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Introduction
Androgens are known to play an important role in
benign prostatic hyperplasia (BPH) and prostatic carcinoma
(PCa)[1,2], but their mechanism have not been fully
understood[3]. Androgens could probably affect the structure of
tissues, but it never been reported that they could change
the mitosis orientation in prostate epithelial cells. The present
study provides a further observation on the role of
androgens in androgen-related diseases, such as BPH and PCa.
The development and maturation of the male urogenital
system depends on the normal function of
androgens[4]. However, it has not been reported how the orientation of the
mitosis in prostate epithelial cells alters after hormonal level
regulation. Androgens may affect not only simple processes,
such as proliferation, cell differentiation, and cell_cell or
cell_extracellular matrix interrelation, but also the structure and
orientation of the tissues or their components. Our study
found that testosterone propionate (TP) could adjust
mitosis orientation in prostate epithelial cells, as discussed later.
Materials and methods
Experimental animals All experiments were performed
in accordance with the Guide for the Care and Use of
Laboratory Animals of the Institute of Laboratory Animal
Resources Commission on Life Sciences, America. Male
Sprague-Dawley rats (190_210 g) were obtained from the
Shanghai SIPPR-BK (Institute of Planned Parenthood
Research-BK Laboratory Animal Limited Company, Shanghai,
China). The animals were housed under the same
conditions with free access to water and food. The experiments
were started at 1 month, and then, all animals were castrated
under anesthesia with ketamine.
Treatments The castrated rats were treated with a single
subcutaneous injection of TP in olive oil at a dose 0.5 mg per
rat or benzogynestry (E2) in olive oil at a dose 5 µg per 100
g body weight. There were 8 rats in the control group and
TP-treated or E2-treated group. Prostate, a specimen of the liver,
a specimen of skin, a segment of the jejunum, and segment
of the colon were removed from untreated and TP- or
E2-injected groups at 24, 36, and 48 h. All of the animals were
injected intraperitoneally with bromodeo-xyuridine (BrdU;
Sigma, St Louis, MO, USA) dissolved in saline 2 h before the
tissues were removed. The dose of BrdU for the rats was 2
mg per 100 g body weight[5]. All tissue specimens were removed
under anesthesia, then fixed and embedded in paraffin. The
paraffin-blocked section was consecutively cut at 5
μm thickness for iron hematoxylin-eosin (HE) and
immunohistochemical staining.
Cell proliferation assay Cell proliferation was determined
using BrdU. BrdU was detected using
immunohistochemical staining as described. After deparaffination and
rehydration of the sections, slides were placed in sodium citrate
solution (0.01 mol/L, pH 6.0) and heated to 96_100
oC for 25 min. After cooling, the sections were put into 5% bovine
serum albumin for 20 min. Then the sections were incubated
for 2 h with a primary anti-BrdU mouse monoclonal antibody
(Boster Biotechnology, Wuhan, Hubei, China), diluted at
1:100 in Tris-buffered saline[6], and then covered with
coverslips. In the sections, positive staining was a red
precipitate localized in the nucleus. Stained BrdU cells were
counted under a light microscope. The sections of obtained
tissues (prostate, liver, skin, jejunum, and colon) were
examined in randomly selected areas. No less than 1000 cells
were viewed in each tissue per animal.
Orientation of mitoses In the tissue sections stained with
iron HE, mitotic figures were viewed under a light
microscope and the orientation of mitosis was determined.
Mitoses with the poles disposed from 0 to 45 degrees to the
basement membrane of prostate epithelia were regarded as
parallel oriented. Mitoses with poles aligned from 45 to 90
degrees to the plane of the basement membrane were
considered as perpendicular oriented. In the prostate, only the
prostate epithelial cells were examined.
Statistics Data were expressed as mean±SD. One-way
ANOVA and χ2-test were used to evaluate significant
differences between the groups.
Results
Iron HE staining of rat prostates The prostate of all of
the castrated rats had typically atrophic morphology. At 24,
36, and 48 h after a single injection of TP, a picture of early
proliferative phenomenon was observed. Mitoses were rarely
found in the prostate epithelial cells of castrated rats.
Therefore, the percentage of mitotic cells was very low.
However, a single injection of TP or E2 increased the
proliferative activity of the prostate. All mitoses found in the prostate
epithelial cells of the castrated rats with TP treatment were
oriented parallel to the basement membrane (Figure
1), but mitoses found in the prostate epithelial cells of the castrated
rats untreated or treated with E2 were oriented perpendicular
to the basement membrane (Figure 1). TP treatment resulted
in marked changes in mitosis orientation in the prostate
epithelial cells of the prostate. TP treatment resulted in marked
changes in mitosis orientation in the prostate epithelial cells.
At 24, 36, and 48 h after TP injection, 7.1%, 7.9%, and 7.5%
cells' mitosis orientation were aligned parallel to the
basement membrane of the epithelial cells in all prostate epithelial
cells, respectively. However, in other tissues (liver, jejunum,
colon, and skin), TP or E2 treatment had no effect on the
orientation of cell division.
Immunohistochemical staining The BrdU-labeled cells
were specific for abnormal
hyperplasia[7]. A number of positive cells were observed in the areas of prostate epithelial
cells and stroma, which correlated with the degree of
proliferation. No positive cells were observed in the
untreated group (control group; Figure 2). At 24, 36, and 48 h
after a single injection of TP, the BrdU-labeled positive cells
could be seen throughout the stroma and epithelial cells of
the prostate (Figure 2); however, at 24, 36, and 48 h after a
single injection of E2, the positive cells could only be seen in
the stroma of the prostate (Figure 2). At 24, 36, and 48 h after
TP injection, BrdU-labeled positive cells were found not only
in the stroma of the prostate, but also in the prostate
epithelial cells (Figures 3, 4), and there were significant differences
between the control and TP groups (P<0.01). In the rats
treated with E2 after 24, 36, and 48 h, BrdU-labeled positive
cells were only found in the stroma of the prostate (Figure 5)
and there was significant differences between the control
and E2 groups (P<0.01). However, BrdU-labeled cells were
hardly seen in other tissues (liver, jejunum, colon, and skin)
treated with TP or E2 (Figure 2).
Discussion
The present study shows a novel effect of TP. TP
remarkably altered the mitosis orientation in the prostate epithelial
cells. A single injection of TP increased the proliferation and
appearance of parallel-oriented mitoses. The effect of TP on
mitosis orientation has also been examined in other tissues,
such as liver, jejunum, colon, and skin tissues through
immunohistochemical examination and iron HE staining. No
noticeable effects were found in these tissues. Therefore,
TP may only affect the mitosis orientation in certain organs.
In this case, the effect of TP on mitosis orientation might be
considered specific for target organs, at least for the prostate.
Both TP and E2 induced proliferation in the prostate, but
the results were different. The BrdU-labeled cells were found
in both the epithelia and stroma of the prostate in TP-treated
rats. BrdU-labeled cells were only found in the stroma of the
prostate in E2-treated rats. Androgens could affect not only
the stroma, but also the epithelia; however, the roles of other
steroids, especially estrogens, are still unknown. Bektic
et al[8] first reported the effects of estrogen on global gene
expression in human prostatic stroma cells. Scarano
et al[9] described the effects of chronic estradiol treatment on guinea
pig prostatic stroma. The present study found that a single
injection of E2 could cause proliferation of prostatic stroma.
To our knowledge, it is has not reported that TP can
cause changes in mitosis orientation in prostate epithelial
cells. Although the underlying mechanism is not fully
understood, the effects are most likely related to the
androgen receptors (AR), because mitosis orientation was not
observed in the control or E2-treated groups. Androgens
enhanced the transcription of genes involved in cellular
proliferation by binding and activation, such as the mitogenic
growth factors epidermal growth factor and insulin-like
growth factor-I[10]. This process may be related to the Wnt
signaling pathway, which controls mitosis
orientation[10,11]. Moreover, a SHya may be a useful biomaterial to regulate
Wnt signaling in tissue engineering[12]. In addition, there
was significant crosstalk between these signaling pathways
in the regulation of proliferation and differentiation in the
prostate cells. For example, β-catenin is not only utilized in
the prostate epithelium as an important co-activator of AR
signaling[13], but also the key component in the Wnt
signaling pathway[14]. Therefore, Wnt signaling,
β-catenin, and the crosstalk with androgen signaling and other pathways
may help us understand the process. Therefore, further studies
are needed to explain the mechanism and these
observations, and finally, for treating androgen-dependent diseases such
as BPH and PCa.
Acknowledgements
We would like to thank Gui-lin HE, Xiu-rong JIANG,
Gui-ming LIU, Shu-wu XIE, Zhi-ling LI, Li MA, Wen-juan HU,
and Ying LU for technical assistance.
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