Immunocytochemical localization of alkaline phosphatase in absorptive cells of rat small intestine after colchicine treatment

Summary The purpose of this study was to investigate the effect of colchicine and vinblastine on the localization of alkaline phosphatase (AlPase) in rat duodenum in relation to structural changes. AlPase was localized on the membranes of rough endoplasmic reticulum, Golgi stacks, cytoplasmic vesicles, microvilli, on lateral plasma membranes, and in some lysosomes of the duodenal epithelial cells of rats treated with either lumicolchicine or 0.9% NaCl alone. Microvilli were most intensely stained, and AlPase-positive Golgi stacks were regularly distributed in the supranuclear regions. After colchicine treatment, microvilli were shortened and the staining intensity became weaker, whereas basal as well as lateral plasma membranes showed stronger staining. The AlPase-positive microvilli appeared not only on the luminal surfaces, but also on the baso-lateral plasma membranes and even on the surfaces of characteristic intracytoplasmic cysts. Golgi stacks became smaller and their distribution became less localized, and the staining intensity of the Golgi stacks became weaker. AlPase localization in rats treated with vinblastine was almost identical with that of rats treated with colchicine. Thus, colchicine and vinblastine appeared to have elicited a disorientation of intracellular transport of intestinal AlPase by inhibiting microtubule organization.     

Absorption of horseradish peroxidase by the small intestinal epithelium in postnatal developing rats.

After an intraluminal injection of horseradish peroxidase into the small intestine, the localization of peroxidase was studied in neonatal developing and adult rats by means of electron microscopy. Until around the 14th day of the neonatal period absorbed peroxidase granules in the duodenal and jejunal epithelium were abundant in the microvillous membrane, the apical tubulo-vacuolar system, and the Golgi apparatus, and on the lateral cell and basal membranes, and the luminal surfaces of the capillary cells. At the weaning period the tubulo-vacuolar system was absent in the duodenal and jejunal epithelial cells, and at that point absorbed peroxidase was observed in the same sites as in the adult rats: the microvillous membrane, the lateral cell and basal membranes, the Golgi apparatus, and the vesicles and vacuoles of the cytoplasm. During the suckling period, in the ileal epithelial cells exogenous peroxidase was found on the microvilli, in the tubulo-vacuolar system, in the supranuclear vacuole, in the Golgi apparatus, on the lateral cell and basal membranes, and also on the luminal surface of the endothelial cells of blood capillaries. When the tubulo-vacuolar system and the supranuclear vacuole were lost from the ileal cells at the weaning period, no exogenous peroxidase uptake was observed in the absorptive cell of the ileal epithelium.     

Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.     

Cytochemical localization of alkaline phosphatase in the ependyma of the rat medulla oblongata

Summary The ependyma of the IVth ventricle and the central canal of the rat medulla oblongata was investigated using the cytochemical technique for alkaline phosphatase (AlPase) which revealed two types of ependymal cells in the medulla. The central canal type of the ependymal cell occupying the dorsal part of the central canal in the lower medulla exhibited intense AlPase activity with light microscopy. These cells had reaction products in all plasma membranes, including the microvilli and the cilia at the luminal cell surface. Some cells appeared to be tanycytes, since the process reached the basement membrane of the parenchymal blood vessel. The ventricular type of ependymal cells, which form the floor of the IVth ventricle and the central canal, contained no reaction products in any structure of the luminal cell surface.The possible relationship between the cerebrospinal fluid and the nervous tissues through the ependymal linings is discussed.     

Fine structure of the rhombencephalic tela of the bullfrog,Rana catesbeiana

Summary The posterior rhombencephalic tela choroidea of the bullfrog was examined by electron microscopy. This membrane, the pia-ependymal roof of the caudal hindbrain, contains a large central region characterized by cuboidal ependymal cells which surround sizable microscopic apertures — the interependymal pores.Ultrastructurally ependymal cells of this area are characterized by infrequent apical microvilli and cilia. They contain irregularly shaped nuclei and few cytoplasmic organelles that are largely apical in position. The most striking feature is an abundance of cytoplasmic filaments forming an extensive cytoskeleton. Laterally these cells are joined by numerous elaborate desmosomes. The majority of the ependymal cells have a basal lamina consisting of single, double, or triple laminae lying parallel to the basal plasma membrane.Several unusual specializations are seen at the margins of the interependymal pores. The ependymal cells have lateral cytoplasmic processes that form the actual border of each pore. These processes originate from the apical surface of the cell and partially enclose an elaborate network of basal lamina associated with the interependymal pores.These findings demonstrate microscopic apertures in the roof of the fourth ventricle in the bullfrog that are associated with an unusual form of supportive ependyma.     

Transforming growth factors α and β–1 are co-expressed in the uterine epithelium during early pregnancy

Semi-quantitative immunoperoxidase light microscopy on unfixed frozen sections of rat uterus was used to examine the expression of transforming growth factors (TGF) alpha and beta before, during and after implantation. TGF beta-1 labelling was present in the lateral plasma membranes and basement membrane from day 3, steadily increased until the time of implantation on day 6 when it formed a dense apical band, but had disappeared by day 7. TGF alpha appeared in the apical epithelium, 3 days prior to TGF beta-1. Ultrastructural immunogold labelling showed the intense TGF beta-1 signal observed at the time of implantation to be present on the apical epithelial surface and microvilli and in the luminal secretions. We suggest that TGF alpha stimulates the expression of TGF beta-1 in rats, as in implanting transgenic mice, and that it is involved in extracellular matrix remodelling in the lateral plasma membranes and basal lamina to inhibit invasion and implantation and to stimulate the production of basement membrane.     

ACTIVE TRANSPORT BY THE CECROPIA MIDGUT : II. Fine Structure of the Midgut Epithelium

A morphological basis for transcellular potassium transport in the midgut of the mature fifth instar larvae of Hyalophora cecropia has been established through studies with the light and electron microscopes. The single-layered epithelium consists of two distinct cell types, the columnar cell and the goblet cell. No regenerative cells are present. Both columnar and goblet cells rest on a well developed basement lamina. The basal portion of the columnar cell is incompletely divided into compartments by deep infoldings of the plasma membrane, whereas the apical end consists of numerous cytoplasmic projections, each of which is covered with a fine fuzzy or filamentous material. The cytoplasm of this cell contains large amounts of rough endoplasmic reticulum, microtubules, and mitochondria. In the basal region of the cell the mitochondria are oriented parallel to the long axes of the folded plasma-lemma, but in the intermediate and apical portions they are randomly scattered within the cytoplasmic matrix. Compared to the columnar cell, the goblet cell has relatively little endoplasmic reticulum. On the other hand, the plications of the plasma membrane of the goblet cell greatly exceed those of the columnar cell. One can distinguish at least four characteristic types of folding: (a) basal podocytelike extensions, (b) lateral evaginations, (c) apical microvilli, and (d) specialized cytoplasmic projections which line the goblet chamber. Apically, the projections are large and branch to form villus-like units, whereas in the major portion of the cavity each projection appears to contain an elongate mitochondrion. Junctional complexes of similar kind and position appear between neighboring columnar cells and between adjacent columnar and goblet cells as follows: a zonula adherens is found near the luminal surface and is followed by one or more zonulae occludentes. The morphological data obtained in this study and the physiological information on ion transport through the midgut epithelium have encouraged us to suggest that the goblet cell may be the principal unit of active potassium transport from the hemolymph to the lumen of the midgut. We have postulated that ion accumulation by mitochondria in close association with plicated plasma membranes may play a role in the active movement of potassium across the midgut.     

Fine structure of the epidermis of the optic tentacle in a slug, Limax flavus L.

The epidermis at the tip of the optic tentacle in Limax flavus is constructed of columnar epithelial cells, distal processes of nerve cells, and scattered processes of the collar cells. The epithelial cells extend stout microvilli called plasmatic processes by Wright perpendicularly from the free surface. Each plasmic process branches into a few terminal twigs embedded in a fuzzy filamentous substance. Most nerve cells have their nuclei under the basal lamina. The distal processes of these nerve cells reach the free surface and send long microvilli to form the spongy layer under a filamentous covering. At the side surface of the tentacle the epithelial cells are cuboidal or squamous and the neural elements are fewer. Here, no spongy layer is formed; and the collar cell processes are replaced by the lateral cell processes. Peculiar secretion granules are contained in the lateral and collar cell processes as well as in their cell bodies situated beneath the basal lamina.     

Bone morphology of weanling rats from dams subjected to fluoride

Epithelial cells and surrounding free cells in the choroid plexus were examined cytochemically using filipin to clarify the distribution pattern of cholesterol within plasma membranes. The apical and basal membranes of the choroid epithelial cell are less susceptible to filipin than the lateral epithelial membrane and plasma membranes of adjacent mesenchymal cells such as macrophages and fibroblasts. Apical and basal domains of the epithelial membranes, which are relatively resistant to action of filipin, appear to have a slightly lower cholesterol content. We suggest that the apical and basal membranes may possess a unique membrane fluidity or stability that differs from that of the lateral epithelial, macrophage or fibroblast membranes.     

The surface characteristics of the plasma membrane of the exocrine pancreas.

Regional differentiation of the plasma membrane and related structures of the exocrine pancreas has been studied ultrastructurally and cytochemically. Fixation with an osmium tetroxide-silver acetate solution produced abundant fine precipitates on the luminal and basal surface of the centroacinar but not the acinar cells. Staining with dialyzed iron (DI) revealed the heaviest concentration of anionic sites on the luminal plasma membrane of the acinar cells, including the surface of both the intercellular canaliculi and the main lumen. The reactive sites on the apical acinar plasmalemma appeared to consist of discrete globules. DI-reactivity of the lateral basal membranes was most prominent in the centroacinar cells and essentially absent in the acinar cells but was weak relative to that of the acinar-cell apical plasmalemma. The lamina lucida of the basement membrane of the duct stained with DI, but that of basement membrane under acinar cells did not. Sialidase digestion prior to DI staining abolished the staining of plasma membranes. These results indicate that duct epithelial cells, including most prominently the centroacinar cells, are chiefly responsible for electrolyte and fluid transport.     

Fine structure of the larval midgut of the fly Rhynchosciara and its physiological implications

The midgut of Rhynchosciara americana larvae consists of a cylindrical ventriculus from which protrudes two gastric caeca formed by polyhedral cells with microvilli covering their apical faces. The basal plasma membrane of these cells is infolded and displays associated mitochondria which are, nevertheless, more conspicuous in the apical cytoplasm. The anterior ventricular cells possess elaborate mitochondria-associated basal plasma membrane infoldings extending almost to the tips of the cells, and small microvilli disposed in the cell apexes. Distal posterior ventricular cells with long apical microvilli are grouped into major epithelial foldings forming multicellular crypts. In these cells the majority of the mitochondria are dispersed in the apical cytoplasm, minor amounts being associated with moderately-developed basal plasma membrane infoldings. The proximal posterior ventriculus represents a transition region between the anterior ventriculus and the distal posterior ventriculus. The resemblance between the gastric caeca and distal posterior ventricular cells is stressed by the finding that their microvilli preparations display similar alkaline phosphatase-specific activities. The results lend support to the proposal, based mainly on previous data on enzyme excretion rates, that the endo-ectoperitrophic circulation of digestive enzymes is a consequence of fluid fluxes caused by the transport of water into the first two thirds of midgut lumen, and its transference back to the haemolymph in the gastric caeca and in the distal posterior ventriculus.     

Diversity in the surface morphology of adjacent epithelial cells of the choroid plexus: an ultrastructural analysis

It is generally known that the luminal surface of the choroidal epithelial cells is covered with a luxuriant coat of slender microvilli and cilia. However, extensive ultrastructural studies on the surface morphology of choroidal epithelial cells are lacking. This study, therefore, is focused on the detailed surface morphology of the choroid plexus of the lateral ventricle of adult Wistar rats using transmission and scanning electron microscopy. The animals were anesthetized, perfused with 0.9% oxygenated saline followed by 3% gluteraldehyde and the choroid plexus was processed for routine electron microscopy. The results of the ultrastructural observations presented in this study show that even the neighboring choroidal epithelial cells may express distinct morphology. In addition to the usually described morphology of choroidal epithelial cells, in this study, the presence of cells with uniform small blebs, crenulated or doughnut shaped structures, large mature blebs, or cells with an extensive network of fibers were observed. Although, dissimilar surface morphology of adjacent choroidal epithelial cells may indicate their distinct functional status, further studies are necessary to understand the physiological relevance of the varied surface morphology of choroidal epithelial cells.     

Ultrastructure of midgut endocrine cells in the adult mosquito, Aedes aegypti

The ultrastructure of endocrine cells in the midgut of the adult mosquito, Aedes aegypti, resembled that of endocrine cells in the vertebrate gastro-intestinal tract. Midgut endocrine cells, positioned basally in the epithelium as single cells, were cone-shaped and smaller than the columnar digestive cells. The most distinctive characteristic of endocrine cells was numerous round secretory granules along the lateral and basal plasma membranes where contents of the granules were released by exocytosis. Secretory granules in each individual cell were exclusively of one type, either solid or 'haloed', and for all cells observed, the range in granule diameter was 60-120 nm. The cytoplasm varied in density from clear to dark. Lamellar bodies were prominent in the apical and lateral cellular regions and did not exhibit acid phosphatase activity. The basal plasma membrane was smooth adjacent to the basal lamina, whereas in digestive cells the membrane formed a labyrinth. Some endocrine cells reached the midgut lumen and were capped by microvilli; a system of vesicles and tubules extended from beneath the microvilli to the cell body. An estimated 500 endocrine cells were distributed in both the thoracic and abdominal regions of the adult midgut. In one midgut, we classified a sample of endocrine cells according to cytoplasmic density and granule type and size; endocrine cells with certain types of granules had specific distributions within the midgut.     

The ultrastructure and histochemistry of the spermathecae of Tubifex tubifex (Annelida: Oligochaeta)

The wall of the spermathecal ampulla in Tubifex tubifex consists of epithelial, muscular and peritoneal layers. The epithelial surface contains closely microvilli while lateral and basal plasma membranes are extensively convoluted. Epithelial cytoplasm exhibits a vertical zonation of subcellular components. The distal zone contains filiform secretory particles which are orientated perpendicular to the apical surface; extrusion occurs by their fusion with the plasma membrane between the bases of neighbouring microvilli. Mitochondiral and Golgi zones, the latter containing the nucleus, subtend the distal zone. The basal zone, composed of vertical compartments formed by the folded plasma membrane, is rich in α-glycogen rosettes. The distal epithelium and lumen material contain neutral mucopolysaccharides and carboxylated acid mucopolysaccharides in conjunction with neutral protein. The ultrastructure of the spermathecal duct wall is comparable with that of the ampulla but is characterized by extremely long microvilli and a prominent musculature.     

Epithelial differentiation in the fetal rat colon: I. Plasma membrane phosphatase activities

During the last week of gestation of the fetal rat, the epithelium of the colon is rapidly remodeled. At 16 days a primitive stratified epithelium surrounds a small central lumen. Over the next 3 days, the main lumen extends narrow clefts down to the basal cell layer and small secondary lumina appear within the stratified epithelium between these clefts. At 19 and 20 days, secondary lumina enlarge but remain discrete; an infusion of cationic ferritin into the main lumen does not enter secondary lumina. During the 2 days prior to birth (21–22), the secondary lumina join the main lumen as superficial cells are sloughed, and the epithelium becomes simple columnar. Freeze-fracture replicas indicate that luminal and nonluminal membrane domains of epithelial cell plasma membranes are separated by continuous tight junctions throughout the conversion process. Cytochemical analysis of tissue slices from 16- to 22-day fetal colon demonstrated the appearance and segregation of two phosphatases on apical and basolateral membrane domains during epithelial conversion. Cysteine-sensitive pH 9.0 (alkaline) phosphatase activity was first detected along the luminal membranes of cells bordering both primary and secondary lumina at 18 days gestation and increased to a maximum at 20–21 days; weaker activity was present on basolateral membranes. Phosphatase activity at pH 8.0 also appeared at 18 days and increased thereafter, but was localized primarily on nonluminal membranes. At pH 8.0, reaction product appeared on both inner and outer sides of the membrane, and was only partially abolished by omission of K⁺ or addition of ouabain; thus the reaction may be only partially due to K⁺-dependent ATPase activity. Biochemical analysis of the cytochemical media confirmed the appearance of phosphatase activities at 18 days. Thus, plasma membrane phosphatase activities appear while the epithelium is still stratified, but are segregated to luminal and nonluminal membrane domains at the onset of activity. Segregation is maintained throughout the process of conversion of a simple columnar epithelium.     

Tao controls epithelial morphogenesis by promoting Fasciclin 2 endocytosis

Regulation of epithelial cell shape, for example, changes in relative sizes of apical, basal, and lateral membranes, is a key mechanism driving morphogenesis. However, it is unclear how epithelial cells control the size of their membranes. In the epithelium of the Drosophila melanogaster ovary, cuboidal precursor cells transform into a squamous epithelium through a process that involves lateral membrane shortening coupled to apical membrane extension. In this paper, we report a mutation in the gene Tao, which resulted in the loss of this cuboidal to squamous transition. We show that the inability of Tao mutant cells to shorten their membranes was caused by the accumulation of the cell adhesion molecule Fasciclin 2, the Drosophila N-CAM (neural cell adhesion molecule) homologue. Fasciclin 2 accumulation at the lateral membrane of Tao mutant cells prevented membrane shrinking and thereby inhibited morphogenesis. In wild-type cells, Tao initiated morphogenesis by promoting Fasciclin 2 endocytosis at the lateral membrane. Thus, we identify here a mechanism controlling the morphogenesis of a squamous epithelium.     

Cellular organization and peritrophic membrane formation in the cardia (Proventriculus) of Drosophila melanogaster

The peritrophic membrane of Drosophila melanogaster consists of four layers, each associated with a specific region of the folded epithelial lining of the cardia. The epithelium is adapted to produce this multilaminar peritrophic membrane by bringing together several regions of foregut and midgut, each characterized by a distinctively differentiated cell type. The very thin, electron-dense inner layer of the peritrophic membrane originates adjacent to the cuticular surface of the stomadeal valve and so appears to require some contribution by the underlying foregut cells. These foregut cells are characterized by dense concentrations of glycogen, extensive arrays of smooth endoplasmic reticulum, and pleated apical plasma membranes. The second and thickest layer of the peritrophic membrane coalesces from amorphous, periodic acid-Schiff-positive material between the microvilli of midgut cells in the neck of the valve. The third layer of the peritrophic membrane is composed of fine electron-dense granules associated with the tall midgut cells of the outer cardia wall. These columnar cells are characterized by cytoplasm filled with extensive rough endoplasmic reticulum and numerous Golgi bodies and by an apical projection filled with secretory vesicles and covered by microvilli. The fourth, outer layer of the peritrophic membrane originates over the brush border of the cuboidal midgut cells, which connect the cardia with the ventriculus.     

Histology and ultrastructure of the gut epithelium of the neotenic cave salamander, Proteus anguinus (Amphibia, Caudata)

Histological, histochemical, and ultrastructural features of the gut of the European endemic cave salamander Proteus anguinus were studied. The gut is a relatively undifferentiated muscular tube lined with a simple columnar epithelium containing numerous goblet cells. The mucosa and underlying lamina propria/submucosa are elevated into a number of high longitudinal folds projecting into the lumen. The enterocytes are covered apically with uniform microvilli. Irregularity in the arrangement of microvilli was observed. Occasionally, irregular protrusions of the cytoplasm appear between groups of microvilli. Pinocytotic activity occurs at the bases of the intermicrovillous space. Mitochondria are numerous in the apical cytoplasm and basally beneath the nuclei. The supranuclear cytoplasm contains most of the cell organelles. The lateral plasma membranes of adjacent cells interdigitate and are joined by junctional complexes. The periodic acid-Schiff (PAS) reaction, indicating neutral mucosubstances, is positive only in the apical brush border of enterocytes and in goblet cells. The goblet cells also stained with Alcian blue (AB), at pH 2.5, thus revealing the presence of carboxylated glycosaminoglycans. Compact aggregations of AB- and PAS-negative cells are situated directly below the epithelium. Mitotic figures are present in individual clusters of cells. The fine structure of cells in these clusters indicated that these cells could be responsible for renewal of intestinal epithelium. Numerous endocrine-like cells could also be seen. The closely packed smooth muscle cells and amorphous extracellular material with collagen fibrils constitute a net-like structure under the basal lamina that is very closely associated with the epithelium. There are numerous acidophilic granular cells between epithelial cells, in the lamina propria/submucosa, and between cells aggregations. They seem to be associated with nematode infections and possibly constitute a humoral defense mechanism.     

Light and electron microscopic studies on the development of the spiral prominence of the cochlear duct of the fetal guinea pig

The anlage of the spiral prominence can be seen on the 37th day of development as a small protrusion of the epithelium towards the lumen of the cochlear duct. During the further progress, the spiral prominence more distinctly protrudes by augmentation of the vascularized connective tissue. In the epithelial cells pinocytotic vesicles near the plasmalemma are seen earliest lateral and basal on the 37th day, apical on the 39th day. The epithelial cells send basal cytoplasmic extensions towards the connective tissue. Starting on the 44th day, small invaginations of connective tissue extend into the epithelium, remaining separated from the epithelial cells by the basal lamina. Until the 48th day, the monostratified epithelium remains columnar, thereafter it changes to cuboidal or flat. Towards the end of the development, the invaginations of the connective tissue nearly reach the surface of the epithelium, being separated from the endolymph by a small epithelial area.     

The saccus dorsalis of the rainbow trout,Salmo gairdneri Richardson

The saccus dorsalis of the brain of the rainbow trout, Salmo gairdneri Richardson, has been investigated by means of histological, cytochemical, enzyme-cytochemical, electron microscopical autoradiographical techniques. The saccus dorsalis is a rostro-dorsal evagination of the diencephalic roof, and consists of a partly folded epithelial wall separating the cerebrospinal fluid from the meningeal matrix fluid. The well-developed vascular system around the epithelial wall, consisting of capillaries with different diameters, seems to be part of the pineal vascular system. No structures were found that may be involved in a possible mechanical or nervous blood flow control. The single-layered epithelium consists of highly specialized cells of one specific type. These cells are mainly characterized by infolded basal membranes, long microvilli of a peculiar shape, non-folded lateral membranes bordering intercellular spaces, apical concentrations of elongate and cup-shaped macromitochondria, a basally located rough endoplasmic reticulum, an apically situated smooth endoplasmic reticulum and apical concentrations of micropinocytotic vesicles. Morphological evidence is presented of a multiple function of these cells: (1) fluid secretion, (2) extrusion of low molecular weight organic substances into the ventricular system, (3) uptake of high molecular weight substances, and (4) uptake of low molecular weight organic substances (aminergic neurotransmitters [GABA]) from the cerebrospinal fluid. The significance of light and dark cells is discussed. Indications of a possible innervation of the saccus dorsalis epithelial cells were not observed. The functional significance of the saccus dorsalis (possible analogue of the choroid plexus?) is discussed.