RESUMO
In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe 'en face' the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.
Assuntos
Corpo Lúteo/citologia , Técnica de Fratura por Congelamento/métodos , Poro Nuclear/ultraestrutura , Parto , Prenhez , Animais , Apoptose , Feminino , Marcação In Situ das Extremidades Cortadas , Masculino , Gravidez , Ratos , Ratos WistarRESUMO
In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe 'en face' the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.
Assuntos
Masculino , Animais , Feminino , Gravidez , Ratos , Corpo Lúteo/citologia , Poro Nuclear/ultraestrutura , Técnica de Fratura por Congelamento/métodos , Marcação In Situ das Extremidades Cortadas , Parto , Prenhez , Ratos WistarRESUMO
In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe en face the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.(AU)
Assuntos
Masculino , Animais , Feminino , Gravidez , Ratos , Corpo Lúteo/citologia , Técnica de Fratura por Congelamento/métodos , Poro Nuclear/ultraestrutura , Marcação In Situ das Extremidades Cortadas , Parto , Prenhez , Ratos WistarRESUMO
Toxoplasma gondii resides in a nonfusogenic parasitophorous vacuole (PV), which provides a safe environment for parasite survival and replication. In this work, we used the freeze-fracture technique to analyze the PV during different times of T. gondii infection in an epithelial cell line. After a short time of interaction with host cell, T. gondii PV membrane already showed a significant quantity of intramembranous particles (IMPs)-293IMPs/microm(2). The IMP density evaluated did not vary until 6h of interaction. As the PV area enlarged with the progression of infection, the density of these particles increased, reaching a stable quantity in the order of 1100particles/microm(2). The IMPs were heterogeneous in size and were found distributed without any special pattern throughout the time of infection studied. The membrane lining the PV presented circular figures, which resembled vesicle fusion areas or attachments of the membranous tubular network, regions free from particles and small depressions, demonstrating to be a dynamic structure. IMPs were found in tubulo-vesicular structures present in the intravacuolar matrix, although rarely observed in elements of the intravacuolar network.
Assuntos
Técnica de Fratura por Congelamento/métodos , Toxoplasma/ultraestrutura , Vacúolos/ultraestrutura , Animais , Linhagem Celular , Células Epiteliais/parasitologia , Interações Hospedeiro-Parasita , Rim/citologia , Rim/parasitologia , Camundongos , Microscopia Eletrônica , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/patogenicidade , Vacúolos/fisiologiaRESUMO
Tritrichomonas foetus, a parasitic protozoon of the urogenital tract in cattle, presents a poorly known cytoskeleton, formed by rootlets and proteinaceous structures, many of which have not yet been characterized. Studies on its skeletal organization sheds light on the evolution of the matrix system, characteristic of higher eukaryotes. The skeletal matrix system of T. foetus in interphasic and dividing cells were studied using whole mount cell procedures observed either in field emission scanning electron microscopy (FESEM) or in transmission electron microscope (TEM) after the cell-sandwich technique, where the plasma membrane was mechanically removed. Three-dimensional-like images of the cell matrix were attained revealing a network of filaments that has not been described previously. Freeze-etching and cytochemistry using acridine orange for TEM, were also used. Membrane-skeleton interactions were examined in the hydrogenosomes, on the nuclear envelope at mitosis and interphase, and in the overall matrix filling of the cytoplasm and nucleoplasm. It was demonstrated that this eukaryote has a complex skeletal matrix other than just the rigid cytoskeletal structures. Our analysis indicated that the nucleus has a defined position, and fibrils perform an anchoring system for the nucleus. The possibility of a mechanism for nuclei fidelity migration during mitosis is discussed.
Assuntos
Citoesqueleto/ultraestrutura , Tritrichomonas foetus/ultraestrutura , Laranja de Acridina , Animais , Bovinos , Técnica de Congelamento e Réplica/métodos , Técnica de Fratura por Congelamento/métodos , Masculino , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Matriz Nuclear/ultraestruturaRESUMO
Magnetotactic multicellular aggregates and many-celled magnetotactic prokaryotes have been described as spherical organisms composed of several Gram-negative bacteria capable to align themselves along magnetic fields and swim as a unit. Here we describe a similar organism collected in a large hypersaline lagoon in Brazil. Ultrathin sections and freeze fracture replicas showed that the cells are arranged side by side and face both the external environment and an internal acellular compartment in the center of the organism. This compartment contains a belt of filaments linking the cells, and numerous membrane vesicles. The shape of the cells approaches a pyramid, with the apex pointing to the internal compartment, and the basis facing the external environment. The contact region of two cells is flat and represents the pyramid faces, while the contacts of three or more cells contain cell projections and represent the edges. Freeze-fracture replicas showed a high concentration of intramembrane particles on the edges and also in the region of the outer membrane that faces the external environment. Dark field optical microscopy showed that the whole organism performs a coordinated movement with either straight or helicoidal trajectories. We conclude that the organisms described in this work are, in fact, highly organized prokaryotic multicellular organisms.
Assuntos
Fenômenos Fisiológicos Bacterianos , Técnica de Fratura por Congelamento/métodos , Bactérias Gram-Negativas/fisiologia , Magnetismo , Bactérias Gram-Negativas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Organelas/ultraestrutura , Água , Difração de Raios XRESUMO
The hydrogenosome is an unusual organelle found in several trichomonad species and other protists living in oxygen poor or anoxic environments. The hydrogenosome behavior in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle, is reported here. The hydrogenosomes were followed by light and transmission electron microscopy during the whole cell cycle. Videomicroscopy, immunofluorescence microscopy, and immunocytochemistry were also used. It is shown that the hydrogenosomes divide at any phase of the cell cycle and that the organellar division is not synchronized. During the interphase the hydrogenosomes are distributed mainly along the axostyle and costa, and at the beginning of mitosis migrate to around the nucleus. Three forms of hydrogenosome division were seen: (1). segmentation, where elongated hydrogenosomes are further separated by external membranous profiles; (2). partition, where rounded hydrogenosomes, in a bulky form, are further separated by a membranous internal septum and, (3). a new dividing form: heart-shaped hydrogenosomes, which gradually present a membrane invagination leading to the organelle division. The hydrogenosomes divide at any phase of the cell cycle. A necklace of intramembranous particles delimiting the outer hydrogenosomal membrane in the region of organelle division was observed by freeze-etching. Similarities between hydrogenosomes and mitochondria behavior during the cell cycle are discussed.
Assuntos
Ciclo Celular/fisiologia , Organelas/fisiologia , Tritrichomonas foetus/citologia , Animais , Imunofluorescência , Técnica de Congelamento e Réplica/métodos , Técnica de Fratura por Congelamento/métodos , Interfase/fisiologia , Malatos/análise , Malatos/imunologia , Metáfase/fisiologia , Microscopia Eletrônica , Microscopia de Fluorescência/métodos , Microscopia de Vídeo/métodos , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Mitose/fisiologia , Organelas/ultraestrutura , Prófase/fisiologia , Telófase/fisiologia , Tritrichomonas foetus/fisiologia , Tritrichomonas foetus/ultraestrutura , Tubulina (Proteína)/análise , Tubulina (Proteína)/imunologiaRESUMO
Hassalstrongylus epsilon is a small nematode, whose adult forms are found among the intestinal microvilli of the water rat Nectomys squamipes, Brants 1827 (Rodentia: Muridae). The external appearance of the cuticle, which presents transversal striations and longitudinal ridges, is described using scanning electron microscopy. Transmission electron microscopy of thin sections and replicas of quick-frozen, freeze-fractured, deep-etched and rotatory shadowed samples showed the presence in the cuticle of struts that arise from the fluid median layer, extending outward to the epicuticle. The cuticle showed the presence of five layers: epicuticle, cortical, fibril-rich, fluid median and fibrous. The cuticle layers were made of an assemblage of fibers that create compartments, which were larger in the fluid region than in the fibril-rich median layer.
Assuntos
Intestino Delgado/parasitologia , Muridae/parasitologia , Trichostrongyloidea/ultraestrutura , Animais , Técnica de Congelamento e Réplica/métodos , Técnica de Fratura por Congelamento/métodos , Intestino Delgado/citologia , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Microvilosidades/parasitologia , Microvilosidades/ultraestrutura , Ratos , Técnica Histológica de Sombreamento/métodosRESUMO
The fine structure of the sheath and the cuticle of microfilariae of the filariid Litomosoides chagasfilhoi is described based on observations made using transmission electron microscopy (TEM) and especially on deep-etched replicas of fully developed intrauterine microfilariae and mature stretched microfilariae released by adult females through cultivation in vitro. TEM showed that the sheath was trilaminated. In contrast, in deep-etching replicas the sheath presented two layers: an inner layer composed of tightly arranged globular material, and an outer layer whose external surface was relatively smooth. Both in thin sections and in classical freeze-fracture and deep-etched replicas, the cuticle presented two distinct regions: an external one, corresponding to the trilaminated epicuticle, and an inner one, corresponding to the inner cuticle. Deep-etching replicas revealed that the epicuticle presented several structures on the annulations of the microfilariae and that the inner region was composed by two parallel rows of globular structures.
Assuntos
Filarioidea/ultraestrutura , Técnica de Congelamento e Réplica/métodos , Técnica de Fratura por Congelamento/métodos , Animais , Feminino , Filarioidea/crescimento & desenvolvimento , Microfilárias/ultraestrutura , Microscopia EletrônicaRESUMO
Samples of albino mice were processed by the cryofracture method for scanning electron microscopy and examined with the field emission scanning electron microscope (FESEM). Freeze-etching direct replicas of mice cerebellar cortex were also studied with the transmission electron microscope (FFTEM), as a complementary technique for obtaining higher resolution, three-dimensional correlative images of cerebellar synaptic contacts. At the granular, Purkinje cells and molecular layers, the cryofracture method for FESEM selectively removed the neuroglial cell investment, facilitating the visualization of the outer and inner surfaces of cerebellar synaptic contacts. In addition, FFTEM showed the real extension of perisynaptic neuroglial investment. The outer surface of mossy fiber rosettes and their digitiform processes were seen at the granular layer, making flat and invaginated synaptic contacts with the granule cell dendrites. At the molecular layer, the longitudinal traject of parallel fibers or nonsynaptic segments and their synaptic varicosities were characterized. These latter established synaptic contacts with Purkinje dendritic spines. Fractured parallel fiber endings showed the SE-I images of clustered spheroidal synaptic vesicles and mitochondria and the surrounding cotton-like appearance of Bergmann glial cell cytoplasm. Climbing fibers showed a characteristic crossing-over bifurcation pattern in the white matter and in the three-layer structure of cerebellar cortex, formation of tendril collaterals in the granular layer, topographical relationship with Purkinje cell soma and retrograde collaterals in the molecular layer. The climbing fiber synaptic relationship with Purkinje dendritic spines was characterized, by means of FFTEM, by the presence of large synaptic endings and aggregation of intramembrane particles at the P and E faces of presynaptic endings, characteristic of excitatory synapses.
Assuntos
Córtex Cerebral/ultraestrutura , Junções Comunicantes/ultraestrutura , Animais , Microscopia Crioeletrônica , Técnica de Fratura por Congelamento/métodos , Camundongos , Microscopia Eletrônica de Varredura/métodos , Células de Purkinje/ultraestruturaRESUMO
In this study we describe the early changes of the myelin sheath following surgical nerve crush. We used the freeze-fracture technique to better evaluate myelin alterations during an early stage of Wallerian degeneration. Rat sural nerves were experimentally crushed and animals were sacrificed by transcardiac perfusion 30 h after surgery. Segments of the nerves were processed for routine transmission electron microscopy and freeze-fracture techniques. Our results show that 30 h after the lesion there was asynchrony in the pattern of Wallerian degeneration, with different nerve fibers exhibiting variable degrees of axon disruption. This was observed by both techniques. Careful examination of several replicas revealed early changes in myelin membranes represented by vacuolization and splitting of consecutive lamellae, rearrangement of intramembranous particles and disappearance of paranodal transverse bands associated or not with retraction of paranodal myelin terminal loops from the axolemma. These alterations are compatible with a direct injury to the myelin sheath following nerve crush. The results are discussed in terms of a similar mechanism underlying both axon and myelin breakdown.
Assuntos
Técnica de Fratura por Congelamento/métodos , Bainha de Mielina/metabolismo , Compressão Nervosa , Nervo Sural/cirurgia , Degeneração Walleriana/cirurgia , Animais , Microscopia Eletrônica , Ratos , Ratos Wistar , Degeneração Walleriana/fisiopatologiaRESUMO
In this study we describe the early changes of the myelin sheath following surgical nerve crush. We used the freeze-fracture technique to better evaluate myelin alterations during an early stage of Wallerian degeneration. Rat sural nerves were experimentally crushed and animals were sacrificed by transcardiac perfusion 30 h after surgery. Segments of the nerves were processed for routine transmission electron microscopy and freeze-fracture techniques. Our results show that 30 h after the lesion there was asynchrony in the pattern of Wallerian degeneration, with different nerve fibers exhibiting variable degrees of axon disruption. This was observed by both techniques. Careful examination of several replicas revealed early changes in myelin membranes represented by vacuolization and splitting of consecutive lamellae, rearrangement of intramembranous particles and disappearance of paranodal transverse bands associated or not with retraction of paranodal myelin terminal loops from the axolemma. These alterations are compatible with a direct injury to the myelin sheath following nerve crush. The results are discussed in terms of a similar mechanism underlying both axon and myelin breakdown
Assuntos
Animais , Ratos , Técnica de Fratura por Congelamento/métodos , Bainha de Mielina/metabolismo , Compressão Nervosa , Nervo Sural/cirurgia , Degeneração Walleriana/cirurgia , Microscopia Eletrônica , Ratos Wistar , Degeneração Walleriana/fisiopatologiaRESUMO
The double replica device was used to obtain freeze-fracture replicas of gently pressed cells, allowing the visualization of a large number of longitudinally fractured epimastigote and trypomastigote forms of Trypanosoma cruzi. This technique revealed large areas of the plasma membrane, the region of attachment of the flagellum to the cell body and the branched mitochondria.
Assuntos
Técnica de Fratura por Congelamento/métodos , Trypanosoma cruzi/ultraestrutura , Animais , Membrana Celular/ultraestrutura , Técnica de Fratura por Congelamento/instrumentação , Microscopia EletrônicaRESUMO
The double replica device was used to obtain freeze-fracture replicas of gently pressed cells, allowing the visualization of a large number of longitudinally fractured epimastigote and trypomastigote forms of Trypanosoma cruzi. This technique revealed large areas of the plasma membrane, the region of attachment of the flagellum to the cell body and the branched mitochondria.
Assuntos
Animais , Técnica de Fratura por Congelamento/métodos , Trypanosoma cruzi , Membrana Celular , Microscopia Eletrônica , Técnica de Fratura por Congelamento/instrumentaçãoRESUMO
The double replica device was used to obtain freeze-fracture replicas of gently pressed cells, allowing the visualization of a large number of longitudinally fractured epimastigote and trypomastigote forms of Trypanosoma cruzi. This technique revealed large areas of the plasma membrane, the region of attachment of the flagellum to the cell body and the branched mitochondria.(AU)
Assuntos
Animais , RESEARCH SUPPORT, NON-U.S. GOVT , Técnica de Fratura por Congelamento/métodos , Trypanosoma cruzi/ultraestrutura , Membrana Celular/ultraestrutura , Técnica de Fratura por Congelamento/instrumentação , Microscopia EletrônicaRESUMO
A new rotary-shadowing process to obtain freeze-drying replicas is described for the analysis of virus ultrastructure, using the inner capsid of human rotavirus as a model. The findings corroborate the icosahedral symmetry with an arrangement pattern of capsomers of T = 13L.
Assuntos
Técnica de Fratura por Congelamento/métodos , Rotavirus/ultraestruturaRESUMO
A new rotary-shadowing process to obtain freeze-drying replicas is described for the analysis of virus ultrastructure, using the inner capsid of human rotavirus as a model. The findings corroborate the icosahedral symmetry with an arrangement pattern of capsomers of T=13L
Assuntos
Rotavirus/ultraestrutura , Técnica de Fratura por Congelamento/métodosRESUMO
The present paper shows the potential contribution of conventional and high resolution scanning electron microscopy (SEM) to trace short intracortical circuits in cryofractured fish, primate and human cerebelli. Conventional SEM slicing technique allowed us to identify afferent mossy and climbing fibers and their synaptic relationship in the granular layer. SEM freeze-fracture method exposed the mossy glomerular synapses and the axo-dendritic connections of climbing fibers. At the Purkinje cell layer, the cryofracture process removed the satellite Bergmann glial cell layer, displaying a partial view of the supra- and infra-ganglionic plexuses of Purkinje cells and the ascending pathways of climbing fibers. High resolution SEM (HRSEM) showed the specimen specific secondary electron (SE-I) image of axosomatic synapses on Golgi cell surface. At the molecular layer, the outer surface of parallel fiber synaptic varicosities were distinguished, establishing the cruciform en passant synaptic contact with the Purkinje cell dendritic spines. HRSEM showed the fractured parallel fiber synaptic varicosities containing spheroidal synaptic vesicles embedded in a high dense extravesicular material. Conventional SEM and gold-palladium coating are useful to trace intracortical circuits. With HRSEM and chromium coating, it is possible to study the outer and inner surfaces of synaptic connections.
Assuntos
Cerebelo/ultraestrutura , Técnica de Fratura por Congelamento/métodos , Microscopia Eletrônica de Varredura/métodos , Adolescente , Adulto , Animais , Cerebelo/citologia , Criança , Peixes , Técnicas Histológicas , Humanos , Macaca mulatta , Fibras Nervosas/ultraestrutura , Vias Neurais/ultraestrutura , Células de Purkinje/ultraestruturaRESUMO
The surface of the cuticle of adult Nippostrongylus brasiliensis has been studied by means of the freeze-fracture technique and by transmission electron microscopy. Some of the surface coat appears to have been shed from the surface of the cuticle of adults fixed in situ in the intestine of its host and from the surface of individuals removed from the intestine and freeze-fractured. Freeze-fracturing the cuticle of individuals removed from the host has shown that this surface coat varies in thickness from 30 to 90 nm. The epicuticle is about 20 nm thick and cleaves readily to expose E- and P-faces. The P-face of the epicuticle possesses a small number of particles, similar to intra-membranous particles, whilst the E-face possesses a few, widely scattered depressions. Despite the presence of these particles the epicuticle is not considered to be a true membrane. Freeze-fracturing the remainder of the cuticle has confirmed its structure as described by conventional transmission electron microscopy. Clusters of particles on the P-face of the outer epidermal (hypodermal) membrane and corresponding depressions on the E-face of the membrane are though to be associated with points of attachment of the cuticle to the epidermis (hypodermis). No differences in appearance of the cuticle and its surface layers were observed in individuals taken from 7-, 10-, 13- and 15-day infections.
Assuntos
Nippostrongylus/ultraestrutura , Animais , Técnica de Fratura por Congelamento/métodos , Intestinos/parasitologia , Larva , Microscopia Eletrônica/métodos , Nippostrongylus/crescimento & desenvolvimento , Especificidade de Órgãos , Ratos , Ratos WistarRESUMO
Purkinje dendrite-parallel fiber spine synapses of human, teleost fishes, Rhesus monkey and mouse cerebellar cortex have been studied by means of conventional scanning electron microscopy (SEM) using ethanol-cryofracturing technique and by high resolution field emission scanning electron microscopy (HRFESEM) for studying the outer and inner surface morphology of pre- and postsynaptic endings. Transmission electron microscopy of mouse cerebellar cortex either by means of ultrathin sections and freeze-etching replicas have been complementarily used for proper identification and comparative observations. Normal teleost fishes showed short neck and neckless Purkinje spines with exhibited spread or extended postsynaptic densities. In pathological human cerebellum, the ethanol cryofracturing technique exposed the outer surface of edematous flat and invaginated Purkinje spine synapses. In fractured presynaptic endings HRFESEM showed in Rhesus monkey cerebellar cortex a homogeneous extravesicular material binded to the synaptic vesicles and joining them to the presynaptic membrane. HRFESEM partially resolved the synaptic cleft as currently observed in high magnification transmission electron microscopy. Round subunits, 25-35 nm in diameter, were observed associated to postsynaptic membrane, apparently corresponding to the localization and distribution of E face postsynaptic intramembrane particles, which suggest that such subunits correspond to the domains of neurotransmitter postsynaptic receptors.