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  • Life and Medical Sciences  (4.835)
  • Cell & Developmental Biology  (3.428)
  • 1995-1999  (3.916)
  • 1965-1969  (919)
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  • 101
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 32 (1995), S. 163-172 
    ISSN: 0886-1544
    Schlagwort(e): actin ; C-terminus ; α-actinin ; myosin ; myofibrils ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A series of deletions was made from the C-terminal end of actin by inserting termination codons into a full length cDNA of human α-skeletal muscle actin. These included deletions of 2, 3, 10, 20, 30, and 40 amino acids. The cDNA clones were transcribed and the resulting mRNA were translated in vitro using 35S-labeled methionine. The 35S-labeled actin and actin mutants were then tested for the ability to coassemble with carrier actin, bind DNAse I, bind myosin S-1, bind a 27 kDa proteolytic fragment of α-actinin, and incorporate into myofibrils in vitro. Removal of the C-terminal two or three amino acids did not grossly alter the properties of actin tested. Deletion of an additional 7 amino acids (10 amino acids total) significantly decreased coassembly, binding to DNAse I, and incorporation into myofibrils, but did not dramatically reduce binding to myosin S-1 or the 27 kDa fragment of α-actinin. Deletion of 20 or more amino acids virtually abolished all normal actin function tested. By examining the structure of actin, we propose that the effect of removing residues 356-365 is due to the important role Trp356 plays in maintaining hydrophobic bonds between three non-contiguous segments of actin. We also suggest that removal of residues 366-372 adversely affected the structure or orientation of the DNAse I binding loop and that this change can account for defects in actin binding to DNAse I, coassembly with wild type actin, and incorporation into myofibrils. © 1995 Wiley-Liss. Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 102
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 32 (1995), S. 226-232 
    ISSN: 0886-1544
    Schlagwort(e): Z-line interconnections ; honey-bee flight muscle ; transverse cytoskeletal network ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Located at the level of the Z-line, the transverse cytoskeletal network of insectflight muscle interconnects adjacent myofibrils with one another, and interconnects peripheral myofibrils with the cell membrane. This network has been presumed to keep myofibrils in register, or to distribute tension laterally among myofibrils. In this study, we used scanning-electron microscopy to reveal details of the three-dimensional arrangement of this network. The network is seen to interconnect longitudinal elements of the cytoskeletal network which surround each myofibril. The arrangement is not unlike that seen in vertebrate skeletal muscle. Interestingly, the transverse network makes contact with cell components such as dense bodies and mitochondria. Such contacts imply potential roles over and above those noted above. The network may be involved not only in mechanical function, but possibly also in intracellular communication. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 103
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 32 (1995), S. 273-288 
    ISSN: 0886-1544
    Schlagwort(e): microtubules ; γ-tubulin ; polarized epithelia ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Microtubules oriented in the apicobasal axis of columnar epithelial cells arranged with a uniform polarity with minus ends toward the apical surface, suggesting that these cytoskeletal filaments might serve as a substrate for polarized movement of membrane vesicles within the cell. It is not known whether hepatocytes, a cuboidal epithelium in which transcellular transport is a requisite step in normal apical membrane biogenesis, contain microtubules arranged with a similar polarity. In the present study, we explore the question of microtubule polarity and possible mechanisms for nucleation in the epithelial cell lines WIF-B (hepatocyte), Caco-2 (intestine), and Madin-Darby canine kidney (MDCK). Caco-2 microtubules in the apicobasal axis had uniform polarity with minus ends nearest the apical surface. After cold and nocodazole-induced depolymerization, microtubule regrowth initiated in the apical region in all three cell types. The apex of WIF-B and Caco-2 cells contained two pools of γ-tubulin: one associated with centrosomes and the other delocalized under the apical membrane. Non-centrosomal γ-tubulin was present in complexes that sedimented between 10S and 29S; both forms could bind microtubules. The presence of both centrosomal and noncentrosomal γ-tubulin in apical cytoplasm suggests multiple mechanisms by which microtubule nucleation might occur in epithelial cells. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 104
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 32 (1995), S. 318-331 
    ISSN: 0886-1544
    Schlagwort(e): cytoskeleton ; cyclic AMP ; vinculin ; E-cadherin ; ZO-1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: In epithelial cells interactions between the actin cytoskeleton and cell-cell junctions regulate paracellular permeability and partcipate in morphogenesis. We have studied the relationship between supracellular morphology and actin-junction interactions using primary cultures of porcine thyroid cells grown either as three-dimensional follicles or as open monolayers. Regardless of morphology, thyroid cells assembled occluding and adhesive junctions containing ZO-1 and E-cadherin, respectively, and showed F-actin staining in apical microvilli and a perijunctional ring. In monolayers, actin stress fibers were also observed in the apical and basal poles of cells, where they terminated in the vinculin-rich zonula adherens and in cell-substrate focal adhesions, respectively. Surprisingly, we were unable to detect vinculin localization in follicular cells, which also did not form stress fibers. Immunoblotting confirmed significantly greater vinculin in triton-insoluble fractions from monolayer cells compared with follicular cells. Incubation of monolayers with 8 chloro(phenylthio)-cyclic AMP decreased the level of immunodetectable vinculin in the zonula adherens, indicating that junctional incorporation of vinculin was regulated by cyclic AMP. In monolayer cultures, cytochalasin D (1 μM) caused actin filaments to aggregate associated with retraction of cells from one another and the disruption of cell junctions. Despite morphologically similar perturbations of actin organization in follicular cultures treated with cytochalasin D, junctional staining of ZO-1 and E-cadherin was preserved and cells remained adherent to one another. We conclude that in cultured thyroid cells structural and functional associations between actin filaments and cellular junctions differ depending upon the supracellular morphology in which cells are grown. One important underlying mechanism appears to be regulation of vinculin incorporation into adhesive junctions by cyclic AMP. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 105
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 1-8 
    ISSN: 1040-452X
    Schlagwort(e): Activin ; Expression ; Fecundity gene ; FecB ; Primer extension ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The 5′ untranslated region (UTR) of βA inhibin mRNA was compared in a variety of sheep tissues, using primer extension. Considerable variation in the length and number of 5′ extended products were noted between tissues. Specific bands were noted in ovarian follicular RNA, which were also present in samples from corpora lutea, stroma, and placental cotyledon RNA. Other extended products were observed in RNA from corpora lutea, stroma, cotyledon, pituitary, bone marrow, frontal cortex, medial basal hypothalamus, adrenal, liver, and kidney, which were not present or weakly represented in follicular RNA, Additional tissue-specific bands were noted in testis and bone marrow RNA. No specific differences in the lengths of the 5′ UTR of the βA inhibin mRNA were observed in sheep homozygous for the Booroola fecundity gene FecB, in any tissue studied. The coding region of ovine βA inhibin cDNA was sequenced and a genetic polymorphism confirmed within or close to the ovine βA inhibin gene.We conclude that the βA inhibin gene is expressed widely in the sheep. Furthermore there is variation in the length of the 5′ UTR of βA inhibin mRNA between male and female gonads and other tissues, implying that expression of this gene is differentially controlled. However, the FecB mutation does not affect mRNA splicing events or the initiation site used in ovarian transcription. The mechanism by which the FecB mutation influences the amounts of βA inhibin mRNA, follicle-stimulating hormone (FSH) secretion and ovulation rate has still to be elucidated. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 106
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 29-35 
    ISSN: 1040-452X
    Schlagwort(e): Cytochrome c oxidase ; F1F0-ATPase ; Mitochondrial biogenesis ; Preimplantation development ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The mouse egg contains about 90,000 mitochondria which undergo a buildup of mitochondrial cristae and increase in respiratory activity during cleavage. The mitochondrial DNA does not replicate during preimplantation development but is transcribed actively from the two-cell stage onward (Pikó and Taylor, 1987: Dev Biol 123:364-374). To gain further insight into mitochondrial biogenesis, we have now determined the steady state amounts of the mRNAs for the cytochrome c oxidase (COX) subunits IV, Vb and VIIc and the H+-ATPase subunit 9(P1) (all encoded by nuclear genes) in slot hybridization experiments of total RNA from oocytes and early embryos. All four mRNAs showed a similar developmental pattern of prevalence, characterized by a steady decline in mRNA copy numbers from the late growth-phase oocyte through the two-cell embryo, and an about 30-fold rise during cleavage through the blastocyst stage. However, the ATPase subunit 9 (P1) mRNA was about three times more prevalent in cleavage-stage embryos than the COX mRNAs. A similar pattern was obtained previously for the mitochondrial-encoded COX I and II mRNAs, but the latter accumulate at a 30-50-fold excess over the nuclear-encoded COX subunit mRNAs during the cleavage stages. The results suggest a coordinated activation and transcription of the mitochondrial and nuclear genes for the components of the respiratory apparatus beginning with the two-cell stage. It is estimated that new respiratory chains are produced at a rate of 50-100 chains hr-1/mitochondrion in the early blastocyst, accounting for 3.5-7% of the total protein synthetic activity at this stage. This rapid buildup of the mitochondrial oxidative phosphorylation system appears to be mostly preparatory for postimplantation development. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 107
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 56-61 
    ISSN: 1040-452X
    Schlagwort(e): Zfy ; Sex ratio ; Morula ; PCR ; Sexing ; Development ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Sex-related growth rate differences in preimplantation mouse embryos were investigated. In experiment I, Day 3 embryos were recovered from reproductive tracts, classified according to developmental stage, and cultured for 24 hr in CZB medium containing glucose. Each embryo was then reclassified and stained for measurement of number of nuclei and finally sexed using the polymerase chain reaction. In experiment II, Day 4 embryos were recovered, classified, stained, and sexed as in experiment I immediately after recovery. Morphologically, there were no differences between the sexes in either of the experiments on Day 4. However, based on number of nuclei, the data showed that in vitro conditions support the development of male embryos to the blastocyst stage compared to female embryos. Furthermore, growth rate differences were observed in vivo on Day 3, as females compacted earlier than males. These results suggest that the increased cell proliferation in cultured male embryos is an artifact caused by the in vitro environment. The variation may be due to sex differences in embryonal energy metabolism during the preimplantation stage. The growth difference implies different in vitro requirements of male and female embryos. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 108
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 91-102 
    ISSN: 1040-452X
    Schlagwort(e): Sertoli cells ; Phagocytosis ; Lysosomes ; SGP-1 ; Prosaposin ; Saposins ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Sulfated glycoprotein-1 (SGP-1) is a polypeptide secreted by Sertoli cells in the rat. Sequence analysis revealed a 76% sequence similarity with human prosaposin produced by various cell types. Human prosaposin is a 70 kDa protein which is cleaved in the lysosomes into four 10-15 kDa polypeptides termed saposins A, B, C, and D. The function of lysosomal saposins is to either solubilize certain membrane glycolipids or to form complexes with lysosomal enzymes and/or their glycolipid substrate to facilitate their hydrolysis. The present investigation dealt with the delivery of SGP-1 into the phagosomes of Sertoli cells; these phagosomes contain the residual bodies which detach from the late spermatids at the time of spermiation. Immunogold labeling with anti-SGP-1 antibody was found over Sertoli cell lysosomes, but was absent from phagosomes formed after phagocytosis of spermatid residual bodies in the apical Sertoli cell cytoplasm in stages VIII and early IX of the cycle of the seminiferous epithelium. The phagosomes found later in the basal Sertoli cell cytoplasm in stages IX and X of the cycle became labeled with the antibody as the components of the residual bodies rapidly underwent lysis and disappeared from the Sertoli cells. Sertoli cell lysosomes isolated by cell fractionation (estimated purity of 80%) were found to contain a 65 kDa form of SGP-1 or prosaposin, as well as the 15 kDa polypeptides or saposins. Thus, it appears that this unique lysosomal form of SGP-1 reached the Sertoli cell phagosomes and that their derived polypeptides, the saposins, must play a role in the hydrolysis of membrane glycolipids found in phagocytosed residual bodies. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 15 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 109
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 69-83 
    ISSN: 1040-452X
    Schlagwort(e): SGP-1 ; Hypophysectomy ; Castration ; Efferent ducts ; Lysosomes ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The objective of this study was to define the factors regulating the endogenous production of sulfated glycoprotein-1 (SGP-1) in nonciliated cells of the efferent ducts. To this end we examined five different groups of animals undergoing the following experimental procedures: (1) hypophysectomized animals at 7, 14, and 28 days, (2) 7-day hypophysectomized rats receiving testosterone implants given at various time intervals thereafter, (3) castration at various time intervals up to 7 days, (4) 7-day castrated rats receiving testosterone implants at various time intervals thereafter, and (5) castrated rats given testosterone implants immediately after castration and sacrificed at different time intervals thereafter. Efferent ducts were fixed by perfusion with 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer for quantitative immunocytochemical analysis at the level of the electron microscope. For each experimental condition and their controls, the number of gold particles/μm2 within the endosomal and lysosomal compartments was calculated taking into account the changes in both the volume of the cell and organelles being quantified and expressed as labeling content. The results revealed that hypophysectomy (up to 4 weeks) caused a marked significant decrease in the SGP-1 labeling content of the endosomal and lysosomal compartments. The labeling content of the lysosomal compartment of efferent ducts from rats castrated for up to 1 week did not change significantly. However, there was a significant decrease in the labeling content of endosomes. This decrease is due to SGP-1, which is secreted by Sertoli cells, not being available for uptake in the efferent aucts. These results suggested that testosterone is not required for maintaining the high labeling content of SGP-1 within lysosomes of nonciliated cells, but that a pituitary factor appears to be needed. The administration of testosterone at different intervals to 7-day castrated animals resulted in a significant decrease of lysosomal SGP-1, suggesting that testosterone under these experimental conditions inhibits the production of a pituitary factor that maintains the high labeling content of SGP-1 within lysosomes of the nonciliated cells. Testosterone administered to 7-day hypophysectomized animals over a 24-hr period had no effect on the labeling content of SGP-1 within lysosomes. However, the administration of testosterone to animals immediately following castration showed no differences in the labeling content of SGP-1 within compared to controls. Together these results suggest that the labeling content of SGP-1 within lysosomes of nonciliated cells of the efferent ducts is not dependent on luminal or circulating androgens, nor is it dependent on a testicular factor entering the lumen of the ducts. It does appear, however, that SGP-1 synthesis and targeting to secondary lysosomes is dependent on a pituitary factor that may have a direct or an indirect effect on the nonciliated cells. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 13 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 110
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 110-115 
    ISSN: 1040-452X
    Schlagwort(e): Aneuploidy ; Cryoprotectant ; Fertility ; Polyploidy ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: In a previous study, we have shown that the cryopreservation of mouse oocytes caused increases in the rates of degeneration and of digynic polyploid embryos, while the fertility of frozen-thawed oocytes was decreased. In this study, we have attempted to determine the different stages in the complete freezing-thawing process which are deleterious for the oocytes and the subsequent zygotes. IVF assays showed that DMSO decreased the fertility of oocytes, whereas cooling to 0°C had no effect. DMSO, used at 0°C, was less deleterious for oocytes. Thus, the prefreezing manipulations seem to be important for the quality and fertility of oocytes. However, neither DMSO nor cooling increased the incidence of chromosomal abnormalities in embryos obtained from inseminated exposed oocytes. Therefore, the increased frequency of polyploidy observed in embryos after the cryopreservation of mouse oocytes must correspond to disruption occurring during the freezing-thawing process.
    Zusätzliches Material: 2 Tab.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 111
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995) 
    ISSN: 1040-452X
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 112
    ISSN: 1040-452X
    Schlagwort(e): Embryonal carcinoma cells ; Differentiation ; Transcription ; Transforming growth factor-beta ; Activating transcription factor ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Previous studies demonstrated that differentiation of embryonal carcinoma (EC) cells increases the expression of the TGF-β2 gene and identified a CRE/ATF-like motif in the TGF-β2 promoter that is necessary for its activity. This suggested that differentiation may increase the transcription of this gene by differential binding of transcription factors to the CRE/ATF-like motif. To test this possibility, we performed gel mobility shift analysis using double-stranded oligodeoxynucleotides containing the TGF-β2 CRE/ATF-like motif and nuclear extracts prepared from F9 EC cells and F9-differentiated cells. We determined that the DNA/protein complexes formed by the EC nuclear extracts, but not the complexes formed by differentiated cell nuclear extracts, are recognized and supershifted by an ATF-1 specific antibody. This observation is consistent with our Western immunoblot analysis that detects ATF-1 in the EC cells, but not in their differentiated counterparts. In addition, we provide evidence that protein phosphorylation influences the formation of complexes between F9 nuclear proteins and the CRE/ATF-like motif. Together, our studies identify a likely role for the CRE/ATF-like motif in the regulation of TGF-β2 and suggest that this site binds one set of nuclear proteins in EC cells, where the gene is not expressed, and a different set of nuclear proteins in the differentiated cells, where the gene is expressed. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 113
    ISSN: 1040-452X
    Schlagwort(e): CD4 ; CDS ; AIDS ; Model ; HIV ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A major obstacle to understanding AIDS is the lack of a suitable small animal model for studying HIV-1 infection and the subsequent development of AIDS, and for testing diagnostic, therapeutic, and preventive modalities. Our goal is to produce a rabbit model for the study of AIDS. Here we report on the generation of transgenic rabbits that express the human CD4 (hCD4) gene. The transgene, which contains the coding region for hCD4 and approximately 23 kb of sequence upstream of the translation start site, was used previously to direct hCD4 expression on the surface of CD4+ T cells of transgenic mice (Gillespie et al., 1993: Mol Cell Biol 13:2952-2958). The hCD4 transgene was detected in five males and two females derived from the microinjection of 271 rabbit embryos. Both hCD4 RNA and protein were expressed in peripheral blood lymphocytes (PBLs) from all five males but neither of the females. Human CD4 was expressed on PBLs from F1 offspring of all founder males. T-cell subset analysis revealed that hCD4 expression was restricted to rabbit CD4 (rCD4) expressing lymphocytes; mature rCD4-rCD8+ lymphocytes did not express hCD4. In preliminary studies, PBLs from hCD4 transgenic rabbits produced greater amounts of HIV-1 p24 core protein following HIV-1 infection in vitro than HIV-1 p24 antigen in nontransgenic rabbit infected cultures. These results extend to rabbits our previous observation that this transgene contains the sequence elements required for high-level expression in the appropriate cells of transgenic mice. Furthermore, these and previous studies demonstrating that expression of hCD4 protein enhances HIV-1 infection of rabbit T cells in vitro, coupled with reports that normal, nontransgenic rabbits are susceptible to HIV-1 infection, suggests that the hCD4 transgenic rabbits described herein will have an increased susceptibility to HIV-1 infection. In vivo HIV-1 infection studies with these rabbits are under way. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 114
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 436-443 
    ISSN: 1040-452X
    Schlagwort(e): Glucose ; Pyruvate ; Preimplantation ; Embryo ; Mouse ; Metabolism ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca × C57BL/6) were cultured from the one-and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D+L-lactate, in the presence and absence of 1 mM glucose (M16+G and M16-G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16-G were transferred to M16+G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16+G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16-G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 ± 2.5 [28] vs. 105 ± 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16-G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16-G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16+G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage. The results show that mouse preimplantation embryos from F1 hybrid mice (CBA/Ca × C57BL/6) need only be exposed to glucose for less than 24 h between 22 and 94 h post hCG in order to develop from the morula to the blastocyst stage in vitro. However, the exposure time needs to be increased to between 24 and 72 h in order that blastocyst cell numbers reach control levels. The importance of glucose before the morula stage may relate to the need to synthesize glycogen for later use. If the obligatory requirement for glucose is fulfilled, embryos are able to utilize pyruvate in the absence of glucose at the later stages of development. These results show that the mouse preimplantation embryo can, to some extent, adapt metabolically to changes in its external environment. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 115
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 467-480 
    ISSN: 1040-452X
    Schlagwort(e): Female meiotic apparatus ; Meiotic spindle ; Mouse oocyte ; Microtubule-associated protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The mouse oocyte expresses an Mr 60,000 (p60) polypeptide that is associated with the first and second meiotic spindles. Immunoreactive p60 was not detectable in the meiotic spindles of male germ cells or in mitotic spindles. P60 was identified with a polyclonal antibody whose predominant activity is directed against ankyrin. However, immunoadsorption experiments demonstrated that p60 is not an ankyrin isoform and represents a secondary activity of the polyclonal antibody. Circumstantial evidence suggest that p60 may be a micro-tubule-associated protein. Since the most obvious difference between the female meiotic spindle and other spindles is the long half-life of the former, we hypothesize that p60 may function in the maintenance of the long-lived female meiotic apparatus. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 116
    ISSN: 1040-452X
    Schlagwort(e): IAP ; Transcription factor IPEB ; Expression ; Lymphoid tissues ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The murine intracisternal A particle (IAP) proviral elements are expressed at low levels in undifferentiated F9 embryonal carcinoma cells but are highly expressed when F9 cells are induced to differentiate into parietal endoderm-like cells. IAP elements are also expressed in parietal endoderm-like PYS-2 cells. We previously identified an IAP proximal enhancer (IPE) element that mediates a F9 differentiation-specific enhancer activity. We also identified a 60 kDa IPE binding (IPEB) protein whose activity is high in PYS-2 cells, where IAP is expressed, but very low in F9 cells.Transcription of IAP elements has also been shown in the adult mouse thymus and in activated splenic B cells. We have now shown by DNA affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and band-shift analysis that the 60 kDa IPEB is expressed in adult T lymphocytes and in resting as well as lipopolysaccharide activated splenic B cells but not in adult liver cells, suggesting an important role for IPEB in IAP transcription in vivo. In addition, we find IPEB expressed in the fetal mouse at sites of lymphoid development, such as the liver, spleen, and thymus, suggesting it may play an important role in gene expression during lymphoid development. In support of this, we find IPEB in the human T cell tumor lines, Jurkat and Molt 13, as well as the Daudi B cell line and in the normal calf thymus and in the thymus and spleen of the chicken and rat. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 117
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 54-62 
    ISSN: 1040-452X
    Schlagwort(e): Cumulus oocyte complex ; Follicle ; Atresia ; IVM ; IVF ; In vitro development ; Cow ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Follicular size, follicular atresia, and oocyte morphology were investigated for the possible relation of these characteristics to the developmental competence of bovine oocytes. Ovaries from a local slaughterhouse were dissected to obtain a heterogeneous population of follicles. Half of each follicle was fixed for histological analysis, and the oocytes were detached carefully and cultured individually. Before in vitro maturation, the oocytes were grouped into six different classes based on the morphology of the cumulus and the ooplasm: classes 1 and 2 represent oocytes with a homogeneous ooplasm plus a compact and complete cumulus, and classes 3-6 represent oocytes with a granulated ooplasm and an incomplete and/or expanded cumulus. Oocytes from class 3 (beginning of expansion in outer cumulus layers and slight granulations in the ooplasm) developed past the 16-cell stage significantly (P〈0.05) more than oocytes with a compact and complete cumulus (classes 1 and 2) and oocytes from classes 4-6 (incomplete and/or expanded cumulus) after 5 days of in vitro culture. Oocytes from follicles measuring 3 mm or less did not develop past the 16-cell stage, whereas follicles of 3-5 mm and 5 mm or larger developed at similar rates (17% and 21% morulae, respectively). The state of the follicle did not affect whether an embryo reached at least the 16-cell stage, as comparable rates were obtained in all three groups of follicles: nonatretic (20%), intermediate (14%), and slightly atretic (16%). We concluded that oocytes acquire developmental competence late in the follicular phase, possibly when the first signs of atresia have appeared, and that oocytes with beginning signs of degeneration (class 3) will develop significantly more than all other classes. Class 3 oocytes originated from follicles that were generally atretic and therefore in later phases of follicular growth, suggesting that these oocytes, having been subjected longer to the follicular microenvironment, are more differentiated (possibly at the cytoplasmic level) than other classes of oocytes. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 118
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 338-344 
    ISSN: 1040-452X
    Schlagwort(e): Zygotes ; Cumulus-oocyte complexes ; Zona pellucida ; Gap junctions ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15-18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures.Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4-26% vs. 93-96%), fertilization (0-9% vs. 91-92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58-60% and 71%, respectively, vs. 91-92% for controls), cleavage development (40-47% and 53-54% vs. 74-78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P 〈 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P 〈 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P 〉 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Tab.
    Materialart: Digitale Medien
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  • 119
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995) 
    ISSN: 1040-452X
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 120
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 371-378 
    ISSN: 1040-452X
    Schlagwort(e): Chemoattraction ; Chemokinesis ; Follicular fluid ; Gamete interaction ; Acrosome reaction ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Atrial natriuretic peptide (ANP), found in mammalian ovarian granulosa cells and oocytes (Kim et al., 1992, 1993), induces the human acrosome reaction (Anderson et al., 1994). The purpose of the present study was to determine whether ANP, as eggderived peptides from sea urchins, can act as a chemoattractant to human spermatozoa. Small lengths of capillary tubing that contained different concentrations of ANP were suspended over a suspension of washed spermatozoa. The number of spermatozoa that entered the tubing was determined. More than twice the number of spermatozoa moved into the tubing that contained a maximally effective concentration of ANP, as compared with tubing that contained only medium. The concentration of ANP that produced a half-maximal effect was 0.7 nM. The effect was blocked by LY83583, an inhibitor of guanylate cyclase. ANP produced more than a twofold increase in the rate of cGMP formation, an effect that was blocked by LY83583. Human ANP (5-27), a fragment of the intact peptide, had no chemoattractant activity. These findings suggest that a specific sperm receptor exists for the chemoattractant activity of ANP that is associated with guanylate cyclase. The chemoattractant activity of ANP is independent of the presence of extracellular calcium ions and is independent of the action of ANP as a stimulus of the acrosome reaction. There is no association between the chemoattractant activity of follicular fluid and the follicular fluid concentration of ANP. These data suggest that factors besides ANP are responsible for the chemoattractant activity of follicular fluid. The present study adds to evidence that human ANP is a potent modulator of human sperm function. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 121
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 379-385 
    ISSN: 1040-452X
    Schlagwort(e): Nomifensine ; EEDQ ; Embryonic development ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: [3H]-dopamine ([3H]-DA) uptake was measured in the presence or absence of the catecholamine uptake inhibitor nomifensine in both unfertilized and fertilized eggs. Specific [3H]-DA uptake depended on time and [3H]-DA concentration; it was high in unfertilized eggs, declined 20-30 min after fertilization, and rose again during cleavage. Irreversible inactivation of dopamine receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) resulted in a complete loss of sensitivity of egg adenylate cyclase to dopamine stimulation. In fertilized eggs treated with EEDQ for 1 hr, restoration of adenylate cyclase activity sensitive to dopamine stimulation could be observed 4 hr after the end of treatment, thus suggesting the appearance of new dopamine receptors in cleaving eggs. Short-term EEDQ treatment on unfertilized eggs, although not impairing fertilization, resulted in cleavage inhibition; the same treatment carried out soon after fertilization, on the other hand, elicited no effect on development. On the contrary, in embryos subjected to continuous treatment with EEDQ, development was impaired independent of the stage at which the treatment was started. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 122
    ISSN: 1040-452X
    Schlagwort(e): Calbindin ; Uterus ; Placenta ; Duodenum ; Estrogen ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Expression of the Calbindin-D9k (CaBP-9k) gene was studied in the baboon. Northern blot analysis using a human CaBP-9k cDNA probe detected expression in duodenum but not in uterus and placenta. Reverse transcription/polymerase chain reaction (RT/PCR) confirmed this expression pattern and indicated a high degree of identity between the baboon and human CaBP-9k mRNAs. PCR was employed to amplify the intron A region of the baboon CaBP-9k gene using human-derived primers and baboon genomic DNA. The baboon intron was closely related to the human CaBP-9k intron A, including the presence a complete Alu-repetitive element. Most significantly, a 13 nucleotide long element at the 5′ end of the baboon intron matched exactly the human sequence. This element represents a nonfunctional variation of an estrogen response element found at the same location in the rat CaBP-9k gene. The rat element functions as an enhancer and mediates uterine and possibly placental CaBP-9k expression in the rat and probably most other mammals. The finding of a modified ERE in baboon as in human suggests that during primate evolution the expression of this mammalian-specific gene has been eliminated in uterus and placenta. This scenario raises the question of the role of CaBP-9k in these reproductive tissues. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 123
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 287-299 
    ISSN: 1040-452X
    Schlagwort(e): SGP-1 ; SGP-2 ; Postnatal development ; Nonciliated cells ; Efferent ducts ; Rats ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Sulfated glycoprotein (SGP) -1 and -2, secretory products of Sertoli cells, are secreted into the lumen of seminiferous tubules where they bind to late spermatids. Once released, the spermatozoa traverse the efferent ducts where these proteins detach from their surface and are endocytosed by the nonciliated cells. In adult animals, SGP-1 and SGP-2 are also synthesized by nonciliated cells and targeted from the Golgi apparatus to lysosomes. The purpose of the present study was to determine the pattern of expression of SGP-1 and SGP-2 within nonciliated cells during postnatal development. The efferent ducts of animals at different postnatal ages were prepared for an electron microscopic immunocytochemical quantitative analysis as well as for Northern blot analysis. The data expressed as labeling content (no. gold particles/μm2 and taking into account the volume of the endocytic or-ganelles and the cell) revealed that anti-SGP-1 labeling in endosomes of nonciliated cells was minimal at 15, 21, and 29 days of age. On the other hand, the lysosomal labeling content showed a significant increase by day 29 compared to 15 and 21-day-old animals indicating that an endogenous form of SGP-1 was being synthesized by nonciliated cells and targeted to lysosomes. By day 39 a significant increase in endosomal labeling occurred; this was attributed to the endocytosis of Sertoli-derived SGP-1 which coincided with the entry of spermatozoa into the lumen of these ducts at this age. Lysosomal labeling showed further significant increases at days 39, 49, and then again at day 90. Northern blot analysis detected SGP-1 mRNA transcripts at all postnatal ages examined. While decreases or increases in transcripts could not be determined due to the greater amount of tissue present with increasing age, these data taken together support the idea of an endogenous form of SGP-1 being synthesized by nonciliated cells and targeted to lysosomes during postnatal development.In the case of SGP-2, endosomal labeling was minimal at 15, 21, and 29 days of age but was significantly increased by day 39, with similar values at all subsequent ages. The high value at day 39 was attributed to the endocytosis of SGP-2 which coincided with the entry of spermatozoa into the lumen at this age. Lysosomal labeling, on the other hand, was low at days 15 and 21 but peaked at day 29 at a time when endosomal labeling was minimal. These results suggested the synthesis of an endogenous form of SGP-2 which was being targeted to lysosomes. Similar values for SGP-2 lysosomal labeling comparable to that at day 29 were obtained at all other ages. Since SGP-2 endosomal labeling was significantly increased at day 39 and maintained thereafter, it is suggested that labeling in lysosomes at this and subsequent ages could also be due to the endocytosis of Sertoli-derived SGP-2. However, Northern blot analysis confirmed the presence of mRNA transcripts for SGP-2 at all postnatal ages examined, although increases or decreases in their amount were not determined. These results thus consolidate the hypothesis of an endogenous form of SGP-2 being synthesized by nonciliated cells and targeted to lysosomes. Finally, since the amounts of endogenous SGP-1 and SGP-2 peak at different ages, it is suggested that different factors are involved in regulation of these two proteins during postnatal development. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 124
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 331-338 
    ISSN: 1040-452X
    Schlagwort(e): Ovary ; Connexin 43 ; Gap junctions ; Cell-to-cell communication ; Follicular development ; Atresia ; Corpus luteum ; Rat ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The present immunocytochemical study examines in the rat ovary the pattern of expression of connexin 43 (Cx43), a subunit of gap junctions. Using a well-characterized specific antiserum against rat Cx43, immunoreactivity was not detected in the fetal ovary, i.e., prior to follicular formation. However, in the ovary of 20-day-old, 35-day-old, and adult rats, strong Cx43-immunore-activity was associated with the cell borders of the follicular epithelium/granulosa cells of all developmental stages (primordial follicles, preantral and antral secondary follicles). In general, immunoreactivity of the granulosa cells of large antral follicles appeared more intense than the one of smaller follicles. Staining was also seen in oocytes (cytoplasmic staining). Theca cells of large antral follicles, but not of small follicles were immunoreactive. Immunoreactive interstitial cells were not seen in ovaries of 20- and 35-day-old animals, but staining in these cells was present in adult rats. In large follicles with signs of atresia, granulosa cells lacked Cx43-immunoreactivity, whereas Cx43-immunoreactivity in their theca interna strikingly increased. Corpora lutea in the cyclic adult rats were heterogeneously stained, with either no detectable immunoreactivity, staining of cell borders of most luteal cells, or with conspicuous staining of only a few cells. In the pregnant animals on gestation days (GD) 12, 14, and 17, all luteal cells stained strongly for Cx43 at the cell surface. Shortly before delivery (GD 21), however, the staining pattern vanished and only few, presumably luteal cells remained immunoreactive. In Western blots (using homogenates of whole ovaries), the Cx43 antiserum recognized a major band of approximate Mr 43 × 103, together with minor bands, which may reflect the presence of several differently phosphorylated Cx43 forms. This is indicated by treatment with alkaline phosphatase, which reduced the banding pattern to one single band. In summary, the gap junction molecule Cx43 is abundantly expressed in all endocrine compartments of the rat ovary. The staining pattern obtained in the present study indicates that Cx43 and presumably gap-junctional communication are associated with follicular development, atresia, and the development of the interstitial gland, as well as with the development and regression of the corpus luteum. The heterogeneous staining within the ovary furthermore hints to a contribution of the local intraovarian factors in the regulation of Cx43 expression. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 125
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 360-367 
    ISSN: 1040-452X
    Schlagwort(e): Pig IVF ; Oviductal cells ; Polyspermy ; Spermatozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: It is known that oviductal cells play an important role in fertilization in vivo. We were interested in the effect of those cells on spermatozoa and their influence on the incidence of polyspermy in pig in vitro fertilization (IVF) when cocultured with spermatozoa. Oviductal cells are believed to select a highly fecund population of spermatozoa. By coculturing spermatozoa with oviductal cells it is possible to reduce the number of spermatozoa confronting the eggs at fertilization, reducing the incidence of polyspermy. In our study, spermatozoa were cocultured with oviductal cells for 30 min so that they could bind to the oviductal cells. Both bound and unbound spermatozoa were used for fertilization. The results show that the spermatozoa that were bound to the oviductal cells were capable of fertilizing the eggs, but the nonbound spermatozoa had a reduced penetration incidence. With the bound sperm, polyspermy incidence decreased and the two-pronuclei proportion increased. We also found that with time the spermatozoa released from the cells had better motility than those that did not bind. Therefore, it is our belief that oviductal cells can be used for porcine IVF resulting in a lower polyspermy incidence and higher pronuclei incidence. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 126
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 374-383 
    ISSN: 1040-452X
    Schlagwort(e): Immunolocalization ; Immunogens ; Spermatogenic cells ; Lectins ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We have previously reported the purification of rat testis galactosyl receptor, an equivalent to the Ca2+-dependent (C-type) minor variant of rat hepatic lectin-2/3 (RHL-2/3). We now report the purification of galactosyl receptor from rat sperm and its immunolocalization in the intact rat testis and sperm by polyclonal antibodies prepared using multiple antigen peptides (MAP) as immunogens. Two MAP antigens (designated 27-mer and 28-mer), corresponding to amino acid sequences of the carbohydrate-recognition domain (galactose) and adjacent Ca2+-binding sites of RHL-2/3, were used for immunization. Anti-RHL-2/3, anti-p27, and anti-p28 sera crossreacted with rat hepatocyte RHL-2/3 and its rat testis and sperm equivalent, galactosyl receptor, purified by chromatofocusing followed by galactose-Hydropore-EP affinity chromatography. Neither anti-p27 nor anti-p28 sera crossreacted with the major hepatocyte variant, RHL-1. A RHL-1-equivalent was not detected in rat testis and sperm. Immunofluorescence studies demonstrated that anti-p27 and anti-p28 sera recognize galactosyl receptor sites at the Sertoli cell-spermatogenic cell interface and on the dorsal surfacae of the sperm head, overlying the acrosome. The characteristic crescent-shaped immunoreactive pattern in sperm was lost after induction of the acrosome reaction. Further studies should determine whether antisera to MAP antigens 27-mer and 28-mer, corresponding to specific protein motifs, can serve as immunological probes for examining cell-cell interaction events during spermatogenesis and at fertilization. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 127
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 398-398 
    ISSN: 1040-452X
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 128
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 449-458 
    ISSN: 1040-452X
    Schlagwort(e): Germinal vesicle ; Nucleolus-like body ; Nucleolus-precursor body ; RNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Mouse preovulatory oocytes, zygotes, parthenogenetically activated pronuclear oocytes, and early embryos, as well as hamster zygotes, were analyzed, by autoradiography, for the distribution of either “maternal” or newly synthesized RNAs. Early mouse embryos were also examined for the distribution of newly replicated DNA. Special attention was attributed to NLBs in oocytes or to NPBs in early embryos. In mouse oocytes, [5-3H]uridine radioactivity accumulated (after a 2-hr pulse) in vitro, in addition to other nuclear compartments, in the central compact material of the NLBs. There was no cytoplasmic labeling. In all parthenogenetic pronuclear embryos developed from similarly labeled oocytes, this label was distinctly detectable in the central compact material of the NPBs; less intensive labeling was seen in the nucleoplasm and cytoplasm. On the contrary, the central compact part of the mouse NPB did not show labeling in DNA after a continuous culture with [6-3H]thymidine. In mouse and hamster pronuclear zygotes, convincing evidence was obtained for a lack of any newly synthesized nucleic acids in the compact material of NPBs using 4- to 10-hr culture with [8-3H]adenosine. Based on these data, it was shown that the NLBs of oocytes or NPBs of early embryos probably contain RNAs synthesized during the last stages of antral follicle oocyte differentiation. This unique pathway of RNAs in the oocyte - embryo system may explain the specific morphology of both oocyte and early embryo “nucleoli.” © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 129
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 479-485 
    ISSN: 1040-452X
    Schlagwort(e): Oogenesis ; Folliculogenesis ; Meiosis ; Chromatin structure ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We investigated the changes in the organization of oocyte nuclear chromatin and nucleolar-associated chromatin throughout folliculogenesis. Zona-free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive-chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing, some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I-III, 10-40 μm in diameter: 100% NSN vs. 0% SN; class VII 70-80 μm in diameter: 47.3% NSN vs. 52.3 SN, in 4-6-week-old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2-week-old females; 11.8% NSN vs. 8.2% SN in 56-week-old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 130
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 530-530 
    ISSN: 1040-452X
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 131
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995) 
    ISSN: 1040-452X
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 132
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 188-199 
    ISSN: 1040-452X
    Schlagwort(e): Hamster ; Embryo ; Amino acids ; Culture media ; HECM-6 ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1-cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids - glutamine, taurine, and glycine - were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium-5. Moreover, addition of another eight amino acids - asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM-6) - had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM-5 and -6 (chemically defined, protein-free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 7 Tab.
    Materialart: Digitale Medien
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  • 133
    ISSN: 1040-452X
    Schlagwort(e): Sperm maturation ; Sperm-egg recognition ; Fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: In the preceding study (Okamura et al., 1992; Biol Reprod 47:1040-1052) we suggested that a 135-kDa protein secreted by porcine epididymis is involved in the sperm maturation. In this work, we have isolated the cDNA clone coding the 135-kDa protein in an effort to investigate its structure and function.The 135-kDa protein was purified from porcine cauda epididymal fluid. Three oligonucleotide probes were synthesized according to the amino acid sequences of N-termini of the native protein and trypsin-digested peptides. A cDNA clone hybridizing with these three probes was isolated from the cDNA library derived from the porcine proximal corpus epididymis. It encodes a novel protein with 1,006 amino acid residues in an open reading frame.Its overall amino acid sequence was significantly homologous (25.7%) to the α-mannosidase precursor of Dictiostelium discoideum (P34098). The 135-kDa protein could digest both p-nitro-phenyl-α-D-mannoside and high mannose oligo saccharide (Man8-GlcNAc2), strongly suggesting that it is an α-mannosidase homologue.The expression of this protein was specific to porcine and was localized to the very narrow parts of epididymis: the border of the caput and corpus epididymis. This protein may serve as a good marker for the functional differentiation in porcine epididymis. A possible role of this protein in the species-specific sperm-egg interaction is discussed. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 134
    ISSN: 1040-452X
    Schlagwort(e): Oviduct ; Sheep ; mRNA sorting ; Secretory protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Data from our laboratory have shown that an estrogen (E2)-dependent Mr 90,000-92,000 protein and its mRNA are synthesized and expressed in abundant amounts at estrus from the fimbria and ampulla, not isthmus, oviduct of the sheep. Immunocytochemical studies have shown that the Mr 90,000-92,000 protein is contained in apical secretory granules of oviduct epithelial cells. The objective of this study was to determine whether the mRNA for the E2-dependent oviduct protein was localized and compartmentalized in similar manner. Fimbria, ampulla, and isthmus oviducts obtained from estrous ewes were flash frozen in liquid nitrogen, cryosectioned, fixed in 4% paraformaldehyde, hybridized with digoxigenin (DIG)-labeled oviduct-specific riboprobes, incubated in anti-DIG antibodies conjugated with alkaline phosphatase, and developed in color substrate. Oviduct protein-specific transcripts were localized to basal perinuclear compartments and, surprisingly, at sites distant from the nucleus in the apical cytoplasm of epithelial cells in the fimbria and ampulla. No specific reaction product was observed in the underlying mucosa or smooth muscle layers. Oviduct protein mRNA was contained predominantly in the apical cytoplasm of epithelial cells at the free margins of mucosal folds and in the basal regions of cells located at the crypts of longitudinal folds. No reaction product was present when sections of the fimbria and ampulla oviduct of estrous ewes were incubated in sense riboprobe to the oviduct protein. In addition, when sections of the isthmus oviduct obtained from estrous ewes or fimbria and ampulla oviducts from long-term ovariectomized ewes were hybridized with antisense riboprobes no specific reaction product was detected. Electron microscopy of oviduct protein mRNA containing areas revealed the presence of secretory granules, rough endoplasmic reticulum (RER) and Golgi in the apical cytoplasm, and RER in the basal regions of epithelial cells. These data show that the mRNA encoding an E2-dependent oviduct-specific protein is distributed in epithelial cells at perinuclear foci and at sites distant from the nucleus, which are also the sites of protein localization and protein synthesizing organelles, implying translation at unique cytoplasmic foci. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 14 Ill.
    Materialart: Digitale Medien
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  • 135
    ISSN: 1040-452X
    Schlagwort(e): Mouse teratocarcinoma ; Oncofetal markers ; Differentiation ; Scintigraphy ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Alphafetoprotein (AFP), a major serum protein synthesized during the embryo-fetal and postnatal period (in the yolk sac, then in the liver), is also an oncoprotein. The intracellular presence of AFP and of serum albumin (SA) in normal and neoplastic neural crest and neural tube derivatives was previously demonstrated. In this work we have studied the comparative expression of AFP and SA in primitive neuroectoblastic structures of mouse embryos (6 and 7 days “post coitum”) and mouse teratocarcinomas (derived from the PCC4 cell line). Using immunofluorescence technique, antibodies to SA gave a positive reaction in embryos of 7 days, while AFP was not detected during this period. By mRNA in situ hybridization, SA mRNA gave a strong signal in both 6 and 7 day embryos, whereas AFP mRNA gave a weak signal only in 7-day embryos. The distribution of SA and AFP and their mRNAs was investigated in primitive neuroectoblastic structures of the teratocarcinomas by in situ hybridization and immunostaining. Only SA protein was detectable by immunostaining. SA mRNA gave a strong signal in differentiating structures as well as in undifferentiated cell clusters. AFP mRNA was observed only in differentiating structures. Dot-blot hybridization indicated that the level of SA transcripts was at least 6-fold higher than that of AFP transcripts in the teratocarcinomas investigated. In teratocarcinoma-bearing mice injected intraperitoneally with 125I-radiolabeled SA and AFP, significant accumulations of both SA and AFP were demonstrated in the tumors, SA being about 3-fold higher than that of AFP after normalization to quantity of uptake in liver. External in vivo photoscanning confirmed this relationship of accumulated radiolabeled proteins. The last observation could be useful in vivo for diagnosis of teratocarcinoma. We conclude that the expression of SA relative to AFP and the external cellular uptake of SA relative to AFP are similar in normal embryonic developing tissues and in the corresponding morphologically neoplastic tissues of the teratocarcinomas. The same SA:AFP relationship constitutes an oncofetal marker of primitive neuroectoblastic structures. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 136
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 415-424 
    ISSN: 1040-452X
    Schlagwort(e): Fibrous sheath ; Testes ; Gene expression ; Spermatogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The fibrous sheath is a major cytoskeletal structure in the principal piece of the mammalian sperm flagellum. Two peptide sequences obtained from a tryptic digest of mouse fibrous sheath proteins exhibited high homology with μ-class glutathione S-transferases (GSTs). Using a DNA probe amplified from degenerate polymerase chain reaction (PCR) primers predicted from these two peptide sequences, a ∼ 1.1 kb cDNA clone for fibrous sheath component 2 (Fsc2) was isolated which had 84% nucleic acid and 89% amino acid sequence identity with a previously reported μ-class human GST gene (hGSTM3; Campbell et al., 1990: J Biol Chem 265:4188-9193). Sequences corresponding to those of the two fibrous sheath peptides were present in the protein encoded by the Fsc2 cDNA. Northern analysis with the full length Fsc2 cDNA detected a ∼ 1.1 kb mRNA in 12 of 15 somatic tissues examined, as well as in testis and isolated spermatogenic cells. However, 5′(nt - 96 to 12) or 3′ (nt 637 to 808) Fsc2 probes, containing mostly noncoding sequences, detected a ∼ 1.1 kb mRNA abundant in testis and isolated spermatogenic cells, but absent or present at low levels in somatic tissues. Northern analysis with RNA from testes of mice of different postnatal ages and purified spermatogenic cell populations indicated that this transcript is first present during the meiotic phase of germ cell development. These results suggest that a previously unreported μ-class GST gene (mGSTM5*) is expressed at a specific time during the development of spermatogenic cells in the mouse. Immunoblot analysis indicated that a μ-class GST protein is associated with the fibrous sheath, suggesting that it becomes an integral part of the mouse sperm cytoskeleton. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 137
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 457-458 
    ISSN: 1040-452X
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 138
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 486-492 
    ISSN: 1040-452X
    Schlagwort(e): Microfilaments ; Oocyte maturation ; Confocal laser scanning microscopy (CLSM) ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The localization and changes in microfilaments (MF) during golden hamster oocyte maturation were examined by an immunofluorescein method and confocal laser scanning microscopy (CLSM). We also studied the relationship between the changes in MF and oocyte nuclear and cytoplasmic maturation. During in vivo maturation, generalized submembranous MF were found initially which gradually became more prominent at the site of the first polar body extrusion. However, 43.7% of the in vitro matured metaphase 2 stage oocytes lacked the submembranous MF structure. This fact may partly account for the low fertilization rate of in vitro matured oocytes. MF were not found in the folicular oocytes cultured in cytochalasin-D-containing medium, and metaphase-like chromosomes were located at the center of the oocyte and first polar body extrusion did not occur. Twenty-five percent of the oocytes, which were arrested at meiosis by hypoxanthine, synthesized submembranous MF structure although the nuclear stage of these oocytes was germinal vesicle. These facts suggest that MF plays a role in nuclear behavior but there are some differences in the changes taking place within the nucleus and MF. MF may play a role in oocyte cytoplasmic maturation although the details of this have yet to be established. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 139
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 513-520 
    ISSN: 1040-452X
    Schlagwort(e): Aerosome reaction ; Calcium binding ; Membrane fusion ; Pyroantimonate ; Vesiculation ; Ram spermatozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Calcium was identified by a pyroantimonate-osmium fixation technique in ram spermatozoa undergoing a spontaneous acrosome reaction induced by incubation of diluted semen at 39°C. Intracellular calcium was only detected in diluted spermatozoa and increased in amount and distribution over 4 hr At 4 hr, the majority of the spermatozoa displayed ultrastructural evidence of an acrosome reaction. Calcium was initially evident on the outer acrosomal membrane in multiparticulate clusters, which were seen to be located on scalloped crests of acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly. As vesiculation advanced, calcium particles became associated with the periphery of the vesicles attached in the region of the fusion between the two membranes, but were never seen inside the vesicles. The equatorial segment was not labelled until much later in the reaction, at which time calcium particles were also evident on the nuclear membrane; vesiculation of the equatorial segment was also noted at this time.Dense labelling of the postacrosomal dense lamina was seen in all incubated spermatozoa. At the anterior margin of this structure the labelling was seen to be in a “sawtooth” arrangement. The disposition of the calcium both temporally and spatially is discussed in relation to its possible mechanisms in bringing about membrane fusion. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 140
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 72-79 
    ISSN: 1040-452X
    Schlagwort(e): Acrosome antigen ; Antigenicity ; Epididymis ; Sperm maturation ; Monoclonal antibody ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We found an intra-acrosomal antigen of about 155,000 daltons (155 kDa) in a survey using the monoclonal antibody MC101 raised against mouse cauda epididymal spermatozoa. Morphological studies by means of indirect immunofluorescence and immunogold electron microscopy localized the antigen to the cortex region of the anterior acrosome. Avidin biotin complex immunocytochemistry initially demonstrated a faint signal at the anterior acrosome in the testis spermatozoa that increased in intensity as the sperm moved toward the distal epididymis. This incremental immunoreactivity was also confirmed by immunoblotting following one-dimensional SDS-PAGE. The 155 kDa protein band was immunostained, and it was much more intense in the cauda epididymal than in the caput and corpus epididymal spermatozoa. Only a trace or no immunostain was evident in the caput or testis spermatozoa. The antigen localization did not change during passage through the epididymis, being confined at the cortex region of the anterior acrosome. The epididymal epithelial cells were not immunostained. These findings suggested that the 155 kDa protein is biochemically modified, further implying that the biochemical alteration of intra-acrosomal material is involved in sperm maturation in the epididymis. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 141
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 80-88 
    ISSN: 1040-452X
    Schlagwort(e): Acrosome reaction ; Follicular fluid ; Spermatozoa ; Corticosteroid-binding globulin ; Progesterone ; Progesterone-binding protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Human follicular fluid (hFF), which has been treated with either unspecific proteases or dextran-coated charcoal (DCC) to remove proteins and/or steroids, cannot successfully induce the acrosome reaction (AR). After the removal of steroids, AR-inducing activity can be restored to hFF by supplementation with exogenous progesterone, but only in the presence of intact protein. Gel filtration experiments with 3H-progesterone-labelled hFF showed elution of the radioactive signal in the high molecular weight range, corresponding to bound progesterone. AR-inducing activity was seen in exactly the same fraction. Based on these results, the acrosome reaction-inducing substance (ARIS) appears to be a complex of progesterone and a progesterone-binding protein, which was shown to be identical with the plasma protein corticosteroid-binding globulin (CBG) by immunological techniques. AR induction was only observed in the presence of both CBG and progesterone, suggesting a combined effect of the two components. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 142
    ISSN: 1040-452X
    Schlagwort(e): Epididymis ; Glycoprotein ; Sperm ; Saliva ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: In the rat, the secretory glycoprotein DE/AEG is one of the main constitutents of the epididymal fluid. We have recently reported the cloning of the cDNA for the related cysteine-rich secretory protein-1 (CRISP-1) from murine epididymis (Haendler et al., 1993; Endocrinology 133:192-198). The protein has now been isolated from the same organ and its N-terminal amino acid sequence has been determined. CRISP-1 exhibited an iso-electric point of ∼6.8. High levels of CRISP-1 antigen were detected in the corpus and cauda of the epididymis, vas deferens, seminal vesicle, prostate, and in the salivary gland by immunohistochemistry. A quantitative analysis of the cauda epididymal fluid by sandwich ELISA revealed that CRISP-1 represented ∼15% of the total protein. For heterologous expression, the CRISP-1 coding sequence was introduced into the pMPSV/CMV vector before transfection of baby hamster kidney (BHK) cells and selection with puromycin and neomycin. Expression in insect cells was achieved by co-transfection of Sf9 cells with a transfer vector and baculovirus DNA. Recombinant CRISP-1 was isolated in quantities sufficient for structural analysis. Ethyl maleimide treatment showed that all 16 cysteines were engaged in disulfide bonds. Proteolytic digestion demonstrated that the six cysteines localized in the N-terminal moiety formed three bonds with each other, suggesting the existence of two discrete domains in the protein. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 13 Ill.
    Materialart: Digitale Medien
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  • 143
    ISSN: 1040-452X
    Schlagwort(e): Milk ; Regulation ; Tissue specificity ; Micro-injection ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3-kb upstream region of the rabbit whey acidic protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was introduced into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0-16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 144
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 298-302 
    ISSN: 1040-452X
    Schlagwort(e): Equine ; Embryo ; Development ; Transcription ; Nucleolus ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The time of activation of the embryonic genome (maternal-embryonic transition) in equine embryos was investigated by assessing incorporation of 3H-uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with 3H-uridine (560 μCi/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 μCi/ml for 0.5-1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FBS and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embryos were spread onto glass slides, dipped into emulsion, and exposed for 8 d, then developed and counter-stained with Giemsa and propidium iodide. Embryos at the blastocyst, morula, eight-cell, and five-cell stages incorporated 3H-uridine into their cell nuclei as detected by autoradiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleolar precursors were found in 12 of 23 embryos examined. Most embryos in the four- to six-cell stage (n = 7) had nucleolar precursor bodies (npb) consisting of homogeneous fibrillar structures. Two five- to six-cell embryos also possessed reticulated nucleoli with both fibrillar and granular components as did all eight-cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granular components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of 3H-uridine and the formation of functional nucleoli with typical fibrillar and granular components occurs between the four- to eight-cell stage in equine embryos. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 145
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 359-368 
    ISSN: 1040-452X
    Schlagwort(e): In situ ; Mitochondria ; Nucleoli ; Oocytes ; RNase MRP RNA ; Xenopus laevis ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: RNase MRP is a ribonucleoprotein endoribonuclease found predominantly in nucleoli, but which has been associated with mitochondria and mitochondrial RNA processing. In order to analyze the intracellular localization of specific RNA components of ribonucleoproteins of this type, a whole-mount method for in situ hybridization in Xenopus laevis oocytes was employed. Results with specific probes (for both mitochondrial and nonmitochondrial RNAs) indicate that this procedure is generally effective for the detection of a variety of nucleic acids that reside in different cellular compartments. Probes used to detect the endogenous RNA component of RNase MRP (MRP RNA) during X. laevis oogenesis revealed a continuous nuclear signal as well as a possible dual localization of MRP RNA in nucleoli and mitochondria at developmental stages temporally consistent with both ribosomal and mitochondrial biogenesis. Genomic DNA encoding MRP RNA was injected into the nuclei of stage VI oocytes and correctly transcribed. The in vivo-transcribed RNA was properly assembled with at least some of its cognate proteins as demonstrated by immunoprecipitation with specific autoantiserum. In addition, detectable levels of the RNA were exported to the cytoplasm. This whole-mount procedure has permitted us to identify MRP RNA in situ at different developmental time points as well as during transcription of the injected gene, and suggests differential localization of MRP RNA during oogenesis consistent with its proposed function in both mitochondria and nucleoli. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 146
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 292-304 
    ISSN: 1040-452X
    Schlagwort(e): nucleus ; replication ; transcription ; MPM-2 ; rabbit embryo ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Mechanisms of nuclear reprogramming and assessment of potential malfunctions that could be deleterious for development were evaluated in rabbit zygotes, parthenotes, and nuclear transfer embryos by analysis of DNA replication, nucleolar fibrillarin label, and localization of nuclear material reactive to the MPM-2 antibody. Nuclear transfer embryos were derived from G1/early S-phase donor nuclei and MII oocytes. In nuclear transfer embryos, DNA rerelication was likely to have occurred because label was incorporated, possibly in the centromeric regions of the chromosomes, prior to premature chromosome condensation and again following pronuclear formation. In parthenotes, DNA replication began very late in the cell cycle, which may be due to deficiencies in the artificial activation stimulus. The presence of fibrillarin label in the nucleolus was used as an indication of nucleolar transcriptional activity. Fibrillarin label was absent in embryos of all types up to the 16-32-cell stage. Although fibrillarin reappeared in nuclear transfer and parthenote embryos at the appropriate stage, not all blastomeres showed label indicating impaired development in these embryos. Labelling of phosphorylated epitopes by MPM-2 antibody showed a change in pattern of labelling during early development. Early cleavage stage embryos did not exhibit labelling over the spindle poles as did blastomeres from 32-cell embryos and tissue culture cells. All cell types exhibited labelling during interphase as dots located primarily over the nucleus in blastomeres from 32-cell embryos and in tissue culture cells, together with cytoplasmic label in embryos at early cleavage stages. Nuclear transplant embryos had a normal pattern of MPM-2 label. In contrast, the appearance of MPM-2 label in parthenotes depended on the type of calcium stimulation. These results demonstrate defects in DNA synthesis, nucleolar activity, and specific phosphorylation events, likely resulting from an improper activation stimulus and chromosome condensation in the transplanted nucleus. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 147
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 481-489 
    ISSN: 1040-452X
    Schlagwort(e): Xenopus oocyte ; Interspersed RNA ; Translation ; Oligodeoxynucleotides ; RNA binding protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: It has been shown that about two thirds of Xenopus oocyte or sea urchin egg cytoplasmic poly(A)+ RNA contains interspersed repetitive sequences. The functional significance of this interspersed RNA has remained unknown. Here the function of a subfamily of interspersed RNA (XR family; McGrew and Richter, 1989: Dev Biol 134:267-270) in Xenopus oocytes was studied. We found that the elimination of T7 XR (one of the two complementary strands of the XR repeat) interspersed RNA by complementary oligodeoxynucleotides significantly inhibited protein synthesis. On the other hand, the injection of in vitro synthesized T7 XR RNA stimulated translation. Moreover, the insertion of the T7 XR RNA sequence into globin mRNA repressed the translation of the globin mRNA. In order to explain these results, we analyzed interactions between the XR interspersed RNA and oocyte proteins. We found that the major XR RNA binding proteins were p56 and p60, which could be the known mRNA “masking” proteins that bind mRNA and inhibit translation. Further, a 42 kD protein has been identified that appears to bind T7 XR RNA relatively specifically, although it interacts with mRNA with a lower affinity. Based on all of these data, we have proposed that interspersed RNA may be involved in regulating translation by competing with mRNA to interact with certain proteins that can regulate translation. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 148
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 100-108 
    ISSN: 1040-452X
    Schlagwort(e): Sperm ; SP-10 ; Intra-acrosomal Quantitative competitive PCR ; Cryptic splicing ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Alternatively spliced mRNAs encoding the human intraacrosomal protein SP-10 were sought by the reverse transcriptase polymerase chain reaction (RTPCR). Eleven RTPCR products were identified, characterized, and found to represent authentic alternatively spliced SP-10 mRNAs. The 11 alternatively spliced SP-10 mRNAs encoded proteins ranging from 81 to 265 amino acids. The 10 smaller variants all resulted from one or two in-frame deletions in exons 2 and/or 3 of the SP-10 genomic sequence. Quantitative competitive RTPCR showed that the four largest SP-10 mRNAs represented the majority (〉99%) of the SP-10 message in testes from each of four men. The relative abundance of each of the four SP-10 mRNAs varied between individuals, but the longest SP-10 mRNA, SP10-1, which encoded a 265 amino acid protein, was consistently the most abundant, comprising 53-72% of the total SP-10 message. This was followed by the second largest SP-10 mRNA, SP10-2, which encoded a protein of 246 amino acids and comprised 15-32%. The third and fourth largest SP-10 mRNAs, SP10-3 and SP10-4, encoded proteins of 210 and 195 amino acids and accounted for 3.4-8.3% and 8.7-12.5% of the total SP-10 messages, respectively. The remaining 7 SP-10 mRNAs combined accounted for〈1% of the total SP-10 message. Within the low abundance group of mRNAs were two that deleted the entire third exon of SP-10. The present study suggests that phenomena of cryptic splicing and exon skipping occur within the SP-10 mRNA. Along with proteolysis, alternative splicing also helps to explain the heterogeneous forms of SP-10 that have been observed on Western blots of human sperm extracts. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 149
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 149-156 
    ISSN: 1040-452X
    Schlagwort(e): Epidermal growth factor receptor ; EGFR ; Receptor regulation ; Alternate mRNA ; Placenta ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Epidermal growth factor receptor (EGFR) plays an important role in growth and differentiation. The human placenta expresses high levels of the receptor. In the placenta, as in many other human tissues, EGFR is encoded by two RNA transcripts of 5.8 kb and 10.5 kb. The placenta also expresses a putative truncated EGFR transcript of 1.8 kb, which encodes only the ligand binding domain of the receptor. The etiology and role of these variant EGFR transcripts is unknown. Using the human placenta as a model to study this area, we report (1) the relationships among these transcripts suggest that the induction of alternate pathways of EGFR RNA processing is involved in their etiologies; (2) the 10.5 kb transcript may be the principal transcript involved in determining the level of the protein receptor; and (3) the isolation of a soluble protein with characteristics consistent with a translational product corresponding to the 1.8 kb transcript, which may act in regulating the activity of EGFR. Together these results suggest that alternate processing of EGFR RNA into variant transcripts may represent a novel mechanism involved in the regulation of the receptor protein. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 150
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 157-166 
    ISSN: 1040-452X
    Schlagwort(e): Gene regulation ; Interferon ; Transcription ; Transcription factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Recent evidence suggests that several processes during mammalian embryogenesis may be regulated by IFNs or IFN-like molecules. With the use of MAPPing, the simultaneous presence of transcripts homologous to IFN-α, IFN-β, IRF-1, and IRF-2 was examined in mouse embryos and in embryonal carcinoma (EC) P19 cells, which are equivalent to epiblast cells of the early postimplantation blastocyst. Transcripts for IFN-α, but not for IFN-β, were detected as maternal transcripts in the ovulated oocyte and persisted over early embryogenesis. IRF-1 transcripts appeared only after the first cell cleavage in the two-cell stage embryo. IRF-2 transcripts were analyzed only in EC P19 cells and were found in both undifferentiated (D-) and differentiated (D+) cells. The IFN-α transcripts present in (D-) P19 cells were cloned and the partial cDNA sequences determined. Mu IFN-αA and a new Mu IFN-α species (Mu IFN-α12) were isolated from (D-) P19 cells. The presence of constitutive IFN-α transcripts in early mouse embryos suggests a role for these molecules during embryogenesis. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
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  • 151
    ISSN: 1040-452X
    Schlagwort(e): Prolactin ; Placenta ; Pregnancy ; Rodent ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Prolactin-like protein C (PLP-C) is a major rat placental protein which is expressed during the second half of pregnancy and belongs to the growth hormone-prolactin family. Here we report on the isolation of overlapping rat placental cDNAs which specify a transcript of 915 base pairs and predict a 205-amino acid translated product. The full-length cDNA shares 93% homology with the nucleotide sequence reported for PLP-C, and the putative protein, which we designate PCRP (prolactin-like protein C-related protein), exhibits 88% homology with the PLP-C precursor protein. PCRP lacks the signal sequence and the first 2 N-terminal cysteine residues present in PLP-C. Northern blot analysis indicated the basal zone-specific expression of PCRP mRNA, with no detectable expression in decidua and labyrinth. Southern blot analysis of rat genomic DNA using PCRP cDNA as a probe demonstrated multiple hybridization bands, suggestive of a family of genes encoding prolactin-like proteins. Western immunoblot analysis of basal zone culture media using a PCRP antipeptide antiserum revealed at least 5 immunoreactive proteins. The existence of a PLP-C family of proteins in rat placenta after midpregnancy suggests their functional significance in the maintenance of pregnancy and fetal development. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 152
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 186-195 
    ISSN: 1040-452X
    Schlagwort(e): Germ cells ; Testis ; Gene expression ; Sperm ; Proacrosin ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A number of hsp70-like proteins are associated with developing male germ cells. One of these molecules, P70, is not sensitive to heat stress and is germ cell-specific, and its expression is developmentally regulated. We have characterized the association of the rat P70(rP70) with differentiating germ cells in the testis and with posttesticular sperm. An antibody originally raised against human sperm proacrosin (designated C3; Siegel et al., 1987: J Reprod Immunol 11:307-319) was found to immunostain rP70 by immunoblot analysis and was used in subsequent studies of the rP70 molecule. The C3 antibody reacted with P70 isoforms in rat, human, mouse, guinea pig, boar, and rooster testicular homogenates. In the developing rat testis, abundant rP70 protein levels were first detected on postnatal day 22, with upregulation to adult levels occurring after postnatal day 28. Purified populations of adult rat pachytene spermatocytes, round spermatids, and elongating spermatids, isolated by unit gravity velocity sedimentation, all expressed rP70. Posttesticular sperm exhibited a loss of the rP70 molecule; caput epididymal sperm were weakly immunoreactive for rP70, but no immunoreactivity was observed in either cauda epididymal sperm or epididymal fluid. In contrast to human ejaculated sperm, rat ejaculated sperm did not express rP70. The loss of P70 from rat posttesticular sperm may reflect species-specific differences in P70 functions, which are thought to include a role in the structural modifications that occur during germ cell differentiation. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 153
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 177-185 
    ISSN: 1040-452X
    Schlagwort(e): Cortical granules ; Oocytes ; Vitrification ; Ultrastructure ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Abattoir-derived oocytes were exposed to a concentrated cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% FCS in TCM199) for 1.5 or 5 min at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Their viability was assessed by in vitro fertilization (IVF) and culture (IVC) to blastocysts. To investigate the effect of DAP213 on the ultrastructure, GV and IVM oocytes were processed for transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound ultrastructural modifications to the microvilli and mitochondria, resulted in large vesicle formation, and, most significantly, caused the premature release of the cortical granules (CG). In IVM oocytes exposed to the cryoproteclant for 5 min, exocytosis of CG into the perivitelline space was common and the IVF rate was reduced (P 〈.05). After exposure for 5 min, GV oocytes displayed clusters of CG comparable to controls, but after IVM-IVF, polyspermy rate was increased (P 〈.05). Furthermore, treated GV oocytes showed a reduced rate of cleavage and blastocyst formation and an increased percentage of oocytes exhibiting alterations in organelles, whereas the viability and ultrastructure of IVM oocytes treated for 1.5 min was not different from controls. These observations demonstrate that (1) cortical granule kinetics is one of the key elements controlling fertilizability of bovine oocytes treated with cryoprotectant, and (2) GV oocytes are more sensitive to the cryoprotectant than those that have already been matured in vitro.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 154
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 221-227 
    ISSN: 1040-452X
    Schlagwort(e): Spermatogonia ; Germ cells ; Spermatogenesis ; Testis ; Differentiation antigen ; Monoclonal antibody ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: To study the mechanism of spermatogenesis during the premeiotic phase, a hybridoma producing monoclonal antibody (mAb) specific for early stages of spermatogenic cells was obtained. In immunohistochemical staining of adult testis, this mAb, designated as EE2, was able to react with type A to B spermatogonia and early meiotic cells, but not with Sertoli cells, Leydig cells, and other somatic tissues. Precursor cells of type A spermatogonia (gonocytes) were also positive for EE2 in perinatal mouse testis. The antigenic molecule recognized by mAb EE2 was a novel glycoprotein with molecular weight of 114 kDa, which had affinity with Con A and WGA lectins, and was susceptible to N-glycanase, suggesting the presence of asparagine-linked sugar chains. Furthermore, EE2 antigen was found to localize on the germ cell surface. The specific expression of this antigenic molecule suggests that it may play an important role in early spermatogenesis, of which only a little information is available at present. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 155
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 259-266 
    ISSN: 1040-452X
    Schlagwort(e): Murine ; Spermatids ; Aneuploidy ; DNA probes ; Fluorescence in situ hybridization (FISH) ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A multicolor procedure employing fluorescence in situ hybridization is described for detecting chromosomal domains and germinal aneuploidy in late-step spermatids in mice using DNA probes specific for repetitive sequences near the centromeres of chromosomes 8 and X. These probes were nick-translated with biotin- or digoxigenin-labeled nucleotides, and were detected with FITC or rhodamine. Probe and hybridization specificities were confirmed using metaphase chromosomes from spleen and bone marrow cells as well as from primary and secondary spermatocytes. Late-step spermatids, identified in testicular preparations by their hooked shape, yielded compact fluorescence domains in ∼ 50% and 〉 99% of cells when hybridized with probes for chromosomes X and 8, respectively. In a survey of 〉 80,000 late-step spermatids from 8 healthy young adult C57BL/6 or B6C3F1 mice, ∼ 3/10,000 spermatids had fluorescence phenotypes indicative of X-X or 8-8 hyperhaploidy. These frequencies are consistent with published frequencies of aneuploidy in meiotic metaphase II and first cleavage metaphases of the mouse, providing preliminary validation of sperm hybridization for the detection of aneuploidy. No significant animal or strain differences were observed. In addition, the hyperhaploidy frequencies for murine spermatids were indistinguishable for those for sperm from healthy men obtained by a similar hybridization procedure. These procedures for detecting aneuploid male gametes are examples of “bridging biomarkers” between human and animal studies. They have promising applications for investigations of the genetic, reproductive, and toxicological factors leading to abnormal reproductive outcomes of paternal origin. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 156
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 286-291 
    ISSN: 1040-452X
    Schlagwort(e): Embryo development ; Growth factor ; Inhibin ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: It is evident that members of several growth factor families are actively involved in embryogenesis from its earliest phases. Several reports also indicate the oviduct as a possible source of growth factors, suggesting an active role of this organ in mammalian embryonic development. The aim of this study was to investigate the presence of activin/inhibin subunits in bovine oviduct since activin is a well-characterised morphogen in amphibian development. The presence of transcripts for α. βA, and βB subunits was investigated by analysing oviduct epithelial cells mRNA with reverse transcription-polymerase chain reaction (RT-PCR). Moreover, antisera specific for the three subunits were used for the Western blot analysis of the proteins secreted by oviduct epithelial cells in vitro and for their immunohistochemical localisation in different oviductal regions. Oviduct epithelial cells expressed only the βA-subunit gene. Immunoreactive material was present among in vitro secreted proteins, indicating that the transcript is translated into a polypeptide that has been localised in the epithelium of both the ampullary and isthmic tract of the organ. Consistent with these results, the antisera for the α and βB subunits did not recognise any specific antigen either among secreted proteins or in the sections. These results indicate that βA subunit gene is expressed in bovine oviduct epithelial cells, and the protein is secreted in vitro and can be found along the whole extension of the organ. In the absence of α or βB subunits, this suggests that activin A is present in bovine oviduct. Such a finding would be consistent with an embry-otrophic activity of this organ, but definitive conclusions on the target tissue and the specific functions of oviductal activin require further studies. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 157
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 311-324 
    ISSN: 1040-452X
    Schlagwort(e): Rat embryos ; ICM ; ES-like cells ; Uterine cells ; Immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: To increase our understanding of rat embryos in culture and to attempt the isolation of blastocyst-derived cell lines, we examinated the initial growth behaviour of rat blastocysts from four strains of rat on four different feeder cell layers. The feeders used were a continuous cell line of murine embryonic fibroblasts (STO), primary mouse (MEF) or primary rat (REF) embryonic fibroblasts, and a continuous cell line of rat uterine epithelial cells (RUCs). A medium that gave optimum plating efficiencies for murine ES cells was used in the rat embryo culture. Each culture system allowed hatching and attachment of the blastocysts, that is, the behaviour was similar on each feeder and each strain for the first 2 days in culture. Subsequently, there was a rapid differentiation of the Inner Cell Mass (ICM) cells on fibroblastic feeder cell layers (STO 〉 MEF 〉 REF), and this was generally complete after 3-6 days in primary culture. On RUCs, the ICM was found to increase in size without differentiation up to and including day 4 and in some cases longer. Embryo-derived cells were obtained by disaggregating and passaging ICMs on REF and RUC feeders. Rounded, refractile, and epithelial-like cells were isolated on REF and colonies of ES-like cells on the RUCs. The ES-like cells were positive for expression of alkaline phosphatase and stage-specific embryonic-antigen 1. This is an important first step towards the derivation and culture of pluripotent ES cells from the rat. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 158
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 84-90 
    ISSN: 1040-452X
    Schlagwort(e): Activation ; Calcium ; Strontium ; Cycloheximide ; Oocyte ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We have examined the ability of the two parthenogenetic agents, strontium (Sr2+) and cycloheximide, to activate mouse oocytes. We demonstrate that Sr2+ and cycloheximide act synergistically to promote parthenogenetic activation up to the pronuclear stage in oocytes collected immediately after ovulation. These two agents appeared to act independently, since incubation in Sr2+ media triggered a series of intracellular Ca2+ rises without affecting protein synthesis and cycloheximide inhibited protein synthesis without causing any intracellular Ca2+ changes. In addition, cycloheximide did not alter the pattern of Ca2+ oscillations induced by Sr2+. In contrast, we show that another commonly used parthenogenetic activation treatment, the Ca2+ ionophore A23187, has dual effects. Exposure of oocytes to the Ca2+ ionophore, A 23187, in Ca2+- and Mg2+-free medium leads to the activation of young oocytes. However, as well as generating a Ca2+ increase, the treatment of mouse oocytes with A23187 and Ca2+- and Mg2+-free media led to a marked inhibition of protein synthesis. Our data show that parthenogenetic agents may have two important loci for activating mammalian oocytes and that the combined effect on Ca2+ release and protein synthesis is most effective. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 159
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 109-125 
    ISSN: 1040-452X
    Schlagwort(e): Paracrine factors ; Retinoic acid ; Gradients ; Imprinting ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Zusätzliches Material: 1 Tab.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 160
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995) 
    ISSN: 1040-452X
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 161
    ISSN: 1040-452X
    Schlagwort(e): Homeobox ; Development ; Preembryos ; Intron spanning primers ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Homeobox gene expression in human preimplantation development has not been established. We used reverse transcriptase-polymerase chain reaction (RT-PCR) with intron spanning primer sets to investigate the presence of mRNA of homeobox genes in human oocytes and preembryos. RT-PCR products obtained from normal and unfertilized oocytes, from cleaving normal and triploid embryos, and from morulae and blastocysts were cloned, sequenced, and analyzed for the presence of homeobox sequences. The presence of mRNA of homeoboxes HoxA4 and HoxA7 was demonstrated; HoxA4 was present in normal and unfertilized oocytes and also in a 4-cell embryo. HoxA7 was present in normal oocytes and cleaving triploid embryos. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 162
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 133-139 
    ISSN: 1040-452X
    Schlagwort(e): Preimplantation mouse embryo ; Gene expression ; mRNA differential display ; TGF-α, TGF-β ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The effect of growth factors on regulating gene expression in the preimplantation mouse embryo was examined, since results of previous experiments revealed a stimulatory effect of exogenously-added growth factors on preimplantation development in vitro. Treatment of early cavitating blastocysts with either 250 pM TGF-α or TGF-β results in changes in the pattern of total protein synthesis as assessed by high-resolution two-dimensional gel electrophoresis. In some cases, the synthesis of a particular polypeptide is either up- or downregulated by each growth factor, whereas in other instances the synthesis of a polypeptide is modulated by one but not the other growth factor. Use of the mRNA differential display method permitted the identification of genes whose expression is either up- or downregulated by these growth factors. Treatment of mouse blastocysts with either TGF-α or TGF-β results in the increased expression of the b subunit of the F0 ATPase. TGF-β also stimulates the expression of the DNA polymerase α. TGF-α treatment results in the increase in expression of a gene homologous to the human HEPG2 cDNA, as well as in a decrease in expression of fibronectin. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 163
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 140-148 
    ISSN: 1040-452X
    Schlagwort(e): Embryonal carcinoma cells ; Differentiation ; Transcriptionα ; Activating transcription factor ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Embryonal carcinoma (EC) cells and embryonic stem (ES) cells provide useful model systems for studying differentiation during early mammalian development. Previous studies have demonstrated that differentiation of two restricted mouse EC cell lines is accompanied by activation of the TGF-β2 gene. Moreover, one negative and two positive regulatory regions upstream of the transcription start site were identified, which appear to play key roles in the transcriptional regulation of the human TGF-β2 gene. In this report, we demonstrate that the same three regulatory regions strongly influence the activity of the TGF-β2 promoter in differentiated cells derived from the multipotent human EC cell line, NT2/D1, and from the murine totipotent ES cell line, CCE. We also determined that the same three regions are active in the regulation of the TGF-β2 gene in the murine parietal endoderm-like cell line, PYS-2. However, an additional negative regulatory region appears to contribute to the regulation of the TGF-β2 gene in PYS-2 cells. Last, mutation of a CRE/ATF element located just upstream of the transcription start site of the TGF-β2 gene reduces significantly the activity of the TGF-β2 promoter in the differentiated cells. However, in contrast to our previous findings, our gel mobility shift analyses demonstrate that this CRE/ATF element is bound by similar proteins in nuclear extracts prepared from undifferentiated and differentiated mouse EC cells as well as from undifferentiated human EC cells. Taken together, our findings suggest strongly that four regulatory regions, including a critical CRE/ATF element, help control the expression of the TGF-β2 gene during early mammalian development. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 164
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 177-183 
    ISSN: 1040-452X
    Schlagwort(e): Placenta ; Decidua ; Endometrium ; Parental imprinting ; Promoter ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: IGF2 is expressed in both placental and decidual tissues, enabling an analysis of the parental imprinting over the fetomaternal boundary. Evidence is provided that IGF2 is monoallelically expressed in both placenta and pregnant, as well as nonpregnant, endometrium. These observations suggest that the maternally derived IGF2 allele is inactivated during germline transmission. Comparison of promoter usage in decidua and placental samples shows that the P3 promoter appears to regulated independently of the others. These observations are discussed with respect to current models of IGF2 imprinting and the hypothesized conflict of parental reproductive interests which bears on the phenomenon of parental imprinting. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 165
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 204-211 
    ISSN: 1040-452X
    Schlagwort(e): Rabbit embryos ; Blastocyst ; Inner cell mass cells ; Trophectoderm ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The proportion of total cells in the blastocyst allocated to the inner cell mass (ICM) and trophectoderm (TE) is important for future development and may be a sensitive indicator to evaluate culture conditions. The number of cells and their distribution within the two primary cell lineages were determined for the rabbit embryo developing in vivo after superovulation or nonsuperovulation or embryo transfer and compared with embryos developing in vitro. Comparisons were made with cultured embryos or embryos grown in vivo until 3.5, 4.0, and 4.5 days of age. Embryos from superovulated rabbits developed in vivo for 3.5, 4.0, and 4.5 days, respectively, had 361, 758, and 902 total cells (P〈0.05), and in nonsuperovulated rabbits 130, 414, and 905 total cells (P〈0.05), with increasing proportions of ICM cells over time (P〈0.05). One-cell embryos recovered from superovulated females and transferred to nonsuperovulated recipients developed more slowly with 70, 299, and 550 total cells after 3.5, 4.0, and 4.5 days of culture (P〈0.05), respectively. The proportion of ICM cells increased with age of the embryo. Corresponding values for one-cell embryos cultured in vitro resulted in 70, 299, and 550 total cells (P〈0.05). However, in vitro culture of morula-stage embryos in the presence of fetal bovine serum for 24 hr did not delay growth. In addition, the proportions of ICM/total cells were 0.17, 0.25, and 0.29 for embryos developing in vitro at 3.5, 4.0, and 4.5 days, respectively, similar to those for embryos developing in vivo at each of the three recovery times. These data establish for the first time the number and proportion of cells allocated to the ICM of the rabbit embryo developing in vivo or under defined conditions in vitro. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 166
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 239-248 
    ISSN: 1040-452X
    Schlagwort(e): Metallic ions ; Hyperactivation ; Motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: An earlier study demonstrated that rabbit sperm incubated for 16 hr under capacitation conditions acquire motility patterns identical to those seen in rabbit sperm capacitated in vivo. We now show that similar motion patterns develop after 0.5 hr incubation in a Trisbuffered medium, medium M. Development and decline of the motion patterns occurred in three phases each recognized by the character of the biphasic motion patterns. Hyperactivated sperm were objectively identified and quantified by a previously developed computer-directed model. The percentage of motile sperm that acquired hyperactivated motility and the period they remained in this state varied among sperm from different rabbits. The decline in hyperactivated motility was paralleled by a decrease in the average sperm curvilinear velocity (VCL) and average amplitude of lateral head displacement (AALH), but was not accompanied by a concomitant decrease in percentage of motile sperm. Pb2+ and Cd2+, at concentrations that did not inhibit motility, prevented development of hyperactivated motility. Inhibition of hyperactivated motility by Pb2+ was time- and concentration-dependent; the average percentage of hyperactivated sperm decreased from ∼ 30% to〈5% (n = 5) in 1 hr at a Pb2+ concentration of 25 μM. Cd2+ inhibition of hyperactivation was dependent only on concentration of the cation. At a concentration of 100 μM, the decrease in the percent of hyperactivated sperm was ∼ 50% (n = 3). Hg2+, Zn2+, and Cr6+ at sublethal concentrations had no effect on hyperactivated motility development. These results suggest that Pb2+ and Cd2+, by virtue of their ability to prevent the wide curvature flagella beating that is characteristic of hyperactivation, can compromise fertilization at concentrations that do not inhibit sperm motility and act as a reproductive toxicant at a level other than spermatogenesis. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 167
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 300-305 
    ISSN: 1040-452X
    Schlagwort(e): XX true hermaphrodite ; XX male ; Sex determination ; Testis ; Ovotestis ; HMG box ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The Sry gene product serves an important function in male sex determination through testis induction. However, testicular development has been reported in SRY-negative XX sex reversed humans. XX sex reversal of the American cocker spaniel, inherited as an autosomal recessive trait, may be a homolog of this disorder. The purpose of this study was to determine whether the Sry high mobility group (HMG) box is present in genomic DNA of affected dogs. Conserved Sry HMG box and hypoxanthine phosphoribosyltransferase (HPRT) sequences were used as primers in polymerase chain reactions. A 167 bp Y-specific canine Sry HMG box sequence was cloned from genomic DNA of normal male dogs. Internal primers generated a 104 bp Sry HMG box product from normal males, but not from females or XX sex reversed dogs. Parallel reactions generated an HPRT product from all dogs. Results indicate that the Sry HMG box is absent in genomic DNA of XX sex reversed dogs. We speculate that activation of the testis differentiation cascade in the absence of Sry in this model is due to a mutant autosomal gene. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 168
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 314-324 
    ISSN: 1040-452X
    Schlagwort(e): Signal transduction ; Maternal mRNA ; Polyadenylation ; G protein ; RT-PCR ; Gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We have developed a novel reverse transcription-polymerase chain reaction (RT-PCR)-based approach for systematically quantifying in a single experiment the abundances of many different mRNAs in preimplantation mouse embryos. With this approach, the entire mRNA population from a small number of embryos is amplified while preserving the relative abundance of each mRNA in the cDNA population. The cDNA is analyzed by quantitative hybridization to radiolabeled probes. The approach is very sensitive and provides reliable, quantitative data regarding changes in mRNA abundance. A major advantage of this method is that estimates of mRNA copy number can be obtained and compared between different mRNAs. With this approach, we analyzed the patterns of expression of nine G protein α subunit mRNAs (Gαs, Gαi, Gαq′, Gαo′, and Gα11-15) in oocytes, eggs, and preimplantation embryos from fertilization to the blastocyst stage. Six α subunit mRNAs were expressed at significant levels, all of which underwent significant temporal alterations in expression. The mRNAs encoding some α subunit types were expressed predominantly in the egg and 1-cell embryo, underwent sharp reductions during the 2-cell stage, and were re-expressed between the 8-cell and blastocyst stages. One α subunit mRNA increased in abundance at the early blastocyst stage. The possible significance of these alterations in G protein mRNA abundance to embryonic development is discussed. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 169
    ISSN: 1040-452X
    Schlagwort(e): ES cells ; Pluripotent ; Bovine embryos ; Nuclear transfer ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Inner cell masses (ICM) from in vitro produced day 8 or 9 bovine blastocysts were isolated by immunosurgery and cultured under different conditions in order to establish which of two feeder cell types and culture media were most efficient in supporting attachment and outgrowth of the bovine ICM cells. The efficiency of attachment and outgrowth of the ICM cells could be markedly improved when STO feeder cells were used instead of bovine uterus epithelial cells, and by using charcoal-stripped serum instead of normal serum to supplement the culture medium. More than 20 stable cell lines were obtained. Some of these lines were examined by immunofluorescence for developmentally regulated markers. From these results we conclude that the cell lines resemble epithelial cells, rather than pluripotent ICM cells. The developmental potential of cells of one of the lines was tested in the nuclear transfer assay. The cell line could support the initial development of enucleated oocytes, but none of the reconstructed embryos passed the eight-cell block. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 170
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 503-512 
    ISSN: 1040-452X
    Schlagwort(e): Checkpoints ; H1 kinase ; Bovine ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Bovine oocytes were irradiated at germinal vesicle (GV) or metaphase II (MI) stage, after Hoechst staining, with chromosomally focused UV-C (254 nm) or UV-A (〉 330 nm). UV-C irradiation at GV stage did not inhibit germinal vesicle breakdown (GVBD) or chromosomal condensation; spindle formation was abolished and maturation promoting factor (MPF) levels failed to increase. UV-A irradiation at GV stage caused meiotic arrest at anaphase I; MPF levels were lower than control. UV-C irradiation at MII stage led to subsequent abnormal parthenogenetic activation when MPF levels failed to decrease. A normal male but no female pronucleus was formed at fertilization. UV-A irradiation at MII stage also caused abnormal activation; MPF levels declined normally. A normal male and abnormal female pronucleus formed at fertilization. UV-A irradiation results have implications for oocyte evaluation during development using Hoechst staining. UV-C irradiation is a potential means for oocyte enucleation in nuclear transfer. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 171
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 530-530 
    ISSN: 1040-452X
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 172
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 274-274 
    ISSN: 1040-452X
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 173
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 28-34 
    ISSN: 1040-452X
    Schlagwort(e): Growth factor ; Oviduct ; Embryogenesis ; In situ hybridization ; Cattle ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: It has become evident that certain growth factors are involved in the regulation of the initial bovine embryogenesis. In the present study, we examined by means of Northern blot and in situ hybridization, the expression and localization in the bovine oviduct of mRNAs encoding for platelet-derived growth factor (PDGF-B), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-I).Northern blot analysis on oviduct tissue demonstrated transcripts for PDGF-B and bFGF, but not IGF-I mRNAs. Two bands with estimated sizes of 5.0 and 1.5 kb were detected for PDGF-B and two bands with sizes of 7.5 and 4.9 kb for bFGF. In situ hybridization analysis demonstrated localization of PDGF-B mRNA in the lamina epithelialis and tunica muscularis of the oviduct whereas bFGF mRNA was detected in the lamina propria. It is concluded that the lamina epithelialis and lamina propria of the oviduct represent sites of synthesis of PDGF-B and bFGF mRNA, respectively. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 174
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 19-27 
    ISSN: 1040-452X
    Schlagwort(e): Differential hybridization ; In situ hybridization ; Mouse development ; Symmetric PCR ; Heat shock ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The visceral yolk sac (VYS), composed of extraembryonic mesoderm and visceral endoderm, is the initial site of blood cell development and serves important nutritive and absorptive functions. In the mouse, the visceral endoderm becomes a morphologically distinct tissue at the time of implantation (E4.5), while the extraembryonic mesoderm arises during gastrulation (E6.5-8.5). To isolate genes differentially expressed in the developing yolk sac, polymerase chain reaction (PCR) methods were used to construct cDNA from late primitive streak to neural plate stage (E7.5) murine VYS mesoderm and VYS endoderm tissues. Differential screening led to the identification of six VYS mesoderm-enriched clones: ribosomal protein L13a, the heat shock proteins hsc 70 and hsp 86, guanine-nucleotide binding protein-related gene, cellular nucleic acid binding protein, and ã-enolase. One VYS endoderm-specific cDNA was identified as apolipoprotein C2. In situ hybridization studies confirmed the differential expression of these genes in E7.5 yolk sac tissues. These results indicate that representative cDNA populations can be obtained from small numbers of cells and that PCR methodologies permit the study of gene expression during early mammalian postimplantation development. While all of the mesoderm-enriched genes were ubiquitously expressed in the embryo proper, apolipoprotein C2 expression was confined to the visceral endoderm. These results are consistent with the hypothesis that at E7.5, the yolk sac endoderm provides differentiated liver-like functions, while the newly developing extraembryonic mesoderm is still a largely undifferentiated tissue. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 175
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 7-18 
    ISSN: 1040-452X
    Schlagwort(e): Maturational competence ; Oocyte ; Ovarian follicle ; Gonadotropin ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Gap junctions (GJ) are aggregates of intercellular channels, composed of connexin (Cx) protein, between adjacent cells. The vertebrate ovarian follicle contains homocellular (granulosa cell-granulosa cell) and heterocellular (granulosa cell-oocyte) GJ. However, the function of GJ during final oocyte differentiation (maturation) is controversial. The objectives of this study are to reexamine the number and identity of Cx genes that are expressed in the Xenopus ovary, and to examine the potential role of GJ in oocyte maturation by determining the temporal association between changes in ovarian Cx mRNA content and the process of maturation. We used reverse transcriptase-polymerase chain reaction to amplify ovarian cDNA fragments using degenerate Cx primers. We amplified three Cx-like fragments: one was novel and two corresponded to known Cx of Xenopus ovaries (Cx38 and 43). The novel fragment was used to screen an ovarian cDNA library. One positive clone was identified and its nucleotide sequence determined. Its deduced amino acid sequence showed that it corresponded to a novel Cx, Cx41, belonging to the Group II class of Cx. Xenopus Cx41 showed the highest homology to rat Cx37 (65% identity, 80% similarity). Also, the last 10 C-terminal amino acids of Cx41 were identical to those of rat, mouse, and human Cx37. Cx41 transcripts were detected by riboprobe mapping in ovarian somatic cells, heart, leg muscle, liver and eye, but not in brain or in oocytes of any developmental stage. Full-grown follicles incubated in vitro with human chorionic gonadotropin became committed to mature within 1-4 hr, and physical signs of maturation (germinal vesicle breakdown) were seen at 4-5 hr. Significant reductions in the levels of Cx41 and 43, but not 38 transcripts were seen at 4 hr, after oocytes had committed to mature. Thus, if availability of Cx mRNA determines availability of Cx protein and GJ, our results would suggest that irreversible commitment to maturation occurred prior to major declines in follicular GJ during the periovulatory period. The present study is the first to report the presence of at least two hormone-responsive Cx gene transcripts (Cx41 and 43 in Xenopus) in ovaries of a single animal species. © 1995 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 176
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 35-52 
    ISSN: 1040-452X
    Schlagwort(e): Bovine ; Blastocyst ; Culture ; Epiblast ; Differentiation ; IVMFC ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Two experiments were conducted to compare the utility of in vitro- and in vivo-derived bovine blastocysts for the isolation of pluripotent epiblasts. In experiment 1, the inner cell masses (ICMs) of in vivo-collected blastocysts yielded a higher proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts (P = .0157). In experiment 2, ICMs of in vivo-collected blastocysts that hatched on day 8 yielded a greater proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts that hatched on day 8. The difference was reversed but smaller for blastocysts that hatched on day 9 (Interaction, P = .0125). Epiblasts from blastocysts that hatched on day 8 regardless of their source generated more differentiated cell lines in extended culture than did blastocysts that hatched on day 9. Extended epiblast culture yielded cells identifiable as products of the three embryonic germ layers that included epithelial cells, fibroblasts, neuronal cells, hepatocyte-like cells, and macrophage-like cells. Alkaline phosphatase activity combined with cell morphology identified the bovine epiblast cells and distinguished them from trophectoderm and endoderm that frequently contaminated epiblast cell cultures. In vivo-derived blastocysts, especially from early-hatching blastocysts, were a superior source of pluripotent epiblasts. Epiblast cells in this study all differentiated or senesced indicating that standard conditions for mouse embryonic stem cell culture do not maintain bovine epiblast cells in an undifferentiated state. © 1995 wiley-Liss, Inc.This artilce is a US Government work and, as such, is in the public domain in the United States of America.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 177
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 58-64 
    ISSN: 1040-452X
    Schlagwort(e): Eggs ; Biflagellate spermatozoa ; Electron microscope ; Fish (Zoarcidae) ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Ultrastructure of sperm and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine teleost, was examined by scanning and transmission electron microscopy. The results showed that the sperm do not have an acrosome but have a very long mid-piece (one to two times the sperm head length) containing numerous well-developed elongated mitochondria. The sperm also have two tails (is biflagellate) each consisting of nine peripheral and one central pair (9 ± 2) of microtubules. This long mid-piece and the biflagellate nature of the sperm appear to be associated with the long life-span of the sperm and with sperm dispersal in the ovary to fertilize the eggs internally. The ocean pout eggs are enveloped by a porous chorionic membrane similar to that found in other teleosts but have two micropyles, a condition likely related to a mechanism of egg fertilization which increases the egg fertlity in the presence of low sperm numbers. Following insemination, some biochemically undefined excretions appeared on the surface of fertilized eggs and led to the acquisition of adherent capability of the eggs which formed a tightly associated egg mass in sea water. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 178
    ISSN: 1040-452X
    Schlagwort(e): β-enolase ; Insulin-like growth factor-II ; Myogenesis in culture ; Gene expression regulation ; 4-Thiouridine labeled RNA isolation ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The glycolytic enzyme enolase (EC 4.2.1.11) exists as dimers formed from three structurally related subunits α, β, and γ, encoded by separate genes. The gene encoding the β-subunit is expressed only in striated muscles. We have previously shown that the β-enolase gene belongs to a small subset of muscle-specific genes showing transcriptional activity in cultured myoblasts, prior to withdrawal from the cell cycle. An increase in the level of β-enolase mRNA occurs during terminal differentiation of myoblasts. To investigate the mechanisms underlying this increase, we have simultaneously estimated, under steady state conditions, the rate of synthesis and the stability of β-enolase mRNA in proliferating C2.7 myoblasts as well as in differentiating myotubes. The method used is based on the isolation of newly synthesized RNA from the total RNA pool, following pulse-labeling of intact cells in the presence of 4-thiouridine. The results described here demonstrate a coordinate increase in newly synthesized and total β-enolase mRNA, while the mRNA half-life, about 4 hr, remains unchanged in the course of terminal differentiation. The expression of the gene for insulin-like growth factor-II (IGF-II), a major positive regulator of myogenesis, was analyzed using the same approach.It is concluded that the up-regulation of β-enolase as well as IGF-II gene expression in differentiating muscle cells reflects an increased rate of entry of newly synthesized mRNAs into the general pool of transcripts without changes in their respective half-lives. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 179
    ISSN: 1040-452X
    Schlagwort(e): Fish ; Estradiol-17β ; Chorion ; Zona radiata ; Vitellogenin ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52, 48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol-17β. As males of both species synthesize these proteins after treatment with estradiol-17β, the origin is not restricted to the ovaries. Vitellogenin of both Eurpoean sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 180
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 348-354 
    ISSN: 1040-452X
    Schlagwort(e): Spermatogenesis ; Germ cells ; Immunoselection ; Monoclonal antibody ; RNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A method was devised for the isolation of round spermatids from the rat using a positive immunoselection technique (panning). A testis suspension was prepared from adult rats by enzymatic digestion of seminiferous tubules with collagenase. Specific mouse monoclonal antibody (97.25) was indirectly attached to Petri dishes and used in a panning protocol to purify spermatids from the testis cell suspension. The quantity and purity of cells isolated were determined by cell counts and histochemical (periodic acid-Schiff stain) or by immunostaining with acrosome-specific antibodies. A mean yield of 1.38 ± 0.15 × 107 cells per dish was obtained with a purity of more than 90%. The viability of the cells was confirmed by epifluorescent microscopy with propidium iodide/carboxyfluorescein acetate probes. Northern blot analysis of RNA extracted directly from the dish indicated good integrity of a spermatid-specific transcript of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 181
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 355-359 
    ISSN: 1040-452X
    Schlagwort(e): Human zona pellucida ; Fertilization ; Human oocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The macromolecular composition of zona pellucida (ZP) isolated from human oocytes and embryos was characterized by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1-D- and 2-D-SDS-PAGE) under reducing conditions after silver staining. ZP specimens obtained after in vitro fertilization were removed from pools of heavily fragmented embryos and inseminated oocytes that failed to fertilize. For unfertilized oocytes, two major bands with an apparent molecular weight of, respectively, 96 and 76-54 kDa were observed after 1-D-SDS-PAGE and silver staining. When ZP were isolated from fragmented embryos, the electrophoretic pattern showed a marked attenuation of the 96-kDa band. Silver-stained 2-D-SDS-PAGE analysis of ZP components from unfertilized oocytes revealed the presence of four protein trains: ZP1 (Mr = 92-80 kDa, pl = 4.9-5.9), ZP2 (Mr = 66-58 kDa, pl = 5.0-6.0), ZP3H (high) (Mr = 72-58 kDa, pl = 3.5-5.1), and ZP3L (low) (Mr = 62-54 kDa, pl = 3.5-5.1). The human ZP3 family (ZP3H and ZP3L) showed marked heterogeneity. Fertilization-associated changes were apparent in the electrophoretic pattern. ZP1 (Mr = 92-86 kDa, pl = 5.0-5.8) displayed a dramatic decrease in intensity, and a new component had migrated to a position similar to that of ZP2. This modification may have been responsible for one aspect of the zona reaction, and could have contributed to a zona block to polysperma. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 182
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 368-373 
    ISSN: 1040-452X
    Schlagwort(e): Monodelphis domestica ; Oocytes Marsupial ; Cortical granules ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The purpose of this study was to characterize the structure of the vestments surrounding unfertilized and cortical granule-reacted oocytes from a marsupial, the grey short-tailed opossum Monodelphis domestica and to determine if a cortical granule envelope (CGE) forms in the perivitelline space (PVS) following the cortical reaction. Unfertilized oocytes collected from mature ovarian follicles and oviducal oocytes that had undergone a cortical reaction were fixed for electron microscopy in the presence of ruthenium red which stabilizes extracellular matrices (ECM) and facilitates demonstration of a CGE. Unfertilized oocytes were surrounded by a zona pellucida and had a PVS which contained a thick ECM comprised of granules and filaments. This matrix appeared to attach to the oolemma and was structurally similar to matrices reported previously in the PVS of unfertilized oocytes from eutherian mammals and two other marsupials, the Virginia opossum and the fat-tailed dunnart. The cortex of unfertilized oocytes contained cortical granules which were absent in oocytes recovered from the oviducts of mated females. Oviducal oocytes which lacked cortical granules exhibited a new coat within the PVS between the zona pellucida and the tips of the oocyte microvilli. This coat, the CGE, appeared structurally similar to CGEs described previously around fertilized eutherian oocytes. The CGE of the grey short-tailed opossum is approximately 1 μm thick and is made up of numerous small dense granules. The coats of the opossum oocyte are compared to those present around other marsupial and eutherian oocytes. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 183
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 398-398 
    ISSN: 1040-452X
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 184
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 416-424 
    ISSN: 1040-452X
    Schlagwort(e): Cytoplasmic activities ; Bisection ; Cleavage cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: One-cell parthenogenetic haploid embryos and blastomeres of the 2- and 4-cell diploid mouse embryos were observed in vitro for the occurrence of two cytoplasmic activities: the cortical activity and the chromatin condensation activity. For this purpose anucleated halves (AHs) and nucleated halves (NHs) were produced by bisection of one-cell embryos and of blastomeres.The cortical activity (manifested by surface deformations) was observed only during the first cleavage cycle. In AHs the surface activity began at the same time as in NHs and disappeared before the time of the cleavage division of nucleated halves. Anucleate fragments of blastomeres from 2- and 4-cell embryos did not exhibit any cortical activity.In the absence of the native nucleus the chromatin condensation activity (assayed by premature chromatin condensation of interphase thymocyte nuclei introduced into cytoplasts by cell fusion) could also have been detected only in the first cleavage cycle. In AHs this activity appeared at the time when NHs started to cleave and disappeared after the NHs finished the first cleavage division. AHs obtained from 2-cell and 4-cell stage blastomeres did not reveal condensation activity. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 185
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 407-415 
    ISSN: 1040-452X
    Schlagwort(e): Serine/threonine kinase ; Spermatogenesis ; Oogenesis ; Meiosis ; In situ hybridization ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: To identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding region shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythroleukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the PlK1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 186
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 41 (1995), S. 435-448 
    ISSN: 1040-452X
    Schlagwort(e): Peri-implantation embryogenesis ; Trophoblast cells ; Recombinant proteins ; Integrins ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits α3, α5, αllb, αv, β1 and β3, but not α4, all bound to trophoblast cells. Antibodies raised against either the β1 or β3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, α3β1, α5β1, αllbβ3, and αvβ3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 187
    ISSN: 1040-452X
    Schlagwort(e): CDC25 ; Dbl ; Ras-related proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Members of the Ras superfamily of proteins function as regulated GDP/GTP switches that cycle between active GTP-complexed and inactive GDP-complexed states. Guanine nucleotide exchange factors (GEFs) stimulate formation of the GTP-bound state, whereas GTPase activating proteins (GAPs) catalyze the formation of the GDP-bound state. We describe three studies that evaluate the mechanism of action of GEFs for Ras (SOS1 and RasGRF/CDC25) or Ras-related Rho (Dbl and Vav) proteins.Growth factor-mediated activation of Ras is believed to be mediated by activation of Ras GEFs (CDC25/GRF and SOS1/2). Although the mechanisms of Ras GEF regulation are unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. Our observation that the addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their transforming and transactivation activities (10-50 fold and 5-10 fold, respectively) suggests that membrane translocation alone is sufficient to potentiate GEF activation of Ras.We have determined that two Ras-related proteins, designated R-Ras and R-Ras2/TC21, can trigger the malignant transformation of NIH 3T3 cells via activation of the Ras signal transduction pathway. Furthermore, like Ras and R-Ras, we observed that TC21 GTPase activity was stimulated by Ras GAPs. However, we observed that both SOS1 and CDC25 were activators of normal TC21, but not R-Ras, transforming activities. Therefore, TC21, but not R-Ras, may be activated by the same extracellular signaling events that activate Ras proteins.Dbl family proteins are believed to function as GEFs and activators of the Ras-related Rho family of proteins. However, one Dbl family oncogene, designated Vav, has been reported to be a GEF for Ras proteins. Therefore we were interested in determining whether Dbl family oncogenes cause transformation by triggering the constitutive activation of Rho or Ras proteins. Our results suggest that Dbl oncogenes cause transformation via a Ras-independent activation of MAP kinases and Rho family proteins. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 188
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 500-506 
    ISSN: 1040-452X
    Schlagwort(e): Ras ; Raf ; Signal transduction ; Kinases ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Ras proteins are members of a superfamily of small GTPases that are involved in many aspects of cell growth control. The ras p21 protooncogene products, H-ras, K-ras, and N-ras, transmit signals from growth factor receptors to a cascade of protein kinases that begins with the Raf protooncogene product, and leads to alterations in transcription factors and cell cycle proteins in the nucleus. This cascade is controlled at several points: Ras p21 proteins are regulated by GAPs and by exchange factors, whose activities are altered by growth factor receptor activation (Boguski and McCormick, 1993: Nature 366:643-654). Transmission of signals from Ras to Raf is regulated by the Ras-related protein Rap1 (a protein capable of reverting cell transformation) and by cAMP. Other aspects of Ras p21 regulation will be discussed, including the existence of RasGDl proteins that inhibit GDP dissociation from Ras, and may thus regulate the level of active Ras in the cell.The role of Ras in activation of Raf kinase appears to be limited to the recruitment of Raf to the plasma membrane, at which time Raf becomes stably modified to render it active (Leevers et al., 1994: Nature 369:411-414; Stokoe et al., 1994: Science 264:1463-1467). The nature of these modifications is unclear. Raf in the plasma membrane becomes associated with insoluble structural cell components that may be part of the activation. Furthermore, Raf is associated with proteins of the 14-3-3 family that appear necessary for kinase activation. The 14-3-3 proteins interact with all three conserved regions of Raf, including the kinase domain.In addition to Raf, Ras proteins interact with two known classes of proteins in a manner consistent with effector functions: these are the GAPs and regulators of the Ras-related protein Ral referred to as RalGDS. These biochemical data suggest that other functional pathways are regulated by Ras, including, perhaps, pathways involved in regulating cell shape and motility.The protein R-Ras p21 is about 50% identical to the Ras p21 protooncogene product. This protein is incapable of transforming cells, even though it interacts with Raf and other putative Ras effectors (Fernandez-Sarabia and Bischoff, 1993: Nature 366:274-275). On the other hand, it has recently been shown that R-Ras binds to the protooncogene product Bcl-2, a protein that transforms B cells by blocking apoptosis. R-Ras is regulated by the same GAP molecules as H-Ras and the other Ras protooncogene products, and may therefore be activated in a manner co-ordinate with these growth-promoting proteins. The possible connection between R-Ras and apoptosis will be discussed. © 1995 wiley-Liss, Inc.
    Materialart: Digitale Medien
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  • 189
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 347-358 
    ISSN: 1040-452X
    Schlagwort(e): Acrosome reaction ; Receptor oligomerization ; Exocytosis ; mAb ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: In the mouse, a 95 kD sperm protein has been identified as a putative receptor for the zona pellucida glycoprotein ZP3. The 95 kD sperm protein is a tyrosine kinase substrate, with phosphorylation on tyrosine stimulated upon zona pellucida binding. The latter finding is observed not only in live cells but also in isolated sperm membranes and in an electroeluted 95 kD protein. Stimulation of 95 kD protein tyrosine phosphorylation by zona pellucida is completely abolished by tyrosine kinase inhibitors, which effectively inhibit the sperm acrosome reaction. Since receptor oligomerization by ZP3 is essential for acrosome reaction triggering, we hypothesized that application of an external crosslinking agent will lead to the acrosome reaction, even in the absence of natural ligand ZP3. Here, we report the generation of a mouse monoclonal antibody (mAb) raised against the 95 kD protein. This antibody, termed LL95, mimics the bioactivities of ZP3 in inhibiting sperm-zona binding and inducing the acrosome reaction. The latter depends on receptor oligomerization. Immunolocalization revealed that the LL95 antigen is restricted to the head surface in the acrosomal region of live sperm. Thus, LL95 fulfills several criteria predicted for an antibody that recognizes a sperm receptor for the zona pellucida. Recently, it was reported that the amino acid sequence of the 95 kD protein we described corresponds to a mouse hepatoma hexokinase (Kalab et al., 1994: J Biol Chem 269:3810-3817). Although both hexokinase and LL95 antigen migrate at 95 kD in nonreducing gels, we show here that LL95 does not recognize hexokinase. Identification of different proteins is clear where hexokinase is a 116 kD protein and LL95 recognizes sperm proteins of 110 and 130 kD. Moreover, mAb anti-phosphotyrosine immunoprecipitates LL95 antigen under conditions where hexokinase is absent. Use of anti-hexokinase antibodies in gamete interaction assays failed to demonstrate any effect on either spermzona binding or acrosome reaction triggering. Finally, anti-hexokinase antibodies bind to a sperm tail antigen, thus direct involvement of hexokinase in gamete interaction seems improbable. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 190
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 397-406 
    ISSN: 1040-452X
    Schlagwort(e): Reverse transcriptase polymerase chain reaction (RT-PCR) ; Tissue-specific gene expression ; Oocyte ; Granulosa cell ; Pituitary ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: In mice, expression of the transcription factor Oct-3 and the proto-oncogene c-mos is limited to germ cells, suggesting a specific role for these factors in gamete physiology and early embryonic development. We have studied the expression pattern of Oct-3 and c-mos in various reproductive as well as control tissues in the cynomolgus monkey, using reverse transcriptase polymerase chain reaction (RT-PCR) and Northern analysis. Analogously with the data from the mouse model, strong expression of Oct-3 and c-mos could be detected in monkey ovary and oocytes. Unexpectedly, strong expression of c-mos was demonstrable in the pituitary gland and the amount of mRNA expression in the pituitary was roughly equal to that found in the ovary. Of the tissues examined, the testicular expression of c-mos was the most intense. Weak signal for c-mos mRNA was also seen in hypothalamus and brain; however, all other tissue types examined were negative for c-mos expression. In addition to the oocytes, expression of Oct-3 mRNA was detected in the ovarian granulosa cells, fallopian tube, myometrium, cervix, breast, liver, adrenal gland, pituitary, hypothalamus, brain cortex, prostate, and in testis. Thus, in the cynomolgus monkey, Oct-3 is predominantly, but not specifically, expressed in reproductive tissues. In the female monkey reproductive organs, the expression of c-mos seems to be germ cell specific. Therefore, further characterization of c-mos and Oct-3 functions in primate reproductive physiology, especially in gametogenesis and early embryonic development, is highly warranted. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 191
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 432-436 
    ISSN: 1040-452X
    Schlagwort(e): Maturation media ; Fertilization ; In vitro culture ; Bovine embryos ; Oxidative stress ; Cysteine ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The present study was carried out to evaluate if the addition of cysteamine to the culture medium during in vitro maturation of bovine oocytes increased the glutathione (GSH) levels in the mature oocytes, and if these changes may promote an improvement on in vitro development to the blastocyst stage. Follicular oocytes from slaughterhouse ovaries were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 (control), 25, 50, or 100 μM of cysteamine for 24 hr. After in vitro maturation the oocytes were fertilized and cultured for 8 days. The percentage of embryos that developed to the blastocyst stage was significantly higher (P 〈 0.01) for oocytes matured in medium containing 100 μM of cysteamine than for those matured in control medium. Moreover, the intracellular GSH levels were increased (P 〈 0.05) in oocytes matured with 100 μM of cysteamine with respect to control. No differences were observed in maturation and cleavage rates, and in the mean cell numbers per blastocyst among treatments (P 〉 0.05). These results indicate that the addition of thiol compounds such as cysteamine to maturation medium increases the efficiency of in vitro blastocyst production from immature bovine oocytes. The higher levels of GSH in oocytes matured in the presence of cysteamine suggest that the beneficial effects of cysteamine on in vitro maturation and subsequent development after in vitro fertilization are mediated by GSH. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 192
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 443-446 
    ISSN: 1040-452X
    Schlagwort(e): Endocytosis ; Yolk ; Lipoprotein ; Chicken ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Apolipoprotein D (apo D) is an unusual apolipoprotein with respect to structure and sites of synthesis. It has been identified in the circulatory system of certain mammals, but its physiological role remains poorly understood. In this report, it is shown that apo D is not exclusively a mammalian apolipoprotein, and evidence is presented which suggests a novel function for this protein during oogenesis in the chicken. The avian apo D which we identify has the same molecular mass (29 kDa) as the human protein and also associates preferentially with the plasma lipoprotein fraction. In addition to the 29 kDa avian apo D species, an immunoreactive 24 kDa protein is observed in chicken serum. The chicken apo D (along with the 24 kDa species) is also demonstrated to be present in the yolk of the rapidly growing chicken oocyte, a cell with high endocytic activity. Clathrin-coated vesicles from chicken oocytes, which we have previously shown to contain specific lipoproteins along with their oocytic receptors (Bujo et al., 1994: EMBO J 13:5165-5175), also contain chicken apo D. Thus, apo D represents a novel candidate for plasma-to-oocyte transport of lipids and/or their mobilization during embryogenesis in oviparous species. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 193
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 477-485 
    ISSN: 1040-452X
    Schlagwort(e): MAP kinase activation pathways ; Inter-pathway signal coordination ; MEK specificity ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: In the budding yeast, Saccharomyces cerevisiae, four separate but structurally related mitogen-activated protein kinase (MAPK) activation pathways are known. The best understood of these regulates mating. Pheromone binding to receptor informs cells of the proximity of a mating partner and induces differentiation to a mating competent state. The MARK activation cascade mediating this signal is made up of Ste 11 (a MEK kinase [MEKK]), Ste7 (a MAPK/ERK kinase [MEK]), and the redundant MAPK-related Fus3 and Kss1 enzymes. Another MAPK activation pathway is important for cell integrity and regulates cell wall construction. This cascade consists of Bck1 (a MEKK), the redundant Mkk1 and Mkk2 enzymes (MEKs), and Mpk1 (a MAPK). We exploited these two pathways to learn about the coordination and signal transmission fidelity of MAPK activation cascades.Two lines of evidence suggest that the activities of the mating and cell integrity pathways are coordinated during mating differentiation. First, cells deficient in Mpk1 are susceptible to lysis when they make a mating projection in response to pheromone. Second, Mpk1 activation during pheromone induction coincides with projection formation. The mechanism underlying this coordination is still unknown to us. Our working model is that projection formation generates a mobile second messenger for activation of the cell integrity pathway.Analysis of a STE7 mutation gave us some unanticipated but important insights into parameters important for fidelity of signal transmission. The Ste7 variant has a serine to proline substitution at position 368. Ste7-P368 has higher basal activity than the wild-type enzyme but still requires Ste 11 for its function. Additionally, the proline substitution enables the variant to transmit the signal from mammalian Raf expressed in yeast. This novel activity suggests that Ste7-P368 is inherently more permissive than Ste7 in its interactions with MEKKs. Yet, Ste7-P368 cross function in the cell integrity pathway occurs only when it is highly overproduced or when Ste5 is missing. This behavior suggests that Ste5, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7 specificity and fidelity of signal transmission. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 194
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 515-522 
    ISSN: 1040-452X
    Schlagwort(e): Receptor tyrosine kinase ; Ras ; Raf ; MEK ; Drosophila ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Cell fate choice at the anterior and posterior embryonic termini of the Drosophila embryo requires the activation of a signal transduction pathway regulated by the receptor tyrosine kinase Torso. When Torso, which is uniformly distributed in the egg cell membrane, becomes activated locally at the termini, it triggers a phosphorylation cascade that culminates with localized expression of the transcription factors, tailless and huckebein. Expression of tailless and huckebein in turn determines terminal cell fates. Several genes have been characterized which encode proteins that are involved in Torso signaling: the adaptor protein Drk, the GTP-binding protein Ras1, the guanine nucleotide exchange factor Son of sevenless, and the kinases D-Raf and D-Mek. Genetic and molecular evidence supports a model in which these proteins lie in the same biochemical pathway. When activated by its ligand the membrane-bound receptor tyrosine kinase Torso initiates a signal transduction pathway mediated by Drk, Sos, and Ras1, which in turn activates a phosphorylation cascade mediated by the kinases D-Raf and D-Mek, which ultimately control the localized expression of the transcription factors tailless and huckebein. Recently, we found that D-Raf can be partially activated by Torso in the absence of Ras1, a finding supported by the phenotype of embryos lacking either Drk or Sos activity, as well as by the phenotype of a D-raf mutation that abolishes binding of Ras1 to D-Raf. These findings indicate that full D-Raf activation requires input not only from Ras1 but also from an as yet uncharacterized Ras1-independent pathway. In addition to these molecules we have characterized the putative protein tyrosine phosphatase Corkscrew as a positive transducer downstream of Torso. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 195
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 447-456 
    ISSN: 1040-452X
    Schlagwort(e): Oocyte development ; Oxygen concentration ; Developmental competence ; Mouse ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The purpose of this study was to assess the effects of oxygen concentration on the developmental competence of mouse oocytes grown in vitro because oxygen has been shown to affect the nuclear maturation of oocytes (Haidri et al., 1971: J Reprod Fertil 26:409-411) and preimplantation embryo development (Whitten, 1971: Adv Biosci 6:129-139). Oocyte - granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in serum-free medium equilibrated with 5, 10, 15, or 20% O2. Five percent CO2 was used for all groups. Oocytes from all groups were then matured and fertilized, and preimplantation embryos cultured using 5% O2. With increased oxygen tension, there were dramatic decreases in the percentage of (1) oocytes that survived in vitro culture, (2) surviving oocytes that could resume meiosis and undergo germinal vesicle breakdown (GVB), and then cleave to the two-cell stage, and (3) two-cell-stage embryos that completed the blastocyst transition. For example, 42% of the oocytes grown in a 5% O2 atmosphere cleaved to the two-cell stage compared with only 3% when the oocytes grew in an atmosphere of 20% O2. These dramatic effects of elevated oxygen were mitigated by either increased numbers of the oocyte-granulosa cell complexes in the culture or increased concentration of oxygen late in the culture period, after the oocytes became surrounded by greater numbers of granulosa cells. When the culture period was extended for 4 days beyond the standard 10 day cultures used in the experiments described above, there was an increased occurrence of precocious GVB or failure of the somatic cells to maintain meiotic arrest. This was prevented by increased oxygen concentration. Nevertheless, extending the time of culture even in the presence of elevated oxygen failed to increase oocyte growth to the equivalent of in vivo-grown oocytes or to improve the developmental competence of the in vitro-grown oocytes. It was concluded that concentrations of O2 above 5% have a deleterious effect on oocyte development during the early stages of culture. However, increasing the concentration on O2 during the later stages, in conjunction with other treatments to promote normal granulosa cell development and function and their interaction with the oocyte, may be critical to promote normal oocyte development in vitro. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 196
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 486-492 
    ISSN: 1040-452X
    Schlagwort(e): MAPK ; MAPKK ; MAPKKK ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: MAP kinase (MAPK) and its activator, MAP kinase kinase (MAPKK), are commonly activated by a variety of extracellular stimuli in mammalian cells and in the process of Xenopus occyte maturation. In order to investigate the function of the MAPK cascade in oocyte maturation, we produced an anti-Xenopus MAPKK which specifically reacts with MAPKK in vitro. When this antibody was microinjected into immature oocytes, MAPK activation induced by progesterone was prevented. Surprisingly, H1 kinase activation and germinal vesicle breakdown were also inhibited in the oocytes injected with this antibody. These results suggest that the MAPK cascade plays an important role in the maturation promoting factor (MPF) activation during the oocyte maturation process. When this antibody together with Mos was micoinjected into Xenopus two-cell embryos, the Mos-induced metaphase arrest (CSF arrest) was prevented. Thus, the MAPK cascade may mediate CSF arrest. During Xenopus early embryogenesis, a low but significant level of MAPK remains active. Injection of mRNA encoding a constitutively active MAPKK resulted in mesoderm induction in animal cap explants. In addition, fibroblast growth-factor (FGF)-induced mesoderm induction was inhibited by expressing CL100 (a MAP kinase phosphatase) in animal cap explants. Thus the MAPK cascade may be involved in the mesoderm induction of Xenopus embryos. The activation pathways and roles of the MAPKK/MAPK cascade in various signaling processes will be discussed. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 197
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 507-514 
    ISSN: 1040-452X
    Schlagwort(e): Signal transduction ; Serine/threonine kinases ; Cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Raf-1 is a key protein involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Biochemical and genetic studies have demonstrated that Raf-1 functions downstream of activated tyrosine kinases and Ras and upstream of mitogen-activated protein kinase (MAPK) and MAPK kinase (MKK or MEK) in many signaling pathways. A major objective of our laboratory has been to determine how Raf-1 becomes activated in response to signaling events. Using mammalian, baculovirus, and Xenopus systems, we have examined the roles that phosphorylation and protein-protein interactions play in regulating the biological and biochemical activity of Raf-1.Our studies have provided evidence that the activity of Raf-1 can be modulated by both Ras-dependent and Ras-independent pathways. Recently, we reported that Arg89 of Raf-1 is a residue required for the association of Raf-1 and Ras. Mutation of this residue disrupted interaction with Ras and prevented Ras-mediated, but not protein kinase C-or tyrosine kinase-mediated, enzymatic activation of Raf-1 in the baculovirus expression system. Further analysis of this mutant demonstrated that kinase-defective Raf-1 proteins interfere with the propagation of proliferative and developmental signals by binding to Ras and blocking Ras function.Our findings have also shown that phosphorylation events play a role in regulating Raf-1. We have identified sites of in vivo phosphorylation that positively and negatively alter the biological and enzymatic activity of Raf-1. In addition, we have found that some of these phosphorylation sites are involved in mediating the interaction of Raf-1 with potential activators (Fyn and Src) and with other cellular proteins (14-3-3). Results from our work suggest that Raf-1 is regulated at multiple levels by several distinct mechanisms. © 1995 wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 198
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 460-466 
    ISSN: 1040-452X
    Schlagwort(e): Spermatogenesis ; Sperm-zona pellucida binding ; Transmembrane animal lectin proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Galactosyl receptor, a cell surface Ca2+-dependent lectin with binding affinity for galactose, was evaluated by immunoblotting, immunoprecipitation, Northern blotting, and immunocytochemistry in human liver, testis, and sperm. Polyclonal antisera raised against the minor asialoglycoprotein receptor variant of rat hepatocytes (designated rat hepatic lectin-2/3, RHL-2/3), and its human liver-equivalent (designated H2), recognize native galactosyl receptor in the testis and sperm in immunoblotting, immunoprecipitation, and immunocytochemical experiments. An equivalent to the major hepatocyte asialoglycoprotein receptor variant (rat RHL-1 and human H1) was not detected. Human testis and sperm galactosyl receptor was resolved, after immunoprecipitation and immunoblotting, as a single protein component of molecular mass 50 kD. The single protein component in human testis and sperm contrasted with the doublet nature of rat testis and sperm galactosyl receptor, consisting of two components of molecular masses of 54 and 49 kD. Northern blotting experiments using radiolabeled H1 and H2 cDNA probes confirmed the presence of H2 mRNA and the lack of H1 mRNA in the human testis. Immunocytochemical studies detected specific antigenic sites on the entire surfaces of spermatogenic cells. However, immunoreactivity in epididymal and ejaculated sperm was confined to head surfaces overlying the acrosome. Results from these studies, and from previous studies in the rat, suggest that the testis/sperm galactosyl receptor is a C-type Ca2+-dependent lectin with possible roles in cell-cell interaction during spermatogenesis and sperm-zona pellucida binding at fertilization. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 199
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 490-502 
    ISSN: 1040-452X
    Schlagwort(e): Heparin ; Image analysis ; Intracellular buffering ; Ammonium chloride ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The effect of heparin-induced capacitation on the intracellular pH (pHi) of individual bovine sperm was determined with image analysis. Sperm were loaded with the acetoxymethyl ester of the pH sensitive fluorescent indicator, 2′,7′-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF). The pHi of 5303 sperm was evaluated from a total of five bulls at .5, 2, 3, 4, and 5 h of incubation. The pHi did not differ between the sperm head and mid-piece (P 〉 0.05). An increase in sperm head pHi was seen in heparin-treated sperm at 3, 4, and 5 h of incubation relative to sperm incubated without heparin (control, P 〈 0.05). At 5 h of incubation, the pHi in heparin-treated sperm was 6.92 ± 0.07, while control-treated sperm pHi was 6.70 ± 0.03. Initially a normal frequency distribution was seen for sperm pHi in both heparin- and control-treated sperm. As the incubation progressed, the frequency distribution began to skew towards higher pHi in both samples but was more dispersed for the heparin-treated sperm. Following an NH4Cl-induced alkaline load, the pHi of both control- and heparin-treated sperm recovered toward the resting pHi with a half-time of recovery of 1.5-1.7 min. The recovery of sperm pHi was not due to leakage of NH4+ into sperm because recovery also occurred with trimethylamine. The instantaneous velocity of the pHi recovery (vi) was dependent on pHi and decreased as pHi decreased. Capacitation by heparin was associated with an 81% decrease in vi at a pHi of 7.00, but there was no effect of capacitation on the proton buffering power of the sperm, which was 87 ± 8 mM/pH unit. Results demonstrate that both the regulation of pHi and resting pHi were altered during capacitation of bovine sperm by heparin. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 200
    ISSN: 1040-452X
    Schlagwort(e): Hamster ; Oocyte ; Ovulation ; Meiosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: This study was undertaken (1) to devise a method of inducing multiple follicular development and subsequent ovulation in the Djungarian or Siberian hamster (Phodopus sungorus) and (2) to assess the quality of ovulated oocytes collected from PMSG/hCG treated animals in comparison to naturally ovulating animals. Hamsters (4-5 weeks; n = 70) received 5 IU PMSG followed 50-52 hr later by 10 IU hCG. Ovulated oocytes were collected 14-20 hr after hCG injection. Ovulated oocytes were flushed from oviducts of cycling animals (7-12 weeks; n = 30) exhibiting two consecutive estrous cycles. Oocytes were fixed and subjected to triple fluorescence immunostaining using anti-tubulin antibodies, fluorescein phalloidin, and Hoechst 33258. The mean number of ovulated oocytes collected from cycling animals was 4.8 ± 0.4 (range 1-7). Ovulation occurred in 73% of the PMSG/hCG-stimulated animals. The mean number of oocytes ovulated from stimulated animals was 9.2 ± 0.8 (range 0-22). The ovaries of animals that did not ovulate or that ovulated few oocytes did respond to PMSG, as indicated by the presence of multiple follicular development and pre-ovulatory stigmata. There was no evidence of a polar body in ovulated oocytes collected from PMSG/hCG-treated or cycling animals, indicating that oocytes were arrested in meiosis I. In the majority (80%) of ovulated oocytes from PMSG/hCG-treated and cycling animals, cortically placed chromosomes were aligned on a metaphase plate equidistant from a bipolar spindle. Sparse f-actin staining was observed in the region of the ooplasm surrounding the chromosomes. As the interval between hCG injection and the time of collection increased, chromosomes lost their proper alignment and migrated away from the cortex of the oocyte concomitant with a disruption of spindle integrity. This collapse of proper chromosome alignment and disruption of spindle architecture also characterized aging oocytes collected from cycling animals. These data show that in the Djungarian or Siberian hamster (Phodopus sungorus), (1) there is individual animal variation in responsiveness to hCG following PMSG treatment, (2) there are no cytological differences in the quality of oocytes collected from hormonally treated animals when compared to cycling animals, and (3) oocytes are ovulated in meiosis I. © 1995 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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