SEARCH

SEARCH BY CITATION

Keywords:

  • gallbladder;
  • cholecystocytes;
  • lysosomes;
  • mucus;
  • Torpedo marmorata;
  • electric ray;
  • elasmobranch fish

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

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.

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).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Fish Collection

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).

Fixation

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.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

General Morphology

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.

Histology

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).

thumbnail image

Figure 1. A–E: SEM (A) and LM (B-E) views of Torpedo marmorata gallbladder. A: Surface epithelium of wide folds or rugae with most of the cholecystocyte cells showing bulging apices. B–E: LM views of the gallbladder wall with 1-μm-thick epoxy sections, after staining by toluidine blue. In B: Paraxial section of the gallbladder depicting the folded wall with lumen (L) and a small area of pancreas (P). The mucosal epithelium and fibromuscular layer (FM) with its external musculature covered by a thin, serosal lining. In C: Cross section of the gallbladder showing all typical layers of the organ: E: mucosal epithelium, the lamina propria or submucosal layer (SM), where diverse blood vessels can be found adjacent to the basement membrane; the fibromuscular layer (FM) where arteries (A) are embedded in the most external musculature; the serosal layer (S) covered by its mesothelium has an underlying subserosal layer (SS) with lymph (l) and blood vessels. In D and E: the epithelium illustrates its crowded, simple columnar epithelium with pseudostratified appearance where numerous bulging apices produce cell debris (d) in the lumen (L). Notice a grayish-marbled appearance of the near apices, and small densely contrasted granulations throughout with the largest ones in the most basal aspects of cholecystocytes. Some surveillance cells (S) can be seen among the epithelium. All arrowheads in lumina indicate accumulated cell debris and apical decapitations. Scale bar in A equals 25 μm; in B–D scale bars equal 25 μm; in E, scale bar equals 10 μm.

Download figure to PowerPoint

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).

thumbnail image

Figure 2. A–C: LM views of Torpedo marmorata gallbladder wall. A: The fibromuscular layer (FM) features a loose musculature, loose connective components, and blood vessels (bv). B: Enlarged area showing the long and wavy smooth muscles of the FM and some wantering and/or fibrocytes-like cells. C: enlarged view of subserosal and serosal (S) layers; f: fibroblast-like cell; l: lymphocytes; m: smooth muscle fiber; P: peritoneal cavity. Scale bar in A is 25 μm; in B and C it equals 10 μm.

Download figure to PowerPoint

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).

Ultrastructural Aspects

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).

thumbnail image

Figure 3. A–C: Three aspects of the surface lining of the Torpedo gallbladder as viewed by SEM and with enlargements of fields similar to Fig. 1A. The apices appear in diverse states of bulging: narrow to large hemispheroids coated with short microvilli from which bursts of mucus-like blebs and other debris are found on their surfaces. All bars equal 5 μm.

Download figure to PowerPoint

Transmission electron microscopy (TEM)
General aspects

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).

thumbnail image

Figure 4. A–B: TEM views of the surface epithelium of Torpedo gallbladder. A: Tall, simple columnar cholecystocytes with apical bulges on all cells show a supranuclear layer rich in highly contrasted lysosomal bodies. Notice the thin basal lamina (arrowhead) invaded by a lymphocyte (L), and example of another surveillance cells (S), as well as apical cell debris. Scale bar is 10 μm. B: Details of excrescent apices emptied from organelles; noting that many pale contrasted, apical mucous vesicles (m) first admixed with a few mitochondria (mt) that become numerous abundant in the supranuclear cytoplasm as more contrasted than mucous vesicles. On the upper left an unexpected exocytotic crypt formed as remnant of mucus exocytosed. Bar equals 1 μm.

Download figure to PowerPoint

thumbnail image

Figure 5. A–D: Gallbladder epithelium of Torpedo marmorata. A: Area of damaged epithelium after repair contains at least three apoptotic cells (a) with cell debris (d). B: the upper region of adjacent cells seen in A demonstrates a healing, dome-like apex filled by a fine granular material, that is, mucus, above an aggregate area filled by numerous mucous vesicles (m) and mitochondria (mt). An arrow indicates remnants of surface microvilli. An alignment of small, spot desmosomes links adjacent cells (6 thick arrows). C: A series of adjacent cell apices with heterogeneous morphology, some bulging with high density of fine granular material and one pale and swollen apex left with poorly preserved microvilli. D: a magnified view of some apices of C filled with granular cytoplasmic material possibly contributed by the subjacent emptying vesicles as suggested by pale structures left by intracytoplasmic discharge of lightly-contrasted mucus; a few, rare but darker contrasted mucous vesicles (m) can be found. L: lumen; arrows indicate disappearing microvilli. Arrowheads mark some of the apical junctional complexes. Bar in A is 10 μm; in B is 1 μm, C and D is 5 μm.

Download figure to PowerPoint

thumbnail image

Figure 6. A–E: Apical regions of the gallbladder epithelium of Torpedo. From A to E, this pane of micrographs illustrates some complex structural changes associated with the maturation and intracellular opening of mucous vesicles (m) having a marbled appearance as containing a fine fibrous-like content (series of arrowheads); their secretion can be either of the swollen cell apices (in A–C) with isolated exocytoses or whilst the apical regions of cholecystocytes are stretched. Ly: Lysosomal body. Thick arrows indicate near apical junctional complexes between adjacent cholecystocytes. In E, apical merocrine-like secretion of mucus and apical swollen apices as apocrine osmotic debris are shown whilst other, adjacent apices depict bulging and exocytosis of mucus containing vesicles. L: lumen; m: mucous vesicle. All bars equal 1 μm.

Download figure to PowerPoint

thumbnail image

Figure 7. A–D: Torpedo gallbladder surface epithelium. In A–D: Diverse morphological aspects of apical regions of cholecystocytes illustrate their changes during and after secretions (mucus as well as apical self-excisions). Those self-excisions contribute to show in the lumen heterogeneous cell pieces or debris as well as mucus. B: A set of arrows mark the disappearance of microvilli whilst an apex is preparing to elongate, they are still barely detectable. In C: Notice the granular cytoplasm and the granular, finely arranged and quasi fibrous arrangement within the most apical, mucous secretory vesicles ready to exocytosis. In D: After excisions, apices appear flat and the apical content is made of smooth, densely contrasted mucous vesicles while deeper into the cells rare, highly contrasted lysosomal bodies are seen. L: Lumen; m: mucous vesicle. Bars in A, B and D equal 5 μm; bar in C equals 1 μm.

Download figure to PowerPoint

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.

thumbnail image

Figure 8. A–D: Apical excisions and lysosomes in cholecystocytes of Torpedo. A and B: Apices undergo luminal (L) discharge of mucus (m) with apocrine detachment producing all sorts of cell debris. Scale bar in A equals 5 μm, in B bar equals 1 μm. C and D: Details of diverse aspects of lysosomal bodies located in the supranuclear region (mainly in C) among mitochondria (mt); some of the largest ones are seen in the more basal aspects of the cholecystocytes (in D) from either autophagocytoses or heterophagocytotic activities. Rare primary lysosomes (ly) and possibly (ly?) can be detected associated to diverse membrane structures. In C and D bars equal 1 μm.

Download figure to PowerPoint

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.

Basal, invading surveillance cells, including lymphocytes or macrophages were noted along with dead cells and cell debris within the tall epithelium (Figs. 1D,E, 4A, and 5A).

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.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

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).

The gallbladder

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).

Mucin secretion

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

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

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

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.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  • Abdel-Aziz SH. 1994. Observations on the biology of the common torpedo (Torpedo torpedo Linnaeus, 1758) and marbled electric ray (Torpedo marmorata Risso, 1810) from Egyptian Mediterranean waters. Austr J Mar Freshw Res 45: 693704.
  • Abele D, Vazquez-Medina JP, Zenteno-Savin T, editors. 2012. Oxidative stress in aquatic ecosysystems. Oxford: University Press, Oxford, UK. p 16.
  • Anderson PM. 1995. 3 Urea cycle in fish: molecular and mitochondria studies. Fish Physiol 14: 5783.
  • Anderson R, Personne P. 1970. The localization of glycogen in the spermatozoa of various invertebrate and vertebrate species. J Cell Biol 44: 2951.
  • Anderson WG, Taylor JR, Good JP, Hazon N, Grosell M. 2007. Body fluid volume regulation in elasmobranch fish. Comp Biochem Physiol A 148: 313.
  • Andrews PLR, Young JZ. 1988. A pharmacological study of the control of motility in the gall bladder of the skate (Raja). Comp Biochem Physiol 89C: 349354.
  • Aparicio SR, Marsden P. 1969. Application of standard micro-anatomical staining methods to epoxy resin-embedded sections. J Clin Path 22: 589592.
  • Ballantyne JS. 1997. Jaws: the inside story: the metabolism of elasmobranch fishes. Comp Biochem Physiol B 118: 703742.
  • Belbenoit P, Bauer R. 1972. Video recordings of prey capture behavior and associated electric organ discharge of Torpedo marmorata (Chondrychthyes). Mar Biol Berlin 7: 9399.
  • Bertin L. 1958. Appareil digestif. In: Pierre-Paul G, editor. Traité de zoologie. Vol XIII fasc. 2. Paris: Masson & Cie. p 12491302.
  • Boyer JL, Schwarz J, Smith N. 1976. Biliary secretion in elasmobranchs. I. Bile collection and composition. Am J Physiol 230: 970973.
  • Bray RN, Hixon MA. 1978. Night-shocker: predatory behavior of the Pacific electric ray (Torpedo californica). Science 200: 333334.
  • Buddington RK, Chen JW, Diamond J. 1987. Genetic and phenotypic adaptation of intestinal nutrient transport to diet in fish. J Physiol 393: 261281.
  • Buisine MP, Devisme L, Degand P, Dieu MC, Gosselin B, Copin MC, Aubert JP, Porchet N. 2000. Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. II. Duodenum and liver, gallbladder, and pancreas. J Histochem Cytochem 48: 16671676.
  • Capapé C. 1979. La torpille marbrée, Torpedo marmorata Risso, 1801 (Pisces, Rajiformes) des côtes tunisiennes: nouvelles données sur l'écologie et la biologie de la reproduction de l'espèce avec une comparaison entre les populations méditerranéennes et atlantiques. Ann Sci Nat Zool Paris, 13ème Série 1: 7997.
  • Capapé C. 1989. Les Sélaciens des côtes méditerranéennes: aspects généraux de leur écologie et exemples de peuplements. Océanis 15: 309331.
  • Capapé C, Crouzet S, Clement C, Vergne Y, Guelorget O. 2007. Diet of the marbled electric ray Torpedo marmorata (Chondrychthyes: Torpedonidae) of the Languedocian coast (South of France, Northern Mediterranean). Ann Sér Hist Nat 17: 1722.
  • Carruba RW, Boers JZ. 1982. Englebert Kaempfer's first report of the torpedo fish of the Persian Gulf in the late seventeenth century. J Histor Biol 15: 263274.
  • Cook JW. 1941. Bile acids of elasmobranch fish. Nature 147: 388388.
  • Cornelius CE. 1991. Bile pigments in fishes: a review. Vet Clin Pathol 20: 106115.
  • Cousquer GO, Patterson-Kane JC. 2006. Cholelithiasis and chronic cholangiohepatitis in a mute swan (Cygnus olor). Vet Rec 158: 166167.
  • Curci S, Casavola V, Lippe C. 1978. Facilitated transport of urea across the gall-bladder luminal membrane. Arch Int Physiol Biochim 86: 243250.
  • Davis SS, Harding SE, Deacon MP, MCGurk S, Roberts CJ, Williams PM, Tendler SJB, Davies MC. 2000. Atomic force microscopy of gastric mucin and chitosan mucoadhesive systems. Biochem J 348: 557563.
  • Deacon MP, McGurk S, Roberts CJ, Williams PM, Tendler SJ, Davies MC, Davis SS, Harding SE. 2000. Atomic force microscopy of gastric mucin and chitosan mucoadhesive systems. Biochem J 348 ( Part 3): 557563.
  • Diamond JM. 1962. The reabsorptive function of the gallbladder. J Physiol 161: 442473.
  • Dorgam JV, de Souza BB, Sarreta SMC, Ferreira MDS, Melo VR, Anselmo-Lima WT. 2004. Histology and ultrastructural study of the mucosa of the maxillary sinus in patients with chronic rhinosinusitis and nasosinusal polyposis. Braz J Otorhinolaryngol 70: 713.
  • Elferink RP, Ottenhoff R, Fricker G, Seward DJ, Ballatori N, Boyer J. 2004. Lack of biliary lipid excretion in the little skate, Raja erinacea, indicates the absence of functional Mdr2, Abcg5, and Abcg8 transporters. Am J Physiol Gastrointest Liver Physiol 286: G762G768.
  • El Kamel O, Mnasri N, Souissi JB, Boumaïiza M, Ben Amor MM, Capapé. 2009. Inventory of elasmobranch species caught in the Lagoon of Bizerte (North-eastern Tunisia, central Mediterranean). Pan-Am J Aquat Sci 4: 383412.
  • Fang LS. 1987. Study of heme catabolism in fish. Comp Biochem Physiol B Comp Biochem 88: 667683.
  • Fang LS, Bada JL. 1990. The blue-green blood plasma of marine fish. Comp Biochem Physiol B 97: 3745.
  • Filho WD, Boveris A. 1993. Antioxidant defenses in marine fish. II. Elasmobranchs. Comp Biochem Physiol 106 C: 415418.
  • Filippich LJ, Kennard CH, Mines JJ. 1984. Vaterite gallstones in a canary (Serinus canarius). Aust Vet J 61: 298.
  • Filiz H, Mater S. 2002. A preliminary study on length-weight relationships for seven elasmobranch species from North Aegean Sea, Turkey. EU J Fish Aquat Sci 19: 401409.
  • Finger S, Piccolino M. 2011. The shocking history of electric fishes from ancient epochs to the birth of electrophysiology. Oxford, UK: Oxford University Press.
  • Forstner JF, Forstner GG. 1994. Gastrointestinal mucus. In: Johnson LR, editor. Physiology of the gastrointestinal tract. 3rd ed. New York: Raven Press. p 12551283.
  • Fowler HW. 1911. Notes on batoid fishes. Proc Acad Nat Sci Phil 62: 468475.
  • Fricker G, Dubost V, Finsterwald K, Boyer JL. 1994. Characteristics of bile salt uptake into skate hepatocytes. Biochem J 299 ( Part 3): 665670.
  • Fricker G, Fahr A, Beglinger C, Kissel T, Reiter G, Drewe J. 1996. Permeation enhancement of octreotide by specific bile salts in rats and human subjects: in vitro, in vivo correlations. Br J Pharmacol 117: 217223.
  • Fricker G, Wössner R, Drewe J, Fricker R, Boyer JL. 1997. Enterohepatic circulation of scymnol sulfate in an elasmobranch, the little skate (Raja erinacea). Am J Physiol 273: G1023G1030.
  • Fricke R. 1999. Annotated checklist of the marine and estuarine fishes of Germany, with remarks on their taxonomic identity. Stutt Beit Naturk A 587: 167.
  • Gaillot HA, Penninck DG, Webster CR, Crawford S. 2007. Ultrasonographic features of extrahepatic biliary obstruction in 30 cats. Vet Radiol Ultrasound 48: 439447.
  • Gelsleichter J, Walker CJ. 2010. Pollutant exposure and effects in sharks and their relatives. In: Carrier JC, Musick JA, Helthaus MR, editors. Sharks and their relatives II. Biodiversity, adaptative physiology, and conservation. Boca Raton, FL: CRC Press. Chapter 12, p. 491537.
  • Gilloteaux J. 1997. Introduction of the biliary tract, the gallbladder and gallstones. Microsc Res Tech 38: 541545.
  • Gilloteaux J, Karkare S, Don AQ, Sexton R. 1997b. Cholelithiasis induced in the Syrian hamster: evidence for an intramucinous nucleating process and down regulation of cholesterol 7α hydroxylase (CYP7) by medroxyprogesterone. Microsc Res Tech 39: 5670.
  • Gilloteaux J, Karkare S, Kelly TR. 1983a. Apical excrescences in the gallbladder epithelium of the female Syrian hamster in response to medroxyprogesterone. Anat Rec 236: 479485.
  • Gilloteaux J, Karkare S, Kelly TR, Hawkins WS. 1997a. Ultrastructural aspects of human gallbladder epithelial cells in cholelithiasis: production of anionic mucus. Microsc Res Tech 38: 643659.
  • Gilloteaux J, Karkare S, Ko W, Kelly TR. 1992. Female sex steroid induced epithelial changes in the gallbladder of the ovariectomized Syrian hamster. Tissue Cell 24: 869878.
  • Gilloteaux J, Kosek E, Kelly TR. 1993b. Epithelial surface changes and induction of gallstones in the male Syrian hamster gallbladder as result of a 2-month sex steroid treatment. J Submicr Cytol Pathol 25: 519533.
  • Gilloteaux J, Miller D, Morrison RL. 2004. Intracellular liposomes and cholesterol deposits in chronic cholecystitis and biliary sludge. Ultrastruct Pathol 28: 123136.
  • Gilloteaux J, Naud J. 1979. The zinc iodide-osmium tetroxide staining-fixative of Maillet: microanalysis and detection of Ca2+-affinity subcellular sites in a smooth muscle. Histochemistry 63: 227243.
  • Gilloteaux J, Oldham CK, Biagianti-Risbourg S. 1995. Ultrastructural diversity of the biliary tract and the gallbladder in fish. In: Datta Munshi, JS, Dutta HM, editors. Fish morphology, horizon of new research. New Delhi: Oxford & IBH Publishing. p 95110.
  • Gilloteaux J, Ott DW, Oldham-Ott CK. 2011. The gallbladder of Uranoscopus scaber L. (Teleost Perciform fish) is lined by specialized cholecystocytes. Anat Rec 294: 18901903.
  • Gilloteaux J, Tomasello LM, Elgison BA. 2003. Lipid deposits and lipo-mucosomes in human cholecystitis and epithelial metaplasia in chronic cholecystitis. Ultrastructural Pathol 27: 19.
  • Giulivi C, Hochstein P, Davies KJ. 1994. Hydrogen peroxide production by red blood cells. Free Rad Biol Med 16: 123129.
  • Glickerman DJ, Kim MH, Malik R, Lee SP. 1997. The gallbladder also secretes. Dig Dis Sci 42: 489491.
  • Goldstein L, Forster RP. 1971. Urea biosynthesis and excretion in freshwater and marine Elasmobranchs. Comp Biochem Physiol 39B: 415421.
  • Griffith RW. 1981. Composition of the blood serum of deep-sea fishes. Biol Bull 160: 250264.
  • Grossbard ML, Boyer JL, Gordon ER. 1987. The excretion pattern of biliverdin and bilirubin in bile of the small skate (Raja erinacea). J Comp Physiol B Biochem Syst Environm Physiol 57: 6166.
  • Hagey LR, Møller PR, Hofmann AF, Krasowski MD. 2010. Diversity of bile salts in fish and amphibians: evolution of a complex biochemical pathway. Physiol Biochem Zool 83: 308321.
  • Hall BA, Ketz-Riley CJ. 2011. Cholestasis and cholelithiasis in a domestic ferret (Mustela putorius furo). J Vet Diagn Invest 23: 836839.
  • Hammarsten O. 1898. Ueber eine neue Gruppe gepäarter Gallensauren. Z Physiol Chem Hoppe-Seyler's 24: 322350.
  • Haselwood GAD. 1964. The biological significance of chemical differences in bile salts. Biol Rev 39: 537574.
  • Haselwood GAD. 1967. Bile salt evolution. J Lipid Res 8: 535550.
  • Haselwood GAD. 1968. Evolution and bile salts. In: Code CF, editor. Handbook of physiology. Washington, DC: American Physiological Society. p 23752390.
  • Hayat MA. 1993. Stains and cytochemical methods. New York: Plenum Press. p 5764.
  • Haywood GP. 1973. Hypo-osmotic regulation coupled with reduced metabolic urea in the dogfish Poroderma africanum. An analysis of serum osmolarity, chloride and urea. Mar Biol 23: 121127.
  • Hazon N, Wellsa AA, Pillans RD, Gooda JP, Anderson WG, Franklin CE. 2003. Urea-based osmoregulation and endocrine control in elasmobranch fish with special reference to euryhalinity. Comp Biochem Physiol B 136: 685700.
  • Hofmann AF, Hagey LR. 2008. Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics. Cell Mol Life Sci 65: 24612483.
  • Holmgren S, Nilsson S. 1999. The digestive system. In: Hamlett WC, editor. Sharks, skates, and rays: the biology of elasmobranch fishes. Baltimore MD: Johns Hopkins University Press. Chapter 6, p 144173.
  • Hughes GM, Johnston IA. 1978. Some responses of the electric ray (Torpedo marmorata) to low ambient oxygen tensions. J Exp Biol 73: 107117.
  • Karkare S, Gilloteaux J, 1995. Gallstones induced by sex steroids in the female Syrian hamster: duration effects. J Submicrosc Cytol Pathol 27: 5374.
  • Karlaganis G, Bradley SE, Boyer JL, Batta AK, Salen G, Egestad B, Sjövall J. 1989. A bile alcohol sulfate as a major component in the bile of the small skate (Raja erinacea). J Lipid Res 30: 317322.
  • Katsoulos PD, Christodoulopoulos G, Karatzia MA, Pourliotis K, Minas A. 2011. Liver flukes promote cholelithiasis in sheep. Vet Parasitol 179: 262265.
  • Kesimer M, Makhov AM, Griffith JD, Verdugo P, Sheehan JK. 2010. Unpacking a gel-forming mucin: a view of MUC5B organization after granular release. Am J Physiol Lung Cell Mol Physiol 298: L15L22.
  • Kinsella JE, German B, Shetty J. 1985. Uricase from fish liver. Isolation and some properties. Comp Biochem Physiol B 82: 621624.
  • Klomp LW, van Rens L, Strous GJ. 1994. Identification of a human gastric mucin precursor: N-linked glycosylation and oligomerization. Biochem J 304: 693698.
  • Ko CW, Schulte SJ, Lee SP. 2005. Biliary sludge is formed by modification of hepatic bile by the gallbladder mucosa. Clin Gastroenterol Hepatol 3: 672678.
  • Krawczyk M, Wang DQ, Portincasa P, Lammert F. 2011. Dissecting the genetic heterogeneity of gallbladder stone formation. Semin Liver Dis 31: 157172.
  • Kuver R, Klinkspoor JH, Osborne WRA, Lee SP. 2000. Mucous granule exocytosis and CFTR expression in gallbladder epithelium. Glycobiology 10: 140157.
  • Lee SP, LaMont JT, Carey MC. 1981. Role of the gallbladder mucus hypersecretion in the evolution of cholesterol gallstones. J Clin Invest 67: 17121723.
  • Lee SP, Scott AJ. 1982. The evolution of morphologic changes in the gallbladder before stone formation in mice fed a cholesterol–cholic acid diet. Am J Pathol 108: 18.
  • Lippe C, Ardizzone C. 1989. Urea transport and its regulation across the gall bladder of Torpedo marmorata. J Exp Zool 252 ( Suppl S2): 143145.
  • Litwin JA. 1985. Light microscopic histochemistry on plastic sections. Prog Histochem Cytochem 16: 184.
  • Louisy P. 2001. Guide d'identification des poissons marins. Europe et Méditerranée. Paris, Ulmer E, editor; p 419420.
  • McDonagh AF, Palma LA. 1982. Heme catabolism in fish. Bile pigments in gallbladder bile of the electric torpedo, Torpedo californicus. Comp Biochem Physiol B 73: 501507.
  • McMaster TJ, Berry M, Corfield AP, Miles MJ. 1999. Atomic force microscopy of the submolecular architecture of hydrated ocular mucins. Biophys J 77: 533541.
  • Madejczyk MS, Boyer JL, Ballatori N. 2009. Hepatic uptake and biliary excretion of manganese in the little skate, Leucoraja erinacea. Comp Biochem Physiol C Toxicol Pharmacol 149: 566571.
  • Martin RA. 2005. Conservation of freshwater and euryhaline elasmobranchs: a review. J Mar Biol Assoc UK 85: 10491073.
  • Maurer KJ, Carey MC, Fox JG. 2009. Roles of infection, inflammation, and the immune system in cholesterol gallstone formation. Gastroenterology 136: 425440.
  • Mellinger J. 1971. Croissance et reproduction de la Torpille (Torpedo marmorata). I. Introduction, écologie, croissance générale et dimorphisme sexuel: cycle, fécondité. Bull biol Fr Belg 105: 165218.
  • Moyes CD, Suarez RK, Brown GS, Hochachka PW. 1991. Peroxisomal beta-oxidation: insights from comparative biochemistry. J Exp Zool 260: 267273.
  • Neuville H. 1901. Contribution à l'étude de la vascularization intestinale chez les cyclostomes et les sélaciens. Ann Sc Nat (Zool) Paris 13: 1116.
  • Nir I, Hall MO. 1974. The ultrastructure of lipid-depleted rod photoreceptor membranes. J Cell Biol 63: 587598.
  • Oldham-Ott C, Gilloteaux J. 1997. Comparative morphology of the gallbladder and biliary tract in Vertebrates: variation in structure, homology in function and gallstones. Microsc Res Tech 38: 571597.
  • Pang PKT, Griffith RW, Atz JW. 1977. Osmoregulation in elasmobranchs. Am Zool 17: 365377.
  • Payan P, Goldstein L, Forster RP. 1973. Gills and kidneys in ureosmotic regulation in euryhaline skates. Am J Physiol 224: 367372.
  • Pazzi P, Gamberini S, Buldrini P, Gullini S. 2003. Biliary sludge: the sluggish gallbladder. Dig Liver Dis 35 ( Suppl 3): S39S545.
  • Pedersen JI. 1993. Peroxisomal oxidation of the steroid side chain in bile acid formation. Biochimie 75: 159165.
  • Perez-Vilar J. 2007. Mucin granule intraluminal organization. Am J Respir Cell Mol Biol 36: 183190.
  • Pica A, Scacco S, Papa F, De Nitto E, Papa S. 2001. Morphological and biochemical characterization of mitochondria in Torpedo red blood cells. Comp Biochem Physiol B 128: 213219.
  • Picton BE, Howson CM. 2000. The species directory of the marine fauna and flora of the British Isles and surrounding seas and CD-ROM. Belfast and Ross-on-Wye, UK. Ulster Museum and the Marine Conservation Society.
  • Poll M. 1947. Faune de Belgique: poissons marins. Genus Torpedo. Brussels, Musee Royal d'Histoire Naturelles de Belgique. 1. p 8083.
  • Qin X. 2007. Inactivation of digestive proteases by deconjugated bilirubin: the possible evolutionary driving force for bilirubin or biliverdin predominance in animals. Gut 56: 16411642.
  • Radii-Weiss T, Kovacevic N. 1970. Influence of low temperature on the discharge mechanism of the electric fish Torpedo marmorata and T. ocellata. Marine Biol Berlin 5: 1821.
  • Rege RV. 2002. The role of biliary calcium in gallstone pathogenesis. Front Biosci 7: 315325.
  • Reus L, Segal Y, Altenberg G. 1991. Regulation of ion transport across gallbladder epithelium. Annu Rev Physiol 53: 361373.
  • Risso A. 1810. Ichthyologie de Nice ou Histoire naturelle des poissons du Département des Alpes Maritimes. F.Schoell, Paris. p. 2122.
  • Roberts BL. 1969. The buoyancy and locomotory movements of electric rays. J Mar Biol Ass UK 49: 621640.
  • Rogers DF. 1994. Airway goblet cells: responsive and adaptable front-line defenders. Eur Respir J 7: 16901706.
  • Romanelli M, Consalvo I, Vacchi M, Finoia MG. 2006. Diet of Torpedo torpedo and Torpedo marmorata in a coastal area of Central Western Italy (Mediterranean Sea). Mar Life 16: 2130.
  • Rondelet G. 1555. De libri piscibus marinis. France: Université de Montpellier. p 1418.
  • Roussel P, Lamblin G, Lhermitte M, Houdret N, Lafitte JJ, Perini JM, Klein A, Scharfman A. 1988. The complexity of mucins. Biochimie 70: 14711482.
  • Saito Y, Tanaka Y. 1980. Glutaraldehyde fixation of fish tissues for electron microscopy. J Electron Microsc 29: 17.
  • Slingluff JL, Williams JT, Blau L, Blau A, Dick EJ, Jr, Hubbard GB. 2010. Spontaneous gallbladder pathology in baboons. J Med Primatol 39: 9296.
  • Smith BF. 1990. Gallbladder mucin as a pronucleating agent for cholesterol monohydrate crystals in bile. Hepatology 12( 3, Part 2): 183S186S; discussion 186S–188S.
  • Solé M, Antó M, Baena M, Carrasson M, Cartes JE, Maynou F. 2010. Hepatic biomarkers of xenobiotic metabolism in eighteen marine fish from NW Mediterranean shelf and slope waters in relation to some of their biological and ecological variables. Mar Environ Res 70: 181188.
  • Sripadi P, Nazarian J, Hathout Y, Hoffman EP, Vertes A. 2009. In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry. Metabolomics 5: 263276.
  • Städeler G. 1860. On the occurrence of urea in the organs of the plagiostomous fishes. Philos Mag Ser 4 19: 7980.
  • Stehmann M, Bürkel DL. 1984. Rajidae. Paris, UNESCO, Fishes of the Northeastern Atlantic and the Mediterranean (FNAM), Whitehead PJP, Bauchot ML, Hureau JC, Nielsen J, Tortonese E, editors. 13:163–196.
  • Storelli MM, Giuliana Perrone V, Barone G. 2011. Organochlorine residues (PCBs and DDTs) in two torpedinid species' liver from the Southeastern Mediterranean Sea. Environ Sci Pollut Res Int 18: 11601165.
  • Theodosiou NA, Hall DA, Jowdry AL. 2007. Comparison of acid mucin goblet cell distribution and Hox13 expression patterns in the developing vertebrate digestive tract. J Exp Zool B Mol Dev Evol 308: 442453.
  • Treberg JR, Speers-Roesch B, Piermarini PM, Ip YK, Ballantyne JS, Driedzic WR. 2006. The accumulation of methylamine counteracting solutes in elasmobranchs with differing levels of urea: a comparison of marine and freshwater species. J Exp Biol 209: 860870.
  • Trischitta F, Faggio C, Torre A. Living with high concentrations of urea: They can! Open J Anim Sci 2012; 2: 3240.
  • Trump BF, Smuickler EA, Benditt EP. 1961. A method for staining epoxy sections for light microscopy. J Ultrastruct Res 5: 343348.
  • Tyner JW, Kim EY, Ide K, Pelletier MR, Towit WT, Morton JD, Battaile JT, Patel AC, Patterson A, Castro M, Spoor MS, You Y, Brody SL, Holtzman MJ. 2006. Blocking airway mucous cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals. J Clin Invest 116: 309321.
  • Vandenhaute B, Buisine MP, Debailleul V, Clément B, Moniaux N, Dieu MC, Degand P, Porchet P, Aubert JP. 1997. Mucin gene expression in biliary epithelial cells. J Hepatol 27: 10571066.
  • VanKlinken BJW, Dekker J, VanGool SA, VanMarle J, Bűller HA, Einerhand AWC. 1998. MUC5B is the prominent mucin in human gallbladder and is also expressed in a subset of colonic goblet cells. Am J Physiol 274 ( Gastrointest Liver Physiol 37): G871G878.
  • Verdugo P. 1991. Mucin exocytosis. Am Rev Respir Dis 144 ( 3, Part 2): S33S37.
  • Verdugo P, Aitken M, Langley L, Villalon MJ. 1987a. Molecular mechanism of product storage and release in mucin secretion. II. The role of extracellular Ca++. Biorheology 24: 625633.
  • Verdugo P, Deyrup-Olsen I, Aitken M, Villalon M, Johnson D. 1987b. Molecular mechanism of mucin secretion. I. the role of intragranular charge shielding. J Dent Res 66: 506508.
  • Vialleton L. 1903. Les lymphatiques du tube digestif de la Torpille (Torpedo marmorata Risso). Arch Anat Microsc 5: 378392.
  • Viehberger G. 1982. Apical surface of the epithelial cells in the gallbladder of the rainbow trout and the tench. Cell Tissue Res 224: 449454.
  • Viehberger G. 1983. Ultrastructural and histochemical study of the gallbladder epithelia of rainbow trout and tench. Tissue Cell 15: 121135.
  • Vilkin A, Nudelman I, Morgenstern S, Geller A, Bar Dayan Y, Levi Z, Rodionov G, Hardy B, Konikoff F, Gobbic D, Niv Y. 2007. Gallbladder inflammation is associated with increase in mucin expression and pigmented stone formation. Dig Dis Sci 52: 16131620.
  • Walsh PJ, Smith CP. 2001. Urea transport. In: Wright P, Anderson P, editors. Fish physiology: nitrogen excretion. San Diego, CA: Academic Press. p 279308.
  • Ward R. 2006. Obstructive cholelithiasis and cholecystitis in a keeshond. Can Vet J 47: 11191121.
  • Watson RR, Dickson KA. 2001. Enzyme activities support the use of liver lipid-derived ketone bodies as aerobic fuels in muscle tissues of active shark. Physiol Biochem Zool 74: 273282.
  • Wickström C, Davies JR, Eriksen GV, Veerman ECI, Carlstedt I. 1998. MUC5B is a major gel-forming, oligomeric mucin of human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage. Biochem J 334: 685693.
  • Wilhelmi M, Mock MR, Zuendt B, Meyer G, Macrides TA, Wright PFA, Jungst D. 2003. The hepatoprotective shark bile sterol, scymnol: a potential nucleation inhibitor of cholesterol crystals in bile. 5th Congress of Toxicology in Developing Countries. R114 (abstract).
  • Windaus A, Bergmann W, König G. 1930. Über einige Versuche mit Scymnol. Z Physiol Chem Hoppe-Seyler's 189: 148154.
  • Withers PC, Morrison G, Hefter GT, Pang T-S. 1994. Role of urea and methylamines in buoyancy of Elasmobranchs. J Exp Biol 188: 175189.
  • Wood CM, Kajimura M, Bucking C, Walsh PJ. 2007. Osmoregulation, ionoregulation and acid–base regulation by the gastrointestinal tract after feeding in the elasmobranch (Squalus acanthias). J Exp Biol 210: 13351349.
  • Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN. 1982. Living with water stress: evolution of osmolyte systems. Science 217: 12141222.
  • Yung E. 1899. Recherches sur la digestion des Poissons. Arch Zool Exp Gen (3e Série) 7: 121201.