New observations on the fine structure of the liver in goldfish (Carassius auratus)

Summary A re-examination of goldfish liver was made through the use of SEM of fractured samples and TEM of ultrathin-sections and freeze-etch replicas. Several new hepatic fine structures described in the present study are morphologically similar to those reported previously in many higher vertebrates including mammals. Hepatic sinusoids of goldfish contain fenestrations which are arranged into sieve plates. Although the hepatic plates are made up of two layers of hepatocytes, the parenchymal cells of goldfish liver are morphologically similar to mammalian hepatocytes, particularly with respect to the sinusoidal surfaces which are studded with numerous microvilli. The intercellular surfaces of hepatocytes have both nexus and desmosomal junctions, similar to those found in various epithelial cells of higher vertebrates, as cell attachments and communication foci. Tight junctions are found mainly between the openings of the intracellular bile canaliculi and the intralobular bile ductules which are situated in the center of the bicellular hepatic plate.Supported in part by Grants # GM92 and ES07017     

Organogenesis of the liver in sea bream

Development of the liver in sea bream, Sparus aurata , was studied using light and electron microscopy from hatching to the age of 23 days. Histochemical reactions were used to monitor changes in lipids and glycogen during this period. During the strictly endotrophic prelarval phase from hatching until mouth-opening, the primordial liver formed by budding on the gut wall and became organized in contact with the vitelline vesicle. The initially undifferentiated cells rapidly polarized and became pyramidal in outline with their apical extremities terminating in cavities corresponding to the future bile canaliculi. Sinusoids formed centripetally. Glycogen was stored in the cytoplasm of differentiating hepatocytes. At the beginning of the larval stage the hepatocytes multiplied, bile canaliculi were completed and the sinusoids acquired their final form. Glycogen reserves decreased strongly as bile secretion started. The end of the endo-extrophic period was marked by distinct recovery of glycogen storage, synthesis of numerous lipoproteins and discharge into the sinusoids.     

鳗鲡肝脏、脾脏显微与超微结构

经光镜和电镜观察发现：鳗鲡肝脏的肝小叶不规则。肝细胞胞质内富含多种细胞器及包含物。胆小管由２—４个肝细胞围成，相邻肝细胞间有连接复合体封闭胆小管。肝血窦为有孔型、孔处无隔膜，内有巨噬细胞。窦周隙明显，未见贮脂细胞。肝细胞向胆小管腔与窦周隙面伸出许多指状微绒毛。脾脏内白髓中淋巴细胞聚集成群，未见明显脾小结、淋巴鞘。红髓由脾索与脾窦组成，动脉分支末端（壁厚的毛细血管）可开放于红髓，无明显巨噬细胞中心。脾窦及脾小动脉内皮细胞通常为长杆状、沿血管纵向平行排列。脾窦为有孔型，孔处可见薄的隔膜。脾小动脉内皮外为２—５层平滑肌（多数为纵行）。     

Fine structure of the liver in the larval lamprey, Petromyzon marinus L.; hepatocytes and sinusoids

The ultrastructure of hepatocytes, bile canaliculi, and hepatic sinusoids of the larval lamprey, Petromyzon marinus, was examined using thin-sectioned and freeze-fractured tissues. The liver is a "tubular gland" with hepatocytes arranged in a tubular fashion around large bile canaliculi. Hepatocytes are roughly conical in shape, with their tapered apices facing a bile canalicular lumen. They possess extensive rough and smooth endoplasmic reticulum, a well-developed Golgi complex, abundant mitochondria, and varying numbers of large secondary lysosomes. Both secondary lysosomes and the Golgi complex are concentrated in the apical or peribiliary cytoplasm, indicating a possible role in bile secretion. The apical surfaces of the hepatocytes bear numerous elongate microvilli and occasional cilia, which project into the bile canaliculi. The hepatocytes are joined, apically, by junctional complexes composed of zonulae occludentes and adhaerentes. In freeze-fracture, the zonulae occludentes are of variable apicobasal depth and consist of honeycomb-like meshworks of fibrils. Spaces of variable width frequently appear in the P-face grooves, indicating that the zonulae occludentes are "leaky." Numerous communicating (gap) junctions join the hepatocytes laterally. Varying numbers of lateral microvilli project into the intercellular spaces and, basally, the plasma membrane is deeply infolded, resulting in the formation of apparently interdigitating basal processes resting upon a thin basal lamina. Sinusoids are composed of both a heavily-fenestrated, continuous endothelium, and phagocytic reticulo-endothelial (Kupffer) cells. Depsite the difference in arrangement of their hepatocytes, the mammalian and lamprey livers show similar ultrastructural features.     

Biochimica et biophysica acta,vol. 646 (1981)

Endocytosis of asialo-glycoproteins in hepatocytes is mediated by a lectin-like receptor with specificity for d-galactose. Early events of receptor-ligand interactions have been studied by ultrastructural analysis. Hepatocytes were isolated from the rat liver by collagenase perfusion and incubated with a galactosylated electron dense marker (gold-Gal-BSA, galactosylated bovine serum albumin adsorbed onto colloidal gold particles). Initial binding of gold-Gal-BSA particles occurs to receptors diffusely distributed at hepatic microvilli of the former space of Dissé. No lectin activity was found in membrane areas that had formed in situ the region of hepatic cell contact or bile canaliculi. Microaggregation of receptor-ligand complexes is seen as an early consequence of particle binding. Microaggregates contain 2–5 particles and are located outside coated pits. After prolonged incubation larger clusters are formed, these are found associated with coated membrane areas. It is concluded that at least three steps precede the uptake of galactosylated proteins by hepatocytes. These are: (i) binding of ligand at diffusely distributed binding sites; (ii) local microaggregation of receptor-ligand complexes; (iii) formation of larger clusters and association with coated pits.     

The passage of radioactive lanthanum from the biliary to the vascular system

Summary Radioactive lanthanum nitrate, an electron opaque tracer, was injected into the common bile duct of rats. Two minutes following the end of injection, samples of blood for radioactivity counts, and of liver and kidney for electron microscopic studies were taken. High levels of radioactivity found in the blood, and demonstration of lanthanum in the kidney by electron microscopy, indicated that this tracer entered the blood stream in vivo. No lanthanum was seen in the cytoplasm of liver cells, and there was no evidence of rupture in bile ducts, junctional ducts, or liver cells. Tight junctions connecting parenchymal cells and cholangiolar cells appeared well preserved. Lanthanum was seen in bile canaliculi, interspaces between liver cells, portions of the extracellular space of the liver, lumina of cholangioles and lumina of portal venules and sinusoids. It is postulated that lanthanum passed from the biliary tract, the site of injection, through the tight junction between liver cells and cholangiolar cells. It is suggested that such passage may represent a physiologic pathway, but the possibility of a chemical action of lanthanum on the tight junction can not be ruled out.This study was supported by the A. D. Williams Fund of Virginia Commonwealth University and the U. S. Veterans Administration. Radiation Counting Equipment was loaned by Dr. F. O'Foghludha, Department of Radiation Physics, Virginia Commonwealth University.     

Novel structures in secreting the androgenic gland hormone

The secretory granules in the androgenic gland of the terrestrial isopod Armadillidium vulgare, which have been indistinct for long time because of vulnerable structures, were revealed by using the rapid-freezing and freeze-substitution method. The fine structure of the androgenic gland is conspicuous by the distribution of numerous particular organelles in the cytoplasm consisting of the endoplasmic reticulum and the Golgi complex, and by having a number of highly organized structures developed between the androgenic gland cells. The structures connect to the intercellular space, which is seen as intercellular canaliculi for exporting the androgenic gland hormone. The plasma membranes near the particular structure of the intercellular canaliculi in the androgenic gland are often specialized to form cellular junctions. The secretory granules including the electron-dense materials, which are supposed to be peptides of androgenic gland hormone, are distributed beside the particular structure of the intercellular canaliculi. Some of the granules are seen to fuse with the plasma membranes. This observation suggests that, in the Armadillidium vulgare, the secretory granules containing androgenic gland hormone are transferred to the extracellular space through the intercellular canaliculi particularly developed for exporting the peptide hormone. This is the first evidence to show the secretory mechanism of the androgenic gland hormone in the Isopoda.     

Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study

When biochemical studies on the liver are interpreted, the cells of the sinusoidal area frequently receive little attention because, compared to hepatocytes, their contribution to subcellular fractions is assumed insignificant. A systematic stereological analysis of liver parenchyma was therefore performed in order to determine the distribution of organelles and membranes between hepatocytic and nonhepatocytic cells, namely endothelial, Kupffer, and fat-storing cells. The livers were fixed by vascular perfusion and the data were corrected for systematic errors dur to section thickness and compression. The extracellular space compartment includes the lumina of sinusoids (10.6%), the space of Disse (4.9%), and the bile canaliculi (0.4%). Hepatocytes constitute 78% of parenchymal volume; the nonhepatocytes account for 6.3% and consist of 2.8% endothelial cells, 2.1% Kupffer cells, and 1.4% fat- storing cells. The nonhepatocytes contribute 55% of the volume of lipid droplets in the liver, 43% of the lysosomes, and 1.2% of the mitochondria. Although the nonhepatocytes account for only 8% of the total surface area of parenchymal membranes, they contain 26.5% of all the plasma membranes, 32.4% of the lysosomal membranes, 15.1% of the Golgi apparatus 6.4% of the endoplasmic reticulum, and 2.4% of the mitochondrial membranes. The data demonstrate the extent to which nonhepatocytic organelles can potentially contaminate subcellular fractions used for biochemical studies. Particularly important for the interpretation of studies on lysosomes, plasma membrane, and Golgi apparatus is the finding that an appreciable part of these organelles may be derived from cell types other than hepatocytes.     

The ultrastructural localization of antigen B10 of the hepatocyte plasma membrane in mouse hepatomas

The ultrastructure of the murine hepatocyte plasma membrane antigen (Ag B10) was studied by immunoelectron microscopy in 5 spontaneous and 3 chemical-induced hepatomas. Ag B10 was associated with plasmalemma of bile canaliculi and membrane of microvilli as in normal liver. Sometimes it was connected with plasmalemma of lateral domain of tumor cells. The availability of Ag B10 in the matrix of bile canaliculi and within microvilli was shown.     

Microanatomy of the digestive system of Supachai's caecilian,Ichthyophis supachaii Taylor, 1960 (Amphibia: Gymnophiona)

The gross anatomy and microanatomy of the digestive system of Ichthyophis supachaii were investigated. The microscopic structures of the digestive system are similar to those in other caecilians. Functional and developing teeth are present in adults. The tongue contains the genioglossus muscle. The digestive tract is elongated and consists of the mucosa, submucosa, muscularis and adventitia/serosa. The oesophagus contains longitudinal folds and lacks oesophageal glands. We report for the first time the caecilian gastric rugae and specific localization of oxynticopeptic cells in the anterior gastric region. The intestinal folds are exclusively present in the anterior intestinal region. The liver comprises 30–40 incomplete hepatic lobes, lying in an imbricate manner. Each lobe is enveloped by haematopoietic tissue that produces and delivers blood cells into sinusoids. Hepatic parenchyma is organized into anastomosing, two‐cell‐thick plates, having sinusoids at the basal domain and bile canaliculi at the apical domain of hepatocytes. Pigment cells are scattered inside sinusoids. The pancreas contains pancreatic acini interspersed with islets of Langerhans. The gallbladder proper is thin and continuous with the cystic duct wall. Neutral and carboxylated acid mucosubstances are secreted along the digestive tract, while sulphated mucosubstances are not produced by the stomach and anterior intestinal regions.     

Appearance of clasmatosis in the normal and pathological liver

It was established that clasmatosis of cytoplasmic fragments toward sinusoids occurred under normal physiological conditions (embryogenesis of chick liver, the liver of starved grass carp and silver carp) and pathological conditions (disturbance of rat hepato-intestinal circulation). The clasmosomes of rat and chick liver cells contained free ribosomes and small vesicles while those in the liver of starved fish consisted of glycogen. It was also shown that mitochondria with the signs of complete clasmatosis appeared in the hepatocyte cytoplasm immediately after the beginning of intensive bile secretion to the bile canaliculus (in liver cells of rat and chick embryo and in those of frogs after complete metamorphosis). Such mitochondria were partially disintegrated and were located near the bile canaliculi. It is assumed that clasmatosis of the fragments of the liver cell cytoplasm or mitochondria takes places where it is necessary to rapidly supply the body or cell with some products of metabolism or to remove something from the cell as is the case with erythroblasts, i. e. clasmatosis is one of the mechanisms of the adaptation of the cell and its organelles to changes in the environment.     

Liver ultrastructure of juvenile Atlantic salmon (Salmo salar)

Light and transmission electron microscopy of the liver of juvenile Atlantic salmon (Salmo salar) reveals a tubular arrangement of parenchymal cells, with biliary passages typically located at the center of tubules. Hepatocytes generally contain a single nucleus surrounded by a cuff of rough endoplasmic reticulum (RER), with many round to elongate mitochondria associated with the perinuclear RER. Whereas glycogen deposits are common and usually lie at the cell periphery, parenchymal cells seldom contain lipid droplets. Golgi complexes and heterogeneous dense bodies also occur in many hepatocytes, often in close proximity to bile canaliculi. Numerous microvilli from hepatocytes extend into the subendothelial space of Disse, which is also the location of stellate fat-storing cells. Interhepatocytic macrophages, sometimes containing prominent phagolysosomes and residual bodies, are common in the liver. The intrahepatic biliary system consists of intercellular canaliculi, bile pre-ductules, ductules, and ducts. In contrast to some other teleosts, the liver of the Atlantic salmon contains no intracellular bile canaliculi or Kupffer cells. The hepatic endothelium, arterioles, and perivenous regions are also described.     

Liver ultrastructure and a new cell type in the Japanese newt, Cynops pyrrhogaster.

The liver of the Japanese newt, Cynops pyrrhogaster, has been investigated using light, scanning, and transmission electron microscopy. Hepatic parenchyma was composed of clusters and cords or tubules of polyhedral cells separated by a sinusoidal net. Hepatocytes had spherical, euchromatic nuclei with one or more nucleoli and stacked mitochondria with sparse cristae and dense bodies. Rough endoplasmic reticula formed peribiliary stacks and diffusely scattered vesicles and tubules. Smooth endoplasmic reticula were more pronounced in glycogen-rich hepatocytes. Most hepatocytes contained peroxisomes, Golgi complexes and large numbers of fat droplets within the cytoplasm along with glycogen. Some cells were mainly glycogen-storing and contained few or no fat droplets. A special feature of the newt liver was biliary atresia. Bile canaliculi had short, stout microvilli which were entirely atretic in some canaliculi. Canaliculi were sealed off by junctional complexes including zonulae occludentes and maculae adherentes. The latter showed extraordinary wider desmosomal gaps in the vicinity of the atretic bile canaliculi. The sinusoid wall was non-distinctive and contained fenestrated endothelial cells connected to Kupffer cells by zonulae occludentes. A distinctive new cell type (OG cell) was observed in the newt liver. These cells were found individually or in small clusters in proximity with the sinusoidal surfaces. They had small nuclei, a paucity of cytoplasmic organelles, but numerous, unique, osmiophilic granules of two distinct types. Less numerous Type I granules contained homogeneous electron-dense material, and a predominant Type II granule contained circumferentially arranged subparticulation. Granules of both types were detected within the cytoplasm of endothelial cells and within sinusoids together with blood elements. The function of this secretory type cell remains obscure, though it may represent a stage of melanophore.     

Binding, uptake, and transcytosis of ligands for mannose-specific receptors in rat liver: an electron microscopic study

We have investigated the initial distribution of mannose-specific binding sites in rat liver as well as the uptake and transcytosis pathways of ligands for this receptor in in situ and in vivo experiments. As ligands we used mannan adsorbed onto colloidal gold particles of sizes 5, 17, and 35 nm (Man-Au5, Man-Au17, or Man-Au35). The in situ binding pattern of Man-Au5 in the prefixed liver is identical to the one described earlier for galactose-exposing ligands in the same organ. With the exception of the binding by hepatocytes, where only scarce binding of Man-Au5 was observed, ligands were found adhering in a preclustered pattern all over the cell surface of liver macrophages and binding in aggregates over the coated pits of endothelial cells. In double-labeling experiments different particle sizes were used for glycoproteins with terminal mannosyl or galactosyl residues. This simultaneous localization of the two binding activities revealed that on endothelial cells the two activities are always found to be present in the same coated pit. On liver macrophages the clustered binding occurred at different membrane areas. Uptake and transcytosis of Man-Au5, 17, 35 were studied after their injection into the tail vein. Three and fifteen minutes after injection most of the Man-Au5 and all of Man-Au17 or Man-Au35 was found in sinusoidal liver cells, i.e., macrophages and endothelial cells. One hour after injection, endocytosed ligand is redistributed from large--presumably lysosomal--vacuoles to small noncoated vesicles that are localized predominantly near the space of Dissé. Between 1 and 40 h after injection, ligands of all sizes are transcytosed and found in the hepatocytes. No ligand accumulation is observed in hepatocytes as an indirect indication for secretion into bile. With this investigation we give evidence for transcytotic activity not only of liver endothelium but also of the resident liver macrophages.     

Plasma membranes of the rat liver. Isolation and enzymatic characterization of a fraction rich in bile canaliculi

A method is described for the rapid isolation of a plasma membrane fraction containing a high concentration of intact bile canaliculi from the rat liver. Isolated bile canaliculi retain most of the ultrastructural features exhibited in the intact liver cell. The final fraction contains 5''-nucleotidase activity at approximately the same concentration as that in previous preparations of plasma membranes. In the presence of 0.01 M Mg⁺⁺, 5''-nucleotidase exhibits a double pH optimum at pH values of 7.5 and 9.5. The activities of glucose-6-phosphatase and alkaline phosphatase are present in low amounts. Cytochrome P-450 is not detectable. Na⁺-K⁺-activation of ATPase is observed to the extent of 20–36% in about half of the assays. The availability of a method for preparation of intact bile canaliculi should prove useful for studying the biochemical events associated with the transport of bile constituents into canaliculi.     

Electron microscopic observations of young rat liver

Summary Electron microscopic observations on normal liver tissue of four-day-old rats reveal the presence of numerous lamellar structures (lamellar bodies). These can be contained within parenchymal cells or in bile canaliculi, Disse's space, and in the lumen of blood sinusoids. Such bodies can also be found in Kupffer and endothelial cells.The lamellar bodies within hepatic cells are generally seen in very intimate relation to glycogen particles and lipide droplets, but in some to agranular endoplasmic reticulum and Golgi membranes as well.On the basis of this intimate relation to intracellular glycogen granules and lipide droplets, it is presumed that lamellar bodies represent a special intermediate stage in carbohydrate and lipide metabolism.Discontinuities in the endothelial layer of intrahepatic sinusoids are described.This work was supported in part by a N.A.T.O. research fellowship of the Consiglio Nazionale delle Ricerche, Roma.Assistant Professor in the Department of Veterinary Anatomy, Histology and Embryology (Dir.: Prof. A. de Girolamo), University of Naples, Naples, Italy.     

Scanning electron microscopy of normal rat liver: the surface structure of its cells and tissue components.

Scanning electron microscopy (SEM) allows the surface ultrastructure of intrahepatic cells and other tissue components of liver to be delineated. Excellent depth of focus of the SEM makes it possible to visualize surfaces of intact cells in their native configurations. This report details the surface characteristics and inter-relationships of hepatocytes and hepatic plates, sinusoidal endothelial cells and sinusoids, presumed Kupffer cells, vessels, bile ducts, connective tissue, and the capsule of rat liver. Hepatocytes present three structurally distinctive faces--the intercellular face containing flat surfaces and bile canaliculus, the sinusoidal face, and the connective tissue face which abuts portal tracts and hepatic veins. Sinusoidal endothelium is penetrated by large (1 to 3 mum) and small (0.1 mum) fenestrae, the latter occurring in clusters of up to 50. The width of bile canaliculi and distribution of large fenestrae vary proximodistally along hepatic plate or sinusoid. The cells of portal bile ductules contain microvilli located in linear rows and sparse cilia. Endothelium of hepatic artery and of portal vein is sparsely fenestrated.     

Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride. A study using electron spectroscopic imaging

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED100; ED = effective dose) and a toxic (LD25; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.     

The effects of dexamethasone on metabolic activity of hepatocytes in primary monolayer culture

The effects of dexamethasone on multiple metabolic functions of adult rat hepatocytes in monolayer culture were studied. Adult rat liver parenchymal cells were isolated by collagenase perfusion and cultured as a primary monolayer in HI/WO/BA, a serum free, completely defined, synthetic culture medium. Cells inoculated into the culture medium formed a monolayer within 24 hr. Electron microscopy showed that the cells in primary culture had a fine structure identical to liver parenchymal cells in vivo, including the observation of desmosomes and bile canaliculi in intercellular space. There was significant gluconeogenesis by the cells 24 hr postinoculation but it had decreased markedly by 48 hr. There was a marked induction of tyrosine aminotransferase (TAT) by dexamethasone, which was maintained for up to 72 hr postinoculation of cells. The transport of alpha-aminoisobutyric acid into the cells in monolayer culture was stimulated by dexamethasone and was dependent on the concentration of dexamethasone. Albumin synthesis and secretion by the cells was measured by a quantitative electroimmunoassay. Albumin production was shown to increase linearly over an incubation period of 24 to 48 hr postinoculation. Dexamethasone depressed the albumin synthesis. The effects of dexamethasone are slow, and at times require more than 6 hr to show variation from the control, indicating that dexamethasone is not a single controlling hormone. Possibly it functions in a cooperative and coordinating role in the regulation of cell metabolism.