The effects of sex steroids on thyroid C cells and trabecular bone structure in the rat model of...

The effects of sex steroids on thyroid C cells andtrabecular bone structure in the rat model of maleosteoporosisBranko Filipovi�c, Branka �So�si�c-Jurjevi�c, Vladimir Ajd�zanovi�c, Jasmina Panteli�c, Nata�sa Nestorovi�c,Verica Milo�sevi�c and Milka Sekuli�c

Institute for Biological Research, University of Belgrade, Belgrade, Serbia

Abstract

Androgen deficiency is one of the major factors leading to the development of osteoporosis in men. Since

calcitonin (CT) is a potent antiresorptive agent, in the present study we investigated the effects of androgen

deficiency and subsequent testosterone and estradiol treatment on CT-producing thyroid C cells, skeletal and

hormonal changes in middle-aged orchidectomized (Orx) rats. Fifteen-month-old male Wistar rats were either

Orx or sham-operated (SO). One group of Orx rats received 5 mg kg�1 b.w. testosterone propionate (TP)

subcutaneously, while another group was injected with 0.06 mg kg�1 b.w. estradiol dipropionate (EDP) once a

day for 3 weeks. A peroxidase–antiperoxidase method was applied for localization of CT in the C cells. The

studies included ultrastructural microscopic observation of these cells. The metaphyseal region of the proximal

tibia was measured histomorphometrically using an IMAGEJ public domain image processing program. TP or EDP

treatment significantly increased C cell volume (Vc), volume densities (Vv) and serum CT concentration

compared with the Orx animals. Administration of both TP and EDP significantly enhanced cancellous bone

area (B.Ar), trabecular thickness (Tb.Th) and trabecular number (Tb.N) and reduced trabecular separation

(Tb.Sp). Serum osteocalcin (OC) and urinary Ca concentrations were significantly lower after these treatments in

comparison with Orx rats. These data suggest that testosterone and estradiol treatment in Orx middle-aged rats

affect calcitonin-producing thyroid C cells, which may contribute to the bone protective effects of sex

hormones in the rat model of male osteoporosis.

Key words: bone; histomorphometry; immunohistochemistry; sex steroids; thyroid C cells; ultrastructure.

Introduction

Sex steroids are important for the maintenance of the

female and male skeletons. In both sexes, age-related

decline in circulating sex hormone levels is associated with

modifications of bone remodeling and this is the main

cause of osteoporosis in older people. Osteoporosis research

is rarely undertaken in men. While there is no dramatic

decline in sex hormones in men as occurs in menopausal

women, the continuous slow reduction in circulating andro-

gens is associated with bone loss (Swerdloff & Wang, 2002).

However, although testosterone is the major male sex hor-

mone, the principal female hormone, estradiol, is also

formed in men in significant amounts. While a small frac-

tion of estradiol production is provided by testicular secre-

tion (Nitta et al. 1993), the remaining estradiol is derived

from aromatization of circulating androgens by the enzyme

aromatase in adipose, skin, muscle, bone and brain tissue

(Simpson & Dowsett, 2002).

Another important hormone involved in bone metabo-

lism is calcitonin (CT). This potent hypocalcemic peptide con-

tributes to calcium (Ca) homeostasis by direct inhibition of

osteoclast-mediated bone resorption and output of Ca from

skeletal tissues (Warshawsky et al. 1980). CT is produced

and secreted by thyroid C cells, the function of which is

affected by gonadal steroids (Foresta et al. 1985; Greenberg

et al. 1986). As with circulating levels of gonadal steroids,

the number of C cells and plasma CT decline with age in

humans. However, in aged rats, together with lower levels

of gonadal steroids, physiological hyperplasia of C cells and

hypercalcitoninemia have been noticed (Delverdier et al.

1990; Sekuli�c et al. 1998; Lu et al. 2000). This hypersecretion

of CT in old rats may be partly caused by hyperprolactin-

Correspondence

Branko Filipovi�c, Department of Cytology, Institute for Biological

Research, University of Belgrade, 142 Despot Stefan Boulevard,

11060 Belgrade, Serbia. T: + 381 11 2078322; F: + 381 11 2761433;

E: [email protected]

Accepted for publication 23 October 2012

Article published online 21 November 2012

© 2012 The AuthorsJournal of Anatomy © 2012 Anatomical Society

J. Anat. (2013) 222, pp313--320 doi: 10.1111/joa.12013

Journal of Anatomy

emia (Lu et al. 2000). The lack of sex hormones in gonadec-

tomized rats of both sexes reduces the synthesis and release

of CT from thyroid C cells (Lu et al. 2000; Sakai et al. 2000).

The mechanisms by which sex hormones affect CT produc-

tion, may include their receptors, as already demonstrated

in thyroid C cells (Naveh-Many et al. 1992; Zhai et al. 2003).

In humans, hypogonadism accelerates bone loss and is

associated with osteoporosis (Hoff & Gagel, 2005). In male

rats, orchidectomy (Orx) leads to androgen deficiency, caus-

ing increased bone resorption (Tuukkanen et al. 1994;

Filipovi�c et al. 2007). For this reason, as ovariectomized

female rats are widely used to mimic postmenopausal oste-

oporosis, Orx rats represent an adequate animal model for

simulation of male osteoporosis (Vanderschueren et al.

1992). Such animal models may be useful in studies examin-

ing bone remodeling rates and for assessing the efficiency

of bone-sparing treatments.

In this study, we evaluated the effects of chronic testoster-

one and estradiol treatment on calcitonin-producing thyroid

C cells, bone structure and bone function in Orx middle-

aged rats. This animal model of male osteoporosis mimics

the hormonal changes that are an important contributing

factor to overall loss of bone in the aging population.

Materials and methods

Animals and experimental design

The experiment involved 32 male Wistar rats, bred at the Institute

for Biological Research ‘Sini�sa Stankovi�c’ (IBISS), Belgrade, Serbia,

and maintained under constant laboratory conditions (22 °C, 12 h

light–dark cycle) with free access to food and water. The animals

were randomly bilaterally orchidectomized (Orx) or sham-operated

(SO) at the age of 15 months under ketamine anesthesia (15 mg

kg�1 b.w.). At 2 weeks post-operation, the rats were divided into

different groups and treated once a day for 3 weeks. One group

(n = 8) of Orx rats received 5 mg kg�1 b.w. testosterone propionate

(TP; Fluka Chemie AG, Buchs, Switzerland) subcutaneously (s.c.).

Another group (n = 8) of Orx animals was injected s.c. with

0.06 mg kg�1 b.w. estradiol dipropionate (EDP; ICN Galenika Phar-

maceuticals, Belgrade, Serbia). The SO group (n = 8) and the third

Orx group (n = 8) received equivalent volumes of sterile olive oil

and served as controls. All animals were sacrificed 24 h after the last

injection. Before killing, 24-h urine samples were collected for Ca

assay. Sera were separated from trunk blood after decapitation and

stored at �70 °C until analyzed biochemically. The experimental

protocols were approved by the Local Animal Care Committee of

IBISS in conformity with recommendations provided in the

European Convention for the Protection of Vertebrate Animals

Used for Experimental and Other Scientific Purposes (ETS no. 123,

Appendix A).

Immunohistochemical studies

The thyroid lobes were excised and fixed in Bouin’s solution for

48 h. The peroxidase-antiperoxidase (PAP) method used for detect-

ing CT in C cells was conducted on 5-lM-thick longitudinal paraplast

sections of the thyroid glands. In this procedure, anti-human CT

antiserum (Dakopatts, Copenhagen, Denmark) diluted 1 : 500

served as the primary antibodies.

Stereological analysis of thyroid C cells

Immunocytochemically stained sections of the thyroid glands were

used for morphometric examination of specifically labeled C cells.

These cells were stereologically analyzed by Weibel’s method

(Weibel, 1979). The C cell volume (Vc) and volume density (volume

of C cells per unit volume of thyroid gland; Vv) were measured

under a light microscope using 50 test fields at 10009magnification

with the multipurpose M42 test system.

Transmission electron microscopy studies of thyroid

C cells

For electron microscopic observation a thyroid gland lobe was

excised, fixed in 4% glutaraldehyde solution in 0.1 M phosphate

buffer (PB) (pH 7.4) for 24 h and postfixed in 1% osmium tetroxide

in the same buffer. Tissue slices were dehydrated through a graded

series of ethanol and embedded in Araldite resin. Ultrathin sections

of thyroid gland were stained with uranyl acetate and lead citrate,

and examined with a transmission electron microscope (MORGAGNI

268; FEI Company, USA).

Bone histomorphometry of the tibia

Right tibiae were used for bone histomorphometric analysis of the

tibial proximal metaphysis. The bones were fixed in Bouin’s solu-

tion, decalcified with 20% ethylenediaminetetraacetic acid disodi-

um salt, routinely processed, embedded in paraplast and sectioned

longitudinally. Sections were stained by the Azan method, as previ-

ously described (Filipovi�c et al. 2007).

An IMAGEJ public domain image processing program was used to

measure bone histomorphometric parameters of the tibial speci-

mens. A standard sampling site was established in the secondary

spongiosa of the proximal tibial metaphysis, 1 mm distal to the

epiphyseal growth plate. All parameters were expressed as recom-

mended by the American Society for Bone and Mineral Research

histomorphometry nomenclature (Parfitt et al. 1987). These data

were used to calculate cancellous bone area (B.Ar) and cancellous

bone perimeter (B.Pm) (Evans et al. 1994). Trabecular thickness

(Tb.Th), trabecular number (Tb.N), and trabecular separation

(Tb.Sp) reflect the spatial distribution of trabeculae and are derived

from B.Ar and B.Pm (Parfitt et al. 1983; Chappard et al. 1999) as

described earlier (Filipovi�c et al. 2007).

Serum and urine biochemical analyses

Blood serum was separated for estimation of CT, osteocalcin (OC),

testosterone, estradiol, Ca and phosphorus (P). Urine was collected

for determination of Ca concentration. Serum CT levels were mea-

sured in an immunochemiluminometric assay (Nichols, USA), using a

mouse monoclonal antihuman CT antibody marker with acridium

ester. The luminescence was quantified with a semiautomated MLA

1 chemiluminescence analyzer (Ciba-Corning). Serum OC was deter-

mined on a Rochle Elecsys 2010 immunoassay analyzer (Roche

Diagnostics GmbH, Mannheim, Germany). Serum testosterone and

estradiol were measured by competitive ACS 180 Plus (Bayer)

and ADVIA immunoassay using direct chemiluminescent technology


Sex steroids affect thyroid C cells and bone, B. Filipovi�c et al.314

and polyclonal rabbit anti-testosterone or anti-estradiol antibody,

respectively, bound to monoclonal mouse anti-rabbit antibody.

Serum Ca and P and urinary Ca were determined on a Hitachi 912

analyzer (Roche Diagnostics GmbH).

Statistical analysis

The data were analyzed using STATISTICA 6.0 software (Statsoft,

Tulsa, OK, USA). The Kolmogorov–Smirnov procedure was used to

test for deviation from normal distribution, followed by one-way

analysis of variance (ANOVA). Duncan’s multiple range test was

employed for post hoc comparisons between groups. The confi-

dence level of P < 0.05 was considered statistically significant. The

data are presented as means � standard error of the mean

(SEM).

Results

Immunohistochemical, morphometric and ultrastru-

ctural assessment of thyroid C cells after sex

hormone treatment

In the SO group, CT-producing thyroid C cells formed

groups, were numerous and developed an intense immuno-

cytochemical reaction for CT (Fig. 1A). At the subcellular

level, most granules of these cells had low density contents.

The mitochondria, round to oval in shape, were dispersed

in the cytoplasm (Fig. 2A). After Orx, C cells were more

individual, smaller and of darker intensity for the CT reac-

tion compared with the same cells in SO animals (Fig. 1B).

At the ultrastructural level we observed a clear difference in

the density of granular content. There were numerous elec-

tron opaque granules and fewer cellular organelles were

present than in SO rats (Fig. 2B). In animals treated with TP

or EDP, C cells were similar to the SO control, with a lighter

granular cytoplasm compared with C cells in the Orx group

(Fig. 1C,D). After TP treatment, the cytoplasm of these cells

contained numerous granules with low density content.

The Golgi apparatus was prominent and composed of large

profiles of smooth membranes. More mitochondria were

dispersed in the cytoplasm than in Orx rats (Fig. 3A). In the

Orx rats treated with EDP, secretory granules were of low

density and few in number when compared with Orx rats.

The cytoplasmic area contained profiles of rough endoplas-

mic reticulum, aggregated into long lamellar arrays

(Fig. 3B).

Previously, we had found a significant decrease in the Vc

and Vv in Orx rats compared with the SO controls (Filipovi�c

et al. 2007). The treatment of Orx rats with TP or EDP

increased Vc by 22 and 16% (P < 0.05), and Vv by 21 and

16% (P < 0.05), respectively, in relation to the Orx animals.

No significant differences in the morphometric parameters

of C cells were detected after treatment with either sex hor-

mone when compared with SO control rats (Fig. 4A,B).

A B

C D

Fig. 1 Calcitonin-immunostained thyroid C cells in (A) sham-operated (SO), (B) orchidectomized (Orx), (C) orchidectomized rats treated with tes-

tosterone propionate (Orx + TP) and (D) orchidectomized rats treated with estradiol dipropionate (Orx + EDP). Note the less voluminous cells with

strong immunocytochemical reaction after Orx and voluminous cells with light cytoplasm, grouped in clusters after TP or EDP treatment.


Sex steroids affect thyroid C cells and bone, B. Filipovi�c et al. 315

Trabecular structure parameters after sex hormone

treatment

Analysis of trabecular structure parameters of the proximal

tibia metaphysis has already shown that Orx induced can-

cellous bone loss and marked decreases of B.Ar, Tb.Th and

Tb.N, whereas Tb.Sp was significantly increased in compari-

son with SO rats (Filipovi�c et al. 2007).

In Orx rats treated with TP, numerous trabecular spicules

were observed as in SO rats (Fig. 5C). When compared with

the control Orx group we found that B.Ar, Tb.Th and Tb.N

had increased by 132, 24 and 26% (P < 0.05), respectively,

whereas Tb.Sp had decreased by 23% (P < 0.05) after TP

treatment. No significant changes in the bone histomorpho-

metric parameters were detected after treatment with

TP when compared with the SO group (Fig. 6A–C). EDP

A

B

Fig. 2 Electron micrographs showing the ultrastructure of thyroid C

cells in (A) sham-operated (SO) and (B) orchidectomized (Orx). N,

nucleus; M, mitochondria; sg, secretory granules; L, lysosomes; FC,

follicular cells.

A

B

Fig. 3 Electron micrographs showing the ultrastructure of thyroid C

cells in (A) orchidectomized rats treated with testosterone propionate

(Orx + TP) and (B) orchidectomized rats treated with estradiol dipropi-

onate (Orx + EDP).

A B

Fig. 4 (A) Cellular volume (Vc) and (B) volume density (Vv) of calcitonin immunoreactive thyroid C cells in sham-operated rats (SO), orchidectom-

ized rats (Orx), orchidectomized rats treated with testosterone propionate (Orx + TP) or estradiol dipropionate (Orx + EDP). All values are

mean � SEM. ●P < 0.05 vs. SO and *P < 0.05 vs. Orx.



treatment restored the trabecular structure and the mor-

phological appearance of tibiae was similar to that observed

in SO rat bone (Fig. 5D). Treatment with EDP enhanced B.

Ar, Tb.Th and Tb.N by 175, 31 and 44% (P < 0.05), respec-

tively, and reduced Tb.Sp by 37% (P < 0.05) in comparison

with the Orx rats. Also, EDP treatment increased Tb.N by

18% (P < 0.05) and decreased Tb.Sp by 19% (P < 0.05) when

compared with the SO animals (Fig. 6A–C).

Serum and urine parameters after sex hormone trea-

tment

We have shown previously that Orx induced a significant

reduction of serum CT, Ca and P levels, whereas serum OC

and urinary Ca concentration in Orx rats were significantly

higher than for the SO group (Filipovi�c et al. 2007). In the

current study, Orx led to a 93% decrease in serum

A B

C D

Fig. 5 Trabecular bone of the proximal tibia in (A) sham-operated, (B) orchidectomized (Orx), (C) orchidectomized rats treated with testosterone

propionate (Orx + TP) and (D) orchidectomized rats treated with estradiol dipropionate (Orx + EDP). Note reduced mass of blue-stained trabecular

bone in Orx rats. TP and EDP treatment prevented loss of trabecular bone; 5-lM sections from the center of the specimen; Azan method stain.

A B

C D

Fig. 6 Histomorphometric analysis of

trabecular bone in proximal tibia metaphysis

of sham-operated rats (SO), orchidectomized

rats (Orx), orchidectomized rats treated with

testosterone propionate (Orx + TP) or

estradiol dipropionate (Orx + EDP). Graphs

provide data for (A) cancellous bone area

(B.Ar), (B) trabecular thickness (Tb.Th), (C)

trabecular number (Tb.N) and (D) trabecular

separation (Tb.Sp). All values are

mean � SEM. ●P < 0.05 vs. SO and

*P < 0.05 vs. Orx.



concentrations of testosterone (P < 0.05) but had no effect

on serum estradiol when compared with SO rats (Table 1).

After TP administration, serum CT concentration was ele-

vated by 98% (P < 0.05) in comparison with Orx rats. This

treatment increased serum testosterone by 99 and 92%

(P < 0.05) in comparison with the Orx and SO groups,

respectively. After TP treatment, serum estradiol was also

raised by 63 and 57% (P < 0.05) when compared with Orx

and SO animals, respectively. Serum OC and urinary Ca lev-

els were respectively 82 and 84% lower (P < 0.05) than in

the control Orx group, and 60 and 64% lower (P < 0.05)

than in the SO group (Table 1).

After EDP treatment, the concentration of serum CT was

60% (P < 0.05) higher than in the Orx animals, but 26%

(P < 0.05) lower than in the SO rats. Administration of EDP

decreased the serum concentration of testosterone by 87%

(P < 0.05) compared with SO, whereas serum estradiol was

13 and 11 times higher than in the Orx and SO animals

(P < 0.05), respectively. In the EDP-treated group, serum OC

concentration was decreased by 87 and 72% (P < 0.05) in

comparison with Orx and SO rats, respectively. Serum P was

18% (P < 0.05) lower after EDP administration compared

with values for the SO group, whereas urinary Ca concen-

tration was 65% lower than in the Orx animals (P < 0.05)

(Table 1).

Discussion

Thyroid C cells produce CT, a hormone that lowers plasma

Ca concentration by suppressing osteoclast activity. Altho-

ugh both estrogen and testosterone may influence CT

secretion, little information is available regarding possible

sex hormonal regulation of these cells. We previously

reported that estradiol deficiency after Ovx or androgen

deficiency after Orx modulated the structure of rat thyroid

C cells and decreased CT synthesis and release (Filipovi�c

et al. 2003, 2007). Other authors have also suggested that

lack of gonadal steroids negatively affect thyroid C cells in

Ovx and Orx rats (Lu et al. 2000; Sakai et al. 2000), as well

as in both female and male human subjects (Isaia et al.

1992; Lu et al. 2000).

In the present study we demonstrated that Orx in male

middle-aged rats induced a marked decrease in the periph-

eral circulating concentration of testosterone, while TP

application to Orx animals elevated not only serum testos-

terone but also the concentration of serum estradiol. The

increase in the estradiol levels may have been due to aro-

matization of exogenous testosterone in extratesticular tis-

sue. On the other hand, serum estradiol concentrations in

Orx rats can be elevated by supplying EDP, without altering

testosterone concentrations. Compared with Orx animals,

treatment of Orx rats with TP or EDP significantly increased

the volume and volume density of thyroid C cells and raised

serum CT concentrations. Also, low density granule contents

after gonadal steroid treatments as well as prominent Golgi

apparatus in C cells of testosterone-treated rats, indicated

secretory activity similar to the SO control. Additionally,

long profiles of rough endoplasmic reticulum after estradiol

treatment implied active protein synthesis in these cells.

These findings clearly indicate stimulatory effects of male

and female sex steroids on the structure and function of

CT-producing thyroid C cells in Orx middle-aged rats. To our

knowledge, in addition to our morphological and hor-

monal data, only one report has described the effects of sex

steroids on C cells in male rats (Sekuli�c & Lovren, 1993).

These authors also demonstrated stimulatory effects of tes-

tosterone and estradiol on the morphology of thyroid C

cells in aged male rats. On the other hand, several studies

have shown that estrogen treatment stimulates CT secretion

in Ovx rats (Catherwood et al. 1983; Filipovi�c et al. 2003)

and women (Isaia et al. 1992). Regulation of the activity of

thyroid C cells by sex steroids apparently involves activation

of their receptors. Namely, receptors for estrogens (ER) have

been detected in both normal and hyperplastic C cells

(Naveh-Many et al. 1992; Blechet et al. 2007) and binding

of estradiol to ERb in these cells can stimulate their activity.

However, as androgen receptors (AR) have been detected

in hyperplasia of C cells and medullary thyroid carcinoma

Table 1 Serum calcitonin (CT), testosterone, estradiol, osteocalcin (OC), calcium (Ca), phosphorus (P) and urine Ca concentrations in

sham-operated rats (SO), orchidectomized rats (Orx), testosterone propionate- (Orx + TP) and estradiol dipropionate-treated (Orx + EDP)

orchidectomized rats.

SO Orx Orx + TP Orx + EDP

CT (ng L�1) 2.60 � 0.10 1.20 � 0.07† 2.37 � 0.02* 1.92 � 0.08†,*

Testosterone (ng mL�1) 1.51 � 0.17 0.11 � 0.02† 19.36 � 0.73†,* 0.20 � 0.02†

Estradiol (pg mL�1) 36.15 � 3.44 31.00 � 2.72 83.15 � 7.57†,* 399.65 � 34.72†,*

OC (ng L�1) 9.02 � 0.31 19.74 � 0.81† 3.60 � 0.20†,* 2.57 � 062†,*

Ca (mM L�1) 2.46 � 0.01 2.32 � 0.02† 2.36 � 0.01 2.38 � 0.03

P (mM L�1) 2.35 � 0.01 2.03 � 0.01† 2.20 � 0.07 1.93 � 0.01†

Urine Ca (mM L�1) 3.49 � 0.09 7.98 � 0.50† 1.25 � 0.22†,* 2.81 � 0.21*

Data expresed as mean � SEM (n = 8).

*P < 0.05 vs. Orx.

†P < 0.05 vs. SO.



(Zhai et al. 2003; Blechet et al. 2007), we cannot be sure

that C cells normally have a functional AR. Further investi-

gations are needed to confirm any presence of a functional

androgen receptor in C cells. Also, in-depth studies of the

molecular events, especially determination of mRNA levels

for CT in C cells might distinguish between different rates

of steroid effects on synthesis, storage and secretion of CT

in these animals.

Evidence is now available that gonadal hormone defi-

ciency is associated with reduced bone mass in both sexes,

and there is general acceptance that androgens maintain

bone structure in the male as do estrogens in the female.

In addition to the influence of gonadal steroids on

CT-producing C cells, we followed their effects on cancel-

lous bone in proximal tibia of male rats. Orx rats represent

an excellent animal model of male osteoporosis (Vander-

schueren et al. 1992). We have already shown that andro-

gen withdrawal induced by Orx in middle-aged rats results

in reduction of trabecular bone mass and increased cancel-

lous bone turnover (Filipovi�c et al. 2007). In our experi-

ment, we simulated a dose of testosterone typically applied

in substitution therapy in andropausal men, and the dose of

estradiol corresponded to one shown to have an osteopro-

tective effect in male orchidectomized mice and rats

(Vandenput et al. 2001; Fitts et al. 2004). After treatment

with both TP and EDP in this study, we detected significant

increases of B.Ar, Tb.Th and Tb.N, whereas Tb.Sp was

decreased markedly compared with Orx rats. Changes in

bone histomorphometric parameters suggest that these

treatments increased trabecular bone mass. The effect of

EDP on these parameters was more pronounced than that

of TP, but this may have been due to a different dosage

rather than the nature of the sex steroid hormone and its

receptor. Also, the decline of serum OC and urinary Ca con-

centrations indicates that both TP and EDP reduced cancel-

lous bone turnover and urinary Ca excretion, which were

raised after Orx. Our findings confirm previous reports that

testosterone and estrogen improve trabecular structure in

Orx rats (Wuttke et al. 2005; Stuermer et al. 2009).

Much evidence suggests that sex steroids have a protec-

tive effect on bone directly through their receptors. AR

expression has been detected in bone cells, including osteo-

blasts and osteoclasts (Colvard et al. 1989; Turner et al.

2008). Thus, androgens may maintain trabecular bone vol-

ume directly via the osteoblasts (Notini et al. 2007) or inhi-

bit bone resorption by suppressing the formation and

activity of osteoclasts (Huber et al. 2001; Michael et al.

2005). Also, the antiresorptive effect of estrogen is due to

direct action on bone, by inducing apoptosis in osteoclasts

(Kousteni et al. 2002), probably through activating ERa in

these bone-resorbing cells (Nakamura et al. 2007) and/or

expression of both ERa and ERb in osteoblasts (Bord et al.

2001). This effect of estrogen may be mediated by inhibit-

ing the synthesis and secretion of some cytokines in osteo-

blasts that act as paracrine mediators in osteoclasts and

increase their activity, as well as stimulating the secretion of

alkaline phosphatase associated with increased bone forma-

tion (Turner et al. 1994; Harris et al. 1996).

In addition to binding to AR receptors in bone cells, tes-

tosterone may also exert significant effects on bone indi-

rectly through aromatization to estradiol and subsequent

activation of the ER (Syed & Khosla, 2005). The skeleton is a

site for aromatase activity and osteoblasts possess aromatase

and other enzymes necessary for the biosynthesis of estro-

gen (Vanderschueren et al. 1996). The increased bone turn-

over and decreased bone mass in aromatase-deficient male

mice demonstrate the importance of estrogen in protecting

the male skeleton (Miyaura et al. 2001). However, although

data from cultured rat osteoblasts indicate the presence of

aromatase activity in bone cells, Orx in aromatase-knockout

mice resulted in bone loss beyond the adverse effects of aro-

matase deficiency alone. This suggests that androgens

significantly attenuate bone turnover and preserve bone

mass independently of aromatization (Matsumoto et al.

2006). Testosterone and the non-aromatizable androgen,

dihydrotestosterone, can independently prevent osteopenia

in rats after Orx, which indicates the importance of the

AR-mediated pathway (Wakley et al. 1991).

In summary, this study showed that both testosterone

and estradiol play important roles in maintaining bone

mass and are included in the potential mechanism(s) of

age-related bone loss in the male sex. Our observations of

the stimulatory effects of sex steroids, at the cellular and

subcellular levels of thyroid C cells in male rats, are pioneer-

ing and represent a solid basis for further molecular studies.

The increase of calcitonin levels after sex hormone applica-

tion in this experimental model of male osteoporosis indi-

cates a possible additional mechanism by which these

hormones may influence bone metabolism.

Acknowledgements

This work was supported by the Ministry of Education, Science and

Technological Development of the Republic of Serbia, Grant No.

173009. The authors wish to express their gratitude to Prof. Dr.

Steve Quarrie for language correction of the manuscript.

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