The postimplantation developmental potential of embryos can be affected by various forms of cell death, such as apoptosis, at preimplantation stages. However, correct assessment of apoptosis is needed for adequate inference of the developmental significance of this process. This study is the first to investigate the independent chronological occurrence of apoptotic changes in nuclear morphology and DNA degradation (detected by the TUNEL reaction) and incidences of nuclei displaying these features at various preimplantation stages of bovine embryos produced both in vivo and in vitro. Different elements of apoptosis were observed at various developmental stages and appeared to be differentially affected by in vitro production. Nuclear condensation was observed from the 6-cell stage in vitro and the 8-cell stage in vivo, whereas the TUNEL reaction was first observed at the 6-cell stage in vitro and the 21-cell stage in vivo. Morphological signs of other forms of cell death were also observed in normally developing embryos produced both in vivo and in vitro. The onset of apoptosis seems to be developmentally regulated in a stage-specific manner, but discrete features of the apoptotic process may be differentially regulated and independently modulated by the mode of embryo production. Significant differences in indices of various apoptotic features were not evident between in vivo- and in vitro-produced embryos at the morula stage, but such differences could be observed at the blastocyst stage, where in vitro production was associated with a higher degree of apoptosis in the inner cell mass.
Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt-signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17+ endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17+ cells more effectively than activin-mediated induction. Notably, activin-induction of Gsc-GFP+ cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17+ cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.
Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.
The complexity of the events which orchestrate mammalian oocyte growth and ultimate acquisition of developmental competence is still unclear and under continuous investigation. Starting from the observation that germinal vesicle (GV) bovine oocytes exhibit different patterns of chromatin configuration (from GV0 to GV3), the present study aimed at analyzing the morphology of the nuclear and the cytoplasmic compartments and to determine the total transcriptional activity of immature oocytes sorted on the basis of their chromatin configuration. The oocyte morphology was analyzed by light microscopy (LM) on semi-thin sections and transmission electron microscopy, and the global transcriptional activity was analyzed by H(3)-Uridine incorporation and subsequent autoradiography on semi-thin sections. LM confirmed the increase in chromatin condensation from GV0 to GV3. Ultrastructurally, the nucleolus was fibrillo granular at GV0 while the other stages displayed an electron-dense fibrillar sphere with the remnant of a fibrillar center on the surface. Organelles were dispersed in the cytoplasm at GV0 while at GV1 and GV2 most of them were homogenously distributed in the oocyte cortex. At GV3 most organelles were found in clusters in the oocyte cortex. Typical features of completion of the oocyte growth phase, like undulation of the nuclear envelope and reduction of the size of Golgi complex were found at GV2 and GV3. Moreover, GV3 oocytes presented cortical granules that displayed varying degrees of degeneration. Autoradiographic labeling showed that oocytes with GV0 configuration exhibited high level of RNA synthesis, GV1 and GV2 stages showed a remarkable decrease of transcription, and the acquisition of GV3 configuration was associated with global repression of transcriptional activity. Our findings suggest a temporal relationship between the chromatin remodeling process and the main morpho-functional events that characterize the final growth phase in bovine oocyte.
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