The tight junctions of rabbit choroid plexus and ciliary body epithelia. A comparative study using the double replica freeze-fracture technique

The spatial arrangement of tight junctions in choroid plexus and ciliary body rabbit epithelia has been determined by studying freeze-fracture complementary replicas. In the choroid plexus epithelium, the interruptions of the junctional P-face fibrils were measured to be 14% of their total length. In the ciliary body epithelium, where the fibrils were found to be more fragmented than in the choroid plexus, the P-face fibril interruptions accounted for 12 % of the total length of the zonulae occludentes sealing the non-pigmented cells and 30% in the focal linear tight junctions connecting the non-pigmented and pigmented cells at their apices. In both epithelia, the interruptions of the ridges are precisely complemented by particles or short bars of similar length found in the E-face furrows. Consequently, it is possible to conclude that the junctional fibrils are continuous in these two epithelia. For the zonulae occludentes, this continuity appears to be inconsistent with the ‘leaky’ properties of these epithelia shown by some physiological investigations.     

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.     

Structural integrity of hepatocyte tight junctions

The significance of discontinuities frequently found in freeze-fracture replicas of the tight junction was evaluated using complementary replicas of hepatocyte junctions from control and bile duct-ligated rats. An extensive analysis of complementary replicas using rotary platinum shadowing indicates that discontinuities in the protoplasmic (P) fracture face do not represent structural breaks in the tight- junctional network. In no case did P-face discontinuities correspond with interruptions in the groove network on the complementary extracellular (E) face. Quantitative analysis of replicas shows that P- face discontinuities result in part from "transfer" of material to the complementary E face (approximately 7% of the junctional length). However, many P-face discontinuities (7-30% of the junctional length) are matched only by a groove on the complementary E face. This finding demonstrates that a significant amount of material can be lost during freeze-fracture. An analysis of junctions from bile duct-ligated rats, which are known to have an increased paracellular permeability, shows comparable transfer and loss of material. However, the number of junctional elements and the tight-junction network density was significantly reduced by bile duct ligation. These observations indicate that discontinuities in tight-junctional elements result during the preparation of freeze-fracture replicas and are not physiologically important features of the junctional barrier. Variation in the number of elements provides the best explanation for observed differences in tight-junction permeability.     

Morphology of tight junctions in the ciliary epithelium of rabbits during arachidonic acid-induced breakdown of the blood-aqueous barrier

Summary A reversible breakdown of the blood-aqueous barrier in the iridial processes of rabbits has been induced by arachidonic acid as demonstrated by the passage of horseradish peroxidase at places through the tight junctions. Freeze-fracture images reveal very discontinuous Pface ridges. However, the analysis of complementary replicas demonstrates that discontinuities of P-face ridges are always complemented by particles or short bars found in the E-face furrows. Though the problem exists of correlating freeze-fracture images of the junctional structure to the focal passage of horseradish peroxidase, the data suggest that the discontinuities of P-face ridges cannot be the structural counterpart of the passage of horseradish peroxidase. Alternative pathways of horseradish peroxidase are discussed in context with the offset bifibrillary model of the junction.Supported by a research fellowship from the Deutsche Forschungsgemeinschaft Present address: Universitäts Augen- und Poliklinik der Freien Universität, Klinikum Steglitz, Hindenburgdamm 30, 1000 Berlin 45This paper was presented in part at the International Symposium on Membrane Transport Mechanisms in the Eye, September 1984, Berlin     

The occluding junctions of mouse duodenal enterocytes during development

Summary The architecture of occluding junctions during the differentiation of the mouse duodenum was studied in freeze-fractured material. Irregular zonulae occludentes (ZO) (Type I) are numerous during fetal life, and are characterized by their irregular width, and by the presence of basal open-ended extensions fused with the discontinuous basal strand of the ZO. Regular ZOs (Type II), typical of the adult villous epithelium, appear after Type I junctions by day 16 of gestation. Two patterns are distinguishable: in the first, parallel strands of ridges and furrows are found without crossing branches; in the second pattern, the junction zone is organized like a network of short branches forming various types of polygons. In fetal and adult mice fasciae occludentes (FO) (Type III) are present on the lateral cell membranes; in unfixed specimens particles are found in the furrows of the E-face and pits on the ridges of the P-face. In fixed tissues, the particles are aligned on the ridges of the P-face. These results indicate that fixation with glutaraldehyde modifies considerably the affinity of junctional particles toward the P-face during the fracture process. Moreover, the presence of numerous large FOs on the lateral cell membranes of enterocytes during late fetal life and in the adult, is possibly related to cell movement along the intestinal villi.     

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)     

Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo

The peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12-13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types. The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.     

Definitive Evidence for the existence of tight junctions in invertebrates

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.     

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.     

A vertebrate-like blood--brain barrier, with intraganglionic blood channels and occluding junctions, in the scorpion

India ink and ionic lanthanum injections have revealed that the central nervous system (CNS) of the scorpion possesses a highly vascularized cephalothoracic ganglionic mass. It, together with other abdominal ganglia which form a ventral nerve cord, are all ensheathed by an outer layer of modified glial, or perineurial, cells. These cells resemble those which line the blood channels permeating the CNS, in exhibiting both inverted gap and tight junctions. Although the latter show close or fused membrane appositions, lanthanum appears to penetrate past a number, but not all, of them. Freeze-fracturing reveals that these junctions are composed of E-face particles aligned into a network of rows, or ridges, which are frequently discontinuous, especially near the periphery of the perineurium. This produces a somewhat 'leaky' system but occlusion to tracers occurs ultimately, for in the CNS none can be found beyond the perineurium. The existence of this perineurial blood-brain barrier is also demonstrable electrophysiologically where cations such as Mg2+ are unable to penetrate beyond the perineurial layer although they can, it seems, leak in via the blood vascular system. Relative differences in tightness between the perineurium and the cells lining the blood channels may be attributed to differences in the relative number of discontinuous ridges. This is borne out by the observation that the peripheral nervous system has a highly attenuated perineurium with many fewer junctions, and some of these nerves tend to be leaky with respect to tracer penetration. In fixed material the junctional ridges may fracture on to the E-face or partly on both the EF and PF, while in unfixed tissue they are usually found on the PF. In both cases they exhibit complementary grooves that are coincident with the ridges across membrane transitions; in such cases the cell membranes are fused with concomitant obliteration of the intercellular space. These tight junctions, often closely associated with EF gap junctional particle aggregates which may be very loosely clustered, appear to form the basis of the observed blood-brain barrier in the scorpion CNS.     

Occluding-like junctions at mesaxons of central myelin in Anolis carolinensis are not 'tight'. A freeze-fracture-protein tracer analysis.

The junctional complexes of the myelin sheath of central nervous system axons in the American chameleon, Anolis carolinensis, exhibit an intramembrane ridge and groove construction in freeze-fracture replicas that has usually been interpreted in other organisms as evidence for an occluding or tight intercellular junction. Close examination of PF fracture face ridges, however, shows them to be made up of discontinuous rows of particles of variable length separated by frequent gaps of non-uniform width. Introduction of horseradish peroxidase into the intercellular milieu of the lizard central nervous system is followed by appearance of this protein in interlamellar spaces of the myelin sheath and in the intercellular spaces containing focal membrane fusions that correspond precisely in position and center-to-center spacing to the ridges and grooves in platinum replicas of the same tissue. Since the junctional ridges on PF fracture faces in these mesaxonal junctional complexes are conspicuously discontinuous and since the areas within the myelin sheath where these junctional complexes are located inner and outer mesaxons) are readily permeated by exogenous protein tracer, it is concluded that the junctional complexes of central myelin mesaxons, heretofore incorrectly interpreted as functionally tight, are actually very leaky and probably contribute only to the structural stability of the myelin sheath architecture.     

Tight junction of sinus endothelial cells of the rat spleen

The fine structure of the tight junctions between sinus endothelial cells of the rat spleen and the permeability of such sinus endothelial cells were examined by transmission electron microscopy, using freeze-fracture, triton extraction, and lanthanum-tracer techniques. In freeze-fracture replicas, the segmented strands and grooves of the tight junctions were frequently observed on the basolateral surfaces of the sinus endothelial cells irrespective of the location of the ring fiber. There were one or two wavy-strands or grooves which were, for the most part, oriented parallel to the long cell axis thus forming networks at places. In addition, some strands or grooves were discontinuous while some networks of the junctional strands were not closed. These strands also occasionally lacked intramembranous particles in the tight junctions. The junctional strands run apicobasically at certain sites. In the vertical sections of the sinus endothelial cells treated with lanthanum nitrate, although no tight junctions were observed wherever the endothelial cells were apposed, most of them were situated on the basal part of the lateral surfaces of the adjacent endothelial cells. Several fusions of the junctional membranes were observed in a vertical section of the lateral surfaces of the adjacent endothelial cells. The intercellular spaces of the adjacent endothelial cells except for the fusion of the junctional membranes, were electron dense and the infiltration of lanthanum nitrate was found not to be interrupted by these tight junctions. Based on these observations, the molecular 'fence' and paracellular 'gate' functions of the tight junctions in the sinus endothelial cells are discussed.     

A novel adhering junction in the apical ciliary apparatus of the rotifer Brachionus plicatilis (Rotifera, Monogononta)

Cultures of the rotifer Brachionus plicatilis were examined with regard to their interepithelial junctions after infiltration with the extracellular tracer lanthanum, freeze-fracturing or quick-freeze deepetching. The lateral borders between ciliated cells have an unusual apical adhering junction. This apical part of their intercellular cleft looks desmosome-like, but it is characterized by unusual intramembranous E-face clusters of particles. Deep-etching reveals that these are packed together in short rows which lie parallel to one another in orderly arrays. The true membrane surface in these areas features filaments in the form of short ribbons; these are produced by projections, possibly part of the glycocalyx, emerging from the membranes, between which the electron-dense tracer lanthanum permeates. These projections appear to overlap with each other in the centre of the intercellular cleft; this would provide a particularly flexible adaptation to maintain cell-cell contact and coordination as a consequence. The filamentous ribbons may be held in position by the intramembranous particle arrays since both have a similar size and distribution. These contacts are quite different from desmosomes and appear to represent a distinct new category of adhesive cell-cell junction. Beneath these novel structures, conventional pleated septate junctions are found, exhibiting the undulating intercellular ribbons typical of this junctional type, as well as the usual parallel alignments of intramembranous rows of EF grooves and PF particles. Below these are found gap junctions as close-packed plaques of intramembranous particles on either the P-face or E-face. After freeze-fracturing, the complementary fracture face to the particles shows pits, usually on the P-face, arrayed with a very precise hexagonal pattern.     

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.     

Development of an apical plasma membrane domain and tight junctions during histogenesis of the mammalian pancreas

The role of tight junctions (zonula occludens) in the formation of apical plasma membrane (PM) domains was investigated in the embryonic rat pancreas. In the present study, lectin-rhodamine (WGA-TRITC and RCAII-TRITC) and lectin-gold (WGA-Au and RCAII-Au) conjugates were used to monitor apical PM domain formation and freeze-fracture analysis was used to monitor tight junction formation in the pancreatic epithelium of embryonic, neonatal, and adult rats. Fluorescent and TEM analysis of WGA and RCAII binding indicated that an apical PM domain is formed as early as Day 13 of gestation in the pancreatic epithelium. While apical WGA binding remained into adult life, RCAII binding was lost by 1 day after birth. In contrast, tight junctions were not observed until Day 14 of gestation. At this time, tight junctions were found to be incomplete in formation and typically consisted of linear arrays of IMPs or discontinuous arrays of sealing strands (focal adherens). Continuous tight junctions were not completely formed until Day 15 of gestation. Continued development of tight junctions during gestation was characterized by (1) an increase in the number of sealing strands and (2) a more parallel arrangement of sealing strands within each junctional complex. By 8 weeks after birth, tight junctions were more loosely organized and contained fewer sealing strands as compared to that observed in the fetus. These results suggest that lateral diffusion of apical PM glycoconjugates may be restricted even in the absence of complete tight junctional complexes during development of the rat pancreas.     

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.     

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 thin-section and freeze-fracture study of microfilament-membrane attachments in choroid plexus and intestinal microvilli

Choroid plexus and intestinal microvilli in thin sections have microfilaments in the cytoplasm adjacent to the membranes, and in replicas have broken strands of filaments in both cytoplasm and on E faces of plasm membranes. The microfilaments contain actin as indicated by their binding of heavy meromyosin (HMM). In sections of choroid plexus, the microfilaments are 7-8 nm in diameter and form a loose meshwork which lies parallel to the membrane and which is connected to the membranes both by short, connecting filaments (8 times 30 nm) and dense globules (approximately 15-20 nm). The filamentous strands seen in replicas are approximately 8 nm in diameter. Because they are similar in diameter and are connected to the membrane, these filamentous strands seen in replicas apparently represent the connecting structures, portions of the microfilaments, or both. The filamentous strands attached to the membrane are usually associated with the E face and appear to be pulled through the P half-membrane. In replicas of intestinal brush border microvilli, the connecting strands attaching core microfilaments to the membrane are readily visualized. In contrast, regions of attachment of core microfilaments to dense material at the tips of microvilli are associated with few particles on P faces and with few filamentous strands on the E faces of the membranes. Freeze-fracture replicas suggest a morphologically similar type of connecting strand attachment for microfilament-membrane binding in both choroid plexus and intestinal microvilli, despite the lack of a prominent core bundle of microfilaments in choroid plexus microvilli.     

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.     

Fine structure of Sertoli cells in the rat testis after hypophysectomy, testosterone treatment and re-involution

Using thin sections and freeze-etch replicas the fine structure of the Sertoli cells of the rat testis was investigated after hypophysectomy, testosterone treatment and re-involution. 41 days after hypophysectomy the Sertoli cells contain numerous dense bodies and remnants of degenerating spermatocytes and spermatids. The Sertoli cell junctions are most prominent. The membranes of neighbouring cells are folded into several layers. Freeze-fracture replicas reveal a normal arrangement of Sertoli cell tight junctions with linear array of membrane particles preferentially on the B-face and complementary grooves on the A-face. The geometric pattern of the ridges is varying with respect to the basal, intermediate and apical portions of the lateral Sertoli cell membranes. Since no major changes of the size, distribution and localization of the Sertoli cell junctions were observed in the different experimental groups these junctions, once formed, are inferred to be independent from hypophyseal hormones.