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Biomedical subjects

J F David-Ferreira

Publications and source records attributed to J F David-Ferreira.

At least 19 recordsLinked to original sources

The inter-Sertoli cell tight junctions in germ cell-free seminiferous tubules from prenatally irradiated rats: a freeze-fracture study.

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood-testis barrier in the normal testis.

Animals↗

Cytokeratin in early hamster embryogenesis and parthenogenesis: reorganization during mitosis and association with clusters of interchromatinlike granules.

In vivo obtained golden hamster embryos were used to study, by immunofluorescence and immunoelectron microscopy, the main cytokeratin pattern rearrangements during completion of meiosis and the first cleavage division. Our results point to three major re-organization steps: (1) diffuse immunofluorescent cytokeratin spots characteristic of recently ovulated oocytes rearrange into large cortical patches interconnected by fibrils in one-cell embryos; (2) during mitosis a homogeneous cytokeratin spotty pattern reappears; (3) in two-cell embryos cortical and perinuclear cytokeratin fibrillar networks become prominent. Parthenogenotic oocytes were able to mimic the major cytokeratin patterns observed until the first embryonic mitosis, supporting the concept of a maternally established common response to activation. Despite the lack of fibrillar immunofluorescent reactivity during mitosis, electron microscopy demonstrates persistence of 10 nm filament meshworks. These cytokeratin meshworks often associate with clusters of interchromatinlike granules, which persist in the cytoplasm for a short period after nuclear envelope reassembly.

Actin Cytoskeleton↗

Ultrastructural and cytochemical studies of nucleolus-like bodies in neurons of rat sympathetic ganglia.

Intracytoplasmic fibrillar inclusions, generally referred to as nucleolus-like bodies (NLBs) were studied by means of ultrastructural cytochemistry. The structure of these bodies was visualized by several different staining procedures: conventional electron microscopy and preferential staining methods for localization of various proteins including ribonucleoproteins, basic proteins, glycoproteins and phosphorylated proteins. The results of the cytochemical tests indicate that NLBs have an essentially proteinaceous nature. They consist of ribonucleoproteins, basic proteins and glycoproteins but do not contain phosphorylated proteins. These findings suggest that NLBs are, at least partially, of the same nature as nucleoli and coiled bodies. The origin of NLBs and their possible functional role is briefly discussed.

Animals↗

Expression of neural cell adhesion molecule immunoreactivity in hypertrophic myocardium.

Myocardial neural cell adhesion molecule (N-CAM) is temporally regulated, being expressed during cardiac morphogenesis and innervation and suppressed in the adult heart. We have investigated the plasticity of N-CAM expression in hypertrophic muscle using the rat model of chronic hypoxia to selectively induce right ventricular hypertrophy over a 14 day time course. Sarcolemmal and intercalated disc N-CAM immunostaining was more extensive in the ventricular myocardium of hypoxic rats compared to normoxic controls. Quantitative assessment of the immunoreactivity in tissue extracts demonstrated a selective increase in the amount of N-CAM immunoreactivity in the hypertrophic myocardium of the right ventricle of rats exposed to hypoxia and this was associated with an increase of the 125 kDa isoform. We conclude that myocardial hypertrophy may be a factor influencing N-CAM expression in the heart and adhesion molecules may have a role in cardiac remodelling.

Animals↗

Peptide-containing nerve fibres in guinea-pig coronary arteries: immunohistochemistry, ultrastructure and vasomotility.

The peptidergic innervation of guinea-pig coronary arteries was investigated by means of immunohistochemical, ultrastructural and in vitro pharmacological techniques. A network of nerves was demonstrated in all major epicardial arteries by means of an antiserum to the neuronal marker protein gene product 9.5. The majority of nerve fibres possessed neuropeptide Y (NPY) and tyrosine hydroxylase (TH) immunoreactivity, the number and distribution of nerves immunoreactive for NPY being similar to that of nerves containing TH immunoreactivity. Numerous nerve fibres displaying immunoreactivity for substance P, neuropeptide K and calcitonin gene-related peptide (CGRP) were also found. In double-stained preparations substance P immunoreactivity was co-localized with CGRP and with neuropeptide K immunoreactivities in the same varicose nerve fibres. Ultrastructural studies revealed the presence of numerous axon varicosities at the adventitial-medial border. NPY immunoreactivity was localized in large granular vesicles in nerve varicosities which also contained numerous small granular vesicles. Large granular vesicle-containing nerves also displayed immunoreactivity for dopamine-beta-hydroxylase. With an in vitro method, the vasomotor responses to perivascular peptides were characterized in epicardial and intramyocardial arteries. In epicardial arteries neither noradrenaline nor NPY elicited a contractile response. Only in some intramyocardial arteries was an NPY-mediated contraction demonstrated. No potentiating effect of noradrenaline and NPY was observed in either epicardial or intramyocardial arterial segments. In contrast, CGRP, substance P and vasoactive intestinal peptide (VIP) all produced a concentration-dependent relaxation of both epicardial and intramyocardial arteries. These results suggest that peptide-containing nerves associated with guinea-pig coronary arteries may predominantly be involved in mediating vasodilation.

Animals↗

Testosterone interferes with the kinetics of endocytosis in the hamster seminal vesicle.

Endocytosis was studied in the seminal vesicle secretory cells of castrated and control hamsters in order to investigate the effect of testosterone withdrawal in the endocytic activity of these cells. Horseradish peroxidase was injected into the glands lumen after removal of their contents, and tracer distribution was qualitatively studied, and the number of labeled endocytic vesicles quantitatively analyzed, following 5, 20, 40 and 60 min incubation. The following compartments are labeled both in castrate and control cells: 1), endocytic vesicles; 2), vacuoles with or without secretory material; 3), multivesicular bodies; 4), Golgi cisternae; 5), intercellular spaces; 6), sub-epithelial space. The pattern of labeling is lighter in castrate than in control cells and the labeling of Golgi cisternae, which correlates with a significant peak in the number of endocytic vesicles, is observed later in castrated animals than in controls: 40 min vs 20 min. Exocytosis, as evaluated through the fraction of secretory protein released in vitro, decreases following castration. Endocytosis performed in castrated, pilocarpine treated animals shows that the Golgi labeling, coinciding with numerous labeled endocytic vesicles, is advanced from 40 to 20 min after stimulation of exocytosis. The results show that, in the seminal vesicle secretory cells a) the endocytic pathway does not depend on testosterone; b) testosterone withdrawal decreases endocytosis and delays the kinetics of labeling and; c) endocytosis couples to exocytosis, probably so regulating the apical cell membrane area.

Animals↗

Interactions of intermediate filaments with cell structures.

Intermediate filaments (IF) are unique components of the cytoskeleton of most eukaryotic cells. Also the nuclear lamins are now recognized to be IF-like proteins, providing the nucleus with a putative skeleton for chromatin attachment. Immunofluorescence and whole-mount electron microscopic studies reveal that IF form a cytoplasmic network that surrounds the nucleus and extends to cell surface, as 'mechanical integrators of cellular space'. It seems however unlikely that IF in the cell accomplish a merely structural role, considering the diversity of IF proteins and the complex regulation of their gene expression. In this work we primarily present electron microscopic data that points to the presence of interactions between IF and several cellular components, namely the nucleus, plasma membrane, other cytoskeletal elements, cytoplasmic organelles and ribonucleoproteins. Although the functional significance of such interactions remains to be demonstrated, assumptions like involvement of IF in information transfer or cytoskeleton-dependent control of gene expression represent attractive hypothesis for future research.

Animals↗

Immunocytochemical study of tissue distribution and hormonal control of chondroitin-, dermatan- and keratan sulfates from rodent uterus.

The distribution patterns of rat and mouse uterine glycosaminoglycans (GAGs), as well as their modulation by estradiol (E2) and/or progesterone (P), were investigated using monoclonal antibodies (MABs) directed against chondroitin- (CS)/dermatan sulfates (DS), keratan sulfate (KS) and a trophoblast GAG. The localization of GAGs in relation to collagens (I, IV and VI) and fibronectin was also analyzed. We found that uterine GAGs are differentially distributed in the endometrium and myometrium, in a pattern that is species-related. CS-containing proteoglycans (PGs) occur between collagen bundles and fibroblasts, at the periphery of the latter, and in basement membrane zones (BMZs), in a pattern resembling that of collagen VI. BMZs contain preferentially CS-PGs bearing 4-sulfated disaccharides adjacent to the core protein. DS-PGs are mostly associated with collagen bundles. E2 and/or P elicit distinct modifications on the above described pattern, which are also species-related. The simultaneous administration of E2 and P changes the prevalent sulfation of the disaccharides adjacent to the core protein of stromal CS-PGs. In the mouse, an unsulfated intracellular epitope appears following E2 (or E2P) administration, mostly in epithelial cells. In the rat, KS and the trophoblast GAG are E2-dependent and down-regulated by P. The functional significance of the hormone-induced GAG changes, namely the possible role of the E2-dependent KS in implantation, are discussed.

Animals↗

Neural cell adhesion molecule is expressed by smooth muscle cells during the development of the rat vascular system.

The presence and distribution of neural cell adhesion molecule (N-CAM) was examined by light and electron microscopical immunocytochemistry in the descending thoracic aorta, the superior mesenteric artery and mesenteric arteries from fetal and adult rats (embryonic day 15 to post-natal day 90). In embryonic and early post-natal rats, N-CAM immunoreactivity was localized in perivascular nerves, in the smooth muscle cell plasma membrane and basal lamina. In nerves, N-CAM-immunoreactive sites were found associated with both the axon and Schwann cell membranes. N-CAM immunoreactivity was also found associated with the surface of adventitial fibroblast-like cells and with collagen fibrils, in regions where these fibrils were in contact with smooth muscle cells. In mature vessels N-CAM immunoreactivity was found to be restricted to the perivascular innervation and the surface of fibroblast-like cells. These observations indicate that N-CAM is expressed transiently in rat vascular tissues during development and is localized not only on the surface of smooth muscle cells but also in association with extracellular matrix components.

Aging↗

Cytokeratin filaments are present in golden hamster oocytes and early embryos.

Light and electron microscope methods were used to study cytokeratin expression in the recently ovulated oocytes, fertilized eggs and early embryos from the golden hamster. Two cytokeratin polypeptides (Mr 51,000 and 58,000) were detected in oocyte lysates by immunoblotting using a polyclonal antiserum to prekeratin. In the oocyte, cytokeratin occurred as patchy aggregates consisting of short anastomosing 10-nm filaments that formed tight meshworks distributed throughout the cytoplasm. After fertilization the aggregates appeared to merge, becoming larger and concentrated at the cortical region. Prominent immunofluorescent fibrils were seen interconnecting the aggregates. In the 2-, 4- and 8-cell embryos, networks of cytokeratin filaments extended throughout the cortical and perinuclear regions, while aggregates progressively disappeared.

Animals↗

Reevaluation of fibronectin-collagen interactions in tissues: an immunocytochemical and immunochemical study.

We studied the distribution of fibronectin (FN) in rat uterus, rat tail tendon, and rooster comb, using LM and EM immunocytochemistry. Special attention was paid to the interaction of FN with collagen (COL). Various labeling protocols and dot-blot experiments were performed to confirm the results. Under conditions of labeling specificity, FN distribution over native COL fibrils was usually sparse, especially when these were organized into thick bundles. No labeling was observed over section surfaces of COL fibrils when postembedding methods were used, which indicates that no FN is present within these fibrils. Under conditions in which exogenous FN could react with tissues, e.g., when preincubation with normal serum for background blocking was performed, artifactual staining appeared over COL. Such a reaction also occurred when anti-FN antiserum completely blocked by liquid-phase adsorption was used. Therefore, the FN present in soluble FN-anti-FN immune complexes must have still been able to react with COL. The artifactual labeling was, in all cases, almost exclusively localized on the section surfaces of COL fibrils. These results suggest that FN has a very low affinity for the surface of native COL fibrils.

Animals↗

Filamentous cross-bridges link intermediate filaments to the nuclear pore complexes.

Intermediate filament-nuclear matrix interactions were studied in cultured rat ventral prostate cells and isolated rat uterine epithelial cells. Cytokeratin filaments were identified by immunoelectron microscopy. In addition to conventional thin section of Triton X-100 treated cells, subcellular residues composed of intermediate filaments and nuclear matrix were critical-point dried and platinum-carbon replicated. The results demonstrate the presence of a previously unrecognized type of filamentous cross-bridges that link intermediate filaments to the nuclear pore complexes.

Animals↗

Different patterns of filipin-sterol labeling in prostate nuclear membranes from normal and castrated rats.

Filipin was used as cytochemical probe for sterol detection in freeze-fractured prostate nuclear membranes from rats under different hormonal conditions. Isolated prostate acini and nuclei were fixed in glutaraldehyde and post-treated with filipin, according to Robinson and Karnovsky (1980). In general, most plasma and intracellular cytoplasmic membranes displayed a marked response to filipin in either epithelial and stromal cells from normal and castrated animals. Nuclear membranes from epithelial secretory cells were systematically negative to filipin labeling in normal animals, although after castration a positive response was detected. Stromal nuclear membranes were labeled both in normal and castrated animals. Filipin-treated isolated nuclei displayed the same overall labeling pattern but there was a different distribution of induced deformations relative to intact cell nuclei. These observations indicate that: a) nuclear membranes from different cell types have different responses to filipin; b) a change in the molecular organization of nuclear membranes from prostate secretory cells follow castration; c) nuclei isolation affects the distribution of filipin induced deformations on the membranes.

Animals↗

Secretory cell activity in the hamster seminal vesicle following castration. A morphometric ultrastructural study.

The ultrastructure of hamster seminal vesicle epithelium was studied 7, 14, 21 and 28 days after castration using a stereological approach. The results show that castration promotes epithelial reorganization, mainly characterized by reduced epithelial cell size and number, decreased rough endoplasmic reticulum and Golgi complex, increased lysosomes and lipid droplets, increased apical secretory granule size and number, and increased intracellular secretory products per average epithelial cell. It is concluded that after testosterone withdrawal the secretory activity of hamster seminal vesicle epithelial cells, although reduced, is not abolished, and that exocytosis is relatively more reduced than secretory protein production. We suggest that an extracellular androgen source is responsible for secretory activity not being lost in the epithelial cells of castrated hamster seminal vesicle.

Animals↗

Testosterone-induced nuclear changes in the rat uterine luminal epithelium. A stereological study.

Testosterone administration to the spayed immature female rat produces significant changes in the nuclear ultrastructure of the uterine epithelial cells. In general, these changes increase when a higher dose is used. The most intense changes occur in the nucleolus: volume density and volume increase; volume increases in all nucleolar compartments; granular and vacuolar volume density increase; fibrillar center number increase, but a reduction of their individual volume is observed. Condensed chromatin volume density and volume decrease. Nuclear volume, nuclear envelope surface, and nuclear body volume density and frequency remain almost the same. The surface density of the interface between condensed and dispersed chromatin, fibrillar centers and fibrillar component, and condensed chromatin and fibrillar component, show significant variations even with a small dose of testosterone (0.5 mg). Such a dose is unable to change the volume density of condensed chromatin or fibrillar centers. Correlations between our results and biochemical or other morphological data are discussed.

Animals↗

Freeze-fracture study of the hamster ovary surface epithelium intercellular junctions.

The apical intercellular junctions between the hamster ovary surface epithelial cells were studied by freeze-fracture. The results showed tight junctions consisting of intertwining ridges on P faces and complementary furrows on E faces. Typical gap junctions, in the form of clusters of particles on P faces and complementary pits on E faces, were also observed associated with the tight junctional elements. Sometimes the gap junction are the outermost element of the junctional complexes. These observations explain data previously obtained in tracer experiments with lanthanum.

Animals↗

Fine structure of seminiferous tubules from prenatally irradiated rats.

The ultrastructure of the seminiferous tubules was studied in rats that had been subjected to whole body irradiation on the 19th day of gestation. The seminiferous tubules from 3 months-old irradiated animals are devoid of germ cells and contain only Sertoli cells. Compared with controls of the same age, the seminiferous tubule basal membrane is thickened and multilayered and several alterations are observed in the Sertoli cells. The most characteristic of these alterations are: (a) an abnormal number of nuclear heterochromatin clumps, (b) the presence of numerous cytoplasmic vacuoles and various sized lipid droplets, (c) elaborate interdigitations and junctions between adjacent cells, and (d) the presence of anomalous ectoplasmic specializations disposed perpendicularly to the Sertoli cell membrane.

Animals↗