The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N 2 • The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.Extra keyword: Cleavage.
In leaves of A. thaliana, there exists an intricate network of epidermal surface layer cells responsible for anatomical stability and vigor of flexibility to the entire leaf. Rho GTPases direct this organization of cell polarity, but full understanding of the underlying mechanisms demands further inquiry. We conduct two experiments: (1) a novel procedure is proposed that could be used in other life and plant science studies to quantify microtubule orientation, and (2) shape analysis. We hypothesize ARK2 as a putative interactor in cell polarity maintenance through stabilization of microtubule ordering. We are the first to automate pavement cell phenotype analysis for cell polarity and microtubule orientation. Breakthroughs in the signaling network regulating leaf cell polarity and development will lead science into the frontier of genetically modifying leaves to dramatically increase Earth's plant biomass; impending food shortages in the 21st century will be well served by such research.
Mouse inner cell masses remained intact when exposed to extreme bsmotic stress (distilled water) for short periods, but the trophectoderm was lysed in one-third of blastocysts. However, these inner cell masses were not viable as they could not fluoresce after incubation in fluorescein diacetate nor continue development in vitro. It was concluded that cells of the inner cell mass are not more tolerant of osmotic stresses than those of the trophectoderm.Inner cell masses were isolated from only 50 % of blastocysts when exposed to the calcium ionophore A23187. Some lysing trophectoderm cells are probably able to restore their normal calcium levels after transfer to fresh culture medium allowing normal blastocyst-like development in vitro. Further evidence which suggests that not all trophectoderm cells are removed after incubation in ionophore is that these inner cell masses produced more trophoblast-like outgrowths in vitro and were able to induce the decidual cell reaction in vivo more often than their immunosurgically isolated counterparts.Irnmunosurgically isolated inner cell masses were viable as they fluoresced brightly after incubation in fluorescein diacetate and developed normally in vitro. There was little or no contamination of these inner cell masses with trophectoderm cells as they formed considerably smaller outgrowths than control blastocysts and were rarely able to induce the decidual cell reaction in vivo.Trophoblast-like giant cells were found less frequently from inner cell masses isolated from 142-h, post-HCG blastocysts and cultured in vitro, than from li8-h, post-HCG blastocysts. These results are discussed in relation to a current theory on the time of inner cell mass determination which states that apparently differentiated inner cell mass cells may reverse their direction of development in response to altered environmental conditions.
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