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  • Articles  (13)
  • Atomic, Molecular and Optical Physics
  • Immunocytochemistry
  • Wiley-Blackwell  (13)
  • Biology  (13)
  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 32 (1992), S. 369-377 
    ISSN: 1040-452X
    Keywords: Spermiogenesis ; Immunocytochemistry ; Confocal microscopy ; Nuclear proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Human autoantibodies were used to localize centromere proteins by immunoelectron microscopy, immunofluorescence, and confocal microscopy in isolated cells and in cryosections of rabbit testis. A computer-assisted three-dimensional reconstruction of the positions and sizes of fluorescent spots allowed us to follow their movements during the different phases of spermiogenesis. In very young spermatids, the centromeres were distributed within a space separated from both the external nuclear limits and the nuclear core. They moved towards the nuclear limits and the nuclear core. They moved towards the nuclear center in cap phase spermatids, where they clustered into a few large centromeric masses. In preelcngating spermatids, the immunolabeled proteins were dispersed within an equatorial area, where they formed one large mass. In late spermatids, the mass moved towards the posterior part of the nucleus, and, in the spermatozoon, the two basal knobs located at the base of the nuclei were the only strongly immunolabeled structures, while no labeling of the main part of the nucleus was observed. Since the number of centromeres remained close to the number of chromosomes until the cap phase of spermatid differentiation, we hypothesize that the labeling of young spermatids corresponds to centromeric proteins associated with their specific DNA counterparts, while the centromere proteins, possibly detached from their DNA loci, were released from nuclei of old spermatids in the same way as are histones and transition proteins.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 35 (1993), S. 261-271 
    ISSN: 1040-452X
    Keywords: Immunocytochemistry ; Ultrastructure ; Perichromatin granules ; Interchromatin granules ; Mouse spermatids ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have studied the ultrastructural distribution of heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoproteins (snRNPs), and ribosomal proteins during mouse spermatogenesis and spermiogenesis by means of specific antibodies and immunocytochemistry.All the above components were detectable from primary spermatocytes until the spermatid elongation phase, when the RNA synthetic activity is known to cease. Ribosomal protein (P1/P2 and L7) labeling disappeared as early as during the acrosome phase, and nucleoli were no longer labeled even during the cap phase. The nucleoplasmic structures labeled with the different anti-nucleoplasmic RNP immunoprobes corresponded, until the acrosome phase, to those previously observed as targets of the same antibodies in the nucleoplasm of somatic cell nuclei. Clusters of interchromatin granules of spermatocyte and early spermatid nuclei exhibit some labeling for hnRNP when compared with nuclei of Sertoli cells or previously analyzed liver or tissue culture cells, where these structural constituents usually remain weakly labeled or unlabeled.In spermatids in step 10, another type of nuclear granule, resembling perichromatin granules, but occurring in aggregates, can be observed. These structural constituents were labeled with antibodies recognizing nucleoplasmic snRNP antigens and therefore suggesting a non-nucleolar origin of these granules.Finally, we have observed nucleoplasmic areas of fibrogranular material, occurring only in primary spermatocytes. These components were labeled with anti-ribosomal protein antibodies but did not contain either hnRNPs or snRNPs. © 1993 Wiley-Liss, Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 311-324 
    ISSN: 1040-452X
    Keywords: Rat embryos ; ICM ; ES-like cells ; Uterine cells ; Immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 25 (1990), S. 14-21 
    ISSN: 1040-452X
    Keywords: Sperm-specific ; Ldh-x ; Testis ; Gene expression ; Immunocytochemistry ; In situ mRNA hybridization ; Northern blotting ; Spermatogenic cell fractionation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Lactate dehydrogenase-X (LDH-X), a glycolytic enzyme found only in mammalian testes and spermatozoa, is encoded by a single gene (Ldh-x) in the mouse haploid genome. Several studies have demonstrated that LDH-X is associated with germ cells at specific stages of development. We have examined the expression of the Ldh-x gene during mouse spermatogenesis and testis maturation using in situ mRNA hybridization and immunocytochemistry. The results showed that transcription and translation of the Ldh-x gene are initiated at the pachytene stage of germ cell differentiation. However, although the amount of LDH-X protein increased as the germ cells progressed to maturation, its mRNA level was greatly decreased. These observations were confirmed by Northern analysis of total RNA derived from fractionated spermatogenic cells and developing testes. Furthermore, Northern studies also indicated two sizes of Ldh-x transcripts among different populations of spermatogenic cells in mature mouse testis.
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  • 5
    ISSN: 1040-452X
    Keywords: Postacrosomal region ; Cytoskeleton ; Ultrastructure ; Immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Monoclonal antibody MN13 raised against mouse spermatozoa specifically recognizes the postacrosomal region of the sperm head in several mammalian species. Colloidal gold-immunoelectron microscopy of demembranated mouse spermatozoa indicated that the antigen is associated with the outer layer of the periodic substructure apparently linking the postacrosomal sheath to the overlying plasma membrane. The antigen recognized by MN13 may cotribute to the intimate association of the postacrosomal sheath with the overlying plasma membrane.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 305-310 
    ISSN: 1040-452X
    Keywords: Fibrillarin ; Nucleolin ; Immunocytochemistry ; Cleavage-stage embryo ; Mouse ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The localization of fibrillarin and nucleolin in the nuclei of mouse two-cell, four-cell, and eight-cell embryos has been studied using immunofluorescent staining with specific antibodies. In all of these cleavage stages, both antigens were associated exclusively with the peripheral region of the nucleolus precursor bodies (NPBs). The original speckled fluorescent staining pattern in the early two-cell stage was progressively changed into a continuous fluorescent-positive layer localized in the cortex of the NPBs in the four-cell embryos. The compact central area of NPBs was never stained. Both proteins were colocalized in the same substructures of developing nucleoli. In order to analyze the interaction of chromatin, with NPBs, DNA structures were specifically immunolbelled. At the time of resumption of nucleolar transcription (in the two-cell mouse embryo), DNA was detected at the periphery of, but not penetrating into, NPBs. Our results confirm the view that the cortical region of NPBs could represent a nucleolonemal area involved in the resumption of nucleolar transcription in the early mouse embryo. © 1995 Wiley-Liss, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 27 (1990), S. 181-190 
    ISSN: 1040-452X
    Keywords: Testis ; Spermatogenic cells ; Bindin mRNA ; In situ hybridization ; Immunocytochemistry ; Electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Expression of the bindin gene was examined in testicular cells of the sea urchin Strongylocentrotus purpuratus. In situ hybridization studies, using an 35S-labeled antisense RNA probe transcribed from a bindin cDNA, reveal that bindin mRNAs are localized in spermatogenic cells displaced towards the lumens of maturing testicular acini. Little or no hybridization is observed in spermatogenic cells displaced towards the perivisceral epithelium or in somatic cells of the testis. A similar localization of the bindin protein itself is observed using a rhodamine-conjugated polyclonal antibody against bindin, which shows a punctate immunofluorescence pattern in late spermatogenic cells. Immunogold labeling of ultrathin sections and electron microscopy reveal that this punctate immunofluorescence is an apparent result of localized deposits of bindin in intracellular vesicles. Through the terminal stages of spermatogenesis, these bindin-containing vesicles apparently fuse to form the single acrosomal vesicle of the mature spermatozoon. These results indicate (1) that bindin mRNAs are transcribed relatively late in spermatogenesis, (2) that bindin is translated soon after production of its mRNA, (3) that bindin quickly associates with intracellular vesicles during or soon after its synthesis, and (4) that these vesicles fuse to form the single acrosomal vesicle during the terminal stage of spermatogenesis.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 35 (1993), S. 244-250 
    ISSN: 1040-452X
    Keywords: Spermadhesin ; Seminal vesicle ; Semen ; Immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: PSP-l, a 13 kDa protein purified from boar seminal plasma, was found to have about 50% amino acid sequence homology with a family of zona pellucida-binding proteins known as spermadhesins. These proteins are produced by the accessory gland(s) of the male reproductive tract and coat the spermatozoa during ejaculation. In this study, we have investigated the possible biological functions of PSP-I using a solid-phase protein binding assay and its site of synthesis using both Western blot and immunocytochemical analyses. PSP-I was found to bind a number of proteins including endo-β-galactosidase digested ZP3, soybean trypsin inhibitor, lgA, lgG and α-casein, indicating that it may have multiple functions. The protein or carbohydrate structures were not critical in the binding, since polyvinyl sulfate could effectively inhibit the binding of PSP-l to these proteins. Western blot analysis using specific antiserum to PSP-l showed that the protein was present in the seminal vesicle but not in the testes, epididymis or prostate. The protein was revealed by immunocytochemical analysis in the epithelium of seminal vesicles but not in the testes or the epididymis. It is concluded that PSP-I is synthesized by the epithelium of the seminal vesicles, secreted into the semen during ejaculation, and may be involved in various reproductive functions, such as preventing premature acrosome reaction and immunosuppression. © 1993 Wiley-Liss, Inc.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 33 (1992), S. 252-258 
    ISSN: 1040-452X
    Keywords: Zfy protein ; Immunocytochemistry ; Fetal mouse testis ; Germ cell ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The zinc finger Y (Zfy) gene is located on the Y chromosome of all placental mammals. Although it is phylogenetically conserved and is expressed in mouse fetal testis, it is not the sex determining Y (Tdy) gene. To address the possible function of the Zfy gene in mice, the distribution of Zfy protein in fetal mice was investigated by immunocytochemical staining using several specific antisera against synthetic peptides of the mouse Zfy protein. Analysis of various fetal tissues at different embryonic stages demonstrated a specific staining only in fetal testis. In particular, reactive protein was initially observed in male fetal gonads at day 11.5 postcoitum (p.c.). The immuno-staining intensified in fetal testes at day 12 and 12.5 p.c., decreased drastically in those at day 13 and 14 p.c. and became undetectable in those at day 15 p.c. and beyond. The reactive molecules were distributed mostly within the seminiferous tubules of the embryonic testis. The present observations confirm the previous findings with RT-PCR analysis and indicate that Zfy or Zfy-like protein is expressed in stage-specific manner during early testis differentiation. Its location in the seminiferous tubules suggests a possible role in early germ cell development. © 1992 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 36 (1993), S. 106-112 
    ISSN: 1040-452X
    Keywords: Basic nuclear proteins ; Male gamete ; Spermiogenesis ; Monoclonal antibodies ; Immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Three monoclonal antibodies against human protamines were obtained by immunization with total human basic nuclear proteins or purified protamine HP3. The specificity of antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. They recognized three distinct epitopes: One was specific for the protamine P1 family, another was specific for the protamine P2 family and the third was common to both families. All were specific for the human species. Antibodies were used to detect protamines in germ cells by indirect immunofluorescence and by immunoelectron microscopy. Protamines appeared in spermtid nuclei at steps 4-5 of spermiogenesis, i.e., during the chromatin condensation process, and were not accumulated in the cytoplasm before entering the nucleus. © 1993 Wiley-Liss, Inc.
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