Three-dimensional imaging of the mucus secretory process in the cryofixed frog respiratory epithelium.

Using the frog palate as a representative model of human mucociliary epithelium, we analyzed, after quick freezing fixation, the three-dimensional (3-D) respiratory mucus secretory release with high voltage (200-300 kV) transmission electron microscopy (TEM). The 3-D vision of the mucus release from the secretory cells was obtained as stereo-pairs and "bas-relief" images after analysis of stereo-pairs using an image analyzer. After standard glutaraldehyde fixation, the secretory cells showed a typical goblet shape with secretory granules heterogeneous in size and electron-density which often fuse together. On the other hand, quick-frozen secretory cells exhibited a columnar shape and their membrane-bound secretory granules contained a homogeneously dark matrix. The expanded gel mucus layer was preserved and its depth never exceeded 2 microns. When the epithelium was immersed in culture medium in presence of cholinergic agonist, a marked discharge of mucus was observed and the granules swelled at the apex of the secretory cell before being discharged in the lumen. In native cryofixed epithelium, the secretory granules exhibited a marked deformability during the process of their extrusion from the secretory cell. Clusters of secretory granules surrounded by cytoplasmic material were observed in the extracellular lumen, suggesting an apocrine-type secretion. These observations indicate that rapid cryofixation and 3-D stereoscopic imaging enable a unique opportunity to analyze, without artifact, the mucous secretory process. We speculate that, apart from the classical merocrine-type secretion mechanism, the respiratory mucus may be released, at least partly by an apocrine-type secretion.     

Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine

The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen.     

The cytomorphology of goblet cells of the fetal intestine. Studies of the large intestine of cattle (Bos primigenius taurus)

In the region of the base of the intestinal crypts undifferentiated goblet cells display a configuration and constellation of organelles and membrane structures that are indicative of their importance for function. These images at this stage of development deliver a scenario of the mechanism of secretory granule production: aggregates of protein vesicles from the "transitional elements" (PALADE) of the granular endoplasmic reticulum are, so to speak, rolled up on the trans side of the Golgi apparatus by inversion of peripheral membrane segments of the innermost Golgi lamellae, thereby forming corpuscles. The origin of the capsulated vacuoles, which contain vesicles as single elements or as conglomerates, is well established. Their capsule consists of a trilaminar external and external and internal membrane; between them lies condensed material of the Golgi apparatus. In the opinion of the present author, the development of the ensheathed vacuoles represents a basic, more general mechanism. In contrast, the further steps of synthesis, for the formation of secretory granules, are more heterogeneous. Condensation of the vesicles and the inner capsular membrane results in the formation of a prosecretory granule, which in the basic element in the process of secretory granule production. The prosecretory granules develop singly or by fusion with other granules to give primary secretory granules. The complexity of this mechanism of secretory granule formation, however, becomes evident when considering the apposition of capsulated vacuoles and prosecretory--primary--secondary secretory granules, of prosecretory and primary secretory granules as well as prosecretory granules and secondary secretory granules. Generally, primary granules show a tendency to become secondary secretory granules or to fuse with them. During maturation of the goblet cells the secretory granules fuse to form larger mucous bodies in the theca by fusion of the laminae of the membranes; a final product, there is a homogeneous mucous mass devoid of membranes.     

Expression of airway secretory epithelial functions by lung carcinoma cells

We examined 12 non-small cell lung carcinoma cell lines for expression of airway goblet, serous, and mucous cell characteristics. The cells expressed some ultrastructural traits of secretory epithelial cells but none contained secretory granules typical of the airway secretory cells. Using immunocytochemistry and cell-specific monoclonal antibodies, we identified heterogeneous expression of goblet, mucous, and serous cell markers among the cell lines. After metabolic radiolabeling, cells incorporated isotope into high molecular weight material. Incubation of pulse-radiolabeled cells with a number of known mucus secretogogues revealed that 5 of the 12 cell lines released radiolabeled material in response to the agonists. However, in each cell line only one of the receptor-activated pathways tested was intact. Although we did not identify a single cell line expressing a phenotype similar to normal airway secretory cells, particular functions retained by some of these cell lines may make them useful for specific studies of mucus production or secretion.     

Differentiation of tracheal epithelium during fetal lung maturation in the rhesus monkey Macaca mulatta

Of the eight categories of epithelial cells identified in pulmonary conducting airways, four are found in the trachea of adult primates: basal, mucous goblet, intermediate, and ciliated cells. While their ultrastructure is well characterized, little is understood about their origin or differentiation. This study describes the pattern of differentiation of the tracheal luminal epithelium in a species of nonhuman primate, the rhesus monkey, Macaca mulatta. Tracheas of 57 fetal and postnatal rhesus were fixed with glutaraldehyde/paraformaldehyde: ten at 29-54 days gestational age (GA), ten at 59-80 days GA (pseudoglandular stage), sixteen at 82-130 days GA (canalicular stage), ten at 141-168 days GA (saccular stage), eight at 1-134 days postnatal, and three adults (2 yr 11 months to 11 yr 11 months). Slices taken proximal to the carina were processed for electron microscopy by a selective embedding procedure. In the youngest fetuses, essentially one population of cells lined the tracheal epithelial surface. These cells were columnar in shape with a central nucleus, few organelles, and large amounts of cytoplasmic glycogen. At 46 days GA, ciliated cells were observed on the membranous side of the trachea. Some nonciliated cells had concentrations of organelles in the most apical portion of their cytoplasm. At 59 days GA, membrane-bound cored granules were intermixed with organelles in the apices of some glycogen-filled cells. They were observed first on the cartilaginous side. Between 59 and 100 days GA, a large number of cell forms which appeared to be transitional between ciliated, secretory, basal, and undifferentiated cells were present. These included ciliated cells with electron-lucent inclusions resembling mucous granules. Mucous secretory cells were more numerous and had more granules and less glycogen in older fetuses. By 105 days GA, few of the secretory cells had significant amounts of glycogen and the cytoplasm was condensed. Secretory granules were very abundant in some cells and minimal in others. The Golgi apparatus was prominent. In animals 120 days GA and older, small mucous granule cells and basal cells resembling these cells in adults were present. By 134 days postnatal age, the epithelium resembled that in adults. We conclude that most of the differentiation of tracheal epithelium in the rhesus monkey occurs prior to birth; the cells differentiate in the following sequences: ciliated, mucous goblet, small mucous granule, basal; and basal and small mucous granule cells do not play a role in ciliated and mucous cell formation in the fetus.     

Immunocytochemical study of pepsinogen 1-producing cells in the fundic mucosa of the stomach in developing mice

Summary Development and maturation of pepsinogen 1-producing cells were studied in the gastric fundic mucosa of the mouse by means of light- and electron-microscopic immunocytochemistry using rabbit anti-rat pepsinogen 1-serum. In the adult mouse, secretory granules in mucous neck cells, transitional mucous neck/chief cells and chief cells are immunolabeled. The numerical density of gold particles on zymogen granules is not significantly altered among different stages of maturation of chief cells. In addition, rough endoplasmic reticulum and Golgi complex of these cell types show a weak labeling. In mice from day 16 of gestation to postnatal day 14 mucous neck cells and chief cells cannot be distinguished, but only one type of pepsinogen 1-producing cell, called primitive chief cell, is identified in the fundic gland. The intensity of immunoreactivity of secretory granules in primitive chief cells is uniform within an individual cell but varies greatly among different cells. The majority of primitive chief cells contains weakly labeled granules regardless of the maturation stage of cells or of animals. On postnatal day 21, mucous neck, transitional and chief cells are distinguishable, and secretory granules in these cells are intensely immunolabeled as in the adult. These results suggest that pepsinogen 1-production rapidly increases with differentiation of mucouse neck and chief cells.     

Cytochemical features of the digestive tract mucosa of Hemisorubim platyrhynchos (Siluriformes: Pimelodidae)

Membranous organelles, acid glycoconjugates and lipids were characterized in the digestive tract mucosa of Hemisorubim platyrhynchos by cytochemistry techniques. Two types of mucous‐secreting cells were observed in the digestive tract epithelium: goblet cells in the oesophagus and intestine and epithelial cells in the stomach. These cells had a Golgi apparatus more developed than the other cell types. The cytochemical analysis revealed that secretory granules are reactive to acid glycoconjugates, varying in reaction intensity according to the region of the digestive tract. Acid glycoconjugate reactions were also observed in oesophageal epithelial cell microridges and in enterocyte microvilli. In the digestive tract, acid glycoconjugates act to protect the epithelial surface, increasing mucous viscosity, which facilitates the passage of food, prevents the binding of parasites and facilitates their removal. Through lipid staining, a coated membrane was observed around each secretory granule of the oesophageal and intestinal goblet cells, while gastric epithelial cells granules were fully reactive. Oxynticopeptic cells of the gastric glands showed lipid droplets in the cytoplasm and also in the mitochondrial matrix, which act as an energy reserve for these cells that have a high energy demand. Enterocytes showed a well‐developed smooth endoplasmic reticulum, especially in the apical region of the cell, being related to absorption and resynthesis of lipids.     

Light and electron microscopic cytochemistry of glycoconjugates in the rectosigmoid colonic epithelium of the mouse and rat

The several cell types in mouse and rat rectosigmoid colon have been examined with light and electron microscopic methods for localizing and characterizing complex carbohydrates. Mucous cells, also termed vacuolated cells, and goblet cells comprised most of the deep crypt epithelium in both species, and absorptive columnar cells and goblet cells mainly populated the more superficial epithelium of the upper crypts and main lumen. Occasional tuft cells and enteroendocrine cells were also encountered. Transitional cells structurally intermediate between mucous cells and absorptive cells contained granules characteristic of mucous cells and vesicles like those of columnar absorptive cells. These intermediate cells supported the concept of replacement of mucous by absorptive cells through transformation of mucous into absorptive cells. The intermediate cells also contained numerous lysosomes often in apparent fusion with mucous granules, indicating crinophagic disposal of mucous granules as a mechanism in the cell transformation. Glycoconjugate in absorptive cell vesicles resembled that coating the apical plasmalemma and appeared to represent the source of the glycocalyx of the brush border. Complex carbohydrate in these vesicles differed cytochemically from that of the mucous cell granules, which release their content into the crypt lumen. The absorptive cell vesicles, therefore, constitute an organelle distinct from the mucous cell granules rather than an atrophic form of the latter in a more mature cell. Goblet cells differed in failing to transform morphologically with age but changed in the cytochemical characteristic of their secretion during migration up the crypts. Terminal N-acetylglucosamine residues diminished, while terminal sialic acid-galactose dimers increased during the upward migration, indicating activation of glycosyl transferase synthesis in relation to goblet cell maturation. Glycoconjugate in secretion of mucous cell granules differed markedly from that in goblet cell granules, and content of both organelles differed from that of absorptive cell vesicles. However, secretion in mucous cell granules appeared generally similar for mice and rats with minor exceptions, and secretion in goblets of mice generally resembled that in goblets of rats. Cells interpreted tentatively as Kulchitsky cells stained for high content of fucose with the Ulex europeus I lectin. Globoid leukocytes infiltrating the epithelium of the rat but not the mouse rectosigmoid colon resembled globoid leukocytes in rat tracheal epithelium and, like the latter, appeared to derive from mast cells.     

Presence of cells combining features of two different cell types in the colonic crypts and pyloric glands of the mouse.

A small number of epithelial cells which combine features of two cell types were observed in the descending colon and pyloric stomach of the mouse. In the descending colon, where the base of the crypts is mainly composed of poorly differentiated "vacuolated" cells, a few of these cells contain, besides the characteristic "vacuoles," mucous globules identical to those in mucous cells or, less frequently, dense granules such as are found in entero-endocrine cells. Because there is evidence that the poorly differentiated vacuolated cells give rise to the other cells of the epithelium, those which also contain mucous globules or dense granules are likely to be differentiating into mucous cells or entero-endocrine cells respectively. In the pyloric stomach, where the glands are mainly composed of mucous cells, some of which are poorly differentiated, a few of the latter exhibit, besides the mucous globules, entero-endocrine type granules or features of caveolated cells. It is likely that the poorly differentiated mucous cells give rise to the other gland cells; and, therefore, those mucous-containing cells which also display dense granules or caveolated cell features are taken to be differentiating into entero-endocrine or caveolated cells respectively. Most of the cells containing two kinds of secretory materials are believed to be stem cells which initially contain a few vacuoles (colon) or mucous globules (pylorus) but are differentiating into a cell containing a different type of secretion. Rare observations of two kinds of secretory materials in a mature cell suggest that the transitional period may be prolonged, perhaps indefinitely.     

The fine structure of sublingual gland acinar cells of the Mongolian gerbil,Meriones unguiculatus,processed by rapid freezing followed by freeze-substitution fixation

Summary We compare the ultrastructure of the gerbil sublingual gland as seen after cryofixation followed by substitution with osmium tetroxide, with the more familiar appearance of material processed by glutaraldehyde-osmium chemical fixation. After primary cryofixation of fresh salivary glands, the nuclei of the mucous cells are found to be spherical in shape and, rather than being displaced toward the cell base, occupy a nearly central position in the cytoplasm, even in the storage phase of the secretory cycle. The mucous secretory granules are seen as membrane-limited inclusions, only rarely partially fused to each other. In both mucous and serous cells the Golgi cisterns have numerous large fenestrae which are aligned to form cytoplasmic channels which extend across the stack.     

Use of transgenic mice to study the routing of secretory proteins in intestinal epithelial cells: analysis of human growth hormone compartmentalization as a function of cell type and differentiation [published erratum appears in J Cell Biol 1990 Jan;110(1):following 227]

The intestinal epithelium is a heterogeneous cell monolayer that undergoes continuous renewal and differentiation along the crypt-villus axis. We have used transgenic mice to examine the compartmentalization of a regulated endocrine secretory protein, human growth hormone (hGH), in the four exocrine cells of the mouse intestinal epithelium (Paneth cells, intermediate cells, typical goblet cells, and granular goblet cells), as well as in its enteroendocrine and absorptive (enterocyte) cell populations. Nucleotides -596 to +21 of the rat liver fatty acid binding protein gene, when linked to the hGH gene (beginning at nucleotide +3) direct efficient synthesis of hGH in the gastrointestinal epithelium of transgenic animals (Sweetser, D. A., D. W. McKeel, E. F. Birkenmeier, P. C. Hoppe, and J. I. Gordon. 1988. Genes & Dev. 2:1318-1332). This provides a powerful in vivo model for analyzing protein sorting in diverse, differentiating, and polarized epithelial cells. Using EM immunocytochemical techniques, we demonstrated that this foreign polypeptide hormone entered the regulated basal granules of enteroendocrine cells as well as the apical secretory granules of exocrine Paneth cells, intermediate cells, and granular goblet cells. This suggests that common signals are recognized by the "sorting mechanisms" in regulated endocrine and exocrine cells. hGH was targeted to the electron-dense cores of secretory granules in granular goblet and intermediate cells, along with endogenous cell products. Thus, this polypeptide hormone contains domains that promote its segregation within certain exocrine granules. No expression of hGH was noted in typical goblet cells, suggesting that differences exist in the regulatory environments of granular and typical goblet cells. In enterocytes, hGH accumulated in dense-core granules located near apical and lateral cell surfaces, raising the possibility that these cells, which are known to conduct constitutive vesicular transport toward both apical and basolateral surfaces, also contain a previously unrecognized regulated pathway. Together our studies indicate that transgenic mice represent a valuable system for analyzing trafficking pathways and sorting mechanisms of secretory proteins in vivo.     

Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells

Summary Chromaffin granules from bovine adrenal medullary chromaffin cells have been found to contain small vesicular structures bounded by unit membranes. Detection of these intragranular vesicles within intact cells requires the use of quick-freezing methods. The intragranular vesicles are labile to fixation by aldehydes which explains why they have not been described in intact cells until now. They are found in approximately 60% of the dense-core chromaffin granules in cells and 85% of isolated granules. They are usually clustered in groups of one to as many as five between the core and the inner surface of the granule membrane. The intragranular vesicles are independent vesicles in that they do not appear as simple invaginations of the granule membrane in either serial thin-section or freeze-etch views. Furthermore, they are released from the cell along with granule contents during nicotine-induced secretion of catecholamines. The structural heterogeneity provided by the intragranular vesicles may be related to the functional heterogeneity of granule contents observed in many recent biochemical studies.     

Zur Cytologie der Rectaldrüsen von Knorpelfischen

The stratified epithelium of the central collecting duct of the elasmobranch(Scylliorhinus canicula, Galeorhinus galeus andRaja batis) rectal gland consists of 3 to 6 layers of cells: one superficial, and several basal cell layers. In the superficial layer normally three different types of cells can be distinguished (a) goblet cells, (b) cells with apical secretory granules and (c) flask-shaped cells. The superficial layer ofScylliorhinus canicula reveals a further cell type, so-called mitochondria-rich cells. The epithelial areas built by these cells are always single-layered. The goblet-cells are very similar to goblet cells found in the intestine of vertebrates. Their dominant structures are a well developed ergastoplasm, a large Golgi-apparatus and mucous granules compactly filling the apical cell region. The cells with apical secretory granules are columnar or dumbbell shaped. They contain a rough-surfaced endoplasmic reticulum and a well developed Golgi-apparatus. The secretory granules are loosely distributed within the Golgi-field and are arranged in one or more rows just below the cell apex. The flask shaped cells are characterized by a cytoplasm rich in small vesicles. They posses few dictyosomes and several small mitochondria. There is some evidence for endocytotic activity. The mitochondria-rich cells are characterized by lateral cell interdigitations, by a basal labyrinth and by numerous mitochondria. They are similar to the excretory cells of rectal gland parenchyma. The cells of the basal epithelium layers are differenciated only to a small extent. They are joined in a loose formation with white blood cells often found in the intercellular spaces. The function of the elasmobranch rectal gland is not restricted to the excretion of concentrated salt solutions. There is also a significant secretion of mucous substances. The tubule glands are primarily excretory, the epithelium cells of the central collecting duct mainly secretory in function.     

Histological features and histochemistry of the mucous glands in ventral skin of the frog (Rana fuscigula)

The glycoconjugate components of secretory granules were analyzed in cells of mucous glands in ventral skin from Rana fuscigula. The analysis was done with standard histochemical methods on semithin glycol methacrylate-embedded tissues. The staining patterns in semithin sections were comparable to those using paraffin-embedded tissue while the cytological detail was better preserved. The mucous glands contained at least two different types of secretory cells lining the lower two-thirds of the mature gland: a principal cell type filled with dense staining secretory granules and a solitary type containing paler staining, globular secretory granules. The principal type of cell contained variable amounts of acid glycoconjugates; predominantly carboxylated but also variably carboxylated and weakly sulfated glycoproteins. Other secretory cells contained mainly neutral glycoproteins. The results indicated that the mucus is a heterogeneous substance and that one cell type may produce different secretory products. We suggested that the variability in histochemical staining might be related to the sequence of biosynthesis of the secretory granule.     

Morphology of the exocrine glands of the frog skin

Frog skin contains three distinct types of exocrine glands: granular (poison), mucous, and seromucous. The granular gland forms a syncytial secretory compartment within the acinus, which is surrounded by smooth muscle cells. The mucous and seromucous glands are easily identifiable as distinct glands. The serous and mucous secretory cells are arranged in a semilunar configuration opposite the ductal end and are filled with granules. Within the acinus, located at the ductal pole of the gland, are distinct groups of cells with few or no granules in the cytoplasm. In both the mucous and seromucous gland there is a cell type with abundant mitochondria; the one in the mucous gland is located in the region adjacent to the secretory cells. The duct of these glands is two-layered, with the individual cells appearing morphologically similar to the layers of the skin epithelium as the duct traverses the skin. The duct appears to be patent throughout its length. The morphological heterogeneity and distinct distribution of the cell types within the gland acinus may be indicative of a functional heterogeneity that allows the production of distinctly different types of secretion from the same gland type, depending on the type of stimulus.     

Inhibition of glycoconjugate secretion by colchicine and cytochalasin B

Summary The effects have been analyzed of cytochalasin B and colchicine on the secretion of glycoconjugates by human bronchial expiants labeled in vitro with radioactive glucosamine. Both cytochalasin B and colchicine had no effect on baseline ¹⁴C-labeled glycoconjugate release but caused a dose-dependent (10^–7–10^–4 M) inhibition of ¹⁴C-glycoconjugate release and discharge of labeled macromolecules from mucous and serous cells induced by 5 · 10^–5 M methacholine.Quantitative autoradiographic analyses showed that neither cytochalasin B nor colchicine inhibited ³H-threonine or ³H-glucosamine incorporation into mucous and serous cells of the submucosal glands or goblet cells of the airway epithelium. Colchicine (10^–5 M) but not cytochalasin B significantly reduced the rate at which labeled macromolecules were transported through mucous, serous and goblet cells but this effect was not observed until 4 h after the addition of colchicine. Neither cytochalasin B nor colchicine affected the basal rate of labeled-macromolecule discharge from mucous, serous or goblet cells. At a concentration of 10^–5 M, both agents completely inhibited the increase in labeled-macromolecule discharge induced in mucous and serous cells by methacholine.Our results suggest that in the submucosal gland of human airways microtubules and microfilaments may be important in secretagogue-induced but not in baseline cellular glycoconjugate discharge, implying that the mechanisms of the two processes differ significantly. Furthermore, a role for microtubules is suggested in the transport of secretory granules through mucous, serous and goblet cells.Supported by National Institutes of Health Research Grant 5R01HL22444. The authors gratefully acknowledge the technical assistance of Mr. Tudor Williams, Mr. Eduardo Quintanilla and Ms. Maureen Hayes     

Heterogeneity of glycoconjugates in the secretory cells of the chinchilla middle ear and eustachian tubal epithelia: a lectin-gold cytochemical study

The present study was conducted to characterize and localize the glycoconjugates in the tubotympanum (auditory or eustachian tube and middle ear cavity) of chinchilla on an ultrastructural level, using lectin-gold complexes with six different lectins: BPA, ConA, RCA-1, WGA, LFA, and SNA. A comparison of the affinity of these lectins demonstrated the heterogeneity of secretory cells. The glandular serous cells and epithelial dark granulated cells produced "serum"-type glycoprotein. The glandular mucous cells and goblet cells produced dominantly "mucin"-type glycoprotein in the light granules, but "serum"-type glycoprotein in the dark cores. The labeling of LFA and SNA showed that sialic acids existed mainly in the mucinous granules of secretory cells and ciliated epithelium glycocalyx, and in the mucous blanket. The results also suggested that the dominant linkage of sialic acids of mucin is a Neu5Ac(alpha 2-6)Gal/GalNAc sequence. Furthermore, the data obtained from ConA and BPA suggested that initial O-glycosylation of mucin took place in the cis side of the Golgi apparatus and that initial N-glycosylation of the serum occurred in the rough endoplasmic reticulum.     

Microanatomy and ultrastructure of the foot of the infaunal bivalve Tegillarca granosa (Bivalvia: Arcidae)

In this study, the morphology and ultrastructure of the foot of Tegillarca granosa was compared with the bivalves from different habitats. The sediment of habitat of T. granosa is mostly a mixture of sand (68.93%) and mud (24.12%). The foot is wedge-shaped with multiple projections on the surface and covered with ciliary tufts. The epithelial layer is simple and composed of ciliated columnar epithelia and mucous cells. Although the mucous cells are distributed mostly in the epithelial layer, they are developed even in the connective tissues and muscle layers, and the mucous cells mostly contain acidic carboxylated mucosubstances. From the TEM observation, secretory cells are classified into three types. Type A secretory cell has a goblet form and is most widely distributed among the three types. Type B secretory cell has an oval form and the secretory granule has fibrous substance. Type C secretory cell has an elongated elliptic form and membrane-bounded secretory granules. The muscle fiber bundles are composed mainly of smooth muscle fibers. The smooth muscle fibers can be divided into two types. Type A muscle fibers have evenly distributed thick microfilaments between the thin microfilaments of cytoplasm. Type B muscle fiber has cluster of condensed microfilaments in the medulla cytoplasm while the cortical cytoplasm has loose distribution of thin microfilaments.     

Morphology and ultrastructure of the venom glands of the northern pacific rattlesnake Crotalus viridis oreganus

The venom gland of Crotalus viridis oreganus is composed of two discrete secretory regions: a small anterior portion, the accessory gland, and a much larger main gland. These two glands are joined by a short primary duct consisting of simple columnar secretory cells and basal horizontal cells. The main gland has at least four morphologically distinct cell types: secretory cells, the dominant cell of the gland, mitochondria-rich cells, horizontal cells, and “dark” cells. Scanning electron microscopy shows that the mitochondria-rich cells are recessed into pits of varying depth; these cells do not secrete. Horizontal cells may serve as secretory stem cells, and “dark” cells may be myoepithelial cells. The accessory gland contains at least six distinct cell types: mucosecretory cells with large mucous granules, mitochondria-rich cells with apical vesicles, mitochondria-rich cells with electron-dense secretory granules, mitochondria-rich cells with numerous cilia, horizontal cells, and “dark” cells. Mitochondria-rich cells with apical vesicles or cilia cover much of the apical surface of mucosecretory cells and these three cell types are found in the anterior distal tubules of the accessory gland. The posterior regions of the accessory gland lack mucosecretory cells and do not appear to secrete. Ciliated cells have not been noted previously in snake venom glands. Release of secretory products (venom) into the lumen of the main gland is by exocytosis of granules and by release of intact membrane-bound vesicles. Following venom extraction, main gland secretory and mitochondria-rich cells increase in height, and protein synthesis (as suggested by rough endoplasmic reticulum proliferation) increases dramatically. No new cell types or alterations in morphology were noted among glands taken from either adult or juvenile snakes, even though the venom of each is quite distinct. In general, the glands of C. v. oreganus share structural similarities with those of crotalids and viperids previously described.