Micron 42 (2011) 642655

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Micron
journal homepage: www.elsevier.com/locate/micron

Hormonal therapy in the senescence: Prostatic microenvironment structure and

adhesion molecules
Fbio Montico a , Amanda Cia Hetzl a , Eduardo Marcelo Cndido a ,
Wagner Jos Fvaro a,b , Valria Helena Alves Cagnon a,
a

Department of Anatomy, Cell Biology, Physiology and Biophysics, Institute of Biology, State University of Campinas (UNICAMP),
P.O. Box 6109, 13083-865 Campinas, So Paulo, Brazil
b
Department of Anatomy, Institute of Biosciences, State University of So Paulo Jlio de Mesquita Filho (UNESP), P.O. Box 510, 18618-970 Botucatu, So Paulo, Brazil

a r t i c l e

i n f o

Article history:
Received 26 January 2011
Accepted 8 March 2011
Keywords:
Prostate
Senescence
Steroid hormones
Ultrastructure
Dystroglycans

a b s t r a c t
Hormonal replacement has been utilized to minimize the harmful effects of hormonal imbalance in
elderly men. The development and progression of prostatic diseases and their relation to hormone therapy
is still unclear. Thus, the aim herewith was to characterize the structure and dystroglycan molecule (DGs)
reactivities in the ventral prostatic lobe from elderly rats submitted to steroid hormone replacement.
Male rats (SpragueDawley) were divided into one Young group and six senile groups. The Young group
(YNG) (4 months old) received peanut oil (5 mL/kg, s.c.). The senile rats (10 months old) were submitted to the following treatments: Senile group (SEN) (5 mL/kg peanut oil, s.c.); Testosterone group (TEST)
(5 mg/kg testosterone cipionate, s.c.); Estrogen group (EST) (25 g/kg 17-estradiol, s.c.); Castrated group
(CAS) (surgical castration); CastratedTestosterone (CT) (surgical castration and treatment similar to
TEST group); and CastratedEstrogen (CE) (surgical castration and treatment similar to EST group). After
30 days treatment, blood samples were collected for hormonal analysis and ventral prostate samples
were processed for light and transmission electron microscopies, morphometrical analysis, immunohistochemistry and Western Blotting. The results showed decreased serum testosterone levels in the
senescence and increased testosterone and estrogen plasmatic levels after hormone administration in
the TEST and EST groups, respectively, highlighting the therapy efciency. Hypertrophied stroma and
inammatory cells were veried in the SEN group. After hormone replacement in the senescence or following castration, atrophic epithelium, epithelial cells with clear cytoplasmic halo around the nucleus,
microacini and maintenance of hypertrophied stroma were seen. Decreased DG levels were veried in
the senescence. After hormonal therapy, increased protein levels of these molecules were observed,
especially in those groups which received estradiol. Thus, the occurrence of inammatory cells, stromal
hypertrophy and the presence of cells with clear halo around the nucleus after hormonal therapy probably
indicated prostatic paracrine signaling imbalance, suggesting a stromal reactive microenvironment favorable to the development of glandular lesions. However, the increase of DG levels characterized positive
effect of steroid hormone replacement on the prostate in the senescence. Thus, it could be concluded that
despite having positive effects on important molecules involved in the maintenance of epithelialstromal
interaction and glandular cytoarchitecture, such as DGs, hormonal therapy enhanced structural changes
associated with senescence, probably due to increased hormonal imbalance between androgens and
estrogens in the prostatic tissue.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Abbreviations: YNG, Young group; SEN, Senile group; TEST, Testosterone group;
EST, Estrogen group; CAS, Castrated group; CT, CastratedTestosterone group; CE,
CastratedEstrogen group.
Corresponding author at: Department of Anatomy, Cell Biology, Physiology and
Biophysics, Institute of Biology, State University of Campinas (UNICAMP), P.O. Box
6109, 13083-865 Campinas, So Paulo, Brazil. Tel.: +55 19 3521 6103;
fax: +55 19 3289 3124.
E-mail addresses: fabio260687@gmail.com (F. Montico),
amandacia12@hotmail.com (A.C. Hetzl), emcandido@gmail.com (E.M. Cndido),
wjfavaro@unicamp.br (W.J. Fvaro), quitete@unicamp.br (V.H.A. Cagnon).
0968-4328/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2011.03.004

The prostate is a male accessory sex gland with important role

in the reproductive process, secreting different nutrients that compose the seminal uid, which is essential to motility and nutrition
of the spermatozoids (Marker et al., 2003). In rodents, the prostate
comprises pairs of lobes: ventral, lateral and dorsal, according to
their relation to the urethra, in addition to an anterior lobe or coagulating gland, localized in the concave face of the seminal vesicle
(Marker et al., 2003; Sugimura et al., 1986). The ventral prostate is
often utilized in carcinogenesis experimental studies due to its pri-

F. Montico et al. / Micron 42 (2011) 642655

mary androgen-dependence, despite having no direct homology to

the human prostate (Slayter et al., 1994). The prostate shows acini
covered by simple epithelium presenting luminal cells, basal cells
and neuroendocrine cells which can be distinguished by differential expression of cytokeratins (CK) and surface antigens (CD) (Berry
et al., 2008; Miki, 2009; Niu and Xia, 2009). The epithelium rests in
a basal membrane, which is composed by laminin, collagen IV and
heparan sulfate (Jorcyk et al., 1998; Knox et al., 1994). The prostatic
stroma is a complex arrangement of broblasts, smooth muscle
cells and extracellular matrix associated with growth factors, regulatory molecules and remodelling enzymes (Taipale and Keski-Oja,
1997; Tuxhorn et al., 2001). In addition, blood vessels, nerves and
immune cells are integral parts of the stroma (Bianchi-Frias et al.,
2010; Tuxhorn et al., 2001).
Adhesion molecules, such as dystroglycans (DGs), make the
connection between epithelial cells and basal membrane, promoting cytoskeleton organization and its link to extracellular milieu
(Brennan et al., 2004; Losasso et al., 2000; Sgambato and Brancaccio,
2005). Initially, DGs were identied as proteins of the dystrophin
glycoproteic complex in the skeletal muscle, having a role in the sarcolemma stabilization during muscle contraction (Sgambato and
Brancaccio, 2005; Singh et al., 2004; Winder, 2001). Currently, it is
known that these adhesion molecules occur in all tissues and cellular types of vertebrates and are responsible for functions such as
cellular adhesion, organization of basal membrane and cytoskeleton, epithelial morphogenesis and extracellular signal transduction
(Cross et al., 2008; Singh et al., 2004). The DGs are composed by
two glycoproteic subunits, -DG and -DG (Brennan et al., 2004;
Sgambato and Brancaccio, 2005). -DG is linked to basal lamina
components and interacts in a non-covalent manner with the extracellular domain of -DG, a transmembrane protein which promotes
the connection between -DG and cytoskeletal actin laments
(Brennan et al., 2004; Losasso et al., 2000; Sgambato and Brancaccio,
2005; Winder, 2001).
The prostate is an androgen-dependent organ and the main
circulating androgens responsible for inducing glandular differentiation and regulating its morphology are testosterone (T)
and dihydrotestosterone (DHT) (Cunha et al., 2002; Hsing, 2001;
Imamov et al., 2005; Leav et al., 2001; Toorians et al., 2003).
The castration is one of the most utilized methods for studying
the mechanisms involving testosterone in prostatic structural and
functional maintenance. Thus, different studies showed that androgen deciency led to prostatic involution, apoptosis activation in
epithelial cells and extracellular matrix remodelling in this organ
(Banerjee et al., 2000; Flrez and Carvalho, 2005; Vilamaior et al.,
2000). In addition to androgens, other hormones such as estrogens act synergistically to testosterone, inuencing both normal
and pathological prostatic processes (Cunha et al., 2002; Weihua
et al., 2001). Estrogens have anti-androgenic effects and regulate negatively the hypothalamus-pituitary-gonadal axis, reducing
androgen production by Leydig cells and leading to prostatic
epithelial involution and stromal growth in adult animals (Weihua
et al., 2002).
Senescence is a period of life in which important changes occur
in the hormonal environment, with functional and morphological
alterations on the prostate of various species, resulting in higher
propension for developing urological disorders such as benign prostatic hyperplasia (BPH) and prostate cancer (PC) (Banerjee et al.,
2000; Krtolica and Campisi, 2002; Lau et al., 2003; Schulman and
Lunenfeld, 2002). According to Krieg et al. (1993), DHT levels in
prostatic epithelium decrease during aging and, on the contrary,
estradiol levels increase.
Nowadays, androgen replacement therapy has been used clinically with the aim of attenuating the harmful effects of hormonal
imbalance in the senescence, improving the quality of life of the
elderly (Lau et al., 2003; Morales, 2002). On the other hand, there

643

are doubts about the effects of hormonal therapy on the development and progression of prostatic malignant lesions. Different
authors pointed that exogen androgen administration frequently
exacerbates pre-existing lesions (Marks et al., 2006; Morales, 2002).
However, there is no direct evidence that hormonal therapy is able
to promote tumor development (Marks et al., 2006; Morales, 2002).
Thus, the aim of this work was to characterize the glandular
structure and the immunoreactivity of and dystroglycans in the
prostatic ventral lobe of senile rats submitted to steroid hormone
replacement, relating the alterations resulting from hormonal therapy to possible prostatic lesions.
2. Materials and methods
2.1. Animals and experimental procedures
A total of 63 male rats (SpragueDawley) were used and divided
into one Young group and six senile groups (9 animals per group).
The Young group (YNG), 4 months old, received a 5 mL/kg dose of
peanut oil subcutaneously (s.c.). The rats in the senile groups (10
months old) were submitted to the treatments: Senile group (SEN):
5 mL/kg of peanut oil (s.c.); Testosterone group (TEST): 5 mg/kg
of testosterone cipionate (Deposteron-Novaqumica, So Paulo, SP,
Brazil) diluted in 5 mL of peanut oil (s.c) (Franck-Lissbrant et al.,
1998; Sttolo et al., 2004); Estrogen group (EST): 25 g/kg of 17estradiol (Sigma Chemical Co., St. Louis, USA) diluted in 25 L of
peanut oil (s.c.) (Prins et al., 2001); Castrated group (CAS): rats
were anesthetized with a 0.25 mL/100 g body weight dose of Francotar/Virbaxyl (1:1, Vibra , Roseira, SP, Brazil) and castrated by
surgical incision; CastratedTestosterone group (CT): surgical castration and after 30 days the rats received the same treatment as
the TEST group; CastratedEstrogen group (CE): surgical castration
followed by the same treatment as EST group after 30 days.
All rats received water and solid ration ad libitum (Nuvilab,
Colombo, PR, Brazil). After 30 days of treatment every other day, the
animals were weighed on a semi-analytical scale (Marte AS 5500)
and sacriced. Samples from ventral lobe of prostate were collected
and processed for light and transmission electron microscopies and
for morphometrical, immunohistochemical and Western Blotting
analysis. Steroid hormone concentrations on blood samples were
also determined.
This study was approved by the institutional Committee for
Ethics in Animal Research (State University of Campinas Unicamp,
protocol no. 1748-1) and the experiments were done in agreement
with the Ethical Principles for Animal Research established by the
Brazilian College for Animal Experimentation (COBEA).
2.2. Serum hormone levels
After 24 h following the last steroid hormone administration,
experimental animals were anesthetized with a 0.25 mL/100 g
body weight dose of Francotar/Virbaxyl (1:1, Vibra , Roseira,
SP, Brazil). Blood samples were collected from ve animals of
each group by cardiac puncture in the left ventricle. Plasma was
isolated by centrifugation (10,000 rpm, 4 C, 10 min) and stored
in 20 C for subsequent analysis. The serum concentrations of
testosterone (ng/mL) and estradiol (pg/mL) were determined by
radioimmunoassay using Coat-a-Count total testosterone/estradiol
kits (Diagnostic Products Corporation, Los Angeles, USA).
2.3. Light microscopy
Samples of the ventral prostate were collected from ve animals in each group and then xed by immersion in Bouins solution
from each one of animals during 24 h. After dehydratation in an

644

F. Montico et al. / Micron 42 (2011) 642655

Table 1
Serum testosterone (ng/mL) and estradiol (pg/mL) levels in the different experimental groups.
Hormone

Experimental groups
YNG

Testosterone
Estradiol

SEN

7.0 0.31 a
3.2 0.34 b
0.06 0.01 a 0.04 0.02 a

TEST

EST

CAS

CT

CE

7.1 0.61 a
0.05 0.03 a

1.4 0.26 c
0.52 0.11 b

0.03 0.02 d
1.7 0.26 c

0.36 0.07 d
0.54 0.04 b

0.15 0.05 d
1.6 0.31 c

Different lowercase letters indicate statistically signicant differences (P < 0.05) among the groups after Tukeys test.

Fig. 1. Photomicrographs of the ventral prostate of animals from Young (AD), Senile (EH), Testosterone (IL) and Estrogen (MP) groups. Young Group: (A) Folded prostatic
acini. Epithelium (Ep); stroma (St), lumen (L). (B) Tall columnar cells with basal nuclei (N) and evident nucleoli. The clear supranuclear areas probably represent the Golgi
complex (G). Basal cells (Bc). (C) Prostatic stroma with smooth muscle cells (Smc) and thin layer of collagen bers (Col) surrounding the glandular acini. Blood vessels (Bv).
(D) Thin layer of reticular bers (Rf) adjacent to prostatic acini. Senile Group: (E) Glandular acini showing folded mucosa. (F) Secretory epithelium with columnar and basal
cells (Bc). Probable location of the Golgi complex (G). (G) Hypertrophic stroma presenting smooth muscle cells (Smc) intermingled by increased amount of collagen bers
(Col). Blood vessels (Bv) and inammatory cells (asterisk). (H) Increased and undulated reticular bers (Rf) around the glandular acini. Testosterone Group: (I) Slightly folded
mucosa and epithelial atrophy. (J) Cubical luminal cells intermingled by atipic phenotype cells (arrowheads). (K) Thickening of collagen bers (Col) and smooth muscle cells
(Smc). Microacinus (Ma), blood vessels (Bv) and inammatory cells (asterisk) in the glandular stroma. (L) Hypertrophic reticular bers (Rf). Estrogen Group: (M) Atrophic
acini and microacinus (Ma). (N) Cubical cells and cells with clear cytoplasmic halo (arrowhead) in the epithelium. Inammatory inltration in the stroma (asterisk). (O)
Hypertrophic prostatic stroma, smooth muscle cells (Smc), collagen bers (Col), blood vessels (Bv) and microacinus (Ma). (P) Increased amount of thick stromal reticular
bers (Rf).

F. Montico et al. / Micron 42 (2011) 642655

645

64,260.9
77,066.8
154,512.8
20.4
42.2
62.6
Different lowercase letters indicate statistically signicant differences (P < 0.05) among the groups after Tukeys test.

Epithelial
Stromal
Luminal
Nuclear
Cytoplasmic
Cellular

105,519.4
63,015.4
127,305.7
28.2
79.5
107.8

11,637.1 ab
10,959.8 a
19,535.2 a
0.97 a
12.9 a
13.8 a

127,964.3
66,485.6
101,390.6
27.7
71.9
99.7

8603.1 a
4419.9 a
10,627.1 ab
2.4 a
5.4 a
7.4 a

85,118.0
91,642.5
119,080.0
26.7
59.4
86.1

22,630.2 bc
13,962.5 a
22,023.2 a
2.8 ab
10.3 ab
13.0 ab

79,303.3
82,812.0
133,725.2
24.9
61.4
85.7

17,926.8 bc
26,106.5 a
30,374.2 a
2.8 ac
10.3 ab
11.8 ab

58,108.6
195,443.6
42,288.3
19.8
29.3
49.1

10,092.1 cd
37,910.7 b
28,527.1 c
1.4 c
2.0 c
3.2 c

CT
CAS
EST
TEST
SEN

The samples of the ventral prostate were collected from ve

animals in each group, the same used in light microscopy analysis. The sections were deparafnized in xylene, hydrated in a
decreasing alcohol series and rinsed under distilled water. Antigens were retrieved by boiling the sections in 10 mM citrate
buffer (pH 6.0) three times for 5 min in a microwave oven. After
that, the sections were incubated in 0.3% H2 O2 for 15 min to
block endogenous peroxidase. Nonspecic binding was blocked
by incubating the sections in a blocking solution (3% BSA in
TBS-T buffer) for 1 h at room temperature. Primary rabbit H300 (sc-28534) (Santa Cruz Biotechnology, California, USA) for
-DG and mouse (NCL-b-DG) (Novocastra Laboratories, Newcastle, UK) for -DG antibodies were diluted in 1% BSA (1:50)
and applied to the sections overnight at 4 C. The sections were
washed for 15 min with TBS-T and secondary labeled polymer
from the Envision HRP Kit (Dako Cytomation Inc., Carpenteria, USA) was applied for 40 min at room temperature. After
washing in TBS-T, peroxidase activity was detected using a
diaminobenzidine (DAB) chromogen from Envision HRP Kit (Dako)
for 10 min. Sections were lightly counterstained with methyl
green, dehydrated in an increasing ethanol series and xylene,
mounted in Entellan (Merck, Darmstadt, Germany) and photographed in the photomicroscope (Nikon Eclipse E-400). Sections

Experimental groups

2.6. Immunohistochemistry

YNG

Four animals from each experimental group were anesthetized

and perfused with Karnovskys solution through the left ventricle (Graham and Karnovsky, 1965; Sprando, 1990). The ventral
lobe of the prostate was collected and xed by immersion in the
same xative, followed by post-xation in 1% osmium tetroxide
for 2 h. The material was then dehydrated in an increasing acetone
series, embedded in resin (Polysciences, Niles, USA) and 0.5 m
thick sections were stained with Toluidine blue and prepared for
light microscopy in order to choose specic areas for transmission
electron microscopy analysis. Ultra-thin sections were obtained
with an ultramicrotome (Ultracult UCT 020 Leica), mounted on 200
Mesh copper grids (Sigma Chemical Company, St. Louis, USA),
and contrasted with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). The specimens were then examined and
photographed with a LEO 906 transmission electron microscope.

Areas

2.5. Transmission electron microscopy

Table 2
Mean standard deviation of epithelial, stromal, luminal, nuclear, cytoplasmic and cellular areas (m2 ) of the ventral prostate of rats from the experimental groups.

Samples of the ventral lobe from ve animals per

group, the same used in light microscopy and stained with
Hematoxylineosin, were submitted to morphometrical analysis.
To quantify epithelial, luminal and stromal areas, ten microscopic
elds were taken at random and analyzed per animal, resulting in
50 elds per group, using a Nikon Eclipse E-400 photomicroscope
(Nikon, Tokyo, Japan) with an X20 objective. Cellular, cytoplasmic
and nuclear areas were measured (10 microscopic elds were
taken at random per animal), using a Nikon Eclipse E-400 photomicroscope (Nikon, Tokyo, Japan) with an X100 objective. All the
areas were measured using the NIS-Elements: Advanced Research
(USA) computerized image analysis system.

17,415.0 cd
15,771.2 a
21,129.6 a
5.4 c
14.4 bc
19.5 bc

2.4. Morphometrical procedures

38,991.5
201,275.2
55,573.8
21.3
34.6
55.9

CE

3138.7 d
45,062.4 b
47,345.7 bc
3.2 bc
9.6 c
12.7 c

increasing alcohol series, the tissues were diaphanized and embedded in paraplast (Paraplast Plus, St. Louis, USA), cut into 5 m
thick sections and submitted to the following staining procedures:
Hematoxylineosin, Massons trichrome (Junqueira et al., 1979)
and Ammoniacal silver (Vilamaior et al., 2000). The slides were
photographed with a Nikon Eclipse E-400 photomicroscope (Nikon,
Tokyo, Japan).

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F. Montico et al. / Micron 42 (2011) 642655

Fig. 2. Photomicrographs of the ventral prostate of animals from Castrated (AD), CastratedTestosterone (EH) and CastratedEstrogen (IL) groups. Castrated Group:
(A) Atrophic acini with reduced lumen, surrounded by a thick layer of collagen bers (Col). Epithelium (Ep); stroma (St). (B) Disorganization and atrophy of the secretory
epithelium, characterized by cubical luminal cells and cells with clear cytoplasm (arrowheads). (C) Prostatic stroma showing thickened collagen bers (Col), blood vessels
(Bv), smooth muscle cells (Smc) and inammatory cells (asterisk). (D) Stromal hypertrophy with great amount of thick reticular bers (Rf). CastratedTestosterone Group:
(E) Slightly folded and atrophic acinar mucosa. Microacinus (Ma); lumen (L). (F) Atrophic epithelium with cubical cells. Nuclei (N). Basal cells (Bc) and cells with clear
cytoplasmic halo around the nucleus (arrowheads). (G) Thickened collagen bers (Col), smooth muscle cells (Smc), blood vessels (Bv) and inammatory cells (asterisk). (H)
Reticular ber (Rf) thickening in the stroma. CastratedEstrogen Group: (I) Acinar atrophy and microacinus (Ma). (J) Epithelial structural disorganization with atrophic
luminal cells, basal cells (Bc) and cells presenting an atypical phenotype (arrowheads). (K) Hypertrophic stroma showing smooth muscle cells (Smc), collagen bers (Col),
blood vessels (Bv) and inammatory cells (asterisk). (L) Increased and thickened reticular bers (Rf).
Table 3
Immunolabelled intensity of alpha and beta dystroglycans (-DG e -DG) in the ventral prostate of rats from the experimental groups.
Molecules

-DG
-DG

Glandular compartments

Epithelium
Stroma
Epithelium/stroma

Experimental groups
YNG

SEN

TEST

EST

CAS

CT

CE

+++
+++
++

+
++
++

++
+++
++

+++
+++
+++

+
+
+

++
+++
++

+++
+++
+++

F. Montico et al. / Micron 42 (2011) 642655

from ventral prostate that were not stained with primary antibody for -DG and -DG were used as negative controls. The
intensity of antigen immunoreactivity in epithelial and stromal compartments was graded as intense (+++), moderate (++),
and weak (+), according to the antigens distribution in the sectioned tissue (altered Markopoulos et al., 2000). Because -DG
immunolabelling occurs in the epitheliumstroma interface, these
compartments were considered together in the evaluation of its
reactivity.

647

Serum estradiol levels in YNG and SEN groups were not statistically different from each other (Table 1). However, estradiol
treatment led to increased plasma concentrations of this hormone
in the EST group in relation to young and senile rats, with similar
levels to those found in animals that received testosterone following castration (Table 1). In addition, greater increase in estrogen
serum concentrations was veried in CAS and CE groups (Table 1).
3.2. Light and transmission electron microscopies,
immunohistochemistry and Western blotting analysis

2.7. Western blotting analysis

Ventral prostate samples from ve animals in each experimental group were collected and frozen. The samples were
weighed and homogenized in a Polytron homogenizer (Kinematica Inc., Lucerne, Switzerland) for 1 min in a 50 L/mg lysis
buffer. The tissue homogenates were centrifuged at 14,000 rpm
for 20 min at 4 C and a sample of each extract was used for
protein quantication with Bradford reagent (Bio-Rad Laboratories, Hercules, USA). The supernatants were mixed (1:1) with 3
sample buffer and transferred to a dry bath at 100 C for 5 min.
Aliquots containing 75 g of protein were separated by electrophoresis in SDS-polyacrylamide gels under reducing conditions.
After electrophoresis, proteins were transferred to Hybond-ECL
nitrocellulose membranes (Amersham, Pharmacia Biotech, Arlington Heights, USA) at 70 V for 3 h. The membranes were blocked
with TBS-T containing 3% BSA for 60 min, rinsed in TBS-T and
incubated at 4 C overnight with the following primary antibodies: rabbit H-300 (sc-28534) (Santa Cruz Biotechnology, California,
USA) for -DG, mouse (NCL-b-DG) (Novocastra Laboratories, Newcastle, UK) for -DG and mouse ACTBD11B7 (sc-81178) (Santa
Cruz Biotechnology, California, USA) for -actin diluted in 1% BSA
(1:500). The membranes were then incubated for 2 h with rabbit or
mouse secondary HRP-conjugated antibody (Promega Corporation,
USA) diluted 1:2500 in 1% BSA. After washing in TBS-T, peroxidase activity was detected by incubation with a diaminobenzidine
(DAB) chromogen (Sigma Chemical Company, St Louis, USA) for
10 min. -Actin quantication was used as an endogenous control for the comparison among groups. The intensity of antigen
bands in each experimental group was determined by densitometry using the software Nis-Elements: Advanced Research (USA)
and was expressed as the mean ratio in relation to -actin band
intensity.
2.8. Statistical analysis
Average data for total testosterone and estradiol serum concentrations; cellular, cytoplasmic, nuclear, epithelial, luminal and
stromal areas (m2 ) and DG protein levels determined by Western
Blotting were compared among groups and analyzed statistically
by analysis of variance (ANOVA) and Tukey multiple range test,
with the level of signicance set at 5% (Montgomery, 1991; Zar,
1999). The results were expressed as the mean standard deviation.
3. Results
3.1. Serum hormone levels
Signicant reduction of plasma testosterone concentrations was
veried in the senile rats in relation to young ones (Table 1). Testosterone replacement was effective in restoring the levels of this
hormone in the senescence, leading to average levels statistically
similar to those found in the YNG group (Table 1). Also, reduced
testosterone concentrations were veried in the animals submitted
to estrogenic therapy and mainly in the castrated groups (Table 1).

3.2.1. Young group (YNG)

The prostatic ventral lobe was characterized by acini with
infolded mucosa (Fig. 1A). The secretory epithelium was simple
and composed by tall columnar cells with basal nuclei intermingled
by basal cells (Figs. 1B and 3A). The cellular cytoplasm presented
attened rough endoplasmic reticulum cisternae and secretory
vesicles showing different electrondensities in the apical region
(Fig. 3B and C). Glandular epithelium area was 1.7 times greater
than stromal one (Table 2; Fig. 2).
The prostatic stroma showed smooth muscle cells intermingled
by thin and organized layer of collagen and reticular bers underlying the glandular acini and blood vessels (Figs. 1C, D and 3D, E).
Intense -DG reactivity was seen in the luminal surface of
epithelial cells as well as in the prostatic stroma. The protein levels
of this adhesion molecule were 128.2% in relation to -actin standard, and were higher in relation to the other groups, except those
which received estradiol (Fig. 5A and H; Table 3). -DG characterized moderate immunolocalization next to basal membrane and
average protein levels of 112.2% (Fig. 6A and H; Table 3).
3.2.2. Senile group (SEN)
The prostatic epithelium from senile rats showed similar
morphological aspects to those observed in the YNG group
(Figs. 1E, F and 3F, G, H). Epithelial area was approximately 1.9
times greater than the stromal one and did not differ statistically in
relation to YNG group. The same occurred with nuclear, cytoplasmic and cellular areas (Table 2). Hypertrophied stroma was veried,
showing increased collagen and reticular bers in addition to the
occurrence of inammatory cells (Figs. 1G, H and 3I, J). However, no
statistical signicance in relation to YNG group stromal area was
observed (Table 2).
In the senile rats, -DG immunolocalization was weak in the
epithelium and moderate in the stroma, with protein levels of
77.2%, which were signicantly reduced in relation to the YNG
group (Fig. 5B and H; Table 3). Moderate -DG reactivity was veried in the stromaepithelium interface, with protein levels of
100.3%. Although these levels were numerically smaller than those
of the YNG group, there was no statistical difference between the
two experimental groups (Fig. 6B and H; Table 3).
3.2.3. Testosterone group (TEST)
The glandular epithelium structural features were similar to
those found in the YNG and SEN groups (Figs. 1I and 3K, L). However,
some acini presented atrophied secretory epithelium with cubical
luminal cells intermingled by cells showing clear cytoplasmic halo
around the nucleus (Fig. 1J). Occasional microacini were veried
(Fig. 1K). Secretory epithelium area was signicantly smaller than
that found in the SEN group and represented 0.9 times the stromal
area (Table 2). Nuclear, cytoplasmic and cellular areas were statistically similar to those found in the YNG and SEN groups, despite
showing smaller numerical values (Table 2).
Maintenance of hypertrophied stroma in relation to YNG group
was veried, with pronounced collagen and reticular ber thickening, in addition to the occurrence of inammatory cells (Fig. 1K
and L). Nevertheless, even though stromal area was numerically

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F. Montico et al. / Micron 42 (2011) 642655

Fig. 3. Electronmicrographs of the ventral prostate of animals from Young (AE), Senile (FJ), Testosterone (KN) and Estrogen (OR) groups. Young Group: (A) Tall columnar
luminal cells, basal nuclei (N) and nucleoli (Nu), secretory vesicles (Sv) in the apical cytoplasm. (B) and (C) Apical and supranuclear regions of luminal epithelial cells showing
secretory vesicles (Sv) and parallel and attened rough endoplasmic reticulum (RER) cisternae. (D) and (E) Stroma (St) with smooth muscle cells (Smc), broblasts (Fb)
and thin collagen bers (Col); blood vessel (Bv); endothelial cell (Ec); basal membrane (Bm). Senile Group: (F) Columnar cells with basal nuclei (N) and prominent nucleoli (Nu); lumen (L). (G) Short and scattered microvilli (Mv). (H) Frequent secretory vesicles (Sv); parallel and attened Golgi complex (arrow) and rough endoplasmic reticulum

F. Montico et al. / Micron 42 (2011) 642655

increased in relation to the YNG group, there was no statistical signicance in this difference (Table 2). Smooth muscle cells showed
small secretory vesicles and folded sarcolemma, conferring them a
spinous aspect (Fig. 3M and N).
Moderate epithelial and intense stromal reactivities were
characterized for -DG, while -DG showed moderate immunolocalization in the basal membrane region (Figs. 5C and 6C; Table 3).
-DG and -DG protein levels represented 90.6% and 119.9%,
respectively, which were signicantly greater than those observed
in senile animals (Figs. 5H and 6H).
3.2.4. Estrogen group (EST)
The majority of glandular acini presented less folded mucosa
(Fig. 1M). Atrophied secretory epithelium was veried, showing
cubical luminal cells and cells with clear cytoplasmic halo around
the nucleus (Figs. 1N and 3O). Occasional points with dilated rough
endoplasmic reticulum and Golgi complex cisternae were veried
in the epithelial cell cytoplasm, as well as mitochondria with ruptured cristae (Fig. 3P). Microacini were frequently seen (Fig. 1M
and O). Similar to the observations in the TEST group, epithelial area
was signicantly reduced in relation to senile animals. On the other
hand, nuclear, cytoplasmic and cellular areas did not show statistical difference (Table 2). Moreover, hypertrophied stroma with
thick collagen and reticular bers and spinous smooth muscle cells
were veried, in addition to the occurrence of inammatory cells
(Figs. 1O, P and 3Q, R). Stromal area was greater than the epithelial
one, however, it was statistically similar to those registered in the
YNG and SEN groups (Table 2).
Intense -DG reactivity occurred in the luminal surface of
epithelial cells and in the stroma, with protein levels of 149.4%,
representing statistical signicant increase in relation to the SEN
group (Fig. 5D and H; Table 3). -DG also showed intense reactivity
in the basal membrane region, and the protein levels represented
198%, which was signicantly greater than the observed in the SEN
group (Fig. 6D and H; Table 3).
3.2.5. Castrated group (CAS)
Intense atrophy and structural disorganization of the glandular
epithelium was observed in the prostatic acini of castrated animals (Figs. 2A, B and 4A). Cubical luminal cells and cells with clear
cytoplasmic halo around the nucleus were veried, especially overlapping the basal membrane (Fig. 2B). In the cellular cytoplasm,
dilated rough endoplasmic reticulum and Golgi complex cisternae
were noticed, in addition to the occurrence of ruptured mitochondrial cristae and digestory vacuoles (Fig. 4AD). Epithelial, luminal,
nuclear, cytoplasmic and cellular areas were signicantly reduced
when compared to young and senile rats (Table 2).
Intense stromal hypertrophy was veried, with a large amount
of thickened and undulated collagen and reticular bers distributed
in concentric layers around frequent microacini (Fig. 2A, C and D).
Stromal area was signicantly increased in relation to the YNG
and SEN groups, and was approximately 3.4 times greater than
the epithelial area (Table 2). Smooth muscle cells showed folded
sarcolemma, giving them a spinous aspect with small secretory
vesicles (Fig. 4E). Inammatory cells were frequently observed in
the stroma (Figs. 2A, C and 4E).

649

Weak -DG reactivity was seen in both epithelium and stroma.

The same reactivity intensity was veried for -DG in the
epitheliumstroma interface (Figs. 5E and 6E; Table 3). -DG and
-DG protein levels were 76.4% and 92.0%, respectively, and were
signicantly reduced in relation to the YNG group. However, these
levels were statistically similar to those veried in the SEN group
(Figs. 5H and 6H).
3.2.6. CastratedTestosterone group (CT)
Partial recovery of acinar atrophy and of the epithelium
structure was characterized following testosterone replacement
(Figs. 2E, F and 4F). However, some regions showed glandular
acini with slightly folded mucosa, epithelial atrophy and microacini
(Fig. 2E). The secretory epithelium presented cubical luminal cells
intermingled by basal cells and cells with clear cytoplasmic halo
around the nucleus (Fig. 2F). In addition, the ultrastructural features
were similar to those observed in the castrated animals (Fig. 4G).
In agreement to the CAS group, epithelial, nuclear, cytoplasmic and
cellular areas showed signicant decrease in relation to the YNG
and SEN groups (Table 2). On the other hand, luminal area presented
no statistical difference in comparison to these groups, indicating
increased acinar area and glandular recovery (Table 2). Despite the
occurrence of hypertrophy and inammatory cells in the stroma
(Figs. 2G, H and 4H and I), there was signicant decrease of stromal
area in relation to the CAS group, with values similar to those found
in the young and senile rats (Table 2).
Moderate epithelial reactivity and intense immunolabelling in
the stroma were detected for -DG, showing protein levels of
91.2%, which were signicantly increased in relation to SEN and
CAS groups and similar to the levels veried in the TEST group
(Fig. 5F and H; Table 3). -DG showed moderate reactivity in the
basal membrane region and protein levels of 116.6%, characterizing greater value in relation to SEN group and statistical similarity
with YNG and TEST groups (Fig. 6F and H; Table 3).
3.2.7. CastratedEstrogen group (CE)
Estrogen therapy after castration did not lead to signicant
recovery of the glandular morphology, characterizing absence of
acinar mucosa infolding and occurrence of microacini (Fig. 2I).
Atrophied secretory epithelium was identied, presenting cubical luminal cells, basal cells and cells with clear cytoplasmic halo
around the nucleus (Figs. 2J and 4J). Epithelial area was similar to
that of CAS group and signicantly reduced in relation to senile and
young animals. Similar behavior was veried regarding nuclear,
cytoplasmic and cellular areas (Table 2). Luminal area, despite being
reduced, did not show signicant statistical difference in relation
to senile animals (Table 2). Stromal hypertrophy, characterized by
thickened brillar elements, and occurrence of inammatory cells
were veried (Fig. 2I, K and L). Similar to castrated animals, stromal area presented signicant increase in relation to YNG and SEN
groups and was 5.2 times greater than the epithelial area (Table 2).
Epithelial and stromal ultrastructural changes were similar to those
described to CAS group (Fig. 4KM).
Intense -DG reactivity was veried in both glandular epithelium and stroma, showing protein levels of 148.4%, which were
signicantly increased in relation to SEN and CAS groups, and

(RER) cisternae. (I) and (J) Stroma (St) with collagen bers (Col) underlying the epithelium and intermingling with smooth muscle cells (Smc) and broblasts (Fb); basal
membrane (Bm). Testosterone Group: (K) Columnar cells with basal nuclei (N) and evident nucleoli (Nu); lumen (L); inset: short and scattered microvilli (Mv). (L) Parallel
and attened Golgi complex (arrow) and rough endoplasmic reticulum (RER) cisternae; frequent secretory vesicles (Sv). (M) and (N) Stroma (St) with abundant collagen
bers (Col) and smooth muscle cells (Smc) showing spinous aspect (arrowhead) and secretory vesicles (v); basal membrane (Bm). Estrogen Group: (O) Columnar luminal
cells with basal nuclei (N) and prominent nucleoli (Nu); basal epithelial cells (Bc); lumen (L). (P) Dilated Golgi cisternae (arrow); mitochondria with ruptured cristae (M). (Q)
and (R) Hypertrophic stroma (St) showing increased amount of collagen bers (Col) and smooth muscle cells (Smc) with spinous aspect (arrowhead); broblasts (Fb); blood
vessel (Bv).

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F. Montico et al. / Micron 42 (2011) 642655

Fig. 4. Electronmicrographs of the ventral prostate of animals from Castrated (AE), CastratedTestosterone (FI) and CastratedEstrogen (JM) groups. Castrated
Group: (A) Atrophic epithelium with cubical cells and irregular nuclei (N). Digestory vacuoles (Dv); lumen (L). (B) Discontinuity of microvilli (Mv); rare secretory vesicles (Sv) and mitochondria with ruptured cristae (M). (C) Dilated Golgi (arrow) and rough endoplasmic reticulum (RER) cisternae. (D) and (E) Basal
epithelial cell (Bc) with numerous digestory vacuoles (Dv) in the cytoplasm; stroma (St) showing abundant collagen bers (Col) and smooth muscle cells (Smc)
presenting spinous aspect and secretory vesicles (v); basal membrane (Bm); inammatory cell (Ic). CastratedTestosterone Group: (F) Atrophic cells; nuclei (N);
stroma (St); lumen (L). (G) Dilated Golgi (arrow) and rough endoplasmic reticulum (RER) cisternae; mitochondria with ruptured cristae (M). (H) and (I) Hypertrophic

F. Montico et al. / Micron 42 (2011) 642655

similar to those found in the EST group (Fig. 5G and H;

Table 3). Also, intense -DG immunolabelling was detected in
the epitheliumstroma interface, characterizing protein levels of
134.8%, which were increased in relation to senile and castrated
animals (Fig. 6G and H; Table 3).
4. Discussion
The results herewith characterized signicant reduction of
testosterone serum levels in the senescence, which were associated
with harmful changes in the prostatic stromal microenvironment.
Hormone replacement was effective, leading to increase of serum
testosterone and estradiol levels in the TEST and EST groups,
respectively. Hormonal therapy in the senescence also caused drastic structural changes in the glandular secretory epithelium, such
as the presence of cells showing clear cytoplasmic halo around
the nucleus, indicating cellular atypia, in addition to increased
stromal hypertrophy. Similar disturbances were veried in castrated groups, with or without hormonal treatment. However in
these cases glandular morphologic disorders were more intense.
In addition, senescence led to molecular changes in the prostatic
microenvironment, showing decreased DG protein levels. On the
other hand, hormonal replacement resulted in recovery of the protein levels of these adhesion molecules. In the castrated animals,
reduced levels of both DG isoforms were observed, with an increase
of these protein levels being registered after hormonal therapy.
Senescence has been showed as a period of signicant changes
in the hormonal environment. In addition to the progressive
decline of circulating androgen levels, which is accompanied by
increased conversion of these hormones to estrogens, decrease of
5-reductase activity in the glandular epithelium was also registered, leading to diminished DHT epithelial levels (Krieg et al., 1993;
Srinivasan et al., 1995). Also, progressive accumulation of estradiol
in the prostatic stroma was characterized, suggesting an enhancement of the pathogenic role of estrogens on the human prostate in
the senescence (Krieg et al., 1993). In elderly rodents, decrease in
serum androgen levels and in prostatic hormonal sensitivity were
veried, probably due to diminished androgen receptor levels and
5-reductase activity in the organ, as well as a result of altered
aromatization rates of testosterone to estradiol (Banerjee et al.,
1998, 2001; Cordeiro et al., 2008; Prins et al., 1996; Scarano et al.,
2006). Moreover, the prostate of elderly rats showed decreased
apoptosis rates following castration than those veried in young
animals, indicating that prostatic cells developed androgenic independence during the senescence process, making the prostate less
sensitive to androgen withdrawal (Banerjee et al., 2000; Morrissey
et al., 2002). Several studies demonstrated the occurrence of progressive morphological changes in the ventral prostate during
senescence, including epithelial atypia and atrophy, epithelial and
stromal hyperplasia, decrease in acinar projections into the lumen,
the presence of amylaceous bodies and inltration of inammatory cells (Acosta et al., 2004; Lau et al., 2003; Morrissey et al.,
2002). As in the present results, Bianchi-Frias et al. (2010) did not
observe structural changes in the epithelial compartment between
young and senile rodents, however senescence led to evident alterations in the stromal microenvironment, such as increased and
disorganized collagen bers, loss of basal membrane rigidity and
of smooth muscle cell orientation, in addition to inammatory cell
inltrates. Also, pronounced inammation process was veried in
elderly mice overexpressing aromatase gene (AROM+ ) in compari-

651

son to wild type controls, suggesting that estrogenic hormones are

able to induce inammation in the prostate (Ellem and Risbridger,
2009). In addition, it is known that cytokines secreted by inammatory cells are able to stimulate prostatic aromatase expression
(Ellem and Risbridger, 2009).
According to Marks et al. (2006), androgen replacement in
elderly men is able to restore serum androgenic concentrations
to standard levels. However, this therapy exerts little inuence
on androgen levels and cell functions in the prostatic microenvironment. Studies with experimental animals registered that the
administration of increasing testosterone doses caused epithelial
hypertrophy and hyperplasia in different prostatic lobes, both in
young and in elderly rodents, whereas stromal changes were not
observed (Acosta et al., 2004; Banerjee et al., 1994). On the other
hand, while androgen treatment preserved prostatic structural
integrity, isolated estrogen administration led to accumulation of
collagen bers and epithelial atrophy (Leav et al., 1989; Suzuki
and Nakada, 1996). It is known that exogen estrogen treatment
results in prostatic epithelial atrophy, since it indirectly induces the
decrease of systemic androgen levels through negative feedback
on hypothalamus-pituitary axis, an event which was also observed
in the present results (Ellem and Risbridger, 2009; Hrknen and
Mkel, 2004). Risbridger et al. (2003) demonstrated that prostatic
malignant alterations are dependent on combined actions of androgens and estrogens and none of these hormones are able to initiate
per se aberrant prostatic growth resulting in glandular malignancies.
Different authors veried that androgen treatment was able to
entirely revert the castration effects on prostatic epithelial cells of
rodents (Oliveira et al., 2007; Pelletier, 2002; Sugimura et al., 1986).
Nevertheless, estrogenic therapy alone induced partial recovery
of prostatic changes resulting from castration, with occurrence
of epithelial hypertrophy and restoration of AR reactivity in the
epithelium (Pelletier, 2002). In contrast, Cordeiro et al. (2008)
observed that androgen replacement was not sufcient to recover
glandular morphology and AR reactivity in the ventral prostate of
rats after castration. According to Hildebrand et al. (1991), contradictory results veried in the literature could be attributed to
methodological differences among studies, considering that several factors, such as strain and age of the animals, may inuence
the prostatic response to hormonal manipulation.
The term proliferative inammatory atrophy (PIA) was proposed to prostatic lesions characterized by atrophy foci, chronic
inammation and increase of proliferation of luminal epithelial
cells (De Marzo et al., 1999). In addition, it was veried that
proliferating epithelial cells in PIA regions showed immature secretory cell phenotype, resembling the cells that occur in prostatic
intraepithelial neoplasia (PIN) and prostate cancer (De Marzo et
al., 1999). Later, Van Leenders et al. (2003) conrmed that cells
with intermediate phenotype between basal and luminal cells
in PIA presented higher proliferative activity in these lesions.
Thus, this cell type would be more susceptible to genetic damages caused by oxidative stress generated by inammatory cells,
suggesting its involvement in the malignant transformation of prostatic epithelium (De Marzo et al., 1999; Van Leenders et al., 2003;
Van Leenders and Schalken, 2003). Wang et al. (2009) characterized direct morphological transition between PIA foci and PIN
and/or cancer, and also showed that atrophic cells from these
mixed lesions had intermediate phenotype and high proliferative
index.

stroma (St) with great amount of collagen bers (Col) and smooth muscle cells (Smc); broblasts (Fb); blood vessel (Bv); occasional digestory vacuoles (Dv) in the basal
cytoplasm; basal membrane (Bm). CastratedEstrogen Group: (J) Cubical luminal cells intermingled by basal cells (Bc). (K) Dilated Golgi (arrow) and rough endoplasmic
reticulum (RER) cisternae; mitochondria with ruptured cristae (M); digestory vacuoles (Dv). (L) and (M) Numerous digestory vacuoles (Dv) in the basal cytoplasm; hypertrophic
stroma (St) with increased collagen bers (Col) and smooth muscle cells (Smc) showing spinous aspect (arrowhead); broblasts (Fb).

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F. Montico et al. / Micron 42 (2011) 642655

Fig. 5. Immunolabelled -DG in the ventral prostate of rats from the experimental groups. (A)(G) -DG immunolocalization in the epithelial and stromal compartments.
The reactivity was graded as weak (+), moderate (++) or intense (+++), according to Table 3. (H) Percentage of -DG protein levels. Signicant reduction of -DG levels was
veried in SEN and CAS groups, showing a recovery after hormonal therapy.

F. Montico et al. / Micron 42 (2011) 642655

653

Fig. 6. Immunolabelled -DG in the ventral prostate of rats from the experimental groups. (A)(G) -DG immunolocalization in the basal membrane region. The reactivity
was graded as weak (+), moderate (++) or intense (+++), according to Table 3. (H) Percentage of -DG protein levels. Decreased -DG levels were detected in senile and
castrated animals, with a recovery of these levels after hormonal therapy.

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F. Montico et al. / Micron 42 (2011) 642655

Interactions between the epithelial compartment and the extracellular matrix are an important factor for the development and
progression of many cancers. Particularly in adenocarcinomas,
the occurrence of disturbances in the basal membrane separating
epithelial and stromal compartments is considered a remarkable characteristic of malignant transformation (Henry et al.,
2001; Sgambato and Brancaccio, 2005). According to Henry et al.
(2001), there is a pronounced reduction in DG expression in
the high grade prostate cancer, leading to abnormal prostatic
cell-extracellular matrix interaction and contributing to tumor
development. Sgambato et al. (2007) suggested that the loss of
-DG immunoreactivity should be an initial event in prostatic carcinogenesis and might already be detected in PIN foci. Later, it was
demonstrated that -DG could also exhibit drastically reduced or
even absent reactivity in various epithelial tumors (Cross et al.,
2008). According to Sgambato et al. (2007), DG expression is
androgen regulated, with stimulation by DHT and inhibition by
anti-androgenic treatment in prostate cancer both in vivo and in
vitro.
Thus, it can be concluded that the occurrence of hypertrophy
and inammatory cells in the prostatic stroma of elderly rats evidenced the structural disorganization and hormonal imbalance of
this organ in the senescence, especially the enhancement of the
estrogenic action in the glandular microenvironment, indicating
the crucial role of pathways involving these hormones in prostatic alterations during this period. In addition, the reduction of
DG protein levels, especially considering -DG, may be associated
with low androgen concentrations in the senescence, pointing to
disturbance of glandular dynamic balance, which probably led to
damage in the epithelialstromal interaction and to impairment in
the organ function. The hormonal therapy utilized in the present
study was not able to reverse the stromal hypertrophy and the
occurrence of inammatory cells, as well as it characterized structural alterations in the epithelial compartment, such as cells with
clear cytoplasmic halo around the nucleus. The presence of these
cells suggested prostatic paracrine signaling imbalance, indicating
that hormonal replacement in the senescence generated a reactive
microenvironment favorable to glandular pathogenesis. Also, the
occurrence of epithelial atrophy not only in animals with reduced
serum androgen levels, but also in those animals which received
testosterone replacement, signaled that circulating levels of steroid
hormones can not be considered as representative of their real concentrations in prostatic tissue. On the other hand, the increase of
DG levels in the prostate of elderly rats after hormonal therapy, in
special with estrogens, characterized an important positive effect
of steroid hormone replacement in restoring the glandular balance in elderly animals, highlighting the estrogenic action on the
prostate. Finally, it can be concluded that despite having positive
molecular effects, contributing to glandular integrity maintenance
through DG stabilization, hormonal therapy accentuated the imbalance between androgens and estrogens in the prostate of elderly
rats, leading simultaneously to tissue remodelling with enhancement of structural changes associated with senescence.

Acknowledgements
This work was supported by FAPESP (2008/56493-7).

References
Acosta, S., Dizeyi, N., Feinstein, R., Pierzynowski, S., Abrahamsson, P.A., 2004. Longterm testosterone stimulation induces hyperplasia in the guinea-pig prostate.
Prostate Cancer Prostatic Dis. 7, 227231.
Banerjee, P.P., Banerjee, S., Dorsey, R., Zirkin, B.R., Brown, T.R., 1994. Age- and lobespecic responses of the Brown Norway rat prostate to androgen. Biol. Reprod.
51, 675684.

Banerjee, P.P., Banerjee, S., Lai, J.M., Strandberg, J.D., Zirkin, B.R., BROWN, T.R., 1998.
Age-dependent and lobe-specic spontaneous hyperplasia in the Brown Norway
rat prostate. Biol. Reprod. 59, 11631170.
Banerjee, S., Banerjee, P.P., Brown, T.R., 2000. Castration-induced apoptotic cell death
in the Brown Norway rat prostate decreases as a function of age. Endocrinology
141, 821832.
Banerjee, P.P., Banerjee, S., Brown, T.R., 2001. Increased androgen receptor
expression correlates with development of age-dependent, lobe-specic spontaneous hyperplasia of the Brown Norway rat prostate. Endocrinology 142,
40664075.
Berry, P.A., Maitland, N.J., Collins, A.T., 2008. Androgen receptor signalling in
prostate: effects of stromal factors on normal and cancer stem cells. Mol. Cell.
Endocrinol. 288, 3037.
Brennan, P.A., Jing, J., Ethunandan, M., Grecki, D., 2004. Dystroglycan complex in
cancer. Eur. J. Surg. Oncol. 30, 589592.
Bianchi-Frias, D., Vakar-Lopez, F., Coleman, I.M., Plymate, S.R., Reed, M.J., Nelson,
P.S., 2010. The effects of aging on the molecular and cellular composition of the
prostate microenvironment. PLoS One 5, Epub ahead of print.
Cordeiro, R.S., Scarano, W.R., Campos, S.G., Santos, F.C., Vilamaior, P.S., Ges, R.M.,
Taboga, S.R., 2008. Androgen receptor in the Mongolian gerbil ventral prostate:
Evaluation during different phases of postnatal development and following
androgen blockage. Micron 39, 13121324.
Cross, S.S., Lippitt, J., Mitchell, A., Hollingsbury, F., Balasubramanian, S.P., Reed,
M.W.R., Eaton, C., Catto, J.W., Hamdy, F., Winder, S.J., 2008. Expression of dystroglycan is reduced or absent in many human carcinomas. Histopathology
53, 561566.
Cunha, G.R., Hayward, S.W., Wang, Y.Z., 2002. Role of stroma in carcinogenesis of
the prostate. Differentiation 70, 473485.
De Marzo, A.M., Marchi, V.L., Epstein, J.I., Nelson, W.G., 1999. Proliferative inammatory atrophy of the prostate: implications for prostatic carcinogenesis. Am. J.
Pathol. 155, 19851992.
Ellem, S.J., Risbridger, G.P., 2009. The dual, opposing roles of estrogen in the prostate.
Ann. N. Y. Acad. Sci. 1155, 174186.
Flrez, M.G., Carvalho, H.F., 2005. Clula Epitelial Prosttica. In: Carvalho, H.F.,
Collares-Buzato, C.B. (Eds.), Clulas: uma abordagem multidisciplinar. Manole,
Barueri, pp. 335345.
Franck-Lissbrant, I., Hggstrm, S., Damber, J.E., Bergh, A., 1998. Testosterone stimulates angiogenesis and vascular regrowth in the ventral prostate in castrated
adult rats. Endocrinology 139, 451456.
Graham Jr., R.C., Karnovsky, M.J., 1965. The histochemical demonstration of
monoamine oxidase activity by coupled peroxidatic oxidation. J. Histochem.
Cytochem. 13, 604605.
Hrknen, P.L., Mkel, S.I., 2004. Role of estrogens in development of prostate
cancer. J. Steroid Biochem. Mol. Biol. 92, 297305.
Henry, M.D., Cohen, M.B., Campbell, K.P., 2001. Reduced expression of dystroglycan
in breast and prostate cancer. Hum. Pathol. 32, 791795.
Hildebrand, R.K., Naslund, M.J., Oesterling, J.E., Coffey, D.S., 1991. Inuence of age,
strain, and the testes on rat prostate hormone sensitivity. Prostate 18, 8189.
Hsing, A.W., 2001. Hormones and prostate cancer: whats next? Epidemiol. Rev. 23,
4258.
Imamov, O., Shim, G.J., Warner, M., Gustafsson, J.A., 2005. Estrogen receptor beta in
health and disease. Biol. Reprod. 73 (5), 866871.
Jorcyk, C.L., Liu, M.L., Shibata, M.A., Maroulakou, I.G., Komschlies, K.L., McPhaul,
M.J., Resau, J.H., Green, J.E., 1998. Development and characterization of a mouse
prostate adenocarcinoma cell line: ductal formation determined by extracellular
matrix. Prostate 34, 1022.
Junqueira, L.C., Bignolas, G., Brentani, R.R., 1979. Picrossirius staining plus polarization microscopy, a specic method of collagen detection in tissue sections.
Histochem. J. 11, 447455.
Knox, J.D., Cress, A.E., Clark, V., Manriquez, L., Afnito, K.S., Dalkin, B.L., Nagle, R.B.,
1994. Differential expression of extracellular matrix molecules and the alpha
6-integrins in the normal and neoplastic prostate. Am. J. Pathol. 145, 167174.
Krieg, M., Nass, R., Tunn, S., 1993. Effect of aging on endogenous level of 5 alphadihydrotestosterone, testosterone, estradiol, and estrone in epithelium and
stroma of normal and hyperplastic human prostate. J. Clin. Endocrinol. Metab.
77, 375381.
Krtolica, A., Campisi, J., 2002. Cancer and aging: a model for the cancer promoting
effects of the aging stroma. Int. J. Biochem. Cell. Biol. 34, 14011414.
Lau, K.M., Tam, N.N., Thompson, C., Cheng, R.Y., Leung, Y.K., Ho, S.M., 2003. Ageassociated changes in histology and gene-expression prole in the rat ventral
prostate. Lab. Invest. 83, 743757.
Leav, I., Merk, F.B., Kwan, P.W., Ho, S.M., 1989. Androgen-supported estrogenenhanced epithelial proliferation in the prostates of intact Noble rats. Prostate
15, 2340.
Leav, I., Lau, K.M., Adams, J.Y., McNeal, J.E., Taplin, M.E., Wang, J., Singh, H., Ho, S.M.,
2001. Comparative studies of the estrogen receptors beta and alpha and the
androgen receptor in normal human prostate glands, dysplasia, and in primary
and metastatic carcinoma. Am. J. Pathol. 159, 7992.
Losasso, C., Di Tommaso, F., Sgambato, A., Ardito, R., Cittadini, A., Giardina, B.,
Petrucci, T.C., Brancaccio, A., 2000. Anomalous dystroglycan in carcinoma cell
lines. FEBS Lett. 484, 194198.
Marker, P.C., Donjacour, A.A., Dahiya, R., Cunha, G.R., 2003. Hormonal, cellular, and
molecular control of prostatic development. Dev. Biol. 253, 165174.
Markopoulos, A.K., Poulopoulos, A.K., Kayavis, I., Papanayotou, P., 2000. Immunohistochemical detection of insulin-like growth factor-I in the labial salivary glands
of patients with Sjgrens syndrome. Oral. Dis. 6, 3134.

F. Montico et al. / Micron 42 (2011) 642655

Marks, L.S., Mazer, N.A., Mostaghel, E., Hess, D.L., Dorey, F.J., Epstein, J.I., Veltri, R.W., Makarov, D.V., Partin, A.W., Bostwick, D.G., Macairan, M.L., Nelson,
P.S., 2006. Effect of testosterone replacement therapy on prostate tissue in
men with late-onset hypogonadism: a randomized controlled trial. JAMA 296,
23512361.
Miki, J., 2009. Investigations of prostate epithelial stem cells and prostate cancer
stem cells. Int. J. Urol. 17, 139147.
Montgomery, D.C., 1991. Design and Analysis of Experiments, 3rd ed. John Wiley &
Sons, New York.
Morales, A., 2002. Androgen replacement therapy and prostate safety. Eur. Urol. 41,
113120.
Morrissey, C., Buser, A., Scolaro, J., OSullivan, J., Moquin, A., Tenniswood, M., 2002.
Changes in hormone sensitivity in the ventral prostate of aging SpragueDawley
rats. J. Androl. 23, 341351.
Niu, Y.N., Xia, S.J., 2009. Stromaepithelium crosstalk in prostate cancer. Asian J.
Androl. 11, 2835.
Oliveira, S.M., Vilamaior, P.S.L., Corradi, L.S., Ges, R.M., Taboga, S.R., 2007. Cellular
and extracellular behavior in the gerbil (Meriones unguiculatus) ventral prostate
following different types of castration and the consequences of testosterone
replacement. Cell Biol. Int. 31, 235245.
Pelletier, G., 2002. Effects of estradiol on prostate epithelial cells in the castrated rat.
J. Histochem. Cytochem. 50, 15171524.
Prins, G.S., Jung, M.H., Vellanoweth, R.L., Chatterjee, B., Roy, A.K., 1996. Agedependent expression of the androgen receptor gene in the prostate and
its implication in glandular differentiation and hyperplasia. Dev. Genet. 18,
99106.
Prins, G.S., Birch, L., Couse, J.F., Choi, I., Katzenellenbogen, B., Korach, K.S., 2001. Estrogen imprinting of the developing prostate gland is mediated through stromal
estrogen receptor alpha: studies with alphaERKO and betaERKO mice. Cancer
Res. 61, 60896097.
Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain
in electron microscopy. J. Cell Biol. 17, 208212.
Risbridger, G.P., Bianco, J.J., Ellem, S.J., McPherson, S.J., 2003. Oestrogens and prostate
cancer. Endocr. Relat. Cancer 10, 187191.
Sttolo, S., Carvalho, C.A.F., Cagnon, V.H.A., 2004. Inuence of hormonal replacement
on the ventral lobe of the prostate of rats (Rattus norvegicus albinus) submitted
to chronic ethanol treatment. Tissue Cell. 36, 417430.
Scarano, W.R., Vilamaior, P.S.L., Taboga, S.R., 2006. Tissue evidence of the testosterone role on the abnormal growth and aging effects reversion in the gerbil
(Meriones unguiculatus) prostate. Anat. Rec. A 288, 11901200.
Schulman, C., Lunenfeld, B., 2002. The aging male. World J. Urol. 20, 410.
Sgambato, A., Brancaccio, A., 2005. The dystroglycan complex: from biology to cancer. J. Cell Physiol. 205, 163169.
Sgambato, A., De Paola, B., Migaldi, M., Di Salvatore, M., Rettino, A., Rossi, G., Faraglia,
B., Boninsegna, A., Maiorana, A., Cittadini, A., 2007. Dystroglycan expression is
reduced during prostate tumorigenesis and is regulated by androgens in prostate
cancer cells. J. Cell Physiol. 213, 528539.
Singh, J., Itahana, Y., Knight-Krajewski, S., Kanagawa, M., Campbell, K.P.,
Bissell, M.J., Muschler, J., 2004. Proteolytic enzymes and altered glycosy-

655

lation modulate dystroglycan function in carcinoma cells. Cancer Res. 64,

61526159.
Slayter, M.V., Anzano, M.A., Kadomatsu, K., Smith, J.M., Sporn, M.B., 1994.
Histogenesis of induced prostate and seminal vesicle carcinoma in LobundWistar rats: a system for histological scoring and grading. Cancer Res. 54,
14401445.
Sprando, R.L., 1990. Perfusion of rat testis through the heart using heparin. In: Russell, L.D., Ettilin, A.P.S., Cleeg, E.D. (Eds.), Histological and
Histopathological Evaluation of the Testis. Cache River Press, Clearwater,
pp. 277280.
Srinivasan, G., Campbell, E., Bashirelahi, N., 1995. Androgen, estrogen and progesterone receptors in normal and aging prostates. Microsc. Res. Technol. 30,
293304.
Sugimura, Y., Cunha, G.R., Donjacour, A.A., 1986. Morphogenesis of ductal networks
in the mouse prostate. Biol. Reprod. 34, 961971.
Suzuki, H., Nakada, T., 1996. Alteration of collagen biosynthesis and analysis of
type I and type III collagens of prostate in young rats following sex hormone
treatments. Arch. Androl. 36, 205216.
Taipale, J., Keski-Oja, J., 1997. Growth factors in the extracellular matrix. FASEB J. 11,
5159.
Toorians, A.W., Kelleher, S., Gooren, L.J., Jimenez, M., Handelsman, D.J., 2003. Estimating the contribution of the prostate to blood dihydrotestosterone. J. Clin.
Endocrinol. Metab. 88, 52075211.
Tuxhorn, J.A., Ayala, G.E., Rowley, D.R., 2001. Reactive stroma in prostate cancer
progression. J. Urol. 166, 24722483.
Van Leenders, G.J., Gage, W.R., Hicks, J.L., Van Balken, B., Aalders, T.W., Schalken,
J.A., De Marzo, A.M., 2003. Intermediate cells in human prostate epithelium are enriched in proliferative inammatory atrophy. Am. J. Pathol. 162,
15291537.
Van Leenders, G.J., Schalken, J.A., 2003. Epithelial cell differentiation in the human
prostate epithelium: implications for the pathogenesis and therapy of prostate
cancer. Crit. Rev. Oncol. Hematol. 46, S3S10.
Vilamaior, P.S.L., Felisbino, S.L., Taboga, S.R., Carvalho, H.F., 2000. Collagen ber reorganization in the rat ventral prostate following androgen deprivation: a possible
role for smooth muscle cells. Prostate 45, 253258.
Wang, W., Bergh, A., Damber, J.E., 2009. Morphological transition of proliferative
inammatory atrophy to high-grade intraepithelial neoplasia and cancer in
human prostate. Prostate 69, 13781386.
Watson, M.L., 1958. Staining of tissues sections for electron microscopy with heavy
metals. J. Biophys. Biochem. Cytol. 4, 475478.
Weihua, Z., Makela, S., Andersson, L.C., Salmi, S., Saji, S., Webster, J.I., Jensen, E.V.,
Nilsson, S., Warner, M., Gustafsson, J.A., 2001. A role for estrogen receptor beta
in the regulation of growth of the ventral prostate. Proc. Natl. Acad. Sci. 98,
63306335.
Weihua, Z., Warmer, M., Gustafsson, J.A., 2002. Estrogen receptor beta in the prostate.
Mol. Cell Endocrinol. 193, 15.
Winder, S.J., 2001. The complexities of dystroglycan. Trends Biochem. Sci. 26,
118124.
Zar, J.H., 1999. Biostatistical Analysis, 4th ed. Prentice Hall Upper, New Jersey.