The Gallbladder of Uranoscopus Scaber L. (Teleost Perciform Fish) is Lined by Specialized Cholecystocytes

Authors

  • J. Gilloteaux,

    Corresponding author
    1. Department of Anatomical Sciences, St George's International School of Medicine, Newcastle-upon-Tyne, United Kingdom
    2. Department of Surgery, Summa Research Foundation, Akron, Ohio
    • Department of Anatomical Sciences, St George's International School of Medicine, Drill Hall 013, Northumbria University—School of Life Sciences, Newcastle upon Tyne, NE1 8ST, United Kingdom
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    • Fax: 191-227-4824; Tel: 44-0-191-227-4855

  • Donald W. Ott,

    1. Department of Biology, The University of Akron, Akron, Ohio
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  • Carla K. Oldham-Ott

    1. School for Professional Studies, Walsh University, Akron, Ohio
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Abstract

The gallbladder of Uranoscopus exhibits a mucosal surface layer of simple columnar epithelium composed of specialized cholecystocytes. The apices show storage and mucous secretions, typical microvilli, and very apical projections extending deep into the luminal contents. Many organelles and heterogeneous vesicles of diverse size fill the cytoplasm, including neutral mucins, mitochondria, peroxisomes, lysosomal bodies, and lipid-rich deposits with cholesterol inclusions. The fibromuscular layer shows little blood supply and contains scattered lymph-like walls with minute cholesterol inclusions. The remaining muscular, subserosal, and serosal or adventitial layers of this species do not show any histologic differences to those of other Vertebrates. It was unexpected to find cholesterol inclusions in the fatty deposits of the cholecystocytes, similar to those noted in human cholesterolosis and in some forms of hypercholesterolemia, in this teleostean. In addition, aggregations of mitochondria and anomalous mitochondrial morphologies were found that resemble oncocytoma-like changes. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.

During our ongoing laboratory research studies to understand the origin of gallstones in humans, a preliminary, comparative morphological study of the bile tract was undertaken in several vertebrate representatives (Oldham-Ott and Gilloteaux, 1997) as part of a literature review of the chordates verifying the presence or absence of a gallbladder in various species. We also studied various fish gallbladders (Gilloteaux et al., 1995, Oldham-Ott and Gilloteaux, 1997) where it was reported that the most complex epithelial morphologies were observed in fishes, probably as adaptations to their respective diets.

More specifically, this report shows that in the teleost genus Uranoscopus, the gallbladder epithelial cells or cholecystocytes contain a complex population of cell organelles that may be involved not only in bile concentration, but also in modification of bile content. The lipid content, abundant to enormous aggregates of mitochondria, lysosomes, peroxisomes, and secretory mucous vesicles are coupled with a mucosal surface of pleiomorphic folds covered with microvilli and long, apical projections. Some lipid deposits contained cholesterol deposits similar to those described in human cholesterolosis (Nevalainen and Laitio, 1972; Koga et al., 1975; Koga, 1985; Gilloteaux et al., 1997, a) and in some forms of cholecystitis (Gilloteaux et al., 1997, b, 2003, 2004).

MATERIALS AND METHODS

Six specimens of Uranoscopus scaber L. (Teleostean, Acanthopterygian perciform fish, also known as U. bufo, U. occidentalis, or Atlantic stargazer), 17–24 cm long, male and female (3:2; male were usually of smaller size) along with other representatives of 12 species of fishes were captured 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 (at the edge of the National Biological Marine Reserve). Following capture, all fishes were kept and transported in running sea water tanks and, less than an hour later, were placed in the laboratory aquaria where, after decapitation, they were dissected following our previous protocol (Oldham-Ott and Gilloteaux, 1997). One specimen was discarded, because it contained a parasitic worm. From all remaining specimens, several organs, including gallbladders, livers and hearts, were excised and cut into small pieces to be fixed for 1.5 h duration in 3% buffered glutaraldehyde (0.1 M sodium cacodylate) diluted in seawater (1:3 [v :v] by distilled water) adopting protocols from Anderson and Personne (1970), Saito and Tanaka (1980), and Griffith (1981). A 30 min wash in the same buffer mixed with diluted sea water was followed by postfixation with 2% OsO4 aqueous solution for 2-hr duration. Tissues were then washed in buffer containing 10% sucrose and dehydrated by graded alcohols. For scanning electron microscopy (SEM), samples were critically point dried and coated with a 15–20-nm thick gold layer. For transmission electron microscopy (TEM) samples were embedded in Polybed 812 epoxy resin (Polysciences, Warrington, PA). Of those 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, 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

General Aspects

In this teleost fish, LM shows a large, 1.5–2.5 cm diameter, spheroid-shaped gallbladder with a wall thickness ranging from 0.5 to 1.5 mm. The 1.0-μm plastic thick sections show a mucosal surface of innumerable, small bulging folds covered by an epithelial surface (Figs. 1, 2). The surface epithelium is a simple columnar epithelium appearing more heavily contrasted with toluidine blue than the other constitutive, adjacent tissues of its wall. The fibromuscular layer (lamina propria and submucosal layer in the gallbladder) is ∼900–1,200-μm thick comprising a lamina propria of 200–500 μm in thickness that appears to occupy more than half of the wall width of the organ and is made of a loose connective tissue matrix containing slender fibrocytes and smooth muscle fibers. A 200–300 μm thick, external muscular layer is made of more or less tightly entwined smooth muscle bundles invaded by few 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 outermost, subserosal layer is less than 15-μm thick and is mainly composed of mesothelium. In the area adjacent to one of the liver lobes, the spheroid gallbladder also shows an adventitial layer. It is also quite common to detect pieces of pancreatic lobes attached and sharing the same adventitial layer and mesothelial covering. The subserosal layer is thin and can be resolved as a few microns in thickness, sometimes displaying superficial blood vessels of varying diameter.

Figure 1.

One-micron thick section of Uranoscopus gallbladder wall stained with toluidine blue reveals the highly contrasted, granular appearance in all upper regions of the rugged epithelium lining the lumen, the thick lamina propria and the fibromuscular layer with its subserosal and serosal layers. Bar = 100 μm.

Figure 2.

A–D: One-micron thick sections of Uranoscopus gallbladder stained with toluidine blue illustrate its mucosal layers. A–C: Transmitted light demonstrates the abundant population of dark-blue purple granules in the upper regions of the cholecystocytes' cytoplasm while the other areas, poorly contrasted, contain flat green inclusions. A loose extracellular matrix and thin fibroblasts are filling the thick lamina propria with a dispersed vascular supply. The irregularly shaped apical surfaces can show elongated extensions (in A) or conical ones with a subjacent narrow, greenish stained zone (in B). C: A complex fibrocytes/macrophage structure contains small dark granules in transmitted light appears in D with phase contrast as brilliant granules of cholesterol (small arrows) located in the same structure. Bars = 10 μm.

Further observations of the thick sections show the epithelial surface of the mucosa composed of cholecystocytes ranging from 25 to 45 μm in height and from 4 to 5 μm in width whose apices appear as if eroded by abundant, exocytotic activities, shaping the bulging apices into irregular, smooth to acute or even jagged, cell shapes (Fig. 2). The cells are darkly contrasted by the presence of heterogeneous, densely stained granules in the supranuclear regions. Some of the apices have long, pointed, fuzzy surface extensions. Most of the apical or supranuclear cytoplasmic regions appear either empty or filled with small, pale green granules, giving a poorly stained aspect to the near apical region (Fig. 2B). Innumerable, darkly purple to blue-stained granules of diverse size, from a fraction of a micrometer to 1.5 μm in width, can be seen throughout most cholecystocytes. In some areas, subnuclear and basal regions of the same cells can contain small vacuolated areas and occasional small to large dark purple to blue granulations (Fig. 2A,B). Among them, one can again detect round to ovoid, flat green inclusions or granules. The euchromatic nucleus is often located in the mid to lower third of the cytoplasm surrounded by a few of the dark, but smaller granules seen earlier (Fig. 2A,B). One can observe the fibromuscular layer constitutive cells and matrices as mainly composed of a loose connective tissue represented by a network of poorly represented lamina propria-like regions supplied by loosely dispersed capillaries and other vessels (Figs. 1, 2A–D). The loose stroma is mainly a spongy matrix type composed of a ground substance admixed with a great deal of faintly contrasted, fuzzy to fibrous material that is organized around what appears to be an entwined network of long and slender fibroblasts or, as seen with the TEM (see next paragraph), slender smooth muscle fibers. In the same layer, one can also detect a few large and branching, fibrocytes or macrophages filled with tiny, brilliant inclusions (Fig. 2C). Most of those appear under immersion oil as small cholesterol or cholesterol ester-rich inclusions with a birefringent Maltese cross appearance under polarized light; Figure 2D illustrates a phase contrast view.

Ultrastructural Aspects (Figs. 3–17)

Viewed with TEM, all the cholecystocytes demonstrate their pleiomorphic apices as bulging into variable, smooth to acute or even jagged, shapes (Figs. 3–6). The cholecystocytes are attached to a thick basal lamina (Figs. 3 and insert, 4, 7) composed of a thin lamina rara of 20–30 nm wide and a thick, 200-nm wide, lamina densa component. Other TEM views show a loose network of narrow smooth muscles or myofibroblast-like cells near a subepithelial blood capillary filled by an erythrocyte, indicating that not all narrow cells found with LM are fibroblasts (Fig. 7).

Figure 3–4.

Fig. 3 (with insert) TEM aspects of Uranoscopus scaber L. gallbladder epithelium. Low magnification of the simple columnar epithelium with a diverse population of organelles and inclusions. Arrows indicate clavate and conical-shaped apices. The insert shows a narrow zone of the apical region with short microvilli and the round, fatty deposits among small, apical mucous secretory vesicles. Notice the patchy-dark contrasted lysosomes, having spheroid, oval, and irregular profiles, located above the nucleus. The remaining, near apical cytoplasmic zones are finely punctuated by minute contrasted, peroxisomes, while the lower cytoplasmic domains have small vacuoles and infrequent, tiny contrasted organelles. A continuous thick basal lamina can be detected below the photomontage line cut while in the lamina propria a few smooth muscle or fibrocytes profiles are noted with a capillary. Bar in 3 and 3 insert = 1 μm. Fig. 4. TEM aspects of Uranoscopus scaber L. gallbladder epithelium. Basal lamina thick lamina is illustrated. Bar = 200 nm.

Figure 5–6.

TEM of Uranoscopus gallbladder. Fig. 5. TEM view of a clavate and adjacent, conical-shaped edges of Uranoscopus cholecystocytes' apices with dispersed mucous containing vesicles (m) and short, thin and thick, long microvilli. Near the apices, the mucus-containing vesicles (m) are surrounded by small, darkly contrasted organelles and intermingle between mitochondria and large fatty deposits (l). A short junctional complex extends into a series of small desmosomes (d arrowed) between adjacent cholecystocytes. Bar = 1 μm. Fig. 6. TEM of Uranoscopus gallbladder. SEM view of apical surfaces showing minute microvilli and at least one long, tall microvillus (open arrow). A suggestive, exocytotic mucous site (crater-like [*]) can be seen among the clavate apices. Scale bar = 1 μm.

Figure 7–9.

TEM of Uranoscopus cholecystocytes: apical organelles and inclusions. Fig. 7. The crowded cells are lined on a thick basal lamina. Numerous fatty droplets can be viewed among mitochondria and more or less densely contrasted lysosomal bodies, usually in the form of stippled vesicles. A loose network of narrow smooth muscles is found near a subepithelial blood capillary filled by an erythrocyte (E). Both Figures 7 and 8 notice the numerous parallel line-like, artifacts created across the micrographs caused by tiny, hard to cut cholesterol microcrystalline deposits located in the lipid inclusions. Fig. 8. TEM of Uranoscopus cholecystocytes: Apical organelles and inclusions. Square-shaped profiles of lipid deposits (arrows). An alignment of irregular and oblong densely contrasted bodies can be found near the cell apices, lysosomes and mitochondria surrounded by innumerable cytoskeletal filaments. Fig. 9. TEM of Uranoscopus cholecystocytes: Apical organelles and inclusions. A detailed view of adjacent cells with lipids, SER and lysosomes attached by sets of desmosomes (d). All scale bars = 1 μm.

Figure 10–13.

TEM of Uranoscopus cholecystocytes. Diverse aspects of lysosomal bodies detected as apical densely contrasted bodies, contained in a tapered, apical cell region showing rare typical microvilli (with enlarged views in Figure 11 and insert) and its tall microvilli, thicker and longer than the typical microvilli. Figures 12 and 13 display other examples of lysosomes located in apical zone. Fig. 13. The cell apical remnant shows peroxisomal bodies located at the edges of areas that had released mucous vesicles, creating a bizarre, fan-like bursting apex. All views contain numerous cytoskeletal filaments, arrowed in Figure 11. Scale bars in Figures 10, 12, 13 = 1 μm; in Figure 11 and insert, the bar = 200 nm.

Figure 14–16.

TEM of Uranoscopus cholecystocytes. Fig. 14. A few short microvilli coat the apex of adjacent cells. An alignment of underlying lysosomal bodies surrounded by cytoskeletal filaments is also covering a large region of the subapical cytoplasm domain with numerous mitochondria with a few fatty droplets. Fig. 15. Enlarged view of 14 showing small and large, oblong lysosomes or residual bodies contacting fatty droplets with cholesterol curved crystals. Two open arrow mark curved, narrow cholesterol deposits in lipid droplets. Fig. 16. Arrows point to a lipid deposit contacting an altered mitochondrion. The adjacent plasma membranes (bottom right) suggest the existence of a narrow junction (possibly gap junction) between the cells. All bars = 1 μm.

Confirming the light microscope observations, the morphology of the upper region of most cholecystocytes depicts irregular folds, with acute profiles, with apices flat to jagged in appearance, or with the apices showing an elongated, clublike profile or clavate aspect (Figs. 3, 5, 10, 13). The apical cell surfaces are usually covered by a discrete glycocalyx and by very short microvilli that are usually not more than 0.5 μm in length (Figs. 3 insert, 6, 8, 14). However, the jagged edges of apices bear long, thick, and slender microvillar projections of more than 10–15 μm in length that reach deep into the gallbladder lumen. These huge microvilli-like projections are tenuous, somewhat twisted or undulating, without microtubules and run along the narrow edges of the cholecystocytes (Figs. 5, 6, 10). The views noted with TEM show a blunt aspect caused at some stage of the SEM preparative procedure, especially at the necessary step of critical-point drying as those reach deep into the more viscous, gallbladder content.

The epithelial cells usually show a typical glycocalyx and a dense cytoskeletal internum (Figs. 10–13, the apical edges appear jagged from the abundant exocytosis of mucous vesicles.

Figure 17.

TEM view of apical and upper perikaryal regions of adjacent, Uranoscopus cholecystocytes. Notice the mucous secretory vesicles and the microvilli surrounding the jagged apical cell edge (seen on the upper right) of the epithelium. Desmosomes (d) enhance the cells' boundaries. Huge aggregates of mitochondria surround rare lipid deposits but also include several large, modified mitochondria (arrowed) containing thick, wavy, branching, or straight (triple headed arrow), distorted tubular cristae produce enormous heterogeneous bodies (e.g., right side of micrograph). Bar = 1 μm.

The overall cytoplasm of the cholecystocytes is very crowded by organelles. Throughout the cholecystocytes, these organelles or inclusions are arranged into discrete cytoplasmic domains that are not well demarcated but possibly show some distinct architectural distribution, as their location may relate to their function(s) as detailed in the following paragraphs. The most superficial and apical of these are often small, densely contrasted organelles that can be curved in shape; others are mucin-containing secretory vesicles, surrounded by mitochondria and other small cell organelles, such as smooth endoplasmic reticulum. Located more deeply are lysosomes and peroxisomes interspersed among small to large fatty inclusions that can reach several micra in diameter. Adjacent to those small dense vesicles, one may see a layer of small or large diameter mucinous vesicles admixed with small mitochondria (Figs. 5, 14, 15). After the mucous vesicles are secreted from these apices, other apical and near apical small vesicles can become more easily differentiated among these crowded edges (Figs. 10, 13).

SEM views (e.g. Fig. 6) confirm the diverse morphology encountered with light and TEM of the mucosal folds and epithelial surface cells and reveals the diverse three-dimensional aspects of the apices that appear as irregular, acute or filiform, rugged, or even cone-shaped. Among the conical or clavate apical surfaces are craters similar to the one labeled in Figure 6 (star-labeled), probably resulting from a large mucinous exocytosis and some slender and long microvilli (Fig. 6, marked by an open arrow).

Near the apices of adjacent cholecystocytes, one can distinguish typical, short junctional complexes (near apical tight junctions with zonulae adherentes) and the basolateral sides of the cholecystocytes connecting with gap junctions. Often long series of small desmosomes that maintain the mechanical integrity of the rugged surface epithelium can be found (labeled d in Figs. 5, 9, 14, 15, 17). The same epithelial cells also interconnect throughout their basolateral domains with short digitations (Figs. 3, 7).

The Organelles and Inclusions of the Cholecystocytes

The crowded population of organelles of these epithelial cells is unique, morphologically. The subnuclear cytoplasmic domain seems to be essentially occupied by typical organelles, including small mitochondria, dilated smooth endoplasmic reticulum that takes the form of more or less large vacuoles, mucous vesicles, and numerous irregularly shaped lysosomal bodies that appear as the darkest contrasted bodies found with LM. In addition, small densely contrasted inclusions, large droplets surrounded by halos, and a myriad of intracellular fine granulations, mainly representing the cytoskeletal components, can be found in the perinuclear and supranuclear locations. It is in the perinuclear and supranuclear locations that the diversity of structure in the cytoplasm makes this fish gallbladder unique and would suggest further detailed, cytochemical investigations.

Peculiarities of Organelles and Inclusions in Uranoscopus Cholecystocytes

Lysosomes.

Because we observed a large number of intensely stained granules in cholecystocytes in LM 1-μm thick sections and TEM using toluidine blue staining (Figs. 1, 2), our attention focused on verifying their morphology and eventually identifying them as lysosomal bodies. The lysosomes appear mainly small and spherical, but can be oblong, densely contrasted bodies of more than 200 nm in diameter intermingling between mitochondria and large fatty deposits. Lysosomal aggregations with lipids also can be seen (Figs. 7–9). The largest of the lysosomes are oblong, with fine to thick, longitudinal to oblique striations as illustrated in Figures 7–12, 15; the densely contrasted contents of the lysosomal bodies make more evident their lightly contrasted needle-like, twisted, or curly needle-like shapes. This heterogeneous content is not aligned in parallel to an axis of the organelle, but instead is haphazardly organized, even comprising curved shapes within these cells' vesicles. The apices of these large lysosomal bodies appear to extend into the cytoplasm with a twisted end (Fig. 8), and their resemblance with those described in the human gallbladder is quite striking (Gilloteaux et al., 1997, a, b, 2004).

Mitochondria.

Most of the cholecystocytes' mitochondria appear small, less than 1 μm in overall length and even smaller in diameter. They are either isolated or in clusters between the lysosomal bodies, the other organelles (Figs. 5–7, 15) and the lipid inclusions (Fig. 10) or, as in some areas of the epithelial cells or also among epithelial cells, as enormous aggregates, resembling those found in oncocytic cells (Figs. 7, 14, 17). It is possible to identify them by their typical cristae. The morphology of these mitochondria does not always correspond to classic morphology and some appear to contact the lipid profiles (Figs. 14–17). Especially in Figures 16 and 17, the delineating boundaries of the organelles or the inner structures are altered whether by fixation and/or processing or by the dynamic process of contact within the cells in which they are located. In Figure 17, some of the aforementioned unusual features are indicated by single and multiple arrows. The triple-headed arrow marks a huge, altered mitochondrion, wherein the cristae appear twisted, branching or with tubular-like aspects; it is found in one of the enormous mitochondrial aggregates.

Peroxisomes or microbodies.

These small organelles, ranging from 100 to 300 nm in diameter, are mainly found aligned and distributed in rows, near the cell apices (Figs. 10–13). We also observed these organelles remaining at the edges of the cytoplasm, after mucous vesicles and other cell components were released. Bizarre tree- or fan-shaped cell apices result, where emptied spaces are encircled by small dense bodies that are not released by the cells (Fig. 13) and probably maintained intracellularly by the abundant cytoskeleton as illustrated in Figures 10, 11. The cells responsible for this peculiar morphology are not apoptotic, and this phenomenon is restricted to the apical regions of those cells; this may reflect some apocrine-like secretion as is found in most vertebrates gallbladders (Oldham-Ott and Gilloteaux, 1997).

Lipid inclusions.

Most lipid deposits appeared greenish when stained with Toluidine blue and were found throughout the mucosal cholecystocytes (Figs. 14–17); these lipid inclusions ranged from 0.2 to 1.5 μm in diameter. Some micrographs of lipid inclusions show an asymmetric, superficial halo (Fig. 3, 4, 15). Their fine morphology suggests they contain triglyceride, but, using higher TEM magnification, there are also numerous, fine to rough, parallel lines across the field of view from the same specimen. These scratches are created by tiny, hard-to-cut microcrystalline cholesterol deposits located in the lipid inclusions (Figs. 7, 8); most of them can be shown to have an electron dense outline, created by the contrasting salts revealing their quasi square-shaped profile (Fig. 8). The contents of the lipid inclusions included extremely hard and poorly contrasted, spheroid to needle-shaped or long whorl-shaped crystals (Figs. 7, 8, 9, 15, 17). The smallest is detected by a contrasted halo enhancing the lack of contrast of the tiny, 20–50 nm deposits (Figs. 8, 9). A curved structure, indicated by an open arrow, in a large fatty inclusion indicates a curved cholesterol deposit within a lipid droplet (arrow in Fig. 8). Interactions with organelles, lysosomes, peroxisomes, and mitochondria were mentioned earlier. In Figure 15, a large electron dense body illustrates the final stages of degradation of a cholesterol/mitochondrion complex, perhaps corresponding to autophagocytosis.

DISCUSSION

Uranoscopus scaber L. is a member of a family of 49 species in the Uranoscopidae. It is a Teleost fish (Actinopterygian Perciform, also called U. bufo, U. occidentalis, or Atlantic stargazer). It is a benthic, stenohaline, demersal species found throughout the sea floor of the continental shelf or in deep waters of the Mediterranean and Black Seas (Demestre et al., 2000) as well as large parts of the Atlantic Ocean. Usually, it buries itself in sandy or muddy bottoms at variable depths ranging between 15 to 400 m. The stargazer has no swim bladder, needing little effort to remain in lower depths. Its main body is totally hidden except for two protruding eyes, often rapidly withdrawn, that can be seen by a diver. It is a uniquely carnivorous feeder (Boundka and Ktari, 1996; Papoutsouglou and Lyndon, 2003). The mouth opens upwardly and attached to the lower jaw is a vascular strip, a projectile appendage, which darts out and serves as a lure to capture prey alive. The prey are commonly small fishes, crustaceans, or, occasionally, mollusks swimming nearby (Young, 1931). All prey is sucked and swallowed at once without chewing. Aiding in its predatory activities, this fish is also equipped with venomous glands and an acoustic apparatus that generates acoustic and electric pulses (Mikhajlenko, 1973; Pietch, 1989).

The specimen size captured in the Banyuls area agrees with data of sizes collected by others, including a recent survey reporting this species as a small part of the fish market in Western areas, though not as much in the Eastern Mediterranean (Rizkalla and Bakhoum, 2009). Although stenohaline, this fish is able to osmoregulate easily after stress or under long-term physiologic experiments; that is, after 30 min, it was shown to adapt from seawater to freshwater (Motais et al., 1960). Like several other demersal fishes, species-specific Acanthostomids parasites can be located in the gallbladder and intestine (Bartoli and Gibson, 2000). In fact, one of our specimens did contain a long Anisocladium fallax Rudolphi and, at the time, that gallbladder sample was discarded from the studied group to avoid describing a potential, abnormal histological structure.

Uranoscopus scaber is exclusively carnivorous; it “relies on protein and lipid rather than carbohydrate digestion for its metabolic needs” (Papoutsoglou and Lyndon, 2003). Analyzed for α-amylase activity, its digestive tract showed the weakest activity when compared with other carnivorous fishes (Papoutsoglou and Lyndon, 2003). At the same time, this species has a large muscular stomach (64% of gut weight) while displaying a typical short intestine but a large pyloric caecum to allow for a slow, digestive transit time (Buddington et al, 1987). These anatomical and physiological adaptations allow this species to manage the digestion of prey swallowed whole, without chewing.

Uranoscopus gallbladder histology is quite typical in its mucosa, fibromuscular, and serosal or adventitial layers, whether or not found attached to the liver adventitial layer. The fibromuscular layer, 200–300 μm thick, has longitudinal, oblique, and cross-sections of smooth muscle bundles, similar to many vertebrate gallbladders (Oldham-Ott and Gilloteaux, 1997). An unusual finding is the presence of a structure, possibly fibroblasts or macrophages laden with lipid enriched by cholesterol, in the same micrographic views as those collected by Roth et al. (1988) in the cornea of patients afflicted by lipid keratopathy and in macrophages of atherosclerosis (Stary, 2000).

The sharp, hard, curved materials found in areas of crowded fatty deposits, that is, the artifacts damaging the ultrathin sections are in all probability cholesterol crystals; these curved objects are similar to crystallizing cholesterol deposits shown by Kaplun et al. (1997) in in vitro bile experiments. Other cholesterol deposits can be fibrillar or curve-shaped accretions in lysosomes (Figs. 15, 17) and resemble morphologically the intracellular types detected in cholecystitis (Kouroumalis et al., 1983b; Gilloteaux et al., 1997, b, 2003, 2004) or cholesterolosis (Nevalainen and Laitio, 1972; Koga et al., 1975; Koga, 1985; Gilloteaux et al., 1997, a, 2003, 2004; Satoh and Koga, 1997). Some of those can reach more than 2 μm in diameter and are usually oblong in shape. The cells also contain mitochondria and peroxisomes in their remaining cytoplasmic spaces as well as in a thin apical zone of each cholecystocyte. Other organelles, heterogeneous in their content, are likely lysosomal bodies.

Although the topographical staining used in 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) and lipids (greenish hue) as well as for lysosomal bodies (dark purple). Deposits appearing dark or flat green in multiple places correspond to observations made by Aparicio and Marsden (1969) and De Martino et al. (1968) of similar fatty deposits stained greenish; in contrast, lysosomes and peroxisomes, rich in proteins and associated charged compounds, usually appear dark violet in many regions of the epithelium (Figs. 15–17) and that mucous bodies are also containing acid polysaccharides (Hirsch and Peiffer, 1957; De Martino et al., 1968; Parsons et al., 1982; Hayat, 1993; Stypmann et al., 2002). Similar bizarre lipid-mucus and lysosomal associations were detected earlier in cholecystitis (Gilloteaux et al., 2003). In Figures 10, 12, 15, the illustrated lysosomal bodies demonstrate their pale-contrasted needle, curved, twisted, or curly needle-like shapes among their electron heterogeneous content; again, these are similar to those observed in cholecystitis (Gilloteaux et al., 2004).

Even though the peroxisomes were not confirmed here by cytochemical methods, their morphology and location is similar to those in the cholecystocytes of other fish species, although they are apically aligned and more abundant. When their electron density is less opaque, one can also find a straight, twisted, or curved, narrow crystal of catalase (Baumgart et al., 1997). Their apical and near apical location is typical for the bile tract as the same kind of peroxisomes are always found associated with the apices of cells in developing and differentiated bile canaliculi (Keller et al., 1993), bile ducts and in the mammalian gallbladder epithelium (Depreter et al., 2002; Kojima et al., 2003); they are similarly located in the gallbladder of other fish species (Kramar et al., 1974; Ruiter et al., 1985, 1988). The peroxisome-mitochondria association is a metabolic requirement in all cells as the peroxisomes offer protection against oxidative species and assist in oxidations of long lipids, while the mitochondria are needed for the oxidative phosphorylation pathways and, in association with peroxisomes, to perform some β-oxidation and other metabolic functions while “protecting” the cells against toxic bile acids in this large gallbladder. One could speculate that their structural and functional association could play several roles: (i) along with mucus, an antioxidant, protective role of the superficial surfaces (tight junctions and associated cytoskeleton; Vassy et al., 1997) vis-à-vis the cytotoxicity of bile salts for the superficial cells' structures; (ii) they are involved in the synthesis of very long chain fatty acids, especially those of C27, which are also highly toxic (Ferdinandusse and Houten, 2006, Ferdinandusse et al., 2009) as components of fish bile; (iii) as in all cells, to harness the β-oxidation of unsaturated long chain fatty acids (Hiktunen, 1991) along with the mitochondria and smooth endoplasmic reticulum, ultimately producing ATP (Nelson and Cox, 2005). However, in areas where mitochondria are numerous, a decreased number of peroxisomes usually occur in lipidosis and the mitochondria in these anomalies show cristae assuming a variety of shapes, length and orientations (Center et al., 1993), similar to our findings (Fig. 17).

Concerning the odd morphology of the mitochondria, they are similar to those from other seawater teleosts (Morrison et al., 1996; Gwo et al., 2004) as shown in several other publications; freshwater fish tissues usually demonstrate a better preservation of organelles than those obtained from sea water, even though care is taken to maintain a proper osmolarity (Ruiter et al., 1985, 1988). Alternatively, the mitochondrial structure could have been altered by their function in this fish's gallbladder; they resemble aggregates observed in oncocytomas, having distorted cristae and a swollen aspect and being located amid mucous vesicles of oncocytic carcinoma cells of the thyroid (i.e., in Fig. 7 in Uccella et al., 2000) or Hürthle cells (Sobrinho-Simões et al., 1985). In addition, the contacts between lipid droplets and mitochondria appear in acute CCl4 poisoning as noted by Cheville (1983) as well as those reported in a former contribution described as lipo-mucosomes (Gilloteaux et al., 2003).

The gallbladder with its high lipid content and its cholesterol deposits may reflect the diet of prey rich in lipids, as seen in the liver metabolism and blood content of this fish during its digestive functions (Center et al., 1993). The fatty acid levels in the blood are relatively low while the diet is lipid-rich (Plisetskaya, 1980), and, probably as in other Perciforms, the main bile components are phospholipids, cholic, and chenodeoxycholic acids along with 5β-bile alcohols (Moschetta et al., 2005; Hagey et al., 2010). Cholesterol nuclei could develop more easily from bile and cholecystocyte transport, because phospholipids appear generally absent in the bile of bony fish and most birds, while in mammals, the phospholipid to bile ratio varies widely among species. In addition, adapted as it is for opportunistic feeding, the liver becomes an adjuvant to the pyloric caeca and could contribute efficiently to maintain low circulatory cholesterol level after meals.

Having studied and surveyed the same organ in omnivorous types of fish and other vertebrates (Gilloteaux et al., 1995; Gilloteaux, 1997; Oldham-Ott and Gilloteaux, 1997), we were curious as to how this gallbladder would be organized morphologically, considering this species with its exclusively carnivorous and predatory habits. This gallbladder histological architecture corresponds well to the classic pattern as described in most vertebrates (Oldham-Ott and Gilloteaux, 1997). However, the peculiar cellular morphology and content of the gallbladder mucosal epithelial cells probably correspond with the physiology and niche habits of this species of perciform.

The peculiar morphology of the mucosa of this fish gallbladder suggests that the organ of this predatory fish may be, as in most vertebrates, just a reservoir of concentrating bile. However, the presence of cholesterol crystals and deposits within fatty droplets and others in the lamina propria makes us raise the possibility that human diseases duplicate some ancient normal structures; further study of the peculiar and complex Uranoscopus cholecystocytes is indicated, as the cells of this organ bears a startling resemblance to alterations found in several human diseases, including cholesterolosis (Nevalainen and Laitio, 1972; Koga et al., 1975; Koga, 1985; Gilloteaux et al., 1997, a, b; Satoh and Koga, 1997) and cholecystitis (Gilloteaux et al., 2003, 2004). It is also probable, as in human pathologic cholecystitis and cholesterolosis, that this species modifies the bile to its advantage by producing large amounts of mucins and, by the local high metabolism (mitochondria, peroxisomes) and liberating membranes, that is, where alkaline phosphatase is usually located in the bile, this reservoir to concentrate bile can alter its lipid composition (e.g., Glikerman et al., 1997; Tilvis et al., 1982; Kouroumalis et al., 1983a).

In fine, one can suggest from our description that this organ has morphologic peculiarities, but it is adapted to concentrate and liberate bile in huge quantities for the irregularly acquired, but always rich in lipids and proteins, feeding bolus, whether the meal is large or small. The chance of acquiring prey on the sea bottom is erratic, but the animal is ready for the opportunity.

Acknowledgements

The authors thank the staff of the Observatoire Océeanologique of Banuyls (France) of the Université Pierre and Marie Curie (Paris VI) in the collection of live specimens. Summa Research Foundation, Akron OH and the Thomas R. Kelly M.D. funds are recognized for financially assisting in this research topic continued as a scholarly activity of J.G. during summers 2010–2011, with the administrative support of S. Schmidt, Ph.D., Director of Research Foundation. Mr. S. Getch, Communication Specialist, Summa Health System, Akron, OH, is also thanked for his expert assistance in the final imagery.

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