ORIG INAL ART ICLE

Oestrogen supplementation following castration promotesstromal remodelling and histopathological alterations in theMongolian gerbil ventral prostate

Wellerson Rodrigo Scarano*, Daniel Emıdio de Sousa�, Silvana Gisele Pegorin Campos�,

Lara Silvia Corradi*, Patricia Simone Leite Vilamaior� and Sebastiao Roberto Taboga�

*Cell Biology Department, Biology Institute, UNICAMP, Campinas, Sao Paulo, Brazil,�Department of Biology, Microscopy and

Microanalysis Laboratory, Sao Paulo State University – IBILCE ⁄ UNESP, Sao Jose do Rio Preto, Sao Paulo, Brazil and�Rio Preto

Universitary Center – UNIRP, Biological Sciences School, Sao Jose do Rio Preto, Sao Paulo, Brazil

Oestrogens regulate the prostate development and func-

tion at several stages by indirect and direct mechanisms.

Prostate growth, differentiation and functions are primarily

controlled by androgens (ARs) but oestrogens modulate

these effects in several ways. The most important routes

of indirect oestrogen regulation are interference in AR

INTERNATIONAL

JOURNAL OF

EXPERIMENTAL

PATHOLOGY

Received for publication:

22 June 2007

Accepted for publication:

22 July 2007

Correspondence:

Sebastiao Roberto Taboga

Department of Biology

IBILCE ⁄ UNESP

R. Cristovao Colombo

2265 - Jardim Nazareth

Sao Jose do Rio Preto, SP 15054-000

Brazil

Tel.: +55 17 32212386

Fax: +55 17 32212390

E-mail: taboga@ibilce.unesp.br

Summary

The effect of oestradiol on the intact and castrated adult gerbil prostate was evalu-

ated by focussing on stromal and epithelial disorders, and hormonal receptor immu-

noreactivity. The experimental animals were studied by histological, histochemical

and immunohistochemical techniques, morphometric–stereological analysis and

transmission electron microscopy. Epithelial alterations in the oestradiol-treated

animals were frequent, with an increase in epithelial cell height, areas of intense dys-

plasia and hyperplasia and formation of prostatic intraepithelial neoplasia (PIN).

Another aspect that did not depend on the presence of testosterone was the arrange-

ment of the fibrillar and non-fibrillar elements of the extracellular matrix among

smooth muscle cells (SMC), suggesting a possible role of these cells in rearrangement

and synthesis of these components, after oestrogenic treatment. In the castrated

animals, an accumulation of extracellular matrix elements under the epithelium was

evident, while in the intact animals the same compounds were dispersed and scarce.

In the groups of intact and castrated animals, SMC and fibroblasts exhibited a secre-

tory phenotype, which was accentuated after oestradiol administration. There was

an increase of the immunoreactivity to a-oestrogen and androgen receptors in hyper-

plastic areas compared to normal epithelium, revealing the involvement of these ste-

roid receptors in the hyperplasia and PIN development.

Keywords

castration, epithelium, gerbil, oestradiol, prostate, stroma

Int. J. Exp. Path. (2008), 89, 25–37

doi: 10.1111/j.1365-2613.2007.00559.x

� 2007 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd 25

production by repression of the hypothalamic-pituitary-

gonadal axis and direct effects on testis. Oestrogens also

clearly have direct effects on the prostate, which may be

elicited by external hormones or by oestradiol produced by

local aromatization of testosterone (Harkonen & Makela

2004). Oestrogen regulation has also been considered as one

of the hormonal risk factors in association with development

of benign prostate hyperplasia and prostate cancer (Bosland

2000; Henderson & Feigelson 2000). Exogenous oestrogens

given during the perinatal period elicit abnormalities in pros-

tate growth (Naslund & Coffey 1986), differentiation (Arai

et al. 1977), function (Prins et al. 1993), AR metabolism

(Santii et al. 1991), expression of AR receptors (Prins et al.

1993) and may lead to prostate cancer (Santii et al. 1990;

Prins 1997).

Squamous metaplasia, dysplasia and prostatic intra-

epithelial neoplasia (PIN) are direct effects of oestrogen on the

prostate induced by long-term exposure to high levels of exo-

genous or endogenous oestrogens (Risbridger et al. 2001;

Scarano et al. 2004). For example, the squamous metaplasia

of the prostate epithelium is characterized by the total replace-

ment of the columnar secretory epithelium by layers of strati-

fied squamous cells (Risbridger et al. 2001). The oestrogen

hormone effects are mediated through a-oestrogen (ERa) sig-

nalling. Stromal–epithelial interactions and a requirement of

both epithelial and stromal ERa contributed to elicit oestro-

gen-induced prostate normal epithelial growth and pathologi-

cal disorders. Presumably, the development of prostate

epithelial disorders involves the stimulation of epithelial pro-

liferation mediated by stromal ERa and an epithelial differen-

tiation mediated by epithelial ERas (Cunha et al. 2002).

Previous studies have indicated that following the oestro-

gen administration the smooth muscle cells (SMC) increased

in size and number, in the rat prostate (Thompson et al.

1979). Castrated and intact adult guinea-pig prostates

showed an increased density and thickness of the collagen

fibrils after oestradiol treatment (Neubauer & Mawhinney

1981; Mariotti & Mawhinney 1982; Scarano et al. 2005).

These effects are directly associated with the presence of oes-

trogen receptors (a-ER) in the stroma and stimulation of the

stromal cells by means of autocrine and paracrine mecha-

nisms (Droller 1997).

The Mongolian gerbil (Meriones unguiculatus) has been

recognized as experimentally useful in some biomedical sci-

ences, such as immunology (Nawa et al. 1994), physiology

(Nolan et al., 1990 ) and morphology (Pinheiro et al. 2003;

Santos et al. 2003, 2006; Corradi et al. 2004; Custodio

et al. 2004 ). More recently, the gerbil has also been sug-

gested as a suitable model for studies on mammalian ageing

and neoplasic prostate lesions (Pegorin de Campos et al.

2006; Campos et al. 2007). The gerbil prostate has compact

lobes, somewhat similar to the human prostate, unlike rats

and mice, which have distinct lobes (Pinheiro et al. 2003;

Goes et al. 2006). Previous data from our laboratory have

demonstrated that histological, histochemical and ultrastruc-

tural features of the adult gerbil ventral prostate are compa-

rable to the human prostate. Also, we have observed that

old gerbils (12 months) may spontaneously develop benign

prostate hyperplasia, cancer and other prostate disorders,

mainly in ventral prostate (Pegorin de Campos et al. 2006).

Morphological studies on the oestrogenic effects in the

prostate gland had been performed in many commonly used

experimental animals, including mouse (Triche & Harkin

1971), rat (Kjaerheim et al. 1974; Thompson et al. 1979)

and guinea-pig (Neubauer & Mawhinney 1981; Scarano

et al. 2005), however, these studies focus on more general

aspects of the gland, related to the epithelial comportment

at most times and they used more simple morphological

methodologies.

In the present study, we evaluated in intact and castrated

gerbils the influence of oestrogen in epithelial and stromal

prostate compartments to establish the effects of this hor-

mone in this new animal model and to compare the results

obtained in other experimental models.

Material and methods

Animals and hormone treatments

Twenty adult (120 days) male M. unguiculatus gerbils were

randomly divided into four experimental groups: intact con-

trol (C), intact oestradiol-treated (E), castrated (Ca) and cas-

trated oestradiol-treated (CaE). The Ca and CaE groups

were submitted to bilateral orchiectomy by abdominal surgi-

cal incision. The deferent ducts were sectioned and tied and

the two testes were removed by abdominal cavity. After the

surgery, the animals of these groups were placed in individ-

ual boxes and submitted to the treatments after 7 days. The

E and CaE groups received, during 21 days, alternately, sub-

cutaneous injections of oestradiol benzoate (Sigma Chemical

Co., St Louis, MO, USA) diluted in corn oil (10 mg ⁄ ml), at

a dose of 0.1 ml ⁄ application ⁄ animal (1 mg ⁄ application),

while C and Ca groups received only vehicle.

After 21 days of treatment, the animals were lightly

anesthetized by CO2 inhalation, weighed and immediately

decapitated for blood collection. The ventral prostate was

removed, weighed in an analytical balance, and immediately

processed for light and electron microscopic studies.

Animal handling and experiments were carried out

according to the ethical guidelines of the Sao Paulo State

26 W.R. Scarano et al.

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Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37

University (Unesp), following the ‘Guide for Care and Use

of Laboratory Animals’. The number of individuals

employed in this work was justified by the large number of

analytical procedures employed.

Serum hormonal levels

Circulating serum testosterone levels were determined by

immunochemical assays. After 21 days of treatment, blood

was collected by decapitation from the ruptured cervical

vessels and the serum was separated by centrifugation (300 g)

and stored at )20 �C for subsequent hormone assay. The

determination of serum testosterone levels was performed

by luminescence immunoassay (mouse anti-testosterone

antibodies; Johnson & Johnson, Orthoclinical Diagnostics

Division, Amersham, UK) in an automatic analyzer:

Vitros-ECi (Johnson & Johnson, Orthoclinical Diagnostics

Division, USA) for ultrasensitive chemiluminescence detec-

tion. The intra-assay and inter-assay variation were 4.6% and

4.3% respectively. The tests are linear from 0 to 30 ng ⁄ ml

(detection level).

Histochemistry

Ventral prostates of the four experimental groups were cut

into fragments of 5 lm, selected from the gland distal seg-

ment. Same ventral prostatic fragments samples were sepa-

rated from these to be proceeded for transmission electron

microscopy. The remaining fragments were immediately

fixed by immersion in Karnovsky’s fixative (0.1 m Sorensen

phosphate buffer pH 7.2 containing 5% paraformaldehyde

and 2.5% glutaraldehyde) for 24 h. Fixed tissue samples

were dehydrated in a graded ethanol series and embedded in

glycol methacrylate resin (Leica historesin embedding kit).

Histological sections (3 lm) were subjected to haematoxy-

lin–eosin (H&E) staining for general studies, to Gomori’s

reticulin (Gomori 1937) staining for collagen and reticular

fibres analyses and to the Feulgen reaction (Mello & Vidal

1980) for nuclear study. Histopathological analyses were

performed on Zeiss-Jenaval or Olympus photomicroscopes,

and the microscopic fields were digitized using the software

Image-Pro�Plus version 4.5 for Windows� software

(Media Cybernetics, Bethesda, MD, USA).

Morphometric and stereological analysis

Using an imaging analysis system (Image-Pro�Plus version

4.5 for Windows� software), H&E and Feulgen sections

were studied. Randomly H&E images of 100 histological

fields per experimental group were captured and analysed by

the stereological method, such that histological fragments of

all animals were evaluated equally (20 per animal). Stereolo-

gical analyses were obtained by Weibel’s multipurpose gra-

ticulate, with 120 points and 60 test lines (Weibel 1979) to

compare the relative volume among the prostate components

(epithelium, stroma and acinar lumen) in the experimental

groups.

Morphometric analysis was performed to evaluate epithe-

lial height, SMC layer thickness surrounding the acini, and

nuclear area and nuclear perimeter (kariometry) of the secre-

tory epithelial cells. For this comparative study, 200 random

group measures in normal acini (free of hyperplasic pro-

cesses) were performed for each parameter.

To quantify the density of cells positive for ERa and AR,

cells from normal and hyperplasic epithelial regions (if pres-

ent) were selected and analysed per experimental animal

(200 ⁄ each region) totalling 1000 cells per group ⁄ region

(n = 5), and the percentage of cells negative and positive for

hormonal receptor immunoreactivity were estimated.

Statistical analysis

The effects of oestradiol on gerbil ventral prostate were evalu-

ated by analysing mean ± standard deviation (SD). The statis-

tical analysis was performed with the statistica 6.0 software

(StatSoft, Tulsa, OK, USA). The anova hypothesis test and

Tukey honest significant difference (HSD) test were employed

and P £ 0.05 was considered statistically significant.

Transmission electron microscopy

Ventral prostates fragments of the experimental groups were

processed for transmission electron microscopy as described

previously (De Carvalho et al. 1994), employing the fixation

procedure of Cotta-Pereira et al. (1976). Briefly, ventral pros-

tate fragments, selected from the distal segment of the gland,

were fixed in 0.25% tannic acid plus 3% glutaraldehyde in

Millonig’s buffer, dehydrated in acetone and embedded in

Araldite resin. After selecting the area of interest in the distal

region of the prostate by trimming of the material using a

glass knife, silver sections (50–75 nm) obtained with a dia-

mond knife were collected and stained by uranyl acetate and

lead citrate. Observation and electron micrographs were made

with a LEO–Zeiss 906 transmission electron microscope

(Leo-Zeiss, Cambridge, UK).

Immunohistochemistry (IHC)

Androgen (SC-816, 1:100 dilution; rabbit polyclonal

antibody), ERa (SC-542, 1:50 dilution; rabbit polyclonal

Oestrogen effects on gerbil’s ventral prostate stroma 27

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Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37

antibody) (Santa Cruz Biotechnology, Santa Cruz, CA, USA)

and chondroitin sulphate-anti-SC56 (Sigma Chemical Co.)

were used for IHC. The immunohistochemical reaction was

performed using the avidin–biotin complex (ABC) kit (Santa

Cruz Biotechnology). For the immunohistochemical tech-

nique, ventral prostate fragments selected from the gland

distal segment were fixed in 10% formaldehyde, dehydrated

in alcohol and embedded in paraplast. The sections (4 lm)

were dewaxed and then rehydrated in graded alcohol and

distilled water. Antigenic recuperation was realized in citrate

buffer at high temperature (100 �C) for 45 min. Endogenous

peroxidase activity was blocked with 0.3% hydrogen perox-

ide in methanol for 45 min, followed by a quick rinse in dis-

tilled water and phosphate-buffered saline (PBS). Sections

were incubated with normal goat and primary antibody at

4 �C overnight. The slides were then incubated with biotiny-

lated anti-rabbit at 37 �C followed by peroxidase-conjugated

ABCs and diaminobenzidine (DAB). The sections were then

counterstained with Harris’s haematoxylin. For negative

control, the primary antibody was replaced with the corre-

sponding normal isotype serum.

Results

Structure and ultrastructure

C group. The prostate of the intact adult gerbil presented

glandular units, with simple cylindrical epithelium, adjacent

to and a fine smooth muscle, which delimited the lumen

of the gland (Figure 1a,b). Among the acini was noted a

dispersed and loose connective vascularized tissue. The epi-

thelial cells of the gland possessed evident secretory charac-

teristics, revealed by the presence of the secretion organelles

above the nucleus (Figure 13). Adjacent to the epithelium

and interspersed with the SMC, there was scanty extra-

cellular matrix fibres (Figures 1b, 5a and 14). Alteration in

body weight was not verified among the experimental

groups (Table 1).

E group. In E animals, absolute and relative weights of the

prostatic gland decreased when compared with the C group

(Table 1). The acinar lumen was also decreased. However,

increases in the epithelial height and in the smooth muscle

layer were noted (Figure 2c). The nuclear area was also sta-

tistically significantly increased, and the nuclear perimeter,

although not statistically significant, was also increased

(Table 1). Ultrastructurally, the epithelial secretory cells

appeared with a large number of dilated cisternae (Figure 22)

and with circular endoplasmic reticulum (Figure 21). Areas

containing frequent hyperplasia and dysplasia presented

elongated epithelial cells with nuclei of different sizes and

heights (Figure 2a,b). In the epithelial compartment, due to

intense dysplasia, areas with cells dislocated to the apical

regions were noted, implying a possible detachment

(Figure 2b). In some acini, the presence of PIN was docu-

mented at the light microscopy (Figure 2d). At the transmis-

sion electron microscopy the epithelial stratification with

loose chromatin and conspicuous nucleolus were observed

Table 1 Quantitative analysis from experimental groups

Experimental groups

C E Ca CaE

Morphometry (lm)

Epithelium height 13.08a ± 2.14 22.32b ± 4.80 10.16a ± 2.73 19.42b ± 3.24

SMC layer thickness 8.51a ± 1.85 11.87b ± 2.00 12.38b ± 2.43 11.75b ± 2.48

Kariometry of secretory cells

Nuclear area (lm2) 23.15a ± 3.84 30.06b ± 4.27 21.15a ± 4.51 27.80b ± 4.96

Nuclear perimeter (lm) 21.27a ± 2.80 22.96a ± 3.20 19.61a ± 2.61 21.94a ± 2.74

Relative volume of tissue components (%)

Epithelium 19.84a ± 2.64 28.15b ± 2.66 17.87a ± 4.64 33.90b ± 12.47

Stroma 38.77a ± 10.04 38.42a ± 5.34 50.13b ± 7.85 41.20a ± 10.25

Lumen 43.89a ± 10.52 33.46b ± 6.19 32.0b ± 11.99 24.90c ± 13.52

Body weight (g) 80.0 ± 8.7 79.5 ± 7.0 78.8 ± 8.5 81.2 ± 6.5

Prostate weight (g) 1.04a ± 0.25 0.55b ± 0.16 0.64b ± 0.11 0.76b ± 0.17

Relative prostate weight (g prostate ⁄ g body weight)2 0.013a ± 0.002 0.007b ± 0.001 0.008b ± 0.001 0.009b ± 0.001

Statistical analyses based on anova and Tukey tests. Lower case letters (a, b, c, d) indicate statistically significant difference (P £ 0.05).

C, control; Ca, castrated; E, oestradiol-treated; CaE, catrated oestradiol-treated animals).

28 W.R. Scarano et al.

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Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37

(Figure 20). These morphological parameters were

considered here as PIN lesions. Morphologically, the stroma

of the E group presented less elongated and more fusiform

SMC compared with the C group (Figure 2c). The ultra-

structure showed hypertrophic SMC with well-developed

secretory organelles (Figure 24). Besides, fibroblasts acti-

vated with large quantities of endoplasmic reticulum and

Golgi complex were observed (Figure 26). Extracellular

(1a)

(1b)

(2a)

(2c)

(2b)

(2d)

(3a)

(3b)

(4a)

(4c)

(4b)

(4d)

Figures 1–4 Histological sections stained by haematoxylin–eosin. 1(a,b): Intact control group (C) – 1(a) general aspect of the prostate

acinus; 1(b) detail of the epithelium (e) and smooth muscle cells layer (SMC) of adult prostate acinus. 2(a–d): Oestradiol-treated

group (E) – 2(a) general aspect of a prostate acinus with high epithelium and dysplasia; 2(b) cells dislocated to the apical regions

(arrow), simulating a possible detachment and secretory granules in the lumen were observed; 2(c) detail of the transition epithelium-

stroma; arrow point the subepithelial stroma and the fusiforms smooth muscle cells. Between the SMC conjunctive tissue was

observed; 2(d) a high-grade prostate intraepithelial neoplasia (PIN). 3(a,b): Castrated group (Ca) – 3(a) prostate acinus with a low

epithelium (ep), SMC and evident subepithelial conjunctive stroma (arrow); 3(b) detail of the prostate acinus where a prominent sub-

epithelial conjunctive stroma (arrow) and SMC with irregular aspect were noticed. 4(a–d): Oestradiol-treated castrated group (CaE) –

4(a) prostate acinus showing the epithelial enfolding (ep). Arrow points to subepithelial connective stroma; 4(b) arrows point to basal

membrane with sinuous aspect (e) abundant conjunctive stroma. Arrow head show the SMC with irregular spine-like cytoplasmic

projections. Notice the high epithelial cells (ep); 4(c) presence of PIN and abundant subepithelial stroma (arrow) and a substantial

layer of SMC; 4(d) detail of epithelium-stroma transition showing the subepithelial connective tissue (arrow), the SMC and the

dysplasic epithelium (ep).

Oestrogen effects on gerbil’s ventral prostate stroma 29

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Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37

matrix compounds appeared in an expressive feature, with

an increase in elastic and collagen fibres mainly adjacent to

SMC. An intimate relationship was observed between syn-

thesized fibrils and stromal cells, including the formation of

cytoplasmic compartments through elongation of fibroblasts

and SMC (Figures 6a, 29 and 30). Furthermore, non-fibrillar

components of chondroitin sulphate had a predominantly

inter-SMC demarcation and distribution (Figure 6b).

Ca group. The prostate gland of the Ca group showed acini

with cubic ⁄ short cylindrical epithelium, surrounded by a

thick connective tissue and SMC concentrically arranged

(Figure 3a,b). The short epithelial cells assumed, a flat aspect

(Figure 3a,b), increasing the nucleus–cytoplasm ratio. An

irregular and wavy basal membrane was noted in most of its

extension, forming a semi-pleated arrangement (Figures 3b,

7a, 15 and 17). Adjacent to the epithelium and filling out

the irregularities of the basal membrane, a thick connective

subepithelial tissue was observed (Figures 3b, 7a, 15 and

17). This connective stroma had abundant extracellular

matrix fibres, mainly collagen (Figures 7a, 15 and 17) and

reticular ones (Figure 7a). Strongly immunoreactive non-

fibrillar components, such as chondroitin sulphate revealed

(9a)

(9b)

(9c)

(10a)

(10b)

(10c)

Figures 9 and 10 Histological sections submitted to ERa IHC –

9(a) intact control group; 9(b–c) oestradiol-treated group; 10(a)

castrated group; 10(b–c) oestradiol-treated castrated group.

Brown stain means positive demarcation.

(11a)

(11b)

(12a)

(12b)

Figures 11 and 12 Histological sections submitted to AR IHC –

11(a) intact control group; 11(b) oestradiol-treated group; 12(a)

castrated group; 12(b) oestradiol-treated castrated group. Brown

stain means positive demarcation.

(5a) (5b)

(6b)(6a)

(7a) (7b)

(8b)(8a)

Figures 5–8 5(a), 6(a), 7(a) and 8(a) Histological sections

stained by Gomori’s reticulin; 5(b), 6(b), 7(b) and 8(b): histolog-

ical sections submitted to chondroitin sulphate IHC – 5(a,b)

intact control group; 6(a,b) oestradiol-treated group; 7(a,b) cas-

trated group; 8(a,b) oestradiol-treated castrated group. Arrows

point positive demarcation to reticulin fibres and chondroitin

sulphate respectively. *collagen fibres; ep, epithelium; l, lumen.

30 W.R. Scarano et al.

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Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37

Table 2 Relative frequencies (%) of AR

and ERa positive cells obtained by anti-

AR and anti-ERa immunohistochemical

method

Experimental groups

C E Ca CaE

AR positive cells

Normal epithelium 49.5 ± 4.5 52.2 ± 5.1 45.8 ± 3.9 51.5 ± 4.8

Hyperplasic epithelium – 72.4* ± 6.0 55.5 ± 5.9 77.2* ± 6.5

ERa positive cells

Normal epithelium 35.2 ± 3.2 42.5 ± 5.2 36.2 ± 4.8 39.5 ± 3.5

Hyperplasic epithelium – 71.5* ± 7.2 37.2 ± 4.5 69.8* ± 7.2

Statistical analyses based on Tukey test. *Statistically significant difference (P £ 0.05).

(13) (14)

(15) (16) (17)

(18)

(21)

(19) (20) (22)

Figures 13–22 Transmission electron

microscopy: ultrastructural aspects –

(13) epithelial cells (ep) from intact con-

trol prostate. Arrow points to secretion

organelles, bar = 2.60 lm; (14) epithe-

lium-stroma transition. Arrow point to

basal laminae; in the stroma the smooth

muscle cells (SMC), fibroblasts (fib) and

scarce collagen fibres (col); intact con-

trol group, bar = 0.94 lm; (15) abun-

dance of bunches of collagen fibres (col)

in the subepithelial stroma and fibro-

blasts (fib), epithelium (ep), Ca group,

bar = 1.56 lm; (16) detail of the acti-

vated fibroblast (fib) with dilated

endoplasmic reticulum cisternae

(arrow), Ca group, bar = 0.72 lm; (17)

sinuous aspect of basal laminae (bl)

with collagen deposition (col) in the

subepithelial stroma, Ca group,

bar = 0.72 lm; (18) irregular arrange-

ment of SMC and collagen fibres (col),

Ca group, bar = 1.56 lm; (19) epithe-

lial cells from oestradiol-treated cas-

trated prostate, bar = 2.60 lm; (20)

PIN in oestradiol-treated prostate. The

epithelial stratification with loose chro-

matin and conspicuous nucleolus were

used here as morphological parameters

for PIN diagnosis, bar = 3.36 lm; (21)

detail of an epithelial cell from

oestradiol-treated prostate, showing the

circular endoplasmic reticulum,

bar = 0.56 lm; (22) a high epithelial

cell from oestradiol-treated group. The

arrow points to dilated cisternae,

bar = 2.01 lm. n, nuclei; ep,

epithelium; st, stroma; SMC, smooth

muscle cells.

Oestrogen effects on gerbil’s ventral prostate stroma 31

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Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37

by IHC, were also noted in this subepithelial region (Fig-

ure 7b). It is interesting that in this experimental group the

extracellular matrix components, despite being markedly dis-

tributed in the periacinar region, were concentrated mainly

in the adjacent area to the basal lamina (Figures 7a, 15 and

17) where fibroblasts presented high synthetic activity

(Figure 16). In the concentrically smooth muscle arranged

around acini, the SMC formed irregular layers (Figure 3a)

among the extracellular matrix fibres (Figure 3b) of the sub-

epithelial stroma. These cells assume a spinous and irregular

phenotype (Figures 3b and 18), different from the elongated

fusiform aspect usually found in the layers of the C group

prostates (Figure 3a).

CaE group. After oestradiol administration for 21 days to

castrated animals, we observed an increase in the amount of

epithelium, besides a decrease in the amount of the lumen,

when compared with the Ca group (Table 1). Significant

alterations in the secretory activity of CaE prostates were

observed. The epithelium was predominantly cylindrical,

which culminated in an increase in the height of the epithe-

lium (Table 1). An increase in the nuclear area of secretory

epithelial cells was also observed (Table 1). After the treat-

ment, dysplastic areas with an increase in cellular size were

observed (Figure 4b,d). Besides, there were areas with occur-

rence of PIN characterized by agglomerated epithelial cells

with heterogeneous phenotypes (Figure 4c). Yet the basal

membrane was observed, as found in the Ca group, with a

wavy and semi-pleated arrangement (Figures 4b,d and 8a).

Accumulation of collagen fibres (Figure 8a) was observed

adjacent to the epithelium, accompanying the confluences

of the basal membrane and interspersed with SMC.

(23)

(25)

(26)

(27) (28) (30)

(29)

(24)

Figures 23–30 Transmission electron

microscopy – ultrastructural aspects –

(23) general aspect of stromal compart-

ment showing the SMC with ER promi-

nent in oestradiol-treated castrated

prostate, bar = 1.21 lm; (24) the SMC

with dilated Golgi apparatus cisternae

(arrow), E group, bar = 0.56 lm; (25)

the SMC with irregular spine-like

cytoplasmic projections surrounded by

collagen fibres (col), CaE group,

bar = 1.21 lm; (26) the activated

fibroblast replete of ER and with

dilated Golgi apparatus cisternae (Ga).

Notice the prominent nucleolus, E

group, bar = 1.21 lm; (27 and 28)

detail of SMC prolongations and colla-

gen (col) arrangement between them.

elastic fibers (el), CaE group; (27)

bar = 1.56 lm; (28) bar = 1.21 lm;

(29 and 30) detail of SMC showing the

intimate contact with elastic fibres (el)

in cytoplasmic prolongations and

collagen fibres (col), E group; (29)

bar = 0.72 lm; (30) bar = 0.94 lm.

n, nuclei; nu, nucleolus.

32 W.R. Scarano et al.

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Apparently, a preferential deposition of collagen fibres

occurred among the SMC in this experimental group

(Figures 8a, 27 and 28). Besides, there was also a discreet

increase in the fibrous bunches that permeate SMC and in

chondroitin sulphate (Figure 8b). The arrangement of the

SMC resembles that observed in the Ca group because they

assumed an elongated fusiform phenotype, forming concen-

tric bunches of fibres (Figure 4a), and irregular spine-like

cytoplasmic projections, unwrapped by the extracellular

matrix (Figures 4b and 25).

Immunohistochemistry

The expression of AR and ERa, in all experimental groups,

presented positive and negative reaction in the epithelial and

stromal cells (Figures 9a, 10a, 11a and 12a). The immuno-

reactivity was heterogeneous, demonstrating that some

cellular clones are more sensitive to the action of those hor-

mones. After oestradiol treatment, the density of ERa and

AR positive cells was greater in hyperplastic and dysplastic

epithelial areas (Figures 9b,c, 10b,c, 11b and 12b; Table 2),

when compared with areas in normal epithelium (Table 2).

The IHC assays for the demarcation of chondroitin sul-

phate, a glycosaminoglycan of the stromal extracellular

matrix, in the C group (Figures 7 and 8) demonstrated a dif-

fuse demarcation, with moderate intensity of reaction, in the

areas adjacent to the epithelial base and SMC. Besides, in

some acini the specific demarcation in the epithelial cells,

especially in the Golgi complex, was observed due to the

production of sulphated residues. Immunoreactivity of chon-

droitin sulphate in E and CaE groups (Figures 6b and 8b)

showed localization less diffuse than in C and Ca groups,

with strong reaction mainly in the areas adjacent to the

basal lamina and among the SMC. A preferential deposition

of chondroitin occurred among the SMC (Figures 6b and

8b), what was similar to the collagen fibres in the E and

CaE groups.

Hormonal levels. Serum testosterone levels varied signifi-

cantly (P £ 0.05) between C, E, Ca and CaE groups, which

confirmed an expected decrease in hormonal concentrations

in each experimental group in comparison to the intact C

group (Figure 31).

Discussion

Understanding the role of oestrogens in the prostate and

how it acts to modulate prostate development growth and

disease is of immense interest. In this work, we investigate

the influence of oestradiol benzoate in the ventral prostatic

stroma and epithelium and demonstrated the direct activity

of oestrogens on stromal cells and extracellular elements

resulting in compositional changes in the stromal

compartment.

Oestrogen has been implicated in the pathogenesis of

prostate diseases by their direct and indirect effects. Promi-

nent among the direct effects is the anti-androgenic action

caused by repression of the hypothalamic-pituitary-gonadal

axis and direct effect on the testis. Oestrogens also clearly

have direct effect on the prostate, which may be elicited by

external hormone or by oestradiol produced by local aroma-

tization of testosterone (Harkonen & Makela 2004).

The effects of orchiectomy have been studied under vari-

ous aspects in several experimental animals, mainly as a

model of studying the rearrangement of extracellular matrix

architecture (Carvalho & Line 1996; Vilamaior et al. 2000;

Antonioli et al. 2004). Along with these studies, researches

using oestrogen have been accomplished to define its role

more precisely in hypoandrogenic prostate tissue (Tam &

Wong 1991; Pelletier 2002) and in physiological conditions

(Scarano et al. 2004).

According to Pegorin de Campos et al. (2006) and Scar-

ano et al. (2006), the prostatic acinus of intact adult gerbil

presents simple cylindrical epithelium, with the presence of

secretion granules in the lumen, and prominent chromo-

phobes areas adjacent to the nucleus, identified as Golgi

apparatus.

Our results showed that the epithelium of the Ca group

was found short and cubic, with scarce secretion vesicles in

Figure 31 Mean ± SD values of serum testosterone levels

(ng ⁄ ml) from experimental groups (C, control; Ca, castrated; E,

oestradiol-treated; CaE, catrated oestradiol-treated animals).

Statistical analysis based on anova and Tukey tests. Different

superscript letters (a, b) indicate statistically significant

difference (P £ 0.05).

Oestrogen effects on gerbil’s ventral prostate stroma 33

� 2007 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37

the luminal border and relatively high nucleus–cytoplasm

ratio. There was no significant decrease in the height of the

epithelium in Ca when compared with C, but it was an evi-

dent morphological and quantitative tendency to this. This

regression of the prostate was frequent in androgenic abla-

tion, which agrees with the data obtained previously by

Pelletier (2002) with mice and after experimental chemical

castration with guinea-pig (Cordeiro et al. 2004) and also

with adult gerbil (Corradi et al. 2004).

The surgical castration process also reveals an increase in

stromal compartment, when comparing with intact animals

(Vilamaior et al. 2000). In the Ca group, a great amount of

extracellular matrix fibres was observed in the subepithelial

stroma, in addition to structural modifications of SMC.

According to Vilamaior et al. (2000), the androgenic defi-

ciency promotes a rearrangement of extracellular matrix

fibres, and possibly, synthesis of constituents of this matrix.

This rearrangement occurs probably due to alterations in

SMC synthetic capacity, which might play a more synthetic,

than contractile, role.

Horsfall et al. (1994) demonstrated in guinea-pigs that

during the ageing process, SMC increases its synthetic capac-

ity, thus modifying its morphological structure. Similarly,

experimental studies using orchiectomy and chemical castra-

tion showed an increase in extracellular matrix components

allied with phenotypic alterations of SMC that started to

exhibit an irregular and spinous arrangement (Antonioli

et al. 2004; Corradi et al. 2004). The phenotypic transition

of SMC, in this case, involves a process denominated dedif-

ferentiation previously described in androgenic blockade

processes (Corradi et al. 2004) and after oestrogen treat-

ments (Zhao et al. 1992), where the muscle cells assume an

essentially secretory phenotype (Vilamaior et al. 2005).

Alterations in prostatic nuclei structure during the castra-

tion, hormonal recovering and in prostatic lesions by quanti-

tative nuclear morphometry (kariometry) may be an

excellent histopathological tool for microscopic diagnosis.

The kariometric data revealed that even the E and CaE

groups had increased the nuclear area, besides an increase of

the epithelial height, as was previously observed by Scarano

et al. (2004) and could be related with increase of the syn-

thetic and ⁄ or metabolic cell activity. Nuclear parameters can

be efficient to compare some epithelial modifications (Martı-

nez-Jabaloyas et al. 2002; Taboga et al. 2003), but it is not

effective in diagnosis of prostate lesions and should be inter-

preted together with other results. Recent research employing

40 quantitative nuclear morphometric parameters suggested

with those parameters as a new biomarker for prostatic can-

cer and histopathological gradation (Veltri et al. 2007). Fur-

thermore, dysplastic processes with alteration of the

epithelial structure and hyperplasia in determinate regions of

the ventral prostate contributed to the relative increase of the

epithelium verified in these animals. Scarano et al. (2004)

described intraepithelial alterations in guinea-pigs treated

with oestradiol, and observed the occurrence of PINs with

relative increase in the epithelial compartment.

The histopathological classification of the epithelial altera-

tions in this work were based to the model proposed by

Campos et al. (2007) to the prostate lesions in the aged ger-

bil that classify and compare normal epithelium, hyperplasic

epithelium, PIN and adenocarcinoma. According to Campos

et al. (2007) and Bar Harbor Classification System for the

mouse prostate, developed by National Cancer Institute

Mouse Models of Human Cancer Consortium Prostate

Steering Committee (Shappell et al. 2004), PIN was charac-

terized by agglomerations of heterogeneous epithelial cells

with probable atypical function and cytoplasmic projections

can extend towards the extracellular matrix, compressing

the basement membrane.

The cytoplasm of epithelial cells presented dilated endo-

membranes in some areas and circular endoplasmic reti-

culum, in E group, was also noted. This circular structure

had been described by Kjaerheim et al. (1974) in castrated

animals, where reduction was observed in the size of

synthesis organelles and number of ribosomes.

Receptors specific for oestrogen were identified in both

the epithelium and stroma (Prins & Birch 1997). Experimen-

tal studies have demonstrated that oestrogen is involved in

the induction of premalignant and malignant alterations

(Weihua et al. 2001; Scarano et al. 2004). According to

Cunha et al. (2002), the epithelial alterations, such as squa-

mous metaplasia, require oestrogen action through stromal

ERa, in a paracrine mechanism, as well as by the ERa epi-

thelial route. Such results concur with the data obtained in

this study, where higher density of ERa-positive cells was

found in hyperplasic areas, showing that oestrogen has

affected routes that do not depend on androgenic levels. In

spite of that, it is important to emphasize that the treatments

with oestradiol exhibited larger AR expression. According to

Droller (1997), oestrogen induces stromal fibroblasts to

express receptors for both epidermal growth factor (EGF-R)

and fibroblast growth factor (FGF-R), besides increasing the

level of AR. The androgenic effects on normal epithelium

are explained by paracrine factors produced by stromal AR-

positive cells (Cunha et al. 2002). However, androgenic reg-

ulation of prostate epithelial cells during malignant transfor-

mation of prostate epithelial cells appears to involve

conversion from a paracrine to an autocrine mechanism

of AR-stimulated growth (Gao et al. 2001). Perhaps this

mechanism of autocrine performance explains the increased

34 W.R. Scarano et al.

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Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37

density of AR-positive cells in the hyperplasic areas and in

PINs.

Droller (1997) identified ER in stromal cells. According to

this author, oestrogen induces receptor expression for spe-

cific growth factors, increasing the synthetic activity of those

cells and also of the SMC. This incentive may be responsible

for the increase in synthesis of extracellular-matrix fibres

and for the apparent increase in the stromal compartment of

the oestrogenized animals, observed in both E and CaE

groups. However the increase in synthetic capacity may be

related to the sensitive decrease in intra-prostate androgenic

levels, which was provoked by the oestrogenic supplementa-

tion previously described by Harkonen and Makela (2004).

Despite verifying that castration increased the stromal

compartment as much as oestrogenic treatments and that

both situations have stimulated the synthesis of fibrillar and

non-fibrillar extracellular matrix components, it should be

emphasized that their distributions were different, castration

promoted a preferential deposition of connective tissue in

the sub-epithelial area while oestrogenic treatments caused

an accumulation of connective tissue around the SMC. This

observation exposes a possible role of SMC in the arrange-

ment of the constituents of the extracellular matrix during

the rearrangement process caused by oestradiol (Scarano

et al. 2005) and in BPH (Cardoso et al. 2004). Besides, it

was observed that in the intact animals treated with oestra-

diol, the frequency of elastic fibres was higher, in agreement

with the data obtained previously by Scarano et al. 2005.

This work revealed two important data: one that the ger-

bil is a good experimental model to study prostatic diseases

of hormonal aetiology and that oestradiol has direct effects

on gerbil prostate, independently of the androgenic levels.

Such effects are represented mainly by the proliferative and

dysplastic epithelial alterations and by the architecture of

the extracellular matrix constituents. This new micro-

environment may be achieved possibly by the fundamental

role of SMC in the post-treatment arrangement. Besides,

hormonal receptors seem to have a fundamental role in the

expression of altered epithelial phenotypes.

Acknowledgements

This paper is part of the thesis presented by WRS to the

Institute of Biology, UNICAMP, in partial fulfilment of the

requirements for a PhD degree, and was supported by grants

from the Brazilian agencies CNPq – Brazilian National

Research and Development Council (fellowship to WRS and

SRT – Proc. Nr. 301111 ⁄ 2005-7) and FAPESP – Sao Paulo

State Research Foundation (Proc. Nr. 02 ⁄ 12942-6 and fel-

lowship to DES and SGPC). The authors wish to thank Mrs

Rosana S. Sousa MS and Mr Luis Roberto Faleiros Jr for

their technical assistance, as well as all other researchers at

the Microscopy and Microanalysis Laboratory. Comments

provided by the anonymous referees helped improve our ori-

ginal manuscript. Special acknowledgement is also due to

Mr James Welsh and Mr Davi A. Pontes for English

language revision of this paper.

References

Antonioli E., Della-Colleta H.H.M., Carvalho H.F. (2004) Smo-

oth muscle cells behavior in the ventral prostate of catrated

rats. J. Androl. 25, 50–56.

Arai Y., Suzuki Y., Nishizuka Y. (1977) Hyperplastic and meta-

plastic lesions in the reproductive tract of male rats induced

by neonatal treatment with diethylstilbestrol. Virchows Arch.

376, 21–28.

Bosland M.C. (2000) The role of steroid hormones in prostate

carcinogenesis. J. Natl. Cancer Inst. Monogr. 27, 39–66.

Campos S.G.P., Zanetoni C., Scarano W.R., Vilamaior P.S.L.,

Taboga S.R. (2007) Age-related histopathological lesions in

the Mongolian gerbil ventral prostate as a good model for

studies of spontaneous hormone-related disorders. Int. J. Exp.

Path. EPub ahead of print; doi: 10.1111/j.1365-2613.2007.

00550.x.

Cardoso L.E.M., Falcao P.G., Sampaio F.J.B. (2004) Increased

and localized accumulation of chondroitin sulfate proteo-

glycans in the hyperplastic human prostate. BJU Int. 93,

532–538.

Carvalho H.F. & Line S.R.P. (1996) Basement membrane asso-

ciated changes in the rat ventral prostate following castration.

Cell Biol. Int. 20, 809–819.

Cordeiro R.S., Scarano W.R., Goes R.M., Taboga S.R. (2004)

Tissular alterations in the guinea pig prostate following anti-

androgen flutamide therapy. BioCell. 28, 21–30.

Corradi L.S., Goes R.M., Carvalho H.F., Taboga S.R. (2004)

Inhibition of 5-alpha-reductase activity induces remodeling

and smooth muscle dedifferentiation in adult gerbil ventral

prostate. Differentiation 72, 198–208.

Cotta-Pereira G., Rodrigo F.G., David-Ferreira J.F. (1976) The

use of tannic acid-glutaraldehyde in the study of elastic

related fibers. Stain Technol. 51, 7–11.

Cunha G.R., Hayward S.W., Wang Y.Z. (2002) Role of stroma

in carcinogenesis of the prostate. Differentiation 70, 473–485.

Custodio A.M., Goes R.M., Taboga S.R. (2004) Acid phospha-

tase activity in gerbil prostate: comparative study in male and

female during postnatal development. Cell Biol. Int. 28, 335–

344.

De Carvalho H.F., Lino Neto J., Taboga S.R. (1994) Micro-

fibrils: neglected components of pressure-bearing tendons.

Ann. Anat. 176, 155–159.

Oestrogen effects on gerbil’s ventral prostate stroma 35

� 2007 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37

Droller M.J. (1997) Medical approaches in the management of

prostate disease. Br. J. Urol. 79 (Suppl. 2), 42–52.

Gao J., Arnold J.T., Isaacs J.T. (2001) Conversion from a para-

crine to an autocrine mechanism of androgen-stimulated

growth during malignant transformation of prostate epithelial

cells. Cancer Res. 61, 5038–5044.

Goes R.M., Zanetoni C., Tomiosso T.K., Ribeiro D.L., Taboga

S.R. (2006) Histological response on dorsal and ventral gerbil

prostate lobes induced by different testosterone withdrawal

procedures. Micron doi: 10.1016/j.micron.2006.06.016

Gomori G. (1937) Silver impregnation for reticulin in paraffin

sections. Am. J. Pathol. 13, 993–1002.

Harkonen P.L. & Makela S.I. (2004) Role of estrogen in devel-

opment of prostate cancer. J. Steroid. Biochem. Mol. Biol.

92, 297–305.

Henderson B.E. & Feigelson H.S. (2000) Hormonal carcino-

genesis. Carcinogenesis 21, 427–433.

Horsfall D.J., Mayne K., Ricciardelli C. et al. (1994) Age-related

in guinea pig prostate stroma. Lab. Invest. 70, 753–763.

Kjaerheim A., Dahl E., Tveter K.J. (1974) The ultrastructure of

accesory sex organs of the male rat: effects of estrogen on the

prostate. Lab. Invest. 31, 391–397.

Mariotti A. & Mawhinney M. (1982) The hormonal mainte-

nance and restoration of guinea pig seminal vesicle fibromus-

cular stroma. J. Urol. 128, 852–857.

Martınez-Jabaloyas J.M., Ruiz-Cerda J.L., Hernandez M., Jime-

nez-Cruz F. (2002) Prognostic value of DNA ploidy and

nuclear morphometry in prostate cancer treated with andro-

gen deprivation. Urology 59, 715–720.

Mello M.L.S. & Vidal B.C. (1980) Praticas de Biologia Celular.

Edgard Blucher-Funamp. Sao Paulo: 57–58.

Naslund M.J. & Coffey D.S. (1986) The differential effects of

neonatal androgen, estrogen and progesterone on adult rat

prostate growth. J. Urol. 136, 1136–1140.

Nawa Y., Horii Y., Okada M., Arizono N. (1994) Histochemi-

cal and cytochemical characterization of mucosal and connec-

tive tissue mast cells of Mongolian gerbil (Meriones

unguiculatus). Int. Arch. Allergy Immunol. 104, 249–254.

Neubauer B. & Mawhinney M. (1981) Actions of androgen

and estrogen on guinea pig seminal vesicle epithelium and

muscle. Endocrinology 108, 680–687.

Nolan C.C., Brown A.W., Cavanagh J.B. (1990) Regional varia-

tions in nerve cell responses to the trimethiltin intoxication in

Mongolian gerbil. Acta Pathol. Microbiol. Immunol. Scand.

81, 204–212.

Pegorin de Campos S.G., Zanetoni C., Goes R.M., Taboga S.R.

(2006) Biological behavior of the gerbil ventral prostate in

three phases of postnatal development. Anat. Rec. 288, 723–

733.

Pelletier G. (2002) Effects of estradiol on prostate epithelial

cells in the castrated rat. J. Histochem. Cytochem. 50,

1517–1523.

Pinheiro P.F.F., Almeida C.C.D., Segatelli T.M., Martinez M.,

Padovani C.R., Martinez F.E. (2003) Structure of the pelvic

and penile urethra-relationship with the ducts of the sex

accessory glands of the Mongolian gerbil (Meriones unguicul-

atus). J. Anat. 202, 431–444.

Prins G.S. (1997) Development estrogenization of the prostate

gland. In: Prostate: Basic and Clinical Aspects, pp. 245–266

(ed. R.K. Naz), Boca Raton, FL: CRC press.

Prins G.S. & Birch L. (1997) Neonatal estrogen exposure up-

regulate estrogen receptor expression in the developing and

adult rat prostate lobes. Endocrinology 139, 874–883.

Prins G.S., Hoodham C., Lepinske M., Birtch L. (1993) Effects

neonatal estrogen on prostate secretory genes and their corre-

lation with androgen receptor expression in the separate pros-

tate lobes of the adult rat. Endocrinology 132, 2387–2398.

Risbridger G., Wang H., Young P. et al. (2001) Evidence that

epithelial and mesenchymal estrogen receptor- alpha mediates

effects of estrogen on prostate epithelium. Dev. Biol. 229,

432–442.

Santii R., Pilkkanen L., Newbold R.R., Mclachlan J.A. (1990)

Development oestrogenization and prostate neoplasia. Int. J.

Androl. 13, 77–80.

Santii R., Newbold R.R., Mclachlan J.A. (1991) Androgen

metabolism in control and in neonatally estrogenized male

mice. Reprod. Toxicol. 5, 149–155.

Santos F.C.A., Goes R.M., Carvalho H.F., Taboga S.R. (2003)

Structure, histochemistry and ultrastructure of the epithelium

and stroma in the gerbil (Meriones unguiculatus) female pros-

tate. Tissue Cell 35, 447–457.

Santos F.C., Leite R.P., Custodio A.M. et al. (2006) Testoster-

one stimulates growth and secretory activity of the adult

female prostate of the gerbil (Meriones unguiculatus). Biol.

Reprod. 75, 370–379.

Scarano W.R., Cordeiro R.S., Goes R.M., Taboga S.R. (2004)

Intraepithelial alterations in the guinea pig lateral prostate

after estradiol treatment at different ages. J. Submicrosc.

Cytol. Pathol. 36, 141–148.

Scarano W.R., Cordeiro R.S., Carvalho H.F., Goes R.M.,

Taboga S.R. (2005) Tissue remodeling in guinea pig lateral

prostate at different ages after estradiol treatment. Cell Biol.

Intern. 29, 778–784.

Scarano W.R., Vilamaior P.S.L., Taboga S.R. (2006) Tissue evi-

dence of the testosterone role on the abnormal growth and

aging effects reversion in the gerbil (Meriones unguiculatus)

prostate. Anat. Rec. A. 228A, 1190–1200.

Shappell S.C., Thomas G.V., Roberts R.L. et al. (2004) Prostate

pathology of genetically engineered mice: definitions and clas-

sification. The consensus report from the Bar Harbor Meeting

of the mouse models of human cancer consortium prostate

pathology committee. Cancer Res. 64, 2270–2305.

Taboga S.R., Santos A.B., Rocha A., Vidal B.C., Mello

M.L.S. (2003) Nuclear phenotypes and morphometry of

36 W.R. Scarano et al.

� 2007 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37

human secretory prostatic cells: a comparative study of

benign and malign lesions in Brazilian patients. Caryologia

56, 313–320.

Tam C.C. & Wong Y.C. (1991) Ultrastructural study of the

effects of 17b-Oestradiol on the lateral prostate and seminal

vesicle of the castred guinea pig. Acta Anat. 141, 51–62.

Thompson A.S., Rowley D.R., Heidger P.M. Jr (1979) Effects

of estrogen upon the fine structure of epithelium and stroma

in the rat ventral prostate gland. Invest. Urol. 17, 83–89.

Triche T.J. & Harkin J.C. (1971) An ultrastructural study of

hormonally induced squamous metaplasia in the coagulating

gland of the mouse prostate. Lab. Invest. 6, 596–606.

Veltri R.W., Marlow C., Kahn M.A., Miller M.C., Epstein J.I.,

Partin A.W. (2007) Significant variations in nuclear structure

occur between and within Gleason grading patterns 3, 4, and

5 determined by digital image analysis. Prostate doi: 10.1002/

pros.20614.

Vilamaior P.S.L., Felisbino S.R., Taboga S.R., Carvalho H.F.

(2000) Collagen fiber reorganization in the rat ventral pros-

tate following androgen deprivation: an possible role for the

smooth muscle cells. Prostate 45, 253–258.

Vilamaior P.S.L., Taboga S.R., Carvalho H.F. (2005) Modula-

tion of smooth muscle cell function: Morphological evidence

for a contractile to synthetic transition in the rat ventral pros-

tate. Cell Biol. Int. 29, 809–816.

Weibel E.R. (1979) Principles and methods for the morphometric

study of the lung and other organs. Lab. Invest. 12, 131–155.

Weihua Z., Makela S., Andersson L.C. et al. (2001) A role for

estrogen receptor beta in the regulation of growth of the ven-

tral prostate. Proc. Natl. Acad. USA 98, 6330–6335.

Zhao G.Q., Holterhus P.M., Dammshauser I., Hoffbauer G.,

Aumuller G. (1992) Estrogen-induced morphological and

immunohistochemistry changes in stroma and epithelium of

rat ventral prostate. Prostate 21, 183–199.

Oestrogen effects on gerbil’s ventral prostate stroma 37

� 2007 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 89, 25–37