Ultrastructural Cytochemical Study Research Articles

Intranuclear sphingomyelin is associated with transcriptionally active chromatin and plays a role in nuclear integrity

Sphingomyelin is one of the major phospholipids in the cell nucleus. However, its intranuclear distribution with regard to different functional nuclear domains as well as its possible involvement in the nuclear functional architecture remains to be elucidated. We carried out an ultrastructural cytochemical study of the intranuclear distribution of SM (sphingomyelin) using an in situ binding assay of neutral SMase (sphingomyelinase) conjugated to colloidal gold particles. The enzymatic labelling was carried out on ultrathin sections of different mammalian cells prepared by means of various fixation and resin-embedding protocols. Transmission electron microscopic analysis revealed preferential localization of SM within the PR (perichromatin region), a functionally important nucleoplasmic domain containing sites of pre-mRNA synthesis and processing. In the nucleolus, SM is mostly associated with the dense fibrillar component containing transcriptionally active ribosomal genes. Microinjection of enzymatically active SMase into living cells resulted in a rapid degradation of intranuclear structure. Our observations, supported by biochemical data, provide evidence for the involvement of SM in important nuclear functions. They bring additional information pointing out the PR as an essential functional nuclear domain. Furthermore, they suggest a role for SM in the internal nuclear architecture.

Distribution of acid phosphatases in the hypopharyngeal glands from workers, queens and males of a Brazilian stingless bee Scaptotrigona postica Latreille: an ultrastructural cytochemical study.

The present study reports the localization of acid phosphatase in the hypopharyngeal gland cells from workers (newly-emerged, nurse and forager), queens (newly-emerged and laying) and males (newly-emerged and mature for mating) of the Brazilian stingless bee, Scaptotrigona postica. The phosphatase activity varied in intensity and localization depending on the individual class, physiological age and the substrate used. In newly-emerged workers, the phosphatase-positive sites suggest the involvement of the enzyme with cellular differentiation that occurs in the presecretory phase, in nurse workers with protein synthesis and in forager workers with changes in cellular activity or glandular regression. In males mature for mating and laying queens, the positive sites are related to secretory activity, showing that the gland maintains some activity in spite of the regressive aspect. Of the substrates used, beta-glycerophosphate gave the least specific localization.

Oxidative Stress and Lipid Peroxides in the Retina of the BBZ/WOR Diabetic Rat: An Ultrastructural Cytochemical Study

Abstract Biochemical studies have documented a role for oxidative stress in complications of diabetes. Our previous cytochemical and immunocytochemical studies demonstrated increased levels of NADH oxidase in retinal endothelial cells and pericytes of the BBZ/Wor diabetic rat which coincided with disruption of the blood retinal barrier. Lipid peroxidation and damage to outer segments was demonstrated in retinas of a streptozotocin-induced rat model of diabetes. Xanthine oxidase has been proposed as a source of superoxide and hydrogen peroxide (H2O2) in oxidative stress mediated complications of diabetes; however, there are no studies which confirm the role and cellular sites of xanthine oxidase in complications of diabetes. In addition, cellular sites of lipid peroxides have not been documented in diabetic retinopathy. Xanthine oxidase was localized in retinas of BBZ/Wor diabetic rat by cerium based histochemistry using hypoxanthine and NADH as substrates.

Glycogen distribution in the larval salivary gland cells during the development of Drosophila melanogaster and Drosophila auraria: an ultrastructural cytochemical study

In this study, glycogen distribution during the development of larval salivary glands in two Drosophila species (D. melanogaster and D. auraria) was examined using the osmium tetroxide–potassium ferrocyanide technique at the ultrastructural level. Glycogen particles are found in big clumps in the salivary gland cells of D. melanogaster during the early third instar, mainly at the basal part of the cells; they are also found as scattered particles in the cytoplasm and very close to the apical plasma membrane during the secretory activity at this developmental stage. During the middle third instar, where the production of ‘glue’ secretory granules begins, glycogen particles are mainly scattered and they are seen near the Golgi complexes and secretory granules. No glycogen particles are seen during the late third instar and secretion of ‘glue’ secretory granules. Re-formation of glycogen begins after spiracle eversion. In the larval salivary gland cells of D. auraria, glycogen particles (scattered and in clumps) are seen in every developmental stage. In the larval fat body cells of both species, many huge clumps of glycogen particles are seen, independent of the developmental stage. These results indicate that glycogen provides an important source of energy during development of Drosophila salivary glands, but different species use glycogen at different metabolic pathways, according to their needs.

Ultrastructural cytochemical characterization of collagen-associated proteoglycans in the endometrium of mice

The decidual reaction in mice is characterized by the transformation of a specific population of endometrial fibroblasts into epithelioid cells, known as decidual cells. An important feature of decidualization in mice is a remarkable modification of the endometrial extracellular matrix. The present work is an ultrastructural cytochemical study of matrix with the purpose of analyzing the arrangement of collagen-associated proteoglycans (PGs) at various regions of nulliparous endometrium and of the antimesometrial decidua of mice using the cationic dye cuprolinic blue associated with enzymatic treatments with chondroitinase ABC, chondroitinase AC, and hyaluronidase. The staining with cuprolinic blue showed PGs as rods and granules of several sizes. Rods measuring 40-60 nm in length (named F2-rods) were apposed to thin collagen fibrils whereas granules were associated with thick collagen fibrils, particularly in the region occupied by mature decidual cells on the 7th day of pregnancy. The amount of granules was higher than that of F2-rods. Both F2-rods and granules were affected by chondroitinase ABC or AC treatment, indicating that they were PGs containing chondroitin sulfate and dermatan sulfate chains. However, the granules associated with thick collagen fibrils were more resistant to chondroitinase AC treatment than F2-rods, indicating the presence of dermatan sulfate chains that contain both L-iduronic and D-glucuronic acid sugar residues. We suggest that the differences of the nature and amount of PGs may be associated with the changes of the thickness of collagen fibrils observed during decidualization of the endometrium in the mouse.

Distribution and space-time relationship of proteoglycans in the extracellular matrix of the migratory pathway of primordial germ cells in mouse embryos

In this paper we present an in situ ultrastructural cytochemical study on the distribution and spatial-temporal expression of proteoglycans (PGs) in the extracellular matrix of the migratory pathway of mouse primordial germ cells (PGCs) during the different phases of migration, by the use of the cationic dye ruthenium hexammine trichloride (RHT). Embryos of 9, 10, 11 and 12 days of development were used. The treatment with RHT revealed PGs as electron dense layers, granules, and filaments. Whereas granules prevailed in the extracellular spaces of the migratory route during the whole migratory process, the amount of filamentous structures increased during the migration phase of PGCs. At the end of the migratory process the surface of the PGCs lost its reaction by RHT. There were differences in the size of the granules of PGs at the initial migratory period (9-day-old embryos) as compared with the other days of gestation. There was a strong reaction for PGs in the extracellular spaces, expressed as a meshwork of granules interconnected by filaments, as well as reaction on the basement membranes during the peak of the PGCs migration in 10-day-old embryos. These results support the hypothesis that these molecules may have an important role in the migration of PGCs, although the precise mechanism involved in this process is not yet clear.

Ultrastructural cytochemical study of proteoglycans in the endometrium of pregnant mice using cationic dyes

Decidualization in rodents is accompanied by remarkable modifications of both fibrillar and non-fibrillar components of the endometrial extracellular matrix. Biochemical studies have shown that the levels of synthesis of hyaluronic acid and sulfated glycosaminoglycans change during decidualization in rodents. As the rodent decidua has regions containing cells in different stages of decidual transformation, we decided to analyse, by an ultrastructural cytochemical technique, the distribution of proteoglycans (PGs) in each region of the decidua of mice on different days of pregnancy. Endometria of mice on days 4, 5 and 7 of pregnancy were processed for electron microscopy in the presence of safranin O, a cationic dye which preserves most of the tissue PGs. The endometrium of non-pregnant mice was used as control. We observed evident differences in the arrangement and distribution of the network of PGs between non-pregnant and 4-day pregnant endometria, as well as between different regions of pregnant endometria. The possible relationship between these modifications and cell transformation that occurs during decidualization is discussed.

Ultrastructural cytochemical study of enzymes expressed by signet ring cells in gastric cancer.

To study the ultrastructural localization of five marker enzymes (ALPase, ACPase, G6Pase, TPPase and CCOase) in gastric cancer signet ring cells to demonstrate their biologic behaviors. Five marker enzymes were examined in signet ring cells of seven gastric cancer patients by ultrastructural enzyme cytochemical techniques. The number of corresponding organelles and the activities of marker enzymes, especially ACPase and TPPase, increased, leading to stronger mucus synthesis, secretion and digestion in gastric cancer signet ring cells. There was a lack of collagenous fibers in the stroma around the cancer nests. Signet ring cell carcinoma is very invasive with metastasis rates due to the secretion of proteolytic enzymes.

The effects of high dose of parathyroid hormone on fetal osteoclasts and their precursors in vivo: An ultrastructural‐cytochemical study

It is not well known how the immediate precursors of osteoclast develop into osteoclasts in the fetus. This ultrastructural-cytochemical study was designed to clarify the formation process of the osteoclasts and their increased activities in the fetal mouse limb buds after administration of high dose parathyroid hormone (PTH). Twenty-four or forty-eight hours after the high doses of PTH were injected into amniotic fluid of the pregnant C3H mice, the femoral limb buds of embryos were dissected out. Tartrate-resistant acid phosphatase (TRAP) reactions were performed while preparing specimens for electron microscopy. Both control and PTH-given preosteoclasts and osteoclasts exhibited TRAP-positivities in dense bodies and vesicles. As effects of PTH, a binucleated preosteoclast of tandem fashion was observed. More osteoclastic hyperactivities were observed in the diaphyseal bone marrow. An osteoclast with a large cytoplasm exhibited two sets of clear zones and ruffled borders. Some osteoclasts demonstrated prominent amoeboid figures, while other osteoclasts developed large cytoplasmic vacuoles, which contained pieces of calcified chondroid bars. Our results revealed the progression of maturation from young preosteoclasts to osteoclasts. An existence of a peculiar binucleated preosteoclasts suggested one of the processes for multinucleation of the osteoclast. Quite remarkable osteoclastic hyperactivities were obviously the effects of high dose PTH. Our results also indicated the endophagocytic ability of the osteoclast. How PTH affected the osteoclasts and their precursors in the diaphyseal bone marrow can be speculated.

Alterations of Na +,K +-ATPase activity after hypoxia and reoxygenation in the perfused rat liver: an electron microscopic cytochemical study

Background/Aims: Using the cerium technique for ultrastructural cytochemical studies, Na +,K +-ATPase activity was investigated in hypoxic and reoxygenated rat liver. Methods: In the control group, the livers were perfused with oxygenated hemoglobin-free Krebs-Henseleit buffer for 1 h. For hypoxia (60 min), the flow rate of the perfusate was decreased and oxygen was replaced by nitrogen. For reoxygenation, the liver was reperfused under oxygenated conditions for 5 min after 60 min of hypoxia. Results: In control livers, a strong Na +,K +-ATPase activity was detected at the basolateral membrane of hepatocytes while the apical membrane forming the bile canaliculi did not display any staining. In hypoxic livers, Na +,K +-ATPase activity had ceased in the plasma membrane of hepatocytes. In reoxygenated livers, Na +,K +-ATPase was rapidly reactivated in the basolateral hepatic membrane. The membrane of blebs generated during the hypoxic phase also showed enzyme activity. In addition, a striking accumulation of reaction product could be observed in about 10% of the apical membranes lining the bile canaliculi. Conclusion: The results indicate a plasticity of the Na +,K +-ATPase in hypoxic and reoxygenated rat liver.

“All‐or‐none” cytochrome C oxidase positivity in mitochondria in chronic progressive external ophthalmoplegia: An ultrastructural—cytochemical study

Teased single muscle fibers from 6 patients with chronic progressive external ophthalmoplegia (CPEO) showed a segmental defect in cytochrome c oxidase (COX) activity. On ultrastructural--cytochemical examination, the majority of mitochondria in COX-positive segments were COX-positive, whereas all mitochondria in COX-negative segments were COX-negative. This "all-or-none" COX positivity in mitochondria in CPEO with deleted mitochondrial DNA can be explained by the "threshold effect," which induces the tissue-specific involvement and clinical heterogeneity.

Remodeling of the Mouse Endometrial Stroma during the Preimplantation Period

An ultrastructural cytochemical study of acid phosphatase activity performed in mouse endometrium on the second day of pregnancy showed that stromal cells which were heavily labeled by the cytochemical reaction had disarranged organelles. On the other hand, the cytoplasm of several stromal cells had collagen-containing phagosomes that were also labeled, indicating that the collagen fibrils were being digested by lysosomal enzymes. It is suggested that cell death and phagocytosis of collagen are events of the remodeling of the mouse endometrium that occur prior to decidualization.

Collagen-associated sulphated proteoglycans. Ultrastructure after formaldehyde-cetylpyridinium chloride fixation

In the course of an ultrastructural cytochemical study of intracellular sulphated proteoglycans involving the addition of cetylpyridinium chloride in the primary aldehyde fixative, a remarkable ultrastructural preservation of the collagen-associated sulphated proteoglycans was observed. Together with the preservation of their localization among the collagen fibrils (with, for some of them, a 50 nm periodic association with d-bands) and of their native elongated shape, previously observed under similar technical conditions, these stick-shaped and chondroitinase ABC-sensitive proteoglycans exhibited a typical pattern with several dense longitudinal parallel tracks (periodicity: 3-4 nm) not described as yet. Readily observable without high iron diamine-staining, the morphology of these cetylpyridinium chloride-precipitated and collagen-associated polyanions was particularly enhanced after incubation in the diamine solution which ascertained their sulphate content. Such a common ultrastructural organization with parallel tracks for both intracellular (i.e., in eosinophilic polymorphonuclear cells and Kurloff cells) and extracellular CPC-precipitated sulphated proteoglycans could correspond to intrinsic properties of the complexed molecules and could be related to 'double track' proteoglycans observed under other technical conditions in basement membranes.

Cytogenetic findings in adult acute leukemia and myeloproliferative disorders with an involvement of megakaryocyte lineage

Cytogenetic analyses were performed on 12 adult patients with abnormal megakaryoblastic proliferation which was detected by ultrastructural cytochemical study (platelet peroxidase) and platelet-megakaryocytes-specific monoclonal antibodies (TP-80, Plt1, AN51, and KOR-77). The patients consisted of two patients with myelodysplastic syndromes (MDS), three with acute megakaryoblastic leukemia (AMKL), six with megakaryoblastic transformation in Philadelphia-positive chronic myelogenous leukemia (CML-meg-BC), and one case of chronic myeloproliferative disorder (CMPD). Among them, an inversion of the long arm of chromosome 3 [inv(3)(q21q26)] was found in one AMKL patient with a normal platelet count. Chromosome change at band 3q26 was also found in one MDS patient without thrombocythemia. Furthermore, the long arm of chromosome 13, where rearrangements in myelofibrosis are clustered (13q12----q22) was seen in one MDS patient. Trisomoy of chromosome 19 was found in one AMKL patient and three CML-meg-BC patients. These findings indicate that cytogenetic abnormalities involving 3q26, 13q, and trisomy 19 are associated with hematologic neoplasia with megakaryocytic lineage in adult patients, although these abnormalities were not related to the survival of the patients. During the period of this study, two acute myelogenous leukemia patients (AML-M2 and AML-M5b) with chromosome rearrangements at band 3q21 and thrombocythemia were found, indicating that chromosome abnormality at band 3q21 is related to quantitative platelet dysfunction, whereas that at 3q26 is related to hematologic malignancies with a proliferation of megakaryocytic lineage.

Chromatin arrangement in mouse sperm nuclei: an ultrastructural cytochemical study.

The arrangement of mouse sperm nuclei chromatin and, in particular, of DNA has been studied by electron microscopic cytochemistry. It had been previously shown that, after a Feulgen-type reaction using an osmium ammine complex (OAC), the OAC-stained DNA was distributed in a spotted pattern in the nucleus (Biggiogera: Basic Appl Histochem 30:501-504, 1986). The present chapter shows that this pattern is characteristic of mouse spermatozoa from testis to vas deferens, with the exception of some testicular spermatozoa, in which DNA was homogeneously stained. DNase digestion of thin-sectioned nuclei resulted in a distribution of residual material complementary to the pattern of the unstained zones after the OAC reaction. These findings are discussed considering the role of -S-S- crosslinks, characteristics of this extremely condensed chromatin, in limiting the availability of DNA to acid hydrolysis.

De novo neuromuscular junction formation on human muscle fibres cultured in monolayer and innervated by foetal rat spinal cord: ultrastructural and ultrastructural--cytochemical studies.

Ultrastructural features of neuromuscular junction formation and transverse tubule development were studied utilizing a newly developed model in which human muscle fibres cultured in monolayer are innervated by foetal rat spinal cord with dorsal root ganglia attached. At early innervation (7-10 days), when distinct 'boutons' are contacting muscle fibres, the contacts of nerve terminals with the muscle fibres are, ultrastructurally, superficial and unorganized, and there is no basal lamina-like material between nerve terminals and muscle fibres. A bouton consists, ultrastructurally, of a cluster of small nerve terminals contacting the muscle fibre. At 2-3 weeks of innervation, shallow 'beds' are formed on the muscle fibre just beneath nerve terminals, and occasionally there are irregular and miniscule fragments of basal lamina-like material in the cleft. There is no Schwann cell apposing the nerve terminal at this stage of innervation. After 4-5 weeks of innervation there is more definite basal lamina material in the cleft and suggestive postsynaptic plasmalemmal densities and invaginations. However, there is no Schwann cell apposing the nerve terminal at this stage. At 6-8 weeks of innervation, deep postsynaptic folds are present, a Schwann cell apposes the nerve terminal, and basal lamina surrounds the entire muscle fibre. At all four stages of innervation examined, ultrastructural cytochemistry of alpha-bungarotoxin binding reveals that nicotinic ACh receptors are located exclusively at the neuromuscular junctions. After 1-2 weeks of innervation, very few lanthanum-positive transverse tubules are observed and only in close proximity to the surface membrane. After 3 weeks of innervation, more lanthanum-positive tubules are present, and they are located deeper within the muscle fibre. Five weeks after innervation, somewhat more elaborated tubules (but no lateral sacs) appear, and honeycomb structures are often present. After 6-7 weeks of innervation the tubular system is very elaborate and lateral sacs are present. Hence, this study describes consecutive stages of the formation of neuromuscular junctions and transverse tubules in innervated cultured human muscle, and provides an important basis to which similar studies related to the diseased human muscle can be compared.

Structure and function of the nematosome

The ultrastructural morphology of human placental and mouse placental nematosomes has been investigated. The description includes a three-dimensional analysis of the shape of the organelles based on serial sectioning, measurements of the repeat distance of the subunit fibre of the organelle derived by optical diffraction analysis and the results of an ultrastructural cytochemical study designed to test whether the organelle contains nucleic acid.

Ultrastructural Cytochemistry of Congenital Basophilic Leukemia

A newborn infant was diagnosed as having congenital basophilic leukemia, an extremely rare disorder, by examination of cord blood. An ultrastructural cytochemical study was conducted on the blasts of the infant. Granules of basophil blasts showed ultrastructural characteristics of immature basophil granules and were similar in size to those of normal basophil granules but were fewer in number in each profile. Histochemical studies showed weak reactions for acid mucopolysaccharides and neutral glycoconjugates in the granules, but they were negative for peroxidase. Acid phosphatase was markedly positive. The findings of congenital basophilic leukemia are different from those of basophils in chronic myelocytic leukemia. The patient underwent induction therapy with vincristine and prednisone and has been in complete remission for 21 months.

Mannose-specific binding sites in synoviocytes of rat synovial membrane: An ultrastructural-cytochemical study

Mannose-specific binding sites in the two types of synoviocytes (A and S) from the synovial membrane of normal rat knee joints were studied by an ultrastructural-cytochemical method using horseradish peroxidase (HRP) as a ligand. The A cells showed mannose-specific binding sites for HRP which were localized in coated pits and coated vesicles (110 nm) as well as in smooth-walled vesicles, vacuoles, and tubules. No mannose-specific binding sites for HRP were detected in the S cells. Together with previous observations on absorption of intraarticularly injected HRP in type A and type S synoviocytes, the present results strongly support the interpretation that the A cells internalize HRP by receptor-mediated endocytosis and that the S cells internalize HRP by fluid-phase endocytosis only. The present results agree with the suggestion that mannose-specific binding sites consist of two pools, the one concerned with internalization of ligand and the other with intracellular targeting of internalized ligand.

Demyelination-induced plasticity in the axon membrane: An ultrastructural cytochemical study of lead neuropathy in the rat

We examined the distribution of ferric ion-ferrocyanide stain (a marker for excitable regions of myelinated fibers) in the lead-induced demyelinating neuropathy of the rat. By electron microscopy, we found that paranodal degeneration resulted in spreading of the reaction product from nodal to internodal axolemma. During repair, nodal-like stained areas formed at the contact zones between preremyelinating Schwann cells. These data suggest that the location and extent of excitable axonal regions are influenced by axoglial relationships. Additionally, some fibers displayed staining at paranodal axolemma adjacent to demyelinated segments, suggesting it might be an alternative site for impulse generation in demyelinated fibers.