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  • Articles  (4)
  • Freeze-substitution  (2)
  • In vitro fertilization  (2)
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 14 (1990), S. 39-45 
    ISSN: 0741-0581
    Keywords: Exocrine pancreas ; Cryofixation ; Cryomicrotomy ; Freeze-drying ; Freeze-substitution ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: This paper describes and compares the morphology of a relatively complex tissue, the exocrine pancreas, prepared by state-of-the-art anhydrous cryoprocedures. Cryopreparative procedures are being used increasingly for a wide range of applications, for example, electron-probe x-ray microanalysis and immunocytochemical localization of antigenic molecules, because they preserve the composition of the specimen better than procedures involving aqueous media. Some doubts have remained concerning the morphology of cryosections and the precise identification of subcellular structures.We show that thin and sufficiently large cryosections of fresh biological tissues can be produced using commercially available hardware. The freeze-dried cryosections display high intrinsic contrast, are stable under the beam, and allow identification of intracellular fine structure.
    Additional Material: 6 Ill.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 16 (1990), S. 298-323 
    ISSN: 0741-0581
    Keywords: In vitro maturation ; In vitro fertilization ; Cytoplasmic phenotype ; Cytoplasmic organization ; Developmental competence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Cellular aspects of reinitiated meiosis, fertilization, and early preimplantation embryogenesis in the bovine species were examined under in vitro conditions. An analysis of the cytoplasmic distribution of mitochondria, lipid droplets and vesicles in over 5,000 living GV-stage oocytes, with subsequent examination by electron microscopy, indicated that the organization of cytoplasm was pleomorphic and that five distinct cellular phenotypes could be identified. Inspection of oocytes during the resumption of arrested meiosis in vivo and in vitro demonstrated that the subcellular organization of the bovine oocyte cytoplasm remains unchanged during maturation to metaphase II. The influence of culture conditions and media on the frequency of maturation, cytoplasmic organization, fertilizability, and early preimplantation embryogenesis was also determined. The findings indicate that meiotic maturation and fertilization in the bovine species can occur at high frequency under comparatively simple and defined conditions. However, the acquisition of developmental competence for fertilization, and the ability of the egg to develop progressively after fertilization appears to be related to the organization of the cytoplasm at the GV stage. The relationship between cytoplasmic organization and conditions of maturation, fertilization, presence or absence of cumulus cells, and the acquisition of developmental competence is discussed with respect to (1) cell biological aspects of mammalian oocyte maturation, (2) the potential influence of extrinsic factors (e. g., differential intrafollicular biochemistry and morphophysiology) on sub-cellular organization of the GV-stage oocyte, and (3) the finding that morphologically equivalent bovine embryos derived from the in vitro fertilization of in vitro-matured oocytes may be developmentally heterogeneous. The studies also revealed that nuclear and cytoplasmic aberrations which could preclude normal embryogenesis can develop shortly after fertilization. The significance of this finding with respect to cytoplasmic phenotype of the oocyte and conditions of maturation, fertilization, and early embryo culture is discussed.
    Additional Material: 44 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 16 (1990), S. 167-173 
    ISSN: 0741-0581
    Keywords: Plunge-freezing ; Cryofixation ; Freeze-substitution ; Monolayers ; Cultured cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A detailed design for a simple and inexpensive variable-speed (1.0-5.8 m s-1) pneumatic plunge-freezing device is presented. Cultured cells, grown on Formvar-coated 75-mesh gold finder grids, are pneumatically driven into a stirring mixture of propane/isopentane (3:1) cooled by liquid nitrogen (LN2). Premature freezing of the sample in the cryogenic vapors above the cryogen is prevented by plunging through an entry tube into an insulating box, to which a partial vacuum is applied. The cryogenic vapors are drafted into the box at the level of the liquid cryogen by the vacuum, thereby preventing a layer of cold gas from collecting above the cryogen. To prevent the sample from thawing during transfer from the cryogen to the substitution medium, the box top is removed and compressed air is forced through a corrugated tube running the length of the box. The resulting boiling LN2 creates an atmosphere below -120°C in which the transfer can be accomplished.
    Additional Material: 5 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 16 (1990), S. 324-346 
    ISSN: 0741-0581
    Keywords: Human oocyte ; Cytoplasmic organization ; Oocyte pathology ; In vitro fertilization ; Ovarian hyperstimulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The extent to which fertilization failure in in vitro inseminated human oocytes obtained from hyperstimulated ovaries was associated with alterations in cellular structure and cytoplasmic organization was examined by a combination of DIC, fluorescence, time-lapse video, and transmission electron microscopy. Detailed analysis of meiotically mature preovulatory (uninseminated) oocytes, and of oocytes that failed to fertilize in vitro, indicated that between 10% and 15% of grossly normal-appearing MII-stage oocytes displayed one of the following cellular perturbations: (1) a subtle change in organelle distribution, (2) a small region(s) of intracellular cytolysis, (3) a massive aggregation of SER tubules, (4) an accumulation of vesicles presumed to be of SER origin, (5) a change in the structural organization of the cortical cytoplasm and overlying plasma membrane, (6) a sudden and rapid internalization of perivitelline fluid by means of an apparently aberrant process of endocytosis, and (7) a premature and partial exocytosis of cortical granules. The description of these disorders is discussed with respect to the developmental consequences for the oocyte.
    Additional Material: 51 Ill.
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