Gap junctions in hemodichorial and hemotrichorial placentae

Summary Gap junctions were found to be a constant feature of chorioallantoic placentae with two or three trophoblastic layers. The gap junctions connect layers I and II in hemodichorial and layers II and III in hemotrichorial placentae. Although the gap junctions vary in form and in the packing density of membrane-associated particles, they cover an extensive surface area in all species examined. The gap junctions always connect adjacent membranes of two trophoblastic layers, which show no evidence of micropinocytotic activity; at least one of these trophoblastic layers is syncytial. It is therefore concluded that the gap junctions play an important role in diaplacental transport. We consider that gap junctions act as molecular sieves, resulting in limitations in the transport of large molecules. The passage of small molecules, on the contrary, would be facilitated by the gap junctions.With the support of the Deutsche ForschungsgemeinschaftDedicated to Prof. Wolfgang Bargmann on his 70. birthdayWe are very grateful to Mrs. B. Brühl, I. Stenull and cand. med. P. Zahn for technical assisstence.We also gratefully acknowledge Prof. Dr. R. Taugner for helping us with freeze-fracturing     

Cell junctions in the renal tubule of a fresh-water teleost,Salmo gairdneri Rich.

Summary Cell junctions in the renal tubule of the fresh-water rainbow trout were studied with thin-section and freeze-fracture techniques. Gap junctions were restricted to the proximal tubule, which is consistent with other vertebrate classes. Segments I and II of the proximal tubule and the collecting tubule/collecting duct system exhibited a well-developed zonula occludens with anastomosing strands. The distal segment showed a narrow zonula occludens composed of few parallel strands. The structure of the occluding junctions along the renal tubule of this teleost displays several similarities with the pattern of the zonulae occludentes in the amphibian and the mammalian nephron. From these observations, in conjunction with available data from other vertebrate classes, it can be concluded that in the proximal tubule the development of a deep and complex zonula occludens is a general feature of cold-blooded vertebrates.     

The ultrastructural organization of the basement membrane of Bowman's capsule in the rat renal corpuscle

Summary The basement membrane of Bowman's capsule (BCBM) of the rat was studied by means of a modified tissue-preservation technique for transmission electron microscopy, which avoids the usual thorough fixation in OsO₄ and applies tannic acid and uranyl acetate for staining (Sakai et al. 1986). At most sites the BCBM is multilayered, consisting of one to seven dense layers separated by electron-lucent layers. The latter, which can be termed laminae rarae, contain fine filaments which connect the dense layers to each other and the innermost dense layer to the basal cell membrane of the parietal epithelium. The laminae densae are basically composed of fine filaments arranged in an anastomosing pattern. Individual filaments ranging from 5 to 15 nm in diameter, combine to form filament bundles up to 100 nm in thickness and 1 to 2 m in length. Within a dense layer, filaments and filamentous bundles are oriented mainly in the same direction. Often the inner dense layers do not form a continuous sheet, and the filamentous bundles are arranged in anastomosing or spiral patterns to form a ribbon-like structure that we call a microligament. These microligaments are often embedded in basal furrows of the parietal epithelium and are best developed around the vascular pole. Intracellular actin bundles of the parietal cells are regularly associated with these extracellular ribbon-like structures of the basement membrane. In conclusion, the BCBM has an unusual structure: the laminae densae are characterized by their filamentous nature and are arranged in different patterns, i.e. as a multilayered mat and as microligaments.Fellow of the Deutscher Akademischer Austauschdienst     

Reformation of gap and tight junctions in regenerating liver after cholestasis

Summary Morphometric analysis of the alterations in interhepatocyte junctions induced by bile duct ligation revealed that after 48 h, during which time the serum bilirubin increased 6 to 8 fold, the membrane area occupied by gap junctions on the apico-lateral and medio-lateral sides decreased from 3.6% in controls to 0.02% in the ligated group. The strands of the zonulae occludentes were reduced in number and showed increased discontinuities.Within 45 min of recanalization of the common bile duct, clusters of particles appeared within and adjacent to the tight junctional areas or in the lateral hepatocyte membrane. Subsequently, the particle aggregations localized in the apico-lateral membrane areas increased in number and size becoming finally indistinguishable from those of controls within 96 h after the onset of recanalization. The zonulae occludentes also rearranged and reestablished their original structure during this period. The serum bilirubin fell to normal within 24 h of recanalization. It is concluded that metabolic and ultrastructural restitution associated with the recanalization of the ligated bile duct have no strict temporal correlation to one another.These studies provide further evidence that alterations in gap and tight junctions induced by pathological processes, e.g. during bile duct ligation, are completely reversible when regeneration occurs.Summer student from Harvard Medical School, Boston (USA)     

Tight junctions and cavitation in the human pre-embryo.

In the human morula, tight junctions are found between all cell pairs, at all levels of cellular apposition, associated with underlying masses of microfilaments. In cavitating morula, lanthanum tracer gained access to the intracellular spaces, except at the intersections with nascent extracellular cavities, marking the first assembly of zonulae occludentes. Presumptive trophectoderm cells contained vacuoles and larger cavities often associated with secondary lysosome-like bodies. Since the vacuoles and intracellular and extracellular cavities contain electron-dense polygranules of about 23 nm diameter, they may have common origins. In trophectoderm cells of the early blastocytes, the large intracellular vacuoles and cavities were absent, and the zonulae occludentes were located apically. Mechanisms for nascent blastocoele formation are discussed.     

Fibrillar and cytoskeletal substructure of tight junctions: Analysis of single-stranded tight junctions linking fibroblasts of the lamina fusca in hamster eyes

Summary The lamina fusca of the hamster eye contains layers of flattened, slightly overlapping fibroblasts. Thin sections of the overlapping margins reveal punctate, tight-junction-like membrane appositions associated with accumulation of cytoplasmic filaments, 5–7 nm in diameter. Intermediate filaments are present in the surrounding cytoplasm. A diffuse dense substance occurs in adjacent intercellular space. Freeze-fracture replicas show that the membrane appositions are mainly single-stranded tight junctions, each composed of two fibrils (micelles), and each continuous or nearly continuous around the fibroblastic perimeter. Fracturing characteristics of these junctions offer a unique opportunity to gain further insight into tight junctional morphology. When exposed, the fibrils adhere to the P-face, measure 9.2±0.3 nm in diameter, and are accompanied by a narrow band of membrane differing in texture from non-junctional membrane. Characteristically, the junctional fibrils themselves mark the deviation line along which fracture planes pass from one membrane of the junction to the other. This pattern exposes, over long distances, the P-face of one membrane on one side of this line and E-face of the adjacent membrane on the other. Analysis of any single junction over such distances reveals that the juxtaposition of the fibrils may gradually twist or undulate over a range of at least 180° within the two involved membranes. The fracture plane appears preferentially to pass between the two junctional fibrils; association of the cytoskeleton with junctional fibrils may govern this route of fracture. Cytoskeletal attachment appears to be to a single fibril and may alternate from one fibroblast to the next depending on which cytoplasmic leaflet is nearest a given fibril.Parts of this work have been presented at meetings of the Association for Research in Vision and Ophthalmology (Kelly and Hageman 1983) and the American Association of Anatomists (Hageman and Kelly 1984)     

Rapid formation of myometrial gap junctions during parturition in the unilaterally implanted rat uterus

Summary In uterine smooth muscles, gap junction plaques rapidly form during the final stages of gestation. To investigate the related mechanisms, regional differences in myometrial gap junction development in rat uterus were examined quantitatively during delivery, using thin-section and freeze-fracture techniques in combination with light- and electron microscopy.Examination of implanted and nonimplanted horns in the unilaterally ligated rat bicornuate uteri, revealed no differences in the occurrence of gap junction plaques, but after 2 to 4 pups had been delivered, the contracted segments contained more gap junction plaques than did noncontracted segments examined immediately before delivery. In all segments, gap junctions were found more frequently in the circular muscle layers than in the longitudinal muscle layers. Gap junctions ranged in size from 0.002 m² to 0.52 m², but two-thirds were less than 0.1 m². The frequency of small gap junction plaques (less than 0.1 m²) was higher in the noncontracted segment.These results suggest that gap junctions are dynamic structures, and that their formation is controlled not only by general hormonal factors, possibly involved in gap junction increases in the myometrium before delivery, but also by local factors, possibly related to the contraction, that may accelerate an increase in gap junction formation during delivery.     

Intercellular junctions in the water-blood barrier of the gill lamella in the adult lamprey (Geotria australis,Lampetra fluviatilis)

Thin sections and freeze-fracture replicas of the water-blood barrier in the gill lamellae of adult lampreys (Geotria australis, Lampetra fluviatilis) demonstrate that the occluding junctions between epithelial pavement cells differ markedly from those between endothelial pillar cells in the structure and arrangement of their strands. The zonulae occludentes between pavement cells typically consist of complex networks of 4–6 strands, the mean number of which undergoes a small but significant decline when the animal is acclimated to seawater. In comparison, the occluding junctions between pillar cells are less elaborate and may represent maculae or fasciae, rather than zonulae occludentes. They do not apparently undergo a change when the animal enters saltwater. The results indicate that the main part of the paracellular diffusion barrier to proteins and ions is located in the epithelium rather than the endothelium. Communicating (gap) junctions are present between adjacent pavement cells, between pavement and basal cells and between pillar cells. These findings suggest that the epithelial cells and the pillar cells in the water-blood barrier of lampreys both form functional syncytia. The results are discussed in the context of ion-transporting epithelia in other aquatic vertebrates.This paper is dedicated to Professor H. Leonhardt on the occasion of his 75^th birthday     

Alterations in intercellular junctions of the uterine epithelium during the preimplantation phase in the rabbit

Summary In the rabbit, the pseudopregnant uterus has been used as a model for studying alterations characteristic of the preimplantation phase. Alterations in intercellular junctions of the uterine epithelium were investigated during early pseudopregnancy (day 0 to day 6) by means of the freeze-fracture technique.In the uterine epithelium of oestrous females the zonula occludens belongs to the tight type of tight junctions. During pseudopregnancy an impressive proliferation of tight junctional belts can be observed. The basal strands proliferate, forming loops perpendicular to the luminal surface, whereas the more or less parallel arrangement of the luminal strands is maintained. At day 4 of pseudopregnancy macular tight junctions begin to develop on the lower portions of the lateral plasmalemma and are extensive by day 6 post hCG.Small gap junctions are infrequent between cells of the uterine epithelium and show no significant changes during the preimplantation phase.The physiological significance of the present morphological observations is discussed in the light of changes occurring during the preimplantation period.Supported by grant Kü 210/9 from the Deutsche Forschungsgemeinschaft     

Cell junctions in early embryos of squid (Loligo pealei)

Summary Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7–8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12–13). During germinal layer establishment (stages 12–13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavagestage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.     

Spontaneous tubular (cuboidal) metaplasia of the parietal layer of Bowman's capsule in Cynomolgus monkeys (Macaca fascicularis)

Tubular (cuboidal) metaplasia of the parietal epithelium of Bowman's capsule was observed in 19 (10 males, 9 females) of 56 adult control Cynomolgus monkeys. Cuboidal epithelium replaced the normally present flattened epithelium to various degrees in animals affected. Cuboidal cells revealed an eosinophilic cytoplasm and vesicular nuclei, thus resembling proximal convoluted epithelial cells in all respects. Continuity of the cuboidal glomerular epithelium with that of the proximal tubule was occasionally observed.     

Gap junctions and impulse propagation in embryonic epithelium of Amphibia

Summary Epithelium of amphibian embryos (Cynops orientalis, Xenopus laevis) was found in preceding experiments to generate and conduct impulses during a limited stage (26–37) of development. In order to elucidate the structural basis of impulse propagation, epithelial cells of four stages were examined by the freeze-etching method: (I) before and (II) during acquisition of conductivity; (III) when propagation was fully established, and (IV) when it was no longer present. Only few gap junctions (GJ) of small size were found in groups I and IV. GJ in epithelia of group III were increased in number and size, and appeared morphologically coupled, i.e., with more loosely arranged connexons. The size of gap-junctional particles did not differ significantly between coupled and uncoupled stages. Zonulae occludentes seemed leaky in stage I, and tight in stages II–IV. Thus, the morphological characteristics of specialized junctions between non excitable cells correlated with the opening and closing of low resistance intercellular current pathways during embryonic development.Gap junctions in particular seem to form an essential link in the non-neural stimulus-response system, which may facilitate the mobility of the embryo during early phases of aquatic life before the reflex pathways have been established. Coupling and uncoupling of gap junctions may also play an important role in the regulation of cell differentiation and morphogenetic movement. The experimental model used in this study provides a useful tool for further investigations of structural correlates of gap junctional permeability under physiological conditions.     

Increased gap junctional area in the rat liver after administration of dibutyryl cAMP

Summary Since cAMP has recently been reported to be a possible physiological modulator of cell-to-cell communication, we performed a quantitative freeze-fracture investigation on the hepatocyte gap junctions after administration of a membrane-permeant derivative of this cyclic nucleotide. For this purpose, male rats received two intraperitoneal injections of 100 mg dibutyryl cAMP/kg body weight with a time interval of 2.5 h. Litter mates were injected with saline only. Five hours after the start of the treatment, tissue blocks of the left lateral liver lobe were fixed by immersion and processed for freeze-fracture. By point counting on negatives projected on a square double-lattice test system the relative gap junctional area on contiguous hepatocyte membranes was determined. As compared to control animals, the proportion of the membrane area occupied by gap junctions in dibutyryl cAMP-treated liver parenchyma significantly increased from 4.9% to 6.1%. Within the gap junctions no changes in shape, particle density or packing pattern were observed. Possibly, the enlarged gap junctional area provides structural pathways for the integration of the response of hepatocytes to messages mediated by cAMP.This investigation was supported by grant nr. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium).Research assistant of the National Fund for Scientific Research (Belgium).     

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     

Junctions between interstitial cells of the renal medulla: A freeze-fracture study

Summary Cell junctions between interstitial cells of the renal medulla were studied in freeze-fracture replicas of kidneys from rat, rabbit, hamster and the tree-shrew Tupaia belangeri. In all species studied a composite type of intercellular junction was found comprising elements of tight junctions and irregular gap junctions of highly variable size and shape. The number of these junctions increased towards the tip of the papilla.Our findings suggest that the composite junctions observed play a role in the maintainance of the ladder-like arrangement of the interstitial cells in the inner zone. The existence of irregular gap junctions raises the possibility that the functions of the interstitial cells are coordinated, especially during alterations of the functional state of the kidney.Supported by the Deutsche Forschungsgemeinschaft     

The formation of intercellular junctions in insect stem cell progeny (cockroach intestinal epithelium)

Summary The stages in the development of intercellular junctions have been followed in the mesenteric caecal cells of the cockroach midgut, where two types of mature cell, the columnar and the secretory, exist. Nests of undifferentiated replacement cells occur at intervals along the basal lamina, consisting of central, dividing cells and peripheral semi-lunar cells; the former act as proliferative stem cells to give rise to either pre-columnar or pre-secretory cells. The semi-lunar cells are pre-columnar and produce an attenuated process which gradually projects up to the luminal surface, producing microvilli and a dense extracellular substance en route. Intercellular gap junctions appear between these maturing columnar cell borders first, while septate junctions differentiate later; these are assembled from two different sets of intramembranous particle which become organized into either plaques or rows in parallel alignment, possibly mediated by actin filaments and microtubules. The pre-secretory cells, which are much fewer in number, remain associated only with the basal lamina and never reach the lumen; they develop into one of three distinct mature secretory cell types which release their secretory product in different ways. Offprint requests to: N.J. Lane