THE ANATOMICAL RECORD 296:79–95 (2013)
The Gallbladder of the Electric Ray
Torpedo marmorata Risso Displays
Excrescent Cholecystocytes with
Merocrine and Apocrine-Like Secretions
1
J. GILLOTEAUX,1,2* DONALD W. OTT,3 AND CARLA K. OLDHAM-OTT4
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
3
Department of Biology, The University of Akron, Akron, Ohio
4
School for Professional Studies, Walsh University, Akron, Ohio
ABSTRACT
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
C 2012 Wiley Periodicals, Inc.
cholelithiasis. Anat Rec, 296:79–95, 2013. V
Key words: gallbladder; cholecystocytes; lysosomes; mucus;
Torpedo marmorata; electric ray; elasmobranch
fish
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,
C 2012 WILEY PERIODICALS, INC.
V
Grant sponsors: The Summa Research Foundation (Akron
OH); Thomas R. Kelly M.D. Funds (Department of Surgery);
St. George’s University School of Medicine, Department of
Anatomy.
*Correspondence to: Professor J. Gilloteaux, St Georges’
University International School of Medicine, Department of
Anatomical Sciences, Drill Hall 013, UNN—School of Life
Sciences, Newcastle upon Tyne, NE1 8ST, United Kingdom.
Fax: 44-191-2274824. E-mail: jgilloteaux@sgu.edu
Received 14 August 2012; Accepted 10 October 2012.
DOI 10.1002/ar.22621
Published online 23 November 2012 in Wiley Online Library
(wileyonlinelibrary.com).
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GILLOTEAUX ET AL.
2003, 2004) and in the Syrian hamster gallbladder
following treatment with sex steroids (Gilloteaux et al.,
1992, 1993a,b).
MATERIALS AND METHODS
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 Beart 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-lm-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
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 lm 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-lm-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 lm in
height and 5-lm 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,
Fig. 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-lm-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
lm; in B–D scale bars equal 25 lm; in E, scale bar equals 10 lm.
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GILLOTEAUX ET AL.
such as lymphocytes, that can be found throughout the
area. These surveillance cells can be found near the basal or in the basolateral aspects of the epithelium (Fig.
1D–E).
The fibromuscular layer was 150- to 250-lm thick,
with the lamina propria of 70–120 lm 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-lm 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 lm in length and 10–
15 lm in width were the dominant components, along
with a few other circulating cells. The smooth muscles
appeared as long tapered cells with undulating features, probably caused by tissue processing and/or the
dissection and preparation for morphologic analysis
(Fig. 2B).
The outermost, subserosal layer ranged from <15 to
50 lm 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
lm 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 selfexcision. Throughout the epithelium little debris or
pieces of cells that were detected were seen loosely dis-
persed near or in the grooves of the gallbladder folded
surface epithelium (Fig. 1B–E) and, at SEM level, similarly located attached to microvillar surfaces (Figs. 1A
and 3A–C).
Transmission electron microscopy (TEM). 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).
The cytoplasm was filled with populations of numerous, mucous-rich vesicles, 0.8–1.9 lm 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 lm, 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
TORPEDO GALLBLADDER MUCOUS EXCRESCENCES
Fig. 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
83
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 lm; in B and C
it equals 10 lm.
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GILLOTEAUX ET AL.
Fig. 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 lm.
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.
Fig. 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 lm. 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 lm.
TORPEDO GALLBLADDER MUCOUS EXCRESCENCES
Fig. 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
85
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 lm; in B is 1 lm, C and D is
5 lm.
Fig. 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: Lysoso-
mal 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 lm.
TORPEDO GALLBLADDER MUCOUS EXCRESCENCES
87
Fig. 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 lm;
bar in C equals 1 lm.
Damage and Repair of the Gallbladder
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
In some rare areas of the gallbladder, TEM observations showed a disorganized epithelium that had
undergone damage and repair. The surface epithelium
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GILLOTEAUX ET AL.
Fig. 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 lm, in B bar equals 1 lm. 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 lm.
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
TORPEDO GALLBLADDER MUCOUS EXCRESCENCES
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
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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
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 marbree’’; 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; Capape, 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;
Capape et al., 2007).
90
GILLOTEAUX ET AL.
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 (Sole 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 (St€
adeler, 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
TORPEDO GALLBLADDER MUCOUS EXCRESCENCES
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 (Wickstr€om
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).
91
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 marbledlike 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; PerezVilar, 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
92
GILLOTEAUX ET AL.
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.
ACKNOWLEDGEMENT
The authors thank the staff of the Observatoire
Oceeanologique of Banuyls (France) of the Universite
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.
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