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  • Cell & Developmental Biology  (3,013)
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  • 1992  (1,697)
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  • 1990-1994  (1,697)
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  • 1
    ISSN: 0886-1544
    Keywords: activation ; fertilization ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 μM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material. © 1992 Wiley-Liss, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 3
    ISSN: 0886-1544
    Keywords: Chloroplast movement ; phytochrome ; near infrared laser ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The cell of the green alga Mougeotia orients its chloroplast by rotation, according to the direction or polarization of incident red light. The mechanics of the rotation is described by the angle of rotation and the angular velocity of the rotator (i.e., the chloroplast). We developed a laser diffractometer to determine the angle of rotation of the chloroplast. The angle of rotation of the chloroplast shifted by a constant angular velocity, and hence, the net torque on the chloroplast was zero. This suggests that the driving torque acting on the chloroplast is always balanced by the viscous torque. The maximal driving force acting on the chloroplast was estimated to be nearly equal to the force generated by an actomyosin system. This is the first measurement of the driving force acting on the chloroplast in Mougeotia. The amplitude of the force supported the anchorage site hypothesis. However, it remains unclear whether or not the angular independence of the force also supports the hypothesis. © 1992 Wiley-Liss, Inc.
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  • 4
    ISSN: 0886-1544
    Keywords: division polarity ; F-actin ; microtubules ; plastids ; preprophase band ; stomata ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Stomatogenesis, the determinate developmental pathway leading to formation of a pair of specialized guard cells, was studied in monoplastidic cells ofSelaginella. Observations of living cells followed by immunofluorescence microscopy of the same cells made it possible to correlate changes in cytoskeletal organization with developmental events. The guard mother cell divides in a plane perpendicular to previous divisions and this shift in polarity is marked by morphogenetic plastid migration, as well as by extensive reorganization of cytoskeletal arrays. The single plastid divides and daughter plastids move to a position opposite each other (incipient spindle poles). The axis defined by the opposing plastids rotates in the cell before becoming fixed in position with polar plastids adjacent to the lateral anticlinal walls. Plastid polarity predicts spindle orientation and the plane of division. Once division polarity is defined by plastid position, which will remain unchanged throughout mitosis and cytokinesis, cortical microtubules become reorganized from radial to longitudinal (relative to the long axis of the leaf). The initially random cortical F-actin also becomes aligned longitudinally. A wide preprophase band of microtubules and F-actin is formed at right angles to the spindle axis. Plastid-based microtubules establish the preprophase spindle and also connect to the preprophase band. The mitotic spindle remains anchored at the polar plastids. After mitosis, a phragmoplast that forms among microtubules emanating from plastids and nuclei develops in the plane marked previously by the preprophase band. Mitosis is completed in 1 h 15 min ± 3 min (mean ± S.E.). © 1992 Wiley-Liss, Inc.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 74-82 
    ISSN: 0886-1544
    Keywords: motion analysis ; sperm activation ; K+inhibition ; Fluo-3 ; eukaryotic flagella ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We investigated the swimming patterns of trout sperm using computer-assisted analyses of video microscopy. Under full activation conditions, in which 80-100% of sperm activate their motility, sperm swim in circular paths for 2-5 sec, followed by 30-60 sec of a more linear swimming, and, finally, cessation of movement, with a straightening of the flagella. Threshold activation, in which 50% of the sperm activate, is characterized by circular patterns of swimming for less than 20 sec, with straightened flagella upon cessation. Full activation and threshold activation are observed in low-K+ solution or in an Mg++ -supplemented K+ solution. Similarities in swimming patterns in low-K+ solution and in a Mg++ -supplemented K+ solution suggest a common underlying mechanism of activation. Initiation of movement in solutions with high Ca++ to K+ ratio is similar to activation in K+ -free solution. However, sperm in Ca++ -supplemented media resume circular swimming within 20-25 sec after activation, and, upon cessation of movement, the flagella are frequently cane shaped or bent. Differences in swimming patterns upon activation by high Ca++ concentration suggest additional effects of Ca++ on regulating swimming patterns. We used the fluorescent Ca++ indicator Fluo-3 to measure changes in intracellular Ca++ concentration upon activation. Intracellular Ca++ concentration transiently increases upon activation, with peak Ca++ concentration coinciding with the period of circular swimming. This transient increase in Ca++ concentration is seen in the absence of external Ca++, providing strong evidence for the released of Ca++ from intracellular stores upon activation.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 101-110 
    ISSN: 0886-1544
    Keywords: F-actin ; silk gland ; phalloin ; periluminal circumferential actin bundles ; actin-coated vacuoles ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Labeling of silk glands with rhodaminyl-phalloin shows that most F-actin is restricted to parallel bundles that form rings around the gland lumen at the apical cell surface. The bundles are lost when larval feeding stops at moulting, and the F-actin is redistributed through the cytoplasm as coats to vacuoles and, occasionally, in variably oriented strands. After moulting there is a return to the distribution of filamentous actin in the apical periluminal rings of bundles. These events occur at the same time as F-actin in the nuclear shell [Henderson and Locke, submitted] undergoes its own set of changes. In silk gland cells two kinds of f-actin deployment take place concurrently.
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  • 7
    ISSN: 0886-1544
    Keywords: myofibrils ; extracellular matrix ; cytoskeleton ; integrins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The influence of the extracellular matrix (ECM) on cell behavior, myofibrillogenesis and cytoarchitecture was investigated in neonatal rat cardiac myocytes in vitro. Cell behavior was examined by analyzing cell spreading on different ECM components under a variety of experimental conditions. Area measurements were made on digitized images of cells grown for various time intervals on fibronectin (FN), laminin (LN), collagens I and III (C I + III), plastic, and bovine serum albumin (BSA). The amount of spreading was varied on the different matrices and was maximal on FN 〉 LN 〉 C I+III 〉 plastic 〉 BSA. Addition of anti-β1 integrin antibodies to myocytes cultured on FN, LN and C I+III blocked spreading outward on the substrates and altered normal myofibrillogenesis, especially on LN. Concomitantly, the integrin antibodies induced the formation of giant pseudopodial processes which protruded upward from the substrates. These pseudopods contained actin polygonal networks which exhibited a regular geometrical configuration.Effects of the ECM on cytoarchitecture was examined by analyzing the temporal and spatial patterns of fluorescence and immunogold labeling of cytoskeletal and integrin proteins as myocytes spread in culture. The first indication of sarcomeric patterns was the appearance at 4 hours of striations formed by lateral alignment of α-actinin aggregates into Z bands. At later times, vinculin at 8 hours and β integrin at 22 hours became co-localized with α-actinin at the Z bands and focal adhesions. These data indicate that ECM components influence myocyte spreading and that myofibril assembly and/or stability is associated with ECM-integrin-cytoskeleton associations.
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  • 8
    ISSN: 0886-1544
    Keywords: cardiac muscle ; actin dynamics ; α-actinin ; vinculin ; microinjection ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, α-actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells'native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, α-actinin was incorporated into Z-bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I-bands at the site of injection. Following a 3-h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z-bands throug hout the cells. Myosin light chains required 4-6 h after injection to become incorporated into the A-bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T-tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC3[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.
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  • 9
    ISSN: 0886-1544
    Keywords: MAP p15 ; microtubule bundling ; trypanosoma brucei ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A protein of 15 kDa (p15) was isolated from Trypanosoma brucei subpellicular microtubules by tubulin affinity chromatography. The protein bound tubulin specifically both in its native form and after SDS-PAGE in tubulin overlay experiments. p15 promoted both the in vitro polymerization of purified calf brain tubulin and the bundling of preformed mammalian microtubules. Immunolabeling identified p15 at multiple sites along microtubule polymers comprising calf brain tubulin and p15 as well as on the subpellicular microtubules of cryosectioned trypanosomes. Antibodies directed against p15 did not cross react with mammalian microtubules. It is suggested that p15 is a trypanosome-specific microtubule-associated protein (MAP) that contributes to the unique organization of the sub-pellicular microtubules.
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  • 10
    ISSN: 0886-1544
    Keywords: microtubules ; vesicles ; cytoplasmic movement ; monoclonal antibody ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A cytoskeletal apparatus is involved in the movement of vesicles, organelles, and gametes in the pollen tube. The function of microfilaments has been defined quite precisely, but the role of microtubules needs to be further clarified. On the basis of immunological and biochemical investigations, we have identified a polypep-tide showing common properties with kinesin, a microtubule-based motor mainly described in nonplant tissues, in the pollen tube of Nicotiana tabacum. Like mammalian kinesin, the kinesin-immunoreactive homolog from Nicotiana tabacum pollen tubes binds to mammalian microtubules in an AMP-PNP dependent manner. The kinesin-like component is likely to be involved in the movement of vesicular material in the growing pollen tube.
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  • 11
    ISSN: 0886-1544
    Keywords: actin ; acidic vesicles ; Ca2+ ; pH ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or pathlength of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.
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  • 12
    ISSN: 0886-1544
    Keywords: bioluminescence ; ATP depletion ; motility ; flagellum ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The initiation of motility and modification of energy metabolism of rat caudal epididymal spermatozoa can be induced by dilution in a saline medium. We have investigated in these cells the relationships between the energy reserve (sperm ATP content measured by bioluminescence) and flagellar movement (high speed videomicrography, 200 frames/sec). A steady state was observed in sperm ATP content, progressive velocity (Vp) and flagellar beat frequence (F) with sperm dilution in a medium with glucose, lactate, pyruvate and acetate substrates after 30 minutes of incubation, without these substrates, changes in metabolic pathways occurred immediately and initially disturbed the relationship between ATP levels and F, suggesting differences in motility initiation when energy is from an endogenous origin via mitochondrial oxidative phosphorylation. This “energy crisis” was reversed by the addition of substrates to the medium.The three-dimensional flagellar movement observed in the presence of substrates quickly became two-dimensional in their absence. The flagellar beat envelope became more splayed, the mean amplitude of lateral head displacement increased and F decreased. The resulting high flagellar beat efficiency can be compared to that observed during hyperactivation which is a physiological event related to a fall in intracellular ATP level. In both media, the displacement of the flagellum in relation to the wave axis varied sinusoidally. The sine period increased with time when the spermatozoa were incubated in the medium without substrates. These results suggest a gradual slowing-down of the velocity of wave formation in the proximal part of the flagellum.
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  • 13
    ISSN: 0886-1544
    Keywords: actin polymerization ; cell elongation ; photoreceptor ; cytoskeleton ; phalloidin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In the retinas of teleost fish, rod photoreceptors elongate in response to light. Light-activated elongation is mediated by the myoid of the rod inner segment and is actin-dependent. Inner segment F-actin filaments form bundles running parallel to the cell's long axis. We examined the mechanism of rod elongation using mechanically-detached rod fragments, consisting of the motile inner segment and sensory outer segment (RIS-ROS). When RIS-ROS are isolated from darkadapted green sunfish and cultured in the light, they elongate 15μm at 0.3-0.6μm/min. Elongation was inhibited 65% by 0.1μM Cytochalasin D, suggesting a requirement for actin assembly. To determine the extent of assembly during elongation, we used three approaches to measure the F-actin content in RIS-ROS: detection of pelletable actin by SDS-PAGE after detergent-extraction of RIS-ROS; quantification of fluorescein-phalloidin binding by fluorimetry, fluorescence-activated cell sorting and image analysis; estimation of total F-actin filament length by electron microscopy. All three assays indicated that no net assembly of RIS-ROS F-actin accompanied myoid elongation. An increase in F-actin content within the elongated myoid was counterbalanced by a decrease in F-actin content within the 13 microvillus-like calycal processes located at the end of the inner segment opposite to the growing myoid. O'Connor and Burnside (Journal of Cell Biology 89:517-524, 1981) showed that minus-ends of rod F-actin filaments are oriented towards the elongating myoid while plus-ends are oriented towards the shortening calycal processes. Our observations suggest that RIS-ROS elongation entails actin polymerization at the minus-ends of filaments coupled with depolymerization at the filament plus-ends.
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  • 14
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 281-292 
    ISSN: 0886-1544
    Keywords: ATPase ; CTPase ; minus-end-directed microtubule motility ; cytoplasmic dynein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Extracts of unfertilized sea urchin eggs contain at least two isoforms of cytoplasmic dynein. One exhibits a weak affinity for microtubules and is primarily soluble. The other isoform, HMr-3, binds to microtubules in an ATP-sensitive manner, but is immunologically distinct from the soluble egg dynein (Porter et al.: Journal of Biological Chemistry 263:6759-6771, 1988). We have now further distinguished these egg dynein isoforms based on differences in NTPase activity. HMr-3 copurifies with NTPase activity, but it hydrolyzes CTP at 10 times the rate of ATP. The soluble egg dynein is similar to flagellar dynein in its nucleotide specificity; its MgCTPase activity is ca. 60% of its MgATPase activity. Non-ionic detergents and salt activate the MgATPase activities of both enzymes relative to their MgCTPase activities, but this effect is more pronounced for the soluble egg dynein than for HMr-3. Sucrose gradient-purified HMr-3 promotes an ATP-sensitive microtubule bundling, as seen with darkfield optics. We have also isolated a 20 S microtubule translocating activity by sucrose gradient fractionation of egg extracts, followed by microtubule affinity and ATP release. This 20 S fraction, which contains the HMr-3 isoform, induces a microtubule gliding activity that is distinct from kinesin. Our observations suggest that soluble dynein resembles axonemal dynein, but that HMr-3 is related to the dynein-like enzymes isolated from a variety of cell types and may represent the cytoplasmic dynein of sea urchin eggs.
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  • 15
    ISSN: 0886-1544
    Keywords: cytoskeleton ; globoside ; vimentin ; desmin ; keratin ; glial fibrillary acidic protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We reported recently that two glycosphingolipids (GSLs), globoside (Gb4)and ganglioside GM3, colocalized with vimentin intermediate filaments of human umbilical vein endothelial cells. To determine whether this association is unique to endothelial cells or to vimentin, we analyzed a variety of cell types. Doublelabel immunofluorescent staining of fixed, permeabilized cells, with and without colcemid treatment, was performed with antibodies against glycolipids and intermediate filaments. Globoside colocalized with vimentin in human and mouse fibroblasts, with desmin in smooth muscle cells, with keratin in keratinocytes and hepatoma cells, and with glial fibrillary acidic protein (GFAP) in glial cells. Globoside colocalization was detected only with vimentin in MDCK and HeLa cells, which contain separate vimentin and keratin networks. GM3 ganglioside also colocalized with vimentin in human fibroblasts. Association of other GSLs with intermediate filaments was not detected by immunofluorescence, but all cell GSLs were detected in cytoskeletal fractions of metabolically labelled endothelial cells. These observations indicate that globoside colocalizes with vimentin, desmin, keratin and GFAP, with a preference for vimentin in cells that contain both vimentin and keratin networks. The nature of the association is not yet known. Globoside and GM3 may be present in vesicles associated with intermediate filaments (IF), or bound directly to IF or IF associated proteins. The prevalence of this association suggests that colocalization of globoside with the intermediate filament network has functional significance. We are investigating the possibility that intermediate filaments participate in the intracellular transport and sorting of glycosphingolipids.
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  • 16
    ISSN: 0886-1544
    Keywords: nonmuscle myosin ; antibodies ; neurons ; blood vessels ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti-peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti-peptide IIA (MIIA2). Polyclonal anti-human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti-peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti-peptide IIA and anti-platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti-peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti-human platelet myosin antibodies. In bovine aorta cryosections, anti-peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti-peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti-platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels. © 1992 Wiley-Liss, Inc.
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  • 17
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 18
    ISSN: 0886-1544
    Keywords: epidermal keratinocytes ; cytoskeleton ; UV induced reorganization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation.In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes. © 1992 Wiley-Liss, Inc.
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  • 19
    ISSN: 0886-1544
    Keywords: spermatozoa ; flagella ; motility ; epididymis ; maturation ; mammal ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Motility and flagellar movement of ram spermatozoa along the epididymis were analysed in vitro. From the caput to the cauda of the epididymis, the percentage of motile and progressive spermatozoa increases. No flagellar bending was observed in spermatozoa from the testis or the epididymal anterior caput. When spermatozoa reached the distal caput of the epididymis, a static curvature, associated with an initiation of the flagellar beating, appeared on the flagella. This curvature normally disappeared during epididymal transit. Its disappearance was associated with an increase in the flagellar beat efficiency. Our results suggest that the initiation of motility is related to two mechanisms involving: (1) the presence of a transient static curvature, and (2) the establishment of a symmetric regular beating of the flagellum. © 1992 Wiley-Liss, Inc.
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  • 20
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 21
    ISSN: 1040-452X
    Keywords: Oncogenes ; Development ; Embryo ; Placenta ; Rabbit ; In situ hybridization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A large variety of proto-oncogenes are known to be of key importance in cellular growth and differentiation during embryonic development. Using quantitative during in situ hybridization, we studied in detail the levels of the proto-oncogenes Ha-ras and c-myc mRNA in embryos and extraembryonic tissues (maternal and embryonic placentas, trophoblast, and endometrial epithelium) during prental life of rabbit. cDNA probes encoding for Ha-ras (fragment Kpn 1-BstE II of 883 bp) and c-myc (fragment Pst 1-Pst 1 of 490 bp) were used to detect specific transcripts in fixed cryostat sections. High levels of Ha-ras and c-myc mRNA were detected in the rabbit embryo as well as in the decidua and in the trophoblast as early as day 9 of gestation. At 12 and 15 days of gestation, Ha-ras and c-myc mRNA levels decreased in both embryonic and maternal placenta while in the embryo a significant increase of Ha-ras and c-myc expression was detected with particular evidence in the central nervous system. Finally, at 25 days of gestation the expression of the two proto-oncogenes, Ha-ras and c-myc, was greatly decreased in both the embryo and extraembryonic tissues, and was undetectable by 30 days of gestation. These results show that in rabbit the expression of the two proto-oncogenes Ha-ras and c-myc is localized in the same tissues with similar intensity and follows an unparallel temporal modulation in the embryo and in the extraembryonic tissues during prental development.
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  • 22
    ISSN: 1040-452X
    Keywords: Spermatogenesis ; Spermiogenesis ; Sperm tail antigen ; CHO cells ; Germ cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A general mammalian expression vector designated pSV2-EP was reconstructed by inserting an oligonucleotide fragment into pSV2-dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2-EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly-A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from Àgt11 expression libraries. A recombinant plasmid (pSVRS-1) was constructed by inserting RSD-1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2-EP vector. Chinese hamster ovarian (CHO) dhfr-negative cells were contransformed with pSV2-dhfr and pSVRS-1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD-1. One of these transformed cell lines stained intensely with anti-rSMP-B antibodies, demonstrating that the RSD-1 was expressed in the transformed CHO cells.
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  • 23
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    ISSN: 1040-452X
    Keywords: Blastocysts ; Serine proteases ; Trophoblast ; Embryonic discs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The type of plasminogen activator (PA) secreted by bovine embryos was identified. Day 12-14 embryos were collected from estrus-synchronized, superovulated, and naturally mated crossbred beef cows. Embryos were left intact (E) or microdissected into component embryonic discs (ED) and trophoblastic vesicles (TV). Intact embryos, ED, and TV were pre-cultured for 2 days in Minimum Essential Medium Alpha (MEMα) with 10% heat-inactivated fetal calf serum, washed in serum-free MEMα, and cultured individually for 5 days in 50 μl microdrops of MEMα with 15 mg/ml bovine serum albumin. At 24 hr intervals, E, ED, and TV were observed for tissue morphology and transferred to fresh microdrops, and medium was recovered and frozen at -20°C. At the end of culture, blastocoelic fluid (BF) and embryonic tissues were recovered and frozen at -20°C. Plasminogen activator concentrations in medium, tissues, and BF were determined by using a caseinolytic assay. Antibodies to urokinase-type PA (anti-uPA) and tissue-type PA (anti-tPA), and the urokinase inhibitor, amiloride (AMR), were used to identify the type of PA produced by bovine embryonic tissues. Intact embryos and TV released more PA (P 〈 0.05) than ED, and tissues exhibiting expanded blastocoels released less PA (P 〈 0.05) than tissues with collapsed blastocoels. Blastocoelic fluid from TV exhibited more PA (P 〈 0.05) activity than from ED. Treatment with anti-uPA decreased PA activity (P 〈 0.05) in pooled medium and tissues from E compared to treatment with nonspecific immunoglobulins and anti-tPA. Amiloride completely eliminated PA activity (P 〈 0.05) in tissues, medium, and BF. These results suggest that day 12-14 bovine embryos secrete a urokinase-type PA.
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  • 24
    ISSN: 1040-452X
    Keywords: Spermatozoa ; Zona pellucida ; Binding ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The hemizona assay is a diagnostic test used to evaluate the binding potential of spermatozoa to zonae pellucida and has been used to predict fertilization potential in the human. In this study, frozen-thawed gorilla spermatozoa were coincubated with human hemizonae to evaluate tight binding and to assess the use of human zonae in evaluating sperm fertility. Matching hemizonae were incubated with human sperm to serve as a control. For evaluation of binding studies in a homologous system, matching halves of gorilla hemizonae were coincubated with both gorilla and human sperm. Whole, intact zonae of both human and gorilla oocytes were also coincubated with heterologous sperm to determine of penetration into the perivitelline space could occur. This study found that gorilla sperm bound well to both gorilla and human hemizonae, with a mean of 112.5 and 81.0 tightly bound sperm, respectively. Human sperm also bound to gorilla (mean 229.5) and human (mean 236.5) hemizonae. Following incubation with intact gorilla zonae, motile human sperm were found within the perivitelline space. However, gorilla sperm were not visible within the perivitelline space of nonviable human oocytes. These findings demonstrate that the zonae of nonviable human oocytes can be used to assess sperm binding of gorilla sperm. Studies continue for optimizing assay condition and correlation of findings with the fertility potential of gorilla sperm.
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  • 25
    ISSN: 1040-452X
    Keywords: Chromatin organization ; Endogenous nicks ; DHR ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: DNase I-hypersensitivity of rat spermatogenic cells was analyzed (1) to establish overall patterns of hypersensitivity in individual cell types, (2) to correlate these patterns with known changes in chromatin organization and function, and (3) to provide a foundation for further analyses examining DNase I-hypersensitivity and the localization of specific genes during spermatogenesis. Parameters for in situ nick translation, using radioactive and fluorescent probes to visualize DNase I-hypersensitive regions (DHR), were established for fixed and sectioned testicular preparations, permeabilized cells, and isolated germ cell nuclei. As anticipated, the pattern of DHR changed in a cell-type specific manner during the course of spermatogenesis, reflective of known stage-dependent alterations in the composition and structure of both the chromatin and the nuclear lamina/matrix as well as changes in gene expression. DHR in preleptotene spermatocytes were primarily peripheral, while in pachytene spermatocytes they were localized along the condensed chromosomes. The pattern of DHR changed from “checkerboard” in steps 7-8 round spermatid nuclei to “lamellar” in steps 10-11 elongating spermatids. In steps 12-13 elongating spermatids, DHR were localized throughout the nuclei or in a graded manner - increasing from anterior to posterior and mirroring the pattern of chromatin condensation. However, unlike the case in other stages, DNA of steps 12-13 elongating spermatids was exquisitely sensitive to nick translation even in the absence of exogenous DNase I. In contrast to the labeling of earlier stages, steps 16-19 spermatids and mature spermatozoa did not demonstrate DNase I-hypersensitivity under any conditions employed. A variety of agents that interact with topoisomerase II and DNA (teniposide, novobiocin, ethidium bromide, and adenosine triphosphate) were tested to determine the basis for the unique sensitivity to nick translation of steps 12-13 elongating spermatids. None of the agents tested, however, affected this unique labeling. The sensitivity of steps 12-13 elongating spermatids to nick translation in the absence of exogenous nuclease indicates the presence of endogenous nicks, which may relieve torsional stress and aid rearrangement as the chromatin is packaged into a form characteristic of the mature spermatozoon.
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  • 26
    ISSN: 1040-452X
    Keywords: Mouse ; Denuded oocytes ; Purines ; Germinal vesicle breakdown ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The potential action of purines, such as hypoxanthine and adenosine, in meiotic arrest was examined using denuded mouse oocytes. The spontaneous meiotic maturation of denuded oocytes was significantly inhibited by hypoxanthine and/or adenosine in a dose-dependent manner. Germinal vesicle breakdown (GVBD) was inhibited even at a low concentration (1 nM) of hypoxanthine, when hypoxanthine was microinjected into the cytoplasm of denuded oocytes. This inhibitory action was potentiated by co-injection with allopurinol, a metabolic blocker of hypoxanthine that can block a metabolic pathway to uric acid. By contrast, a microinjection of adenosine was no longer effective in inhibiting GVBD. Inhibitory action of purines in meiotic maturation was correlated with sustaining intracellular cAMP levels. GVBD was resumed by econazole, one of the nitroimidazole derivatives which act as inhibitors of catalytic subunit of adenylate cyclase. This compound was effective in counteracting the effect of adenosine, but not the action of 3-isobutyl-1-methylxanthine (IBMX) on GVBD, indicating that adenosine is probably exerted at the level of oocyte plasmalemma. These data suggest that the inhibitory action of hypoxanthine and adenosine in oocyte meiotic maturation may be involved in the regulation of cAMP metabolism in a differential manner.
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  • 27
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 28
    ISSN: 1040-452X
    Keywords: Acrosome reaction ; Signal transduction ; Pertussis toxin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mammalian sperm possess a guanine nucleotide-binding regulatory protein (G protein), with properties similar to Gi, that appears to be involved in the signal transduction pathway required for zona pellucida (ZP)-mediated acrosomal exocytosis. Mouse sperm treated with pertussis toxin (PT), a toxin that functionally inactivates Gi proteins, bind to the ZP of mouse eggs but are inhibited from undergoing acrosomal exocytosis. We have measured high-affinity GTPase activity and GTPγ[35S] binding in mouse sperm homogenates incubated in the absence and presence of ZP glycoproteins isolated from either ovulated eggs or from ovarian homogenates to determine whether this extracellular matrix can activate the sperm-associated Gi protein. An increase in GTP hydrolysis (∼50% over basal activity) and GTPγ[35S] binding (∼25-60% over basal activity) is observed when sperm homogenates are incubated in the presence of solubilized ZP glycoproteins, and the increase in GTPase activity is dependent on the concentration of ZP added to the homogenates. Accompanying this increase is a reduction in the ability of PT to catalyze in vitro [32P]ADP-ribosylation of a Mr = 41,000 sperm Gi protein, suggesting that the increase in GTPase activity and GTPγ[35S] binding is associated with the activation of a PT-sensitive sperm G protein(s). The ability of the ZP to stimulate high-affinity GTPase activity in these homogenates appears to be dependent on the capacitation state of the sperm from which the homogenates are prepared. These data suggest that a component(s) of the ZP may function in a manner similar to that of other ligands by binding to a sperm surface-associated receptor and subsequently activating a G protein coupled to an intracellular signal transduction cascade(s) required for induction of acrosomal exocytosis.
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  • 29
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    Molecular Reproduction and Development 31 (1992), S. 287-296 
    ISSN: 1040-452X
    Keywords: Phosphorylation ; Protein synthesis ; Pronuclei ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nuclear restructuring that occurs between insemination and full pronuclear formation in pig eggs is accompanied by posttranslational changes to specific egg proteins. Sperm penetration begins in vitro at 3 hr postinsemination (hpi). By 5 hr, decondensing sperm heads and anaphase II plates are observed in 50% of eggs, and, by 8 hpi, both male and female pronuclei have formed. Three consistent changes to the pattern of newly synthesised proteins are triggered in this period; they affect the 46K, 25K, and 22K polypeptides. Changes are also triggered in the 180-200K polypeptides and in the 14K polypeptides, but these are highly variable. The same changes in the prefertilization pattern were observed when prelabelled eggs were used and new protein synthesis was suppressed. The first and most abrupt change involves the apparent catabolic elimination of a group of 46K unphosphorylated polypeptides (pl 7.3-6.4), whose synthesis was greatest before germinal vesicle breakdown but declined slowly in the final phase of maturation, then declined precipitously after activation. Ageing (beyond maturation) also leads to the disappearance of these polypeptides. The progressive disappearance of a set of 25K polypeptides and the concomitant appearance of a dominant 22K polypeptide is the most characteristic fertilization-induced modification to porcine egg proteins. These modifications begin within 1 hr of sperm penetration or activation, are specific to the pig, and involve heavily phosphorylated polypeptides (25K, pl 6.7-6.0) whose synthesis is begun in the early metaphase I stage. Dual ([35S] and [32P]) labelling, protein blocking experiments, and use of alkaline phosphatase suggest that dephosphorylation selectively affects these 25K polypeptides and is mainly or wholly responsible for converting them (completely within 6 hr) to a single, new (22K, pl 7.6) species that is positively charged. The 25K/22K polypeptide modification has a close temporal relationship with the formation of the male and female pronuclei.
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  • 30
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    New York, NY [u.a.] : Wiley-Blackwell
    ISSN: 1040-452X
    Keywords: Meiotic maturation ; Germinal vesicle breakdown ; Cycloheximide ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The regulation of meiotic events of goat oocytes from prophase I to metaphase II was studied by inhibiting protein synthesis at different times of the transition and by analyzing the changes in the protein synthesis pattern during maturation. Protein synthesis was required for germinal vesicle breakdown (GVBD). Nevertheless, the concomitant event to the rupture of germinal vesicle, i.e., chromosome condensation, took place even in a cycloheximide-containing medium. The transition from metaphase I to metaphase II was also protein synthesis dependent as evidenced by experiments using this protein synthesis inhibitor. The inhibition was partly reversible, i.e., after removal of the drug, oocytes were able to progress until metaphase I but could not proceed beyond this stage. Changes in the protein synthesis pattern were studied by radiolabelling of oocytes with [35S]methionine. These changes were correlated with the nuclear status of the oocyte: At GVBD, a polypeptide of 25 kD disappeared, while one of 27 kD appeared. At the same time, a polypeptide of 33 kD appeared, whereas concomitantly one of 34 kD became barely detectable and finally disappeared as the maturation progressed. During maturation, the synthesis of a 67 kD polypeptide increased and became predominant at the end of the maturation process. The synthesis of actin decreased after 18 hr of culture from a very high to a low level of synthesis. © 1992 Wiley-Liss, Inc.
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  • 31
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    New York, NY [u.a.] : Wiley-Blackwell
    ISSN: 1040-452X
    Keywords: Activation ; Calcium ; Fura-2 ; Electrical pulse ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Electrical stimulation is known to cause activation in mammalian oocytes, possibly by eliciting an elevation in intracellular calcium (Ca2+). This study reports intracellular Ca2+ concentrations in mature rabbit oocytes using the Ca2+ indicator fura-2. Calcium levels were determined prior to, during, and after the administration of an electrical pulse (3.6 kV/cm for 60 μsec). Baseline Ca2+ levels ranged from 30 to 90 nM. The intracellular Ca2+ transient evoked by a pulse, peaked at 11 sec, was highly variable in amplitude (40-300 nM) and returned to prepulse levels within 300 sec. Electrically stimulated oocytes did not exhibit repetitive Ca2+ transients. The size of the cytoplasmic Ca2+ rise was influenced by the duration of the pulse, the field strength and the concentrations of external Ca2+ (P 〈 0.05). Oocytes electrically stimulated in the presence of 100 μM CaCl2, which evoked Ca2+ transients with a mean magnitude of 120 nM, activated at a higher rate (P 〈 0.05) than oocytes stimulated in the presence of either higher or lower levels of external Ca2+. Although oocytes electrically shocked at 16-18 hr after administration of human chorionic gonadotropin (hphCG) activated at a lower rate than oocytes stimulated at 22-24 hphCG (P 〈 0.05), their intracellular Ca2+ response to the pulse was similar (P 〈 0.05). These results indicate that electrical pulse parameters and extracellular Ca2+ concentrations can be used to modulate intracellular Ca2+ levels and optimize oocyte activation rates. Furthermore, the data suggest that as the oocyte ages it becomes more responsive to a given intracellular Ca2+ elevation. © 1992 Wiley-Liss, Inc.
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  • 32
    ISSN: 1040-452X
    Keywords: Sperm-egg interaction ; Glycolipid binding protein ; Capacitation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Sulfolipid-immobilizing protein 1 (SLIP1) is a germ cell plasma membrane protein that binds specifically to sulfogalactosylglycerolipid, a sulfoglycolipid found preferentially in mammalian male germ cells (Lingwood, Can. J. Biochem. Cell. Biol. 63:1077-1085, 1985b). SLIP1 in mouse and rat sperm exists on the periacrosomal membrane, where sperm initially bind to eggs. Using the in vitro mouse sperm-egg binding assay with in vitro-capacitated sperm, we obtained results previously suggesting that sperm SLIP1 is involved in mouse sperm-zona pellucida interaction. In this study, using the in vitro sperm-egg binding assay, we showed that SLIP1 in uterine sperm was similarly engaged in this process. Involvement of mouse sperm SLIP1 was also shown to be important in the vivo fertilization process. Superovulated females inseminated with caudal epidididymal and vas deferens sperm preexposed to anti-SLIP1 lgG yielded only 20% fertilized zygotes, while 80% fertilization was observed in females inseminated with sperm preincubated with preimmune serum lgG. The lower fertilization rate was not due to changes in the sperm capacitation rate as assessed by chlortetracycline staining. © 1992 Wiley-Liss, Inc.
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  • 33
    ISSN: 1040-452X
    Keywords: Assay ; Lectin ; binding ; Fluorescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The use of fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) was evaluated for its ability to distinguish acrosome-intact from acrosome-damaged stallion spermatozoa. Incubation of fresh (acrosomeintact) and frozen-thawed (acrosome-damaged) spermatozoa with FITC-PSA resulted in acrosome-intact spermatozoa that exhibited no fluorescence, while acrosome-damaged spermatozoa exhibited fluorescent staining over the rostral portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome-intact and acrosome-damaged spermatozoa determined the precision (intrasample coefficient of variation), and linearity (increased percentage of spermatozoa with PSA binding, with increased percentage of frozenthawed spermatozoa in a sample) of FITC-PSA binding. The binding of FITC-PSA increased in samples as the portion of frozen-thawed (acrosome-damaged) to fresh (acrosomeintact) spermatozoa increased. A positive correlation existed (r = 0.98, P 〈 0.05) between the percentage of FITC-PSA bound sperm and the proportion of damaged spermatozoa added to a sample. Location of PSA lectin binding on acrosome-damaged spermatozoa, determined by electron microscopy using gold-conjugated PSA, was to components of the outer acrosomal membrane and acrosomal matrix. These results demonstrate that FITC-PSA binding may be useful in determining acrosomal integrity of fresh and frozen-thawed stallion spermatozoa. © 1992 Wiley-Liss, Inc.
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  • 34
    ISSN: 1040-452X
    Keywords: tPA ; Cortical granule reaction ; Sperm penetration ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The resumption of meiosis results in synthesis of tissue-type plasminogen activator (tPA) in the rat and mouse oocytes (Haurte et al., Cell 43:551-558, 1985). The present study demonstrates that freshly ovulated rat oocytes released their tPA into the surrounding medium upon in vitro activation by sperm penetration or treatment with a calcium ionophore. The presence of a neutralizing monoclonal anti-tPA antibody during in vitro activation by the calcium ionophore inhibited the activation-induced zona hardening and also preserved the ability of the oocyte to be penetrated by sperm subsequent to activation. Rat oocytes undergo zona hardening during in vitro maturation in the absence of serum, presumably as a result of spontaneous cortical granule release, based on findings in mice and hamsters. In the present study, the anti-tPA antibody prevented the zona hardening and enhanced partition by spermatozoa of rat oocytes that were matured in vitro without serum. Collectively, the observations reported here suggest a possible role of tPA released during the cortical granule reaction in the zona reaction, which contributes to the block to polyspermy.
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  • 35
    ISSN: 1040-452X
    Keywords: Fluo-3 ; Intracellular Ca2+ transient ; QNB ; Population kinetics ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The acrosome reaction induced by the zona pellucida in mouse sperm has been shown to proceed in two stages experimentally distinguishable by the fluorescent probe chlortetracycline. Entry into the first stage of sperm bound to isolated, structurally intact zonae pellucidae is blocked by the compound 3-quinuclidinyl benzilate. In this study, we show, utilizing the fluorescent Ca2+ indicator fluo-3, that the first stage of the zona-induced acrosome reaction is characterized by an increase in intracellular Ca2+, followed by a decrease as the acrosome reaction proceeds. This calcium transient is completely suppressed by 3-quinuclidinyl benzilate. We conclude that the Ca2+ transient is induced by the zona pellucida and is required for the zona-induced acrosome reaction. Blockage of this sperm intracellular Ca2+ transient provides a mechanism for the inhibitory action of 3-quinuclidinyl benzilate on the zona-induced acrosome reaction in mouse sperm. © 1992 Wiley-Liss, Inc.
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  • 36
    ISSN: 1040-452X
    Keywords: Oocyte growth ; Preimplantation development ; Zona pellucida hardening ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The first objective of this study was to determine whether oocyte growth in serum-free medium affects the solubility of the zona pellucida to α-chymotrypsin digestion, which is an index of zona pellucida “hardening” and reflects the potential penetrability of the zona pellucida by sperm. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in medium containing 5% fetal bovine serum (FBS) or in serum-free medium. The zonae pellucidae of oocytes grown in serum-free medium were four times as hard as freshly isolated germinal vesicle (GV)-stage oocytes grown in vivo or oocytes grown in vitro in FBS-containing medium. The hardening of the zonae pellucidae of oocytes grown in serum-free medium was prevented by addition of fetuin. The second objective was to compare the competence to undergo embryogenesis of oocytes that grew in serum-free vs. FBS-containing medium. Approximately 70% of the oocytes underwent maturation regardless of whether the medium was serum-free or contained FBS. Of the mature ova grown in medium containing FBS, 53% cleaved to the two-cell stage after insemination compared with only 6% of the ova grown in serum-free medium. Addition of fetuin to the serum-free medium used for oocyte growth increased the frequency of cleavage to the two-cell stage. Of the embryos derived from oocytes that grew in FBS-containing medium, 70% completed the two-cell stage to blastocyst transition. In contrast, only about 40% (P 〈 0.01) of the embryos derived from oocytes that grew in serum-free medium completed this transition regardless of whether the medium for oocyte growth was supplemented with fetuin. Nevertheless, 12% of the two- to four-cell-stage embryos derived from oocytes that grew and matured in serum-free medium containing fetuin developed to live young after transfer to the oviducts of foster mothers. These results suggest that serum-borne growth factors may function during oocyte growth and maturation to promote events leading to successful fertilization and embryogenesis.
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  • 37
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    Molecular Reproduction and Development 32 (1992), S. 152-159 
    ISSN: 1040-452X
    Keywords: Growth factor ; Morphogenesis ; Antisense RNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A critical process during early heart development is the formation of mesenchymal cells which will contribute to valves and septa of the mature heart. These cells arise by an epithelial-mesenchymal transformation of endothelial cells in the atrioventricular (AV) canal and outflow tract areas of the heart. Adjacent endothelial cells in the atrium and ventricle remain epithelial. A three-dimensional collagen gel culture system has been exploited to examine the interactions that mediate this transformation. The AV canal myocardium produces a stimulus that is transmitted through an intervening extra-cellular matrix to the AV canal endothelium. This interaction is regionally specific, such that ventricular myocardium does not provide an adequate stimulus and ventricular endothelium does not respond to the AV canal myocardial stimulus. Exogenous TGF-β1 (or TGF-β2) can complement ventricular myocardium to produce transformation by AV canal endothelium. A blocking antibody, effective against several TGF-β, prevents cell transformation. To identify the specific member of the TGF-β family that functions in situ, antisense oligonucleotides for each of the numbered TGF-β were topically added to AV canal explant cultures. Only the oligonucleotide targeted to TGF-β3 was an effective inhibitor of mesenchymal cell formation. Studies have been undertaken to localize specific mRNas by in situ hybridization and RNase protection assays. These assays have concentrated on the regional and temporal appearance of TGF-β2 and 3. Surprisingly, RNase protection assays with a TGF-β3 sense probe showed the presence of a transcript complementary to TGF-β3. Further analysis of this tissue interaction included the testing of a variety of signal transduction mechanisms including kinases, G-proteins, and intracellular calcium. Tissue interaction in the heart is a complex interaction in which regulation of the induction process occurs in both the inducing and target tissues. © 1992 Wiley-Liss, Inc.
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  • 38
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    Molecular Reproduction and Development 32 (1992), S. 160-167 
    ISSN: 1040-452X
    Keywords: BMP ; TGF-beta superfamily ; Bone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The BMPs (bone morphogenetic proteins) are a group of related proteins originally identified by their presence in bone-inductive extracts of demineralized bone. By molecular cloning, at least six related members of this family have been identified and are called BMP-2 through BMP-7. These molecules are part of the TGF-beta superfamily, based on primary amino acid sequence homology, including the absolute conservation of seven cysteine residues between the TGF-betas and the BMPs. The BMPs can be divided into subgroups with BMP-2 and BMP-4 being 92% identical, and BMP-5, BMP-6, and BMP-7 being an average of about 90% identical. To examine the individual activities of these molecules, we are producing each BMP in a mammalian expression system. In this system, each BMP is synthesized as a precursor peptide, which is glycosylated, processed to the mature peptide, and secreted as a homodimer. These reagents have been used to demonstrate that single molecules, such as BMP-2, are capable of inducing the formation of new cartilage and bone when implanted ectopically in a rodent assay system. Whether each of the BMPs possesses the same inductive activities in an animal is the subject of ongoing research. Based on the chondrogenic and osteogenic abilities of the BMPs in the adult animal, the expression of the mRNAs for the BMPs has been examined in the development of the embryonic skeleton by in situ hybridization. These studies demonstrate that the BMP mRNAs are spatially and temporally expressed appropriately for the proteins involved in the induction and development of cartilage and bone in the embryonic limb bud. Furthermore, primary preparations of limb bud cells respond to BMP-2, as do several cell lines of the osteoblastic lineage. In addition to expression in the skeletal system, various of the BMP mRNAs are expressed in distinct tissues, suggesting additional roles during development. © 1992 Wiley-Liss, Inc.
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  • 39
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    Molecular Reproduction and Development 32 (1992), S. 173-178 
    ISSN: 1040-452X
    Keywords: Vertebrate probes ; Phenotypes ; Homology ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Many Drosophila genes have now been identified with substantial sequence similarity to vertebrate protooncogenes and growth factors. Some of these have been isolated directly by cross-hybridization with vertebrate probes and some have been recognized in the sequences of genes cloned because of their intiguing mutant phenotypes. An example of a gene isolated for its interesting development functions but with homology to a vertebrate growth factor is the Drosophila decapentaplegic gene (dpp). An example of a Drosophila gene isolated by virtue of its sequence conservation is the vgr/60A gene. Both dpp and vgr/60A are members of the transforming growth factor-β family and are most similar to the human bone morphogenetic proteins. The regulation of the dpp gene by several different groups of pattern formation genes including the dorsal/ventral group, the terminal group, the segment polarity genes, and the homeotic genes indicates that many events in embryogenesis require the cell to cell communication mediated by the secreted dpp protein. The temporal and spatial pattern of vgr/60A expression differs from that of dpp indicating that it may be regulated by different pattern information genes. The experimental advantages of the Drosophila system should permit a better understanding of the importance of growth factor homologs in specific developmental events, aid in establishing the functional interactions between these regulatory molecules, and identify new genes that are important for the biological functions of growth factors. It is likely that some of the newly identified genes will have vertebrate homologs and the analysis of these may be helpful in studies on vertebrate development and tumor biology. © 1992 Wiley-Liss, Inc.
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  • 40
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    Molecular Reproduction and Development 32 (1992), S. 185-185 
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 41
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 32 (1992), S. 168-172 
    ISSN: 1040-452X
    Keywords: MIS ; Ovarian cancer ; Fetal testes ; Ocular melanoma ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: During embryogenesis normal male phenotypic development requires the action of Müllerian Inhibiting Substance (MIS) which is secreted by Sertoli cells of the fetal testis. As testes differentiate in genetic (XY) males, they produce MIS which causes regression of the Müllerian ducts, the anlagen of the female reproductive tract. Soon thereafter, testicular androgens stimulate the Wolffian ducts. In females, on the other hand, MIS is not produced by grandulosa cells until after birth, before which, estrogens induce Müllerian duct development, while the Wolffian ducts passively atrophy in the absence of androgenic stimulation. High serum MIS levels in males are maintained until puberty, whereupon they fall to baseline levels. In females MIS is undetectable in serum until the peripubertal period when values approach the baseline levels of males. This distinct pattern of sexual and ontogenic expression presupposes and requires tight regulation.MIS may play a role in gonadal function and development. Our laboratory has shown that an important role for ovarian MIS is to inhibit oocyte meiosis, perhaps providing maximal oocyte maturation prior to selection for ovulation and subsequent fertilization. Furthermore, Vigier et al. (Development 100:43-55) have recently obtained evidence that MIS may influence testicular differentiation, coincident with inhibition of aromatase activity. Current structure-function studies demonstrate that MIS, like other growth regulators in its protein family, requires proteolytic cleavage to exhibit full biological activity. MIS can be inhibited by epidermal growth factor. This antagonism, which is common to all MIS functions so far investigated, is associated with inhibition of EGF receptor autophosphorylation. We have provided evidence that bovine MIS can inhibit female reproductive tract tumors arising in adults. More recent work with highly purified recombinant human MIS (rhMIS) has extended these early observations, showing that this recombinantly made fetal inhibitor can also suppress tumors of Müllerian duct origin and thus may prove to be useful in the clinical management of gynecologic neoplasms. Recent investigations have identified ocular melanoma as another potential target for rhMIS therapy in humans. © 1992 Wiley-Liss, Inc.
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  • 42
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 43
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 32 (1992), S. 179-184 
    ISSN: 1040-452X
    Keywords: Cell proliferation ; c-myc ; Retinoblastoma gene ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The predominant effect of TGF-β1 on cell proliferation is inhibition. Earlier studies demonstrated that TGF-β1 inhibition of skin keratinocyte proliferation involves suppression of c-myc transcription and indirect evidence suggested that the protein product of the retinoblastoma gene (pRB) may be involved in this process. Skin keratinocytes transformed by SV40 and human papilloma virus-16 (HPV-16) or HPV-18 resisted growth inhibition and suppression of c-myc mRNA by TGF-β. Transient expression of HPV-16 E7 gene, adenovirus E1A, and SV40 large T antigen (TAg) blocked the TGF-β1 suppression of c-myc transcription. Studies with transformation-defective mutants of E1A and TAg suggested that a cellular protein(s) that interacts with a conserved domain of the DNA tumor virus oncoproteins mediates TGF-β1 suppression of c-myc transcription and keratinocyte growth. Transient expression of pRB in skin keratinocytes repressed human c-myc promoter/CAT transcription as effectively as TGF-β1. The same c-myc promoter region, termed the TGF-β Control Element (TCE), was required for regulation by both TGF-β1 and pRB. TCE bound a cellular protein of approximately 106 kDa and this binding was decreased by TGF-β1 treatment. Our data indicate that pRB can inhibit c-myc transcription and suggest the involvement of cellular factor(s) in addition to pRB in the TGF-β1 pathway for the suppression of c-myc transcription and growth inhibition. The possible involvement of pRB in the TGF-β1 pathway for suppression of c-myc transcription has a number of implications; c-myc is a cellular proto-oncogene involved in positively regulating cell proliferation. TGF-β1 may therefore act through the tumor suppressor gene product, pRB, to negatively regulate c-myc transcription and subsequently cell growth. This would implicate tumor suppressor genes in the response pathway for diffusible growth inhibitors, perhaps analogous to nuclear proto-oncogene involvement in the growth factor pathway. © 1992 Wiley-Liss, Inc.
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  • 44
    ISSN: 1040-452X
    Keywords: Differentiation ; Development ; RNA ; In situ ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The expression of the ras protooncogene was investigated in Xenopus laevis, throughout development, by in situ hybridization using a 35S-labelled antisense RNA probe. During oogenesis, the ras RNA was strongly expressed in the cytoplasm of previtellogenic oocytes and further diluted between yolk platelets; no specific localization of transcripts was observed. The signal density was particularly weak over embryo sections until the tailbud stage. On the other hand, a high level of ras RNA expression was detected on sections through the young tadpoles. An intense labelling was observed in several areas, including the branchial apparatus, gut, somites, nervous system, and lens. It is noteworthy that the heterogeneity of labelling increases as tadpoles grow older. Together, these results are discussed in relation to cellular events appearing throughout the early development.
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  • 45
    ISSN: 1040-452X
    Keywords: H19 ; Human placenta ; Cytotrophoblast differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Placental differentiation is closely correlated with the appearance of specific proteins, yet factors regulating cytotrophoblast differentiation are unknown. One strategy employed to search for such factors makes use of differential screening of cDNA libraries. For this purpose, cytotrophoblasts were isolated from human term placentae and cultured for 24 and 120 hr. cDNA libraries were constructed from the cell's RNA, and differential screening resulted in the isolation of three identical clones highly expressed after 120 hr. A DNA sequencing of 139 bp at the 3′ end of these clones and a search of the data bank revealed that the sequence was identical to the parallel domain in the human H19 gene. This highly conserved gene is unusual in that it may not encode a protein. In the mouse, its RNA was shown to accumulate to high levels in embryonic tissues of endodermal and mesodermal origin. Our present findings imply that, in humans, the H19 gene is efficiently expressed in placental tissue and differentiated cytotrophoblasts, which are of ectodermal origin. RNA blot hybridization revealed a unique bimodal pattern of expression for the H19 gene in cultured cytotrophoblasts. The modulation in expression of H19 during cytotrophoblast growth was not due to the increase in the number of multinuclear cells. Size fractionation of cytotrophoblasts by centrifugal elutriation revealed that H19 expression is correlated with the stage of cell differentiation. We therefore propose that H19 transcripts might play a regulatory role in the process of cytotrophoblast differentiation.
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  • 46
    ISSN: 1040-452X
    Keywords: DNA ; RNA ; P1 ; PI1 ; PI2 ; Lamin B ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nuclear matrix is thought to be responsible for DNA organization, DNA replication, RNA synthesis, and RNA processing. We have looked for the presence of nuclear matrix antigens during early mouse embryogenesis. Antibodies to peripheral and interior antigens (P1, PI1, PI2, and lamin B) were used to immunolocalize nuclear matrix antigens in germinal vesicle oocytes, metaphase II oocytes, zygotes, two-cell-stage embryos, and eight-cell stage embryos. All antibodies reacted with the nuclei of germinal vesicle oocytes, and two- and eight-cell-stage embryos; however, only P1 and lamin B were present at the pronuclear stage. In eggs collected at the pronuclear stage and cultured to the late two-cell stage in the presence of α-amanitin, the matrix morphology was altered for PI1 and PI2. α-Amanitin had no affect on the distribution of P1 or lamin B antigens. If α-amanitin was added 2 hr after cleavage to the two-cell stage, the normal staining pattern of PI2 was retained. These results suggest that the presence of specific components of an internal matrix is correlated with normal genomic activity.
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  • 47
    ISSN: 1040-452X
    Keywords: Transcription ; Transcription factor ; Protein phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Protein phosphorylation catalyzed by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is implicated in regulating zygotic gene activation in the two-cell mouse embryo (Poueymirou and Schultz; Dev Biol 133:588-599, 1989). We now provide evidence that H8, which is a PKA inhibitor, inhibits expression of an hsp70-driven β-galactosidase reporter gene and that the concentration-dependence of this inhibition is similar to that for inhibiting expression of a stage-specific gene(s) that is a product of zygotic gene activation. We also demonstrate that neither cAMP nor serum can stimulate the expression, as detected by a histochemical assay, of a cAMP response element (CRE)- or serum response element (SRE)-driven β-galatosidase reporter gene, respectively in either germinal vesicle-intact oocytes or aphidicolin-arrested one-cell embryos that are chronologically at the tw-cell stage. In contrast, although 12-O-tetradecanoyl phorbol-13-acetate (TPA) does not stimulate expression of a TPA response element (TRE)-driven β-galatosidase reporter gene in germinal vesicle-intact oocytes, it stimulates such expression in aphidicolin-arrested one-cell embryos. Moreover, TPA can stimulate the expression of either a CRE- or an SRE-driven β-galatosidase reporter gene in such embryos. Results of these studies further implicate protein phosphorylation in regulating zygotic gene activation, along with its role in modulating enhancer function in the early mouse embryo.
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  • 48
    ISSN: 1040-452X
    Keywords: Sperm entry site ; Fertilization ; Actin ; Fertilization cone ; Sperm incorporation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The sperm entry site (SES) of zebrafish (Brachydanio rerio) eggs was studied before and during fertilization by fluorescence, scanning, and transmission electron microscopy. Rhodamine phalloidin (RhPh), used to detect polymerized filamentous actin, was localized to microvilli of the SES and to cytoplasm subjacent to the plasma membrane in the unfertilized egg. The distribution of RhPh staining at the SES correlated with the ultrastructural localization of a submembranous electrondense layer of cortical cytoplasm approximately 500 nm thick and containing 5- to 6-nm filaments. Actin, therefore, was organized at the SES as a tightly knit meshwork of filaments prior to fertilization. Contact between the fertilizing sperm and the filamentous actin network was observed by 15-20 sec postinsemination or just before the onset of fertilization cone formation. Growing fertilization cones of either artificially activated or inseminated eggs exhibited intense RhPh staining and substantial increase in thickness of the actin meshwork. Collectively, TEM and RhPh fluorescence images of inseminated eggs demonstrated that the submembranous actin became rearranged in fertilization cones to form a thickened meshwork around the sperm nucleus during incorporation. The results reported here suggest that activation of the egg triggers a dramatic polymerization of actin beneath the plasma membrane of the fertilization cone. Furthermore, the actin involved in sperm incorporation is sensitive to the action of cytochalasins.
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  • 49
    ISSN: 1040-452X
    Keywords: Preimplantation embryo ; Development ; Transcription ; UDP/UTP incorporation ; Hamster 2-cell embryo ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The earliest time of onset of embryonic genome activation in golden hamsters was investigated. The inhibition of transcription by α-amanitin (11 μg/ml) in cultured embryos resulted in a total arrest of development of early 2-cell embryos (26 hr post-egg activation); under similar conditions, immediate cleavage divisions of 1-, late 2-, 4-, and 8-cell embryos were not affected. Electrophoretic analysis of [35S]methionine-labeled embryonic proteins showed that α-amanitin treatment apparently inhibited transcription-dependent protein synthesis in early 2-cell and, to some extent, in late 2-cell when compared to 4-cell embryos. Analysis of total RNA synthesis, using [α32P]-UTP or [32P]-orthophosphate, showed that there was a high proportion of radioactivity associated with the macromolecular fraction (RNA) at the early and late 2-cell stages and at the 4-cell stage compared to that at the 1-cell stage. These results indicate that the de novo synthesis of RNA, encoded by the embryonic genome, occurs at the 2-cell stage and that the second and subsequent cleavage divisions of hamster preimplantation embryos are dependent on new transcriptional activity. This initial activity of the embryonic genome in hamsters is coincident with several characteristic features of in vitro development such as a block to development, synthesis of major proteins, change in energy substrate preference, phosphate-inhibition of development and a requirement for amino acids.
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  • 50
    ISSN: 1040-452X
    Keywords: Blastocyst ; Pre-implantation ; EGF ; Electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have established a monolayer culture system for human fallopian tube epithelial cells. The cells were isolated from tubes using collagenase digestion, and were cultured in Ham's F-10 supplemented with 15% fetal calf serum. The epithelial cells derived from culture were characterized using immunocytochemical staining and electron microscopy. These cells were stained with antikeratin and anti-epithelial membrane antigen, but showed no staining after treatment with antivimentin. Electron microscopy showed many microvilli on the cell surface and tight junctions or desmosomes at areas of cell-cell contact. Cell proliferation was enhanced by epidermal growth factor, but not by fibroblast growth factor, insulin, transferrin, estradiol-17β, or progesterone.The 2-cell ICR mouse pre-embryos were co-cultured for 4 days with tubal epithelial cells (A) (n = 98), in cell-conditioned medium (B) (n = 83), or in medium alone (C) (n = 72). During the first 24 h in culture, for groups A and B, the rates of cleavage to the 4-cell stage were 90.9% and 81.9%, respectively. Cleavage rates in these two groups were significantly higher (P =0.0012, P 〉 0.00001) than in group C (56.9%). After 72 h in culture, the rates of development to the blastocyst stage were significantly higher for groups A and B compared to group C (89.6% and 73.5% vs. 54.5%, P 〉 0.00001, P = 0.0002). These results suggest that factor(s) from tubal epithelial cells may facilitate the development of mouse pre-embryos throughout the pre-implantation stages.
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  • 51
    ISSN: 1040-452X
    Keywords: Receptors ; PAF ; Phospholipid ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF) is a very potent phospholipid, which has been demonstrated to stimulate smooth muscle and change vascular permeability. PAF has been detected in the rabbit preimplantation uterine endometrium and has been demonstrated to bind specifically to rabbit uterine membranes. To evaluate the possible role of PAF in maternal-embryonic chemical communication, we report here that rabbit blastocysts can accumulate [3H]PAF from their environment. Blastocysts were able to accumulate [3H]PAF as time-, buffer-, age-, and concentration-dependent functions. The accumulation was inhibited by some PAF receptor antagonists, such as U66985, as well as by unlabeled PAF and lyso-PAF, indicating that the accumulation process may be receptor mediated. The data support the current model of PAF as a paracrine factor in preimplantation stages of reproduction.
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  • 52
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 32 (1992), S. 251-258 
    ISSN: 1040-452X
    Keywords: Diploid embryos ; Tripronucleate embryos ; Cell doubling time ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The postimplantation development of human and animal triploid embryos is well documented, but there is little informative data on their preimplantation development. An analysis of cell number at appropriate times during this period and thus their cleavage rate would give an indication of the potential triploids have for further development and may explain some problems associated with their postimplantation development. To rule out any effects of technical procedures on cleavage rate, appropriate controls were used. Diandric triploid embryos were produced using standard micromanipulatory techniques, which involved the injection of a male pronucleus into a recipient one-cell-stage embryo. The karyoplast was fused to the cytoplasm by electrofusion, and the resulting tripronucleate diandric triploid embryos were transferred to appropriate pseudopregnant recipients. At specific times after the transfer, the embryos were recovered and cell numbers established. The results were plotted and regression lines drawn. Three controls were used (1) micromanipulated diploid embryos from which the male pronucleus had been removed and immediately reinserted and fused to restore diploidy, (2) diploid embryos that had been briefly incubated in cytochalasin D and colcemid to find out the effects these agents had on development, and (3) diploid embryos that had been isolated and briefly incubated in tissue culture medium. All embryos were subsequently transferred to recipients. After isolation at specific times during the preimplantation period, cell numbers were also established and the results plotted. The cell doubling time of the diandric triploid embryos was 13.55 hr (± 1.25), and this was not significantly different from the various controls. The cell doubling time of (1) the micromanipulated controls was 12.12 hr (± 1.16), (2) the control embyros incubated in cytoskeletal inhibitors 10.87 hr (± 0.75), and (3) the group that was briefly incubated in tissue culture 12.43 hr (± 0.74). There was no significant effect of manipulation or incubation in cytoskeletal inhibitors on cleavage rate. Our findings indicate that triploid embryos divide at the same rate as diploid embryos during the preimplantation period.
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  • 53
    ISSN: 1040-452X
    Keywords: PCR ; Primer ; Sexing ; Growth rate ; Blastocyst ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Bovine in vitro-fertilized embryos at the blastocyst stage were collected at days 7, 8, and 10 postinsemination and sex was determined via the polymerase chain reaction (PCR) to compare the embryonic development with the sex of the embryos. The percentages of males (sex ratio) after division of the embryos into three developmental groups were 68%, 48%, and 35% in the fast, intermediate, and slow groups, respectively (P = 0.014). The percentages of males on days 7, 8, and 10 were 60%, 40% and 33%, respectively (P = 0.043). The average sex ratio for the whole material was 50%. It is thus concluded that male bovine preimplantation embryos develop faster than female embryos.
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  • 54
    ISSN: 1040-452X
    Keywords: Egg ; Oocyte ; Pronuclei ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The distribution of microtubles was studied during fertilization of the rabbit oocyte by immunofluorescence microscopy after staining with an anti-α-tubulin antibody. In ovulated oocytes, microtubules were found exclusively in the meiotic spindle. At fertilization, the paternal centrosome generated sperm astral microtubules. During pronclear development, the sperm aster increased in size, and microtubules extended from the male pronucleus to the egg center and towards the female pronucleus. These observations indicate that microtubules emanating from the sperm centrosome were involved in the movements leading to the union of the male and female pronuclei. At late pronuclear stage, microtubules surrounded the adjacent pronuclei. The mitotic spindle that emerged from the perinuclear microtubules contained broad anastral poles.
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  • 55
    ISSN: 1040-452X
    Keywords: Differential cDNA screening ; Spermatogenesis ; Germ cell differentiation ; Stage-specific gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The analysis of genes expressed in a restricted temporal and spatial manner during spermatogenesis has given insights into different gene-regulatory mechanisms active in germ cells. However, very few genes have so far been described that are predominantly active in spermatogonia and the early meiotic cell types of testis. To isolate a battery of such genes, more than 100 different mRNA molecules were isolated from a mouse prepubertal testicular cDNA library, and their expression patterns in different tissues analyzed. Thirty mRNAs, 26 of them previously not described in the literature, were found to be predominantly expressed in mouse testis. A detailed analysis of their expression patterns identified a number of mRNA molecules differentially expressed in testicular cell types, including both germ cells and somatic cell types. Characterization of these mRNAs also revealed five distinct temporal phases of gene expression during prepubertal germ cell development. Three different genes, mainly active in the spermatogonial and the early meiotic cell types of testis, were isolated and will be used to characterize further stage-specific gene expression during germ cell differentiation. © 1992 Wiley-Liss, Inc.
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  • 56
    ISSN: 1040-452X
    Keywords: Zfy protein ; Immunocytochemistry ; Fetal mouse testis ; Germ cell ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The zinc finger Y (Zfy) gene is located on the Y chromosome of all placental mammals. Although it is phylogenetically conserved and is expressed in mouse fetal testis, it is not the sex determining Y (Tdy) gene. To address the possible function of the Zfy gene in mice, the distribution of Zfy protein in fetal mice was investigated by immunocytochemical staining using several specific antisera against synthetic peptides of the mouse Zfy protein. Analysis of various fetal tissues at different embryonic stages demonstrated a specific staining only in fetal testis. In particular, reactive protein was initially observed in male fetal gonads at day 11.5 postcoitum (p.c.). The immuno-staining intensified in fetal testes at day 12 and 12.5 p.c., decreased drastically in those at day 13 and 14 p.c. and became undetectable in those at day 15 p.c. and beyond. The reactive molecules were distributed mostly within the seminiferous tubules of the embryonic testis. The present observations confirm the previous findings with RT-PCR analysis and indicate that Zfy or Zfy-like protein is expressed in stage-specific manner during early testis differentiation. Its location in the seminiferous tubules suggests a possible role in early germ cell development. © 1992 Wiley-Liss, Inc.
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  • 57
    ISSN: 1040-452X
    Keywords: IGF-1 ; Receptor ; B-10 Fab fragment ; Life and Medical Sciences ; Cell & Developmental Biology