The fine structure of sensory receptor processes in the auricular epithelium of the planarian,Dugesia tigrina

Summary The marginal epithelium of the lateral auricles of the planarian, Dugesia tigrina, includes a cell type with surface cilia and microvilli, a basal nucleus, and dense cytoplasm containing secretory vacuoles, Golgi elements, mitochondria and ribosomes. Through channels within the epithelial cytoplasm, cellular processes, interpreted as extensions of neurosensory receptor cells located in the subepidermis, project to the surface. The receptor processes, containing microtubules, mitochondria, vesicles and an agranular tubular reticulum, project beyond the epithelial cell surface; one or two cilia each emerge from a basal body in the apex of the projection. Close to the point of emergence to the epithelial surface, each cylindrical receptor process is surrounded by a collar-like septate junction between adjacent plasma membranes. The cilia of the projections differ from those of the epithelial cells in diameter, density of matrix and in the banding patterns of the rootlets. A few projections appear with the apex and basal body retracted below the epithelial surface. The possible function of these ciliated processes in sensory reception is discussed.This work was supported by Grant No. SO 1 FR 5369 from the U.S. Public Health Service to the University of Illinois at the Medical Center.I thank Dr. J. P. Marbarger, Director of the Research Resources Laboratory, for use of the electron microscope facilities, Miss Irena Kairys for technical help, Miss Marie Jaeger for assistance with photography, and Mr. Robert Parshall for the drawing.To Professor Arthur Wagg Pollister, I respectfully dedicate this article on the occasion of his retirement from Columbia University.     

Implantation in vitro: co-culture of rat blastocyst and epithelial cell vesicles

Implantation of blastocysts involves conversion of maternal and embryonic cell surfaces from a nonadhesive to an adhesive state in response to the internally driven developmental program or to externally generated factors. However, the intricacies of the cellular and subcellular changes that promote the attachment are not known, because these changes are difficult to determine in situ because of the nonaccessibility of the site. To overcome this, an in vitro model of implantation was developed by co-culturing rat blastocysts and uterine epithelial cells of the same gestational age (day 5 postcoitum; plug day as day 1) in drops hanging from the lid of a Petri dish. The system was used to study the changes on the surface membranes of the cells of the trophectoderm and uterine epithelium and to evaluate the antiadhesive activity of the newly designed test substances. The isolated epithelial cell vesicles were co-cultured with zona-free blastocysts in the microdrops (40–50 µl) hanging from the lid of a 60-mm Petri dish. The lid was placed over the lower dish, which was presaturated with the medium. The culture was examined 48 h later to determine the site of adhesion of epithelial cell vesicles with the trophoblasts lining the blastocyst. The cell-cell adhesion was monitored on a computerized image analyzer. To validate the adhesion of blastocysts and epithelial cell vesicles in co-culture, the expression of a cell adhesion molecule, uvomorulin, was studied using immunocytochemical technique after incubating with antiuvomorulin antibody. Intense staining was noted on the membrane surfaces at the site of attachment of the blastocyst and cell vesicles.The authors express their sincere thanks to the Ministry of Health and Family Welfare, Government of India, for their financial support     

Ultrastructural differentiation of the intestinal epithelium in the bandicoot Perameles nasuta

Summary The principal cells of the epithelium in the small intestine of the marsupial Perameles nasuta were studied with the electron microscope. The cells in the lower parts of the crypts are undifferentiated and have a high nucleo-cytoplasmic ratio and an abundance of free ribosomes. As the cells move upwards to take their place in the surface epithelium covering the mucosal folds their nucleo-cytoplasmic ratio and the number of free ribosomes decrease, the cells elongate and develop a brush border, a system of microtubules in the apical cytoplasm, a terminal web, terminal bars and desmosomes.The brush border develops from a series of cell processes interdigitating with those from the opposite cell. Spaces arising between the cell processes gradually separate the contiguous cells and the cell processes become microvilli which increase in number and become uniform in size and shape. The Golgi complex gives rise to small vesicles with a different membrane structure than that of the Golgi membranes themselves. It is suggested that the microtubules do not arise as tubular invaginations of the surface membrane but that they develop from the Golgi vesicles.     

Electron microscopic studies of coated membranes in two types of gill epithelial cells of lamprey

Summary Coated membranes in two types of gill epithelial cell of adult lamprey, Lampetra japonica, were studied by electron microscopy. The type 3 gill epithelial cells possess well-developed microvilli or microfolds, apical vesicles and abundant mitochondria. The cytoplasmic surface of the microvillous plasma membrane is covered by a coat of regularly spaced particles with a center-to-center distance of about 15 nm. Each particle consists of a bulbous free end, about 10 nm in diameter, and a connecting piece, about 5 nm long. Apical vesicles are covered by a surface coat which consists of fine filamentous material but lack any special coating on their cytoplasmic surface.The type 4 cells (chloride cells) are characterized by apical vesicles, abundant mitochondria and cytoplasmic tubules. These tubules possess a coat on their luminal surface which consists of spirally wound parallel rows of electron-dense materials. The rows are about 16 nm apart and wound at a pitch of about 45°. The cytoplasmic surface of these tubules does not display a special coat. These coated membranes are assumed to be the sites of active ion transport across the plasma membrane. In particular, particles in type 3 cells and linear coat materials in chloride cells may be either loci of transport enzymes or energy generating systems. Apical vesicles lack any coating on their cytoplasmic surface but a fine filamentous coat is present on their luminal surface. They contain intraluminal vesicles and are continuous with apical ends of cytoplasmic tubules.     

STRUCTURE OF THE TOAD'S URINARY BLADDER AS RELATED TO ITS PHYSIOLOGY

The structure of the urinary bladder of the toad Bufo marinus was studied by light and electron microscopy. The epithelium covering the mucosal surface of the bladder is 3 to 10 microns thick and consists of squamous epithelial cells, goblet cells, and a third class of cells containing many mitochondria and possibly representing goblet cells in early stages of their secretory cycle. This epithelium is supported on a lamina propria 30 to several hundred microns thick and containing collagen fibrils, bundles of smooth muscle fibers, and blood vessels. The serosal surface of the bladder is covered by an incomplete mesothelium. The cytoplasm of the squamous epithelial cells, which greatly outnumber the other types of cells, is organized in a way characteristic of epithelial secretory cells. Mitochondria, smooth and rough surfaced endoplasmic reticulum, a Golgi apparatus, "multivesicular bodies," and isolated particles and vesicles are present. Secretion granules are found immediately under the plasma membranes of the free surfaces of the epithelial cells and are seen to fuse with these membranes and release their contents to contribute to a fibrous surface coating found only on the free mucosal surfaces of the cells. Beneath the plasma membranes on these surfaces is an additional, finely granular component. Lateral and basal plasma membranes are heavily plicated and appear ordinary in fine structure. The cells of the epithelium are tightly held together by a terminal bar apparatus and sealed together, with an intervening space of only 0.02 mµ near the bladder lumen, in such a way as to prevent water leakage between the cells. It is demonstrated in in vitro experiments that water traversing the bladder wall passes through the cytoplasm of the epithelial cells and that a vesicle transport mechanism is not involved. In vitro experiments also show that the basal (serosal) surfaces of the epithelial cells are freely permeable to water, while the free (mucosal) surfaces are normally relatively impermeable but become permeable when the serosal surface of the bladder is treated with neurohypophyseal hormones. The permeability barrier found at the mucosal surface may be represented, structurally, either by the filamentous layer lying external to the plasma membrane, by the intracellular, granular component found just under the plasma membrane, or by both of these components of the mucosal surface complex. The polarity of the epithelial sheet is emphasized and related to the physiological role of the urinary bladder in amphibian water balance mechanisms.     

Translocation of dimeric IgA through neoplastic colon cells in vitro.

We studied the translocation of dimeric IgA across epithelium, using neoplastic human colon cells in culture as a source of epithelial cells, and immunoelectronmicroscopy with peroxidase-labeled antigens and antibodies. The cells had some of the ultrastructural characteristics of normal, mature epithelial cells, i.e., polarity, desmosomal junctions, and secretory component on their basal and lateral plasma membranes. Horseradish peroxidase-labeled dimeric IgA, exposed to the cells at 0 degrees C, bound selectively to secretory component on the cell surfaces. At 37 degrees C, the bound dimeric IgA was taken into the cells by endocytosis and transported apically through the cytoplasm in vesicles. After 30 min, IgA was discharged across the apical surface. Neither colchicine (10(-4) M) nor cytochalasin B (10(-5) M) interfered with binding or endocytosis of dimeric IgA, but colchicine inhibited intracellular transport of the IgA-containing vesicles. These experiments demonstrated that dimeric IgA can be transported through living intestinal epithelial cells in vitro. The transport includes 1) specific binding of IgA dimers to secretory component on plasma membranes, 2) endocytosis of IgA in vesicles, 3) transcytoplasmic transport of the IgA-containing vesicles by a process involving microtubules, and 4) discharge of IgA at the apical surfaces.     

Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae)

Glycoproteins (GPs) were visualised histochemically in the secretory cells – the mucous goblet cells (the type A and the type B), the serous goblet cells, the club cells and the epithelial cells in the gill epithelium of Rita rita. The type A mucous goblet cells, the type B mucous goblet cells and the epithelial cells elaborate GPs with oxidizable vicinal diols and GPs with sialic acid residue without O-acyl substitution. In addition, GPs with O-sulphate esters are elaborated by the type A and GPs with O-acyl sugars by the type B mucous goblet cells. GPs are absent in the serous goblet cells and are with oxidizable vicinal diols in low moieties in the club cells. The analysis of the results elucidates interesting differences in the composition and concentration of GPs in the mucus elaborated by the epithelium of the gill arches and the gill rakers; and the gill filaments and the secondary lamellae indicating the potential importance of the glycoproteins at these locations. GPs elaborated on the surfaces of the gill arches and the gill rakers could be associated to assist in feeding activities and on the surfaces of the gill filaments and the secondary lamellae in the respiratory activity.     

The fine structure of gill 'podocytes' in Panulirus argus (crustacea)

A cell type structurally resembling the podocyte of the renal glomerulus is situated in the gill of the crustacean Panulirus argus. These cells adjoin the medial septum of the gill filament and invariably face the efferent haemolymph channel. The basal cell surface is produced into a series of regular ridges, between which are inserted elongated cell processes, together constituting a palisade that includes narrow slits (250 A or more in width) resembling the filtration pores between the foot process of the glomerular epithelium. In each instance, the slit is traversed by a diaphragm which in the crustacean 'podocyte' is ca. 30 A in width and contiguous with the outer leaflet of the unit membrane limiting the cell. Numerous coated vesicles originate from the cell surface beneath the diaphragms. The possible role of these cells in detoxification by withdrawal of materials from the circulation is discussed.     

Rhombic particle arrays in gill epithelium of a mollusc, Aplysia californica.

Gill epithelia from adult and juvenile Aplysia were examined by conventional thin section and freeze-fracture methods. Freeze-fracture replicas of adult gill epithelium revealed septate and gap junctions, which served as membrane markers for the epithelial cells. In these same cell membranes, non-junctional rhombic arrays of intramembranous particles were observed on prominent ridges on the membrane P fracture face of some epithelial cells. In thin sections of adult epithelium, nerve terminals were observed abutting the lateral plasma membranes near the basal lamina of some epithelial cells. Correlative areas of plasma membrane in freeze-fracture replicas showed a close association between rhombic particle arrays and abutting nerve terminals. In thin sections of juvenile Aplysia, nerve terminals abutting the epithelial cells were not recognizable, and rhombic arrays were not observed in freeze-fracture replicas. This suggested that a developmental association existed between the appearance of rhombic arrays in adult epithelia and their innervation. It is not known with certainty if, in invertebrates, rhombic arrays are an essential structural entity of all innervated cell membranes; however, in the cells thus far studied, there appears to be an associative condition. In the case of the gill epithelium of Aplysia, rhombic arrays are located in the same vicinity as the abutting nerve terminals. Similar arrays of intramembranous particles have been observed in myoneural postjunctional complexes of other invertebrates and have been interpreted to be the morphological expression of neurotransmitter receptors. An analogous explanation is put forth, namely that rhombic arrays may represent the structural correlates of neurotransmitter receptors and/or ionic channels in innervated membranes of invertebrates.     

Ordered arrays of intramembrane particles on the surface of fish gills

Freeze fracture of euryhaline fish gill epithelium reveals large smooth surfaces with intramembrane particles or corresponding pits often aggregated into hexagonal arrays. They belong to the outer plasma membranes of respiratory cells, the main surface of contact between the internal and external milieu of the fish. Possible functions for the arrays are discussed in relation to the fine structure of the respiratory cell.     

Surface specializations of the epithelial cells at the tip of the optic tentacle,dorsal surface of the head and ventral surface of the foot in Helix aspersa

Summary Morphologically the surface specializations of the epithelium covering the dorsal head and ventral foot regions in Helix aspersa consists either of cilia or microvilli respectively. The epithelium at the tip of the optic tentacle is a simple one. Each epithelial cell has a number of cilia-like projections from their free surfaces. These projections usually branch at their tips into two or three slender, microvilli-like structures. From the bases of the cilia-like projections arise numerous, tubular processes which form a thick, spongy layer interspersed between these projections. The microvilli-like structures are immersed in a fine, fibrous mat; unlike the fibrous mats on the dorsal head and ventral foot epithelia this material does not autofluoresce. It is suggested that it arises from the collar cells and not from typical mucocytes. The functional relationship between these surface specializations of the optic tentacle epithelium and the abundance of sensory axons in this region is discussed. These epithelial cell projections on the tentacle probably function not only as a protective covering but also to create a fluid trap for odours in the ambient air. The various contacts between epithelial cells serve to maintain the integrity of the epithelium while allowing for stretching due to protrusion of the tentacle.This work has been supported by the Australian Research Grants Committee.     

FINE STRUCTURE AND MORPHOGENIC MOVEMENTS IN THE GASTRULA OF THE TREEFROG, HYLA REGILLA

The blastoporal groove of the early gastrula of the treefrog, Hyla regilla, was examined with the electron microscope. The innermost extension of the groove is lined with invaginating flask- and wedge-shaped cells of entoderm and mesoderm. The distal surfaces of these cells bear microvilli which are underlain with an electron-opaque layer composed of fine granular material and fibrils. The dense layer and masses of vesicles proximal to it fill the necks of the cells. In flask cells bordering the forming archenteron the vesicles are replaced by large vacuoles surrounded by layers of membranes. The cells lining the groove are tightly joined at their distal ends in the region of the dense layer. Proximally, the cell bodies are separated by wide intercellular spaces. The cell body, which is migrating toward the interior of the gastrula, contains the nucleus plus other organalles and inclusions common to amphibian gastrular cells. A dense layer of granular material, vesicles, and membranes lies beneath the surface of the cell body and extends into pseudopodium-like processes and surface undulations which cross the intercellular spaces. A special mesodermal cell observed in the dorsal lining of the groove is smaller and denser than the surrounding presumptive chordamesodermal cells. A long finger of cytoplasm, filled with a dense layer, vesicles and membranes, extends from its distal surface along the edge of the groove, ending in a tight interlocking with another mesodermal cell. Some correlations between fine structure and the mechanics of gastrulation are discussed, and a theory of invagination is proposed, based on contraction and expansion of the dense layer and the tight junctions at distal cell surfaces.     

Effects of Diazinon on bluegill sunfish, Lepomis macrochirus, gills: scanning electron microscope observations

Gills of bluegill sunfish, Lepomis macrochirus, exhibited varied degrees of structural damage following a 24-h exposure to sublethal concentrations (15 μg/l, 30 μg/l, 45 μg/l, 60 μg/l and 75 μg/l) of Diazinon [O,O-diethyl-O-(2-isopropyl-6-methyl-4 pyrimidinyl ester or phosphorothioate]. Exposure to 15 μg/l and 30 μg/l resulted in exocytosis of some material to the cell surface and perforations of the microridges. At higher doses (above 45 μg/l), the extrusion was reduced and the cells were swollen. Compared to control values, the thickness of the microridge on the gill arch and on the gill filament generally increased with exposure to Diazinon. Also, the distance between microridges decreased with increased exposure concentrations. At 60 μg/l, gill arch microridges fused and some ridges of gill filaments disappeared. At 75 μg/l exposure, epithelial cells of the gill arch became obscured with severe cellular extrusions and the lamellar surfaces swelled. The mucus extrusion, lamellar swelling and reduced microridges may be related to a defence mechanism which reduces the water surface around the gill and increases the barrier distance for diffusion of toxicants from outside to the blood capillaries. Although this mechanism protects the fish from toxicants, it also reduces the oxygen supply which leads to suffocation of the fish.     

Function of the cytoskeleton in cells with microridges from the oral epithelium of the carp Cyprinus carpio

Superficial cells of the oral mucosal epithelium in the carp and the cytoskeleton of the epithelial cells are examined by scanning and transmission electron microscopy. Microridges are formed on the surface of the epithelium. Epithelial cells contain two types of vesicles: mucous secretory vesicles and coated vesicles. Most of the mucous vesicles are situated in the center of the cell near the Golgi apparatus. In freeze-fracture replicas, intramembranous particles are abundant in the membranes of the secretory vesicles but rare in the apical plasma membrane. Coated vesicles are situated in the apical and subapical cytoplasm. A great number of thick filaments, considered to be keratin filaments, run randomly throughout the cell to form a meshwork. Thick filaments, which are sparse in the central cytoplasm, are connected to the membranes of the secretory vesicles and other membranous organelles. A layer of closely packed thin filaments, considered to be actin filaments, is found just beneath the apical plasma membrane. Microtubules also occur in the apical cytoplasm and run almost parallel to the cell surface. Both kinds of vesicles are connected to the thin and thick filaments. Their functional significance in the regulation of membrane at the free surface is discussed.     

Organization of a phyllobranchiate gill from the green shore crab Carcinus maenas (Crustacea,Decapoda)

Summary The phyllobranchiate gills of the green shore crab Carcinus maenas have been examined histologically and ultrastructurally. Each gill lamella is bounded by a chitinous cuticle. The apical surface of the branchial epithelium contacts this cuticle, and a basal lamina segregates the epithelium from an intralamellar hemocoel. In animals acclimated to normal sea water, five epithelial cell types can be identified in the lamellae of the posterior gills: chief cells, striated cells, pillar cells, nephrocytes, and glycocytes. Chief cells are the predominant cells in the branchial epithelium. They are squamous or low cuboidal and likely play a role in respiration. Striated cells, which are probably involved in ionoregulation, are also squamous or low cuboidal. Basal folds of the striated cells contain mitochondria and interdigitate with the bodies and processes of adjacent cells. Pillar cells span the hemocoel to link the proximal and distal sides of a lamella. Nephrocytes are large, spherical cells with voluminous vacuoles. They are rimmed by foot processes or pedicels and frequently associate with the pillar cells. Glycocytes are pleomorphic cells packed with glycogen granules and multigranular rosettes. The glycocytes often mingle with the nephrocytes. Inclusion of the nephrocytes and glycocytes as members of the branchial epithelium is justified by their participation in intercellular junctions and their position internal to the epithelial basal lamina.     

The Ulrastructure of the Gill of the Lugworm Arenicola marina (L.) (Annelida,Polychaeta)

Arenicola marina gills are hollow, branched, body outgrowths with a central coelomic cavity and afferent and efferent vessels. The gill surface area per unit body weight is about 4 cm²/g wet weight. The blood vascular system anatomy differs from the tip to the base of the gill. In the distal branches of the gill the superficial afferent and efferent vessels are joined by connecting vessels. All vessels arise as spacings between the basal laminae of the thin epidermis and of the coelomic myoepithelium. The contractile part of this epithelium mainly borders the afferent and efferent vessels, whereas pedicel-like cytoplasmic processes extend from the cell bodies and mainly line the connecting vessels. In the proximal branches of the gill the afferent and efferent vessels located in the coelomic cavity are surrounded by the coelomic myoepithelium, and a peripheral blood plexus is present below the epidermis. The gill epidermis is everywhere thin and does not exhibit the characters of a transporting epithelium. The gill coelomic myoepithelium has several functions: (i) periodic contractions of the gill, propelling blood and coelomic fluid toward the central vascular and coelomic compartments; (ii) blood ultrafilration toward the coelomic cavity; (iii) probably transport, suggested by the specialized structures of the lateral membranes of the cells.     

A correlated light and electron microscopic study of the structure and secretory activity of the accessory salivary glands of the marine gastropods,Conus flavidus and C. vexillum (neogastropoda,conacea)

The structure and secretory activity of the accessory salivary gland in two species of Conus were examined using routine and histochemical techniques of light, scanning and transmission electron microscopy. The composite layers of the accessory salivary gland of Conus are a luminal epithelium, fibromuscular layer, submuscular layer, and a capsule. In C. flavidus and C. vexillum, the luminal epithelium is formed by epitheliocytes and cytoplasmic processes extending from the secretory cells, whose perikarya form the submuscular layer. The processes carry secretory cell products (chiefly Golgi-derived glycoprotein) across the fibromuscular layer and terminate between epitheliocytes (at the bases of the secretory canaliculi) or beyond the surface of the epithelial cells. Conus vexillum is distinguished from C. flavidus by its high content of lipofuscin. Epitheliocytes are the only microvillated cells in the accessory salivary gland of Conus. In C. flavidus, epitheliocytes extrude secretory granules, various types of cytoplasmic blebs and clear vesicles by apocrine “pinching off”. Clear vesicles are shed from the tips of microvilli. The luminal epithelial cells of C. vexillum similarly egest clear vesicles, but normally undergo additional holocrine secretion to release lipofuscin. The secretions of epitheliocytes appear to be major products of the accessory salivary gland: consideration of secretory activities by both epitheliocytes and secretory cells will therefore be necessary when directly investigating accessory salivary gland function in Conus.     

Primary cell culture from gill explants of rainbow trout

Primary cultures of gill cells were initiated from gill filament explants of rainbow trout, Oncorhynchus mykiss . The explants were cultured in Leibovitz l -15 medium with 5, 10 or 20% foetal calf serum (FCS) and l -glutamine. The attachment efficiency was serum-dependent though increased FCS concentration did not stimulate further outgrowth of cells. The explants produced cell outgrowth 24 h after attachment as a sheet of cells which exhibited characteristics of gill pavement epithelial cells as indicated by surface microridges revealed by scanning electron micrographs. There was high proliferation for the first 14 days then a stable plateau for 30 days followed by a decline phase from 45 days. Following removal of cells, the explants produced further cell outgrowth which was especially active at the proliferation phase (14 days). Removal of these cells caused the explants to produce a further proliferation of cells reaching confluence in 10–14 days. After the third cell removal cell outgrowth from explants showed migratory activity but did not develop to resemble gill epithelial cells. The use of gill explants to establish primary cultures of fish gill cells has advantages which include longevity of the culture and successive proliferations from explants which could provide a useful tool for the investigation of long-term processes in cellular biology and reduce the number of culture preparations.     

Ultracytochemistry of cholera-toxin binding sites in ciliary processes

Summary Cholera toxin reduces the rate of formation of aqueous humor in concentrations (10^–11 M) that do not disturb the morphology of the aqueoushumor forming epithelial cells of the ciliary processes of the rabbit eye. The search for an endogenous mediator of aqueous-humor formation comparable to cholera toxin in its mode of operation prompted us to map the distribution of cell surface receptors for cholera toxin in the ciliary processes of the eyes of rabbits. Cytochemical studies were carried out with the use of conjugates of cholera toxin to fluorescein isothiocyanate (CT-FITC) and to horseradish peroxidase (CT-HRP), and of the B subunit of cholera toxin to horseradish peroxidase (B-HRP). Multiple fluorescent CT-FITC binding sites were observed on the outer nonpigmented epithelial layer near the crests of the processes. Processes incubated with CT-HRP in vitro showed surface staining of 30–40% of the nonpigmented epithelial cells. A prominent reaction product was observed along the basal and lateral plasma membranes of these cells. In vivo studies carried out after arterial infusion of B-HRP showed a reproducible dense reaction product between the apical surfaces of the pigmented epithelium (PE) and of the nonpigmented epithelium (NPE) facing each other. Aggregations of reaction product were observed with the electron microscope in the extracellular space between the apices of PE and NPE. The apical plasma membrane of the endothelium of the blood vessels near the crests of the ciliary processes was stained after either in vivo or in vitro exposure to peroxidase conjugates. These findings indicate that the cell-surface receptors which mediate the action of cholera toxin on aqueous humor formation are very likely localized in the apical plasma membranes of the epithelium of the ciliary processes.Supported in part by USPHS grant # EY-00237, the Connecticut Lions Eye Research Foundation, Inc., and Research to Prevent Blindness, Inc.