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Endocrinology, doi:10.1210/en.2007-1413
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Endocrinology Vol. 149, No. 9 4421-4427
Copyright © 2008 by The Endocrine Society

Estrogen Receptor-{alpha} Signaling in Growth of the Ventral Prostate: Comparison of Neonatal Growth and Postcastration Regrowth

Yoko Omoto

Department of BioSciences and Nutrition, Karolinska University Hospital, Huddinge, Karolinska Institutet, SE-14186 Huddinge, Sweden

Address all correspondence and requests for reprints to: Yoko Omoto, Department of BioSciences and Nutrition, Karolinska University Hospital, Huddinge, Karolinska Institutet, SE-14186 Huddinge, Sweden. E-mail: yoko.omoto{at}mednut.ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A role for estrogen receptor (ER)-{alpha} in branching morphogenesis in the ventral prostate (VP) has previously been demonstrated; in the VP of ER{alpha}–/– mice, there are fewer side branches than in wild-type littermates. In the present study, we show that in the postnatal VP, fibroblast growth factor 10 (FGF10) is expressed in wild-type mice but not in ER{alpha}–/– mice, and because branching involves proliferation pathways also used in malignant growth, we investigated whether branching during regrowth of the VP after castration involves ER{alpha} and FGF10. ER{alpha} was not detectable in the prostates of sham-operated or castrated mice but was expressed in the prostatic epithelium between d 3 and 5 after testosterone replacement. Blocking either ER{alpha} or ERβ with ICI 182,780 had no detectable effects on epithelial cell proliferation during regrowth by testosterone. The ER{alpha} agonist, propylpyrazoletriol, did not induce regrowth by itself, but exposure to propylpyrazoletriol on d 3–5 of testosterone replacement resulted in cyclin D1-positive cells in the ductal epithelium, invasion of FGF10-positive immune cells in the regrowing prostate, and budding 14 d later. Testosterone replacement alone did not induce cyclin D1, FGF10, or bud formation. These results indicate that stimulation of ER{alpha} is essential for ductal branching during postnatal prostate growth. During regrowth after castration, there is a window in time when selective stimulation of ER{alpha} can also induce ductal branching. The FGF10 for this growth comes from the immune system, not from the prostatic mesenchyme.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GROWTH AND DEVELOPMENT of the prostate are regulated by androgen and estrogen and their receptors, androgen receptor (AR) and estrogen receptors (ERs), ER{alpha} and ERβ (1, 2, 3, 4, 5). The prostate requires androgenic steroids for its embryological formation and development and postpubertal growth. Androgens are not only ligands for AR but also precursors of estrogens; 17β-estradiol (E2) is produced from testosterone (T) and 5{alpha}-androstane-3β,17β-diol (3βAdiol) from 5{alpha}-dihydrotestosterone (DHT) (6, 7). There is evidence that 3βAdiol rather than E2 is the most abundant estrogen in the ventral prostate (VP). The adult prostate remains dependent on a continuous supply of androgens for its function. Reduction of androgen levels rapidly induces apoptosis of the prostate epithelium, leading to extensive glandular regression (8). In the adult prostate, ER{alpha} is present in some stromal cells but not in epithelial cells, whereas ERβ is present in epithelial cells. In several tissues, including the prostate (5, 9, 10), mammary gland (11, 12), and colon (13, 14), ER{alpha} appears to mediate the proliferative functions of E2, whereas ERβ represses proliferation and mediates differentiation. ER{alpha} has key functions in prostate development; exposure of estrogenic agents during the fetal and/or neonatal period predisposes mice to prostatic intraepithelial neoplasia and prostate cancer in older males (15, 16, 17). This early developmental effect, which leads to permanent programming of the gland, is called imprinting. Imprinting is totally dependent on ER{alpha} and does not occur in ER{alpha}–/– mice (18).

Ductal branching morphogenesis in the prostate gland involves participation of both the epithelial and mesenchymal compartments and is influenced by steroid and peptide growth factors and their receptors (19, 20). Estrogen signaling is associated with branching morphogenesis. ER{alpha} is important for branching morphogenesis in various epithelial tissues, e.g. mammary glands and lung. Recently, we have reported that ER{alpha} is present in the prostate epithelium in mice during wk 2–4 of postnatal life and suggested that ER{alpha} is involved in branching morphogenesis of the ventral prostate. Fibroblast growth factor 10 (FGF10) is one of the key morphogens in the prostate mesenchyme (21, 22, 23). In the present study, we investigated whether ER{alpha} and FGF10 are involved in T-induced prostatic regrowth after castration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
C57BL/6J wild-type (wt) mice from our colony were used. ER{alpha}–/– mice on a C57BL/6J background were purchased from Taconic (Ry, Denmark). These mice were housed in Huddinge University Hospital Animal Facility in a controlled environment with an illumination schedule of 12 h light and 12 h dark under the Guideline for Care and Use of Experimental Animals issued by Stockholm’s Södra Djurförsöksetiska Nämnd. They were fed a standard pellet diet with water provided ad libitum. Animals were asphyxiated by CO2, and tissues were fixed in 4% paraformaldehyde for immunohistochemical studies.

Chemicals and antibodies
Testosterone propionate was purchased from Sigma (Stockholm, Sweden), the ER{alpha} agonist propylpyrazoletriol (PPT) (24, 25) and ICI 182,780 (ICI) (26) from Tocris (Ellisville, MO), T-releasing pellets (1.5 mg/21 d) from Innovative Research of America (Sarasota, FL), and bromodeoxyuridine (BrdU) from Roche (Mannheim, Germany). The polyclonal anti-ER{alpha} (MC-20), anti-AR (N-20), and anti-FGF10 (C-17) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-BrdU antibody was from PharMingen (BD, Franklin Lakes, NJ), anti-cyclin D1 (Ab-3) antibody was from Oncogene (Boston, MA), and biotinylated conjugated secondary antibodies were from Vector Laboratories (Burlingame, CA). Chicken polyclonal anti-ERβ503 antibody was produced in our laboratory (12).

Operation and treatment procedure
Castration was performed via scrotal incision while mice were under Avertin anesthesia. Implantation of T-releasing pellets under the backside of neck skin was performed using a precision trochar. Hormones, e.g. T or PPT, were delivered by sc injection once daily for the time periods indicated. BrdU was given by ip injection (100 mg/kg) 2 h before killing. There were three mice in each treatment group.

Whole-mount analysis of prostate
VP was dissected into chilled Hanks’ balanced salt solution (Ca and Mg free) under a microdissection microscope. The dissected VPs were incubated in 0.5% collagenase in Hanks’ balanced salt solution for 30 min at 37 C to remove stroma. The ducts of VP were carefully unfolded on a glass slide. The specimens were then dried completely and mounted after staining with 4% Giemsa solution overnight and washed with 1% acetic acid in 95% ethanol.

Immunohistochemical staining
Representative blocks of paraffin-embedded tissues were cut in 4 µm thickness, dewaxed, and rehydrated. Antigens were retrieved by microwaving in 10 mM citrate buffer (pH 6.0) for 15 min. The sections were incubated in 0.5% H2O2 in PBS for 30 min at room temperature to quench endogenous peroxidase and then incubated in 0.5% Triton X-100 in PBS for 15 min. For BrdU staining, sections were additionally incubated in 2 M HCl for 10 min and in solution mixed equally with 0.05 M sodium tetraborate (pH 8.5) and 0.05 M NaCl in 0.2 M boric acid for 15 min at room temperature after blocking endogenous peroxidase activity and then incubated in 0.5% Triton X-100 in PBS for 5 min at room temperature. To block the nonspecific binding, sections were incubated with BlockAce (Dai-Nippon Pharmaceutical, Osaka, Japan) for 20 min at room temperature. Then sections were incubated with the following antibodies: anti-ER{alpha} (1:200), anti-ERβ (1:100), anti-AR (1:300), anti-FGF10 (1:50), anti-cyclin D1 (1:20), and anti-BrdU (1:100) in PBS overnight at 4 C. After washing, sections were incubated with the corresponding secondary antibodies (all in 1:200 dilutions) for 1 h at room temperature. The Vectastain ABC kit (Vector) was used for the ABC method following the manufacturer’s instructions. Peroxidase activity was visualized with 3,3'-diaminobenzidine (Dako, Glostrup, Denmark). The sections were lightly counterstained with hematoxylin. Negative controls were incubated without primary antibody.

Counting of positive staining
From the sections stained by anti-BrdU antibody, six individual views from each mouse sample under the microscope (x400) and then the total number of epithelial cells and the number of positive cells were counted in the field. Additional areas were chosen if the total cell number per mouse did not reach 1000. The data are expressed as a percentage of BrdU-positive epithelial cells.

Antigen absorption
FGF10 blocking peptide (sc-7375P), from Santa Cruz, was incubated with activated CH Sepharose at 4 C overnight. The Sepharose pellet was recovered by centrifugation and washed with PBS. Then anti-FGF10 antibody was incubated with FGF10-coupled Sepharose gel at 4 C overnight to remove FGF10-interacting antibodies.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FGF10 expression in postnatal prostate of wt and ER{alpha} knockout mouse
FGF10 was visualized by immunohistochemistry in VP of postnatal mice from 1 to 5 wk of age. Weak staining was observed in cytoplasm of mesenchymal cells in stroma at 1 wk of age (Fig. 1AGo). Staining in the mesenchymal cells became stronger at 2 wk (Fig. 1BGo), weakened in 3-wk-old mice (Fig. 1CGo) and disappeared after 4 wk of postnatal life (Fig. 1Go, D and E). In epithelial cells, staining was not observed in 1-wk-old mice (Fig. 1AGo). It became detectable at wk 2 of postnatal life (Fig. 1BGo) increased in 3-wk-old mice (Fig. 1CGo) and 4-wk-old mice (Fig. 1DGo) and disappeared by the time the mice were 5 wk old (Fig. 1EGo). FGF10 could not be detected in either stromal or epithelial cells in 1-wk-old (Fig. 1FGo) and 2-wk-old (Fig. 1GGo) ER{alpha}–/– mouse VP.


Figure 1
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  		FIG. 1. Immunohistochemical staining of FGF10 in neonatal wt and ER{alpha}–/– mouse VP. A–E, The VPs of postnatal mice from 1–5 wk of age were probed with an FGF10 antibody. Weak staining was observed in cytoplasm of mesenchymal cells in stroma in 1-wk-old mice (A). Staining was strong at 2 wk (B), weakened at 3 wk (C), and disappeared from the VP after 4 wk (D and E). In epithelial cells, staining could be observed from 2 wk (B), was intense at wk 3 (C) and 4 (D), and disappeared at wk 5 (E). F and G, FGF10 staining in 1-wk-old (F) and 2-wk-old (G) ER{alpha}–/– mouse VP. There was no detectable FGF10 staining of VP.

 
Ductal branching morphology in wt and ER{alpha} knockout mouse
There are clear differences in the structure of the VP between 1-yr-old ER{alpha}–/– mice and their wt littermates. The ER{alpha}–/– mouse VP has fewer side branching, and many branches sprout from single points (Fig. 2Go, C and D), whereas wt VP has many distinct branching points (Fig. 2Go, A and B) (Fig. 2Go, B and D, shows the scheme of branching of Fig. 2Go, A and C, respectively).


Figure 2
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  		FIG. 2. Ductal branching morphology in 1-yr-old ER{alpha}–/– mouse VP. A and B, Whole mounts of VP from wt (A) and scheme of branching structure (B); C and D, whole mount of VP from ER{alpha}–/– mice (C) and schematic view of its branching (D). The branch of ER{alpha}–/– VP looks like a broom, has primary branch, but not secondary, and budding from tip only, whereas wt VP has primary and secondary branches.

 
Changes in ductal length and structure by castration and T replacement
By 14 d after castration, the distal and lateral ducts in the VP were shorter (Fig. 3CGo) than that seen in sham-operated mice (Fig. 3AGo). This change was not as pronounced 9 d after castration (Fig. 3BGo). The number of ducts and overall ductal structure were not changed by castration at 9 (Fig. 3FGo) and 14 d after castration (Fig. 3GGo) (Fig. 3Go, F and G, shows the scheme of branching of Fig. 3Go, A and C, respectively). Castrated mice were treated with T by daily sc injection. As expected, T replacement after castration caused rapid growth and morphological restoration of the VP. Treatment with T for 6 d after 9 d of castration (Fig. 3DGo) or for 14 d after 14 d of castration (Fig. 3EGo) totally restored the length of ducts but had no effect on ductal structure (Fig. 3HGo; the scheme of branching of Fig. 3EGo).


Figure 3
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  		FIG. 3. Morphological change of mouse VP by castration and T replacement. A–E, Whole mount of VP 9 d after sham operation (A), 9 d after castration (B), 14 d after castration (C), 6 d of T treatment after 9 d of castration (D), and 14 d of T pellet implantation after 14 d of castration (E). Ductal length was shortened by 14 d of castration and returned to normal by T replacement. All pictures are shown in the same scale. One graduation represents 1 mm in the picture. F–H, Schematic view of ductal branching 9 d after sham operation (F), 14 d after castration (G), and 14 d after T pellet implantation after 14 d of castration (H), which were identical samples to A, C, and E, respectively. Although ductal length was shortened by castration and recovered by T replacement, the number of ducts and overall ductal structure were not changed by castration and T replacement.

 
Expression of AR, ERβ, and ER{alpha} during regrowth
AR is present in luminal cells, and ERβ is present in both luminal and basal cells of the epithelium of adult mouse VP. Nine days after castration, expression of both AR and ERβ in the mouse VP was reduced to levels barely detectable above background (Fig. 4Go, A and B). Both AR and ERβ staining returned 1 d after T replacement, and intensity of staining was as strong as in a normal prostate after 2 d of T replacement (Fig. 4Go, A and B). AR staining was observed in some basal cells 2–6 d after castration (Fig. 4AGo). ER{alpha} was not present in epithelial cells of VP of intact mice or sham-operated mice or in mice 9 d after castration. However, on d 3–5 after T replacement began, ER{alpha} was expressed in the luminal epithelium. Staining intensity was highest at 5 d after treatment but diminished to undetectable levels by d 6. No ER{alpha} was detected in basal cells (Fig. 4CGo).


Figure 4
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  		FIG. 4. Immunohistochemical staining of AR and ERs in mouse VP. VP was obtained from sham-operated mice, from mice 9 d after castration, and from mice that had T treatment (2.5 mg/kg·d) from d 1–6 after 9 d of castration. A and B, AR (A) or ERβ (B) staining of VP from sham-operated mice, from mice 9 d after castration, and from castrated mice 1, 2, 4, or 6 d after T treatment. Strong nuclear staining was observed in sham-operated mouse VP. Although this staining disappeared after castration, it was recovered after T treatment. C, ER{alpha} staining in VP from sham-operated mice, mice 9 d after castration, and castrated mice 1–6 d after T treatment. No staining was observed in either sham-operated mice or mice 9 d after castration. However, after 3–5 d of T replacement, ER{alpha} was present in nuclei of luminal cells.

 
Epithelial cell proliferation during regrowth
Nine days after castration, each treatment was begun. VPs were removed from these mice for analysis every day for 6 d of treatment. BrdU was administered to each mouse 2 h before killing. Sections of VPs were stained using BrdU antibody, and the number of positively staining cells was counted as we described in Materials and Methods. The combination of mouse and treatment was wt mice with T (2.5 mg/kg·d), wt mice with T (2.5 mg/kg·d) plus ICI (2.5 mg/kg·d), and ER{alpha}–/– mice with T (2.5 mg/kg·d). The percentages of BrdU-positive cells in the epithelium of VPs were 0.1 ± 0, 1.5 ± 0.3, 6.1 ± 0.7, 12.9 ± 1.0, 8.0 ± 0.5, and 4.6 ± 0.2% (mean ± SD) at 1–6 d after T treatment in wt mice (Fig. 5AGo). The wt mice treated with a combination of T plus ICI showed 0 ± 0, 3.4 ± 0.2, 9.9 ± 1.5, 10.8 ± 2.5, 5.4 ± 1.0, and 3.4 ± 0.4%, respectively (Fig. 5BGo). ER{alpha}–/– mice, treated with T showed 0 ± 0, 1.2 ± 0.7, 5.6 ± 1.5, 8.5 ± 3.4, 3.6 ± 1.1, and 2.3 ± 1.5%, respectively (Fig. 5CGo).


Figure 5
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  		FIG. 5. Epithelial cell proliferation during regrowth. Nine days after castration, treatment was begun. VPs were removed from these mice for analysis every day for 6 d of treatment. BrdU was administered to each mouse 2 h before killing. Sections of VPs were stained using BrdU antibody, and the percentage of BrdU-positive cells in the epithelium of VPs was counted as indicated in Materials and Methods. Columns, means; bars, SD. A, The wt mice treated with T (2.5 mg/kg·d) were 0.1 ± 0, 1.5 ± 0.3, 6.1 ± 0.7, 12.9 ± 1.0, 8.0 ± 0.5, and 4.6 ± 0.2% (mean ± SD) at 1–6 d after treatment. B, The wt mice treated with a combination of T (2.5 mg/kg·d) plus ICI (2.5 mg/kg·d) showed 0 ± 0, 3.4 ± 0.2, 9.9 ± 1.5, 10.8 ± 2.5, 5.4 ± 1.0, and 3.4 ± 0.4%, respectively. C, ER{alpha}–/– mice, treated with T (2.5 mg/kg·d) showed 0 ± 0, 0.7 ± 1.2, 5.6 ± 1.5, 8.5 ± 3.4, 3.6 ± 1.1, and 2.3 ± 1.5%, respectively.

 
Effects of ER{alpha} stimulation on d 3, 4, and 5 after T replacement
To examine the function of ER{alpha} in prostate regrowth, mice were treated with the ER{alpha}-specific agonist PPT on d 3, 4, and 5 after T stimulation. In these experiments, mice received T by T-releasing pellets from 14 d after castration. Three days after implantation of the T-releasing pellet, mice were administered PPT (5 mg/kg) or vehicle for 3 d. Fourteen days after the start of T treatment, mice were killed to observe branching morphology and histology. There were three mice in each group.

Morphological observations revealed that the recovery of length of branches was similar in mice that were injected with T and in mice that received the T-releasing pellet. Regrowth was similar in mice treated with T alone (Fig. 6AGo) and in those with T plus PPT (Fig. 6BGo). The difference between the two groups was the development of many small buds in T- plus PPT-treated mice (Fig. 6BGo) but not in mice treated only with T. There was strong positive cytoplasmic staining for FGF10 in the epithelium in T- plus PPT-treated mice (Fig. 6DGo) but not in T-treated mice (Fig. 6CGo). This strong staining was mainly observed in the peripheral ducts of prostate. The specificity of the immunostaining was confirmed by abolition of the signal upon antigen preabsorption of the antibody with FGF10 (Fig. 6EGo). In the T- plus PPT-treated mice but not in the T-treated mice, there were many infiltrating FGF10-positive lymphocytes (Fig. 6Go, F and G, red arrow). No FGF10 signals were observed in the stromal cells. Many cyclin D1-positive cells could be observed in the thick layer of epithelial cells in T- plus PPT-treated mice (Fig. 6Go, I and J); however, there was very little staining in cells in T-only-treated mice (Fig. 6HGo).


Figure 6
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  		FIG. 6. FGF10 and cyclin D1 expression in regrowing mouse VP. A and B, Ductal branching morphology 14 d after T-releasing pellet implantation (A) and after PPT treatment on d 3, 4, and 5 after T-releasing pellet implantation (B). The areas indicated by red circles are side branching formations, shown in small panels on a larger scale. The side branching was not observed in control mice. C–E, FGF10 staining in a sample from T-treated mice (C) and T- plus PPT-treated mice (D). Negative controls were samples stained with antibody after preadsorption with FGF10 (E). Strong FGF10 staining was observed in T- plus PPT-treated mice. F and G, FGF10 staining in a samples from T- plus PPT-treated mice. Red arrows show FGF10- positive infiltrating lymphocytes in the stroma. H–J, Cyclin D1 staining in VP from T-treated mice (H) and T- plus PPT-treated mice (I and J). Many cyclin D-positive cells can be seen in the multilayered epithelial cells in T- plus PPT-treated mice. However, only a few stained cells were seen in T-treated mice. Red arrows show representative stained cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Steroid hormone receptors are important for growth and development in hormone-sensitive organs, including the prostate. In the normal adult rodent VP, AR and ERβ are expressed in prostatic epithelial cells and ER{alpha} in stroma (1, 3, 27, 28, 29). In the early postnatal VP, ER{alpha} is abundant for a brief period in the epithelium of the mouse VP. There were high levels of ER{alpha} in almost all stromal cells in VP in 1-wk-old mice. Expression declined after wk 1, and by wk 2, very few stromal cells were ER{alpha} positive. In the epithelium, ER{alpha} was not detectable in the first week of life, but both ER{alpha} and ERβ were expressed in the VP epithelium of 2- and 3-wk-old-mice. After 3 wk of age, ER{alpha} levels declined, and no ER{alpha}-positive epithelial cells were detectable by wk 4 (30). This neonatal and postnatal period is the most crucial time for morphogenesis in the prostate. It was reported that during the first 15 d after birth, 80.7% of tips and 76.4% of branch points of the adult gland formed in the VP, and ductal branching was completed by 60–90 d (31).

The morphology of young ER{alpha}–/– mouse prostate has been described previously (30). In the absence of ER{alpha}, the number of secondary branches is reduced and ducts are extremely dilated and surrounded by very loose stroma. In the present study, in VP of 1-yr-old ER{alpha}–/–, mice there are clearly fewer branching points than in wt littermates (Fig. 2Go). These data suggest that ER{alpha} has a function in branching morphogenesis of the VP. In the mammary gland, ductal morphogenesis is clearly under ER{alpha} control, and branching does not occur in ER{alpha}–/– mammary glands (32, 33). In FGF10–/– mice, most of the male secondary sex organs are absent or atrophic, including the prostate, seminal vesicles, bulbourethral gland, and caudal ductus deferens (22). Therefore, both ER{alpha} and FGF10 are thought to be important for prostatic branching morphogenesis. In the present study, we found that FGF10 is present in stromal cells around VP buds in 1-wk-old mice. Between 2 and 4 wk of age, FGF10 was detected in epithelial cells (Fig. 1Go, B–D). Because this expression pattern is similar to that of ER{alpha}, the question arose as to whether ER{alpha} takes part in branching morphogenesis together with FGF10.

Previous studies using animal models have reported that castration causes prostatic atrophy. Sugimura et al. (34) reported that ductal tips and branch points were lost 14 d after castration and were recovered after T replacement. Our present study shows that the fundamental structure of ductal branching was not much changed by castration but that all ducts were reduced in size. As expected, the hormone-depleted prostate was very sensitive to T and showed rapid regrowth and restoration of branches after T replacement (Fig. 3Go).

In the prostate, T is the precursor of both a potent androgen, DHT, and the estrogens E2 and 3βAdiol, which can stimulate both ER{alpha} and ERβ. In the prostate, aromatase activity is quite low (35, 36, 37), and there is evidence that 3βAdiol is a physiological ligand of both ER{alpha} and ERβ (5, 7). Expression of AR is reduced upon activation of ERβ with 3βAdiol (5, 38) and increased by activation of ER{alpha} (30). To evaluate the role of ERs in regrowth of the prostate, we used selective ER{alpha} and ERβ agonists PPT and diarylpropionitrile (39). Neither of these agents alone stimulated regrowth of the castrated prostate (data not shown). Therefore, as expected, stimulation of AR is necessary for regrowth. However, T replacement might provide ERs with ligands that stimulate ERs in prostate. To investigate the function of ERs on proliferation of epithelial cells, we used wt mice with T and ICI treatment and ER{alpha}–/– mice with T treatment and compared with wt mice with T treatment (Fig. 5Go). The pattern of cell proliferation was quite similar in these three groups; with most of the proliferation occurring during 3–5 d after treatment. Inhibition of ERs after activation of AR had no clear effect on epithelial cell proliferation.

In the T-replaced prostate after castration, ER{alpha} was briefly and abundantly expressed in the luminal epithelium on d 3–5 of T treatment (Fig. 4CGo). Staining intensity was highest at d 5 of treatment and thereafter diminished to undetectable levels after d 6. No ER{alpha} was detected in the basal cell layer (Fig. 4CGo). To investigate the effect of ER{alpha} on androgen- induced proliferation, we replaced castrated mice with T for 2–3 d and then added an ER{alpha} agonist to the regimen. Treatment with PPT for 3 d after 2 d of T treatment resulted in formation of small side branching points that were most prominent 2 wk after starting PPT treatment (Fig. 6BGo). This result indicates that there is a window in time after androgen replacement of castrated mice when ER{alpha} can induce branching morphogenesis. In T-replaced mouse prostates, 3βAdiol should be present, and this could lead to activation of both ERs. PPT treatment targets ER{alpha} specifically and does not activate ERβ. Thus, it appears that ER{alpha} induces proliferation, but when both receptors are activated, ERβ opposes the proliferative activity of ER{alpha}.

Side branch formation was observed in whole mounts of VP in T- plus PPT-treated regrowing prostate but not in T-treated prostate (Fig. 6Go, A and B). Although it is difficult to identify side branching in the paraffin-embedded sections, places with thick epithelial layers that were positive for FGF10 (Fig. 6DGo) and cyclin D1 (Fig. 6Go, I and J) were considered to be side branching points. Cyclin D1, the marker of nuclear DNA synthesis and considered as a proliferation marker, was not observed in VP of mice 14 d after T-only treatment (Fig. 6HGo). By this time, regrowth was completed. These data showing that FGF10-positive and cyclin D1-positive epithelial cells could be observed in T- plus PPT-treated prostate indicate that stimulation of ER{alpha} increases proliferation of stem cells and induces FGF10 localization, resulting in side branch formation.

Development of the male reproductive tract is dependent upon androgens and mesenchymal-epithelial interactions. It has been shown that it is the mesenchymal ARs, not the epithelial AR, which are essential for prostatic ductal development (40). This has led to the hypothesis that paracrine factors, which are produced by the mesenchyme and regulated by androgens, control the development of the male reproductive tract. Two FGFs, FGF7 and FGF10, promote prostatic growth and branching morphogenesis. Both of these growth factors are secreted from mesenchymal cells and act on their receptor, FGFR2iiib, which is exclusively epithelial (41, 42, 43). In adult mouse VP, there are very few stromal cells. The source of the FGF10 appeared to be FGF10-positive lymphocytes that infiltrated the stroma in T- plus PPT-treated but not in T-only-treated mice (Fig. 6Go, C, D, F, and G). The stromal cells themselves were negative for FGF10. Infiltrating T lymphocytes are known to provide FGF7 and FGF10 to local target cells in psoriasis (44). {gamma}{delta} T lymphocytes are believed to play a role in maintaining the normal configuration of epithelial tissue by providing FGF9 (45). These infiltrating lymphocytes are the source of FGF10, which stimulates reconstruction of the gland. Because FGF10 does not appear to be regulated by androgens (23), we suggest that the ER{alpha} agonist activated lymphocytes and induced their infiltration into VP. It is possible that ER{alpha} increased expression of FGFR2iiib, but this idea could not be tested because no specific antibodies to detect FGFR2iiib are commercially available.

FGF10 was observed at 9 d after PPT stimulation, a time when prostate side branching were evident. From both the estrogen-induced prostate imprinting of the neonate (15, 16, 17) and the castration-regrowth model, estrogen via ER{alpha} appears to be essential for induction of FGF10 and for prostate branching.


   Acknowledgments
 
I thank Prof. Jan-Åke Gustafsson and Margaret Warner for valuable suggestions and discussion and Jóse Inzunza for managing the ER{alpha}–/– mice.


   Footnotes
 
This study was supported by grants from the Swedish Research Council, Karolinska Institutet Research Grant, the Swedish Cancer Fund, the European Commission-funded PIONEER STREP (FOOD-CT-2004-513991) and KaroBio AB.

Disclosure Statement: The author has nothing to disclose.

First Published Online June 5, 2008

Abbreviations: 3βAdiol, 5{alpha}-Androstane-3β,17β-diol; AR, androgen receptor; BrdU, bromodeoxyuridine; DHT, 5{alpha}-dihydrotestosterone; E2, 17β-estradiol; ER, estrogen receptor; FGF10, fibroblast growth factor 10; ICI, ICI 182,780; PPT, propylpyrazoletriol; T, testosterone; VP, ventral prostate; wt, wild type.

Received October 15, 2007.

Accepted for publication May 23, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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