Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.     

Freeze-fracture analysis of gap and septate junctions in embryos of a moth

Gap and septate junctions were examined in embryos of Manduca sexta (tobacco hornworm). The junctions observed were similar in structure to those reported for adult insect tissues. In the epidermis typical pleated septate junctions were found. Associated with the pleated septate junctions were inverted gap junctions which had irregularly arranged particles and pits. In the midgut typical smooth septate junctions were found. Associated with these septate junctions were gap junctions which had a regular hexagonal packing pattern. This codistribution of gap and septate junction types is discussed in light of current theories that the gap junction types are alternative forms of the same structure in different metabolic environments. In addition to these gap and septate junctions a new junction, perhaps a modified pleated septate junction, is described.     

Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. III. A morphometric analysis of the number and diameter of intramembrane particles

The intramembrane particles on the presynaptic membrane and on the membrane of synaptic vesicles were studied at freeze-fractured neuromuscular junctions of the frog. The particles on the P face of the presynaptic membrane belong to two major classes: small particles with diameters less than 9 nm and large particles with diameters between 9 and 13 nm. In addition, there were a few extralarge particles with diameters greater than 13 nm. Indirect stimulation of the muscle, or the application of black widow spider venom, decreased the concentration of small particles on the presynaptic membrane but did not change the concentration of large particles. Three similar classes of particles were found on the P face of the membrane of the synaptic vesicles. The concentrations of large and extralarge particles on the vesicle membrane were comparable to the concentrations of these particles on the presynaptic membrane, whereas the concentration of small particles on the vesicle membrane was less than than the concentration of small particles on the presynaptic membrane. These results are compatible with the idea that synaptic vesicles fuse with the presynaptic membrane when quanta of transmitter are released. However, neither the large nor the extralarge particles on the P face of the presynaptic membrane can be used to trace the movement of vesicle membrane that has been incorporated into the axolemma.     

Freeze-fracture,ultrastructural and autoradiographic analysis of the ventral prostate glands in castrated mice

Summary Ultrastructure of the ventral prostate glands was studied in mice castrated for 1 through 60 days and for 11 and 17 months and in age-matched normals. We have described freeze-fracture and ultrastructural characteristics of acinar epithelial cells in addition to the patterns of thymidine incorporation in the cells of castrates and normal animals. Our study has shown a biphasic pattern of prostatic involution in the long-term castrated mice. In castrates the initial atrophy of prostate glands occurred by sloughing of the apical portions of columnar cells, autophagia of the cytoplasmic organelles as well as by occasional sloughing of the individual cells into the acinar lumen. Concurrent with the initial atrophy, the glands and stroma were infiltrated by neutrophils and lymphocytes. The cell loss by sloughing and leucocyte infiltration of glands became infrequent in 7- to 21-day castrates. However, the cell loss by sloughing increased secondarily in mice castrated for 21 to 37 days along with the increased leucocyte infiltration of the glands. The cell loss became minimal in castrates of 60 days and beyond. Our evidence suggests that the cell loss by sloughing was an active process in the involution of prostate glands which also showed differential sensitivity to castration stimuli in mice.This research was supported by Medical Research Service of Veterans Administration     

Body fluid volume regulation in elasmobranch fish

This review addresses an often overlooked aspect of elasmobranch osmoregulation, i.e., control of body fluid volume. More specifically the review addresses the impact of changes in blood volume in elasmobranchs exposed to different environmental salinities. Measurement of blood volume in the European lesser-spotted dogfish, Scyliorhinus canicula, following acute and chronic exposure to 80% and 120% seawater (SW) is reported. In 80%, 100% and 120% SW-adapted S. canicula, blood volume was 6.3+/-0.2, 5.6+/-0.2 and 4.6+/-0.2 mL 100 g(-1) body mass, respectively. Blood volume was significantly higher and lower in 80% and 120% SW-acclimated animals compared to 100% SW controls. Comparisons are made between these results and previously published data. The role of drinking and volume regulation in elasmobranchs is discussed. For the first time measured water reabsorption rates and solute flux rates across the elasmobranch intestinal epithelia are presented. Water reabsorption rates did not differ between 100% SW-adapted bamboo shark, Chiloscyllium plagiosum, and fish acutely transferred to 140% SW. For the most part net solute flux rates and direction for both the 100% and 140% SW groups were the same with the exception of a net efflux of chloride and potassium in the 140% group and influx of these ions in the 100% adapted group. The significance of the intestine as part of the overall elasmobranch osmoregulatory strategy is discussed as is the role of the kidneys, rectal gland and gills in the regulation of body fluid volume in this class of vertebrates.     

Carbonic anhydrase localization in the elasmobranch rectal gland

Carbonic anhydrase (CA) activity was histochemically localized in the elasmobranch rectal gland at the light and electron microscopic levels. Reaction product in the secretory tubules was localized coincident with that reported for sodium-potassium activated adenosine triphosphatase (Na-K-ATPase): along the highly amplified basolateral plasma membranes of the epithelial cells. Reaction product was also localized along the plasma membrane of adjacent central canal epithelial cells. The results suggest that CA plays a role in modulating the environment of the intercellular space which in the secretory tubule is believed to be the paracellular pathway for sodium. The results also draw attention to the possible role of the central canal epithelium in modification of the secreted fluid.     

Structure of occluding junctions in ileal epithelial cells of suckling rats

Summary Two kinds of occluding junctions are found between ileal epithelial cells of suckling rats: apical zonulae occludentes (ZO) and fasciae occludentes (FO) which are associated with the lateral plasma membranes of many epithelial cells. In unfixed preparations, glycerol treatment induces the further proliferation of extensive fasciae occludentes. Both kinds of junction have identical structural elements when visualized in freeze fracture replicas, although the arrangement of these elements differs. Zonulae occludentes consist of networks of branching and anastomosing linear ridges or rows of 10 nm particles with 20–30 nm spaces between the rows which form narrow belt-like structures around the apical region of adjacent cells. Fasciae occludentes, on the other hand, consist of similar linear ridges or rows of particles but the junction strands are often discontinuous, open ended and only occasionally intersect with each other. Several different fracture planes through the plasma membrane in the region of the occluding junctions have been observed and these provide further evidence that two components, one from each membrane, fused at the level of the extracellular space, form the junction sealing element. Furthermore, we present evidence which indicates a staggered rather than an in-register arrangement of these two components.This study was supported in part by National Institutes of Health Program Project No. NS10299 and National Institutes of Health Sciences Advancement Award No. RR06148 (J.D.R.) and by the Cancer Research Campaign (S.K.) and Medical Research Council (A.R.L.)     

Membrane specializations in flagellar ribbons of elasmobranch fish

Specialized epithelial cells lining the elasmobranch nephron bear numerous flagella which are organized into closely-packed, parallel rows forming ribbons (Lacy et al., 1989a). The compact arrangement of the adjacent flagella comprising each ribbon suggests they are structurally bound together, forming a single unit which functions to force urine along the nephric tubule. In the present study, the structural basis of the interflagellar connections was investigated by scanning electron microscopy (SEM) and by transmission electron microscopy (TEM) of thin sections and freeze fracture replicas. Various fixatives and histochemical stains were used to elucidate the structure and composition of the interflagellar adhesive material. SEM of the luminal cell surface showed the organization of the flagella in ribbons. In TEM, fixation in a solution containing glutaraldehyde and tannic acid, Ruthenium red or Alcian blue, or postfixation in reduced OsO(4) revealed that the plasma membrane of each flagellum of a ribbon was surrounded by a thin layer of surface coat composed of very short filaments more prominent at sites where adjacent flagella were in close apposition. In comparable locations, freeze-fracture replicas disclosed small aggregates or plaques of particles arranged in an irregular, discontinuous line on both faces P and E of the flagellar membrane. In areas where the flagella were not arranged into ribbons (most frequently after immersion fixation), the surface coat was thick and expanded and, in replicas, the intramembranous particles were randomly scattered. All of these plasma membrane specializations appear to function in binding adjacent flagella and thus facilitate a coordinated flagellar ribbon beat.     

Freeze-fracture replica studies of tight junctions in normal human bronchial epithelium

Using freeze-fracture techniques, tight junctional networks were observed in the human normal bronchial epithelium. They were morphologically classified into three types: type I was a loosely interconnected, most complicated network consisting of 7-11 roughly parallel wavy strands and situated between ciliated cells; type II was a randomly anastomosing, simple network made up of 2-4 strands and present between goblet cells; type III was an irregularly anastomosing network composed of 4-7 strands and located between a ciliated cell and a goblet cell. Type III junctions, when a goblet cell was strongly bulged, were located on the swollen ridge, the upper surface of which was separated by a deep groove from the bulged apical surface, around the lateral surface of the cell at the level of the luminal surface. The possible relation between the orientation of strands of these networks and extra- or intracellular stress was discussed.     

Homologs of CD83 from elasmobranch and teleost fish

Dendritic cells are one of the most important cell types connecting innate and adaptive immunity, but very little is known about their evolutionary origins. To begin to study dendritic cells from lower vertebrates, we isolated and characterized CD83 from the nurse shark (Ginglymostoma cirratum (Gici)) and rainbow trout (Oncorhynchus mykiss (Onmy)). The open reading frames for Gici-CD83 (194 aa) and Onmy-CD83 (218 aa) display approximately 28-32% identity to mammalian CD83 with the presence of two conserved N-linked glycosylation sites. Identical with mammalian CD83 genes, Gici-CD83 is composed of five exons including conservation of phase for the splice sites. Mammalian CD83 genes contain a split Ig superfamily V domain that represents a unique sequence feature for CD83 genes, a feature conserved in both Gici- and Onmy-CD83. Gici-CD83 and Onmy-CD83 are not linked to the MHC, an attribute shared with mouse but not human CD83. Gici-CD83 is expressed rather ubiquitously with highest levels in the epigonal tissue, a primary site for lymphopoiesis in the nurse shark, whereas Onmy-CD83 mRNA expression largely paralleled that of MHC class II but at lower levels. Finally, Onmy-CD83 gene expression is up-regulated in virus-infected trout, and the promoter is responsive to trout IFN regulatory factor-1. These results suggest that the role of CD83, an adhesion molecule for cell-mediated immunity, has been conserved over 450 million years of vertebrate evolution.     

Gap junctions in human sebaceous glands

Summary An examination of human sebaceous glands by transmission electron microscopy has revealed the presence of gap junctions. The junctions are found in abundance between differentiating cells, and annular forms are also seen. The possible significance of this new finding is briefly discussed.We thank the ENT consultant staff of Royal Prince Alfred Hospital, Sydney, for making tissue available. The co-operation of the director, Dr. D.J.H. Cockayne, and staff of the University of Sydney Electron Microscope Unit is gratefully acknowledged. Mr. E. Foster provided excellent assistance with the photography. -The project was supported by the Consolidated Medical Research Funds, University of Sydney, and the National Health and Medical Research Council of Australia. One of us (N.K.) was the recipient of the Phyllis Anderson Medical Research Fellowship awarded by the Faculty of Medicine, University of Sydney     

Renin-angiotensin system in elasmobranch fish: A review.

The renin-angiotensin system (RAS) has been identified recently in elasmobranch fish, and the structure of angiotensin II (ANG II) is unusual ([Asp(1),Pro(3),Ile(5)]-ANG II) compared to other vertebrates. Receptors for ANG II have been identified in blood vessels and in a variety of osmoregulatory tissues including the gill, kidney and rectal gland. In addition, there is considerable binding to the interrenal gland and the stimulation of 1alpha-hydroxycorticosterone production in vitro suggests a physiological role in corticosteroidogenesis. ANG II is a potent vasoconstrictor and this effect does not appear to be mediated by sympathetic activation or catecholamine release. Although the RAS may not be involved in maintaining basal blood pressure, it may be important in situations in which blood pressure is reduced. Understanding of the role of ANG II as an osmoregulatory hormone is only just emerging with putative roles in the control of gill, rectal gland and perhaps, drinking. In addition, the stimulation of corticosteroid secretion may provide another means of controlling osmoregulation. J. Exp. Zool. 284:526-534, 1999.     

Postlarval muscle growth in fish: a DNA flow cytometric and morphometric analysis

The mechanism of postlarval fish myotomal growth was investigated in trout (Salmo gairdneri) by means of morphometric and cytofluorometric analysis. The mechanism by which new fibres are added during postlarval growth (hyperplasia) is not fully understood. In histological cross sections these new fibres have a small diameter which give the muscle a "mosaic" appearance. One hypothesis suggested that they could be derived from the proliferative activity of satellite cells. DNA cytofluorometric analysis of nuclei suspensions obtained from trout white myotomal muscle during different developmental stages (eleutherembyronic; alevin; yearling and adult) showed a consistently low S-cytometric phase during all stage in which myofibres of small diameters were present. The percentage of such small fibres, determined by morphometric analysis, suggested that satellite cells are the proliferative population. In fact, their percentages, as determined by morphometric analysis in histological section, bear a linear relationship with the S-cytometric phase percent nuclei (R = 0.927). Only in adults (67 cm in size) there was a significant decrease in the S-cytometric phase. At this stage, in histological sections, the myotomal muscle no longer had a "mosaic" appearance because of the disappearance of the small fibres. It may, therefore, be supposed that in the cm 67 adult specimens, the proliferative population is entering the G0 phase. It is known, in fact, that muscle growth proceeds only by fibre hypertrophy in trout longer than 70 cm in length (Stickland, 1983).     

Freeze-fracture studies of gap junctions in the developing and adult amphibian cardiac muscle.

The freeze-fracture technique has been used to characterize the junctional devices involved in the electrical coupling of Ambystoma cardiac tissue. These cells are connected by junctions formed by either linear or circular arrays of particles. Such structures can be interpreted as a special type of gap junction. Gap junctions have also been investigated during the growth and differentiation of two amphibians, Rana and Xenopus. In both genera the earliest stage of junctional assembly is characterized by linear rows of particles. Later, a gradual transformation of these linear rows into circles was found. Finally, in the fully formed gap junctions, these circles appeared to join together into clusters. In summary, in the adult amphibian myocardial cells, three different types of gap junctions can be described. The first type, which has been observed in all embryonic stages and in adults in all three genera, consists of linear or circular arrays of particles: this is the only type of gap junction seen at any age in Xenopus. The second type, consisting of a variable number of anastomosing circles forming regular networks, is never observed in embryonic cells. It is typical of the adult frog heart and may also be seen in Ambystoma. The third type is characteristic only of adult Ambystoma heart and consists of geometrically packed particles identifiable with classic communicating macula. The fact that only the first class of structure is observed in Xenopus heart strongly supports the conclusion that such linear arrays of intramembranous particles really represent true functional electrical junctions.     

AMP-deaminase in elasmobranch fish: a comparative histochemical and enzymatic study

AMP-deaminase activity was measured in white muscle from a wide range of fish, including one cyclostome, 13 chondrosteans, and one teleost to elucidate the pattern of the AMP-deaminase activity in white muscle of fish. Compared to a mammalian (rat) muscle extract, low enzyme activities are found in the cyclostome and two elasmobranchs from two families (Scyliorhinidae, Hexanchidae). In contrast, higher AMP-deaminase activities, similar to mammals, are expressed in Squalidae, all families of skates, Chimaeridae and in the teleostean fish. We then compared AMP-deaminase activities in red and white muscles from two representative elasmobranch fish, the dogfish (Scyliorhinus canicula) and the thornback ray (Raja clavata). The fibre type composition and distribution of the locomotory musculature were determined in these two elasmobranchs to establish a relationship between the morphology, the type of fibres of the locomotion-implicated muscles and the AMP-deaminase activity. Experimental data are discussed with respect to the layout of fibres in the myotome. In both species, three fibre types were identified. In the two fish myotomes, most of the axial muscles are white fibres while red fibres constitute a thin sheet. Some differences were observed between the two species in the distribution of intermediate fibres: in dogfish, these are located between the red and white fibres; in thornback ray, some are dispersed within the white fibre region, while others form an intermediary layer like in dogfish. These results suggest that in the course of evolution, an amplification of the AMP-deaminase activity in muscle was coupled with increase of complexity of the muscular structure.     

Freeze-fracture replicas of rat ameloblasts reveal unusual membrane domains around gap junctions

Specimens of aldehyde-fixed and glycerol-impregnated tooth germs obtained from 1-2 day old rats were prepared for ultrathin section studies and for freeze-fracture, with the purpose of studying the structural organization of membranes of developing ameloblasts. In this report we describe unusual membrane domains which were found surrounding several ameloblast gap junctions. Developing ameloblasts - when examined in ultrathin sections - exhibit gap junctions which appear straight, curved or invaginated. In freeze-fracture replicas, in addition to their typical appearance, several gap junctions were found to be surrounded by a membrane margin which was undulating and devoid of intramembrane particles (IMP's). We believe that these hitherto unreported particle-free membrane margins are associated with the formation of curved or invaginated gap junctions. It is possible that these membrane margins are particle-free because plasma membrane proteins (presumably IMP's) become transiently detached from the cytoskeleton and move laterally. It is therefore likely that these margins are pure lipid domains which are more flexible, thus providing a transient hinge-like mechanism which facilitates the movement required for the formation of the curved or invaginated ameloblast gap junctions.     

Freeze-fracture surface of salivary glands of rat observed by scanning electron microscopy

The structure of parenchymal components of rat submandibular glands observed with scanning electron microscopy is presented. The glands were fixed, submerged in liquid nitrogen, cryofractured, dehydrated and critical-point-dried. The fracture surface displayed the acini, granular ducts and striated ducts. Acini exhibit a typical sponge-like pattern of irregular and empty cavities. Granular ducts are lined by bulging cells laden with numerous secretory granules. Their diameter ranged from 0.2 to 3.2 microns, with a mean value of 1.240.4 microns. Measurement of the actual granules was rendered possible by direct observation. Striated ducts exhibited a cribiform pattern near the basal cell region which corresponds to infoldings of the basal cell membrane.     

Macromolecules can pass through occluding junctions of rat ileal epithelium during cholinergic stimulation

Summary Crypt, but not villus, goblet cells in the ileum accelerate their secretion of mucus within 5 min following cholinergic stimulation. This study was done to determine whether the macromolecular permeability and structure of occluding junctions in the ileum are altered during accelerated secretion. Rats were injected intravenously with horseradish peroxidase followed by carbachol (250 g/kg, subcutaneous) and the intestinal mucosa was fixed 3–12 min later. In control mucosa (saline-injected), peroxidase filled lateral intercellular spaces up to the occluding junctions of both crypt and villus epithelium, but did not enter occluding junctions or pass into the lumen. In 3 of 8 carbachol-stimulated rats, peroxidase was present within occluding junctions in crypt epithelium and in the crypt lumen, although all intermembrane junctional fusion sites appeared intact. Villus epithelial occluding junctions, in contrast, continued to exclude peroxidase. In freeze-fracture replicas of crypt cells prepared after carbachol stimulation, we detected no structural changes in strand networks of occluding junctions that could account for increased paracellular permeability.     

Effects of extracellular calcium depletion on membrane topography and occluding junctions of mammary epithelial cells in culture

Ca2+ dependence of occluding junction structure and permeability, well documented in explanted or cultured epithelial sheets, presumably reflects inherent control mechanisms. As an approach to identification of these mechanisms, we induced disassembly of zonulae occludentes in confluent monolayers of mouse mammary epithelial cells by exposure to low concentrations of the chelators, EGTA or sodium citrate. Stages in disassembly were monitored during treatment by phase-contrast microscopy and prepared for transmission and scanning electron microscopy. Cellular response included several events affecting occluding junctions: (a) Centripetal cytoplasmic contraction created tension on junction membranes and displaced intramembrane strands along lines determined by the axis of tension. (b) Destabilization of junction position, probably through increased membrane fluidity, augmented tension-induced movement of strands, resulting in fragmentation of the junction belt. (c) Active ruffling and retraction of freed peripheral membranes remodeled cell borders to produce many filopodia, distally attached by occluding-junction fragments to neighboring cell membranes. Filopodia generally persisted until mechanically ruptured, when endocytosis of the junction and adhering cytoplasmic bleb ensued. Junction disassembly thus resulted from mechanical tensions generated by initial centripetal contraction and subsequent peripheral cytoskeletal activity, combined with destabilization of the junction's intramembrane strand pattern.