Cortisol mobilizes mineral stores from vertebral skeleton in the European eel: an ancestral origin for glucocorticoid-induced osteoporosis?
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241
Cortisol mobilizes mineral stores from vertebral skeleton in the
European eel: an ancestral origin for glucocorticoid-induced
osteoporosis?
Miskal Sbaihi1, Karine Rousseau1, Sylvie Baloche1, François Meunier1, Martine Fouchereau-Peron1,2
and Sylvie Dufour1
1
Muséum National d’Histoire Naturelle, DMPA USM 0401, UMR 7208 CNRS BOREA ‘Biologie des Organismes et Ecosystèmes Aquatiques’, 7 rue Cuvier,
CP 32, 75231 Paris Cedex 05, France
2
Marine Station of Concarneau, 29182 Concarneau Cedex, France
(Correspondence should be addressed to S Dufour; Email: dufour@mnhn.fr)
Abstract
Endogenous excess cortisol and glucocorticoid (GC) therapy and image analysis of ultrathin microradiographs showed the
are a major cause of secondary osteoporosis in humans. induction by cortisol of different mechanisms of bone
Intense bone resorption can also be observed in other resorption, including periosteocytic osteolysis and osteoclas-
vertebrates such as migratory teleost fish at the time of tic resorption. Specificity of cortisol action was investigated
reproductive migration and during fasting when large by comparison with the effects of sex steroids. Whereas,
amounts of calcium and phosphate are required. Using a testosterone had no effect, estradiol induced vertebral skeleton
primitive teleost, the European eel, as a model, we demineralization, an effect related to the stimulated synthesis
investigated whether cortisol could play an ancestral role in of vitellogenin (Vg), an oviparous specific phospho-calcio-
the induction of vertebral skeleton demineralization. lipoprotein. By contrast, the cortisol demineralization effect
Different histological and histomorphometric methods were was not related to any stimulation of Vg. This study
performed on vertebral samples of control and cortisol- demonstrates GC-induced bone demineralization in an
treated eels. We demonstrated that cortisol induced a adult non-mammalian vertebrate, which undergoes natural
significant bone demineralization of eel vertebrae, as shown bone resorption during its life cycle. Our data suggest that the
by significant decreases of the mineral ratio measured by stimulatory action of cortisol on bone loss may represent an
incineration, and the degree of mineralization measured by ancestral and conserved endocrine regulation in vertebrates.
quantitative microradiography of vertebral sections. Histology Journal of Endocrinology (2009) 201, 241–252
Introduction have provided many data on the deleterious effects of GC
excess on bone (rat: Lindgren et al. 1983, Jowell et al. 1987,
Osteoporosis is a major disease affecting human bone. It is Goulding & Gold 1988; mouse: Altman et al. 1992, Weinstein
commonly divided into primary osteoporosis, which is an et al. 1998, 2002; ewes: Chavassieux et al. 1997; dogs: Quarles
inheritable metabolic bone disease, and secondary osteoporo- 1992). However, in vitro studies in rodents have been
sis, which is caused by endocrinological disorders and drugs. contradictory with either stimulation (rat: Gronowicz et al.
Glucocorticoid-induced osteoporosis (GIO), in patho- 1990; mouse: Reid et al. 1986, Conaway et al. 1996,
logical conditions of endogenous hypercortisolism such as Weinstein et al. 2002) or inhibition (rat: Stern 1969, Raisz
Cushing’s syndrome (Mancini et al. 2004) or after cortico- et al. 1972, Tobias & Chambers 1989, Dempster et al. 1997) of
therapy, has been well demonstrated in human bone bone resorption.
(Mazziotti et al. 2006, Canalis et al. 2007). GIO is the most Intense bone resorption can also be observed in non-
common form of secondary osteoporosis and represents mammalian vertebrates such as migratory teleost fish during
increasing concern for human health and therapy (Tamura sexual maturation. This was shown in naturally matured
et al. 2004, Mazziotti et al. 2006, Canalis et al. 2007). Chronic conger eels (Conger conger: Lopez & Deville-Peignoux 1974)
treatments with synthetic glucocorticoids (GC) are com- and salmons (Salmo salar: Kacem et al. 2000, Kacem &
monly used in autoimmune, pulmonary, and gastrointestinal Meunier 2003), as well as in experimentally matured
diseases (Mazziotti et al. 2006). Many studies have been European eels (Anguilla anguilla: Lopez & Martelly-Bagot
performed to seek a suitable animal model that could mimic 1971, Lopez 1973) and Japanese eels (Anguilla japonica:
bone disorder observed in patients with GIO. In vivo models Yamada et al. 2002). It is well known that sexual maturation
Journal of Endocrinology (2009) 201, 241–252 DOI: 10.1677/JOE-08-0492
0022–0795/09/0201–241 q 2009 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org
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via free access242 M SBAIHI and others . Cortisol-induced eel bone osteoporosis
is characterized in fish as in other vertebrates, by high plasma female Japanese eels, the calcium content of the skin (which
levels of sex steroids. However, other hormones including contains the scales) did not decrease, while the vertebral
cortisol may also show large variations during sexual skeleton constituted the principal source for calcium and
maturation. Studies in salmon (Sower & Schreck 1982, phosphate. Furthermore, the eel has a cellular bone
Martin et al. 1986, Carruth et al. 2000a) showed that very high (containing osteocytes in its matrix), in contrast to most
plasma levels of cortisol are observed during the final phase of teleosts which present acellular bone, but similar to other
reproduction in males as well as in females, pointing out the vertebrates (Lopez 1970a, Francillon-Vieillot et al. 1990,
occurrence of a syndrome similar to that of Cushing’s. Meunier & Huysseune 1992, Sire & Huysseune 2003),
In fish, as in other vertebrates, cortisol is known as a major represents a good experimental model to study the different
hormone of the intermediary metabolism, as well as the types of bone resorption. Finally, as a representative of a
principal mediator of the response to stress. Its role in the vertebrate group of ancient origin (Elopomorphes), this
mobilization of energy and metabolite reserves, in particular species could also provide important information on ancestral
during periods of fasting and reproduction, was demonstrated regulation of bone resorption.
in teleosts (for review: Mommsen et al. 1999). By contrast, its In our study, we investigated whether cortisol could play a
potential role in the mobilization of mineral reserves has major physiological role in bone remodeling in a non-
received so far little attention. In teleosts, cortisol is in mammalian vertebrate. We undertook histological and
addition a key actor in osmoregulation, being involved in histophysical (qualitative and quantitative microradiographs)
both ion uptake and secretion (for reviews: Boeuf & Payan studies on vertebral bone of control and cortisol-treated
2001, McCormick 2001). In eels, cortisol may act on the gills silver eels, according to the methods we recently developed in
of freshwater fish to aid in the absorption of sodium and the eel (Sbaihi et al. 2007). We also investigated the specificity
chloride (European eel: Mayer et al. 1967, American eel: of cortisol action by comparing its effects with those of
Perry et al. 1992), while it is a sodium excreting factor in fish sex steroids.
adapted to seawater (Mayer et al. 1967).
It is known that the need for calcium and phosphate is
strongly increased during sexual maturation and migration in
Materials and Methods
fish (Persson et al. 1997, 1998, Kacem et al. 2000, Yamada et al.
2002). Calcium and phosphate are usually taken from the
Animals
external environment (food and water), but if the availability
in the external medium is insufficient, they can be drawn from Two batches of female European silver eels (A. anguilla) were
the mineral-bearing elements i.e. scales and bone (A. anguilla: caught from ponds in the north of France (Somme, France) by
Lopez & Martelly-Bagot 1971; Carassius auratus: Mugiya & professional fishermen at the time of their downstream
Watabe 1977; Oncorhynchus mykiss: Carragher & Sumpter migration (mid-November) during two consecutive years. At
1991). In migratory fish, the time of reproductive migration the silver stage, eels have ended the juvenile growth phase and
synchronizes with a period of starvation, and consequently, are initiating their reproductive migration towards the ocean.
the organic matter as well as minerals (calcium and phosphate The animals were immediately transferred to the laboratory
in particular) is mobilized from the internal medium. Eels (MNHN, Paris, France) and kept in running aerated
have a complex and particular life cycle. During juvenile freshwater (15G2 8C). As eels undergo a natural starvation
growth that occurs during the freshwater phase of their life period at the silver stage, they were not fed during
cycle (called yellow stage), eels grow and accumulate experiments. Animal manipulations were performed under
metabolic and mineral reserves. At the end of the yellow the supervision of authorized investigators.
phase, they metamorphose into silver eels (Aroua et al. 2005),
stop feeding and growing, and initiate their long oceanic
Hormonal treatment
reproductive migration during which they will mobilize their
reserves. In female European eels, the concentrations of both Two weeks after their transfer at Muséum National d’ Histoire
estradiol (E2) and testosterone increase between yellow and Naturelle (December), eels were divided into experimental
silver stages (Sbaihi et al. 2001, Aroua et al. 2005) and rise groups in separate tanks (15G2 8C; 4 eels/100-L tank; 8
further during experimental maturation (Leloup-Hatey et al. eelsZ2 tanks/treatment) and hormonal treatments were
1988, Peyon et al. 1997). Van Ginneken et al. (2007) reported started (weekly i.p. injections of steroid hormones or
that plasma cortisol levels in European eels also increased saline over a 3 month period). Two independent experiments
between yellow and silver stages. were done on independent eel batches for two consecutive
Considering that scales in eel are poorly developed years.
compared with other teleosts (Zylberberg et al. 1984), this
species provides a relevant model for investigating the role of Experiment 1 Sixteen eels (mean body weight (BW) 385G
the internal skeleton, and in particular the long vertebral 9 g) were divided into two groups: eight control eels and eight
skeleton, in the storage and mobilization of minerals. Yamada eels treated with cortisol (F; Sigma-Aldrich Corp.). Animals
et al. (2002) found that during experimental maturation in received a chronic treatment with cortisol (one i.p. injection
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via free accessCortisol-induced eel bone osteoporosis . M SBAIHI and others 243
per week of 2 mg steroid suspended in 0.9% NaCl/g BW), (Leloup-Hatey 1976), which means that administration of
according to the protocol by Huang et al. (1999). The control 2 mg/g BW per week corresponds to 3–10 times the basal
group was injected with saline alone (0.9% NaCl). production of cortisol.
Experiment 2 In order to compare the effect of cortisol
Calcium and phosphate assays
with those of sex steroids, 32 eels (mean BW 400G12 g) were
divided into four groups: eight control eels, eight eels treated Plasma levels of calcium and phosphate were measured
with cortisol (F), eight eels treated with testosterone (Sigma) spectrophotometrically at the end of Experiment 2, using
and eight eels treated with E2 (Sigma). As in Experiment 1, the Ca-Kit 61041 and PHOS UV 61571 respectively
animals received a chronic treatment with steroids (one i.p. (bioMérieux, Craponne, France). Each sample was measured
injection per week of 2 mg steroid suspended in 0.9% NaCl/g in duplicate and data expressed as mg/dl.
BW) according to the protocol by Huang et al. (1999) and
Weltzien et al. (2006). The control group received saline Immunoassay of vitellogenin
alone.
At the end of both experiments, fish were killed by Vg was assayed in plasma samples at the end of Experiment
decapitation 1 week after the last injection. Blood was 2, using a homologous ELISA for European eel Vg (Sbaihi
collected on heparin and plasma, obtained after centri- et al. 2001).
fugation, was stored at K20 8C until steroid, calcium,
phosphate, and vitellogenin (Vg) assays. For each animal, a Histology of vertebrae
portion of vertebral skeleton was sampled 2 cm behind the
anal region according to Sbaihi et al. (2007). Vertebrae were fixed in 70% alcohol and stained using basic
fuchsine at 1% (Frost 1959). They were dehydrated in a
graded series of ethanol and in xylene, and subsequently
Immunoassays of steroids embedded in prepolymerized methacrylate (Reinhold 1997).
Slides were cut into 30G5 mm and mounted in Canada balm.
Plasma levels of steroids were measured at the end of Observations were made under a light microscope (LEICA
Experiment 2 using ELISA (AbCys S.A., Paris; E 2: MZ APO).
DNOV003; testosterone: DNOV002; cortisol: DNOV001).
At the end of the 3-month treatment, plasma testosterone
levels were higher in testosterone-treated eels (17.02G Measurement of mineral ratio by incineration
5 ng/ml) than in controls (2.5G0.5 ng/ml). Plasma E2 levels Vertebral samples were cleaned, dried, and weighed (dry
were also higher in E2-treated eels (80.57G9.3 ng/ml) than weight) to a precision of 0.01 mg (Mettler AC 100),
in controls (4.2G0.8 ng/ml). These increases were similar to incinerated for 7 hours at 850 8C and the ashes weighed
those previously observed (Weltzien et al. 2006, Aroua et al. (mineral weight) to a precision of 0.01 mg, as described
2007). By contrast, plasma cortisol levels at the time of killing earlier (Sbaihi et al. 2007). The mineral ratio (MR) which is
did not differ between treated and control eels (32.74G the quantity of mineral per unit mass of dried material was
7.47 ng/ml in cortisol-treated eels versus 34.08G7.80 ng/ml calculated as followed: MR (%)Z(mineral weight/dry
in controls). In order to further investigate the impact of weight)!100.
cortisol injections, a kinetic study of plasma cortisol levels
after i.p. injection of cortisol (2 mg/g BW) or of saline was
performed (four eels/group). Blood samples were taken from Measurement of mineralization degree by quantitative
the caudal vasculature at 4, 8, 24, 48 h, 4, and 7 days after microradiography
injection. Throughout the kinetic study, plasma cortisol levels This method has been previously developed (Sissons et al.
remained between 20 and 150 ng/ml in control eels. Eight 1960, Boivin & Baud 1984) and recently used in studies on
hours after injection, plasma cortisol levels peaked at human bone biopsies (Boivin et al. 2000) and fish vertebrae
1000–1500 ng/ml in cortisol-injected eels, decreased to (Salmo salar: Kacem & Meunier 2003; European eel: Sbaihi
300–500 ng/ml at 24 h, and returned to basal levels at 48 h, et al. 2007). Briefly, slices (100G1 mm) of embedded (Stratyl:
as well as at 4 and 7 days. Previous studies showed that the chronolite 2195) vertebrae were microradiographied with a
metabolic clearance rate for cortisol in European eel was 1500 CGR Sigma 2060 X-ray generator. The settings of the X-ray
to 2500 ml/kg BW/day (Leloup-Hatey 1976), a value much unit were 20 kV, 7 mA, 60 min exposure time and a distance
higher than those for testosterone (41 ml/kg BW/day; of 40 cm between the beam source and the X-ray film (Sbaihi
Quérat et al. 1985) and E2 (12 ml/kg BW/day; Quérat et al. et al. 2007). The microradiographied vertebrae were
1985). These clearance rates are in agreement with the compared with the optical density of 99.9% pure foil
respective plasma levels observed 7 days after the last injection aluminium standard (Strems Chemical Ltd, Strasbourg,
in the present study. It was also reported that the secretion rate France) of increasing thickness (Boivin & Baud 1984,
for cortisol in European eel was 210 to 560 ng/g BW/7 days Meunier & Boivin 1997, Kacem & Meunier 2003, Sbaihi
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via free access244 M SBAIHI and others . Cortisol-induced eel bone osteoporosis
et al. 2007). Data could be converted from gray-level values responsible for osteoclastic resorption in higher vertebrates,
(optical densities) to degree of mineralization, i.e. the quantity could be observed in these lacunae. Such large lacunae were
of mineral substance present in a unit of bone volume, and not observed in vertebral sections of control eels. These results
expressed as equivalent of g mineral/cm3 bone (Sissons et al. suggested that chronic treatment with cortisol stimulated
1960, Boivin et al. 2000, Kacem & Meunier 2003, Sbaihi et al. osteoclastic resorption.
2007). On each microradiograph, surface areas 100G1 mm
thickness were digitalized, selected surface areas were edited
Effect of cortisol on MR
and analyzed by NIH-Image 1.61 program. Eight zones in the
mineralized regions were measured in each section, and the Measurement of MR% by the method of incineration showed
average was considered as the value of mineralization degree a significant (P!0.001) decrease in the mineral content of
(MD) for the corresponding vertebra. vertebrae from cortisol-treated eels (42.76G0.23%) as
compared with controls (46.07G0.56%; Fig. 2). This indi-
cated an important mineral loss after treatment with cortisol.
Measurement of the periosteocytic lacunae surface area
Ultrathin slices (!20 mm) of embedded vertebrae were
Effect of cortisol on MD
prepared and microradiographied with a CGR Sigma 2060
X-ray without nickel filter. The settings of the X-ray unit The measurement of the MD, which represents the quantity
were 10 kV, 5 mA, 5 min exposure time, and a distance of of mineral per unit of bone volume, was carried out by
5 cm between the beam source and the X-ray film (Sbaihi image analysis of the quantitative microradiographs (sections
et al. 2007). Microradiographs were digitalized and selected 100G1 mm). The distribution frequency of the MD
surface areas edited and analyzed by NIH-Image 1.61 measurements made on eight zones of each microradiograph
program. The surface area of 40 osteocytic lacunae was of 100G1 mm vertebral section is shown in Fig. 3A. The shift
measured on each slide and the average was considered as the of the peak of MD in eels treated with cortisol compared
value of lacunae surface area for the corresponding vertebra. with control eels indicated a global reduction of the MD in
The results were expressed in mm2. treated eels. A significant (P!0.001) decrease in mean values
of MD was observed between vertebrae from cortisol-
treated eels (1.18G0.01 g/cm3) and vertebrae from control
Statistical analysis
eels (1.26G0.01 g/cm3; Fig. 3B).
Data from control and steroid-treated eels were expressed as
meanGS.E.M. Data were analyzed by Student’s t-test or one-
Effect of cortisol on periosteocytic osteolysis
way ANOVA followed by Student-Newman-Keuls multiple
comparison tests, using InStat (GraphPad Software). Microradiographs of ultrathin vertebral sections (20 mm;
Differences were considered significant at P!0.05. Fig. 4A) revealed that the osteocytic lacunae observed in
cortisol-treated eels were larger than those in control eels.
The distribution frequency of the lacunae surface area
measured on forty osteocytic lacunae for each microradio-
Results
graph of !20 mm vertebral section is shown in Fig. 4B. The
shift of the peak in eels treated with cortisol compared with
Vertebral histological structure and effect of cortisol on osteoclastic
control eels indicated a global increase of the lacunae surface
resorption
area in treated eels. This indicated an activation of the
The microscopic examination of the vertebrae sections of periosteocytic osteolysis in cortisol-treated eels. The mean
control eels showed primary bone (primary mineralized surface area of osteocytic lacunae from vertebra sections of
tissue) and at the periphery of this one, zones of secondary control and cortisol-treated eels is shown in Fig. 4C. In
bone (new mineralized bone), which differed by the cortisol-treated eels, the average surface area of osteocytic
orientation of the bone structure (Fig. 1A). In all sections, lacunae was significantly increased as compared with controls
the osteocytes were easily identifiable and strongly stained (56.93 mm2G1.2 vs 44.47G0.8 mm2 P!0.001; Fig. 4C).
with fuchsine. The existence of osteocytes inside the bone
structure means that eel bone belongs to the cellular type
Comparison of the effects of cortisol and sex steroids on MR
(osteocytic bone), as first mentioned by Lopez (1973). High
magnification showed that osteocytes present a spangled form In agreement with Experiment 1 (Fig. 2), vertebral mineral
(the shape of spider; Fig. 1B). content was significantly decreased (P!0.01) by cortisol
Histological study revealed the presence of large cavities of treatment (42.54G0.32%) as compared with controls
resorption (Howship’s lacunae) at the periphery of the (45.59G0.22%) in Experiment 2 (Fig. 5). A significant
vertebral bone in cortisol-treated eels (Fig. 1C). These decrease in MR was also observed in E2-treated eels (39.93G
lacunae were strongly stained by fuchsine in contrast to the 0.22%, P!0.001; Fig. 5). By contrast, no significant change
non-resorbed bone. Pluri-nucleated osteoclasts, known to be was observed with testosterone (45.26G0.40%; Fig. 5).
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Figure 1 Histology of eel vertebral bone. Vertebrae were stained with basic fuchsine,
embedded in methacrylate and cut at 30G5 mm (A) Vertebral section from a control eel
showing primary bone (PB) and secondary bone (SB) indicating bone remodeling. Many
osteocytes (O) are included in the bone structure demonstrating a cellular (osteocytic) bone
(Scale barZ75 mm). (B) Higher magnification of a vertebral section from a control eel
showing the spangled form of the osteocytes (O; Scale barZ20 mm). (C) Vertebral section of
a cortisol-treated eel showing a large osteoclastic lacuna (OL) and the presence of
osteoclasts (Ocl; Scale barZ75 mm).
Comparison of the effects of cortisol and sex steroids on calcium, A drastic increase (more than !105) of plasma Vg
phosphate, and Vg plasma concentrations concentrations was observed after E2 treatment (576G
170 mg/ml vs !2 mg/ml for controls; P!0.001). By contrast,
Plasma calcium concentrations (Fig. 6A) were unchanged in
plasma Vg concentrations remained low (!2 mg/ml) in
cortisol- and testosterone-treated eels (9.29G1 mg/dl and
testosterone- and cortisol-treated eels (Fig. 6B).
10.16G0.45 mg/dl respectively) as compared with controls
(10.20G0.4 mg/dl). By contrast, E2 treatment induced a
large and significant increase in calcium plasma levels (70.9G
0.7 mg/dl; P!0.001; Fig. 6A). Discussion
Similarly, plasma phosphate concentrations (Fig. 6A) were
unchanged in cortisol- and testosterone-treated eels (6.42G The strong vertebral bone demineralization, observed in the
0.66 mg/dl and 7.17G0.58 mg/dl respectively) as compared present study, after chronic cortisol treatment in a primitive
with control eels (6.31G0.64 mg/dl), while E2 treatment teleost suggests that GC induction of bone loss may represent
induced a large and significant increase in phosphate plasma an ancestral regulatory mechanism, conserved during
levels (47.93G3.72 mg/dl; P!0.001; Fig. 6A). vertebrate evolution.
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Kacem et al. 2000), for studying bone demineralization. In the
Japanese eel, Yamada et al. (2002) demonstrated by this
method that the mineral content (phosphorus and calcium) in
bone tissue decreased during experimental sexual maturation.
In the European eel, we previously showed by this method
the effect of thyroid hormone on vertebral demineralization
(Sbaihi et al. 2007).
The demineralization of eel vertebrae after cortisol
treatment was further demonstrated by measuring the
MD, using quantitative microradiographies. A reduction
of MD has been previously shown in eel vertebra during
experimental maturation (Lopez et al. 1970, Lopez &
Martelly-Bagot 1971) or after chronic treatment with thyroid
Figure 2 Effect of cortisol on mineral ratio (MR) of eel vertebrae.
MR is measured by incineration. MRZ(mineral weight/dry hormones (Sbaihi et al. 2007), as well as in Atlantic salmon
weight)!100. Results are expressed as meanGS.E.M. (nZ8 during the anadromous reproductive migration (Kacem &
eels/group). ***P!0.001 as compared with controls. Meunier 2003). Our data suggest that cortisol, the levels
of which increase during sexual maturation in fish
Cortisol-induced eel vertebral demineralization (Donaldson & Fagerlund 1972, Pickering & Christie 1981,
In the European eel, chronic treatment with cortisol induced Sower & Schreck 1982, Ueda et al. 1984), may be involved
a significant demineralization of the vertebral skeleton, as physiologically in the maturation-related bone deminerali-
shown by the reduction of MR measured by incineration. zation. Measurement of MD has also been previously used in
This method has already been used in rat (Wink & Felts mammals in experimental osteoporosis studies (Jowsey et al.
1980, Ortoft & Oxlund 1988) and fish (Casadevall et al. 1990, 1965, Baud et al. 1980, Meunier & Boivin 1997) and in post-
menopausal osteoporotic women on transiliac bone biopsies
(Boivin et al. 2000).
Recently, zebrafish larva was proposed as a rapid in vivo
model of GIO (Barrett et al. 2006) using staining mineralized
tissue with fluorescent dye as a method to quantify bone size
and density (Du et al. 2001). The authors exposed zebrafish
larvae in 96-well plates to different doses of GC pharma-
cological agonist (prednisolone) for 5 days. The data revealed
a marked bone loss in larvae treated with prednisolone.
Furthermore, this prednisolone-induced bone loss was
prevented by RU486, a glucocorticoid receptor (GR)
antagonist (Barrett et al. 2006). Our present study demon-
strates that GIO also occurs in the adult European eel. These
data suggest that the bone demineralization effect of GCs may
represent a general regulatory process in teleosts, since it may
occur at different life stages (larvae or adults) and in species
representative of different teleost groups (cypriniformes for
zebrafish and elopomorphs for eel). Cortisol mobilization of
skeletal mineral stores likely reflects a physiological process
especially in the case of long migratory and fasting fish, such as
eels and salmons. In addition, this regulatory pathway could
lead to stress-induced pathological impairment of skeleton
development and homeostasis in fish. For instance, further
investigations could decipher whether GIO may participate in
skeletal abnormalities largely observed in aquaculture
(Deschamps et al. 2008).
Figure 3 Effect of cortisol on mineralization degree (MD) of eel
vertebrae. MD (g mineral/cm3 of bone) is measured by quantitative
microradiography of vertebral sections (100G1 mm) of control and
cortisol-treated eels. (A) Distribution of MD values in vertebrae
Cortisol-induced osteocytic osteolysis
from control and cortisol-treated eels (eight vertebral zones The histological observation of eel vertebral bone showed the
measured per eel vertebral section; eight control eels and eight
treated eels). (B) Mean MDGS.E.M. of vertebrae from control and
presence of numerous osteocytes inside the bone structure,
cortisol-treated eels (nZ8 eels/group). ***P!0.001 as compared demonstrating that eel vertebra, as in higher vertebrates but
with controls. different from the majority of other teleosts, is a cellular
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Figure 4 Effect of cortisol on periosteocytic resorption of eel vertebrae. (A) Microradiographs
of ultrathin vertebral sections (!20 mm) from control and cortisol-treated eels, showing
periosteocytic lacunae (arrows). The surface areas of lacunae are measured by image
analysis of microradiographies. Osteocytic lacunae are larger in cortisol-treated eels than in
controls. (Scale barZ10 mm). (B) Distribution of surface area of periosteocytic lacunae in
vertebral sections of control and cortisol-treated eels (40 osteocytic lacunae measured/eel;
eight control eels and eight treated eels). (C) Mean surface area of periosteocytic lacunae in
vertebrae from control and cortisol-treated eels. Results are expressed as meanGS.E.M. (nZ8
eels/group). ***P!0.001 as compared with controls.
bone (osteocytic bone; Weiss & Watabe 1979, Meunier & treatment with thyroid hormones (Sbaihi et al. 2007), and in
Huysseune 1992, Eckhard-Witten 1997). The eel thus the salmon during natural maturation (Baud 1962, Kacem &
provides an interesting comparative model for the study of Meunier 2000). Periosteocytic osteolysis resorption has
the osteocytic mechanisms of bone resorption. We showed in also been observed in the vertebrae of a snake, Vipera aspis,
the present study, using image analysis of ultrathin micro- during reproduction (Alcobendas & Baud 1988, Alcobendas
radiographs, that treatment with cortisol induced a significant et al. 1991).
increase in the surface area of the osteocytic lacunae. This Numerous studies in mammals have demonstrated that
indicates a stimulation of osteocytic osteolysis by cortisol in osteocytes are highly active cells and that mature osteocytes
eel vertebral bone. are capable of resorbing mineral substance as well as organic
In fish, osteocytic resorption was previously shown to matrix from their surrounding bone under physiological and
increase in the eel during experimental maturation (Lopez pathological conditions (for reviews: Kogianni & Noble
1970b, Lopez & Martelly-Bagot 1971) and after chronic 2007). In humans, osteoporosis caused by long-term GC
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via free access248 M SBAIHI and others . Cortisol-induced eel bone osteoporosis
vertebrate. Our data suggest that cortisol-induced osteocytic
osteolysis may represent an ancient and conserved cellular
mechanism of bone resorption in vertebrates.
It is not yet known whether the perilacunar resorption is
due to a direct action of GC on the eel bone cells. The
presence of GR on osteocytes was demonstrated in rat
(Silvestrini et al. 1999) and in human (Abu et al. 2000). This
suggests that the action of GC on osteocyte activity may be
directly mediated via the GR. In fish, two distinct functional
GR were cloned in rainbow trout (Ducouret et al. 1995, Bury
et al. 2003). GR have been demonstrated in a variety of
salmonid tissues (Maule & Schreck 1991, Maule et al. 1993,
Knoebl et al. 1996, Carruth et al. 2000b, Bury et al. 2003), but
Figure 5 Comparison of the effects of cortisol and sex steroids on
until now, there is no information on the presence of GR in
mineral ratio. MR is measured by incineration. MRZ(mineral the bone tissue in fish. Further investigation in the eel could
weight/dry weight)!100. Results are expressed as meanGS.E.M. address this question.
(nZ8 eels/group). ***P!0.001 as compared with controls.
treatment is accompanied by a significant enlargement of the Cortisol-induced osteoclastic resorption
periosteocytic lacunar surface area (Baud & Boivin 1978,
The microscopic observation showed large lacunae of
Wright et al. 1978). In GC-treated mice, larger osteocyte
osteoclastic resorption at the periphery of the vertebral
lacunae were also observed on vertebral bone sections (Lane
bone of cortisol-treated eels. This type of resorption has
et al. 2006).
already been described in the female European eel under
The present study is the first demonstration of a stimulatory experimental maturation and in the female conger
effect of cortisol on osteocytic osteolysis in a non-mammalian during spontaneous maturation (Lopez 1970b, Lopez &
Martelly-Bagot 1971).
In mammals, GC treatments have a wide spectrum of
effects on osteoclastic resorption according to models and
studies. In humans, a biphasic effect of GC on osteoclasts
has been hypothesized: GC could have an acute inhibitory
effect on osteoclast synthesis without significant modifi-
cation of bone resorption; on the contrary, in the long-
term, the GC-mediated stimulation of osteoclast synthesis
might be coupled with an increase in bone resorption (for
review: Manelli & Giustina 2000). In rodents, GC was
shown to decrease basal activity of osteoclasts (Wong 1979),
to impair osteoclastogenesis (Weinstein et al. 1998) and to
reduce the number of osteoclasts (in vivo: Lindgren et al.
1983, Jowell et al. 1987; in vitro: Tobias & Chambers 1989,
Dempster et al. 1997). By contrast, other authors indicated
a stimulatory effect of GC on osteoclastic resorption in
rodents. Some studies have suggested a possible direct
effect of GC on osteoclast-mediated bone resorption, as
stimulation of bone osteoclastic resorption by GC was
observed in organ cultures of fetal rat parietal bones and
neonatal mouse calvariae (Gronowicz et al. 1990, Conaway
et al. 1996). Furthermore, GC has been shown to stimulate
osteoclast formation in bone marrow cultures in the mouse
(Shuto et al. 1994) and to extend the life span of pre-
existing osteoclasts in murine osteoclast cultures (Weinstein
Figure 6 Comparison of the effects of cortisol and sex steroids on et al. 2002). In another model, the dog, as in the mouse, a
plasma concentrations of calcium, phosphate (A) and vitellogenin significant decrease of trabecular bone volume was observed
(B) in the European eel. Plasma concentrations of calcium and
phosphate are measured spectrophotometrically and vitellogenin
in vivo after treatment with GC (Altman et al. 1992, Quarles
by homologous ELISA. Results are expressed as meanGS.E.M. (nZ8 1992, Weinstein et al. 1998, McLaughlin et al. 2002).
eels/group). ***P!0.001 as compared with controls. Our present study indicates that GC-induced osteoclastic
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via free accessCortisol-induced eel bone osteoporosis . M SBAIHI and others 249
resorption would also occur in a non-mammalian and other oviparous and non-oviparous vertebrates, including
vertebrate, in which it may represent a physiological mammals.
mechanism of mobilization of mineral stores. By using various histological and histomorphometric
methods, we showed that cortisol could act through different
mechanisms of bone resorption, including osteoclastic
E2 action on bone demineralization: an oviparous feature
resorption and periosteocytic osteolysis. This is the first
To investigate the specificity of cortisol action, we compared demonstration of the role of cortisol in the induction of bone
its effects with those of sex steroids. Our data showed that, like loss in an adult non-mammalian vertebrate, which undergoes
cortisol, E2 induced a decrease of vertebral MR. However, in natural bone demineralization during its life cycle.
contrast to cortisol, E2 also induced a drastic increase in The stimulatory role of cortisol on bone demineralization,
plasma Vg concentrations, as well as in plasma calcium and observed in this study in a representative of a vertebrate group
phosphate concentrations. The increase in calcium and of ancient origin (Elopomorphes), the European eel, could
phosphate plasma concentrations under E2 treatment reflects be considered as an ancestral regulation for mobilization of
their involvement in the composition of Vg, which is a mineral stores in vertebrates. Nevertheless, as opposed to
phospho-calcio-lipoprotein (for review: Polzonetti-Magni most vertebrates, eels die following spawning; this may led
et al. 2004). In the eel, the large increase in plasma Vg to a divergence in vertebrate evolution, in that in species such
concentrations after E2 treatment results from both the as eel, bone demineralization could potentially occur to
stimulation of Vg liver production and the very low Vg a greater degree (‘point of no return’) as post-spawning
uptake by oocytes at the early silver stage (Burzawa-Gérard mortality is pre-determined.
& Dumas-Vidal 1991). The specificity of E2 on both bone In conclusion, we demonstrated cortisol-induced demi-
demineralization and Vg induction was confirmed by the lack neralization of the eel vertebral bone, a phenomenon
of effect of testosterone. observed in human under pathological conditions such as
By contrast, some previous studies in other teleosts showed secondary osteoporosis after hypercortisolism or GC thera-
that E2 did not induce any bone demineralization. In rainbow pies. This suggests that the stimulatory action of GC on
trout, Armour et al. (1997) reported that following E2 bone loss may reflect an ancestral endocrine regulation
treatment, calcium and phosphate contents decreased in scales for mobilization of mineral stores from vertebral skeleton,
while they increased in bone. E2 treatment induced calcium a regulation that would have been conserved throughout
mobilization from scales in goldfish, killifish, and rainbow vertebrate evolution. Non-mammalian vertebrates, such as
trout (Mugiya & Watabe 1977, Carragher & Sumpter 1991, the eel, can thus provide comparative models for experi-
Persson et al. 1994). Teleost scales are calcified tissues, which mental studies of the mechanisms of osteoporosis and
may contain up to 20% of the total body calcium in some bone loss.
species and serve as a functional mineral reservoir during
periods of increased mineral demand, such as sexual
maturation and starvation (Takagi et al. 1989, Bereiter-Hahn Declaration of interest
& Zylberberg 1993, Persson et al. 1998). However, scales in
The authors declare that there is no conflict of interest that could be perceived
eel are poorly developed which differs from other teleosts as prejudicing the impartiality of the research reported.
(Zylberberg et al. 1984) and cannot serve as a mineral
reservoir. Yamada et al. (2002) found that during sexual
maturation in female Japanese eels, the calcium and Funding
phosphorus contents of the skin (which contains the few
scales) remained unchanged, while they decreased in the This work was supported in part by grants from MNHN (BQR) and
European Community (EELREP Q5RS-2001-01836). Dr M Sbaihi was a
vertebral skeleton, which constitutes the principal source for recipient of a post-doc fellowship from the European community (EELREP).
calcium and phosphorus. Estrogen receptors were reported in
bone and scales of rainbow trout (Armour et al. 1997, Persson
et al. 2000), regenerating scales of goldfish (Yoshikubo et al. Acknowledgements
2005) and bone of sea bream (Socorro et al. 2000). This
suggests a possible direct effect of E2 on bone and scale We would like to thank Drs J-Y Sire and J Castanet (CNRS, Universities
Paris 6 and Paris 7) for facilitating access to their laboratory and the use of
demineralization in teleosts, related to the strong demand in their equipment concerning the realization of microradiographs and image
minerals for Vg production in oviparous vertebrates. analyses.
Cortisol action on bone demineralization: a general vertebrate
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