Schistosoma mansoni: Development of acetabular glands of cercaria at ultrastructural level

Cercariae of Schistosoma mansoni in daughter sporocysts in Biomphalaria glabrata were studied with the electron microscope to observe the maturing process of their acetabular glands. The undifferentiated acetabular gland displays its enlarged basal area (fundus) and extended narrow process (duct) before other organ systems are recognized. Its fundus contains a prominent nucleus and subcellular organelles typical of active secretory cells.The secretory granules of the postacetabular glands are formed in a milieu of dilated rough endoplasmic reticulum and Golgi. Two morphologically different secretory granules are produced: (1) homogeneously granular ones, and (2) other granular ones with electron dense bodies in their matrices. Mostly, the homogeneously granular ones are produced first in the fundus and are forced into the ducts as the other type is formed.The secretory granules of preacetabular glands are formed from translucent vacuoles which arise from an environment of endoplasmic reticulum and Golgi. Two morphologically different secretory granules are produced: (1) one type has a homogeneous dense matrix, and (2) the other type has a less dense matrix containing electron-lucid bodies.The duct of an undifferentiated acetabular gland has either filamentous material or microtubules dispersed in its cytoplasm. Once microtubules are formed, they persist during the life of the cercaria. The microtubules are believed to have possibly two functions: (1) to support the long duct, and (2) to assist the movement of the secretory granules into the channels of the ducts where they remain until released during host penetration.Few of the subcellular organelles associated with secretory granules formation are seen in the duct except the area in close proximity to the fundus; thus, the few secretory granules produced in the duct are in this region.     

Cellular compartmentation of lysozyme and alpha-amylase in the mouse salivary glands. Immunogold approaches at light and electron microscopy level

The research was planned to study the subcellular distribution of enzymatic secretory products within the secretory structures of the mouse major salivary glands at light and electron microscopy level by immunogold silver stain (IGSS) technique and double-sided post-embedding immunogold binding and silver amplification in order to speculate about their compartmentation. In particular, we experimented the above immunogold labeling approaches to localize the lysozyme and to verify its distribution patterns in relation to another secretion enzyme, alpha-amylase. Co-presence of lysozyme and alpha-amylase was observed in the convoluted granular tubule cells of the submandibular gland and in the demilunar cells of the sublingual gland as well as in the electron-dense regions of the mottled secretory granules in the parotid gland. Exclusive binding patterns of lysozyme were observed in the acinar cells of the submandibular and sublingual glands where alpha-amylase did not occur.     

Nerve-induced secretion of parotid acinar granules in cats

Summary Electron microscopy of cat parotid glands revealed great heterogeneity in the secretory granules of normal unstimulated acinar cells. Electrical stimulation of the parasympathetic nerve to the gland evoked a copious flow of parotid saliva which was accompanied by an extensive depletion of the secretory granules from the acinar cells. Exocytosis was captured as it was occurring by means of perfusion-fixation, and showed that the events occur in a conventional manner. Stimulation of the sympathetic nerve caused only a very small flow of saliva, and no acinar degranulation was detected. It can be concluded that the parasympathetic secretomotor axons provide the main drive for parotid acinar degranulation in the cat. This contrasts with the rat in which sympathetic impulses provide the main stimulus for parotid acinar degranulation. These dissimilarities serve to emphasise how extensively species differences may influence autonomic responses in salivary glands.     

Exocytosis in living salivary glands: direct visualization by video-enhanced microscopy and confocal laser microscopy

Although exocytosis is widely believed to involve granule movement, membrane fusion and the emptying of granule content, direct study of these processes has been difficult in living cells because of the limited resolution of conventional light microscopy. Using video-enhanced microscopy and confocal laser microscopy, we have now studied these processes in living rat parotid and submandibular gland acinar cells. Under a differential interference contrast (DIC) microscope equipped with a CCD camera and a high speed image processor, secretory granules were in general stationary even after secretory stimulation with isoproterenol (IPR). Following IPR stimulation, however, there were abrupt changes in light intensity of secretory granules, and many granules disappeared. Confocal microscopy was then performed to confirm whether the observed changes in granules were related to membrane fusion and content release. For this, cells were perfused with the fluid-phase tracer Lucifer Yellow; confocal images thus obtained clearly demonstrated the appearance of fluorescence in omega-shaped invaginations of the apical plasma membrane which corresponded to the sites at which changes were observed in DIC images. The time sequence analyses of confocal images showed that there was a repetitive appearance and disappearance of omega-shaped fluorescent foci at the apical plasma membrane until most of the granules were depleted. During this time, there did not appear to be any significant expansion of the apical plasma membrane and if endocytic uptake of the tracer occurred, it was below the limit of detection. These observations provide new insights into the exocytotic process in salivary glands and are at variance in some respects with previous interpretations made from electron microscopy.     

Monoclonal Antibodies to the Esophageal Glands and Stylet Secretions of Heterodera glycines

Three monodonal antibodies (MAbs) that bound to secretory granules within the subventral esophageal glands of second-stage juveniles (J2) of the soybean cyst nematode (SCN), Heterodera glycines, were developed from intrasplenic immunizations of a mouse with homogenates of SCN J2. Two MAbs to the secretory granules within subventral glands and one MAb to granules within the dorsal esophageal gland of SCN J2 were developed by intrasplenic immunizations with J2 stylet secretions. Stylet secretions, produced in vitro by incubating SCN J2 in 5-methoxy DMT oxalate, were solubilized with a high pH buffer and concentrated for use as antigen. Three of the five MAbs specific to the subventral esophageal glands bound to stylet secretions from SCN J2 in immunofluorescence and ELISA assays. Two of these three MAbs also bound to secretory granules within both the dorsal and subventral esophageal glands of young SCN females. All five of the subventral gland MAbs bound to the subventral glands of Heterodera schachtii and one bound to the subventral glands of Globodera tabacum, but none bound to any structures in Meloidogyne incognita or Caenorhabditis elegans.     

The histology and ultrastructure of the salivary glands of Neopanorpa longiprocessa (Mecoptera: Panorpidae)

The salivary glands of Panorpidae usually exhibit distinct sexual dimorphism and are closely related to the nuptial feeding behavior. In this study, the salivary glands of Neopanorpa longiprocessa were investigated using light microscopy and transmission electron microscopy. The salivary glands are tubular labial glands and consist of a scoop-shaped salivary pump, a common salivary duct, and a pair of salivary tubes. The male and female salivary glands are remarkably different in the bifurcation position of the common salivary duct and the length and shape of the secretory tubes. Compared with the simple female salivary glands, the male’s are more developed as their paired elongated salivary tubes can be divided into two parts, the glabrate anterior tube and the posterior tube with many secretory tubules. The ultrastructural study shows that the male salivary tubes have strong secretory function. The existence of different secretion granules indicates that there are some chemical reactions or mixing occurring in the lumen. Based on the ultrastructural characteristics, the functions of the different regions of the salivary tube have been speculated. The relationship between the salivary glands and nuptial feeding behavior of N. longiprocessa has been briefly discussed based on the structure of the salivary glands.

     

Involvement of Aquaporin-5 Water Channel in Osmoregulation in Parotid Secretory Granules

Aquaporins (AQPs) are a family of channel proteins that allow water or very small solutes to pass, functioning in tissues where the rapid and regulated transport of fluid is necessary, such as the kidney, lung, and salivary glands. Aquaporin-5 (AQP5) has been demonstrated to localize on the luminal surface of the acinar cells of the salivary glands. In this paper, we investigated the expression and function of AQP5 in the secretory granules of the rat parotid gland. AQP5 was detected in the secretory granule membranes by immunoblot analysis. The immunoelectron microscopy experiments confirmed that AQP5 was to be found in the secretory granule membrane. Anti-AQP5 antibody evoked lysis of the secretory granules but anti-aquaporin-1 antibody did not and AQP1 was not detected in the secretory granule membranes by immunoblot analysis. When chloride ions were removed from the solution prepared for suspending secretory granules, the granule lysis induced by anti-AQP5 antibody was inhibited. Furthermore, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, an anion channel blocker, blocked the anti-AQP5 antibody-induced secretory granule lysis. These results suggest that AQP5 is, expressed in the parotid gland secretory granule membrane and is involved in osmoregulation in the secretory granules.     

Ultrastructural investigations of the salivary glands in adults of the microtrombidiid mite Platytrombidium fasciatum (C. L. Koch, 1836) (Acariformes: Microtrombidiidae)

The organization of the salivary glands in ad libitum-fed adult females of the microtrombidiid mite Platytrombidium fasciatum (C. L. Koch, 1836) was observed using transmission electron microscopy. In all, four pairs of large simple alveolar salivary glands were determined, which have been named due to their position as posterior, ventral, medial and dorsal. These glands occupy a body cavity behind, around the base and partly inside the gnathosoma. The posterior glands are largest and possess large nuclei with greatly folded nuclear envelope. Secretory granules are electron-light, containing fine granular material and are partly provided with various lamellar inclusions inside the granules. The latter tend to be placed predominantly in the middle parts of the gland around the central (intra-alveolar) cavity. The remaining glands, conversely, are typically filled with tightly packed electron-dense secretory granules, except for the ventral glands, the granules of which may show a compound organization. The nuclei of all these glands occupy a peripheral position and are mostly pressed between the granules. No prominent endoplasmic reticulum or conspicuous Golgi bodies were observed within the salivary glands. The salivary glands are provided with a complex apparatus of the intra-alveolar cavity (acinar lumen) with the excretory duct base provided by a system of branched special cells producing the duct walls. The ventral glands open by separate ducts into the most posterior part of the subcheliceral space. Ducts of the posterior glands immediately fuse with the ducts of the tubular (coxal) glands. The common duct of each side of the body joins with the ducts of the medial and dorsal glands respectively, and opens into the subcheliceral space far anterior to that of the ventral glands.     

Immunocharacterization of the DNA puff BhC4-1 protein of Bradysia hygida (Diptera: Sciaridae)

The DNA puff BhC4-1 gene is amplified and highly expressed in the salivary gland of Bradysia hygida late larvae. Using affinity-purified polyclonal antibodies we have identified the product of the BhC4-1 gene as a 43 kDa polypeptide which is present in extracts of salivary glands from late fourth instar larvae and in the corresponding gland secretion, but not in glands from earlier stages. We also demonstrate that this protein is produced mainly in the S1 and S3 regions of the salivary gland, where BhC4-1 amplification levels are more pronounced and larger amounts of mRNA are produced. By immunoelectron microscopy the BhC4-1 protein was detected in secretory granules of the S1 and S3 regions, and localized in fibrous structures present in the saliva.     

Basic fibroblast growth factor in rat salivary glands

We studied the occurrence and localization of basic fibroblast growth factor (bFGF) in rat salivary glands using a specific monoclonal antibody. It was shown that the extract of rat salivary glands has a pronounced stimulatory activity on the growth of bovine capillary endothelial cells, which is blocked by the addition of an antibody against bFGF. The concentration of bFGF in the submandibular/sublingual gland, as determined by radioimmunoassay, was 80% that in the brain. Immunocytochemistry revealed bFGF-immunoreactivity localized primarily in the epithelial cells lining the striated ducts and excretory ducts of the parotid, sublingual and submandibular glands. In addition, intense bFGF-immunoreactivity was observed in the granular convoluted tubule of the submandibular gland, localized predominantly in the agranular pillar cells, which lay in small numbers among the majority of weakly immunostained cells containing many apical secretory granules. At the electron-microscopic level, the immunoreactive material was distributed diffusely in the cytoplasmic matrix and nuclei of all immunoreactive cells, whereas it was absent from all cytoplasmic organelles including the secretory granules. These results indicate that bFGF is localized in different cellular and subcellular compartments from those of other growth factors in the duct system of rat salivary glands.     

Monoclonal Antibodies to Secretory Granules in Esophageal Glands of Meloidogyne Species

Monoclonal antibodies to secretory granules in the dorsal or subventral esophageal glands were generated by injecting BALB/c mice with immunogens from preparasitic second-stage juveniles (J2) of Meloidogyne incognita. Antibodies specific for secretory granules in the J2 subventral esophageal glands or the dorsal gland were identified by indirect immunofluorescence microscopy. Only antibodies that reacted with granules in the J2 dorsal gland reacted with the esophageal gland lobe ofM. incognita adult females. The antibodies also reacted with secretory granules in both types of esophageal glands in M. javanica and M. arenaria J2 but not with granules in esophageal glands of Heterodera glycines J2.     

VAMP8/endobrevin as a general vesicular SNARE for regulated exocytosis of the exocrine system

The molecular mechanism governing the regulated secretion of most exocrine tissues remains elusive, although VAMP8/endobrevin has recently been shown to be the major vesicular SNARE (v-SNARE) of zymogen granules of pancreatic exocrine acinar cells. In this article, we have characterized the role of VAMP8 in the entire exocrine system. Immunohistochemical studies showed that VAMP8 is expressed in all examined exocrine tissues such as salivary glands, lacrimal (tear) glands, sweat glands, sebaceous glands, mammary glands, and the prostate. Severe anomalies were observed in the salivary and lacrimal glands of VAMP8-null mice. Mutant salivary glands accumulated amylase and carbonic anhydrase VI. Electron microscopy revealed an accumulation of secretory granules in the acinar cells of mutant parotid and lacrimal glands. Pilocarpine-stimulated secretion of saliva proteins was compromised in the absence of VAMP8. Protein aggregates were observed in mutant lacrimal glands. VAMP8 may interact with syntaxin 4 and SNAP-23. These results suggest that VAMP8 may act as a v-SNARE for regulated secretion of the entire exocrine system.     

Ultrastructure and cytochemistry of salivary glands of the predator Podisus nigrispinus (Hemiptera: Pentatomidae)

Podisus nigrispinus Dallas (Hemiptera: Pentatomidae) is a zoophytophagous insect with a potential for use as a biological control agent in agriculture because nymphs and adults actively prey on various insects by inserting mouthparts and regurgitating the contents of the salivary glands inside the prey, causing rapid paralysis and death. However, the substances found in saliva of P. nigrispinus that causes the death of the prey are unknown. As a first step to identify the component of the saliva of P. nigrispinus, this study evaluated the ultrastructure and cytochemistry of the salivary glands of P. nigrispinus. The salivary system of P. nigrispinus has a pair of principal salivary glands, which are bilobed with a short anterior lobe and a long posterior lobe, and a pair of tubular accessory glands. The principal gland epithelium is composed of a single layer of cells enclosing a large lumen. Epithelial cells of the principal salivary gland vary from cubic to columnar shape, with one or two spherical and well-developed nuclei. Cells of the anterior lobe of the principal salivary gland have an apical surface with narrow, short, and irregular plasma membrane foldings; apical and perinuclear cytoplasm rich in rough endoplasmic reticulum; and mitochondria with tubular cristae. The basal portion of the secretory cells has mitochondria associated with many basal plasma membrane infoldings that are short but form large extracellular canals. Secretory granules with electron-dense core and electron-transparent peripheral are dispersed throughout the cytoplasm. Cells of the posterior lobe of the principal salivary gland are similar to those of the anterior lobe, except for the presence of mitochondria with transverse cristae. The accessory salivary gland cells are columnar with apical microvilli, have well-developed nucleus and cytoplasm rich in rough endoplasmic reticulum, and have secretory granules. Cytochemical tests showed positive reactions for carbohydrate, protein, and acid phosphatase in different regions of the glandular system. The principal salivary glands of P. nigrispinus do not have muscle cells attached to its wall, suggesting that saliva-releasing mechanism may occurs with the participation of some thorax muscles. The cytochemical and ultrastructural features suggest that the principal and accessory salivary glands play a role in protein synthesis of the saliva.     

Light and electron microscopy study of the salivary glands of the carnivorous opisthobranch Philinopsis depicta (Mollusca, Gastropoda)

Cephalaspideans are a group of opisthobranch gastropods that comprises carnivorous and herbivorous species, allowing an investigation of the relationship between these diets and the morphofunctional features of the salivary glands. In this study, the salivary glands of the carnivorous cephalaspidean Philinopsis depicta were observed by light and electron microscopy. The secretory epithelium of these ribbon-shaped glands is formed by ciliated cells, granular cells and cells with apical vacuole. In ciliated cells the nucleus and most cytoplasmic organelles are located in the wider apical region and a very thin stalk reaches the base of the epithelium. These cells possess significant amounts of glycogen. Granular cells are packed with electron-dense secretory granules and also contain several cisternae of rough endoplasmic reticulum and Golgi stacks. The other type of secretory cell is mainly characterized by the presence of a large apical vacuole containing secretion. These cells possess high amounts of rough endoplasmic reticulum cisternae and several Golgi stacks. Vesicles with peripheral electron-dense material are also abundant, and seem to fuse to form the apical vacuole. The available data point out to a significant difference between the salivary glands of carnivorous and herbivorous cephalaspidean opisthobranchs, with an intensification of protein secretion in carnivorous species.     

Correlative light and electron microscopy (CLEM) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited. On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM), affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy. This powerful technique has been implemented to study many aspects of cell biology. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from ≤5 s up to several hours, depending on the nature of the microinjected sample.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Salivary glands in Cicadidae (Hemiptera: Cicadoidea): comparative morphology, ultrastructure, and their phylogenetic significance

The present investigation provides information on gross morphology and ultrastructure of salivary glands of species in Cicadidae in detail. The structure of the salivary glands of 11 representative species from 10 genera belonging to three subfamilies of Cicadidae was studied using light microscopy and transmission electron microscopy. In the examined species, the salivary glands are paired structures, and each of which is comprised of a principal gland (pg) and an accessory gland (ag). The pg is divided into anterior and posterior lobes, and both of which consist of numerous long digitate lobules. The lobules at the base of the long digitate lobules of posterior lobe are greatly short; here, we named as “short lobules.” All the lobules vary in size, disposition, length, and shape. The anterior lobe and posterior lobes are connected by an anterior–posterior duct (apd). Two efferent salivary ducts (esd), derived from corresponding posterior lobes, fuse to form a short common duct which enters into the saliva syringe. The ag is composed of a greatly tortuous and folded accessory salivary tube, a gular gland (gg) constituting of several acini, and an accessory salivary duct (asd). The asd joins the esd at the place where the latter emergences. Constituents and arrangement of the salivary glands, the number and shape of the long digitate lobules in the anterior and posterior lobes, and the visibility of the apd were promising characters for the taxonomic and phylogenetic analysis of Cicadoidea. The variations of secretory granules in size, shape, and electron density in lobule cells of pg of Platypleura kaempferi probably indicating different materials are synthesized. The absence of the infoldings of basal plasma membrane in the basal area of the cells and the presence of electron-lucent vesicles in the cytoplasm of the gg cells of P. kaempferi might suggest that the secretions of gg are more watery.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin.

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.