This paper describes the ultrastructure of the commoner myofibroblastic tumours and tumour-like lesions. The objective is to complement mainstream pathology texts, which have concentrated on the clinical and light microscopy features of these lesions and which have arguably but understandably somewhat neglected electron microscopy as an ancillary diagnostic tool and a technique for investigating tumour cell biology. Ultrastructural features are described of nodular fasciitis, the myofibromatoses (including Dupuytren's disease), inflammatory myofibroblastic tumour, post-operative spindle cell nodule, fibroma of tendon sheath, fibrous pseudotumour, benign fibrous histiocytoma, atypical fibroxanthoma, dermatofibrosarcoma protuberans, myofibrosarcoma (myofibroblastic sarcoma), malignant fibrous histiocytoma (pleomorphic myofibrosarcoma), epithelioid sarcoma and spindle-cell carcinoma. Fibrosarcoma and leiomyosarcoma are illustrated for comparison. The fibronexus is emphasised as an important marker for the most confident diagnosis of myofibrosarcoma. Some pathologists accept a light microscope definition, which includes alpha-smooth-muscle actin positivity, h-caldesmon negativity and, in some cases, desmin positivity. Caution in the interpretation of desmin staining in a possible myofibroblastic lesion is urged, since, in combination with an ultrastructurally identified lamina, it more probably suggests true smooth-muscle differentiation. Myofibroblastoma and angiomyofibroblastoma are examples of tumours argued on the basis of ultrastructural findings (sometimes in combination with desmin staining) to be primitively differentiated smooth-muscle cell rather than myofibroblastic proliferations.
Mitochondrial encephalomyopathies (MEs) are a group of clinically and genetically heterogeneous diseases. They can be caused by defects in both mitochondrial or nuclear coded genes. Their phenotypic expression is governed by unique biological phenomena such as the dual genetic control, mitotic segregation, heteroplasmy and threshold effects. Currently, the correct diagnosis of ME relies on a multidisciplinary approach which includes clinical information as well as laboratory data from muscle morphology, biochemistry and molecular genetics. Among the morphological methods, histology, histochemistry and electron microscopy were historically instrumental in the diagnosis of MEs. However, with the development of molecular genetics, the diagnostic value of morphology and of electron microscopy in particular have been questioned. The aim of the present review is to present a comparative assessment of the diagnostic contribution of histology, histochemistry and electron microscopy in a group of 48 patients with a diagnosis of ME.
Eggs from the bivalve mollusc Chamelea gallina were transfected in vitro. The p-GeneGrip gene construction that expresses the green fluorescent protein (GFP) was employed. It was necessary to remove the jelly coat which covers the egg surface for a successful transfection, and then 44.2% of gametes appeared transfected after using naked DNA. On the other hand, cationic liposomes (Lipofectamine) and neutral lipids (GenePORTER) were employed as gene vectors. After the employ of Lipofectamine 35.6% of eggs were transfected and 41.4% after using GenePORTER. Fluorescence analysis showed that the foreign gene appeared principally located in the egg cytoplasm, but laser confocal microscopy showed that it was also present in the nucleus. Furthermore, PCR analysis demonstrated that the foreign DNA appeared in the DNA extracted from the treated eggs. This simple method for the transfection of mollusc eggs would be interesting for future biotechnological applications in species of commercial interest.
Different steps of sperm activation such as acrosomal reaction and capacitation are described in details. The molecules involved in sperm-egg interaction are also reported.
Electron microscopic examinations are sometimes limited due to the small number of cells available for analysis. The purpose of this study was to determine the limit of cell concentration for a successful transmission electron microscopic preparation. Various concentrations of monocyte cell suspension were fixed in glutaraldehyde and osmium tetroxide according to the standard methods. Cell preparations were made on silane-coated glass slides in a cytospin centrifuge. The attached cells to the glass slides were dehydrated, and embedded in epoxy resin by routine electron microscopic technique. By this method, cell suspensions containing as low as 2x10(3) cells could show approximately 5 to 10 cells in each hole of the 150-mesh grids which was designated as the lowest limit for the successful preparation with detectable cells for evaluation. The fine structure of cells was clearly evident and the preparations were uniformly free from artifacts, similar or superior to those of cell pellet preparations. This method is useful whenever dealing with the samples containing a low number of cells, particularly those of clinical samples.
The Golgi apparatus of spinal ganglion neurons was studied in 1, 3.6, 6.7, and 8.8-year-old rabbits. The structure of this organelle did not differ in the four age groups examined. While the mean volume of the neuronal perikaryon increased progressively with age, the total volume of the Golgi apparatus remained stable throughout life. As a consequence, the mean percentage of perikaryal volume occupied by this organelle decreased significantly with age. Since the percentage of perikaryal volume occupied by lipofuscin remained at low levels throughout life, the ratio of the total volume of the Golgi apparatus to the functionally active volume of cytoplasm decreased with age. It is possible that this decrease is related to the reduction in neuronal metabolism that occurs in senescence. The age-related quantitative changes in the Golgi apparatus were very similar in large light and in small dark neurons. Finally, neither fragmentation, nor peripheral displacement of the Golgi apparatus was observed with advancing age.
Histomorphology and ultrastructure of the testis during breeding and nonbreeding phases of the reproductive cycle of the lizard Mabuya carinata are studied. Observations of the ultrastructural features of the testis during breeding and nonbreeding phases of the reproductive cycle reveal a prenuptial type of spermatogenesis and a clearcut discontinuous spermatogenic cycle. Seminiferous tubules are enlarged and there is active spermatogenesis as shown by the presence of all the stages of spermatogenesis (spermatogonia to spermatids) and spermatozoa during the breeding phase (November). During the nonbreeding phase (April) only spermatogonia and Sertoli cells are seen in the shrunken seminiferous tubules. Leydig cells and Sertoli cells show distinct changes in the morphological appearance with hypertrophy of the cells in breeding phase and atrophy of the cells in the nonbreeding phase of the reproductive cycle. The present study suggests that Sertoli cells and Leydig cells functions are synchronous in the lizard M. carinata.
Type V and VI collagen were capable to joint each other and with type I and IV collagen, as well as mucopolysaccharides. This capability suggested that these collagens function for cohesion of fibrillar tissue components of dermis. This study demonstrated the locality of these types of collagen in dermis. Fresh specimens of normal skin were fixed in 2% paraformaldehyde in phosphate-buffered saline, overnight. Besides, in order to loosen the twist of collagen fibril, some pieces of the skin specimens were treated by citrate buffer pH 3.0, prior to fixation. The specimens were embedded in Technovit 4100 and the ultrathin sections were stained by antibody to type V collagen and followed by antibody to type I, III, IV and VI collagen. The immune reactant was visualized by gold particles for electron microscopic observation. Type V and VI collagen formed networks in dermis and jointed to collagen fibrils, elastic fibre and basal lamina. Type V collagen was found inside collagen fibrils, broad elastic fibres and junctions. Dermo-epidermal junction showed type V collagen on the dermal aspects of basal lamina and at the sites where anchoring filaments joint to basal lamina, while in junction of mesenchymal tissues, no precise structural components for type V collagen were identified. Type VI collagen wove with type V collagen in dermis and associated with mucopolysaccharides. In conclusion, type V collagen formed networks in dermal interfibrillar space and participated in assembling collagen fibrils and forming broad elastic fibres. Epithelial and mesenchymal cells cohered to the underlying dermal matrix in the junction by type V collagen. Type VI collagen interwove with type V collagen in the interfibrous space and associated with mucopolysaccharides. Types V and VI collagen preserved architecture of dermal matrix.
The objective of the present study was to assess the possible toxic effects of chronic alcohol ingestion on the ultrastructure of the glandular epithelium of the prostate of the rodent Calomys callosus, in order to contribute to the understanding of the consequences of alcohol abuse for the morphology of the male reproductive apparatus. Sixteen adult animals aged three months were divided into two experimental groups. The control group received a solid diet and tap water, and the alcoholic group received the same solid diet and ethanol P.A. diluted 20% in water (v/v). After 120 days of treatment, all animals were anesthetized, weighed and sacrificed. At the end of treatment, mean body weight did not differ between control and alcoholic animals. The prostate epithelial cells of the alcoholic group showed intense atrophy and ultrastructural alterations such as the presence of lipid droplets, altered nuclei, ruptured mitochondrial cristae, and intense dilatation of the cisterns of the granular endoplasmic reticulum. It was concluded that 20% ethanol provokes marked lesions on the epithelium of the prostate probably interfering on the glandular secretion.
Two types of autophagy in the podocytes were found in renal biopsy specimens by electron microscopy. Type I autophagy (about 1 microm in diameter) was found in 10 out of the 100 cases with renal diseases, and showed a condensed ribosome area with a limiting membrane. The origin of limiting membrane appeared to be from degenerated mitochondria. During type I autophagy formation, the thickness of limiting membrane changed from 5-6 nm to about 8-10 nm thickness. Type I autophagy did not transform to autophagosomes and autophagic vacuoles. On the other hand, many cases (90 out of the 100 cases) showed type II autophagy. Type II autophagy (3-8 microm in diameter) showed that many ribosomes were aggregated, formed condensed ribosome area, which always included many aggregated lipid droplets at first. Next, during the formation of autophagosome, rough ER connected to condensed ribosome area, and partly formed limiting membranes from dilated ER membrane. Finally, the limiting membrane of autophagic vacuoles was completely formed, and this membrane changed from about 5-6 nm to 8-10 nm thickness. Ribosomes and lipid droplets were resolved in autophagic vacuoles. Thus, type II autophagy might play a significant role in clearance of proteins and lipids in comparison with type I autophagy. The occurrence of type I autophagy in the renal biopsy specimens was not clearly associated with age, sex or pathological diagnosis. However, cases with type I autophagy may show a tendency to poor prognosis.
An infant of African-American descent presented in the immediate newborn period with secretory diarrhea, the cause of which turned out to be microvillus inclusion disease (MID). Small intestinal mucosal biopsies at 6 weeks of age were diagnostic for MID by electron microscopy and repeat biopsies from the small intestine at 15 months demonstrated the seeming relentless progression of this disorder, when a normal structure and organization of small intestinal mucosa was no longer recognizable. Since the child could not tolerate any form of enteral nutrition, a small intestinal transplant was contemplated, but could not be done. The patient did not survive the consequences of an overwhelming sepsis, which resulted in multi-organ failure.
Malaria, a common health problem in certain parts of the world, has a considerable morbidity and mortality. This work reports under electron microscopy studies serious ultrastructural kidney damage such as extensive cytoplasmic vacuolation, vesiculation and autophagic vacuoles in proximal tubular cells. A thickened endothelial wall on peritubular capillary, interdigitation disorganization and significant decrease of their number in some areas were detected. Swollen rough endoplasmic reticulum, swollen mitochondria, and parasitized erythrocytes were observed. Many epithelial cells exhibited cytoplasmic areas of autophagia and a myelin-like form. A tubular cell presented severe cytoarchitecture alterations. Abundant lipid droplets were noticed. Almost total loss of interdigitations, rough endoplasmic reticulum vesiculation, peritubular capillaries with endothelial cells thickened cytoplasm, papillary processes projected to the lumen, and an inflammatory infiltrate of macrophages were also observed. These ultrastructural kidney changes could cause, on the basis of their clinical and pathologic expressions, a fat accumulation, an acute temporary reversible glomerulonephritis, a chronic progressive irreversible glomerulonephritis, and an acute renal failure (ARF).
Submicroscopic alterations in the cytoskeletal structure of sperm flagellum are associated with severely reduced or completely absent motility in subfertile or infertile men. Sometimes these alterations can be related to well known genotypic defects when the same anomaly affects the whole sperm population. Transmission electron microscopy (TEM) is the only tool able to specifically characterize the morphological features of genetic sperm defects. In this study, the frequencies of aneuploid and diploid spermatozoa were identified in three patients showing specific flagellar anomalies, each of them affecting the whole sperm population: dysplasia of the fibrous sheath, primary ciliary dyskinesia and absence of fibrous sheath. All these defects were highlighted by TEM. Fluorescence in situ hybridization (FISH) analysis was performed on decondensed sperm nuclei for chromosomes 18, X and Y, highlighting higher diploidies and sex chromosome disomies in cases of dysplasia of the fibrous sheath and primary ciliary dyskinesia, in agreement with other reports. We have also described FISH results in spermatozoa with absence of fibrous sheath. In this case, the only one reported due to the rarity of this defect, the aneuploidies and diploidies were within normal range. These data contribute to the growing evidence that genetic sperm defects of sperm flagella are generally correlated with meiotic segregation derangement. For this reason, genetic counseling is advisable, although all the genes involved and the possible mechanisms of these mutations have not yet been fully characterized.
The purpose of this study was to elucidate using transmission electron microscopy (TEM) the ultrastructural changes that occur within the cortical gray matter of a novel reproducible model of congenital hydrocephalus in mice created to overexpress the cytokine transforming growth factor-beta1 (TGF-beta1) in the central nervous system. Brain tissue was obtained from mice from a colony engineered to overexpress TGF-beta1 at two days postpartum and compared to a wild-type aged-matched control. This tissue was fixed using a solution containing 1.25% paraformaldehyde and 1.25% glutaraldehyde in phosphate buffer at least 3-4 h and then cut into 40-50 microm sections. Randomly selected thin sections were stained with uranyl acetate and lead citrate, and then analyzed using a JEOL-100CX or 1200EX transmission electron microscope at accelerating voltage 80 kV. Dramatic neuronal and glial pathology was observed throughout the cortical neuropil in TGF-beta1 mice. The most striking change in the hydrocephalic mice was severe edema with extracellular fluid, possibly due to cerebrospinal fluid (CSF) penetration into the cortex. In addition, severe disruption of the cytoplasmic matrix was seen throughout the cortex, with damage to cellular organelles and particularly severe damage to mitochondria. Our results suggest that congenital hydrocephalus may be associated with significant damage to cortical tissue.
Biochemical studies demonstrate that the NO-releasing-aspirin derivative (NCX4016) stimulates soluble guanylate cyclase (sGC) activity and increases cyclic GMP (cGMP) in human platelet and monocytes by releasing NO. In the present study, an ultracytochemical technique for electron microscopy was used to investigate the effects of NCX4016 (2 mM) on sGC activity in rat thoracic aorta, using sodium nitroprusside (0.01 mM) as reference NO-donor. Guanylyl-imidodiphosphate sodium salt [Gpp(NH)p], a synthetic non-hydrolyzable analogue of GTP, was used as sGC substrate. NO-activated sGC released imidodiphosphate ions which were precipitated with lead ions, giving rise to deposits of electron-dense granules (reaction product). Ultracytochemistry allowed us to demonstrate that NCX4016 stimulated sGC activity in smooth muscle cells, and particularly in vascular endothelial cells, as sodium nitroprusside did. This result could explain the protective effects of chronic treatment with NCX4016 on aortic endothelium of diabetic rats demonstrated by scanning and transmission electron microscopy.
The epididymal epithelium of Agouti paca, a wild South American rodent, was basically formed by principal and basal cells. Principal cells were closely related to processes of adsorptive endocytosis, phase-fluid endocytosis and also secretion originating from their cytoplasmic ultrastructural features. Principal cells were also characterized by the presence of vesicles of several shapes, sizes and internalized content occurring in smaller pits, pale small vesicles next to the apical brush border of microvillus, as well as coated vesicles, smooth surface vesicles and great vesicles. Multivesicular bodies, endosomes and lysosomes were mainly observed in supranuclear position. Moreover, presence of an apocrine secretory process was demonstrated by the occurrence of apical cytoplasmic expansions projecting into the vas deferens luminal compartment. Basal flattened cells without luminal surface contact occurred next to the basement membrane of the ductus, and did no exhibit special ultrastructural features.
Morphological, cytochemical and ultrastructural studies are important to demonstrate the function of the blood cells, which is very little understood in teleosts. In peripheral blood of 'piracanjuba' Brycon orbignyanus, thrombocytes, lymphocytes, monocytes, neutrophils and heterophils were studied and characterized. Thrombocytes had a fusiform or oval shape with PAS-positive granules. Lymphocytes presented small size with sparse basophilic cytoplasm. Monocytes were large in size, presented basophilic cytoplasm that may be foamy or vacuolated, with non-specific esterase staining. The neutrophils presented lightly neutrophilic granule cytoplasm, with positivity for PAS and peroxidase. The heterophils were large in size, with eosinophilic and basophilic granules cytoplasm and PAS-positive. Transmission electron microscopy study demonstrated that the thrombocytes, lymphocytes and monocytes features were similar to other teleosts. In ultrastructural study only one type of neutrophils was observed. Cytochemical findings indicated that neutrophils and monocytes of B. orbignyanus may be involved in phagocytosis, and neutrophils play an important microbicidal role.
Nestin is a neuroepithelial precursor cell marker expressed in a variety of human cell types during development. However, no information exists on the expression of nestin in mature glomeruli as well as during the glomerular development. Here, we examined nestin expression in rat and human glomerular tissues in quiescent states using RT-PCR and immunohistochemical methods. Nestin mRNA was detected in the rat glomeruli in parallel with its expression in developing rat brains. In the normal mature rat glomeruli, WT-1 positive cells expressed nestin. Co-expression of nestin and vimentin was observed in mature rat podocytes. Immunoelectron microscopy revealed nestin localization in the cell bodies and primary processes of podocytes. A similar expression pattern was observed for vimentin. In matured glomeruli, nestin was not expressed by mesangial and endothelial cells. In the newborn rat, early developing glomeruli (metanephric cap, metanephric vesicle, comma-shaped vesicle and S-shaped body phases) expressed nestin. In the capillary loop stage, Bowman's capsules also expressed nestin. Immunoelectron microscopy demonstrated that developing podocytes and endothelial cells in S-shaped phase glomeruli expressed nestin. Additionally, in immature glomeruli, the mesangial cells in capillary stage of glomerulus also expressed nexin. As in the rat, WT-1 positive cells in human glomeruli also expressed nestin and immunoelectron microscopy confirmed nestin expression in human glomerular podocytes. These results reveal that in normal condition nestin is expressed in several glomerular cell types at early stage of development and becomes confined to podocytes in mature glomeruli, thus implicating nestin in podocyte functions.
Beta-keratins form large part of the corneous material of scales and feathers. The present immunocytochemical study describes the fine distribution of scale- and feather-keratins (beta-keratins) in embryonic scales of the alligator and in avian embryonic feathers. In embryonic scales of the alligator both scale-keratin and feather-keratin can be immunolocalized, especially in the subperiderm layer. No immunolabeling for feather keratin is instead present in the adult scale after the embryonic epidermis is lost. The embryonic epidermis of feather folds into barb ridges while subperiderm or subsheath cells are displaced into two barbule plates joined to the central ramus. Subperiderm cells react with an antibody against feather keratin and with lower intensity with an antibody against scale keratin. The axial plate is colonized by barb ridge vane cells, which surround subperiderm cells that become barb/barbule cells. The latter cells merge into a branched syncitium and form the micro ramification of feathers. The lengthening of barbule cells derives from the polymerization of feather keratin into long bundles coursing along the main axis of cells. Keratin bundles in feather cells are however ordered in parallel rows while those of scales in both alligator and birds are irregularly packed. This observation indicates a different modality of aggregation and molecular structure between the feather keratin of subperiderm cells versus that of barbule/barbs. Barb vane ridge cells among barbule cells degenerate at late stage of feather development leaving spaces that separate barbules. Barb vane ridge cells contain alpha-keratin and lipids, but not beta-keratin. Cells of marginal plates do not contain beta-keratin, and later degenerate allowing the separation of barbs. The latter become isolated only after sloughing of the sheath, which cells contain bundle of keratin not reactive for both scale- and feather-keratin antibodies. The study confirms morphological observations and shows that subperiderm or subsheath cells differentiate into barb and barbule cells. The morphogenesis of barb ridges has to be considered as an evolutionary novelty that permitted the evolution of feathers from a generalized archosaurian embryonic epidermis.
The light microscopy, histochemical and TEM studies of the epididymis and the vas deferens revealed the presence of PAS positive secretory granules in the epithelial cells lining the lumen of these organs. One dimensional SDS gel electrophoretic pattern of luminal fluid proteins and the total protein content of the testis, three regions of the epididymis and the vas deferens of the lizard, Mabuya carinata were studied during breeding and nonbreeding season of the reproductive cycle. During breeding season, 25 protein bands in the testicular luminal fluid, 26 in the anterior epididymal luminal fluid and 28 in the middle and posterior epididymal luminal fluid were found. Ten new protein bands appeared in the anterior epididymal region whereas five new protein bands appeared in the middle region of the epididymis indicating regional difference in protein secretions of the epididymis. Vas deferens luminal fluid showed the highest number of protein bands (32) and the highest total protein content (9.07 mg/ml) compared to the testis and the epididymis. Four new protein bands appeared in the vas deferens. Number of protein bands in the luminal fluids of testis, epididymis and the vas deferens were significantly reduced during nonbreeding season compared to those of the breeding season. Consistent with the decrease in the number of protein bands, there was a significant reduction in the total protein concentration in all the tissue samples during nonbreeding season. The results indicate seasonal differences in number of proteins secreted and quantity of proteins in the luminal fluid of male reproductive tract of M. carinata. This is the first study in reptiles revealing appearance of new proteins in epididymis, and vas deferens by conducting simultaneous electrophoretic profile of testicular, epididymal and vas deferens luminal contents.
