Reproductive Toxicology 58 (2015) 238–245

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Reproductive Toxicology
journal homepage: www.elsevier.com/locate/reprotox

Bisphenol-A promotes antiproliferative effects during neonatal

prostate development in male and female gerbils
Rodrigo Fernandes de Lima a , Daniel Andrés Osório Rodriguez a , Mônica Souza Campos b ,
Manoel Francisco Biancardi a , Iana Figueiredo Ferreira Roriz dos Santos a ,
Wendyson Duarte de Oliveira a , Gláucia Maria Cavasin a , Mara Rubia Marques a ,
Sebastião Roberto Taboga b , Fernanda Cristina Alcantara Santos a,∗
a
Department of Histology, Embryology and Cell Biology, Federal University of Goiás, Samambaia II, Goiânia, Goiás 74001970, Brazil
b
São Paulo State University – UNESP, Department of Biology, Laboratory of Microscopy and Microanalysis, Rua Cristóvão Colombo, 2265, São José do Rio
Preto, São Paulo 15054000, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to evaluate the development of male and female neonatal gerbil prostate under
Received 3 February 2015 normal conditions and exposed to bisphenol-A (BPA). Normal postnatal development of the female gerbil
Received in revised form 20 October 2015 prostate occurs earlier than and is morphologically distinct from that occurring in males. In BPA-exposed
Accepted 26 October 2015
PND8 gerbils, we have not observed evidence of alterations in the ductal branching in either gender.
Available online 31 October 2015
However, the exposure to BPA alters the immunolabeling pattern of AR, ER␣, and PCNA. In males, the
exposure to high dosages of BPA resulted in a decrease in the proliferative status of the developing
Keywords:
ventral prostate. In females, both high and low dosages were sufficient to decrease the proliferation of
Prostatic morphogenesis
Gerbil
paraurethral buds in the branching process by more than 50%. Therefore, the obtained data indicate that
Endocrine-disrupting chemicals BPA promotes antiproliferative effects during the neonatal development of the gerbil prostate, with more
AR antagonists sensitivity to this endocrine disruptor in females.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction
The prostate is an accessory gland in the reproductive tract and
its main function includes the production of a glycoproteic secre-
tion that is responsible for the maintenance of nutrition and the
survival of the spermatozoon and the promotion of coagulation
and liquefaction of the semen [1]. This non-male exclusive gland
can be also found in females of mammals other than humans: the
primates, rodentia, carnivora, and lagomorfa [2–5].
The prostate gland arises from the endodermal urogenital
sinus (UGS) during morphogenesis as urogenital epithelial (UGE)
cells penetrate into the surrounding urogenital sinus mesenchyme
(UGM) [6–8]. The UGS is present in mammals of both genders and
occurs between fetal days 16.5 and 17.5 in mice, and at fetal day
18.5 in rats [8].
∗ Corresponding author at: Department of Histology, Embryology and Cell Biology,
Federal University of Goiás, Campus II Samambaia, Goiânia, Goiás, 74001970, Brazil.
E-mail addresses: fernanda alcantara@ufg.br, fer-alcantara@hotmail.com
(F.C.A. Santos).
Prostate development and growth begin in fetal life and are
complete at sexual maturity [7]. In rats, prostate morphogenesis
is a continuous process which can be categorized into five stages
that occur at different periods of the ontogenetic development.
These stages are: (1) determination and (2) budding (fetal period),
(3) branching morphogenesis (neonatal period), (4) differentia-
tion (neonatal and pubertal phases), and (5) glandular maturation
(pubertal life) [8].
Prostatic organogenesis is dependent on androgens stimulus via
androgen receptor (AR). Androgens are necessary to initiate the
gland development, and to continue its embryonic and neonatal
growth. This steroid is also essential to initiate the prostatic secre-
tory activity in puberty, as well as to maintain the gland during adult
life [9]. During the initial development, ARs are expressed in mes-
enchymal cells prior to expression in the UGE. In this way, specific
paracrine factors secreted by mesenchymal cells act in the emerg-
ing epithelial buds [10]. Estrogens, which act via estrogen receptors
alpha (ER␣) and beta (ER␤) are able to modulate the androgen sig-
nalization. ER␣ is expressed in both UGM and UGE during early
prostate development, and it is suppressed by the action of andro-
gens when the production of this hormone increases. In contrast,
ER␤ is not expressed during the initial prostate development, being

http://dx.doi.org/10.1016/j.reprotox.2015.10.016
0890-6238/© 2015 Elsevier Inc. All rights reserved.
 R.F.d. Lima et al. / Reproductive Toxicology 58 (2015) 238–245
important during the maturation and maintenance of the prostate
during adulthood [11].
The prostatic morphogenesis is a stage of development when
branching and organ formation occur. During this phase, mor-
phoregulatory genes regulated by steroids hormones are expressed
in specific locations and times. Thus, exogenous interferences, such
as an exposure to environmental endocrine disrupting chemicals
(EDCs), especially in neonatal period, can alter prostate develop-
ment, predisposing this gland to the irreversible alterations during
aging [8,12].
A great number of endocrine disruptors mimic endogenous
hormones [13]. An example is bisphenol-A (BPA), a synthetic
monomer-like chemical applied in the production of polycarbonate
plastics, which is spread widely in the environment [14]. BPA is a
crystalline, solid white component very soluble in oils but not very
soluble in water [15]. It was first synthesized in 1891 by Alexander
P. Dianin and, in 1930,studies began to investigate its estrogenic
action [16]. Nowadays, it is known that BPA acts as an endocrine
disruptor and competes with estradiol for the ER␣ and ER␤ [15].
Recent studies have shown that BPA is found in different concen-
trations in food cultivated with contaminated water or in heated
polycarbonate dishes [16]. This compound is used in the production
of food and beverage containers, toys, bottles, medical and elec-
tronic devices, dental products, CD/DVDs, antioxidant stabilizers,
vinyl chloride, and thermal papers [16,17].
The primary source of BPA exposure occurs via ingestion, and
secondary routes include transdermal absorption and inhalation
[18]. According to the American Environmental Protection Agency
(EPA), the safe exposure to BPA for humans is about 50 ␮g/kg/day,
and its migration limit for food packaging is 3 mg/kg. The European
Union regulates that the amount of BPA must not exceed 0.1 ng/ml
in food and water [19–20].
Biomonitoring studies have demonstrated that about 92% of U.S.
[21] and Canadian [22] citizens have significant levels of BPA in their
urine. In experimental research with rodents, BPA has been asso-
ciated with several diseases, including diabetes [23], obesity [24],
reproductive alterations [25], and hepatotoxicity [26]. Many stud-
ies suggest that exposure to BPA presents effects on the prostate
gland. There is substantial evidence indicating that this endocrine
disruptor leads to permanent epigenetic alterations in the pro-
static genome, increasing the risk of cancer development with aging
[20,27]. However, variable results can be found based on dose and
timing of BPA exposures [28]. Hence, many questions need to be
answered as regards how BPA can influence prostatic development,
increasing the risk of cancer [29].
Therefore, it is very important to evaluate the effects of exposure
to BPA during the neonatal prostatic development. The Mongo-
lian gerbil (Meriones unguiculatus) is considered an ideal model for
such a study since it presents a significant homology to the human
prostate and has shown crucial outcomes to a variety of hormonal
therapies and/or to exposure to environmental chemicals [30–33].
The aim of this study was to evaluate morphologically the neonatal
development of the prostate gland of male and female gerbils in
normal conditions and under exposure to different concentrations
of BPA.
2. Material and methods
2.1. Experimental design
The animals were provided by the São Paulo State University
(UNESP; São José do Rio Preto) and maintained under controlled
conditions of light and temperature. To avoid exposing the animals
to EDCs such as BPA, all animals were housed in new polyethylene
cages and filtered water was provided from glass bottles. Gerbils
239
were fed with rodent food ad libitum (Presence, Labina-Purina® );
composition: 23% proteins, 4% fats, 5% fibers and 12% minerals. Ani-
mal handling and experiments were performed according to the
ethical guidelines of the Federal University of Goiás (UFG; Ethical
committee number 024/13CEUA), following the Guide for the Care
and Use of Laboratory Animals.
We used 30 adult female and 30 adult male (between 3 and
4 months old) gerbils (M. unguiculatus, Muridae: Gerbillinae) for
mating. We matched, randomly, one male and one female to form
independent litters. The mating day was determined by the pres-
ence of spermatozoa in the vaginal smears; this day was considered
day 0, being the initial day of the pregnancy period [34]. After birth,
the males and females of the litter were destined to form six exper-
imental groups as follows: (1) male control; (2) female control; (3)
male low BPA; (4) female low BPA; (5) male high BPA; (6) female
high BPA; (n = 10 animals/group). Only one pup of each litter was
randomly employed to compose each subgroup.
Control groups (C) received from the 1st until the 7th postnatal
day (PND), by gavage, daily doses of vehicle dilution (10 ␮L of min-
eral oil, Nujol/Mantecorp); Low BPA groups (LBPA) received from
the 1st until the 7th PND, by gavage, environmental doses of BPA
(Sigma, 40 ␮g/kg/day diluted in 10 ␮L of mineral oil); and high BPA
groups (HBPA) received from the 1st until the 7th PND, by gavage,
high doses of BPA (Sigma, 4 mg/kg diluted in 10 ␮L of mineral oil).
The protocol of BPA treatment was adapted from Prins et al. [29]. To
prepare BPA in oil solution, BPA was completely dissolved in min-
imum quantity of 100% ethanol, and then diluted to appropriate
mineral oil volume. Fresh BPA was prepared every other day.
All pups were euthanized in the 8th PND (PND8) by a lethal
dosage of anesthesia (Ketamine/Xylazine— Syntec; 0.1 mL/pup).
The body weight, prostatic complex weight (PrC—urethral segment,
ventral, dorsolateral and dorsal prostate lobes in males, and corre-
spondent urethral segment plus paraurethral glands in female) and
anogenital distance (AGD) were measured, and, then, the develop-
ing prostate tissues were dissected out using a Leica stereoscopic
microscope (Leica, Germany).
2.2. Light microscopy
The prostate of the PND8 animals were fixed by immersion in
methacarn (proportions: methanol 60%, chloroform 30% and acetic
acid 10%) for three hours or 4% paraformaldehyde (buffered in 0.1 M
phosphate, pH 7.2) for 24 h. After fixation, the tissues were washed
in water, dehydrated in ethanol series, embedded in paraffin (His-
tosec, Merck, Darmstadt, Germany) and sectioned at 5 ␮m on a
Leica microtome (Leica RM2155, Nussloch, Germany). The sections
were stained by hematoxylin-eosin (HE). The specimens were ana-
lyzed using an Olympus BX43 light microscope (Olympus, Japan),
and the images were digitalized using CellSens Standard software
v1.6 (Olympus, Japan).
2.3. Three-dimensional reconstruction
The serial sections (n = 1 animal/group) were analyzed using the
Olympus BX43 light microscope (Olympus, Japan) and digitalized
with CellSens Standard software v1.6 (Olympus, Japan). The align-
ment and the reconstruction of the structures were performed with
the Reconstruct software [35]. Digital images were submitted to
morphometric analysis of the developing prostate buds. We deter-
mined number, area and perimeter of epithelial outgrowths buds
(ventral in males and paraurethral in females). These parameters
of male and female prostate epithelial buds were determined as
the sum of all structures in a central section of each gland (n = 5
prostates/group).
240 R.F.d. Lima et al. / Reproductive Toxicology 58 (2015) 238–245

Table 1
1
Body and prostate complex (PrC) weight and anogenital distance (AGD) measurement in control and BPA-treated gerbils (n = 6 animals/group). Values are means ± SEM.
2
Stereological data obtained for the male and female prostate in all experimental groups (mean ± SEM; n = 30 fields in 5 animals/group).

Male Female

C LBPA HBPA C LBPA HBPA

1
Biometry
Body weight (g) 6.4 ± 0.4 7.4 ± 0.4 6.5 ± 0.6 5.9 ± 0.5 6.4 ± 0.6 6.5 ± 0.6
PrC weight (g) 0.02 ± 0.003 0.02 ± 0.002 0.03 ± 0.004 0.02 ± 0.003 0.02 ± 0.005 0.02 ± 0.005
AGD (mm) 1.1 ± 0.1 1.3 ± 0.1 1.2 ± 0.1 0.6 ± 0.04 0.6 ± 0.03 0.6 ± 0.08
2
Stereology (%)
Epithelial buds* 19.1 ± 1.9 19.2 ± 1.3 19.9 ± 1.3 12.4 ± 0.7a 13.8 ± 0.7a 19.9 ± 1.3b
Mesenchyme* 48.2 ± 1.6a 55.4 ± 1.6b 46.8 ± 2.3a 62.5 ± 1.8a 56.1 ± 1.4b 52.4 ± 2.1c
Smooth muscle 29.5 ± 2.4 23 ± 2 29.6 ± 3 20.2 ± 1.6 24.5 ± 1.6 23.6 ± 2.5
Blood vessels* 2.3 ± 0.4 1.8 ± 2 3 ± 0.4 2.4 ± 0.4a 4.3 ± 0.6b 3.1 ± 0.4a
Lumen* 0.9 ± 0.3 0.6 ± 0.2 0.8 ± 0.3 2.5 ± 0.5a 1.3 ± 0.3a 1.1 ± 0.3b

(a, b, c) superscript letters represent statistically significant differences between the experimental groups.
*
Statistically significant differences between control and treatment groups (p ≤ 0.05).

2.4. Stereology
The stereological analyses were carried out using Weibel’s mul-
tipurpose graticulate with 130 points and 10 test lines [36], to
compare the relative frequency of each component of develop-
ing prostate (epithelial buds, mesenchyme, smooth muscle, blood
vessels and lumen) as described by Huttunen [37]. We chose 30
microscopic fields at random from each experimental group (six
fields per animal; n = 5). Briefly, we determined the relative val-
ues by counting the coincident points in the test grid and dividing
them by the total number of points. Stereological analysis was per-
formed using Image-Pro Plus software v6.1 for Windows (Media
Cybernetics Inc., Silver Spring, MD, USA).
2.5. Immunohistochemistry
Tissue sections were subjected to immunohistochemistry to
detect the androgen receptor (AR—rabbit polyclonal IgG, N-20, sc-
816, Santa Cruz Biotechnology, Santa Cruz, CA, USA), the estrogen
receptor-alpha (ER␣—rabbit polyclonal IgG, MC-20, sc-542, Santa
Cruz Biotechnology), and the proliferating cell nuclear antigen
(PCNA—mouse monoclonal IgG2a, SC 56, Santa Cruz Biotechnol-
ogy, CA, USA). Primary antibodies were employed at a dilution of
1:100 overnight at 4 ◦ C, as described in protocols applied to the
prostate [38] NovoLink Polymer Detection System kit (RE7150-K,
United Kingdom) was employed as secondary antibodies. The sec-
tions were stained with Novocastra DAB Chromogen and NovoLink
DAB Substrate Buffer, and counterstained with Novocastra Hema-
toxylin. The histological sections were analyzed using an Olympus
BX43 light microscope (Olympus, Japan).
of 1:100 overnight. The next morning, the sections were incubated
with fluorochrome-conjugated specific secondary antibodies (anti-
mouse, sc-2010, IgG-FITC, Santa Cruz Biotechnology, CA, USA) for
2 h at room temperature. DAPI was employed to identify the cell
nuclei [34]. The histological sections were analyzed with a Zeiss
Imager M2 fluorescence microscope (Zeiss, Germany) coupled to
the AxioVision (Zeiss, Germany) software.
2.8. Statistical analyses
The hypothesis tests employed to determine statistical sig-
nificance were the Kruskal–Wallis test for non-parametric
distributions and the ANOVA for parametric distributions. Fur-
ther determination of the significant statistical differences between
experimental groups was done using Dunn’s test for non-
parametric distributions and the Tukey’s test for parametric
distributions. The data were analyzed using Statistica 7.0 (Star-
Soft, Inc., Tulsa, OK, USA). The level of significance was set at 5%
(p ≤ 0.05). Values are presented as mean ± standard error of the
mean (SEM).
3. Results
3.1. Biometry
According to Table 1, the exposure to BPA did not promote sig-
nificant alterations in the body and PrC weight and in the AGD of
both sexes in the different experimental groups.
3.2. Three-dimensional reconstruction

2.6. AR and PCNA quantification
For AR and PCNA quantification, 30 microscopic fields (n = 3
animals/group; magnification of 400×) were employed for each
experimental group. In each field, the total number of positive cells
was obtained as a relative frequency (%) in relation to the total num-
ber cells. In the AR and PCNA quantification, between positive and
negative cells, we counted ∼95,000 cells. All these analyses were
performed using the image analysis system previously described.
2.7. Immunofluorescence
In order to evaluate whether BPA exposure alters the differen-
tiation and structure of the smooth muscle cells, tissue sections
were subjected to immunofluorescence for the detection of smooth
muscle ␣-actin (mouse monoclonal IgG2a , sc-32251, IA4, Santa
Cruz Biotechnology, CA, USA), which was incubated at a dilution
In the PND8 male control group (Fig. 1a,b), it was observed that
the buds of the ventral prostate emerge from the lateral region of
the urethral epithelium. These epithelial buds (PeB) elongate ven-
trally toward the ventral mesenchyme pad (VMP), presenting a
‘V’-shaped elongation pattern (Fig. 1a). The smooth muscle (SM)
becomes thin in the VMP adjacent region. Based on the model
analysis, it is possible to notice a gap in the SM, which allows the
interaction of PeBs with the VMP, resulting in the formation of the
ventral lobe of the male gerbil prostate.
In the female control group, paraurethral prostatic lobes (PaL)
could be seen in the cranial region of the urethra (Fig. 1g,h). The
3D-reconstruction models demonstrated that the females PeBs can
emerge from one or both lateral regions of the caudal urothelium.
These PeBs elongate cranially inside the periurethral mesenchyme
(PeM) until reaching an SM gap, which is adjacent to the parau-
rethral mesenchyme (PaM). Thus, the PeBs cross the SM gap and
penetrate into the PaM, thereby initiating the ductal canalization
 R.F.d. Lima et al. / Reproductive Toxicology 58 (2015) 238–245 241

Table 2
Number, area and perimeter of epithelial buds (EB) in PND8 male and female gerbils (n = 5 prostate/group). Values are means ± SEM.

Male Female

C LBPA HBPA C LBPA HBPA

EB number (units) 10.4 ± 2.1 7.6 ± 1.6 10.0 ± 1.7 2.4 ± 0.2 3.2 ± 0.6 3.4 ± 0.2
EB area (␮m2 ) 10,970.9 ± 1368.9 13,991.2 ± 4254.4 15,909.3 ± 3300.1 3,021.6 ± 414.0 3,148.4 ± 686.9 3,389.6 ± 489.7
EB perimeter (␮m) 1,394.5 ± 224.8 1,327.1 ± 342.5 1,650.0 ± 294.5 354.3 ± 32.5 408.8 ± 73.9 438.9 ± 35.4

Fig. 2. PrC sections of PND8 male gerbils. Hematoxylin-eosin method. Urethra (U),
periurethral mesenchyme (PeM), ventral mesenchymal pad (VMP), smooth muscle
layer (SM), periurethral buds (PeB, arrowheads), ventral buds (VB, arrows).

four times greater that the paraurethral EB of females. Still, the

morphometric data showed that there are no substantial differ-
ences in the number, area or perimeter of EB between the control
and treated groups of both sexes (Table 2).

3.3. Morphology and stereology

Male ventral prostate of the control gerbils presented two dis-

Fig. 1. Three-dimensional reconstruction of the PND8 male and female prostates.
Blue: urethra (UR); Yellow: periurethral buds (PeB) and ventral buds (VB) in males,
and PeB and paraurethral buds (PaB) in females; Red: smooth muscle layer (SM);
Light blue: ventral mesenchymal pad (VMP) in males and paraurethral mesenchyme
(PaM) in females. Scale bars: 100 ␮m. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)
and branching (Fig. 1h). At this stage of development, elongation of
PeBs was not observed and all formed epithelial buds were inside
the PaM (Fig. 1h).
There were no significant alterations in the development pat-
tern of the ventral prostate in males exposed to the BPA (Fig. 1c–f),
although in the female group, an apparent reduction of the PaM
in both LBPA and HBPA was observed. However, we found no
alterations in the budding pattern in the females of these groups
(Fig. 1i–l).
Comparatively, in this stage of development, the male ventral
prostate presented up to three times more EB occupying an area
tinct components: a periurethral region, composed of periurethral
buds (PeB) and periurethral mesenchyme in differentiation (PeM);
and ventral region constituted of ventral buds (VB) and VMP
(Fig. 2a–c). Between these two regions occurs a discontinuous and
thick SM layer (Fig. 2a, b). In this stage of prostatic development,
the PeB and VB showed an absent or reduced lumen and were
surrounded by a thin SM layer.
There was no evidence of morphological alterations in the ven-
tral prostates of males exposed to different concentrations of BPA
during the neonatal development (Figs. 2d–i). Furthermore, stereo-
logical data (Table 1) showed a significant increase in the relative
frequency of the mesenchymal compartment in the LBPA group
when compared with the other groups (p ≤ 0.05).
The female prostate gland of the PGD8 control groups (Fig. 3)
showed several morphological differences when compared with
the ventral prostate of males at the same age. Although this gland
presented similar PeB and PeM to those observed in males, the
epithelial cords that crossed through the SM did not elongate
ventrally. These paraurethral epithelial cords, here referred to
paraurethral buds (PaB), invaded the PaM, becoming less branched
than the VB of the male prostate (Fig. 3c). The PeB and PaB
formed solid buds of epithelial cells surrounded by a thin SM layer
(Fig. 3b,c). In females exposed to the BPA (Fig. 3d–i), only the
242 R.F.d. Lima et al. / Reproductive Toxicology 58 (2015) 238–245
Fig. 3. PrC and adjacent vagina sections of PND8 female gerbils. Hematoxylin-eosin
method. Urethra (U), vagina (V), periurethral mesenchyme (PeM), paraurethral mes-
enchyme (PaM), smooth muscle layer (SM), periurethral buds (PeB), paraurethral
buds (PaB), blood vessels (arrows).
prostates from the HBPA group presented a PaB that was appar-
ently more developed (Fig. 3i). Such morphological evidence was
supported by the stereological analysis of the HBPA group (Table 1),
which shows a significant increase in the frequency of prostatic
epithelial buds with consequent reduction in the luminal and mes-
enchymal frequency (p ≤ 0.05).
3.4. Immunohistochemical analyses
3.4.1. AR
Nuclear AR immunostaining were observed in the PeB, PeM, VB,
and SM of male prostate PND8 gerbils of all experimental groups
(Fig. 4a–i). The nuclear immunolabeling became significantly more
frequent in the PeM, VB, and SM of LBPA (Fig. 4d–f) and HBPA
(Fig. 4g–i) groups (Table 3, p ≤ 0.05).
The female PND8 gerbils of both control and treated groups
showed nuclear AR-positive immunostaining in all regions
(Fig. 5a–f). In the LBPA group, an increase was observed in the
frequency of AR-positive cells in the PeM and a decrease in PeB
and PaM (Fig. 5c,d; Table 4; p ≤ 0.05). In the HBPA group, the AR-
positive cells were more frequent in the PeM and PaB, though with
a reduction in the PeB and PaM (Fig. 5e,f; Table 4; p ≤ 0.05).
3.4.2. PCNA
Proliferative cells were observed in all prostatic components of
the PND8 males in the C, LBPA, and HBPA groups (Fig. 4j–r). How-
ever, there was a reduction in the number of PCNA-positive cells
in the PeB and SM of the LBPA group (Fig. 4m–o; Table 3; p ≤ 0.05).
In the HBPA group, all prostatic tissue components in development
became significantly less proliferative (Fig. 4p–r; Table 3; p ≤ 0.05).
In the female control group, all prostatic compartments and
mainly the cells of PeB and PaM presented a pattern that was highly
proliferative (Fig. 5 g,h; Table 4). The postnatal exposure to BPA
resulted in a significant decrease in the cellular proliferation of PaB
in the LBPA and HBPA groups (Fig. 5i–l; Table 4). Furthermore, we
observed a discreet reduction in PCNA-positive cells of PaM in the
LBPA and HBPA groups (Table 4).
Fig. 4. Immunohistochemical reactions in the PND8 ventral prostate for all exper-
imental groups. (a–i) Immunohistochemistry for the androgen receptor (AR).
Arrowheads indicate the AR-positive cells. The details (b1, e1 and h1) highlight
AR-positive cells, with nuclei stained in brown (arrows), either in the epithelium
or in the stroma. Bars: 5 ␮m. (j–r) Immunohistochemistry for the proliferation
cell nuclear antigen (PCNA). Arrowheads indicate the PCNA-positive cells. (s–u)
Immunofluorescence for ␣-actin. Green: alpha-actin (arrows); Blue: DAPI. Peri-
urethral buds (PeB), periurethral mesenchyme (PeM), ventral buds (VB), ventral
mesenchymal pad (VMP), smooth muscle layer (SM). (For interpretation of the ref-
erences to color in this figure legend, the reader is referred to the web version of
this article.)
3.4.3. ER˛
For this study, the immunolabeling for ER␣ in the PND8 male
gerbils was unsuccessful. However, nuclear ER-␣ immunostaining
were observed in PeM, PaM, and SM in the female prostates in
all experimental groups (Fig. 5m–r). We did not find ER␣-positive
nuclei in the EB of the female groups. The LBPA prostate presented
an immunolabeling reduction in PeM and SM. Moreover, in the
HBPA groups, immunolabeling occurred only in the PaM (Fig. 5o–r;
Table 4; p ≤ 0.05).
3.4.4. ˛-actin
The immunofluorescence analysis for ␣-actin in the ventral
prostate of the PND8 male control group demonstrated that this
protein is abundant in the SM and rare around the VB (Fig. 4s).
The LBPA and HBPA male groups, when compared with the control
group, presented VBs surrounded by a thicker SM layer with intense
labeling for ␣-actin (Fig. 4t,u). In the female prostate, no changes
were observed in the immunolabeling patterns for ␣-actin in any
experimental groups (Fig. 5s–u).
4. Discussion
This is the first study to compare the postnatal development of
male and female gerbil prostates under normal conditions and upon
exposure to different bisphenol-A concentrations. In the PND8 male
group, it can be observed that growth of the epithelial buds under-
goes the same ‘V’-shaped elongation pattern that was previously
demonstrated by Sanches et al. [34] in one day old male gerbils.
Nevertheless, in the PND8 males, several prostatic buds elongat-
ing from the urethra toward the VMP were still observed, which
indicates that the branching and elongation of the gerbil ventral
prostate takes place throughout the whole first week of postnatal
development, until the beginning of the second week. This branch-
 R.F.d. Lima et al. / Reproductive Toxicology 58 (2015) 238–245 243

Table 3
Frequency (%) of AR and PCNA-positive cells in the male gerbil prostate of all experimental groups.

AR PCNA

C LBPA HBPA C LBPA HBPA

PeB 68.7 ± 2.7 70.1 ± 3.2 70.6 ± 2.1 77.8 ± 2.1a 72.6 ± 2b 68.1 ± 2.4b
PeM 60.6 ± 3.6a 72.2 ± 1.6b 73.9 ± 1.8b 63.2 ± 3.1a 58 ± 2.2a 48.1 ± 2.1b
VB 29.4 ± 3.3a 41.5 ± 4b 39.3 ± 3.5b 88.3 ± 2.4a 89.4 ± 2.5a 55.8 ± 3.7b
VMP 68 ± 2.7 70.5 ± 3.5 75.3 ± 2.8 65.7 ± 2.8a 68 ± 1.9a 51.1 ± 2.3b
SM 31.6 ± 1.3a 39.4 ± 2.1b 39.1 ± 1.8b 74.1 ± 1.8a 66.5 ± 1.5b 47.2 ± 1.9b

Periurethral buds (PeB), periurethral mesenchyme (PeM), ventral buds (VB), ventral mesenchymal pad (VMP), smooth muscle layer (SM). Values are means ± SEM. (a, b, c)
superscript letters represent statistically significant differences (p ≤ 0.05) between the experimental groups (n = 30 fields in 3 animals/group).

Table 4
Frequency (%) of AR, PCNA and ER␣-positive cells in the female gerbil prostate of all experimental groups.

AR PCNA ER-␣

C LBPA HBPA C LBPA HBPA C LBPA HBPA

PeB 86.5 ± 3.3 a

63.4 ± 7.7b
56.9 ± 3.6 b
80.4 ± 4.1 73.5 ± 5.7 75.3 ± 2.5 – – –
PeM 27.4 ± 4.2a 56.4 ± 5b 73.3 ± b.3c 75.2 ± 2.7 67.9 ± 2.1 78.8 ± 2.3 38.3 ± 3.1a 22.3 ± 2.2b 44.9 ± 4.8a
PaB 70.6 ± 3.1a 56.3 ± 9.2a 95.9 ± 2.4b 73.0 ± 9.1a 33.3 ± 6.2b 35.5 ± 7.1b – – –
PaM 76.5 ± 4.3a 37.6 ± 7.1b 16.4 ± 4.8c 77.3 ± 4.0a 68.0 ± 3.5b 66.0 ± 1.6b 29.6 ± 2.2a 24.0 ± 2.5a 15.2 ± 1.5b
SM 51.1 ± 5.3 47.7 ± 6.5 49.9 ± 7.8 60.1 ± 2.4a 66.0 ± 3.6a 70.8 ± 2.9b 16.4 ± 1.2a 9.8 ± 1.6b 18.1 ± 1.1a

Periurethral buds (PeB), periurethral mesenchyme (PeM), paraurethral buds (PaB), paraurethral mesenchyme (PaM), smooth muscle layer (SM). Values are means ±nSEM.
(a, b, c) superscript letters represent statistically significant differences (p ≤ 0.05) between the experimental groups (n = 30 fields in 3 animals/group).

ing and elongating pattern of the prostate observed in the male

Fig. 5. Immunohistochemical reactions in PND8 female prostate of all experimental
groups. (a–f) Immunohistochemistry for the androgen receptor (AR). The details (b1,
e1 and f1) highlight AR-positive cells, with nuclei stained in brown (arrows), either
in the epithelium or in the stroma. Bars: 5 ␮m. (g–l) Immunohistochemistry for
the proliferation cell nuclear antigen (PCNA). (m–r) Immunohistochemistry for the
estrogen receptor alpha (ER␣). Inserts (n1, p1 and r1) show nuclear immunostaining
in periurethral mesenchyme (PeM), paraurethral mesenchyme (PaM), and smooth
muscle layer (SM). Bars: 5 ␮m. (s–u) Immunofluorescence for ␣-actin. Green: alpha-
actin (arrows); Blue: DAPI. Periurethral buds (PeB), paraurethral buds (PaB), (SM).
(For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
gerbils is similar to that described in rats in which the prostatic mor-
phogenesis is finalized between the 15th and 30th day of postnatal
life [8].
The occurrence of prostate-like glands in adult females has been
described for various species of mammals [39–41]. However, little
has been reported about the intrauterine and neonatal develop-
ment in females, with only a few reports related to different Wistar
rat strains [42,43]. In this study, we observed that, although the
female PND8 gerbil presents prostate structures homologous to
those in the males of the same age, there are some spatial, temporal,
and structural differences with respect to the elongation processes
and ductal branching between both sexes. In female gerbils, the
PeBs arising from the urothelium do not have an elongation in a
‘V’-shaped pattern. These lateral buds extend linearly across the
urethra to reach the PaM, which is located in the cranial region of
the urethra near the base of the bladder. Moreover, in the female
PND8 gerbil, all PeBs in elongation have already reached PaM and
begun the branching process and ductal canalization. These results
demonstrate that the postnatal development of the female ger-
bil prostate is earlier than and morphologically distinct from that
occurring in males of the same species. Additionally, the prostate
buds of PND8 females occupy, in comparison, only 25% of the area
of the ventral EBs of male gerbils.
In general, the data collected from the BPA exposure suggests
that this compound did not change the AGD, body weight, and PrC
weight of male and female gerbils in both LBPA and HBPA groups.
Similarly previous studies that evaluated the effects of different
dosages of BPA in other rodent species found no alterations in
these same endpoints [44–46]. However, in our study, we evaluated
different parameters, including morphometric and stereological
analysis as well as immunohistochemistry. In spite of not finding
alterations in the glandular morphogenesis pattern and prostatic
morphology in both sexes of PND8 gerbils, we observed changes
in the immunolabeling patterns for AR and PCNA of all animals of
LBPA and HBPA groups.
In the males of LBPA and HBPA groups, an increase in the fre-
quency of AR-positive cells in the PeM, VB, and SM was observed. On
the other hand, PCNA-positive cells became less frequent in the PeB
and SM in the LBPA animals as well as in all compartments of the
ventral prostate in the HBPA. In the VBs of HBPA, which are struc-
244 R.F.d. Lima et al. / Reproductive Toxicology 58 (2015) 238–245
tures that will differentiate into the prostatic alveoli of adult males,
a reduction of about 35% in the rate of epithelial cell proliferation
occurred. It has been shown that BPA can bind to AR, antagonizing
its functions [15,29,47]. Therefore, these results indicate that BPA
has triggered an antiproliferative effect in the ventral prostate of
PND8 male gerbils, especially in the group exposed to high dosages
(HBPA).
In the present study, we were unable to detect ER␣ in PND8 male
prostates. This receptor has been shown in the mesenchyme and
emerging prostate buds of male gerbils with 23 days of intrauter-
ine development [34]. In mice, the ER␣ profile exhibited important
and rapid changes during the first weeks of postnatal development,
becoming frequent in the mesenchyme during the first week, but
exclusively epithelial in the second week [48]. Therefore, our fail-
ure to demonstrate immunolabeling to ER␣ in PND8 male gerbils
may be due to no expression of this marker in this period of male
prostate postnatal development. This hypothesis seems acceptable,
since PND8 females showed immunolabeling to ER␣ in prostate
mesenchymal cells.
In the PND8 females, the effects of BPA were more pronounced
on immunohistochemical endpoints than in the male prostate. In
the LBPA, the AR frequency increased in the PeM, and, for PeB and
PaM, a decrease was observed. In the HBPA group, this receptor
became more frequent in the PeM and PaB and less frequent in the
PeB and PaM. Furthermore, a reduction in the ER␣-positive cells in
the PeM and SM of the LBPA group and in the PaM of the HBPA group
was also observed. Thus, it was found that the different regions of
the female prostate of PND8 gerbils expressed AR and ER␣ recep-
tors that responded in distinct manners to the BPA exposure. It is
also noted that this complex interaction network can be related
to alterations in the cellular proliferation pattern observed in the
present study. The immunohistochemistry for PCNA in females
demonstrated a significant reduction of cell proliferation in the
LBPA and HBPA groups. This antiproliferative pattern was espe-
cially pronounced in the PaBs that presented a reduction of more
than 50% of the proliferative rate in both treatments. Interestingly,
the relative frequency (%) of epithelial buds significantly increased
in the female prostate of the HBPA group. In this respect, we believe
that the bud growth occurred earlier in development and the pro-
liferative effects are no longer seen in PND8 when the tissues are
labeled for PCNA. Finally, it is possible that the reduction of postna-
tal proliferation of PaBs, structures that will differentiate into the
paraurethral alveoli in adult females, can compromise the physi-
ology of the organ during the aging process. Thus, it is suggested
that, in PND8 female gerbils, BPA, even in low dosages, can also bind
to ARs, inhibiting their androgenic function as can be observed in
other studies [47,49].
Studies which evaluated the toxicity of reproductive organs in
terms of the environmental dosages of BPA during critical peri-
ods of ontogenetic development (fetal, neonatal, and pubertal) are
controversial, and, generally, the different outcomes are associ-
ated with the experimental design applied [15,50]. Many studies
demonstrate that dosages considered ‘safe’ by the US Environmen-
tal Protection Agency (EPA, <50 ug/kg/day) [47,51] can still cause
epigenetic alterations in many organs and systems, leading to a
predisposition to serious lesions throughout adult life [29,52–56].
In this study, we did not observe any discernible changes in the
ductal branching pattern or in prostate morphology in either gen-
der at PND8. However, this does not preclude adverse effects later
in life, as previously reported. Indeed, our results demonstrate that
exposure to BPA alters the expression pattern of AR, ER␣, and PCNA,
which may predispose this hormone-regulated tissue to abnormal-
ities later in life. In males, the exposure to high dosages of BPA
resulted in a decrease of the proliferative status of all compart-
ments of the developing ventral prostate. In females, both high and
low dosages were sufficient to decrease the proliferation of parau-
rethral buds in the branching process by more than 50%. Therefore,
the collective data indicate that BPA promotes antiproliferative
effects during the neonatal development of the animals, with more
sensitivity to this endocrine disruptor in females.
5. Conclusion
This study revealed that the normal postnatal development of
the gerbil female prostate occurs earlier than and is morpholog-
ically distinct from to what occurs in males of the same species.
Furthermore, it can be seen that the BPA exerted an antiprolifer-
ative effect on the prostate gland of male and female PND8, with
females being more susceptible to this environmental chemical.
Based on the methodologies used in this study, it is not possible
to say whether the changes observed in the prostates of newborn
gerbils are activational (transient) or organizational (permanent).
Although a decrease in PCNA expression was observed in female
prostates of treated groups, the glands showed normal morphol-
ogy at PND8. The reason for this remains unclear, but suggests
that the decrease in PCNA-positive cells observed in this study was
insufficient in causing effects on the prostate observable at the mor-
phological level at this stage of development. It does not, however,
preclude the possibility that earlier changes in proliferation status
may cause more prominent morphological alterations later in life.
Therefore, these results have great importance for public health
since all individuals, from fetal development to senescence, are
exposed to increasing levels of EDCs such as BPA.
Transparency document
The Transparency document associated with this article can be
found in the online version.
Acknowledgements
We are very grateful to Luiz Roberto Falleiros Júnior as well
as to the other researchers from the Laboratory of Microscopy
and Microanalysis for their technical assistance. This paper was
supported by a grant from the Brazilian agency CNPq (Brazil-
ian National Research and Development Council, Procs. Nr.
475148/2012-6) and FAPEG (Goiás Research Foundation, Procs Nr.
05/2012).
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