A small set of extra-embryonic genes defines a new landmark for bovine embryo staging.

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.

Myogenesis is delayed in bovine fetal clones.

We have recently reported that maturation of the skeletal muscle is delayed in cloned calves during their first year postnatally. This delay could originate from perturbations in fetal myogenesis. The aim of this study was to evaluate the developmental characteristics of muscle in clones versus animals derived from conventional reproduction. We have characterized the anatomical and biochemical properties of the Semitendinosus muscle of clones versus controls at day 60 and day 260. We have analyzed the contractile and metabolic properties of muscle fibers by measuring the abundance of myosin heavy chain (MyHC) isoforms and activities of metabolic enzymes (LDH, PFK, COX, CS, ICDH), respectively. The spatial repartition of some components of the extracellular matrix (collagen types I, IV, VI, chondroitin-6-sulfate, decorin, and tenascin-X) was also studied. At day 60 we found lower numbers and structural organization of fibers, and a delay in the setup of the extracellular matrix. IGF-2 transcript abundance was lower in clones than in their controls. There was no difference in the expression of VEGF (a growth factor regulating vascularization and myogenesis) and its receptor. At day 260 the muscles of fetal clones have not reached the same degree of differentiation than controls as shown by their lower energy metabolisms and their MyHC pattern. These results show for the first time that disturbances in myogenesis occur early in fetal life in cloned cattle.

The immune status of bovine somatic clones.

Agronomical applications of cloned livestock produced by somatic cell nuclear transfer (SCNT) have been authorized in the United States and the European Food Safety Authority published that there was no evidence of risks associated with the use of cloned animal in the breeding industry. Both assessments, however, underlined that complementary data are needed to update their conclusions. SCNT is associated with a high incidence of perinatal losses. After birth, cloned cattle appear to possibly present subtle immune defects, requiring extensive studies to be properly evidenced. Twenty-five cloned Holstein heifers from five distinct genotypes and their contemporary age- and sex-matched controls were compared. An extensive survey of leukocyte subsets was performed and the humoral and T-cell immune responses to exogenous antigens were studied. Cloned cattle presented a normal representation of leukocyte subsets. Functional immunity was not modified in cloned heifers, as they were able to raise an antibody response and to develop B and T cell-specific responses against the model antigen OVA (ovalbumin) and against a rotavirus vaccine as in controls. Thus, this extensive analysis supports previous data suggesting that cloned cattle have a normal immunity.

Endometrium as an early sensor of in vitro embryo manipulation technologies.

Implantation is crucial for placental development that will subsequently impact fetal growth and pregnancy success with consequences on postnatal health. We postulated that the pattern of genes expressed by the endometrium when the embryo becomes attached to the mother uterus could account for the final outcome of a pregnancy. As a model, we used the bovine species where the embryo becomes progressively and permanently attached to the endometrium from day 20 of gestation onwards. At that stage, we compared the endometrial genes profiles in the presence of an in vivo fertilized embryo (AI) with the endometrial patterns obtained in the presence of nuclear transfer (SCNT) or in vitro fertilized embryos (IVF), both displaying lower and different potentials for term development. Our data provide evidence that the endometrium can be considered as a biological sensor able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process. Compared with AI, numerous biological functions and several canonical pathways with a major impact on metabolism and immune function were found to be significantly altered in the endometrium of SCNT pregnancies at implantation, whereas the differences were less pronounced with IVF embryos. Determining the limits of the endometrial plasticity at the onset of implantation should bring new insights on the contribution of the maternal environment to the development of an embryo and the success of pregnancy.