83). In mammalian species presenting an invasive implantation, decidua restrains the invasion of the embryo in a spatiotemporal manner (20). The expression and the regulation of some factors involved in the apposition, adhesion, and invasion aspects of implantation have been reported in ruminants (62, 79), but, overall, the comparative cascade of molecular mechanisms remains largely unknown.The sequence of events occ...

In this study we investigated expression of the two isoforms of the prostaglandin-forming enzyme, cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2), in sheep embryos. Using Western blot and immunohistochemical analyses, we demonstrated that Cox-2 was highly expressed in embryos from Day 8 to Day 17 of development whereas Cox-1 was undetectable during this time. The expression of Cox-2 was developmentally regulated. It was maximal between Days 14 and 16. There was a 30-fold increase in Cox-2 content per protein extract between Day 10 and Day 14, corresponding to a 50,000-fold increase in the whole embryo. The expression of Cox-2 declined after Day 16 to become undetectable by Day 25 of pregnancy. Cox-2 was localized in the trophoblastic cells and was not detected in the inner cell mass. The [3H]arachidonic acid metabolites synthesized by Cox-2-rich conceptuses were analyzed by HPLC after short-term embryo culture. Day 14 conceptuses released mainly cyclooxygenase metabolites and to a lesser extent lipoxygenase derivatives. Cyclooxygenase products were 6-keto-prostaglandin (PGF)1alpha 18.2% (+/- 4.2), thromboxane-B2 22.51% (+/- 15.9), PGF2alpha 21% (+/- 11), PGE2 14.5% (+/- 7.4), and PGD2 2.7% (+/- 2.6). Taken together, these results suggest an important role for the Cox-2-dependent cyclooxygenase metabolites during embryo development.

We investigated the lysophosphatidic acid (LPA) pathway during early pregnancy in sheep. LPA was detected in the uteri of early-stage pregnant ewes. Using quantitative RT-PCR, the expression of autotaxin, the LPA-generating enzyme, was found in the endometrium and conceptus. In the latter autotaxin, transcript levels were low on d 12-14 and increased on d 15-16, in parallel with the level of LPA. Autotaxin was localized in the luminal epithelium and superficial glands of the endometrium and in trophectoderm cells of the conceptus. The expression of G protein-coupled receptors for LPA was also examined in the ovine conceptus. LPA receptor LPAR1 and LPAR3 transcripts were expressed during early pregnancy and displayed a peak on d 14, whereas the highest level of protein for both receptors was observed at d 17. LPAR1 was localized in cellular membranes and nuclear compartments of the trophectoderm cells, whereas LPAR3 was revealed only in membranes. LPA activated phosphorylation of the MAPK ERK1/2 in ovine trophectoderm-derived cells. Moreover, the bioactive lipid increased the proliferation of trophectoderm cells in culture, as shown by thymidine and bromodeoxyuridine incorporation. Furthermore, LPA induced changes to the organization of beta-actin and alpha-tubulin, suggesting a role for it in rearrangement of trophectoderm cells cytoskeleton. Because a link had previously been established between prostaglandin and LPA pathways, we analyzed the effect of LPA on prostaglandin synthesis. LPA induced an increase in the release of prostaglandin F2alpha and prostaglandin E2, with no significant modifications to cytosolic phospholipase A2alpha and prostaglandin synthase-2 expression. Taken together, our results suggest a new role for LPA-mediated signaling in the ovine conceptus at the time of implantation.