Confocal laser scanning light microscopy of the extra-thecal epithelia of undecalcified scleractinian corals

The extra-thecal epithelia of cryofixed undecalcified, freeze-substituted polyps of the scleractinian corals Galaxea fascicularis and Tubastrea faulkneri and axial and basal polyps of Acropora formosa have been examined, in anhydrously prepared thick slices, by confocal laser scanning light microscopy. The avoidance of chemical fixation and decalcification makes it possible to determine whether previously seen structures are real or artefactual products of swelling, shrinkage and distortion. All of the epithelia of all the corals examined are characterised by well defined intercellular spaces. Mucocytes are present in all cell layers in Galaxea and Tubastrea but are not present in any cell layers in the axial polyp of Acropora although they are abundant in the oral ectoderm of the basal polyps in this coral. Zooxanthellae are absent in Tubastrea, the epithelia of the exert septa of Galaxea and the axial polyp of Acropora. The calicoblastic ectoderm is generally composed of thin squamous cells with large intercellular spaces. At rapidly calcifying regions such as the tips of the exert septa of Galaxea, the calicoblastic cells are elongated with extensive arborisation of the basal regions of the cells. They are separated by large intercellular spaces and contain numerous fluorescent granules. The apical regions of these cells appear to be closely applied to the surface of the skeleton. There is no evidence of a space between the apical region of the calicoblastic cells and the skeleton.     

Calcium associated with a fibrillar organic matrix in the scleractinian coral Galaxea fascicularis

Summary.  Field emission scanning electron microscopy of frozen-hydrated preparations of the scleractinian coral Galaxea fascicularis revealed organic fibrils which have a diameter of 26 nm and are located between calicoblastic ectodermal cells and the underlying CaCO₃ skeleton. Small (37 nm in diameter) nodular structures observed upon this fibrillar organic material possibly correspond to localised Ca-rich regions detected throughout the calcifying interfacial region of freeze-substituted preparations by X-ray microanalysis. We propose that these Ca-rich regions associated with the organic material are nascent crystals of CaCO₃. Significant amounts of S were also detected throughout the calcifying interfacial region, further verifying the likely presence of organic material. However, the bulk of this S is unlikely to be derived from mucocytes within the calicoblastic ectoderm. It is suggested that in the scleractinian coral G. fascicularis, nodular crystals of CaCO₃ establish upon a fibrillar, S-containing, organic matrix within small but distinct extracellular pockets formed between calicoblastic ectodermal cells and skeleton. This arrangement conforms with the criteria necessary for biomineralisation and with the long-held theory that organic matrices may act as templates for crystal formation and growth in biological mineralising systems. Received April 30, 2002; accepted September 11, 2002; published online March 11, 2003     

Morphological studies of the soft tissues involved in skeletal dissolution in the coralFungia fungites

Light and transmission electron microscopy were used to study mechanisms involved in the separation of the disc from the stalk in juvenileFungia fungites (Scleractinia, Fungiidae). Separation occurs because the skeleton is weakened by dissolution across a distinct plane at the junction of the stalk and disc. The tissue layer adjacent to the skeleton in the stalk was composed of typical, squamose, calicoblastic cells. In contrast, calicoblastic cells in the region of skeletal dissolution were tall and columnar. They contained many microvilli, abundant mitochondria and several different types of vesicles. It is assumed that these calicoblastic cells are actively involved in skeletal dissolution.     

Acid polysaccharides in the skeletal matrix and calicoblastic epithelium of the stony coral Mycetophyllia reesi

Like many corals the skeletal organic matrix and associated epithelium of Mycetophyllia reesi is physico-chemically unstable to preparative procedures for electron microscopy. Ethanol cryofracture of mineralized and demineralized material is accompanied by delamination of tissue and skeleton. Filamentous algae occur in the interface and account for some but not all of the separation artifact. Transmission microscopy accompanied by decalcification requires embedment in glycerol jelly to preserve the skeletal organic matrix. Even then, the matrix is not fixed and is not retained within the gel using standard double fixation with or without tannic acid as an additive. Ruthenium red, in combination with osmium, prevents the matrix from physical disruption, although positional artifacts relative to the calicoblastic epithelium are still evident. Inclusion of other glycan precipitating agents in the fixative sequence (Alcian blue, iron diamine or the detergent cetylpyridinium chloride) are more useful in preserving an acid polysaccharide-rich, fibrillar, extracellular matrix after demineralization. This material is not observed in SEM preparations. The calicoblast cells appear to be the source of this extracellular material that also appears to contribute to the composition of the mineralizing matrix. Moreover, a hyaluronan-like substance appears to play a significant role in matrix structure as suggested by its degradation by hyaluronidase.     

Observations of the tissue-skeleton interface in the scleractinian coral <Emphasis Type="Italic">Stylophora pistillata</Emphasis>

Recent micro-analytical studies of coral skeletons have led to the discovery that the effects of biology on the skeletal chemical and isotopic composition are not uniform over the skeleton. The aim of the present work was to provide histological observations of the coral tissue at the interface with the skeleton, using Stylophora pistillata as a model, and to discuss these observations in the context of skeletal ultra-structural organization and composition. Several important observations are reported: (1) At all scales of observation, there was a precise morphological correspondence between the tissues and the skeleton. The morphological features of the calicoblastic ectoderm correspond exactly to the shape of individual crystal fiber bundles in the underlying skeleton, indicating that the calicoblastic cell layer is in direct physical contact with the skeletal surface. This is consistent with the previously observed chemical and isotopic composition of the ultra-structural components in the skeleton. (2) The distribution and density of desmocyte cells, which anchor the calicoblastic ectoderm to the skeletal surface, vary spatially and temporally during skeletal growth. (3) The tissue above the coenosteal spines lack endoderm and consists only of ectodermal cell-layers separated by mesoglea. These findings have important implications for models of vital effects in coral skeletal chemistry and isotope composition.     

Comprehensive characterization of skeletal tissue growth anomalies of the finger coral Porites compressa

The scleractinian finger coral Porites compressa has been documented to develop raised growth anomalies of unknown origin, commonly referred to as “tumors”. These skeletal tissue anomalies (STAs) are circumscribed nodule-like areas of enlarged skeleton and tissue with fewer polyps and zooxanthellae than adjacent tissue. A field survey of the STA prevalence in Oahu, Kaneohe Bay, Hawaii, was complemented by laboratory analysis to reveal biochemical, histological and skeletal differences between anomalous and reference tissue. MutY, Hsp90a1, GRP75 and metallothionein, proteins known to be up-regulated in hyperplastic tissues, were over expressed in the STAs compared to adjacent normal-appearing and reference tissues. Histological analysis was further accompanied by elemental and micro-structural analyses of skeleton. Anomalous skeleton was of similar aragonite composition to adjacent skeleton but more porous as evidenced by an increased rate of vertical extension without thickening. Polyp structure was retained throughout the lesion, but abnormal polyps were hypertrophied, with increased mass of aboral tissue lining the skeleton, and thickened areas of skeletogenic calicoblastic epithelium along the basal floor. The latter were highly metabolically active and infiltrated with chromophore cells. These observations qualify the STAs as hyperplasia and are the first report in poritid corals of chromophore infiltration processes in active calicoblastic epithelium areas.     

Uptake of horseradish peroxidase from CSF into the choroid plexus of the rat,with special reference to transepithelial transport

Summary Protein uptake from cerebral ventricles into the epithelium of the choroid plexus, and transport across the epithelium were studied ultrastructurally in rats. Horseradish peroxidase (HRP, MW 40,000) was used as protein tracer. Steady-state ventriculo-cisternal perfusion with subatmospheric pressure (-10cm of water) in the ventricular system was applied. HRP dissolved in artificial CSF was perfused from the lateral ventricles to cisterna magna for various times, and ventriculo-cisternal perfusion, vascular perfusion or immersion fixation with a formaldehyde-glutaraldehyde solution was performed.Coated micropinocytic vesicles containing HRP were seen both connected with the apical, lateral and basal epithelial surface and within the cells. Heavily HRP-labeled vesicles were often fused with the lining membrane of slightly labeled or unlabeled intercellular spaces. Since the apical tight junctions of the epithelium never appeared open or never contained HRP in the spaces between the fusion points, and since the intercellular spaces between adjacent epithelial cells below the junctions only infrequently contained tracer after 5 min, by increasing amounts after 15–60 min of HRP perfusion, a vesicular transport of HRP from the apical epithelial surface to the intercellular spaces, bypassing the tight junctions, is suggested.In addition to the transepithelial transport, micropinocytic vesicles also transported HRP to the lysosomal apparatus of the epithelial cells. With increasing length of exposure to HRP, a sequence of HRP-labeled structures could be evaluated, from slightly labeled apical vacuoles and multivesicular bodies to very heavily labeled dense bodies.     

Dense-core vesicles and non-synaptic exocytosis in the central body of the crayfish brain

Summary The central body in the median protocerebrum of the brain of the crayfish Cherax destructor is a distinctive area of dense neuropile, the nerve fibres of which contain three main types of vesicles: electronlucent vesicles (diameter 35 nm), dense-core vesicles (diameter 64 nm), and large structured dense-core vesicles (diameter 98 nm, maximum 170 nm). Different vesicle types were found together in the same neurons. Electronlucent vesicles were seen at presynaptic sites and rarely observed in the state of exocytosis. Exocytosis of densecore and structured dense-core vesicles was a regular feature on non-synaptic release sites either close to, or at some distance from pre- and subsynaptic sites. Non-synaptic exocytotic sites are more often observed than chemical synapses. Different forms of exocytosis seen at non-synaptic sites included the release of single densecore vesicles, packets of dense-core vesicles, and rows of dense-core vesicles lined up along cell membranes and around fibre invaginations. Swelling and the enhanced electron density of extracellular non-synaptic spaces may mark the positions of prior exocytotic events. In vitro treatment of the brain with tannic acid buffer solution followed by conventional double fixation resulted in the augmentation of non-synaptic exocytosis. Electron microscopy of proctolin- and serotonin-immunoreactive nerve fibres shows them to contain dense-core and electron-lucent vesicles and to be surrounded by many unlabelled profiles similarly laden with dense-core vesicles and electron-lucent vesicles, indicating the presence of other, not yet identified, neuroactive compounds.     

The calcifying interface in a stony coral primary polyp: An interplay between seawater and an extracellular calcifying space

Stony coral exoskeletons build the foundation for the most biologically diverse marine ecosystems on Earth, coral reefs, which face major threats due to many anthropogenic–related stressors. Therefore, understanding coral biomineralization mechanisms is crucial for coral reef management in the coming decades and for using coral skeletons in geochemical studies. This study combines in–vivo imaging with cryo-electron microscopy and cryo–elemental mapping to gain novel insights into the biological microenvironment and the ion pathways that facilitate biomineralization in primary polyps of the stony coral Stylophora pistillata. We document increased tissue permeability in the primary polyp and a highly dispersed cell packing in the tissue directly responsible for producing the coral skeleton. This tissue arrangement may facilitate the intimate involvement of seawater at the mineralization site, also documented here. We further observe an extensive filopodial network containing carbon-rich vesicles extruding from some of the calicoblastic cells. Single-cell RNA-Sequencing data interrogation supports these morphological observations by showing higher expression of genes involved in filopodia and vesicle structure and function in the calicoblastic cells. These observations provide a new conceptual framework for resolving the ion pathway from the external seawater to the tissue-mineral interface in stony coral biomineralization processes.     

REGULAR STRUCTURES IN MEMBRANES : I. Membranes in the Endocytic Complex of Ileal Epithelial Cells

An "apical endocytic complex" in the ileal lining cells of suckling rats is described. The complex consists of a continuous network of membrane-limited tubules which originate as invaginations of the apical plasma membrane at the base of the microvilli, some associated vesicles, and a giant vacuole. The lumenal surface of this tubular network of membranes and associated vesicles is covered with a regular repeating particulate structure. The repeating unit is an ~7.5-nm diameter particle which has a distinct subunit structure composed of possibly nine smaller particles each ~3 nm in diameter. The ~7.5-nm diameter particles are joined together with a center-to-center separation of ~15 nm to form long rows. These linear aggregates, when arranged laterally, give rise to several square and oblique two-dimensional lattice arrangements of the particles which cover the surface of the membrane. Whether a square or oblique lattice is generated depends on the center-to-center separation of the rows and on the relative displacement of the particles in adjacent rows. Four membrane faces are revealed by fracturing frozen membranes of the apical tubules and vesicles: two complementary inner membrane faces exposed by the fracturing process and the lumenal and cytoplasmic membrane surfaces revealed by etching. The outer membrane face reveals a distinct array of membrane particles. This array also sometimes can be seen on the outer (B) fracture face and is sometimes faintly visible on the inner (A) fracture face. Combined data from sectioned, negatively stained, and freeze-etched preparations indicate that this regular particulate structure is a specialization that is primarily localized in the outer half of the membrane mainly in the outer leaflet.     

Electron microscopic research on the mechanism of intestinal secretion

Electron microscopic investigation of the rat small intestine revealed a great number of vesicles 50-75 nm in diameter with enterocyte microvilli. The number of vesicles increased with the increase of digestive activity in the small intestine. Vesicles were formed by gemmation of enterocyte microvilli from the lateral membrane in contraction of microvillous actin skeleton. Simultaneously with the production of exocytotic vesicles, the formation of pinocytotic vesicles in the base of microvilli was observed. There is a supposition that the vesicle gemmation is a natural process of the intestinal secretion to fulfil numerous important function: it promotes the penetration of enterocyte hydrolases into the parietal layer; equilibrates an increase in the enterocyte volume during absorption. This is a possible way of translocation of synthesized enzymes into the cytoplasm and of transport proteins on the apical surface of epithelial cells.     

Transtubular transport of proteins in rabbit proximal tubules

The purpose of the present experiments was to study possible different pathways of intracellular transport of proteins after luminal and basolateral uptake in isolated rabbit proximal tubules. Tubules were exposed to cationized ferritin (CF) in the perfusion fluid and horseradish peroxidase (HRP) in the bath simultaneously or to HRP in the bath alone for 30 min. The peritubular fluid (bath) and perfusion fluid were then exchanged and the tubules either fixed immediately or allowed to function during chase-periods for 10, 20, 30, or 60 min before fixation to follow the migration of the proteins through the cells. The proteins were to a large extent found separated in different vacuoles and lysosomes at all time periods studied, indicating separate pathways after uptake via the luminal and basolateral membranes respectively. About 0.5% of the CF taken up by the cells was transported through the cells and became located in the intercellular spaces. HRP was transported from the peritubular fluid to the apical cytoplasm of the tubules indicated by a gradual accumulation of small HRP-containing vesicles, first in the basal part of the cells and then in the apical cytoplasm. In tubules perfused with both CF and HRP in the perfusate, the CF and HRP were found together in apical vacuoles and lysosomes. After perfusion with HRP alone, this tracer was found in similar large vacuoles and lysosomes in the apical cytoplasm, in contrast to the small HRP-filled vacuoles seen after uptake from the bath.     

The number of secretory vesicles remains unchanged following exocytosis.

Earlier studies using electron microscopy demonstrate that there is no loss of secretory vesicles following exocytosis. Depletion however, of vesicular contents resulting in the formation of empty or partially empty vesicles is seen in electron micrographs, post exocytosis, in a variety of cells. Our studies using atomic force microscopy (AFM) reveal that following stimulation of secretion, live pancreatic acinar cells having 100-180 nm in diameter fusion pores located at the apical plasma membrane, dilate only 25-35% during exocytosis. Since secretory vesicles in pancreatic acinar cells range in size from 200 nm to 1200 nm in diameter, their total incorporation at the fusion pore, would distend the structure much more then what is observed. These earlier results prompted the current study to determine secretory vesicle dynamics in live pancreatic acinar cells following exocytosis. AFM studies on live acinar cells reveal no loss of secretory vesicle number following exocytosis. Parallel studies using electron microscopy, further confirmed our AFM results. These studies demonstrate that following stimulation of secretion, membrane-bound secretory vesicles transiently dock and fuse to release vesicular contents.     

Structure of the novel membrane-coating material in proton-secreting epithelial cells and identification as an H+ATPase

Specialized proton-secreting cells known collectively as mitochondria-rich cells are found in a variety of transporting epithelia, including the kidney collecting duct (intercalated cells) and toad and turtle urinary bladders. These cells contain a population of characteristic tubulovesicles that are believed to be involved in the shuttling of proton pumps (H+ATPase) to and from the plasma membrane. These transporting vesicles have a dense, studlike material coating the cytoplasmic face of their limiting membranes and similar studs are also found beneath parts of the plasma membrane. We have recently shown that this membrane coat does not contain clathrin. The present study was performed to determine the structure of this coat in rapidly frozen and freeze-dried tissue, and to determine whether the coat contains a major membrane protein transported by these vesicles, a proton pumping H+ATPase. The structure of the coat was examined in proton-secreting, mitochondria-rich cells from toad urinary bladder epithelium by rapidly freezing portions of apical membrane and associated cytoplasm that were sheared away from the remainder of the cell using polylysine-coated coverslips. Regions of the underside of these apical membranes as large as 0.2 micron2 were decorated by studlike projections that were arranged into regular hexagonal arrays. Individual studs had a diameter of 9.5 nm and appeared to be composed of multiple subunits arranged around a central depression, possibly representing a channel. The studs had a density of approximately 16,800 per micron2 of membrane. Similar arrays of studs were also found on vesicles trapped in the residual band of cytoplasm that remained attached to the underside of the plasma membrane, but none were seen in adjacent granular cells. To determine whether these arrays of studs contained H+ATPase molecules, we examined a preparation of affinity-purified bovine medullary H+ATPase, using the same technique, after incorporation of the protein eluted from a monoclonal antibody affinity column into phospholipid liposomes. The affinity-purified protein was shown to be capable of ATP-dependent acidification. In such preparations, large paracrystalline arrays of studs identical in appearance to those seen in situ were found. The dimensions of the studs as well as the number per square micrometer of membrane were identical to those of toad bladder mitochondria-rich cells: 9.5 nm in diameter, 16,770 per micron2 of membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

The ultrastructure of the cardiac ganglion of the desert scorpion,Paruroctonus mesaensis (Scorpionida: Vaejovidae)

Light and electron microscopy of the pacemaker ganglion of the scorpion heart indicate that it is about 15 mm long and 50 μm in diameter and extends along the dorsal midline of the heart. The largest cell bodies (30–45 μm in diameter) occur in clusters along the length of the ganglion. The ganglion appears to be innervated with fibers from the subesophageal and first three abdominal ganglia. The cardiac ganglion is surrounded by a neurilemma and a membranous sheath. The latter is apparently derived from connective tissue cells seen outside the ganglion. Nerve fibers other than those in the neuropil areas are usually surrounded by membrane and cytoplasm of glial cells. Often there are several layers of glial membrane, forming a loose myelin. The cardiac nerves to the heart muscle are also surrounded by a neurilemma, and the axons are surrounded by glia. The motor nerves contain lucent vesicles 60–100 nm and opaque granules 120–180 nm in diameter. In the cardiac ganglion, some nerve cell bodies have complex invaginations of glial processes forming a peripheral trophospongium. In the neuropil areas, nerve cell processes are often in close apposition. The septilaminar configuration typical of gap junctions is common, with gap distances of 1–4 nm. In tissues stained with lanthanum phosphate during fixation, we found gaps with unstained connections (1–2 nm diameter) between nerve-nerve and glial-nerve cell processes. Annular or double-membrane vesicles in various stages of formation were also seen in some nerve fibers in ganglia stained with lanthanum phosphate. Nerve endings with electron-lucent vesicles 40–60 nm in diameter are abundant in the cardiac ganglion, suggesting that these contain the excitatory transmitter of intrinsic neurons of the ganglion. Less abundant are fibers with membrane-limited opaque granules, circular or oblong in shape and as much as 330 nm in their longest dimension. Also seen were some nerve endings with both vesicles and granules.     

The fine structure of nerve endings on rat thyroid follicular cells

Summary Axon profiles in thyroid glands obtained from adult male Wistar rats were studied electron-microscopically, using common and serial thin sections.Bouton profiles of nerve fibers, resembling the terminal or en passant type, often appeared closely associated with vascular smooth muscle cells via basement membranes. These structures are probably adrenergic, since they contained mainly small-core vesicles (mean diameter: 41.2 nm), in addition to a few large-core (mean diameter: 88.4 nm) and flattened vesicles.Nerve fibers containing microtubules and sometimes mitochondria and vesicles were seen lying between basement membranes and follicular cells. The incidence of nerve fiber contacts on profiles of follicular cells was 0.0177±0.0092 (S.D.). Using serial sections, follicles were seen to have up to two nerve endings, separated from the plasma membranes of the follicular cells by a gap of 22 nm. They contained mainly flattened vesicles and several large-core vesicles (mean diameter: 95.1 nm). Small-core vesicles were rarely seen in these nerve endings. Furthermore, subsurface cistern-like rough endoplasmic reticulum was found immediately under the plasma membranes of follicular cells facing membranes of nerve endings. These results suggest that the nerve fibers in contact with follicular cells are different from the adrenergic type.     

Ultrastructural aspects of the hyphal tip ofSclerotium rolfsii preserved by freeze substitution

Summary The hyphal tip ofSclerotium rolfsii was examined after fixation by freeze substitution. The Spitzenkörper consisted of a dense mass of apical vesicles and microvesicles surrounding a vesicle-free zone. Linear arrangements of microvesicles were occasionally observed within the Spitzenkörper. Abundant microfilaments were seen within the Spitzenkörper region, often in close association with apical vesicles and microvesicles. Microtubules passed through the Spitzenkörper and terminated at the plasmalemma at the extreme hyphal apex. Filasomes were mostly observed within the apical region and were in close proximity to the plasmalemma. Rough ER, mitochondria, microtubules, and vacuoles were abundant in the subapical region and were usually oriented parallel to the long axis of the hypha. Ribosomes were aligned on the outer surfaces of mitochondria. Golgi body equivalents were observed throughout the subapical region and appeared as inflated cisternae of varying shapes and electron opacities. Relationships to other basidiomycetous hyphal tip cells are discussed.Abbreviations AV apical vesicle - C Celsius - diam diameter - f filasome - G Golgi body equivalent - h hour - nm nanometer - M mitochondria - ME membranous elements; min minute - MV microvesicle - MVB multivesicular body - N nucleus - OsO₄ osmium tetroxide - R ribosome - ER endoplasmic reticulum - S Spitzenkörper - Va vacuole - m micrometer     

Ultrastructure of pinealocytes of the cotton rat,Sigmodon hispidus

Summary Fine structural features of pinealocytes of cotton rats (Sigmodon hispidus) were examined. Golgi complexes, mitochondria, endoplasmic reticulum and polysomes are usual organelles seen in the perikaryonal cytoplasm of pinealocytes. Many non-granulated vesicles (40 to 80 nm in diameter) and a few granulated vesicles (about 100 nm in diameter) are associated with the Golgi cisternae. Occasionally, the cisternae contain granular materials. The perikaryonal cytoplasm of pinealocytes is characterized by the presence of inclusion bodies. These bodies are usually round in shape, not bounded by a limiting membrane and composed of fine granular or filamentous materials of high electron-opacity, which are similar in appearance to the substance seen in the nucleolonema. Pinealocyte processes, filled with abundant non-granulated vesicles and some granulated vesicles, are mainly found within the parenchyma and occasionally in perivascular spaces.Supported in part by NSF grant no. PCM 77-05734 and NIH grant no. HD-10202 (Morphology Core)     

Effect of hydrocortisone on the ultrastructure of the small,intensely fluorescent,granule-containing cells in cultures of sympathetic ganglia of newborn rats

Summary Sympathetic chain ganglia of newborn rats were cultured in Rose chambers with or without hydrocortisone. After one week, the cultures were examined by light microscopy for formaldehyde-induced catecholamine fluorescence and by electron microscopy after fixation in 5% glutaraldehyde solution and thereafter in 1% osmium tetroxide. Hydrocortisone (10 mg/l) caused a great increase in the number of the small, intensely fluorescent (SIF) cells in the ganglion explants, and the fluorescence intensity of these cells was also increased. The SIF cells corresponded to small, granule-containing (SGC) cells in the electronmicros copic preparations, and in addition to an increase in their number there was also an increase in the size and number of granular vesicles in the presence of hydrocortisone. In control cultures the granular vesicles were round (about 100 nm in diameter) or elongated (40–150 nm in cross section and 150–250 nm in length); both types of vesicles contained electron dense cores. In hydrocortisone-containing cultures round granular vesicles up to 200 nm in diameter were also observed; the cores of these vesicles were of variable electron density. It is concluded that in tissue culture, hydrocortisone causes an increased formation of catecholamine-containing granular vesicles in SIF-SGC cells and their precursors and an increase in the number of these cells.This work was supported by grants from the National Heart Foundation, the Australian Research Grants Committee and the Sigrid Juselius Foundation.University of Melbourne Senior Research Fellow, September, 1971 – August, 1972; present address: Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki, Finland, 00170.Holder of a grant from the National Health and Medical Research Council of Australia.Sunshine Foundation and Rowden White Research Fellow in the University of Melbourne, September, 1971 – August, 1972; present address: Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki, Finland, 00170.     

Ultrastructure of the intima in WHHL and cholesterol-fed rabbit aortas prepared by ultra-rapid freezing and freeze-etching

Intima from aortas of normal Watanabe Heritable Hyperlipidemic (WHHL) and cholesterol-fed (10 days - 3 months) rabbits were examined by ultra-rapid freezing without chemical fixation followed by rotary shadow freeze-etching. The extracellular matrix in areas devoid of cells was seen in extraordinary detail and consisted of a reticulum of thick filaments, finer branching filaments, collagen fibrils, and granules of varying sizes. No lipid deposits were seen in normal intima. However, the subendothelial region of WHHL intima was filled with collagen fibrils surrounding and entwined between clusters of discrete lipid vesicles that ranged in size from 23 to 169 nm. Approximately 80% of the lipid vesicles in the WHHL rabbit intima measured between 70 and 169 nm. The lipid particles in the WHHL intima always appeared in clusters, many of which appeared to be fusing into larger size vesicles. These aggregates were clearly linked to the matrix filaments. A similar deposition of lipid particles was seen in the extracellular matrix of cholesterol-fed rabbits but in contrast to the particle size distribution of the WHHL intima, more than 75% of the particles in the cholesterol-fed intima had a diameter between 23 and 68 nm and 51% were between 23 and 45 nm. We conclude that in cell-free areas of WHHL and after only 10 days of cholesterol feeding, lipoprotein-derived lipid is present in the intima as clusters of vesicles enmeshed in the complex extracellular matrix.