Freeze-fracture Technique Research Articles

Freeze-fracture observations on changes in the distribution of Filipin-sterol complexes during epididymal maturation and capacitation of boar spermatozoa

It is believed that i ntramembra.nous sterols play an essential role in membrane stability and permeability. To investigate the distribution changes of sterols in sperm membrane during epididymal maturation and capacitation, filipin has been used as a cytochemical probe for the detection for membrane sterols. Using this technique in combination with freeze fracturing, we examined the boar spermatozoa under various physiological conditions.The spermatozoa were collected from: 1) caput, corpus and cauda epididymides, 2) sperm rich fraction of ejaculates, and 3)the uterus 2hr after natural coition. They were fixed with 2.5% glutaraldehyde in 0.05M cacodylate buffer (pH 7.4), and treated with the filipin solution (final concentration : 0.02.0.05%) for 24hr at 4°C with constant agitation. After the filipin treatment, replicas were made by conventional freeze-fracture technique. The density of filipin-sterol complexes (FSCs) was determined in the E face of the plasma membrane of head regions.

Characterization of germ cell nuclei in freeze-fracture replicas using seminiferous tubules isolated by transillumination

The present investigation was under taken, combining the identification of specific germ cell types present in a particular stage of the seminiferous wave with freeze-fracture techniques to follow the changing pattern of their nuclear pore distribution during spermatogenesis. Male albino adult rats were used. Segments of seminiferous tubules were separated (stages VII-VIII; IX-XI; XII-XIV) by a technique described by Parvinen and Vanha-Perttula where the stages of the cycle were recognized and isolated by transillumination under a dissecting microscope. The tubular segments were immediately fixed in glutaraldehyde, immersed in glycerol, freeze-fractured in a Balzers BAF 301 apparatus at-110°C and examined under a Siemens Elmiskop I EM. The only spermatogonia located in the studied stages (type A) are ovalcells in contact with the basal lamina. Pore distribution is non-random with a relatively in conspicuous aggregation. Preleptotene spermatocytes (stages VII-VIII) are easily identified by being numerous, their location over the basal lamina and the irrelatively small size. They exhibit a striking clustering of pores, in groups of 15-30 separated by pore-free areas (fig.1). Leptotenes (fig.2, 3) (stages IX-XI) and zygotenes (fig.4) (XII-XIII) change the pattern to a more even distributional though clearly maintaining some degree of clustering.

Electron microscopic study of a glass-forming water/oil pseudo-three-component microemulsion system

The development of microemulsion systems that do not break down during cooling and in which neither dispersed nor matrix phases crystallize during the cooling process opens the way to direct studies of the microemulsion structure and also the investigation of the dispersed liquid in unusual states. The authors report the first water-in-oil example of this type of system. It was obtained by partial replacement of water by glycerol and total replacement of normal paraffin by ethylcyclohexane, in the water/oil/didodecyldimethylammonium bromide three-component system. The phase diagram at 25{degree}C indicates a remarkably wide range of clear-phase compositions. A dispersed droplet structure for the water-rich range is unequivocally established by direct electron microscope imaging of the vitrified microemulsion, using the freeze-fracture technique.

Ultrastructure of airway epithelial cell membranes among patients with cystic fibrosis

Alterations in transepithelial ion fluxes are a primary pathophysiologic feature in cystic fibrosis (CF). Chronic respiratory infections and host responses are secondary aggravating pulmonary complications of this disease. In the present study, the application of the freeze-fracture technique to samples of nasal and tracheal epithelium from patients with CF has provided a perspective of large areas of cell membrane for the evaluation of possible structural correlates to the pathophysiology of this disease. A variety of aberrant configurations in stranding pattern and disorganization of the epithelial tight junctional complexes in CF airway epithelium are described. Additionally, examination of ciliary membranes revealed the presence of compound cilia and dysmorphology of ciliary necklace configuration. These features are thought to represent acquired structural lesions possibly derived from chronic infection and/or host responses which may further exacerbate abnormal ion transport properties and decrements of ciliary function that appear to be associated with the airway epithelium of individuals with CF.

An Optimized Freeze-Fracture Replication Procedure for Human Skin

This in vitro study aimed at substantial modification of the freeze-fracture replication technique (FFRT) which should result in an optimal visualization of the ultrastructure of human skin. The technique was modified in two ways: firstly, the conventional sample holders such as gold cups and copper plates were replaced by silver cylinders (83.5% silver, 16.5% copper) resulting in almost perpendicular cross fractures through the skin. Secondly, the replica cleaning procedure was optimized through the following sequence of treatments. Firstly, a mild tissue destruction was obtained by simultaneous lipid solvation and water extraction with absolute methanol (20 h), followed by protein denaturation with dimethyl sulfoxide (DMSO, 24 h). Subsequently, a final treatment was given using an alkaline sodium hypochlorite solution (20% KOH/13% NaClO; 1:3 v/v, 4 d). After rinsing the replicas for 45 min in aqua bidest, they were mounted on copper grids and examined in the transmission electron microscope (TEM). The combination of the unorthodox fracturing method and the optimized cleaning procedure yielded large, practically undamaged and very clean replicas of near perpendicular cross fractures through human skin. Common handicaps related to current freeze-fracture procedures when applied to skin, such as incomplete cleaning and fragmentation of replicas and oblique or irregular fracturing planes, can largely be avoided in this way. In this paper a complete description of the method is given, and a number of advantages are illustrated with the aid of TEM micrographs.

Rabbit retinal Müller cells in cell culture show gap and tight junctions which they do not express in situ

Retinae of early postnatal rabbits were enzymatically dissociated and explanted in a culture system. The prospective myelinated region was discarded in order to avoid the presence of astrocytic or mesenchymal cells. After about 14 days in vitro (DIV), outgrowing glial (Müller) cells formed what light optically appeared to be confluent monolayers but by electron microscopy was shown to consist of flat epitheloid cells which overlapped considerably by extension of cytoplasmic tongues. Applying the freeze-fracture technique, apposed membranes of these cells were demonstrated to express infrequently but consistently both gap and tight junctions. This kind of junctions has never been observed on the membrane of rabbit Müller cells in situ. In comparison with Müller cell membranes in situ, the density of intramembrane particles was considerably reduced. Orthogonal arrays of particles which are characteristic elements of Müller cells in situ were not detected. Our results suggest that in homogeneous cell culture, Müller cells form some kind of epithelium-like specialized intercellular junctions. This situation resembles that of closely related glial cell types which form homogeneous layers in situ as e.g. retinal pigment epithelium cells expressing tight junctions, and marginal astrocytes being coupled by extensive gap junctions.

Orthogonal arrays of intramembranous particles in the M�ller cell and astrocyte endfoot membrane of rabbit retina. Postnatal development and adulthood.

The freeze-fracture technique was applied to the retina of early postnatal and adult rabbits to investigate the distribution and density of orthogonal arrays of intramembrane particles (OAP) within the vitread endfoot membranes. In adult animals, two distinct types of endfoot membranes were observed within the central myelinated retina but not in the retinal periphery. One type of endfoot membranes contained low density of individual 'background' particles, and a more or less stripe-like pattern of OAP; this type was found only within the myelinated centre and is concluded to represent membranes of retinal astrocytes. The other type of endfoot membranes was rather tightly packed with individual 'background' particles, and contained OAP which formed rows only at the margins of footplates; this kind of membrane was found throughout the whole retina, and represents Müller cell endfeet. The density of OAP in both types of endfoot membranes was higher within the central myelinated retina than in the Müller cell endfeet of the retinal periphery. In early postnatal retinae, a discrimination between the two types of endfeet was impossible. At the day of birth, only very few OAP were observed, and the majority of footplates were free of OAP. Within the next 10 days, both density and size of OAP were found to increase but fail to reach adult levels. Quantitative data are presented with the hope of providing a basis for future correlation with functional maturation of rabbit retinal glia.

Androgen‐induced changes in nuclear pore number and in tight junctions in rat seminal vesicle epithelium

The numbers of nuclear pore complexes and tight junctions were quantified in the seminal vesicle epithelial cells of castrated and castrated-plus-androgen-treated male rats, which received subcutaneous pellets of testosterone propionate (1 mg/kg body weight) for 1 week. Seminal vesicle weights were 0.284 +/- 0.02 g for castrated, 1.006 +/- 0.006 g for androgen-treated, and 0.918 +/- 0.04 g for untreated groups. Tissue samples were processed for light or electron microscopy and for freeze-fracture techniques. Nuclear areas were measured: controls were 279.34 +/- 8 microns 2; these increased significantly (P less than .001) in castrated-plus-androgen-treated rats (324.66 +/- 11 microns 2) and decreased (P less than .001) in castrated animals (173.14 +/- 6.3 microns 2). Nuclear pore density increased (P less than .001) in castrated-plus-androgen-stimulated rats (5.38 +/- 0.24 pores/microns 2) (control: 4.78 +/- 0.14 pores/microns 2), and decreased (P less than .001) in castrated rats (3.16 +/- 0.14 pores/microns 2). A significant (P less than .001) increase in numbers of tight junction strands that extended in the lateral cell membranes was detected in castrated-plus-androgen-treated rats vs. controls or castrated-only animals.

Orthogonal Arrays Are Absent from the Membranes of Human Glioblastomatous Tissues

Human astrocytic gliomas were studied with the freeze fracture technique. Orthogonal arrays of particles were noted in the plasma membranes of low-grade astrocytoma tissues. However, no such arrays were found in the plasma membranes of anaplastic glioma or glioblastoma tissues. Gap junctions were rarely seen in the membranes of these higher-grade gliomas; when seen, they consisted of relatively few particles in poorly organized plaques. These plasma membranes were dominated by randomly distributed single particles. These findings constitute aspects of the loss of differentiation in these malignant tumors.

Inhibitory innervation of a lobster muscle

Inhibitory neuromuscular synapses formed by the common inhibitor (CI) neuron on the distal accessory flexor muscle (DAFM) in the lobster, Homarus americanus, were studied with electrophysiological and electron-microscopic (thin-section and freeze-fracture) techniques. Postsynaptic inhibition as indicated by inhibitory junctional potentials was several-fold stronger on distal compared to proximal muscle fibers. This difference correlated with the results of serial thin-section studies, which showed more inhibitory synapses on distal fibers than on their proximal counterparts. Effects of postsynaptic inhibition on excitatory junctional potentials via current shunting had a morphological correlate in the spatial relationship between inhibitory and excitatory synapses on the distal fibers. Inhibitory synapses were larger than their excitatory counterparts and had fewer glial processes. In freeze-fracture views, inhibitory synapses did not appear as raised plateaus in the P-face as do excitatory synapses, and their active zones were more widely scattered. The intramembrane particles in the inhibitory postsynaptic membrane - representing neurotransmitter receptors - are arranged in parallel rows in the sarcolemmal P-face and have complementary furrows in the sarcolemmal E-face. Altogether, our findings help to describe a population of inhibitory neuromuscular synapses formed by the CI neuron in lobster muscle.

Tetanus toxin blocks potassium-induced transmitter release and rearrangement of intramembrane particles at pure cholinergic synaptosomes

We have studied the action of tetanus toxin on the release of acetylcholine from a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. We have also studied the morphological changes induced by chemical stimulation on the presynaptic plasma membrane of poisoned synaptosomes. These changes were studied by means of freeze-fracture techniques. We found that tetanus toxin blocks the release of acetylcholine from isolated nerve terminals in a dose-dependent manner. The maximal inhibition is achieved at a concentration of 12.5 nM in 10 min. This effect is prevented by tetanus toxin antiserum. Tetanus toxin also blocks the rearrangement of intramembrane particles at plasma membrane of poisoned synaptosomes, specifically the decrease of small (≤ 9.5 nm diameter) intramembranous particles at the protoplasmic hemimembrane leaflet and the increase of large ( > 9.5 nm diameter) intramembrane particles at the external hemimembrane leaflet induced by potassium stimulation. These results suggest that intramembrane particle rearrangement could be related to acetylcholine secretion.

Applications of freeze-fracture replication to problems in materials and colloid science.

Understanding the relationship between the molecular structure and the macroscopic properties of polymer solutions and gels, oil-water-surfactant emulsions, lyotropic and thermotropic liquid crystals, colloidal dispersions, detergents, and other such "microstructured fluids" is essential to the optimal use of these commercially important materials. Modern rapid-freezing methods followed by freeze-fracture replication techniques are ideally suited to allow the direct visualization of the three-dimensional structure of the particles or units that make up the dispersion, while simultaneously revealing their orientation and distribution with molecular resolution. This paper reviews the necessary experimental conditions required to successfully exploit the freeze-fracture technique as it applies to microstructured fluid systems. The benefits and limitations of structural studies by freeze-fracture techniques as opposed to the more commonly used light, X-ray, and neutron-scattering methods are discussed. Freeze-fracture replicas can also be imaged by scanning tunneling microscopy to reveal directly three-dimensional fracture contours with improved resolution.

Freeze-fracture investigation of the red pulp of human spleen.

This study concerns the morphology of the human spleen in freeze-fracture replicas and compares this with the findings in ultrathin sections. The material investigated consisted of two spleens resected at gastrectomy and one resected because of splenomegaly in a case of hairy cell leukaemia. The current concepts concerning the ultrastructure of the spleen were generally confirmed with the freeze-fracture technique. The sinus walls were found, as expected, to consist of closely fitting endothelial cells, which were identifiable in freeze-fracture replicas by numerous caveolae of the cell membrane. Contrary to the opinion upheld in the literature, the sinus endothelial cells were occasionally found to be connected by desmosomes or maculae adhaerentes. Corresponding to the finding of desmosomes in ultrathin sections, focal collections of intramembranous particles were observed in freeze-fracture replicas and a positive immunohistochemical reaction for desmoplakin in the sinuses was found at the light microscopic level. The view generally held in the literature that sinus endothelial cells can exhibit tight junctions was not confirmed. However, such junctions were found between vascular endothelial cells. The ring fibres of the sinuses, which are closely connected to the sinus endothelial cells through contractile fibres, apparently have various functions. Firstly, they contribute towards maintaining mechanical stability. Secondly, they represent basement membranes through which exchange occurs between the sinus endothelial cells and their surroundings. This is indicated by the caveolae and vesicles that are often found here in large numbers and in focal collections. Hairy cells exhibit no features in freeze-fracture replicas to suggest a cytogenetic relationship to interdigitating reticulum cells.

A study of substitutional disorder phenomena in Y 2Fe 17C x (0≤x≤1.6) by high energy electron diffraction

The ultrastructural organization of nuclear matrix, purified from intact or membrane-denuded rat liver nuclei, has been analysed by means of freeze-fracturing technique. This method avoids dehydration and embedding which, in conventional thin sectioning, partly distort or mask the matrix ultrastructure. The various matrix components, and mainly the peripheral lamina and the inner network revealed complex arrangements undetectable with conventional techniques. Morphometric analyses performed with a Texture Analysis System (TAS) Leitz, allowed to obtain precise information on the matrix constituents, based on the histograms of their size distribution. These textural characteristics have been utilized in order to identify, by means of a particular computer programme, the putative matrix localization within intact freeze-fractured nuclei.

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles.

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.

Application of freeze-fracture technique for analyzing the structure of lubricant greases

The freeze-fracture technique has been developed to study solid-liquid dispersions. In this technique, a sample of dispersion is frozen, then fractured. A replica of the fracture surface is made and observed using a scanning electron microscope. The physical form of the solid thickener in dispersion can be studied, and the structure of the thickener can be revealed as well as a combination of rupture properties of the solid and the frozen liquid. The application of this technique is on lubricant greases. Greases are two-phase dispersed systems composed of mineral oil gelled with solid particles of soap. Three lithium 12-hydroxystearate greases are examined. Two different kinds of washing of the replica are used, the first eliminating the liquid phase. The configuration of soap particles and the network formed by them are observed, and artifacts such as twisting soap fibers are avoided. The second washing eliminates the solid and liquid phase. Examination of the fracture surface of the soap provides information on crystalline arrangement. The observed structures by freeze fracture are related to the thermal treatments during grease manufacture. The results are compared with the observations obtained from an electron transmission microscope analysis after preparation by the usual dispersion technique.

A NEW MODEL FOR CAPILLARY FILTRATION BASED ON RECENT ELECTRON MICROSCOPIC STUDIES OF ENDOTHELIAL JUNCTIONS

Existing models to predict the filtration and permeability of hydrophilic solutes in the capillary interendothelial cleft have been based largely on one-dimensional models of channels with constrictions that are represented by long rectangular slits of 6 to 8 nm gap width that run along the length of the junction and do not interact with one another [1,7–10]. It has been commonly assumed based on these simple models that the water and solute fluxes are proportional to the area of the junction that is open. Recent electron microscopic studies [7, 11] using freeze fracture and ultrathin serial sectioning techniques indicate that the pores in the tight junctions might be numerous much narrower disruptions in the protein strands of 10 to 20 nm length. A new model is proposed in which the pores are short periodically distributed equivalent cylindrical holes in the junctional strands that have dimensions comparable to individual missing proteins. Since these pores have spacings which are small compared to the d...

Comparative study of neuronal and glial gap‐junctions in crayfish nerve cords

Electrical synapses (neuronal gap-junctions) and glial gap-junctions were compared by using thin sectioning, freeze fracturing, and negative staining techniques. Neuronal and glial gap-junctions differed in the length of the extracellular domains of the channels, in the presence of a cytoskeleton associated to neuronal gap-junctions, and in their unit cell dimensions. The difference in length of the channels and the fact that both glial and neuronal gap-junctions had the same particle diameter suggest that the proteins forming glial and neuronal gap-junctions might have different molecular weights. The cytoskeleton associated to neuronal gap-junctions consisted of a beaded layer of densities located parallel to the membrane in the synaptic regions. Synaptic vesicles associated to neuronal gap-junctions were attached to this cytoskeleton, which was in turn anchored to the synaptic membrane through densities about 20 nm apart, a spacing similar to the neuronal unit cell dimension. These results suggest that the cytoskeleton might be responsible for the association of vesicles to neuronal gap-junctions and for maintaining the crystalline appearance of neuronal gap-junctions in situ.

Changes in the distribution of intramembranous particles and filipin-reactive membrane sterols during in vitro capacitation of golden hamster spermatozoa.

Membrane alterations accompanying in vitro capacitation of hamster spermatozoa were examined using the freeze-fracture technique with or without use of filipin, a sterol-binding probe. In the spermatozoa prior to or at 10 min after start of incubation in capacitating medium, large (about 11 nm) and small (8-9 nm) intramembranous particles (IMPs) were present in the periacrosomal region of the sperm plasma membrane (PAPM). Filipin sterol complexes (FSCs) were densely (about 500/micron 2) distributed in the PAPM prior to incubation. The density of FSCs in the PAPM was reduced by 70-80% of the original density by 2 hr of incubation. At the same time, small patches of IMP-free areas appeared in the plasma membrane above the equatorial and middle segments of the acrosome. By the end of 3 hr of incubation, the majority of small IMPs had disappeared from the PAPM. Remaining large and small IMPs tended to aggregate in the PAPM. During incubation in capacitation medium, "cords," or linear arrangements of closely packed IMPs, appeared near the posterior ring of the sperm head. These observations strongly suggest that the acrosome reaction of the hamster spermatozoa is preceded by the removal (deletion) of filipin-reactive sterols (FRSs) and the disappearance of small IMPs from the lipid bilayer of PAPM.

Rheological and electron microscopic characterization of aqueous carboxymethyl cellulose gels Part II: Visualization of the gel structure by freeze-fracturing

The spatial structure of gels of sodium carboxymethyl cellulose (NaCMC-gel) and carboxymethyl cellulose in the free acid form can be imaged in the transmission electron microscope (TEM). The freeze-fracturing technique is suitable for this. Experiments with test preparations (10 % aqueous glycerol solution) show that cooling rates during freeze-fixing are decisive for visualization, and that cooling rates can be improved substantially by using a cryojet. The increase of the cooling rate to more than 15 000 K/s makes it possible to obtain extremely fine network structures with a mesh width of 5–25 nm and with a filament thickness of 2–3 nm. The results obtained after jet-freezing show differences in the structure of the two gels: quasi-crystalline microaggregates in the HCMC-gel can be seen, and they cause an increased elasticity and opalescence of the aged HCMC-gels.