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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 26 (1990), S. 253-260 
    ISSN: 1040-452X
    Keywords: Microinsemination ; Micromanipulation ; Fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Micronsemination sperm transfer (MIST) is a technique whereby sperm are transferred into the perivitelline space (PVS) with the aid of a micromanipulator. MIST is now used to investigate whether blastomere membranes of early human embryos are capable of fusing with the sperm as in the metaphase II oocyte. Between 10 and 30 sperm were transferred into 11 donated human embryos between pronuclear and 16 cell stage. After culture for 6-24 hr in vitro, the embryos were fixed for transmission electron microscopy (TEM). Both acrosome-intact and acrosome-reacted sperm were located in the PVS and between blastomeres. Sperm in the PVS were sometimes penetrating the inner regions of the zona. Sperm-blastomere membrane fusion was not observed, but sperm tail incorporation by phagocytosis was occasionally evident. Sperm heads incorporated into blastomeres were often located in membrane-bound vesicles. Both acrosome-intact and acrosome-reacted sperm heads were found in vacuoles. Acrosome-reacted sperm heads were lying passively in vacuoles or were undergoing degenerative changes at their surfaces. Sperm chromatin decondensation was not observed in any of the sperm heads that were detected in the blastomeres. The evidence presented clearly shows that sperm heads are incapable of expanding their chromatin to form typical male pronuclei following MIST into early human embryos.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 90 (1977), S. 361-374 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: 5-azacytidine at concentrations higher than 5 μM inhibited the differentiation of a rat myoblast line in vitro. It was also somewhat cytotoxic at this level. Variants resistant to the cytotoxic effect of 5-azacytidine were obtained which were simultaneously unable to differentiate into myotubes and exhibited altered morphology. These characteristics were retained by the variants when subcultured in the absence of the drug for over 700 generations. Several of the azacytidine resistant cells were more susceptible than the parental line to the lethal action of 5-bromodeoxyuridine and adenosine, but not that of cytosine arabinoside, ouabain or 8-azaguanine.The variants were capable of transporting uridine, thymidine and 5-azacytidine. The uridine kinase activity was one-half to one-third of that in the parental cells but it was not missing completely in any of the variants. Two independently isolated variants selected for detailed study showed a 2- to 3-fold increase in the activity of orotidylic acid decarboxylase. This enzyme in the variants in contrast to that of the parental cells was completely insensitive to the inhibitory effect of a nucleotide generated from ATP and 5-azacytidine in cell extracts (probably 5-azacytidine monophosphate). These observations point to the possibility that 5-azacytidine resistance arises in myoblasts due to an alteration of the components of two target pathways of this drug, viz., the de novo pyrimidine pathway and an undefined sequence leading to the synthesis of membrane components.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 15 (1986), S. 317-326 
    ISSN: 0148-7280
    Keywords: in vitro fertilization ; sperm-egg fusion ; polyspermy ; sperm incorporation ; electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The early events of gamete membrane fusion and sperm incorporation are portrayed, which are possibly the earliest pictures of human conception on record.Oocytes recovered at laparoscopy from an in vitro fertilization programme using clomiphene and human menopausal and chorionic gonadotrophin stimulation were zona-punctured and examined for polyspermic penetration 3-6 hours after insemination. Three eggs were penetrated, and one donor became pregnant in the same cycle.Fusion occurred between the oolemma and the midsegment of the sperm cell membrane extending from the equatorial vestige of the acrosome to the anterior region of the postacrosomal segment. Only acrosome-reacted sperm were capable of fusing with the egg. Fusion was followed by incorporation of the spermhead into the ooplasm by a process akin to phagocytosis. Sperm chromatin decondensation occurred by progressive inflation and disorganization of the original nuclear envelope.The mechanism of gamete fusion and sperm incorporation resembles that observed during human monospermic fertilization and generally conforms to that reported for eutherian mammals.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0148-7280
    Keywords: spindles ; oocytes ; embryos ; microtubules ; cryopreservation ; electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33-34%) compared to that of 2-cell embryos (78-79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7-15% for oocytes and 79-80% for the embryos.Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.
    Additional Material: 20 Ill.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 19 (1988), S. 253-263 
    ISSN: 0148-7280
    Keywords: cryopreservation ; IVF ; in vitro fertilization ; embryo-freezing ; ultrastructure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Previous studies by a French group (Fertil Steril 44:645-651, 1985) have shown that two-to eight-cell human embryos can survive slow freeze-thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase-contrast microscopy. We assessed the structure of 25 frozen-thawed one- to 12-cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase-contrast and electron microscopy, using the same method of cryopreservation. One-fourth of the embryos examined had all cells intact, and more than one-half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell-to-cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268-272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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