A freeze fracture and thin section study of intestinal cell membranes and intercellular junctions of a nematode, Ascaris

The microvillar and lumenal plasma membrane P-face of Ascaris intestinal cells is shown to be covered by relatively large (13 nm) particles at a fairly high density (1000/μm²), while the E-face has virtually none. The P-face of the lateral cell membranes, those separating the cells, have fewer and smaller (8 nm) particles. The intestinal cells are also shown to be connected by an apical complex of smooth septate and tricellular junctions similar to those found between some insect midgut cells. A periodic layer of tannic acid staining material is found on the cytoplasmic sides of the smooth septate junction, and when the intercellular space is filled with lanthanum, smoothly curved, 10 nm wide septal walls can be seen. Below the belt of septate junctions are a large number of gap junctions. These have closely packed arrays of particles on the P-face with some particle aggregates adhering to the closely packed pit arrays on the E-face.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions. I. Larval development

In the central nervous system (CNS) of full-grown larvae of the blowfly Calliphora erythrocephala, the glial-ensheathed nerve cells are completely surrounded by a layer of perineurial cells which form a “blood-brain barrier” between the circulating haemolymph and the CNS. A variety of intercellular junctions, including gap and tight junctions, are found between adjacent perineurial cells and some also between apposing glial cells; these have been characterized by freeze-fracturing as well as by tracer studies and analysis of thin sections. They are found not to be present between such cells in the undifferentiated CNS in the newly hatched larvae, nor are the nerve cells encompassed by glial cells; ionic lanthanum can penetrate to the axonal surfaces at this stage. However, over the 5 days of larval growth and development the glial cells produce attentuated cytoplasmic processes that ensheath the nerve cells, and the perineurium is formed; junctional complexes are assembled and a larval blood-brain barrier is produced which excludes tracers. Freeze-fracture preparations suggest that the inverted gap junctions which develop have done so by migration of individual intramembranous EF particles to form, at first, linear arrays and small clusters and, ultimately, macular aggregations in the perineurium; these lie between the undulating rows of PF particles forming the septate junctions. These septate junctions are formed by the organization of arrays of PF particles into multiple rows. Extensive PF particles fusing into ridges with EF grooves to form perineurial “tight” junctions are also observed, seemingly in the process of development; entry of exogenous lanthanum followed by its exclusion parallels the completion of ridge formation. These ridges are simple linear arrays of particles which may be discontinuous, lying in parallel with one another and the surface. Clustered particle arrays as well as scattered short ridges on the axonal PF, however, appear to be present unchanged throughout larval life; their role may therefore be associated with neural membrane function although there are suggestions that some may form axo-glial junctions. This is the first report on the lateral migration of intramembranous particles as the mode of formation of gap junctions in the nervous system of an invertebrate.     

Changes occurring in plasma membranes and intercellular junctions during the process of carcinogenesis and in squamous cell carcinoma

A 0.5% mineral-oil solution of 9.10-dimethyl-1.2-benzanthracene (DMBA) was applied to artificial cecal pouches in the lower lips of rats. Ultrastructural studies were made of plasma membranes and intercellular junctions during the process of malignant transformation in the oral mucosal epithelium and after squamous cell carcinoma had been induced by the carcinogen. After the administration of DMBA, the inner leaflet of the membranes where the microfilaments are attached showed high electron density and intramembranous particles on the P-face of basal cells decreased to about half that of controls. However, on the E-face the number of intramembranous particles increased by approximately 10% compared with controls. Though the normal size range for intramembranous particles was 9-12 nm, the administration of DMBA caused aggregations of from three to six particles on the P-face. In squamous cell carcinomas, only the outer leaflet of the membranes showed high electron density; the number of intramembranous particles was 30% higher on the P-face and approximately three times higher on the E-face compared with controls and the morphology of the intramembranous particles, which formed irregular aggregates of from five to 20 particles, was specific. In animals treated with DMBA, the number of gap junctions decreased by between 50% and 70%, although no structural changes occurred. In squamous cell carcinomas, the area of gap junctions was about 50% lower and the number of gap junctions about 40% lower than in controls. Changes in the number and area of desmosomes were similar to those of gap junctions both in the DMBA-treated animals and in squamous cell carcinomas.     

A freeze-fracture study of two types of collagen-phagocytosing cell in the post-partum rat endometrium

In the post-partum rat endometrium, ultrastructural distinction could be made between stromal cells (fibroblast-like cells) and macrophages, especially by the freeze-fracture technic. The stromal cells were characterized by a well-developed rough-surfaced endoplasmic reticulum (RER) and intercellular junctions, while the macrophages had many vacuoles and vesicles, but no intercellular contact with each other. The freeze-fracture image showed that the stromal cells had many low linear elevations and gap junctions on the cleaved plane of the cell membranes, while the macrophages had no linear elevations or intercellular junctions. The cell membranes of the stromal cells had more intramembranous particles (IMP) (P-face 697 +/- 63/micrometers 2, E-face 303 +/- 52/micrometers 2) than those of the macrophages (P-face 467 +/- 50/micrometers 2, E-face 217 +/- 35/micrometers 2). It was confirmed that these two types of cell phagocytosed collagen fibrils.     

Membrane specializations in the paired spermatozoa of dytiscid water beetles

The paired spermatozoa of the dytiscid beetles Dytiscus marginalis and Hydaticus seminiger were studied by electron microscopy with the aim of examining whether the regions of the cell membrane in the zones of sperm conjugation might differ from other regions and to explore whether these cells had any other specialized domains of the cell membrane that could be recognized by the freeze-fracturing technique. The spermatozoa are conjugated along one side of the sperm head and proximal tail portion, called the ventral side. The cell membrane was seen to contain tightly packed intramembranous particles (IMPs) that were predominantly located in the external membrane face (the E-face). In thin sections the cell membrane had a ladder-like appearance at these regions and a specialized type of glycocalyx seen as a fluffy material containing granules. Other specialized membrane domains could also be recorded: a ribbon of particles in the protoplasmic face (P-face) of the dorsal side of the spermatozoon at the proximal tail portion and regularly arranged particle rows in the P-face of the distal tail portion. These domains corresponded to regions where the glycocalyx is prominent. Both the E-face and the P-face of the cell membrane were seen to contain numerous intramembranous particles, which suggests an active function for both membrane leaflets; this is in contrast to the situation in most cells where the particles are mainly in the P-face. The functions of the intramembranous particles in the specialized domains of the cell membrane remains unknown. Some particles may represent receptors or ion gates, others proteins with a mechanical function.     

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.     

Two different forms of gap junctions within the same organism, one with cytoskeletal attachments, in tunicates

The cells of the intestinal tract and the stigmatal cells of the branchial basket have been studied in a range of tunicates including phlebobranch, aplousobranch and stolidobranch ascidians, as well as the doliolid and pyrosomatid thaliaceans. The intercellular gap junctions between gut cells appear conventional in thin section as do those found in the lower part of adjacent stigmatal cells. However, save for the stolidobranchs, the stigmatal cells also have a second kind of gap junction which exhibit an unusual fibrous density in association with their junctional cytoplasmic surfaces; these are found in the apical region of the cells. The fibrous density is particularly well demonstrated in specimens treated with tannic acid during fixation, and subsequent en bloc uranyl acetate staining. In the branchial basket the position of these apical gap junctions is at regular intervals between adhaering junctions, which have a more substantial paramembranous fibrous mat; these two kinds of junctions alternate along deeply undulating membrane appositions. With freeze-fracture, after chemical or cryo-fixation, the gap junctions of the gut and those of the lower part of the stigmatal cells appear typical, with P-face connexons, while in the apical part of cells of the branchial basket the two faces of the gap junctions are very difficult to cleave apart. Frequently the P- and E-faces are found to adhere together in replicas, so that in these apical gap junctional regions, plaques of E-face with pits overlie the PF particles. In addition, regions of cytoplasm, into which the dense fibres project, often cleave over these adhaering E-faces of the apical gap junctions. The presence of these unusual gap junctional features in the apical region of the stigmata in the vicinity of cilia is discussed as regards their functional role.     

Formation of tight and gap junctions in the inner enamel epithelium and preameloblasts in human fetal tooth germs

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.     

Gap junctions between the oocyte and companion follicle cells in the mammalian ovary

Tracer and freeze-fracture electron microscopy of the ovaries of neonatal rat and adult mouse, rat, rabbit, and primate have revealed the presence of gap junctions between follicle cells and oocytes. The junctional connections are found at the ends of follicle cell projections which traverse the zona pellucida and terminate upon microvilli and evenly contoured nonmicrovillar regions of the oolemma. Gap junctions are often seen associated with a macula adherens type of junction. The gap junctions occasionally consist of minute ovoid plaques, but nore frequently appear as rectilinear single- or multiple- row aggregates of particles on the P-face or pits on the E-face. The functional significance of follicle cell-oocyte gap junctions is discussed with respect to the regulation of meiosis and luteinization.     

Freeze-fracture analysis of gap junction disruption in rat ovarian granulosa cells

The effects of chemical dissociation on rat ovarian granulosa cell gap junctions has been studied using freeze-fracture electron microscopy. Sequential exposure of granulosa cells within follicles to solutions containing 6·8 mM EGTA [ethylene-bis-(β-aminoethyl ether)-N,N′-tetra acetic acid] and 0·5 M sucrose results in extensive cellular dissociation of the follicular epithelium. Freeze-fracture replicas made from fixed, control or EGTA-treated ovarian follicles exhibit extensive gap junctions between granulosa cells that are characterized by a range of packing order of constituent P-face particles or E-face pits. In contrast, exposure to 0·5 M sucrose containing 1·8 mM EGTA for as little as 1 min results in a consistently close packing of particles or pits which is accompanied by splitting of gap junctions between granulosa cells. The process of junction splitting was studied in detail in replicas prepared from follicles treated sequentially for various periods of time with EGTA and sucrose solutions. Initially, large gap junctions lose their regular shape and fragment into numerous tightly packed aggregates of P-face particles or E-face pits which are separated by unspecialized areas of plasma membrane. Subsequent to junction fragmentation, individual junction plaques separate at sites of cell contact and generate hemijunctions that border the intercellular space, Hemijunctions undergo particle dispersion of the P fracture face which results in an increased density of large intramembrane particles; no corresponding change in E-face pits is discernible at this stage. Morphometric analysis of replicas of tissue undergoing junction splitting indicates that junctional surface area decreases to 10–20% of control levels during this same treatment and so further supports the qualitative observations on junction fragmentation. Viabilities of granulosa cells obtained by these techniques also agree with the sequence observed in the morphometric analysis of the replicas. Finally, within 15 min after placing ovaries in isotonic, Ca²⁺-containing salt solutions, gap junction reformation occurs by aggregation of particles at sites of intercellular contact. These sites are distinguished by the appearance of short surface protrusions or indentations on their respective P and E fracture faces. The data suggest a mechanism for EGTA-sucrose mediated cellular dissociation in the follicular epithelium in which gap junctional particles are free to move in the plane of the plasma membrane and may be re-utilized to form gap junctions in the presence of extracellular calcium.     

Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells

Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.     

Morphological variations in gap junctions of ovarian granulosa cells.

Granulosa cells in ovarian follicles of rat, mouse, rabbit and hamster were studied by lanthanum tracer and freeze-fracture techniques. Abundant gap junctions exhibited striking intraspecific variation in size and pattern of particle aggregation. The smaller gap junctions showed close packing of the intramembranous A face particles. In large gap junctions, ranging up to 6 mu in diameter, particles were packed in rectilinear arrays separated by a labyrinthine network of particle-free 'aisles'. Small clusters of particles in a particle-poor circumferential zone suggested enlargement of junctions by peripheral accretion. Linear intramembranous structures, resembling those of occluding junctions, occasionally bounded large gap junctions. Spherical intracytoplasmic structures limited by gap junctional membranes were shown by tracer studies to arise by invagination of the cell surface. These were intrepreted as a means of disposal of junctions by interiorization.     

Cyclic modulation of Sertoli cell junctional complexes in a seasonal breeder: the mink (Mustela vison)

The development and modulation of Sertoli cell junctions was studied in newborn and adult mink during the active and inactive spermatogenic phases. The techniques used were electron microscopy of freeze-fractured replicas and thin sections of tissues infused with horseradish peroxidase as a junction permeability tracer. In the newborn, freeze-fractured developing junctions had either spherical or fibrillar particles. In addition, junctional domains where particles were associated preferentially with the E-face, and others where particles were associated preferentially with the P-face, were found developing either singly or conjointly within a given membrane segment, thus yielding a heterogeneous junctional segment. Coincidently with the development of a tubular lumen and the establishment of a competent blood-testis barrier, junctional strands were composed primarily of particulate elements associated preferentially with the E-face. In adult mink during active spermatogenesis, cell junctions were found on the entire lateral Sertoli cell plasma membrane from the basal to the luminal pole of the cell. In the basal third of the Sertoli cell, membranous segments that faced a spermatogonium or a migrating spermatocyte displayed forming tight, gap, and adherens junctions. In the middle third, abutting membrane segments localized above germ cells were involved in continuous zonules and in adherens junctions. In the apical or luminal third, the zonules were discontinuous, and the association of junctional particles with the E-face furrow was lost. Gap junctions increased in both size and numbers. Junctional vesicles that appeared as annular gap and tight-junction profiles in thin sections or as hemispheres in freeze-fracture replicas were present. Reflexive tight and gap junctions were formed through the interaction of plasma membrane segments of the same Sertoli cell. Internalized junctional vesicles were also present in mature spermatids. During the inactive spermatogenic phase, cell junctions were localized principally in the basal third of the Sertoli cell; junctional strands resembled those of the newborn mink. During the active spermatogenic phase, continuous zonules were competent in blocking passage of the protein tracer. During the inactive phase the blood-testis barrier was incompetent in blocking entry of the tracer into the seminiferous epithelium. It is proposed that modulation of the Sertoli cell zonules being formed at the base and dismantled at the apex of the seminiferous epithelium follows the direction of germ cell migration and opposes the apicobasal direction of junction formation reported for most epithelia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions

In the central nervous system (CNS) of pupal Calliphora, dramatic alterations occur in the perineurial and glial gap junctions. Having formed macular plaques by late larval stages, in early pupae cell migration causes the EF intramembranous junctional particles to disaggregate and move apart into linear and then disorganised arrays as shown by freeze-fracture. After nerve and glial cell reorganisation into the adult pattern, the gap junctions begin to reform in the late pupae, again seemingly by particle migration into linear arrays and clusters. Ultimately the particles form numerous macular plaques between both perineurial and glial cells. Statistical analyses support the contention that these are performed EF particles which undergo translateral movement from macular larval junctions into the disaggregated particles of early pupae and that the same particles appear to undergo realignment and reclustering in late pupae to form the mature gap junctions of adults. This is the first report to indicate breakdown and reformation of gap junctions in vivo involving reutilisation of the same intramembranous particles. Perineurial “tight” junctions are not to be found in early pupal stages and their absence can be correlated with the free entry of ionic lanthanum into the CNS observed during that period. In late pupae, when the tight junctional moniliform ridges have apparently reformed, the entry of the tracer lanthanum becomes restricted to the level of the perineurium, penetrating no deeper. This is also the case in the adult, where the blood-brain barrier is maintained. PF particles in the form of short linear ridges and clustered particle arrays in nerve cell membranes are present throughout pupal and adult stages; their continued presence throughout the whole of development suggests some role in neuronal function, as yet unclear.     

Junctional diversity in two regions of the epidermis of Oikopleura dioica (Tunicata,Larvacea)

Summary A simple continuous epithelium surrounds the body of the pelagic larvacean. It consists of two zones of cells: oikoplast cells and flattened cells. The oikoplast cells are columnar and produce a thick extracellular house that ensheathes the body of the organism. These cells are joined laterally by wide tight junctions (zonulae occludentes). The tail of the animal is surrounded by exceedingly thin cells which are joined by narrow tight junctions under which lie intermediate junctions (zonulae adhaerentes) and gap junctions. A web of fibrous material inserts into the intermediate junctions. The transitional cells between the two epithelial zones have one lateral border with a wide tight junction, and the other lateral border with a narrow tight junction and a wide intermediate junction. In freeze-fracture replicas, the wide tight junction has a number of anastomosing ridges, in comparison with the narrow tight junction, which usually consists of only a single row of intramembranous particles. In replicas, the thin epithelial cells show unusual parallel arrays of particles in clusters on their apical plasma membranes. This simple epithelium, therefore, exhibits striking differences between the two cellular zones, in the structural characteristics of both the lateral borders and the apical membrane.     

Structure of the plasmalemma of human spermatozoa after treatment with antispermatozoa serum and concanavalin A

Human ejaculated spermatozoa were treated with antispermatozoa serum as well as with the same serum and concanavalin. A. At both experimental conditions clusters of intramembranous particles both on P-face and on E-face in the acrosomal and postacrosomal regions were seen. The clusters were with different form and size and were built up from different number of intramembranous particles. Large areas with single particles or in some cases devoid of these particles were found out.     

The structure and function of the smooth septate junction in a transporting epithelium: The malpighian tubules of the new zealand glow-worm Arachnocampa luminosa

Junctional complexes between the epithelial cells in the four distinct regions of the glow-worm Malpighian tubule were investigated by electron microscopy using thin sectioning, freeze-fracturing, osmotic disruption and tracer techniques. The lateral plasma membranes of all four cell types are joined by smooth septate junctions but the extent of the complex across the cell depth varies in the four different regions. The width of the septa, the interseptal spacing and the separation between the outer leaflets of the adjacent plasma membranes are different for each cell type. Gap junctions were identified only in the junctional complex between Type IV cells and were intercalated amongst large lateral sinuses. In oblique sections of lanthanum infiltrated tissue, the electron-lucent septa at the basal side of the junction are outlined by the tracer as it penetrates. In the Junctional complexes of all four regions the septa appear as short, distinct, linear bars. In tangential sections of gap junctions between Type IV cells, the junctions appear as a hexagonal array of intermembrane particles with a centre to centre spacing of 18 nm. Horseradish peroxidase did not penetrate the junctional complexes very far but readily passed through the basal lamina into the spaces between extracellular invaginations of the basement membrane of the cells. Junctional complexes in all four areas of the tubule have similar freeze-fracture faces. In freeze-fracture replicas of fixed tissue continuous ridges of fused particles are seen on the P face and complementary furrows are found on the E face. Junctional response to osmotically adjusted Ringer solutions was similar in all four cell types. Distortion or ‘blistering’ of the intercellular space between the septa of the junction occurred when the tissue was bathed in or injected with a hypertonic Ringer solution. The structure of these junctions, visualized by the different techniques, and the role of the septate junction in a transporting epithelium, are discussed.     

Effects of cytochalasin D on the integrity of the Sertoli cell (blood-testis) barrier

Ectoplasmic specializations (ES) containing packed actin microfilaments are associated with the numerous parallel rows of occluding junctions which form the Sertoli cell (blood-testis) barrier. To determine if ES regulate the structure of the occluding junctions and/or barrier permeability, we experimentally disrupted ES microfilaments in vivo with intratesticularly injected cytochalasin D (CD). Electron microscopic observations of seminiferous tubules from CD-treated (150-500 microM CD; 0.5-12 hr) animals indicated that ES was absent from regions where the Sertoli cell barrier is located. Seminiferous epithelial sheets from uninjected or vehicle-injected animals (1 DMSO: 1 saline) stained with NBD-phallacidin demonstrated the presence of patterned ES actin surrounding the basolateral regions of adjacent Sertoli cells. After exposure to CD, epithelial sheets exhibited increasingly patchy fluorescence indicating progressive F-actin disruption. Freeze-fracture replicas of CD-injected testes revealed numerous focal alterations in the region of occluding junctions which included disorganization of the parallel arrangement of junctional rows, the presence of free-ending rows, clustering of intramembranous particles (IMPs) between rows, reduction in the number of rows, and loss of IMPs on both the P-face and E-face. Tracer experiments, following CD exposure, were conducted to test the integrity of occluding junctions: lanthanum hydroxide, dextrose, or filipin was added, in separate experiments, to the fixative during perfusion-fixation. In another study, serum containing an antibody against adluminal germ cells was injected intratesticularly, and frozen sections were processed for immunofluorescence study. A final study consisted of simultaneous intratesticular infusions of CD and radiolabelled inulin with subsequent intraluminal and peritubular fluid sampling. In animals which were injected with CD, lanthanum was found to enter the adluminal compartment; fixative made hypertonic by addition of dextrose caused germ cells within the adluminal compartment to shrink and produce exaggerated intercellular spaces; filipin-cholesterol perturbations were present between some Sertoli cell junctional rows and on spermatid plasma membranes; and IgG was detected within the adluminal compartment of many seminiferous tubules. None of these adluminal manifestations was noted in control animals or those which received vehicle. Quantitatively, in the in vivo micropuncture experiments, significantly more radiolabelled inulin entered the lumen of seminiferous tubules from CD-treated animals than from those exposed to vehicle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prokaryotic-eukaryotic cell junctions between spiral-shaped bacteria and cecal epithelium of the guinea pig

Summary Scanning electron microscopy demonstrated that the cecum of the guinea-pig is colonized by numerous spiral-shaped bacteria; these microorganisms, which adhere to mucosa at one end, were found exclusively on the brush border of the surface epithelium. The membranes of sectioned bacteria have a set of electron-dense bands girdling the tip adhered to epithelium. Freeze-fracture replicas of the bacteria revealed the prokaryote-eukaryote junction as a set of ridges on the P-face of outer membrane; the numerous particles of E-face were arranged in parallel rows; on the other hand, the apical plasma membrane and subjacent cytoplasm of epithelium occupied by the spiral-shaped bacteria did not show a structural counterpart. Observations suggest that one end of the spiral-shaped bacteria possesses specialized membrane components that permit specific attachment to the apical surface of epithelial cells.