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  • 1
    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.
    Additional Material: 13 Ill.
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  • 2
    ISSN: 0741-0581
    Keywords: BrdU incorporation ; Cultured cells DNA replication ; Electron microscopy ; EM immunocytochemistry ; Immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In the present study, we have optimized an immunocytochemical ultrastructural approach for in situ localization of newly synthesized DNA in unsynchronized as well as in synchronized human HeLa cells and in exponentially growing mouse P815 cells, which had incorporated bromodeoxyuridine (BrdU) during short pulses varying from 1 to 20 minues. The incorporated BrdU was detected in hydrolyzed ultrathin cryosections or Lowicryl sections by means of a monoclonal antibody, revealed by secondary colloidal gold-labeled probes. The results demonstrate our ability to study, with high resolution and reproducibility, DNA replication during consecutive periods of the S-phase, which is monitored by the incorporation of tritiated thymidine. In addition, this approach allows one to perform a concomitant mapping of replicated DNA and various enzymes of the replisome.
    Additional Material: 14 Ill.
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  • 3
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Adrenocortical cell nuclei of the dormouse Muscardinus avellanarius were investigated by electron microscopic immunocytochemistry in hibernating, arousing and euthermic individuals. While the basic structural constituents of the cell nucleus did not significantly modify in the three groups, novel structural components were found in nuclei of hibernating dormice. Lattice-like bodies (LBs), clustered granules (CGs), fibrogranular material (FGM) and granules associated with bundles of nucleoplasmic fibrils (NF) all contained ribonucleoproteins (RNPs), as shown by labeling with anti-snRNP (small nuclear RNP), anti-m3G-capped RNA and anti-hnRNP (heterogeneous nuclear RNP) antibodies. Moreover, the FGM also showed immunoreactivity for the proliferation associated nuclear antigen (PANA) and the non-snRNP splicing factor SC-35. All these nuclear structural components disappeared early during arousal and were not found in euthermic animals. These novel RNP-containing structures, which have not been observed in other tissues investigated so far in the same animal model, could represent storage and/or processing sites for pre-mRNA during the extreme metabolic condition of hibernation, to be quickly released upon arousal. NFs, which had been sometimes found devoid of associated granules in nuclei of brown adipose tissue from hi-bernating dormice, were present in much higher amounts in adrenocortical cell nuclei; they do not contain RNPs and their role remains to be elucidated. The possible roles of these structures are discussed in the frame of current knowledge of morpho-functional relationships in the cell nucleus.
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  • 4
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The fine structure of the nuclear components was studied following mild lysis of mouse or Drosophila tissue culture cells and spreading of nuclear material. Particular attention was paid to nuclear ribonucleoprotein (RNP) constituents, which were analysed by high resolution autoradiography after [3H]uridine pulse labelling of cells. Comparison with the labelling kinetics of various in situ nuclear RNP constituents described previously suggests strong similarities between in situ constituents and structures observed within spread nuclear components. The present observations suggest that the nucleolar dense fibrillar component, shown previously in ultrathin sections of [3H]uridine-labelled intact cells as carrying rapidly labelled pre-rRNA, in fact consists of highly compacted transcribing ribosomal genes. The growing RNP fibrils appearing in transcription complexes of extranucleolar active genes and the in situ observed perichromatin fibrils also show the same labelling properties. This confirms that the two structures indeed represent the same nucleoplasmic constituents. As for the nuclear structures involved in post-transcriptional events, our observations demonstrate the occurrence of a rapidly labelled RNP fibro-granular network. Its granular elements correspond, in size and perichromatin location, to the perichromatin granules seen in in situ preparations and suggest similarities between the two constituents. The results are discussed in the light of other data providing information on the role of various nuclear structural constituents.
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  • 5
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Dense granular bodies (DGB) are particular structural constituents observed in cell nuclei of different tissues—liver, pancreas, brown adipose tissue, adrenal cortex—of hibernating dormice. They appear as strongly electron-dense clusters of closely packed granules, with thin fibrils spreading out at their periphery. DGB always occur in the nucleoplasm, sometimes making contact with other nuclear structural constituents typical of the hibernating state, such as coiled bodies, amorphous bodies and nucleoplasmic fibrils. DGB are present only during deep hibernation and rapidly disappear upon arousal from hibernation. Cytochemical and immunocytochemical analyses showed that DGB contain ribonucleoproteins and several nucleoplasmic RNA processing factors, suggesting that DGB can represent accumulation sites of splicing factors which are provided to splicing sites when normal metabolic activity is rapidly restored during arousal.
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  • 6
    ISSN: 1432-0878
    Keywords: Key words Pancreas ; exocrine ; Hibernation ; Amylase ; Immunocytochemistry ; Muscardinus avellanarius (Rodentia)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Pancreatic acinar cells of euthermic, hibernating and arousing individuals of the hazel dormouse Muscardinus avellanarius (Gliridae) have been observed at the electron-microscopic level and analysed by means of ultrastructural morphometry and immunocytochemistry in order to investigate possible fine structural changes of cellular components during periods of strikingly different degrees of metabolic activity. During hibernation, the cisternae of the rough endoplasmic reticulum (RER) flatten assuming a parallel pattern, the Golgi apparatus is extremely reduced and the mitochondria contain many electron-dense particles. The cell nuclei appear irregularly shaped, with deep indentations containing small zymogen granules. They also contain abundant coiled bodies and unusual constituents, such as amorphous bodies and dense granular bodies. Large numbers of zymogen granules occur in all animals. However, the acinar lumina are open and filled with zymogen only in euthermic animals, whereas, in hibernating and arousing individuals, they appear to be closed. Morphometrical analyses indicate that, in pancreatic acinar cells, nuclei and zymogen granules significantly decrease in size from euthermia to hibernation, probably reflecting a drastic decrease of metabolic activities, mainly protein synthesis and processing. In all the studied animals, immunocytochemistry with specific antibodies has revealed an increasing gradient in α-amylase content along the RER-Golgi-zymogen granule pathway, reflecting the protein concentration along the secretory pathway. Moreover, during deep hibernation, significantly larger amounts of α-amylase accumulate in RER and zymogen granules in comparison to the other seasonal phases analysed. Upon arousal, all cytoplasmic and nuclear constituents restore their euthermic aspect and all morphometrical and immunocytochemical parameters exhibit the euthermic values, thereby indicating a rapid resumption of metabolic activities.
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  • 7
    ISSN: 1059-910X
    Keywords: Ultrastructure ; Immunocytochemistry ; Chromatin structure ; Nuclear proteins ; Testis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: We have followed the fine structural distribution of two nucleosomal core histones, H2B and H3, and of protamines in the course of mouse spermiogenesis by means of specific antibodies and ultrastructural immunocytochemistry.Our results demonstrate that the nuclear labeling density of histone H2B decreases during steps 6-8 and then increases again in step 9-10 spermatids, while the labeling for histone H3 is constant throughout this period. In step 12 spermatids, the anti-H2B antibody labels mainly the central area of the nucleus. The first signs of protamine labeling are present in step 12 spermatids, where the gold grains can be found over the periphery of the nucleus. Later on, protamine labeling constantly increases and, by the end of spermiogenesis, the whole nucleus is labeled.We suggest that the morphological and structural differences between the central area and the periphery of mouse spermatids are, at least partly, due to a difference in the protein moiety associated with DNA. The central area, which is peculiar to the mouse and has been previously considered as a focus of chromatin condensation, represents, however, the last nuclear region containing histones and consequently the last area where the substitution of histones by protamines takes place.
    Additional Material: 8 Ill.
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  • 8
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Adrenocortical cell nuclei of the dormouse Muscardinus avellanarius were investigated by electron microscopic immunocytochemistry in hibernating, arousing and euthermic individuals. While the basic structural constituents of the cell nucleus did not significantly were found in nuclei of hibernating dormice. Lattice-like bodies (LBs), clustered granules (CGs), fibrogranular material (FGM) and granules associated with bundles of nucleoplasmic fibrils (NF) all contained ribonucleoproteins (RNPs), as shown by labeling with anti-snRNP (small nuclear RNP), anti-m3G-capped RNA and anti-hnRNP (heterogeneous nuclear RNP) antibodies. Moreover, the FGM also showed immunoreactivity for the proliferation associated nuclear antigen (PANA) and the non-snRNP splicing factor SC-35. All these nuclear structural components disappeared early during arousal and were not found in euthermic animals. These novel RNP-containing structures, which have not been observed in other tissues investigated so far in the same animal model, could represent storage and/or processing sites for pre-mRNA during the extreme metabolic condition of hibernation, to be quickly released upon arousal. NFs, which had been sometimes found devoid of associated granules in nuclei of brown adipose tissue from hibernating dormice, were present in much higher amouts in adrenocortical cell nuclei; they do not contain RNPs and their role remains to be elucidated. The possible roles of these structures are discussed in the frame of current knowledge of morpho-functional relationships in the cell nucleus.
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  • 9
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The nucleolus is a very dynamic structure able rapidly to adapt its activity to the cellular metabolic state. An interesting physiological model characterized by drastic modifications of cellular metabolism is represented by hibernating animals. In the present study we investigated the hepatocyte nuclei of euthermic and hibernating edible dormice (Glis glis) with the aim of revealing, by means of ultrastructural and immunocytochemical analyses, possible modifications of nucleolar components during hibernation. Our observations demonstrate that, in deep hibernation, nucleoli undergo structural and molecular modifications: (a) they show numerous nucleoplasmic invaginations and clumps of dense fibrillar component extend from the nucleolar surface; (b) they are frequently in contact with coiled bodies and fibro-granular material, two nuclear bodies usually occurring in the nucleoplasm; (c) the dense fibrillar component contains significant amounts of small nuclear ribonucleoproteins, splicing factors usually distributed in the nucleoplasm. Taken together, these results suggest that during hibernation complex relationships are established between the nucleolus and nucleoplasm, probably related to functional activities peculiar to this physiological phase. However, since no evident nucleolar modification was found in early hibernating dormice, it seems likely that the particular structural and molecular arrangement of nucleoli establishes progressively during hibernation, becoming evident only in the deepest phase, and then disappears upon arousal.
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