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Postnatal Development of Mongolian Gerbil Female Prostate:

An Immunohistochemical and 3D Modeling Study

Department of Structural and Functional Biology, State University of Campinas, Av. Bertrand Russel s/n, Campinas, S~
ao Paulo, Brazil
Department of Biology, Laboratory of Microscopy and Microanalysis, University of Estadual Paulista – UNESP, Rua Cristov~ ao
Colombo, S~ao Jose do Rio Preto, S~
ao Paulo, Brazil
Department of Morphology, Federal University of Goias, Samambaia II, Goiania, Goias, Brazil

KEY WORDS prostate development; ventrolateral mesenchyme; branching morphogenesis;

female prostate

ABSTRACT The development of the prostate in male rodents, which involves complex epithelial–
mesenchymal interactions between the urogenital sinus epithelium (UGE) and the urogenital sinus
mesenchyme (UGM), has been deeply studied. In females, however, this process is not very clear. In this
study, the postnatal development of the prostate in female Mongolian gerbils employing three-
dimensional (3D) reconstructions, histochemical, and immunohistochemical techniques was character-
ized. It was observed that prostatic branching and differentiation in females was induced by a single
mesenchyme localized at a ventrolateral position, which was named as ventrolateral mesenchyme
(VLM); furthermore, the canalization of solid buds began on the third postnatal day (P3) and the branch-
ing morphogenesis on P5. We observed secretions in the acini at the end of the first month, and, on P45,
the acini were completely differentiated. The strong cell proliferation phase in the first week coincided
with the mesenchymal expression of estrogen receptor 1 (ESR1). The expression of androgen receptor
(AR) paralleled cell differentiation, and, on P30, immunolabelling with p63 was restricted to basal cells.
This study serves as a baseline parameter for future research on disruptions that could affect the devel-
opment of the female prostate. Microsc. Res. Tech. 79:438–446, 2016. V 2016 Wiley Periodicals, Inc.

INTRODUCTION is that the gland maturation in females occurs before

The development of the prostate in males is a consid- than that observed in males (Custodio et al., 2004, 2010)
erably conserved process during mammalian evolution and the prostate of adult females expresses AR and
(Timms, 2008), which initially involves epithelial–mes- ESR1 in periductal region, moreover, gerbil female pros-
enchymal interactions between the urogenital sinus epi- tate present great sensitivity to hormonal fluctuations
thelium (UGE) and the urogenital sinus mesenchyme (Fochi et al., 2013). In addition, these animals has been
(UGM). During the prenatal period, solid epithelial pro- shown to be an excellent experimental model for the
jections (prostatic buds) from the UGE invade the peri- study about several aspects of prostate biology due to
urethral mesenchyme (PM) and the UGM originates the proximity morphofunctional with human prostate
three distinct compartments: the PM, a layer of smooth (Rochel et al., 2007; Rochel-Maia et al., 2013).
muscle named periurethral smooth muscle (PSM), and Because of the low occurrence of prostate in female
a peripheral mesenchyme adjacent to the PSM. The rats and mice, there is a substantial knowledge gap in
development of prostatic buds (PB), or budding process, this subject (Thomson et al., 2002). The Mongolian ger-
is induced by these peripheral urogenital sinus mesen- bil is a promising experimental model to study the
chymes (Cunha, 1973; Sanches et al., 2014; Thomson female prostate because females have a large amount
and Marker, 2006). Such mesenchymes induce the of prostate and this rodents are easy to breed and
branching of the PB in a lobe-specific way. The most
studied of these mesenchymes, the ventral mesenchyme *Correspondence to: Dr. Sebasti~
ao R. Taboga; Department of Biology, Laboratory
of Microscopy and Microanalysis, S~ ao Paulo State University, Rua Cristov~ ao
pad (VMP), is responsible for the development of the Colombo 2265, Jardim Nazareth, S~ao Jose do Rio Preto, S~
ao Paulo, Brazil.
prostate ventral lobe (Timms et al., 1995), whereas E-mail: taboga@ibilce.unesp.br
other mesenchymes induce the development of the The authors declare that there is no conflict of interest.
Bruno D.A. Sanches and Bruno C. Zani contributed equally to this work.
remaining lobes (Timms and Hofkamp, 2011). In addi-
Received 25 November 2015; accepted in revised form 20 February 2016
tion to the branching process, canalization and morpho-
REVIEW EDITOR: Prof. Alberto Diaspro
functional differentiation of the acini also occur in these Contract grant sponsor: S~ ao Paulo Research Foundation (FAPESP); Contract
mesenchymes. In mice, these changes terminate by the grant number: 2013/15939-0, 2013/16443-9; Contract grant sponsor: Brazilian
end of the first postnatal month (Prins and Putz, 2008). National Research and Development Council (CNPq); Contract grant number:
In female rodents, on the other hand, the develop- DOI 10.1002/jemt.22649
ment of the prostate has not been well studied. Evidence Published online 12 March 2016 in Wiley Online Library (wileyonlinelibrary.com).


handle in laboratory (Jesus et al., 2015; Santos and cleared in xylene, and finally embedded in paraffin
Taboga, 2006; Sanches et al., 2014). The description of (Histosec, Merck Dermstadt, Germany) for 3 hours. The
the main events in the development of the prostate in prostatic complexes fixed in paraformaldehyde 4% were
female Mongolian gerbils generates an important com- evaluated for the presence of ESR1, proliferating cell
parative parameter for studies evaluating hormone nuclear antigen (PCNA), AR, and p63 by immunohisto-
manipulation, development disruptions, and the sus- chemistry assays. Samples fixed in Metacarn were
ceptibility to prostatic diseases; topics that have been employed for morphological analyses and 3D reconstruc-
under intense scrutiny recently (Jesus et al., 2015; tions. All prostatic complexes were serially sectioned
Perez et al., 2011, 2012; Zanatelli et al., 2014). (5 lm) in a rotary microtome and mounted on histologi-
The importance of the analysis of the postnatal pros- cal slides. Tissue sections were subjected to hematoxylin-
tatic development in the female Mongolian gerbil is eosin (H&E) staining for general morphological analyses
supported by similarities between the female gerbil or were used for immunohistochemical assays. The
and women prostates (Custodio et al., 2010, Dietrich specimens were analyzed with an Olympus BX60 light
et al., 2011; Santos and Taboga, 2006) and by the ethi- microscope (Olympus, Japan) and the resulting images
cal and practical issues associated with the study of were digitalized by using DP-BSW V3.1 (Olympus) and
the prostate development in humans (Zaviačič, 1999). the Image-ProPlus 6.1 for Windows (Media Cybernetics,
Additionally, increased exposure of women to xenoes- Silver Spring, MD) software.
trogens has raised their susceptibility to pathological
conditions, a phenomenon that has drowned the atten- Immunohistochemistry
tion of many researchers (Perez et al., 2012; Santos Tissue sections were subjected to immunohistochem-
and Taboga, 2006). Thus, the female Mongolian gerbil istry assays to detect AR, ESR1, PCNA, and p63, as
is an important experimental tool to understand pros- described in the protocol applied to the prostate tissue
tatic pathologies in women (Santos and Taboga, 2006). (Fochi et al., 2013). The following antibodies were used
Given the importance of the description of the events at a dilution of 1:100: AR (rabbit polyclonal IgG, N-20,
of female prostate development and the information sc-816, Santa Cruz Biotechnology, CA), ESR1 (rabbit
gap in the literature about this subject, we used three- polyclonal IgG, MC 20, sc-542, Santa Cruz Biotechnol-
dimensional (3D) reconstructions, histochemical, and ogy), PCNA (monoclonal mouse IgG2a, PC10, SC-56,
immunohistochemical techniques to describe the early Santa Cruz Bio- technology), and p63 (rabbit polyclo-
postnatal development of the prostate in the female
nal IgG, H-300, SC-367 333, Santa Cruz Bio-technol-
Mongolian gerbil.
ogy). PolyK4061 (Dako Dako Envision, North America,
MATERIAL AND METHODS Carpinteria) was used as a secondary antibody.
Animals and Experimental Design Samples were incubated for 45 minutes at 378C and
Animals used in this study were provided by the were counterstained with diaminobenzidine (DAB) and
University of Estadual Paulista (UNESP), S~ ao Jose do Harris hematoxylin. Finally, histological sections were
Rio Preto, SP, Brazil. They were maintained in polyethyl- examined under an Olympus BX60 Light Microscope
ene cages under controlled conditions of light (light/12-h (Olympus). Negative controls were obtained by omitting
dark) and temperature (258C) 12-h and were provided the step with the primary antibody incubation. In order
with filtered water and rodent food ad libitum. Handling to assess cellular proliferation, two different stages were
of animals and experiments were conducted in accord- evaluated, the peak of the branching morphogenesis on
ance with the UNESP ethical guidelines (Ethical com- P7 and the acini morphofunctional differentiation on
mittee CEUA number 116/2015) and the Guide for Care P30, a quantification of PCNA immunohistochemistry
and Use of Laboratory Animals (The National Academies assay was performed according to Fochi et al. (2013), in
Press, 2011, Washington, DC). We used 21 adult female which the total number of cells was counted in 30 micro-
and 21 adult male Mongolian gerbils between 3 and 6 scopic fields randomly selected from each experimental
months old, which were randomly distributed to form 21 group, which were separated into positively and nega-
pairs. Subsequently, they were divided into seven experi- tively stained. A minimum of 1,000 cells were counted,
mental groups composed of three pairs each. The groups and then the percentage of immunolabelling was calcu-
were distributed in a way that covered the early post- lated as the number of positive cells divided by the total
natal development of the female prostate: P1, P3, P5, P7, number of cells. The computation of immunostainings
P14, P30, and P45, where “P” represents the postnatal was performed for both prostatic compartments (epithe-
period and the numbers represent the day in that period. lium and stroma). The immunohistochemistry assays for
The animals were later sacrificed by lethal injection con- AR, ESR1, and p63 were qualitatively analyzed.
taining a mixture of an anesthetic, ketamine (100 mg/kg
bw, Dopalen, Vetbrands, Brazil), and a muscle relaxant, Three-Dimensional Reconstruction
xylazine (11 mg/kg bw, Rompun, Bayer, Brazil). Finally, Three-dimensional reconstructions were performed
six puppies from three different pairs were obtained for for all experimental groups in order to obtain detailed
each experimental group and their prostatic complexes 3D images of the branching and morphofunctional dif-
were dissected and removed after sacrifice. ferentiation of the female prostate. Serial female pros-
tatic complex histological sections (5 lm) were analyzed
Light Microscopy and photographed with an Olympus BX60 light micro-
The female prostatic complexes were fixed in parafor- scope (Olympus, Japan). The resulting images were dig-
maldehyde 4% during 24 hours or Metacarn (methanol, italized with the DP-BSW V3.1 (Olympus, Japan) and
chloroform, and acetic acid in the ratio 6:3:1) for 12 the Image-ProPlus 6.1 for Windows (Media Cybernetics,
hours. Then, samples were dehydrated in ethanol series, Silver Spring, MD) software. Later, the images were

Microscopy Research and Technique


Fig. 1. Histology of the sequential early development of the Gerbil P5, branching morphogenesis occurs concomitantly with canalization.
female prostate. Histological sections of the prostatic complex develop- (G, H) Branching increases and reaches P7. PM (periurethral mesen-
ment were stained with hematoxylin-eosin to cover the early postnatal chyme), UE (urethral epithelium), Arrows (prostatic budding), insertion
development period of the gland. (A, B) On P1, prostatic buds growing (details of prostate development), VLM (ventrolateral mesenchyme),
parallel to the axis and in a proximal region of the urothelium. (C, D) PSM (periurethral smooth muscle), CN (canalization), Arrow Head (cel-
On P3, prostatic buds undergoing canalization. Cellular death is present lular death). [Color figure can be viewed in the online issue, which is
in the region that will originate from the lumen of the acini. (E, F) On available at wileyonlinelibrary.com.]

reconstructed by using Reconstruct software (Fiala, RESULTS

2005). The Postnatal Development of the Female
Prostate Depends on Prostatic Buds That Reach
Statistical Analysis a Specific Inductive Mesenchyme, Which we
All quantitative results are expressed as mean 6 SD. Denominated Ventrolateral Mesenchyme (VLM)
The data were initially studied by the analysis of var- We observed that the prostatic buds of the developing
iance (one-way ANOVA) and, subsequently, by Tukey’s female prostate grew parallel to the axis of the urethra
test for multiple comparison, with a 5% significance on P1, at the same time, these buds reached the VLM
level (P  0.05). The statistical tests were performed (Figs. 1A and 1B) and underwent canalization (Figs. 1C
with Statistica 7.0 software (StarSoft Inc., Tulsa, OK). and 1D). Cell death in the center of the distal portion of
The quantitative comparisons were carried out only buds opens spaces that will consist in the lumens of the
between groups for PCNA immunohistochemistry assays. prostatic acini. The prostatic development proceeded in

Microscopy Research and Technique


Fig. 2. Histology of the sequential late development of the Gerbil entiated epithelium and periductal smooth muscle. Similarly, the
female prostate. (A, B) On P14, developing acini are identified and prostatic stroma has high amounts of stromal connective fibers. PS
canalization generates the lumina of the acini. The presence of stromal (prostate stroma), insertion (details of prostate development), L (lumen
connective fibers suggests the differentiation of the prostatic stroma. (C, of the acini), arrow head (cellular death), SF (stromal connective fibers),
D) On P30, secretions are present in the developing prostatic acini. large arrows (prostatic acini), SM (periductal smooth muscle), PE (pros-
Well-defined lumina are observed in addition to the periductal muscula- tatic epithelium), Asterisk (prostatic secretion). [Color figure can be
ture. (E, F) On P45, acini increase in size and show a completely differ- viewed in the online issue, which is available at wileyonlinelibrary.com.]

the VLM, which differentiated progressively into the of morphological and functional differentiation of the
prostate stroma. On P5, branching morphogenesis acini, secretions were observed on P30. At this stage,
occurred concomitantly with the gland canalization we could see well-defined lumen, and the presence of
(Figs. 1E and 1F), and this process continued up to P7. differentiated muscle cells forming the periductal mus-
Increased branching volume was also observed on P7 culature (Figs. 2C and 2D). Finally, on P45, the acini
(Figs. 1G and 1H). increased in size and their epithelium and periductal
smooth muscle cells were completely differentiated.
Differentiation of the Prostatic Epithelia Similarly, the prostatic stroma contained large amounts
Occurs Simultaneously with the Differentiation of stromal connective fibers (Figs. 2E and 2F).
of the Prostatic Stroma
Few Prostatic Buds Generate the Majority
We detected that the prostatic development occurred
simultaneously in both the epithelial and stromal com- of the Branching and, Consequently,
partments. On P14, we noted the presence of develop- the Acini in the Female Prostate
ing acini and the occurrence of canalization, which led The 3D reconstructions of the developing prostate
to the generation of lumina. At the same time, we con- complex pointed out to the existence of few prostatic
firmed the presence of stromal connective fibers, indi- buds, usually two, which grew parallel to the urethra
cating the progressive differentiation of the prostatic axis in its proximal portions and presented a cranio-
stroma (Figs. 2A and 2B). Along with the progression caudal growth pattern in the more distal regions of the

Microscopy Research and Technique


Fig. 3. Three-dimensional reconstructions of the developing pros-

tatic complexes showing branching morphogenesis. (A) On P1, the
growth of prostatic buds occurs parallel to the urethra in its proximal
region. In the distal portions, buds grow in a caudal–cranial axis and
their distal ends reach the VLM, which is located beyond the periure-
thral musculature layer that surrounds the urethra. (B) In the VLM,
the distal ends of the buds undergo branching morphogenesis as they
reach the VLM. On P5, the extremity of one bud has already branched
and another extremity is still dilating. PSM (periurethral smooth mus-
cle), Green (urethral epithelium), Dark Blue (urethral lumen), Cyan
(buds/prostatic branches), Arrow (distal end of a prostate bud in the
VLM region), Broad Arrow (caudal–cranial growth), brackets (prostatic
branching) bar (200 lm). [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]

urethra. In Figure 3A, on P1, two buds reached the

VLM. On P5, we noted that one bud had already
branched multiple times. The second bud had not
branched and reached the VLM later (Fig. 3B). The
prostatic buds reached the VLM, the single female
inductive mesenchyme, located beyond the periure-
thral smooth muscle layer. This muscle layer was
already closed on P1 (Fig. 4A), implying that the pros-
tatic buds reached the VLM still at the prenatal
period. The prostatic buds dilated in the VLM (Fig. 4B)
and progressively branched (Figs. 4C and 4D). On P14
and P30, ramifications continued to increase in size
and gradually differentiated into acini (Figs. 4E and
4F). On P45, we observed differentiated acini. At this
point, the VLM was arranged bilaterally and could be
reached by one or more buds (Fig. 4G).

Most Prostate Development in Females

Occurs at the Postnatal Period
At birth, prostatic buds had already reached the
VLM; however, their differentiation into acini required
a period of over 6–7 weeks. The main events of the post-
natal development consisted of canalization, already Fig. 4. Three-dimensional reconstructions of the developing pros-
confirmed on P3, in the extremities of the branches that tatic complexes of all groups. (A) On P1, the distal ends of the prostatic
buds have reached the VLM, which is located beyond the periurethral
had reached the VLM, observed on P5, the formation, smooth muscle that surrounds the urethra. (B) In the VLM, on P3, the
morphological, and functional differentiation of the distal ends of the buds are enlarged. (C) On P5, branching begins at
acini, observed after P14. As mentioned before, the epi- the extremities of the buds. (D) On P7, there is a sharp increase in the
number of branches. (E, F) On P14 and P30, the branches increase in
thelial and stromal differentiation occurred simultane- size and differentiate progressively to form the acini. (G) On P45, dif-
ously. The VLM present in the early stages of the ferentiated acini are detected. PSM (periurethral smooth muscle),
prostatic postnatal development became the prostatic Green (urethral epithelium), Dark Blue (urethral lumen), Cyan (buds/
stroma. The differentiation took place in the VLM branchings/developing prostatic acini), Arrow (distal end of a prostate
budding in the VLM region), brackets (branching region), Large
(Fig. 5) and the progressive differentiation of fibroblasts Arrows (acini) bar (200 lm). [Color figure can be viewed in the online
occurred with high deposition of stromal connective issue, which is available at wileyonlinelibrary.com.]
fibers (Figs. 2B and 2F).

Microscopy Research and Technique


Fig. 5. Schematic design depicting the early development of the gland. Dark Blue (VLM/prostatic stroma), light blue (periurethral
female prostate. Buds reach the VLM on P1, undergo canalization on mesenchyme), Magenta (buds/branches/prostate acini epithelium),
P3, branching starts on P5, and progressive morphofunctional differ- Red (periurethral smooth muscle), Green (urothelium/urethral epi-
entiation of acini occurs after P14. Simultaneously with the epithelial thelium). [Color figure can be viewed in the online issue, which is
differentiation, the VLM differentiates into the stroma of the mature available at wileyonlinelibrary.com.]

Fig. 6. Immunohistochemistry assay showing the localization of lium. VLM (ventrolateral mesenchyme), PB (prostatic buds), Arrows
PCNA in the developing female prostate on P7 and P30. (A, B) On P7, (positive mesenchymal cells), Arrow head (positive epithelial cells),
PCNA is highly expressed in both the prostate mesenchyme and the insertion (PCNA expression detail). [Color figure can be viewed in the
branching epithelium. (C, D) On P30, PCNA is less expressed which online issue, which is available at wileyonlinelibrary.com.]
shows weaker cell proliferation in both the mesenchyme and epithe-

Cell Proliferation in the Female was related to weaker proliferative activity (Figs. 2C
Prostate Decreases as the Gland and 2D).
Differentiation Occurs
The High Expression of ESR1 in the Prostatic
Strong PCNA staining in both the mesenchymal Mesenchyme Coincides With the Largest
and epithelial compartments of the gland on P7 (Fig.
Epithelial Proliferation
6A) points out to a large proliferative activity, which
corroborates the strong branching process observed On P7, ESR1 was highly expressed in the prostate
in this period (Figs. 1E and 1H). There was, however, mesenchyme, but less expressed in the branching epi-
reduced PCNA expression on P30 (Fig. 6B), when the thelium (Figs. 7A and 7B). The expression of ESR1 coin-
female prostate already had acini undergoing strong cided with the high proliferative activity of the prostate
cellular differentiation. Reduced PCNA expression during this period (Fig. 6A).

Microscopy Research and Technique


Fig. 7. Immunohistochemistry assay showing the presence of stem cells of the branches. On P30, the expression of this protein is
ESR1, RA, and P63 in the developing female prostate. (A, B) On P7, restricted the epithelial basal cells of the acini. VLM (ventrolateral
ESR1 is highly expressed in the prostate mesenchyme, but weaker mesenchyme), PB (prostatic bud), Arrows (positive mesenchymal
markings occur in P30. (C, D) On P7 and P30, AR is weakly cells), AC (Prostatic acini), Arrow head (positive epithelial cells),
expressed in the mesenchyme. Epithelial immunostaining is also insertion (ESR1 expression detail). [Color figure can be viewed in the
noticed on P30. (E, F) On P1, p63 is expressed in prostatic epithelial online issue, which is available at wileyonlinelibrary.com.]

In the Developing Female Prostate, laboratory rodents. Prostatic buds develop in a V

as the Prostate Differentiates, AR is Expressed shaped pattern to reach the VMP. On P1, buds branch
in the Epithelium and p63 Expression is in the VMP (Sanches et al., 2014). In females, we could
Restricted to the Basal Cells not identify the presence of the VMP. Their prostate
has a single lobe that presents morphological and
On P7 and P30, the expression of AR was low in the physiological similarities with the ventral lobe of the
mesenchyme (Figs. 7C and 7D). Epithelial staining was male prostate (Santos and Taboga, 2006) and is located
seen on P30 (Fig. 7D). On P1, the expression of p63 protein ventrolateral to the urethral axis. We named this lobe
was found in the epithelial cells of the undifferentiated VLM. It appears condensed at the initial prenatal
branches. As early as P30, the expression of p63 protein period and will progressively differentiate along with
was restricted to the basal epithelial cells of the acini the gland epithelium development. In short, prostatic
(Figs. 7E and 7F). The expression of AR in the epithelium buds in females, as seen in males, grow toward the
and of p63 limited to the prostatic basal cells coincided VLM inductive mesenchyme.
with the strong morphological differentiation of the acini Unlike in males, the periurethral smooth muscle in
that occurred on P30 (Figs. 2C and 2D). females is completely closed on P1, and the prostatic
buds migrate to the VLM still in the prenatal period.
DISCUSSION This finding contradicts the smooth muscle hypothesis
In male Mongolian gerbils, the initial development (Thomson et al., 2002; Thomson and Marker, 2006;
of the prostate occurs later than that observed in other Timms and Hofkamp, 2011), which predicts that the

Microscopy Research and Technique


cause of the absence of prostate in female rats is the P30, the presence of epithelial AR concurred with the
rapid closure of the smooth muscle still in the embry- morphofunctional differentiation of the gland. Corrobo-
onic period. The reason for the existence of a functional rating this finding, the epithelial AR has been associ-
prostate in female Mongolian gerbils remains unclear, ated with the prostate epithelial differentiation,
however, their hormone dynamics, which is character- especially with the secretory activity (Donjacour and
ized by high levels of circulating steroids in the blood Cunha, 1993; McNamara et al., 2013).
stream in the early postnatal period, may be a key fac- Results from previous studies with prostates of female
tor for the understanding of such occurrence (Custodio Mongolian gerbils have confirmed the rapid maturation
et al., 2008). of the gland. The acid phosphatase activity reaches
The development of the female gerbil prostate pro- higher levels earlier in females than that in males (Cus-
ceeds in the VLM and the canalization process is todio et al., 2004; Santos et al., 2006), which can be corre-
detected in the developing gland on P3. In rats, this lated with an earlier expression of the epithelial AR and
phenomenon is observed on P5 (Prins and Birch, with a lower expression of the mesenchymal AR. In the
1995). In female gerbils, the early prostate branching same way, with the progression of the prostate differen-
begins on P5 and continues up to P30. On P14, lumeni- tiation, the expression of p63 is confined to the basal
zation occurs in some prostate branches. In male rats, cells, which are already distinct from the luminal cells
the lumenization is observed on P12 and the secretory on P30. Researchers have shown that p63 is as an impor-
activity of the aciny is already observed between P10 tant marker of basal cells and of the prostate differentia-
and P15 (Prins and Birch, 1995). In female gerbils, the tion (Kurita et al., 2004; Shi et al., 2014.).
secretory activity is observed only on P30. The faster dif-
ferentiation of the male rat prostate is probably due to a CONCLUSION
short generation time (Prins and Putz, 2008) and an ear- In this study, we have described in detail for the first
lier development than that seen in gerbils (Sanches time the initial postnatal development of the Mongo-
et al., 2014), which have a longer generation time. In lian gerbil female prostate. One of the main character-
general, the prostate development in female gerbils hap- istics of this development is the presence of a single
pens earlier than that seen in males of the same species. inductive mesenchyme arranged laterally, which we
Hence, evidence is that the morphological and functional named VLM. Furthermore, unlike what is observed in
differentiation of the gland occurs first in females (Custo- rats (Sanches et al., 2014), we confirmed that, at birth,
dio et al., 2008), but more accurate comparative analyses there is no longer a discontinuity in the periductal
are needed. smooth muscle, making it impossible for the buds to
In rats and gerbils, the epithelial and mesenchymal reach the VLM in the postnatal period. This implies
differentiation occurs simultaneously (Prins and Birch, that the female prostate is derived from usually one or
1995; Sanches et al., 2014.). In this study, we identified two buds that have reached the VLM still in intrauter-
developing acini undergoing lumenization on P14. At ine life. Buds in the VLM are channeled and suffer a
the same time, we confirmed the presence of stromal rapid process of branching. Developing acini are pres-
connective fibers, indicating the differentiation of the ent in the second week of the postnatal development
prostatic mesenchyme into prostatic stroma. On P30, and, by the end of the first month; they already have a
differentiated muscle cells formed the periductal mus- secretory activity. Broadly, our study generated an
culature. Finally, on P45, the acini increased in size and important comparative parameter for new research on
the secretory epithelium was fully differentiated. Like- alterations that can affect the development of the
wise, the glandular stroma also showed a similar degree female Mongolian gerbil prostate.
of differentiation. The mesenchymal differentiation also
occurs earlier in rats (Prins and Birch, 1995). ACKNOWLEDGMENTS
Another peculiarity of the postnatal prostatic devel- The authors would like to thank the researchers from
opment in female Mongolian gerbils is the existence of the Microscopy Laboratory and Microanalysis, Luiz
few prostatic buds, usually two, which achieve the Roberto Falleiros Junior and Rosana Silistino de Souza
VLM. In males, there are a lot of prostatic buds in (Univ. Estadual Paulista – UNESP, S~ ao Jose do Rio
the VMP (Sanches et al., 2014), which explains why Preto) for their technical support and Rodrigo P. A.
the male gland is bigger than that of the female. The Barros, MD, PhD, for assisting with preparation of the
sooner the distal edges of the buds reach the VLM, the manuscript. Author contribution: BDAS and BCZ con-
faster they undergo the branching process, and there- tributed equally to this work. All authors (BDAS, BCZ,
fore the faster the morphofunctional differentiation of JMS, MFB, FCAS, RMG, PSLV and SRT) contributed
the gland occurs. to the design and interpretation of results and to the
Researchers have found that cell differentiation in review of the manuscript. BDAS, BCZ and JMS per-
the female prostate opposes proliferation and that the formed the experiments. BDAS wrote the manuscript
PCNA immunostaining is weak during the progression and SRT conducted the final review of the manuscript.
of development in both the mesenchymal and epithelial
compartments of the gland. The ESR1 expressed in the REFERENCES
prostate mesenchyme has been identified as an impor- Cunha GR. 1973. The role of androgens in the epithelio-mesenchymal
tant promoter of the epithelial proliferative activity in interactions involved in prostatic morphogenesis in embryonic
the male (Ellem and Risbridger, 2009; Sanches et al., mice. Anat Rec 1:87–96.
2014) and female prostates (Rochel-Maia et al., 2013). Custodio AMG, G oes RM, e Taboga SR. 2004. Acid phosphatase activ-
ity in gerbil prostate: Comparative study in male and female dur-
In our study, we observed strong ESR1 staining in the ing postnatal development. Cell Biol Int 28:335–344.
prostatic mesenchyme, which coincided with a period of Custodio AM, Santos FCA, Campos SG, Vilamaior PS, G oes RM,
strong epithelial proliferation in the gland on P7. On Taboga SR. 2008. Aging effects on the mongolian gerbil female

Microscopy Research and Technique


prostate (Skene’s paraurethral glands): Structural, ultrastructural, Prins GS, Putz O. 2008. Molecular signaling pathways that regulate
quantitative, and hormonal evaluations. Anat Rec 291:463–474. prostate gland development. Differentiation 76:641–659.
Custodio AMG, Santos FCA, Campos SGP, Vilamaior PSL, Oliveira Rochel SS, Bruni-Cardoso A, Taboga SR, Vilamaior PS, G oes RM.
SM, Goes RM, Taboga SR. 2010. Disorders related with ageing in 2007. Lobe identity in the Mongolian gerbil prostatic complex: A
the gerbil female prostate (Skene’s paraurethral glands). Int J Clin new rodent model for prostate study. Anat Rec (Hoboken) 290:
Exp Pathol 91:132–143. 1233–1247.
Dietrich W, Susani M, Stifter L, Haitel A. 2011. The human female Rochel-Maia SS, Santos FCA, Alonso-Magdalena P, G oes RM,
prostate —immunohistochemical study with prostate-specific anti- Vilamaior PSL, Warner M, Gustafsson J-A, Taboga SR. 2013.
gen, prostate-specific alkaline phosphatase, and androgen receptor Estrogen receptors alpha and beta in male and female gerbil pros-
and 3-D remodeling. J Sex Med 8:2816–2821. tates. Biol Reprod 88:1–7.
Donjacour AA, Cunha GR. 1993. Assessment of prostatic protein Sanches BDA, Biancardi MF, Santos FCA, G oes RM, Vilamaior PSL,
secretion in tissue recombinants made of urogenital sinus mesen- Taboga SR. 2014. Budding process during the organogenesis of the
chyme and urothelium from normal or androgen-insensitive mice. ventral prostatic lobe in mongolian gerbil. Microsc Res Tech 77:
Endocrinology 132:2342–2350. 458–466.
Ellem SJ, Risbridger GP. 2009. The dual, opposing roles of estrogen Santos FCA, Taboga SR. 2006. Female prostate: A review about the
in the prostate. Ann N Y Acad Sci 1155:174–186. biological repercussions of this gland in humans and rodents. Anim
Fiala JC. 2005. Reconstruct: A free editor for serial section micros- Reprod 3:3–18.
copy. J Microsc 218:52–61. Santos FC, Leite RP, Custodio AM, Carvalho KP, Monteiro-Leal LH,
Fochi RA, Santos FCA, Rejane M, Goes RM, Taboga SR. 2013. Proges- Santos AB, Goes RM, Carvalho HF, Taboga SR. 2006. Testosterone
terone as a morphological regulatory factor of the male and female
stimulates growth and secretory activity of the female prostate in
gerbil prostate. Int J Exp Path 94:373–386.
Jesus MM, Negrine AC, Taboga SR, Pinto-Fochi ME, G oes RM. 2015. the adult gerbil (Meriones unguiculatus). Biol Reprod 75:370–379.
Histopathological alterations in the prostates of Mongolian gerbils Shi X, Gipp J, Dries M, Bushman W. 2014. Prostate progenitor cells
exposed to a high-fat diet and di-n-butyl phthalate individually or proliferate in response to castration. Stem Cell Res 13:154–163.
in combination. Reprod Toxicol 52:26–39. Thomson AA, Marker PC. 2006. Branching morphogenesis in the
Kurita T, Medina RT, Mills AA, Cunha GR. 2004. Role of p63 and prostate gland and seminal vesicles. Differentiation 382–392.
basal cells in the prostate. Development 131:4955–4964. Thomson AA, Timms BG, Barton L, Cunha GR, Grace OC. 2002. The
McNamara KM, Nakamura Y, Sasano H, Handelsman DJ, role of smooth muscle in regulating prostatic induction. Develop-
Simanainen U. 2013. Prostate epithelial AR inactivation leads to ment 129:1905–1912.
increased intraprostatic androgen synthesis. Prostate 73:316–327. Timms BG. 2008. Prostate development: A historical perspective. Dif-
Perez APS, Biancardi MF, G oes RM, Santos FCA, Taboga SR. 2011. ferentiation 76:565–577.
Exposure to ethinylestradiol during prenatal development and Timms BG, Hofkamp LE. 2011. Prostate development and growth in
postnatal supplementation with testosterone causes morphophy- benign prostatic hyperplasia. Differentiation 82:173–183.
siological alterations in the prostate of male and female adult ger- Timms BG, Lee CW, Aumuller G, Seitz J. 1995. Instructive induction
bils. Int J Clin Exp Pathol 92:121–130. of prostate growth and differentiation by a defined urogenital sinus
Perez APS, Biancardi MF, Vilamaior PSL, G oes RM, Santos FCA, mesenchyme. Microsc Res Tech 30:319–332.
Taboga SR. 2012. Microscopic comparative study of the exposure Zanatelli M, Silva DAL, Shinohara FZ, G oes RM, Santos FCA,
effects of testosterone cypionate and ethinylestradiol during prena- Vilamaior PSL, Taboga SR. 2014. Actions of oestradiol and proges-
tal life on the prostatic tissue of adult gerbils. Microsc Res Tech 75: terone on the prostate in female gerbils: Reversal of the histological
1084–1092. effects of castration. Reprod Fertil Dev 26:540–550.
Prins GS, Birch L. 1995. The developmental pattern of androgen Zaviačič M. 1999. The female prostate: From vestigial Skene’s para-
receptor expression in rat prostate lobes is altered after neonatal uretral glands and ducts to woman’s functional prostate. Brati-
exposure to estrogen. Endocrinology 136:1303–1314. slava, Slovakia: Slovack Academic Press. 171 p.

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