During a comparative morphological study of the bile tract in several vertebrates, we observed the gallbladder morphology of various fish species (Gilloteaux et al., 1995; Oldham-Ott and Gilloteaux, 1997). To date, the most complex gallbladder epithelial morphology was observed in the stargazer (Gilloteaux et al., 2011), probably adapted to an unusual ethologic and predatory diet niche. This study was undertaken to study the morphologic peculiarities of the Torpedo marmorata gallbladder, also a predator but an Elasmobranch. It is noted that the gallbladder secretory activity resembles the complex morphologic changes observed in cases of human cholecystitis and cholelithiasis (Gilloteaux et al., 1997a, b, 2003, 2004) and in the Syrian hamster gallbladder following treatment with sex steroids (Gilloteaux et al., 1992, 1993a, b).
The gallbladder of Torpedo marmorata exhibits a mucosal surface layer of simple columnar epithelium with very tall cholecystocytes. The apical domain of each cell has few microvilli, but many mucous vesicles that are secreted by exocytosis at the cell apices. The apical regions may also elongate and undergo self-excision while shedding mucus and cell debris into the gallbladder lumen in a manner similar to that described in mammals as a result of sex steroid treatment to induce gallstones and to that found in the cholecystitis associated with cholelithiasis. Numerous small mitochondria, spherical to elongated, are distributed throughout the cells, while the nuclei are often located in the lower third of each cell. In the lower part of the cholecystocytes, large and very densely contrasted lysosomes can be found. All cells are tightly joined by junctional complexes, including long, highly contrasted desmosomes. The fibromuscular layer is made of a loose stroma with a limited muscular component and a poor blood supply. Large diameter blood vessels can only be found in the subserosal layer. It is hypothesized that the obligatorily carnivorous diet of this ureotelic fish has resulted in the evolution of a gallbladder ultrastructure resembling that found in cholecystitis but without the associated cholelithiasis. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.
MATERIALS AND METHODS
Six specimens of Torpedo marmorata (Risso 1810), an elasmobranch of the Torpedinidae family, were captured along with other representatives of 12 species of fishes outside the Banyuls Bay (France, Mediterranean Sea, Gulf of the Lion) by net trailing between 50 and 60 m of depth along a NNW-SSE line facing Cape Béart to Cape de l'Abeille (outside and at the edge of the National Biological Marine Reserve). Specimens had a 35–50 cm body length and a male to female ratio of 4:2. Following capture, all fishes were kept and transported in running sea water tanks on the boat and, about 30 min later, were placed in the laboratory aquaria of the Arago Marine Biological Station of the University of Paris where they were kept in aerated sea water aquaria for 2 days to acclimate. After decapitation, they were dissected following our previous protocol (Oldham-Ott and Gilloteaux, 1997).
From all specimens several organs, including gallbladders, livers and hearts, were excised and cut into smaller pieces to be fixed for 1.5-hr duration in 3% buffered glutaraldehyde (0.1 M sodium cacodylate) dissolved in distilled water: seawater (1v :3 v) at 4°C and by adapting protocols from Anderson and Personne (1970), Saito and Tanaka (1980), and Griffith (1981), taking into account the high tissue osmolarity (ca. 1100 mOsM) of the internal milieu of this species (Goldstein and Forster, 1971). A 30-min wash in the same buffer mixed with sea water was followed by postfixation with 2% OsO4 aqueous solution for 2-hr duration both were done at 4°C. Tissues were then washed in buffer containing 10% sucrose and dehydrated by graded alcohols.
For Scanning Electron Microscopy (SEM)
Three gallbladders and pieces of three others were critically point dried in a Polaron E3000 apparatus (Polaron/Biorad, Cambridge, MA). All the samples were coated with a 25- to 40-nm-thick gold layer and examined in a Jeol JSM-35C SEM.
For Transmission Electron Microscopy (TEM)
Three gallbladders and samples of three others were embedded in Polybed 812 epoxy resin (Polysciences, Warrington, PA). From the TEM-embedded samples, 1-μm-thick sections were stained by Toluidine blue to be examined with light microscopy (LM) (Trump et al., 1961; Hayat, 1983). From chosen areas of the LM examined slides, thin sections were collected on 100 mesh hexagonal copper grids, contrasted with uranyl acetate and lead citrate, and observed through a Jeol S100 TEM.
Dissection of the viscera showed a brownish, dark colored liver with four incompletely separated lobes, with two elongated, wing-shaped lobes, bilaterally located along the abdominal side. The liver is ∼4.0–4.5% of total body weight. The liver appeared to have poor buoyancy as only a few hepatic deposits were found in this species, unlike other Torpedo species or Selachians (Roberts, 1969) where one can observe a rich oily fluid seeping out of the tissues while dissecting the liver, usually between the right long lobes and the stomach where the gallbladder is often located. When filled with bile, as was found in most captured fishes, the gallbladder ranged from 2.5 to 3.5 cm in diameter; it was spheroid to oblong-shaped, and often partly covered by some surrounding pancreatic tissues. Observations with a magnifying glass and scanning electron microscopy (SEM) revealed the gallbladder surface delineating the lumen to appear finely folded. No difference was found between male and female specimens.
SEM shows the folded aspect of the organ's mucosal surface with its bulging apical surface cells (Fig. 1A). Figure 1B–E displayed LM aspects of Torpedo gallbladder showing a typical histology and its epithelium. The gallbladder wall structure resembled that of the mammalian gallbladder. Its wall thickness ranges from 0.25 to 0.40 mm. The mucosal epithelium was more strongly contrasted than the other remaining tissues of the gallbladder wall (Fig. 1B,C). Both SEM and LM views revealed the mucosal appearance as wavy coating of undulating folds of 150–200 μm in height. In areas adjacent to the pancreas, the folds were shallow and the lamina propria was reduced to depict a more compact musculature (Fig. 1B). In all the fields examined with the 1-μm-thick sections stained with toluidine blue, the cholecystocytes were organized as a simple columnar epithelium with primarily darkly stained cells in addition to a few palely stained cells. The tightly aligned cholecystocytes ranged in size between 50 and 85 μm in height and 5-μm wide or less on their basement membrane (Fig. 1E). The columnar cells showed bulging and clavate apices with less contrast that the main cell body (Fig. 1D,E). Some of the hemispherical-shaped bulging apices appeared as dissociated from the main cell body and probably have a rich mucinous content, as they contrast poorly with toluidine blue. The nuclei usually appeared in the lower third of the cells and were situated at various basal levels, contributing to an overall epithelium falsely appearing as pseudostratified (Fig. 1B–E). The upper cytoplasmic region, just beneath the self-excising pale apices, appeared greenish to blue in color, caused by a mixed population of numerous mitochondria, maturing mucinous vesicles and dark granules. The supranuclear and perinuclear zones revealed fine, dark blue granules while the lower region of the cytoplasm, beneath the nucleus, contained variably sized, isolated or aggregated, highly contrasted granules likely lysosomal in nature (Fig. 1D–E). The pseudostratified epithelial morphology is also affected by cell deaths and renewal with invading immune cells, such as lymphocytes, that can be found throughout the area. These surveillance cells can be found near the basal or in the basolateral aspects of the epithelium (Fig. 1D–E).
The fibromuscular layer was ∼150- to 250-μm thick, with the lamina propria of 70–120 μm in thickness appearing to occupy about half of the wall width of the organ. This layer was composed of a loose connective tissue matrix containing slender cells, probably fibrocytes, myofibrocytes, and delicate, undulating smooth muscle fibers. A 50- to 120-μm thick external muscular layer was organized of more or less tightly entwined smooth muscle bundles invaded by blood vessels. The orientation of the bundles seems to be typically longitudinal to oblique, although proximally, near the cystic duct, the bundles occur with an internal circular and outer longitudinal architecture. The smooth muscle bundles were seen either tightly bundled (Fig. 1B) or more loosely arranged, leaving spaces for the arteries (Fig. 1C) and other large vessels (Fig. 2A–C) containing numerous blood cells. Nucleated erythrocytes of 30–35 μm in length and 10–15 μm in width were the dominant components, along with a few other circulating cells. The smooth muscles appeared as long tapered cells with undulating features, probably caused by tissue processing and/or the dissection and preparation for morphologic analysis (Fig. 2B).
The outermost, subserosal layer ranged from <15 to 50 μm in thickness and was primarily composed of mesothelium and a thin subserosal connective tissue (Figs. 1C and 2B,C). In the area adjacent to one of the liver lobes, the gallbladder also showed a typical but thin adventitial layer. It was also quite common to detect large pieces of pancreatic lobes attached and sharing the same adventitial layer and mesothelial covering (Fig. 1B). The subserosal layer sometimes displayed superficial blood vessels of varying diameter. The adventitial layers between gallbladder and pancreas occasionally appeared noncontiguous, probably caused by a dissection artifact (Fig. 1B).
Scanning electron microscopy (SEM).
Most of the mucosal surface of the gallbladder exhibited cholecystocytes with bulging apices ranging from 4 to 10 μm in width, delineating each cell apex, although not providing the actual cell diameter (Figs. 1A and 3A–C). Cell apices were found at different states of extrusion and dilation. The apices were coated with numerous, short microvilli. As an exocytotic event was initiated, the apex morphology became distorted; resulting in a bulging appearance and the local microvillar coat disappeared. Following this, one, or eventually more than one, knob-like swelling appeared as a major elongation of the apical region. The entire apex then elongated irregularly and finally the entire apical surface became smooth surfaced and more reflective by SEM. The elongated apex was expelled from the cell as a self-excising spherical excrescence in some cases (Fig. 3A–C). In other cells, the cell apices secreted and extruded mucous material without self-excision. Throughout the epithelium little debris or pieces of cells that were detected were seen loosely dispersed near or in the grooves of the gallbladder folded surface epithelium (Fig. 1B–E) and, at SEM level, similarly located attached to microvillar surfaces (Figs. 1A and 3A–C).
Transmission electron microscopy (TEM)
Using TEM, the surface epithelium of Torpedo gallbladder was again shown as a very tall, simple columnar epithelium. In some views the epithelium appeared pseudostratified (Fig. 4A) as a result of not only the invading lymphocytes or other surveillance cells, but also the presence of some basal cells above the basal lamina. Each cholecystocyte showed an apical bulge and densely contrasted junctional complexes, with some patchy desmosomes. The apical membrane domain of the cholecystocytes was seen as either a smooth surface, poorly coated by glycocalyx and microvilli, or with apical disruptions characteristic of stages of typical mucous exocytosis with or without apocrine decapitation (Figs. 4A,B, 5A, 6A–D, 7A). The apices seldom contained organelles, and, in some cases, only a few mucous vesicles and tiny, empty appearing vesicles of 80–120 nm in diameter (Fig. 5C,D).
The cytoplasm was filled with populations of numerous, mucous-rich vesicles, 0.8–1.9 μm in diameter. The mucinous vesicles were differentiated by their either tightly granular or finely fibrillar content, appearing somewhat marbled but with less electron density than the adjacent cytoplasm (Figs. 5A–D and 7A–D). The mucus-containing vesicles' outlines ranged from irregular to spheroid-shaped, but some were also seen to be somewhat polyhedral. In some, at higher magnification and especially during the exocytotic events noted as in Fig. 6 A,B,D,E, a microfibrillar content ranging between 12 and 15 nm in thickness was apparent, not only in the exocytotic material but also in the cytoplasm where several vesicles had the opportunity to fuse with each other (Fig. 6B). We also observed mucinous vesicles to contain a contrasted, proteinaceous core or condensation similar to those found in mucous secretory vesicles of mammalian tissues that are usually made of lysozyme and other associated negatively charged molecules.
Amid the mucous vesicles, rare but small, spherical to elongated mitochondria can be seen (Figs. 5A,B, 6A, and 8C) with a few highly contrasted lysosomal bodies that can reach up to 1 μm, as previously seen with LM (Fig. 1D–E). These dense bodies were also observed in other electron micrographs (Figs. 7A,B,D and 8A,C,D). Most dense bodies, at high magnification, were revealed to have a heterogeneous microstructure of membrane whorls amid a highly electron dense matrix also containing tiny granular artifacts that can be seen throughout the electron densities. The organelles are similar to residual bodies originating from both auto- and heterophagocytosis. Those located in the lowest part of the cytoplasm were usually the largest and contained patchy but granular deposits more intensely contrasted than the heterogeneous content; this could be caused by content high in ionized calcium. Electron dense vesicles, 150–300 nm in diameter, could be detected among the mitochondria, the residual bodies and other membrane bound, spheroid organelles; they could be the primary lysosomes (Fig. 8C). The morphology of the nuclei suggests that these are very active cells; they show an overall euchromatic aspect with a few heterochromatic areas along the inner membrane of the nuclear envelope as well as in the nucleoplasm.
Damage and Repair of the Gallbladder Epithelium
In some rare areas of the gallbladder, TEM observations showed a disorganized epithelium that had undergone damage and repair. The surface epithelium alignment was disrupted and appeared pseudostratified (Fig. 4). In Fig. 5A,B, an area of damaged epithelium exhibits a repaired or reactive cell growing an apical dome of mucous-free cytoplasm while a few adjacent cells have undergone apoptotic cell death and debris from these cells can be seen. In Fig. 5A,D, apices with mucous vesicles appear swollen as a result of decapitation, oncotic stress, or damage. Mucins and apical cytoplasm remnants are visible as part of a sludge-like debris in the lumen, produced by apocrine secretion (Figs. 4A,B, 7A–D, 8A,B). After repair, as shown in Fig. 5 A,B,D, a dome-like apex covers each cholecystocyte. These dome-shaped apices are crowded with a faint, densely packed granular cytoplasm filled with numerous mucous vesicles, with some fibrillar content, small, round mitochondria and a few densely contrasted lysosomal bodies. Adjacent cells (Fig. 5B,D) are attached by punctuate desmosomes. The apices have a poorly preserved or absent glycocalyx and microvilli, particularly when the bulging of the apex is initiated or when oncotic damage is ongoing. The micrographs in the panel of Fig. 6A–E show the apices of the cholecystocytes are crowded with tightly, packed cytoplasm with a granular material, and also shows mucus vesicles and residual bodies. A characteristic basal lamina of the epithelium can also be found.
A loose network of narrow smooth muscles or myofibroblast-like cells can be found in the lamina propria along with bundles of collagen, extracellular matrix materials and small blood vessels, while in the outer subserosal layers, larger diameter vessels were found, usually filled by erythrocytes and other blood elements (Fig. 2C).
The Cholecystocyte Secretory Activity is Cyclically Both Merocrine and Apocrine
Figures 4A,B, 5A,C,D, 6A–E, 7A–C, and 8A,B summarize most of the dynamic, morphologic changes of the surface epithelium accompanying the cholecystocyte secretory process. Each epithelial cell acquires an apical, bulging, mammillary-like appearance, elongates for dozen of microns while preparing for merocrine secretory events, and culminates in self-excision, in an apocrine fashion. This was suggested in Fig. 3A–C and noted in Figs. 4A,B, 6D,E, 7A–C, and 8A,B where the apical region of the cholecystocyte appears to secede from the epithelial cell, during which the excrescent apex, apparently emptied of organelles, leaves the cell. Along with these events the junctional complexes that delineate each epithelial cell from one another remain strongly contrasted, continuing intact throughout the secretory cycles (Figs. 6A–D and 7A–D).
During exocytosis in a merocrine fashion, mucous vesicles reaching the most apical cytoplasm release their content while the microvilli have disappeared (Figs. 6A,B,D,E and 7C). It is noticeable that the lateral apices also release mucous secretory vesicles (Fig. 6B,C,E). Exocytotic crypts can be found along the apical edge (Fig. 4 B). The lateral secretory events appear to be accompanied by multiple intervesicular fusions that fill most of the apical region of these cells with dispersed mucus. As a result, only a few mucous vesicles of higher density and contrast remain (Fig. 6C). These abundant secretory events may cause the apical region of the secreting cholecystocytes to lose their integrity, as shown by multiple, apical membrane defects, and an osmotic burst can occur, liberating the apical content into the gallbladder lumen (Fig. 6C–E as in Figs. 4B, 7A, and 8A,B). The elongation of the cell apices can then become more lucent to electrons (Figs. 6C–E, 7A,B, and 8A,B). Other such events are also recorded in Fig. 7A–C, showing the ruptured apices of cholecystocytes associated with cell corpses and debris in the lumen. However, by examining Fig. 7B, one can detect the loss of integrity of the intercellular, junctional complexes (Fig. 7C), cholecystocyte cell debris with heterogeneous secretion products in the lumen of the gallbladder; the apical, conical shaped surfaces suggest that healing and repairs have occurred (Fig. 7B,C). At the end of the excision process, epithelial apices sealed appear flattened and showed a new apical crowding of organelles, mainly consisting of an alignment of cytoplasmic mucous vesicles with smooth, densely contrasted content and lysosomal bodies, as noted in Figs. 7D and 8A. Microvilli are then restored after cells assume a “resting” shape before beginning a new secretory event (Fig. 7B,D). Lysosomal bodies found in the cholecystocytes appear to result from autophagocytoses or heterophagocytotic activities whilst a few rare primary lysosomes (Fig. 8C), others can only be problematic without histochemistry, even though they appear to become part of the complex phagosomes (ly? In Fig. 8D) associated with complex membrane structures.
Torpedo marmorata (Risso, 1810) is a species of the family of Torpenidae; it belongs to the class Chrondrochthyes, subclass Elasmobranchii (Vialleton, 1903; Poll, 1947; Bertin, 1958; Louisy, 2001; Martin, 2005). It is also commonly known as the marbled electric ray (e.g., in French “torpille marbrée”; in Spanish “tembladera”). It is a benthic, aplacental and viviparous, euryhaline species found throughout the rocky reefs or sea grass beds of sandy or muddy shallow sea floors of most coastal waters of the Eastern Atlantic Ocean from the North Sea to S. Africa as well as the entire Mediterranean Sea (Stehmann and Bürkel, 1984; Martin, 2005). The specimens captured corresponded to the average adult size in this sea (Poll, 1947; Filiz and Mater, 2002).
Originally described by Rondelet (1555) the name Torpedo originated from the Latin for “torpor,” because of its electrogenic capability as it jolts its enemies and prey, resulting in numbness or “torpor”; these electric “shocks” were used medically by ancient Greeks and Romans against pain and gout (Carruba and Boers, 1982; Finger and Piccolino, 2011). The speciation was finalized according to the Linnean nomenclature as Torpedo marmorata by Risso (1810) as “marmorata” refers to the marbled pattern seen on the body. Risso's original drawing was designated as the lectotype for this species (Fricke, 1999). Torpedo is adapted to live in cool temperature (<20°C) and therefore the species can be found as a bottom-dweller in warm seas at depths from 10 to 30 m and as deep as 370 m (Risso, 1810; Fowler, 1911; Radii-Wess and Kovacevic, 1970; Capapé, 1979, 1989; Abdel-Aziz, 1994; ElKamel et al., 2009). It can also survive in poorly oxygenated waters such as tidal pools or in extreme hypoxia using the glycolytic pathway (Hughes and Johnson, 1978; Ballantyne, 1997). The species is often solitary, carnivorous, and hunts mainly during the night, as does a related species, T. californica (Bray and Hixon, 1978). It can bury itself in the sea floor, leaving only the eyes and spiracles visible as it ambushes its prey, which includes mostly small fishes and, to a lesser extent, cephalopods and subdues them with electric bursts (Mellinger, 1971; Belbenoit and Bauer, 1972; Abdel-Aziz, 1994; Picton and Howson, 2000; Romanelli et al., 2006; Capapé et al., 2007).
Torpedo species are predators and have little or no nutritional importance and economic value. However, their ethologic niche with longevity, size, slow metabolism, polyunsaturated-rich livers, and so forth can make them a strong, bioconcentrating marker species of environmental pollutants or toxins (Solé et al., 2010). Because the detoxification of these xenobiotics involves the liver and gallbladder, the normal structure of the gallbladder as described here may be of use to allow fisheries and biologists to verify, understand and survey the marine, coastal environments of the continental shelves. Torpedo species have been already used as warning signals of toxicity of organochlorine (Gelsleichter and Walker, 2010; Storelli et al., 2011), and of metal residues (Madejczyk et al 2009).
In Torpedo, as in most carnivorous Elasmobranchs, homeostasis relies on the availability of food rich in proteins and lipids (Haywood, 1973; Pang et al., 1977; Ballantyne, 1997; Buddington et al., 1987; Holmgren and Nilsson, 1999) to maintain an internal milieu with a high urea content (Städeler, 1860; Walsh and Smith, 2001; Hazon et al., 2003) and a higher osmolarity than sea water (Goldstein and Forster, 1971; Sripadi et al 2009); urea is accompanied by trimethylamine oxide or TMAO (Yancey et al., 1982; Anderson 1995; Watson and Dickson, 2001; Anderson et al., 2007; review in Ballantyne, 1997; Treberg et al., 2006; Trischitta et al., 2012); TMAO provides a positive buoyancy to these Elasmobranch fishes (Withers et al., 1994). This ureosmotic strategy, in which the gallbladder actively participates (Lippe and Ardizzone, 1989) along with the gills and the kidneys (Payan et al., 1973), calls for a high purine diet and probably results in a very slow digestive transit (Yung, 1899) as is found in sharks (Wood et al., 2007). As in most vertebrates, a major part of the enzymatic digestion of the stomach bolus reaching the small intestine is achieved by a combination of both pancreatic and bile secretions.
Observations of mammals and humans show that the gallbladder is not just a reservoir for and concentrating storing bile (or gall), but also modifies bile. Bile is composed of a solution of bile salts, originating from the pigments of erythrocyte turnover essentially made by the spleen (bilirubin, biliverdin) and from the hepatic transformation of digested foods or foreign products (fats, cholesterol and its ester, inorganic salts and xenobiotics (Diamond, 1962; Gilloteaux, 1997; Gilloteaux et al., 1997a, b; Oldham-Ott and Gilloteaux, 1997). Bile secreted into the small intestine emulsifies and increases the absorption of fats and fat-soluble vitamins. The gallbladder also secretes a solution of bicarbonate, cations and mucus (Gilloteaux et al., 1997b; Glickerman et al., 1997) that can be modified through concentration (Reus et al., 1991) and, along with the pancreatic enzymes and its alkaline secretion, bile facilitates the intestinal absorption of lipids as chylomicrons obtained through the acid stomach bolus. Concentration of bile certainly occurs, as indicated by the presence of abundant blood and lymphatic vessels in its wall as shown by Neuville (1901) and confirmed by Figs. 1C and 2A–C; these micrographs are astonishingly similar to the drawings of T. marmorata gallbladder wall histology illustrated by Planche XIII made with a camera lucida by Vialleton (1902– 1903)!
The gallbladder epithelium
Although the topographical staining used in the epoxy-cut thick sections offers only a limited histochemical interpretation, as chemical moieties available for toluidine blue could have been masked or altered after fixation, especially the osmium fixation step (Litwin, 1985) and contrasting by heavy metal salts, it appears that a quite good staining pattern and high contrast were shown for both mucus (does not stain or poorly stains the apical regions of the cholecystocytes) as well as for lysosomal bodies (dark purple). Lysosomes, organelles rich in proteins and associated charged compounds, usually appeared as small, dense, dark purple-violet droplet-like deposits in the upper regions of cholecystocytes and as more numerous, somewhat thick spheroids in the perikaryal and lower regions of those epithelial cells. Numerous exocytoses of mucus need recycling of membranes and probably those many lysosomes are related to these events. We also noted that peroxisomes were not present, as they are in the teleost gallbladder (Gilloteaux et al., 2011) and other organs (Moyes et al., 1991). This absence may well correlate with the poikilothermic metabolism (Pica et al., 2001) and poor cytochrome content in large nucleated erythrocytes, confirming the low respiratory activity of elasmobranches (Hughes and Johnston, 1978). A high level of lipid peroxidation can be reached in most tissues (at least 14 times higher than in mammals or five times more than in teleosts [Filho and Boveris, 1993]). Tissues in Elasmobranchs may have few or no peroxisomes. Other antioxidant adaptations can be found in these fish: high SH-rich hemoglobin, catalytic activity of erythrocytes (Giulivi et al., 1994) and high levels of squalene containing low-density polyunsaturated fatty acids to also support their buoyancy (Ballantyne, 1997; Abele et al., 2012).
Contrary to the clear absence of peroxisomes, mitochondria can be found throughout the cholecystocytes, closely associated with mucus but at a distance from the apical zones that will be decapitated. The high level of nitrogen wastes and urea metabolism is associated with the mitochondrial enzymatic armamentarium. It is not surprising to find these organelles abundantly present, as ATP is needed to carry on transport against the gradient of urea and other ions as well as to manage the maturation and transport of the mucous vesicles and local cell repairs. However, respiration makes large amounts of free radicals, and one can see how, with high disulfides, omega lipids and TMAO, this species is adapted to high peroxidation; it is possible that this extra load may be a factor in altering the apical areas of the cholecystocytes that are preparing to secrete; it may also be responsible for the poor preservation of membranes, as shown in reports using a mixture of glutaraldehyde and urea as a fixative (Nir and Hall, 1974).
As in most vertebrates, Torpedo bile also contains mucus provided by the cholecystocytes that serves as a significant barrier protecting the wall of the bile “reservoir.” Conjugated bile salts would be injurious to the epithelium wall as they are amphipathic and strong detergents; damage would occur if the wall was free from a mucous coating (Forstner and Forstner, 1994). The mucins secreted on the surface epithelium of the gallbladder are probably needed to protect the organ's wall from the strongly detergent scymnol bile (Karlaganis et al., 1989; Fricker et al., 1997). Normal human and rodent gallbladder mucus is mainly mucin 5B, abbreviated MUC5B (Verdugo, 1991; Vandenhaute et al., 1997; VanKlinken et al., 1998; Buisine et al., 2000; Vilkin et al., 2007; Kesimer et al., 2010). It is similar to the major mucin produced by the respiratory tract, salivary, and endocervical glands (Wickström et al., 1998), and the colon goblet cells (VanKlinken et al., 1998). Mucus has been shown to consist of secretions of high molecular weight glycoproteins made of an O-glycosylated coat attached to the serine or threonine residues of the core peptide (Roussel et al., 1988; Buisine et al., 2000; Kuver et al., 2000). Elasmobranchs secrete acid mucins comparable to those of mammalians (Theodosiou et al., 2007) but we do not yet know the molecular form. In mammals, a mucus molecule has been estimated at 150 × 106 Da, with a size of not >15 nm as seen by atomic force microscopy, with the average size being between 11 and 15 nm (McMaster et al., 1999; Davis et al., 2000; Deacon et al., 2000; Kesimer et al., 2010). This value fits the measurements estimated in viewing the enlarged mucous vesicles that can be noticed in the micrographs A and D of Fig. 6.
Mucus in the human gallbladder is secreted by apical vesicles (Klomp et al., 1994; Gilloteaux et al., 1997a) in a merocrine fashion. This mode of merocrine exocytosis was noted by Verdugo (1991) and Rogers (1994) at the interface between the cell surface and surface of the epithelium as not just merocrine but also apocrine. In Torpedo marmorata mucus is secreted in a merocrine and an apocrine mechanism. Torpedo gallbladder mucus secretion resembles that found in many other species of vertebrates, inclusive of teleosts (Viehberger, 1982, 1983; Gilloteaux et al., 1995; Oldham-Ott and Gilloteaux, 1997; Gilloteaux et al., 2011). However, we observed a striking resemblance of the apical modifications and apical extrusion of mucus to those shown with SEM (Gilloteaux et al., 1993a, b) and TEM in cases of cholelithiasis induced in the Syrian hamsters (Gilloteaux et al., 1997a, b), as well as in human pathologies, such as in cholecystitis (Gilloteaux et al., 1997b, 2003, 2004). These studies found a modification in the composition of the mucus, whether in Syrian hamster or human gallbladder, in that it became more electron-negative, that is probably as a result of the carbohydrate moieties of the mucus becoming charged, perhaps by becoming sulfated and charged with ionized calcium, exchanged for osmium salt during fixation procedure (Gilloteaux and Naud, 1979).
While SEM shows little cell debris among the excrescent mucus, debris could have been washed away during preparation of the samples (especially the dehydrations and critical-point drying) unlike the TEM views, where the fixation and embedding steps have better preserved exocytotic events and images of debris from apocrine secretions is seen along with other morphologic changes. These exocytotic findings are similar to those made in the Syrian hamster and in cholecystitis gallbladders (Gilloteaux et al., 1993a, b, 1997a, b) where electron microscopy of cholescystocytes showed mucus expelled along with pieces of cell apices or debris, indicating remnants of apocrine secretion, similar to what is described in chronic sinusitis (Dorgam et al., 2004).
The processing of the mucus has not been evaluated by biochemical studies in Elasmobranchs, but the highly conserved molecular structures found throughout vertebrates leads us to conclude that the process of mucous secretion is caused by a similar macromolecular annealing processing, influenced by an osmotic shock bringing loads of cations to form an entwined packing of glycoprotein that becomes charged with the incoming ionized calcium to anneal as a film, forming a delicate microfibrillar morphology that expands outwardly into the luminal surface of the epithelium, as is seen in airway mucus release (Tyner et al., 2006). Because of the high osmolarity and fixatives needed to fix the samples, a “salting-out” effect has rendered their content marbled-like with a kind of granular-to-microfibrillar structure of the mucous vesicles, forming a microfibrillar structure aforementioned in an above paragraph. The mucus appears to fill the secretory vesicles with a homogeneous, fine, grainy content. This highly hydrated, gel-like material forms a film or tight, networked blanket of mucins over the entire surface of the gallbladder mucosa with a net negative charge caused by sialic acid and sulfate residues; those charges are usually balanced with cations such as calcium (Verdugo et al., 1987a, b; Perez-Vilar, 2007).
The bile composition of Elasmobranchs differs from that found in teleosts and most other vertebrates, including humans, in four main components: (1) the main bile salt is an acid ester of scymnol or scymnol sulfate; it is a 5-alpha C27 bile alcohol, named after the shark Scymnus borealis from which it was first purified (Hammarsten, 1898; Cook, 1941; Haselwood, 1967, 1968; Hagey et al., 2010) and is an end product of cholesterol metabolism (Windaus et al., 1930; Fang, 1987); (2) there are significantly less phospholipids and (3) cholesterol than in all mammals (Elferink et al., 2004); (4) there is also less bicarbonate and Cl- than in mammals (Boyer et al., 1976; Elferink et al., 2004). In the small skate, Raja erinacea, a related species, it has been shown that bile produced by the liver flows slowly through the bile tract, controlled by a low portal vein blood pressure, and producing only about 1% of that measured in rats (Fricker et al., 1996, 1997). This low liver output could be caused by low metabolism, as this fish has a low body temperature (Boyer et al., 1976). Furthermore, in Elasmobranchs, the enterohepatic circulation of bile has been found efficient and comparable to that of mammals (Fricker et al., 1994, 1996, 1997). Cornelius (1991) verified that the concentration of biliverdin and conjugated bilirubin increases with time and fasting. Grossbard and collaborators (1987) found that Raja erinacea excreted approximately equal amounts of bilirubin and biliverdin and McDonagh and Palma (1982) found that biliverdin (unconjugated) was the predominant pigment in the gallbladder of Torpedo californicus. This does not support Qin's hypothesis that the feeding habits of carnivorous fish would result in the development of a higher bilirubin liver secretion than biliverdin, a pigment essentially associated with herbivorous diets (Qin, 2007).
It was also demonstrated that the gallbladder has a quasi constant volume while bilirubin and biliverdin increased three to fourfold; bilirubin monoglucuronide accounted for 65% of the bilirubin content, while biliverdin accounted for the blue-green color of the blood in some marine fish (Fang, 1987; Grossbard et al., 1987; Fang and Bada, 1990). Fricker and collaborators (1994) found the preference for unconjugated bile salts to be associated with the scymnol salts. The making of bile salts unlike those in other vertebrates may be caused by the lack or quasi absence of peroxisomal activity (Pedersen 1993). The release of bile depends on a cholecystokinin-like peptide (Andrews and Young 1988).
Gallstones have been found in many mammals, among them being humans, baboons, sheep, cows, ferrets, dogs, and cats (Oldham-ott and Gilloteaux, 1997; Ward, 2006; Gaillot et al., 2007; Slingluff et al., 2010; Hall and Ketz-Riley, 2011; Katsoulos et al., 2011), and in birds (Filippich et al., 1984; Cousquer and Patterson-Kane, 2006). Diet and lifestyle have been implicated as causative agents in gallstone formation, as have inflammation and infection (Maurer et al., 2009) and a genetic predisposition (Krawczyk et al., 2011). Commonalities in gallstone formation appear to be hypersecretion of mucus and formation of a biliary sludge, and a high concentration of cholesterol in the bile as well as calcium salt deposition (Gilloteaux et al., 1997b; Rege, 2002; Ko et al., 2005). Morphological changes have been reported in the gallbladder epithelium prior to gallstone formation (Lee and Scott, 1982; Gilloteaux et al., 1993b).
Gallstones have not been reported in sharks or rays. Although we found that the gallbladder epithelium in Torpedo resembles that found in pathologic gallbladder epithelium (Lee and Scott, 1982; Gilloteaux et al., 1993b), we saw no indication of pathology in this organism. Apical debris issued from cholecystocytes could become pronucleating agents for cholesterol monohydrate to crystallize in bile (Smith, 1990), but as the bile of elasmobranchs is low in cholesterol (Elferink et al., 2004), it is unlikely that nucleation of gallstones would occur. Increased mucus secretion is a common factor in gallstone formation. However, Torpedo bile does not appear to stimulate gallbladder mucus hypersecretion, as was found in cholesterol-fed prairie dogs (Lee et al., 1981). The presence of scymnol sulfate in the bile of elasmobranchs deserves further investigation; Wilhelmi and others (2003) found that scymnol may possibly inhibit the nucleation of cholesterol crystals in bile. It is possible that scymnol or a derivative thereof could be used in the treatment of gallstones, much as the ursodeoxycholic acid found in bear bile is now chemically synthesized and used in the treatment of cholesterol gallstones. Further investigations of the normal anatomical structures and chemistry of the hepato-biliary tract of Torpedo marmorata, and other Elasmobranchs, in the future may assist in clarifying human gallbladder pathologic conditions.
The authors thank the staff of the Observatoire Océeanologique of Banuyls (France) of the Université Pierre and Marie Curie (Paris VI) for their assistance in the collection of live specimens. Also they thank the staff of the Library of the Institut Royal des Sciences Naturelles, Brussels (Belgium) for its assistance in accessing books for our oldest literature references. Steve Getch (Summa, Communication Specialist) and Jeff Workman (Northumbria University Graphics) are recognized for their help in imaging the submitted illustrations, originally captured with photomicrographs into electronic version.