Confocal laser scanning microscopic analysis of ectopic sublingual gland‐like tissue inside the hamster submandibular gland

Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3′‐diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO‐positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous‐like cells were found in resin‐embedded semi‐thin sections of the DAB‐reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance‐mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity‐negative secretory granules and seromucous cells with PO activity‐positive spot‐like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron‐dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron‐dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland‐like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct. Microsc. Res. Tech. 76:1284–1291, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructure of submandibular salivary glands of mouse: TEM and HRSEM observations

The fine structure of submandibular glands of mouse were analyzed using light microscopy (LM), high resolution scanning electron microscopy (HRSEM), and transmission electron microscopy (TEM) methods. For LM, the specimens were embedded in Spurr resin, stained by toluidin blue solutions. For TEM, the tissues of submandibular salivary glands were fixed with modified Karnovsky solution and postfixed with osmium tetroxide. For HRSEM, the tissues were fixed with 2% osmium tetroxide solution in 1/15M sodium phosphate buffer (pH 7.4). The samples were immersed successively in dymethylsulphoxide and freeze cracked. The maceration was made in diluted osmium tetroxide for 24-48 h. The samples were examined by high resolution scanning electron microscopy. The intracellular components of acinar and ductal cells revealed clearly the Golgi apparatus, rough endoplasmic reticulum, secretory granules, and mitochondria. The end bulbs of Golgi lamellae and flattened cisterns of rough endoplasmic reticulum showed the luminal surface. A few mitochondria were identified intermingling between the rough endoplasmic reticulum and the mitochondriales cristae in three-dimensional HRSEM images. Secretory granules were numerous and presented different sizes. Small granules of ribosomes were attached on cistern surface, measuring 20-25 nm in diameter. Numerous arranged microvilli were found on the luminal surface of secretory canaliculus. The contact surfaces of acinar cells revealed complicated interdigitations by cytoplasmic processes. The mitochondria of duct cells were disposed vertically and surrounded by basal infoldings of plasma membranes. Basement membrane showed a spongy-like structure having an irregular surface with various strands and meshes of fine collagen fibrils.     

Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over‐expression of enhanced green fluorescent protein‐tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge‐coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K⁺ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.     

Morphological filter improve the efficiency of automated tracking of secretory vesicles with various dynamic properties

Membrane trafficking is a very important physiological process involved in protein transport, endocytosis, and exocytosis. The functions of vesicles are strongly correlated with various spatial dynamic properties of vesicles, including their types of movements and morphology. Several methods are used to quantify such dynamic properties, but most of them are specific to particular populations of vesicles. We previously developed the so-called PTrack system for quantifying the dynamics of secretory vesicles near the cell surface, which are small and move slowly. To improve the system performance in quantifying large and fast-moving vesicles, we firstly combined morphological filter with two-threshold image processing techniques to locate granules of various sizes. Next, Kalman filtering was used to improve the performance in tracking fast-moving and large granules. Performance evaluation by using simulation image sequences shown that the new system, called PTrack II, yields better tracking accuracy. The tracking system was validated using time-lapse images of insulin granules in βTC3 cells, which revealed that PTrack II could track better than PTrack, averaged accuracy up to 56%. The overall tracking results indicate that PTrack II is better at tracking vesicles with various dynamic properties, which will facilitate the acquisition of more-complete information on vesicle dynamics. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Cytochemistry of sialoglycoconjugates,lysozyme, and β‐defensin in eccrine glands of porcine snout skin as studied by electron microscopy

In most mammals except for humanoid primates, eccrine glands are confined to the skin of a series of specific body regions. Sialic acids and antimicrobial substances exhibit various functional properties and serve as a component of nonspecific defense against micro‐organisms, respectively. In this study, the distribution of these moieties was studied by electron microscopic histochemical methods. The eccrine glandular acini consisted of two types of dark cells as well as clear cells. The secretory granules and Golgi apparatus of both types of dark cells contained sialic acid residues linked to α2‐6Gal/GalNAc. On the other hand, sialoglycoconjugates with Siα2‐3Galβ1‐4GlcNAc sequence were confined to those of the Type II dark cells. In addition, lysozyme and β‐defensin were mainly detected in the secretory granules of the Type II dark cells. These secretory products may create a defensive barrier against microbial invasion and play an essential role in preservation of the integrity of porcine snout skin as a sensory organ. Microsc. Res. Tech. 2013. © 2012 Wiley Periodicals, Inc.     

Mitotic cell division in the extraadrenal chromaffin system of various species

Mitotic activity often has been reported in embryonic and fetal sympathetic neuroblasts, principal sympathoblasts, and primitive sympathetic cells in various species at different stages of development. Postnatal adrenal medullary cells also are known to undergo mitosis, but such dividing capabilities rarely have been observed in the true postnatal extraadrenal chromaffin system. Although few in number, this work nevertheless has clearly identified such cells in varying stages of the mitotic cycle in the young dog, Syrian hamster, mouse, rabbit, and rat. The dividing cells were noted in paraaortic chromaffin organs, paraganglia, and within the inferior mesenteric ganglion as well. They displayed the morphological character usually associated with their adrenal medullary catecholaminergic counterparts, including numerous dense-cored vesicles known to be the harbingers of catecholamines and various peptides. Nerve endings were not noticed upon the mitotic cells. The phenomenon of dividing extraadrenal chromaffin cells augments existing data and perhaps suggests that these cells are more endocrine than neural in type and subservient to the adrenal medulla in its classic endocrine function.     

Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: a study using photonic force microscopy

It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 microm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.     

Fine structure of the male accessory glands of Triatoma rubrofasciata (De Geer, 1773) (Hemiptera, Triatominae)

Male of Triatoma rubrofasciata has four elongated sac-like reproductive mesodermic accessory glands, lined by an inner single layer of secretory cells, with basal plasma membrane infolds and short apical microvilli, and externally enveloped by a thin visceral muscle layer. The secretory cells have a well-developed rough endoplasmic reticulum, Golgi complex, mitochondria, and secretory granules. In one day old adult the gland cells are poorly developed, presenting small, electron-transparent secretory granules scattered among the rough endoplasmatic reticulum, whereas in three days old adult these cells have the cisternae of the rough endoplasmatic reticulum varing size degree, filled with granular electrondense content. In five days old males the secretory granules increase in diameter, being released to the gland lumen. Therefore, there is an increase of the secretory activity according to male maturation.     

Cytopathological changes induced by selected Brazilian flaviviruses in mouse macrophages

Scanning and transmission electron microscopy were used to analyse the ultrastructure of peritoneal mouse macrophage cells infected with Brazilian flavivirus (yellow fever, Rocio, Bussuquara and Saint Louis encephalitis viruses). Macrophage cells collected 3 days after viral infection had a flattened shape, with an increased number of large spikes of cytoplasm prolongations, giving an appearance of hairy cells. Cytopathological changes to the macrophage cells were similar regardless of the infecting flavivirus. Rough and smooth endoplasmic reticulum of the macrophage cells infected with flavivirus were abundant, hypertrophic and enlarged. A large number of free ribosomes were seen in the cytoplasm of these infected cells. Spherical particles approximately 50–70 nm in diameter, some of which were empty, were observed in the cytoplasm, generally inside vesicles. These particles probably correspond to viral particles.     

Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitation

The real-time uptake of serotonin, a neurotransmitter, by rat leukemia mast cell line RBL-2H3 and 5-hydroxytryptophan by Chinese hamster V79 cells has been studied by fluorescence lifetime imaging microscopy (FLIM), monitoring ultraviolet (340 nm) fluorescence induced by two-photon subpicosecond 630 nm excitation. Comparison with two-photon excitation with 590 nm photons or by three-photon excitation at 740 nm shows that the use of 630 nm excitation provides optimal signal intensity and lowered background from auto-fluorescence of other cellular components. In intact cells, we observe using FLIM three distinct fluorescence lifetimes of serotonin and 5-hydroxytryptophan according to location. The normal fluorescence lifetimes of both serotonin (3.8 ns) and 5-hydroxytryptophan (3.5 ns) in solution are reduced to approximately 2.5 ns immediately on uptake into the cell cytosol. The lifetime of internalized serotonin in RBL-2H3 cells is further reduced to approximately 2.0 ns when stored within secretory vesicles.     

Statistical analysis of the quantal basis of secretory granule formation

ABSTRACT The size distribution of vesicles exocytosed from secretory cells displays quantal nature, vesicle volume is periodic multi‐modal, suggesting that these heterogeneous vesicles are aggregate sums of a variable number of homogeneous basic granules. Whether heterogeneity is a lumping‐together artifact of the measurement or an inherent intra‐cell feature of the vesicles is an unresolved question. Recent empirical evidence will be provided for the quantal nature of intra‐cell vesicle volume, supporting the controversial paradigm of homotypic fusion: basic cytoplasmic granules fuse with each other to create heterogeneously sized vesicles. An EM‐algorithm‐based method is presented for the conversion of multi‐modal to quantal data that provides as by‐product estimates of means and variances of basic granule packaging. Microsc. Res. Tech. 77:1–10, 2014. © 2013 Wiley Periodicals, Inc.     

Ultrastructural immunocytochemistry of the adenohypophysis in the rat: A review

Immunocytochemistry has made great strides in the morphology of endocrine glands, especially the adenohypophysis, because the localization of hormones can be clearly demonstrated by this method in the microscopic preparations both for light and electron microscopy. In the adenohypophysis, electron microscopic immunocytochemistry is useful for identifying the producer cell of each hormone. The second contribution is its application to the cell biology of secretion mechanisms. The pituitary hormones, their precursors, derivatives, and fragments were artificially synthesized and their antibodies were produced. Using these antibodies the intracellular sites of synthesis, condensation, processing, and sorting were studied under the electron microscope. The ultrastructure of each cell organelle and its alteration due to the changing function was studied. It was proved that the intracisternal granules in the thyroidectomy cells contain thyroid-stimulating hormone (TSH). The trans-Golgi network or GERL contains a peculiar supporting structure, intracisternal skeleton. Transport of secretory granules may be performed in relation to the microtubules, actin, and some related substances. The most frequently observed mode of hormone release in the adenohypophysis is exocytosis. Sometimes multigranular exocytosis occurs. Vesiculation of membrane around the secretory granules often occur inward or outward. The inward vesiculation forms pinocytotic vesicles, through which the membrane material may be retrieved. The outward vesiculation forms vesicle-like fragments of cytoplasm being discarded to the extracellular space. By these mechanisms the surface area of the cell is maintained constantly.     

Cell secretion machinery: studies using the AFM

A new field in biology, 'nano-cell biology', has emerged from the successful use of force microscopy in understanding the structure and dynamics of cells and biomolecules, at nm resolution and in real time. Atomic force microscopy, in combination with conventional tools and approaches (electron microscopy, electrophysiology, X-ray diffraction, photon correlation spectroscopy, mass spectroscopy, biochemistry, and molecular biology), has revealed for the first time, the universal molecular machinery and mechanism of secretion in cells. Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them, dock and fuse at plasma membrane-associated supramolecular structures called Porosome, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release utilize a highly regulated secretory process. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the porosome as the universal secretory machinery, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution into artificial lipid membrane, have been determined. The molecular mechanism of secretory vesicle swelling, and the fusion of opposing bilayers, i.e., the fusion of secretory vesicle membrane at the base of the porosome membrane, has also been resolved.     

Morphology and cytochemistry of acinar secretory granules in normal and isoproterenol-treated rat submandibular glands.

Several fixation procedures have been utilized in a fine-structural study of rat submandibular glands with the aim of correlating the morphology of granule substructure with its composition. Procedures included fixation with aldehyde in a variety of buffers, with or without fixation additives such as tannic acid or calcium chloride. Both immersion and perfusion fixation studies were performed. Osmium tetroxide postfixed tissue was compared with non-postfixed tissue. Thin sections were stained for carbohydrate-containing constituents by either periodic acid-thiocarbohydrazide-silver proteinate or tannic acid-ferric chloride sequences. The morphology and cytochemistry of acinar secretory granules were highly dependent upon the fixation procedure utilized. In postfixed tissue, fine filaments and vesicles were the major granule constituents. Filaments often aggregated into fibrils in fixatives containing phosphate buffer or calcium, whereas vesicles were prominent with fixatives containing collidine buffer. Tannic acid, as a fixation additive, imparted enhanced density to the peripheral rim of aggregated filaments. If post fixation was eliminated, an amorphous meshlike material was the major granule component. This stained readily with methods for carbohydrate whereas the granules of post-osmicated tissue did not. Following chronic isoproterenol treatment the degree of filament aggregation in postfixed tissue was augmented, and this corresponded to an increase in amount of material stainable for carbohydrate in non-post-osmicated tissue.     

Freeze-fracture organization of hair cell synapses in the sensory epithelium of guinea pig organ of Corti

The fine structure of both the afferent and efferent hair cell synapses in the sensory epithelium of guinea pig organ of Corti was examined by freeze-fracture electron microscopy. In the afferent synapse, barlike aggregates of intramembrane particles (IMPs) of about 10 nm in diameter were seen on the P-face of the afferent presynaptic membrane directly beneath the presynaptic dense projection which is located in the active zone of the presynaptic membrane. Small and large depressions have been seen on the presynaptic membrane. The former were observed in the proximity of the barlike aggregates, while the latter were observed some distance from the aggregate. In outer hair cells, IMPs of about 10 nm in diameter were seen on the P-face of the afferent postsynaptic membrane at a density of 3,000/μm². In the efferent synapse, many aggregates composed of from several to tens of large IMPs of 13 nm in diameter were observed on the presynaptic membrane. These aggregates were localized to small membrane depressions, which tended to be deeper as particle number per aggregate increased. Dense populations of IMPs of about 9 nm in diameter were observed on the P-face of the efferent postsynaptic membrane at a density of 4,000/μm². A fenestrated subsynaptic cistern completely covers the efferent postsynaptic membrane. Moreover, the subsynaptic cistern spans several efferent postsynaptic membranes when efferent synapses are gathered in a group. In the afferent and efferent synapses of hair cells, specializations of the synaptic membranes were represented by marked aggregates characteristic of IMPs. In the efferent synapse, IMP movement inside the synaptic membrane was proposed in relationship to retrival of synaptic vesicle membrane. Structural relationship between the subsynaptic cistern and efferent postsynaptic membrane was revealed.     

Electron microscopic study of the morphology and morphogenesis of virus of hemorrhagic fever with renal syndrome

The morphology and morphogenesis of the virus of hemorrhagic fever with renal syndrome (HFRS) and the associated ultrastructural changes in neurons of the infected mouse brain were examined by electron microscopy. The primary location of the infection in large neurons was in the Golgi apparatus, which had highly proliferated laminar and vesicular profiles. A small number of matured virus particles were found later individually or in small groups within the distended Golgi cisternae and vesicles. Most of the virus particles were round, oval, or elongated and measured about 70–110 nm in diameter. A lipid bilayered viral envelope with an external fringe of surface projections could be resolved at high magnification. The maturation (budding) of the virus occurred exclusively at smooth membrane vesicles, and predominantly at membranes in, or adjacent to, Golgi cisternae. Viral inclusion bodies containing fine filamentous material were seen frequently in close proximity to sites of virus maturation. The known morphological and morphogenetic characteristics of the virus particles observed in infected mouse brain gave further evidence for taxonomic identification of HFRS virus as a member of the family of Bunyaviridae.     

Dynamics of subcellular organelles, growth hormone, Rab3B, SNAP-25, and syntaxin in rat pituitary cells caused by growth hormone releasing hormone and somatostatin

Rab3B is involved in the exocytosis of synaptic vesicles and secretory granules in the central nervous system and the anterior pituitary cells. The aim of this study was to elucidate both the role of rab3B in GH secretion and the mutual relationship of rab3B and SNARE proteins. Adult male rats were injected intravenously with 10 microg of growth hormone releasing hormone (GHRH) or 10 microg of somatostatin (SRIF). Untreated rats were used as controls, and their pituitary glands were sectioned for histochemical examination. Rab3B is localized on the limiting membrane of the secretory granules and the cytosol. Confocal laser scanning microscopic observation of immunohistochemical double staining of rab3B and GH revealed that immunoreactivity of rab3B increased in GHRH-treated rats and decreased in SRIF-treated rats. Confocal laser scanning microscopic observation of immunohistochemical double staining of SNAP-25, syntaxin, and rab3B revealed the co-localization of rab3B and these SNARE proteins in GHRH-treated rats, and their dissociation in SRIF-treated rats. These results suggest that rab3B plays a principal role in GH secretion in the anterior pituitary cells and that SNAP-25 and syntaxin act as co-workers with rab3B in the functional regulation of GH secretion.     

Cell biology of the adipokinetic hormone-producing neurosecretory cells in the locust corpus cardiacum.

The adipokinetic cells are neuron-like unipolar cells, the cell bodies and cell processes of which are intermingled within the glandular part of the corpus cardiacum. In Schistocerca gregaria, they produce two adipokinetic hormones, AKH-I and -II, whereas in Locusta migratoria an additional hormone, AKH-III, is present. The three AKHs are produced by the same cells and are co-localized in secretory granules. The biosynthesis and processing of the AKH prohormones to the bioactive hormones, which has been elucidated in detail for AKH-I and -II in S. gregaria, takes less than 75 min and goes on continuously. In older locusts in particular, the adipokinetic cells contain intracisternal granules, widely dilated cisternae of the rough endoplasmic reticulum, which function as stores of prohormones of AKH-I and -II, not of AKH-III. The adipokinetic cells are subjected to regulation by a number of neural and humoral substances, neural influences coming from secretomotor cells in the lateral part of the protocerebrum. Flight activity is the only natural stimulus unequivocally shown to induce the release of AKHs, which in L. migratoria results in parallel secretion of all three AKHs. During secretory stimulation, young secretory granules containing newly synthesized hormones are preferentially released over older granules. Secretory stimulation is not accompanied by a clear increase in the levels of the AKH mRNAs and the AKH prohormones and in the rate of synthesis of the (pro-)AKHs. Apparently, a coupling between release and biosynthesis of the AKHs in the adipokinetic cells is very loose or does not even exist.     

Effects of loud noise exposure on mouse myocardium: a comparison with the rat

Loud noise is an environmental stressor of everyday life, which affects different organs and apparati, in particular the cardiovascular system. We have already reported that noise exposure produces significant alterations in the rat myocardium, consisting of mitochondrial damage, which is evident as lysis of the cristae and dilution of the matrix. Since there are high similarities between mouse and human species, the aim of our study was to investigate the effects of acute noise exposure on the mouse heart. We found that noise exposure affects mouse myocardium at similar subcellular sites to those already described in the rat; nonetheless, quantitative analysis of the percentage of altered mitochondria in both species disclosed a clear difference between mouse and rat myocardium, which strongly suggests a different sensitivity to noise stimulus. We hypothesize that the species differences on the extent of myocardial alterations here observed might be due to the zonal pattern of cardiac noradrenergic receptors, which should be the final effectors for noise-induced myocardial changes.     

Mineralization patterns in elasmobranch fish

This article reviews current findings on the organic matrix and the mineralization patterns in elasmobranchs, including an analysis of the role of the dental epithelial cells and the odontoblasts during odontogenesis. Our electron micrographs demonstrated that tubular vesicles limited by a unit membrane occupied the bulk of the elasmobranch enameloid matrix during the stage of enameloid matrix formation. It is likely that the tubular vesicles originated from the odontoblast processes. Two types of electron-dense fibrils, with cross-striations at intervals of approximately either 17 nm or 55 nm, respectively, were detected in the enameloid matrix. These data suggest that odontoblasts were strongly involved in enameloid matrix formation and in initial enameloid mineralization. Two types of odontoblasts, dark and light cells, were recognized during the stage of dentinogenesis. The light cells contained numerous mitochondria, intermediate filaments, and microtubules that extended their processes into the dentin. The dark cells possessed a well-developed Golgi apparatus and many cisternae in the rough endoplasmic reticulum, which suggests that the dark cells are involved in the formation of dentin. The inner dental epithelial (IDE) cells exhibited a well-developed Golgi apparatus, many mitochondria, cisternae of smooth endoplasmic reticulum, vesicles, vacuoles, and granules during the mineralization and maturation stages. During the stages of mineralization and early maturation, ACPase-positive granules were visible in the IDE cells and ALPase and Ca-ATPase activities were found at the lateral and proximal cell membrane of the IDE cells, suggesting that the IDE cells are involved in the removal of enameloid organic matrix and in the process of mineralization during later stages of enameloid formation. Our data indicate that elasmobranch enameloid is distinct from teleost enameloid, based on its organic content, on the mechanisms of its mineralization, and on the role of IDE cells concerning enameloid formation.