Diffusion barriers in the vaginal epithelium during the estrous cycle in guinea pigs

Summary In the present study the permeability barriers of the multilayered vaginal epithelium were examined using tracer perfusion techniques, freeze-fracture and thin sectioning. During diestrus and proestrus the upper layers of mucified epithelial cells exhibit tight-junctional belts, which restrict tracer molecules such as lanthanum and horseradish peroxidase. When the highly mucified cells begin to degenerate toward the end of proestrus the underlying epithelium is already keratinized as typical for estrus. The keratinized epithelial cells have a tight-junctional network that joins the basal plasma membranes with the apical membranes of subjacent cells and blocks paracellular diffusion of the tracer molecules. During conversion of the cornified epithelium to a mucified epithelium in metestrus the intercellular space of the epithelium is stained by tracer molecules even though tight-junctional belts can be observed.These results indicate that during cyclic changes of the vaginal epithelium tight junctions can, in general, be considered for the restriction of paracellular diffusion. In metestrus, however, junctions become functionally leaky although they remain morphologically intact.Intercellular lipids, which are normally common in cornified epithelia, are extremely rare and cannot constitute an effective barrier to diffusion in the vagina of the guinea pig. The significance of a strategy that bases the regulation of the permeability on tight junctions rather than on intercellular lipids is discussed.     

PERMEABILITY OF SERTOLI CELL TIGHT JUNCTIONS TO LANTHANUM AFTER LIGATION OF DUCTUS DEFERENS AND DUCTULI EFFERENTES

The permeability of Sertoli cell tight junctions to lanthanum administered during fixation has been compared in rats after ligation of the ductus deferens and after ligation of the ductuli efferentes. In both control and vasoligated testes, lanthanum penetrated only short distances into the Sertoli cell tight junctions before stopping abruptly. The tight junction, consisting of numerous pentalaminar fusions of contiguous Sertoli cell membranes, prevented diffusion of lanthanum into the adluminal compartment of the seminiferous epithelium. In rats with ligated ductuli efferentes, lanthanum completely permeated many Sertoli cell tight junctions and occupied intercellular spaces of the adluminal compartment. In spite of their newly acquired permeability to lanthanum, tight junctions retained characteristic ultrastructural features, including numerous membrane fusions. When lanthanum-filled tight junctions were sectioned en face, membrane fusions appeared as pale lines in lakes of electron-opaque tracer. These linearly extensive fasciae occludentes occasionally ended blindly, suggesting that lanthanum may have traversed the junction by diffusing around such incomplete barriers. The increased permeability of Sertoli cell tight junctions after efferent ductule ligation, which caused rapid testicular weight gain followed by atrophy, indicates that tight junctions are sensitive to enforced retention of testicular secretions inside the seminiferous tubules. The apparent normalcy of Sertoli cell tight junctions after vasoligation, which had no effect on testis weight, supports the view that blockage of testicular secretions distal to the epididymis is relatively innocuous.     

A freeze-fracture and lanthanum tracer study of the complex junction between Sertoli cells of the canine testis

What appear to be true septate junctions by all techniques currently available for the cytological identification of intercellular junctions are part of a complex junction that interconnects the Sertoli cells of the canine testis. In the seminiferous epithelium, septate junctions are located basal to belts of tight junctions. In thin sections, septate junctions appear as double, parallel, transverse connections or septa spanning an approximately 90-A intercellular space between adjacent Sertoli cells. In en face sections of lanthanum-aldehyde-perfused specimens, the septa themselves exclude lanthanum and appear as electron-lucent lines arranged in a series of double, parallel rows on a background of electron-dense lanthanum. In freeze-fracture replicas this vertebrate septate junction appears as double, parallel rows of individual or fused particles which conform to the distribution of the intercellular septa. Septate junctions can be clearly distinguished from tight junctions as tight junctions prevent the movement of lanthanum tracer toward the lumen, appear as single rows of individual or fused particles in interlacing patterns within freeze-fracture replicas, and are seen as areas of close membrane apposition in thin sections. Both the septate junction and the tight junction are associated with specializations of the Sertoli cell cytoplasm. This is the first demonstration in a vertebrate tissue of a true septate junction.     

Ultrastructural study of cholestasis induced by longterm treatment with estradiol valerate. I. Tight junctional analysis and tracer experiments

Over a period of 20 weeks estradiol valerate (1.5 mg/kg body weight/week) was administered subcutaneously to male Wistar rats from which the livers were examined at four week intervals employing a freeze-fracture technique and colloidal lanthanum tracer studies. In connection with intrahepatic cholestasis, distinct alterations in the tight junctions were observed, consisting of disorganization, rarification and proliferation. Disruption of the tight junctions was not seen and colloidal lanthanum did not penetrate into the bile canalicular lumen. Holding the view that the term "leakiness" of tight junctions should be defined with reference to the tracer employed, we conclude that in the liver one tight junctional strand is sufficient to prevent the escape of larger bile constituents such as bile acids and that a back diffusion of bile acids over the tight junctional barrier does not play a role in the pathogenesis of the estrogen-induced cholestasis. Interruptions of tight junctions, as described by other authors, are interpreted as a secondary mechanical effect. On the other hand, we consider an increased permeability of the tight junctions to water and small solute molecules as probable; possibly this increased permeability is brought about by alterations in the microfilaments. A model for the pathogenesis of the estrogen-induced intrahepatic cholestasis is proposed.     

The response of junctional complexes to induced desquamation in mouse bladder urothelium

During desquamation, the cells of mouse urinary bladder epithelium undergo detachment. In this process we examined the disconnection of cell adhesion molecules. Two proteins of cell junctions were studied: ZO1 of tight junctions and desmoplakin of desmosomes. Desquamation was induced by intravesical injection of LPS, constant illumination of mouse for 96 h, application of a combination of stress hormones hydrocortisone and norepinephrine or by removal of calcium with EGTA. All the inducers caused penetration of lanthanum tracer through the tight junctions, indicating paracellular permeability. Dilatation of extracellular spaces between neighboring cells was seen whenever desquamation was induced in bladders containing urine. Desquamation of single cells as well as groups of cells was observed. Contrary to obvious disconnection of cell junctions, as a precondition for desquamation, the distribution of junctional proteins did not change either in urothelial tissue or in desquamated cells. This study demonstrates that all the inducers of desquamation cause first an extensive dysfunction of a blood urine barrier and after that an occasional mechanical disconnection of adhesive junctions which consequently leads to desquamation.     

Expression and function of tight junctions in the crypt epithelium of human palatine tonsils.

The human palatine tonsils have surface and crypt stratified epithelium and may be initiated via the epithelium to mount immune responses to various presenting antigens. Here we investigated the expression and function of tight junctions in the epithelium of human palatine tonsils from patients with tonsillar hypertrophy or recurrent tonsillitis. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, -7, -8, and -14 mRNAs were detected in tonsillar hypertrophy. Occludin and claudin-14 were expressed in the uppermost layer of the tonsil surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the epithelium. In the crypt epithelium, claudin-4 was preferentially expressed in the upper layers. In freeze-fracture replicas, short fragments of continuous tight junction strands were observed but never formed networks. In the crypt epithelium of recurrent tonsillitis, the tracer was leaked from the surface regions where occludin and claudin-4 disappeared. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, and -14, but not claudin-7, mRNAs were decreased in recurrent tonsillitis compared with those of tonsillar hypertrophy. These studies suggest unique expression of tight junctions in human palatine tonsillar epithelium, and the crypt epithelium may possess an epithelial barrier different from that of the surface epithelium.     

Opening of blood-brain barrier in Triturus cristatus carnifex by hyperosmolar mannitol solutions

The permeability of the newt cerebral capillaries to lanthanum ion has been studied after perfusion with mannitol solutions of increasing molarity. In the control specimens lanthanum deposits were limited to the luminal side of the capillaries and tracer did not spread to the pericapillary spaces due to the tight junctions. Treatment with hypertonic solutions of mannitol (0.25M, 0.5M, 1M) caused opening of the blood brain barrier with a progressive increase in lanthanum between the endothelial cell edges, in the basal lamina and in the extracellular spaces of the nervous parenchyma in relation to the molarity of the mannitol solution. The spread of lanthanum is probably due to opening of the tight junctions between the endothelial cells, since pinocytotic vesicles labelled with tracer were not evident.     

Fine structure of the monkey epididymis: A correlated thin-section and freeze-cleave study

Summary The epithelium of the monkey epididymis was studied by means of freeze-fracture techniques and conventional electron microscopy. For the study of transepithelial permeability lanthanum hydroxide was used as an intercellular tracer. The epididymal epithelium consists mainly of tall columnar cells. The long stereocilia at the apical surface, similarly to microvilli, exhibit after freeze-fracture, two distinct faces: the E face, concave and with fewer membrane-associated particles, and the complementary convex P face. In the lumen unusual groups of smooth-surfaced vacuoles are present. A tight junctional network, which shows some permeability to the lanthanum tracer, is located at the apical end of the cells. Supranuclear cross-fractures clearly show the well developed Golgi cisternae and numerous vacuole profiles. The highly infolded, centrally located nucleus exhibits, after freeze-fracture, an even distribution of nuclear pores. In the perinuclear region the rough endoplasmic reticulum, which also presents pores, displays a sheet-like organization. The basal cytoplasm is filled by numerous globular profiles of membrane-bounded granules. Freeze-cleave exposes large cytoplasmic areas where the types and amount of organelles indicate an intense metabolic activity.Supported by Grant No 104.193.1.78 from PLAMIRHInvestigator of the National Council for Scientific and Technical Research (CONICET) Argentina     

Modeling Cell Dynamics in Colon and Intestinal Crypts: The Significance of Central Stem Cells in Tumorigenesis

Colon and intestinal crypts have been widely chosen to study cell dynamics because of their fairly simple structures. In the colon and intestinal crypts, stem cells (SCs) are located at very bottom of the crypt, fully differentiated cells (FDs) are located in the top of the crypt, and transit-amplifying cells (TAs) are in the middle of the crypt between FDs and SCs. Recently, it has been discovered that there are two types of stem cells in the intestinal crypts: central stem cells (CeSCs) and border stem cells. To investigate dynamics of mutants in colon and intestinal crypts, we develop a four-compartmental stochastic model, which includes two SC compartments, and TAs and FDs compartments. We calculate the probability of the progeny of marked or mutant cells located at each of these compartments taking over the entire crypt or being washed out from the crypt. We found that the progeny of CeSCs will take over the entire crypt with a probability close to one. Interestingly, the progeny of advantageous mutant TAs and FDs will be washed out faster than disadvantageous mutants. Saliently, the model predicts that the time that the progeny of wild-type central stem cells will take over the mouse intestinal crypt is around 60 days, which is in perfect agreement with an experimental observation.     

Differentiation of the plasma membrane of hepatic cells in monolayer cultures

Hepatocytes from rats were isolated by treatment with trypsin and cultured. Plasma membranes at different culture stages were observed by electron microscopy. The activities of 5' nucleotidase and adenosinetriphosphatase on the plasma membranes were examined. The cell coat was also studied by use of the concanavalin A-peroxidase technique. The surfaces of single cells, covered with microvilli, are the site of adenosinetriphosphatase activity only and are devoid of 5'-nucleotidase activity. After a few h of culture, the cells are grouped together in tight clusters or long trails and are separated by an intercellular space of 250 A, partially permeable to lanthanum nitrate. The juxtaposed plasma membranes on which 5'-nucleotidase and adenosinetriphosphatase activities occur also delimit spaces similar to bile canaliculi. The formation of junction complexes and their permeability to lanthanum nitrate was also studied. No enzymatic activity is observed at the junctions. The numerous tight junctions, impervious to the tracer, are always accompanied by a profusion of microfilaments. Mature desmosomes are rare, and are present only in the form of "maculae adhaerentes diminutae." The gap junctions, nearly always permeable to the tracer, form rapidly and assume a variety of shapes (trail, bulge and ring-like), the significance of which is open to discussion. The use of concanavalin A permits localization of the free sugar sites on the surface of the cells, in the pinocytotic vesicles and in the internal space of the gap junctions.     

Permeability barrier in the mantle epithelium lining the testis in the apple-snail Pomacea canaliculata (Gastropoda: Ampullariidae)

Intercellular junctions are studied in the epithelium lining the testis of the freshwater snail Pomacea canaliculata by conventional staining and lanthanum tracer techniques. The junctional complex consists of belt desmosomes and septate junctions. Septate junctions are of the pleated-sheet type and they are constantly associated with mitochondria. Gap and tight junctions appear to be absent. These septate junctions seem to be the structural correlate of an epithelial permeability barrier that separate the testis from the extrapallial space where the shell elements are deposited. These junctions may contribute to a functional barrier in the male gonad of Pomacea canaliculata. The results indicate that freshwater prosobranchs have junctional structures very close to those found in other molluscs.     

Structure and permeability of goblet cell tight junctions in rat small intestine

Summary Two major cell types, goblet and absorptive cells, dominate the epithelial lining of small intestinal villi. We used freezefracture replicas of rat ileal mucosa to examine the possibility that tight junction structure, known to relate to transepithelial resistance, might vary with cell type. Tight junctions between absorptive cells were uniform in structure while those associated with villus goblet cells displayed structural variability. In 23% of villus goblet cell tight junctions the strand count was less than 4 and in 30% the depth was less than 200 nm. In contrast, only 4% of absorptive cell tight junctions had less than 4 strands and only 9% had depth measurements less than 200 nm. Other structural features commonly associated with villus goblet cell tight junctions but less commonly with absorptive cell tight junctions were: deficient strand cross-linking, free-ending abluminal strands, and highly fragmented strands. Bothin vivo ileal segments and everted loops were exposed to ionic lanthanum. Dense lanthanum precipitates in tight junctions and paracellular spaces were restricted to a subpopulation of villus goblet cells and were not found between villus absorptive cells. After exposure of prefixed ileal loops to lanthanum for 1 hour, faint precipitates of lanthanum were found in 14% of tight junctions and paracellular spaces between absorptive cells compared to 42% of tight junctions and paracellular spaces adjacent to villus goblet cells. When tested in Ussing chambers, the methods used for lanthanum exposure did not lower transepithelial resistance. Everted loops exposed to ionic barium and examined by light microscopy showed dense barium precipitates in the junctional zone and region of the paracellular space of villus goblet cells but not in these regions between absorptive cells. However, the macromolecular tracers, microperoxidase, cytochromec and horseradish peroxidase, were excluded from both villus goblet cell and absorptive cell paracellular spaces inin vivo segments. These findings suggest that a subpopulation of villus goblet cells may serve as focal sites of high ionic permeability and contribute to the relatively low resistance to ionic flow which characterizes the small intestinal epithelium.     

The Use of Lanthanum Chloride as a Marker for Intercellular Junctions in Rat Exocrine Pancreas

A simple technique of perfusion and immersion of tissue in fixative containing lanthanum chloride as an extracellular tracer is described. In addition to functioning as a tracer, the lanthanum chloride appears to enhance electron staining. In rat exocrine pancreas, intercellular spaces between exocrine and centroductular cells were outlined clearly by electron dense material and, at cellular interfaces, spot desmosomes, gap junctions, and tight junctions were demonstrated. The technique proved simple and effective and should prove useful in studies of epithelial and other tissues.     

The use of lanthanum chloride as a marker for intercellular junctions in rat exocrine pancreas

A simple technique of perfusion and immersion of tissue in fixative containing lanthanum chloride as an extracellular tracer is described. In addition to functioning as a tracer, the lanthanum chloride appears to enhance electron staining. In rat exocrine pancreas, intercellular spaces between exocrine and centroductular cells were outlined clearly be electron dense material and, at cellular interfaces, spot desmosomes, gap junctions, and tight junctions were demonstrated. The technique proved simple and effective and should prove useful in studies of epithelial and other tissues.     

Evidence for tight junctions between odontoblasts in the rat incisor

Odontoblasts are known to be involved in the process of dentinogenesis but it is not clear whether substances may also be deposited in predentine and dentine by passing between these cells. Although tight junctions have been described, it is not clear if they are macular or "leaky" as opposed to continuous or "tight". In this study use has been made of the permeability of fenestrated capillaries amongst the odontoblasts to deposit the penetrative tracer lanthanum in the interodontoblastic space. This was done by perfusion of anaesthetized rats with physiological solutions containing lanthanum nitrate at 37 degrees C. Immersion fixation of transverse segments of mandibular incisors and examination with an electron microscope showed that lanthanum could permeate 40-50 microns between the odontoblasts to reach the peripheral pulp. Towards the predentine, often less than 10 microns from the capillaries, its progress was abruptly and completely halted by the junctions at the apical ends of the odontoblast cell bodies. Lanthanum was not found in the predentine. The mature secretory odontoblasts in the rat incisor have therefore been shown to be joined by continuous tight junctions. In the process of dentinogenesis this means that all substances deposited in predentine and dentine must arrive by passing through the odontoblasts.     

The gastric mucosal barrier: structure of intercellular junctions in the dog

The canine gastric mucosa consists of two regions, the surface mucous cells and gland area cells including parietal, chief, and mucous-containing cells. We have used quantitative freeze-fracture methods in conjunction with thin-section extracellular tracers to document and correlate tight junction morphology with epithelial permeability. The number of strands in the tight junction complexes of the surface cells and gland cells is the same, but differences in strand arrangement exist. The surface cells have an interwoven tight junction configuration which is impermeable to extracellular tracers. The gland cell junctions are regularly arranged and often permeable to extracellular lanthanum. The possibility that the observed difference in permeability between the tight junctions of the surface mucous cells and those of the gland cells is related to their structural configuration is discussed.     

The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development.

Junctions in developing mammalian embryos were investigated with lanthanum tracer and freeze-fracture methods. The outermost blastomeres of mouse morulae possess focal tight junctions which become zonular and exclude lanthanum, thereby separating the “inner” cells from the maternal environment. This compartmentalization, creating a microenvironment inside the embryo, may be required for cell determination and for the accumulation of fluid during blastocoel expansion. Desmosomes appear for the first time at the blastocyst stage in the trophoblast junctional complex which also is characterized by gap junctions and a zonula occludens with underlying microfilament-like material and microtubules. Both gap and tight junctions have been visualized in freeze-fracture replicas of rabbit blastocysts. The zonula occludens forms a permeability barrier which is consistent with the high transtrophoblast electrical resistance. Mouse presumptive and mature inner cell mass (ICM) cells were associated by frequent gap junctions whereas junctional complexes were absent. Trophoblast gap and adhering junctions and cytoplasmic processes appeared to fix the ICM to one pole of the embryo and partially isolate it from the blastocoel. These findings support the idea that the ICM and trophoblast communicate upon implantation and require that the intercellular junctions between them be dissembled if the ICM is to migrate to a mesometrial position.     

Stand by me: Fibroblasts regulation of the intestinal epithelium during development and homeostasis

The epithelium of the small intestine is composed of a single layer of cells that line two functionally distinct compartments, the villi that project into the lumen of the gut and the crypts that descend into the underlying connective tissue. Stem cells are located in crypts, where they divide and give rise to transit-amplifying cells that differentiate into secretory and absorptive epithelial cells. Most differentiated cells travel upwards from the crypt towards the villus tip, where they shed into the lumen. While some of these cell behaviors are an intrinsic property of the epithelium, it is becoming evident that tight coordination between the epithelium and the underlying fibroblasts plays a critical role in tissue morphogenesis, stem-cell niche maintenance and regionalized gene expression along the crypt-villus axis. Here, we will review the current literature describing the interaction between epithelium and fibroblasts during crypt-villus axis development and intestinal epithelium renewal during homeostasis.     

The permeability of nonhuman primate vaginal epithelium: a freeze-fracture and tracer-perfusion study

The lining of the vaginal mucosa in primates is a keratinized stratified squamous epithelium. As in other structurally similar epithelia, one function of the vaginal epithelium is to provide a barrier between the external environment and the underlying tissues. The vaginal lining is aglandular and the source of true vaginal fluid has been suggested to be the intercellular channels of the epithelium. On the other hand, other structurally similar epithelia have been shown to have a barrier to the movement of water-soluble molecules through these channels. In the present study, we have examined the permeability of rhesus monkey vaginal epithelium to lanthanum and horseradish peroxidase. Both tracer molecules penetrated the intercellular channels in the lower layers of the epithelium, but were excluded from the channels at and above the granular layer. Neither tracer penetrated significantly between cells at the free surface of the epithelium and usually did not penetrate between cells in the upper layers to any degree from the cut edges of the biopsy. These results are consistent with tracer studies in other structurally similar epithelia and strongly suggest that the upper layers of vaginal epithelium present a barrier to the movement of water-soluble molecules through the intercellular channel system. Freeze-fracture analysis of the epithelium revealed gap junctions and desmosomes between cells in the lower layers, but the former disappear in the upper layers. Unlike other keratinizing epithelia that have been described, random intramembranous particles do not disappear from the plasma membranes of the fully differentiated cells. Fracture planes through the upper layers reveal particle-free lamellae in the intercellular spaces, supporting the idea that intercellular lipids may be one of the components that limits the permeability of the intercellular spaces in this epithelium.     

A blood-brain barrier without tight junctions in the fly central nervous system in the early postembryonic stage

Summary The anatomical basis of the vertebrate blood-brain barrier is a series of tight junctions between endothelial cells of capillaries in the central nervous system. Over two decades ago, tight junctions were also proposed as the basis of the blood-brain barrier in insects. Currently there is a growing understanding that septate junctions might possess barrier properties in various invertebrate epithelial cells. We now examine these two views by studying the blood-brain barrier properties of the early postembryonic larva of a dipteran fly (Delia platura) by transmission electron microscopy. Newly hatched larvae possess a functioning blood-brain barrier that excludes the extracellular tracer, ionic lanthanum. This barrier is intact throughout the second instar stage as well. The ultrastructural correlate of this barrier is a series of extensive septate junctions that pervade the intercellular space between adjacent perineurial cells. No tight junctions were located in either nerve, glial or perineurial cell layers. We suggest that the overall barrier might involve septate junctions within extensive, meandering intercellular clefts.