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Endocrinology Vol. 144, No. 6 2269-2278
Copyright © 2003 by The Endocrine Society

Prostate Hyperplasia in a Transgenic Mouse with Prostate-Specific Expression of Prolactin

Jon Kindblom, Karin Dillner, Lena Sahlin, Fiona Robertson, Christopher Ormandy, Jan Törnell and Håkan Wennbo

Department of Physiology (J.K., K.D., J.T., H.W.), Göteborg University, Göteborg 405 30, Sweden; Department of Molecular Medicine (K.D.), Karolinska Institutet, Stockholm 171 77, Sweden; Department of Woman and Child Health (L.S.), Karolinska Hospital, Stockholm 171 76, Sweden; Cancer Research Program (F.R., C.O.), Garvan Institute of Medical Research, Sydney 2010, Australia; and Astrazeneca Transgenic Center (J.T.) and Integrative Pharmacology (H.W.), Astrazeneca R&D Mölndal, S-431 83 Mölndal, Sweden

Address all correspondence and requests for reprints to: Jon Kindblom, M.D., Department of Physiology, Box 434, Göteborg University, S-405 30 Göteborg, Sweden. E-mail: jon.kindblom{at}medic.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prolactin (PRL) is one of several polypeptide factors known to exert trophic effects on the prostate. We have previously reported a dramatic prostate enlargement with concurrent chronic hyperprolactinemia and elevated serum androgen levels in a PRL transgenic mouse (Mt-PRL) with ubiquitous expression of the transgene. To address the role of local PRL action in the prostate, a new transgenic mouse model (Pb-PRL) was generated using the prostate-specific rat probasin (Pb) minimal promoter to drive expression of the rat PRL gene. Pb-PRL transgenic males developed a significant enlargement of both the dorsolateral and ventral prostate lobes evident from 10 wk of age and increasing with age. Expression of the transgene was restricted to the prostate and detected from 4 wk of age. Low levels of transgenic rat PRL were detectable in the serum of adult Pb-PRL animals. Serum androgen levels were normal. The Pb-PRL prostate displayed significant stromal hyperplasia, ductal dilation, and focal areas of epithelial dysplasia. Quantitative analysis of prostatic tissue cellularity demonstrated a marked increase in the stromal to epithelial ratio in all lobes of Mt-PRL and Pb-PRL transgenic prostates compared with controls. Microdissections demonstrated an increased ductal morphogenesis in dorsolateral and ventral prostate lobes of Mt-PRL prostate vs. Pb-PRL and controls. In conclusion, this study indicates the ability of PRL to promote, directly or indirectly, ductal morphogenesis in the developing prostate and further to induce abnormal growth primarily of the stroma in the adult gland in a setting of normal androgen levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PROLACTIN (PRL) hormone was first discovered more than 70 yr ago. It was found to be of pituitary origin, with stimulatory effects on mammary gland development and lactation. A multitude of additional physiological effects have since been attributed to activation of the PRL receptor (PRLR) in various species (1, 2). The membrane-bound PRLR is a member of the cytokine receptor superfamily and is expressed in virtually all organs and tissues. Recent lessons learned from the PRL-related knockout models have mainly proved its irreplaceable role in functions of lactation and reproduction (3, 4, 5). The lack of other major phenotypic features suggests that most of its other reported target tissues are presumably modulated by, rather than strictly dependent on, PRL. Although the large majority of circulating PRL is of pituitary origin, the existence of extrapituitary PRL production in several peripheral tissue types, including the prostate gland (6, 7), uterus (8), ovaries (9), and myeloid leukemic cells (10), as well as normal (11, 12) and malignant (13, 14) mammary gland epithelium, is now well established in several species (15). These observations have initiated new efforts to further the understanding of whether local production of PRL is involved in human pathophysiology.

Over the last three decades, numerous in vivo studies have demonstrated diverse PRL effects on the prostate gland. These chiefly include actions related to proliferative activity, secretory function, and regulation of specific metabolic functions (16, 17). We have previously generated a transgenic mouse (Mt-PRL) with ubiquitous expression of a rat PRL (rPRL) transgene under control of the metallothionein I gene (Mt-1) promoter. The Mt-PRL transgenic males were shown to develop a progressive enlargement of the prostate gland (18). The Mt-PRL males exhibited hyperprolactinemia and elevated serum androgen levels. The presence of rPRL and endogenous mouse PRL, as well as PRLR expression, was detected locally in the prostate gland. Further studies of the Mt-PRL animals revealed that the elevated levels of circulating androgen in the adult male were not necessary for the abnormal prostate growth (19). In addition, androgen treatment of wild-type males of this strain, C57BL/6J x CBA, was found not to significantly affect growth of the adult prostate (19). A separate study of differentially expressed transcripts in the Mt-PRL transgenic prostate, using representational difference analysis, indicated some molecular similarities between the prostate hyperplasia in PRL transgenic mice and benign prostate hyperplasia (BPH) in human prostate (20).

These results prompted us to further explore the role of locally produced PRL, possibly acting via an auto-/paracrine mechanism, in promoting abnormal prostate growth. We now report the generation of prostate-specific rPRL transgenic expression in a mouse model. In this transgenic model, the minimal probasin (Pb) promoter was used to direct expression of the rPRL transgene to the epithelial cells of the dorsal (DP), lateral (LP), and ventral (VP) prostate lobes. Pb is an androgen-dependent basic secretory protein, abundantly localized in the lumen and acinal regions of the rat prostate epithelium (21). Studies have demonstrated that the Pb minimal promoter (458 bp) can target heterologous gene expression specifically to the prostate in a developmentally and hormonally regulated fashion (22). In contrast to the Mt-1 promoter, activation of the Pb promoter is androgen dependent, and it is thus activated by the increasing androgen levels seen in late prepubertal stages (22). The Pb promoter is consequently not active until after the most essential period of ductal morphogenesis in the neonate prostate gland (23). The Pb-PRL transgenic phenotype was characterized by analysis of transgenic mRNA expression, serum hormone levels, prostate lobe weights, total DNA content, and morphology. Distribution of androgen (AR) and estrogen (ER) receptors in the prostate was investigated by immunohistochemistry. In addition, ductal morphogenesis and relative tissue components of the prostate gland were analyzed and compared with the Mt-PRL transgenic model previously presented (18).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of the Pb-rPRL plasmid
The construct was based on the pRPRL-HindIII A and B plasmids described earlier (18) and the pBH500 plasmid containing the Pb minimal promoter (a gift from Dr. R. Matusik, Vanderbilt University, Nashville, TN). The 458-bp Pb promoter fragment was excised from pBH500 as a HindIII-BamHI restriction enzyme fragment and blunted at the HindIII site. This fragment was ligated into the pRPRL-HindIII plasmid A cut with HindIII-BsmFI (blunted site). The Pb-exon 1 fragment was excised by BamHI and KpnI and ligated into the rPRL-containing plasmid cut by KpnI/HpaI. The Pb-rPRL injection fragment was excised by restriction enzyme cleavage with BamHI and Asp.

Transgenic mice
Transgenic mice were generated in C57BL/6J x CBA-f2 embryos by standard pronuclear microinjection procedure (24). The DNA fragment described above was separated by gel electrophoresis through a 0.8% agarose gel, cut out, isolated using standard isotachophoresis, and precipitated with ethanol. Founder animals were identified by PCR analysis. Tissue specimens from tail sections obtained at the age of 2 wk were digested with 10 mg/ml proteinase K in lysis buffer at 56 C overnight. Genomic DNA was collected by isopropanol extraction and centrifugation. The DNA pellet was washed in 70% ethanol and finally eluted in 50 µl of distilled (d)H2O. Primers used in the PCR (94 C for 5 min, and 30 cycles of sequential incubations at 94 C for 30 sec, 56 C for 30 sec, and 72 C for 120 sec) were complementary to the cDNA sequence in the Pb minimal promoter (5'GACAGGCATTGGGCATTG-3') and in exon 1 of the rPRL gene (5'GCAGCACATACCTTTCCG-3'). The founder animals, one male and one female, were mated to normal mice for establishment of transgenic lines. Offspring were genotyped by PCR from tail DNA at 2–3 wk of age. All experiments were performed on heterozygous transgenic animals, and nontransgenic littermates served as controls. Mt-PRL males used in this study are derived from transgenic line 2 previously described (18). All animal experimentation was conducted in accord with accepted standards of humane animal care and approved by the local ethical committee at Göteborg University.

RT-PCR
Prostatic tissue for mRNA assays was dissected submerged in RNA stabilization reagent, RNAlater (QIAGEN, Valencia, CA), and thereafter frozen in RNAlater. Total RNA was isolated from frozen tissues by the RNAeasy mini kit (QIAGEN) according to the manufacturer’s instructions. An RT-PCR assay was used to detect specific RNA. The RT reaction was performed with 0.2 mg of RNA template using the One-Step RT-PCR kit (QIAGEN) according to the manufacturer’s protocol. Briefly, the RT reaction was performed for 30 min at 50 C, immediately followed by PCR (initial activation for 15 min at 95 C, then 30 cycles of sequential incubations at 94 C for 30 sec, 54 C for 30 sec, and 72 C for 60 sec) and final extension at 72 C for 10 min. A sense primer located in exon 4, bases 844–863 (5'-TCCATGAAGC TCCTGATGCT-3'), and an antisense primer located in exon 5, bases 1646–1666 (5'-AATCCCTGCGC AGGCACCGAA-3'), specific for the rPRL gene were used. The PCR products were analyzed by electrophoresis in 1% agarose gel. The expected size of the fragment amplified from spliced mRNA was 222 bp.

Microdissection and analysis of prostate ductal morphogenesis
The genital tract was removed en bloc to a Petri dish containing calcium- and magnesium-free Hanks’ solution and cleared of periprostatic fat and connective tissue under a stereomicroscope. The individual prostate lobes were dissected free, with a minimal amount of urethra in the case of the DP, and weighed using an analytical four-decimal-place balance. Individual lobes were microdissected in depression slides in calcium- and magnesium-free Hanks’ solution containing 1% collagenase (Sigma Blend C-8051, Sigma, St. Louis, MO) under the stereomicroscope using very fine forceps to grasp and dissociate the loose interductal connective tissue. Microdissected lobes were illuminated using an oblique light source to produce a pseudodark field image that was captured with a digital camera (Coolpix 990, Nikon, Tokyo, Japan) mounted to the stereomicroscope. Ductal morphology was quantified by counting the number of ducts emerging from the urethra and the number of ductal tips, allowing calculation of the number of duct branch points. Three or four age-matched animals were included in each group.

Histology and morphometric analysis
Transgenic and wild-type prostate lobes were removed as described and thereafter fixed in 4% paraformaldehyde in PBS (pH 7.4) for 6 h, processed, and embedded in paraffin blocks for sectioning. Fixation times were identical in transgenic and control groups. Serial sections (5 µm) were then stained with hematoxylin/eosin for histological analysis, and images were obtained in a Nikon E1000 microscope 10x magnification using an Optronics DEI 750 charge-coupled device camera (Optronics, Goleta, CA) running proprietary software on a PC system. Fields were selected by a systematic random sampling scheme, and genotype information was not recorded so that the subsequent analysis was blind to genotype. Tissue component areas (epithelium, stroma, and lumen) were manually defined, and areas measured using NIH Image version 1.62 software (by Wayne Rasband, National Institutes of Health, Bethesda, MD) were calibrated against a hemocytometer. Epithelial and stromal cell nuclei in defined areas were manually counted using images captured at 20x magnification allowing calculation of area density (nuclei per 0.1 mm2).

Immunohistochemistry
Transgenic and wild-type prostate lobes were removed as described and thereafter were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 6 h, processed, and embedded in paraffin blocks for sectioning.

Fixation times were identical in transgenic and control groups. All tissues from wild-type and transgenic mice were processed in the same assay to minimize discrepancies due to variability in staining intensity. Analysis was performed blind to genotype. Images were digitally obtained as described in the previous paragraph.

ER
All tissue sections were dewaxed with Bio-Clear (Bio-Optica, Milan, Italy), rehydrated in graded ethanol, and washed consecutively in double dH2O and 0.01 M PBS (pH 7.4). After washing, the sections were transferred to plastic jars containing 0.01 M citrate buffer (pH 6.0) and boiled in a microwave oven for 10 min at 700 W. Samples were allowed to cool for 20 min before washing in PBS. Nonspecific endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in methanol for 10 min at room temperature. Nonspecific blocking was performed for 30 (ER{alpha}) or 60 (ERß) min with 1.5% normal goat serum (plus 2% BSA for ERß). Excess liquid was removed, and slides were incubated with the respective primary antibody. The rabbit polyclonal ER{alpha} antibody (ZS18–0174, Zymed Laboratories, Inc., South San Francisco, CA), diluted 1:1500 in PBS, was added to the slides and incubated overnight at 4 C. The rabbit polyclonal ERß antibody (PA1-310B, Affinity BioReagents, Inc., Golden, CO), diluted 1:500 in PBS plus 2% BSA, was applied and incubated without cover slips overnight at 4 C. The primary antibodies were substituted with a normal rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to obtain negative controls. After washing in PBS, sections were incubated for 30 min at room temperature with goat antirabbit biotinylated secondary antibody (Vector Laboratories, Inc., Burlingame, CA) diluted 1:200 in 1.5% normal goat serum (+2% BSA for ERß). After an additional wash in PBS, the sections were incubated for 30 min at room temperature with avidin and biotinylated horseradish peroxidase macromolecular complex (Vectastain Elite ABC Reagent, Vector Laboratories, Inc.). After further washing in PBS, the sections were developed with 3,3-diaminobenzidine (DAKO Corp., Carpinteria, CA), then washed with dH2O for 2 x 5 min and counterstained with hematoxylin. The sections were then rewashed in tap water, dehydrated by graded alcohol, cleared with Bio-Clear, and permanently mounted using Pertex mounting medium (Histolab Products, Gothenburg, Sweden).

AR
After deparaffination and rehydration in graded alcohol, sections were first subjected to heat treatment using 10 mM sodium citrate buffer (pH 7.6), 10 min at 95 C, allowed to cool in buffer for approximately 15 min, and then washed in dH2O. The endogenous peroxidase activity was blocked by incubating in 0.5% hydrogen peroxide in dH2O for 5 min. As a negative control, normal rabbit IgG was substituted for primary antibody on separate sections of all of the tissues analyzed to determine nonspecific binding. AR immunoperoxidase staining was performed using the AR (N-20) antibody and the ImmunoCruz staining system (both from Santa Cruz Biotechnology, Inc.), according to the manufacturer’s instructions. All sections were lightly counterstained with Mayer’s hematoxylin.

DNA content analysis
Total nucleic acids were extracted by homogenization of frozen tissues in 1% sodium dodecyl sulfate, 20 mM Tris-HCl (pH 7.5), and 4 mM EDTA, followed by a 45-min digestion with proteinase K at 45 C and standard phenol-chloroform extraction. The DNA content in the total nucleic acid preparations was measured with a fluorescence spectrophotometer (450-nm excitation and 555-nm emission) after addition of Hoecht's dye H 33258 [0.2 mg/ml in 2 M NaCl, 1 mM EDTA, and 10 mM Tris (pH 7.4)].

Measurement of rPRL
Serum levels of rPRL were measured by rPRL RIA (Amersham Pharmacia Biotech, Little Chalfont, UK) according to the manufacturer’s instructions. Serum samples were obtained from heart puncture at the specified animal age. Endogenous mouse PRL was not cross-reactive with the antibody raised against rPRL. Detection range was 0.8–50 ng/ml. All samples were analyzed in duplicates.

Measurement of testosterone
Serum testosterone was measured by a testosterone 125I RIA (ICN Biomedicals, Inc., Costa Mesa, CA), according to instructions provided by the manufacturer. Serum samples were obtained from heart puncture at the specified animal age. The RIA detection range was 0.2–20 ng/ml. All samples were tested as duplicates.

Statistical procedures
Data are presented as mean ± SEM unless otherwise indicated. Statistical analysis was performed by using a two-tailed Student’s t test. Significance was established at levels of 5% and 1% (P < 0.05 and P < 0.01, respectively).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the Pb-rPRL transgene was restricted to dorsolateral (DLP), VP, and anterior prostate lobes
Specific mRNA for the Pb-rPRL transgene was detected by RT-PCR in the DLP, VP, and anterior (also known as the coagulating gland) prostate lobes from 4 wk of age (Fig. 1Go). Weak expression could also be observed in seminal vesicles. Pb-PRL transgene mRNA was not present in other organs and tissues tested, including testis, liver, spleen, kidney, and muscle (Fig. 1Go). At 2 wk of age, transgenic mRNA was not detectable in the prostate or any other organ included (data not shown). Primers were selected so that cDNA corresponding to expression of the mouse PRL gene could not be amplified.



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  		Figure 1. Expression of the Pb-PRL transgene. A panel of organs was analyzed using RT-PCR as described in Materials and Methods. Tissue samples were collected from line L1 at 4 wk of age. Lane 1, H2O negative control; lane 2, DLP; lane 3, VP; lane 4, anterior prostate; lane 5, seminal vesicles; lane 6, testis; lane 7, kidney; lane 8, liver; lane 9, spleen; lane 10, muscle. An expected 222-bp fragment was detected in DLP, VP, and anterior prostate lobes, and weak expression was also seen in seminal vesicles. Appropriate lanes from the same ethidium bromide-stained gel were selected and shown.

 
Low levels of rPRL were detectable in serum of adult transgenic males
A rPRL-specific RIA was used to determine whether local production of transgenic rPRL in the prostate resulted in detectable serum levels. At 4 wk of age, rPRL was not detectable in serum of Pb-PRL transgenic males. However, from 10 wk of age, low levels of transgenic rPRL in serum of adult Pb-PRL transgenic males were detectable. Serum levels subsequently doubled from 10–25 wk of age but did not increase further thereafter (Table 1Go). In addition, the relative expression levels of PRLR mRNA in the liver of Pb-PRL transgenic and control animals were determined by real-time RT-PCR. This analysis demonstrated no significant difference in liver PRLR expression, a known target gene for the PRL ligand (data not shown).


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  		Table 1. Serum testosterone and transgenic rPRL levels determined by RIA in Pb-PRL and controls at different ages

 
Serum androgen levels were not altered in Pb-PRL transgenic males compared with controls
Serum testosterone levels were measured by RIA in both Pb-PRL and wild-type control animals at four different time points (4, 10, 25, and 60 wk of age). Results showed no significant difference in testosterone levels between transgenic and wild-type at any time point (Table 1Go).

Pb-PRL transgenic males developed a marked enlargement of the prostate gland
Prostate lobe weight, total DNA content, and gross morphology were assessed at four different time points in the Pb-PRL transgenic males: 4, 10, 25, and 60 wk. Lobe weights showed no significant difference at 4 wk of age (data not shown). A significant enlargement of the Pb-PRL prostate gland (DLP and VP) was evident at 10 wk of age (DLP, 29.5 ± 7 mg vs. 17.4 ± 4 mg; and VP, 22.2 ± 7 mg vs. 10.4 ± 3 mg in controls; P < 0.01; Fig. 2Go). The relative difference in prostate gland weight increased further with age, and at 60 wk DLP and VP wet weights were on average five or six times those of controls (Fig. 2Go). In addition, the DNA content in the prostate gland was measured at 20 wk of age. The total DNA content in the DLP was increased 3.3 times (119 ± 27 µg DNA/lobe vs. 36 ± 4 µg DNA/lobe in controls; P < 0.01; n = 3) and in the VP was increased 2.7 times (68 ± 9 µg DNA/lobe vs. 25 ± 5 µg DNA/lobe in controls; P < 0.01; n = 3). Gross morphology of Pb-PRL transgenic gland at 6 months of age showed marked enlargement of DP, LP, and VP lobes characterized by dilated and elongated ducts (Fig. 3AGo). No significant difference in body weight or other major organ weights was found (data not shown). In addition, no apparent fertility problems have been noted in heterozygous Pb-PRL transgenic animals.



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  		Figure 2. DLP and VP lobe weights of Pb-PRL transgenic (TG) and wild-type (WT) animals. Values represent mean lobe-pair wet weights at 10, 25, and 60 wk (w) of age. Age-matched nontransgenic littermates were used as wild-type controls. Error bars indicate SEM. A standard two-tailed Student’s t test was used. **, P < 0.01 vs. age-matched wild-type control group (n = 7).

 


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  		Figure 3. Prostate morphology and ductal dissections. A, The VP, LP, and DP lobes were dissected free of periprostatic fat and connective tissue but left attached to a small section of the urethra (U). Seminal vesicles and coagulating glands were removed and not included because of their much larger size. Prostates shown are from animals 25 wk of age. Scale bar, 3 mm. B, Individual lobes from transgenic and matching wild-type littermate control animals were microdissected further in a collagenase blend under a dissecting microscope to segregate the interductal stoma, allowing the ducts to be disentangled and arranged two-dimensionally, revealing ductal branching patterns. All ductal branching data reported are from microdissections of 12-wk-old animals. Quantification of ducts (D) attached to the urethra, ductal tips (T), and duct branch points (B) are given with the number of lobes (n) analyzed and P value from a two-tailed Student’s t test, vs. control. n/a, No mean difference between groups. Depicted controls are from Pb-PRL nontransgenic littermates. Mt-PRL controls are not shown.

 
Ductal branching morphogenesis was increased in Mt-PRL transgenic prostate
To reveal possible phenotypic differences in ductal architecture due to different onset of transgenic rPRL expression, microdissection technique was used to examine branching morphogenesis of individual lobes in Mt-PRL and Pb-PRL transgenic prostate. Quantification was made by counting primary urethral ducts as well as duct branch-points and terminal ductal tips at 12 wk of age (Fig. 3BGo). This analysis defined the distinctive ductal branching patterns of each lobe. The VP and LP lobes were attached to the urethra by two or three main ducts that showed extensive oak tree branching morphology, whereas the DP lobe consisted of multiple ducts attached to the urethra that showed less extensive, palm tree branching morphology (23). In 12-wk-old Pb-PRL prostate, no statistically significant differences were detected in the number of branch points per duct and the number of ductal tips present in each lobe compared with wild-type controls (Fig. 3BGo). However, marked ductal dilation and elongation was seen in the Pb-PRL from an early age, and complete microdissection was not achievable in animals over 20 wk of age due to the formation of a densely fibrous interductal stroma that abrogated its normally high susceptibility to collagenase.

In contrast, counting of ducts and tips in Mt-PRL VP and LP lobes at the same age demonstrated a significant increase, with approximately a doubling in the number of branching points and terminal tips compared with wild-type, whereas the number of main urethral ducts remained unchanged (Fig. 3BGo). In the Mt-PRL DP lobe, the number of ducts emanating from the urethra showed a nonsignificant increase (145% of control; P = 0.561) with significantly increased number of branch points and tips (Fig. 3BGo). The ducts were also elongated and more dilated compared with controls. In prostate lobes of older Mt-PRL animals, microdissection was also prevented by formation of a densely fibrous stroma.

Histological alterations in the Pb-PRL transgenic prostate
A dramatically increased cellularity of the stromal compartment and secretion-filled distended ducts with flattened epithelium characterized the DP, LP, and VP lobes of the Pb-PRL prostate (Fig. 4Go, A, C, and E) compared with controls (Fig. 4Go, B, D, and F). In addition, focal areas of mild to moderate chronic inflammation, exhibiting stromal mononuclear (primarily lymphocytes and macrophage) infiltrate, were frequently observed in both VP and LP lobes in Pb-PRL (Fig. 4GGo). No signs of inflammatory activity were noted in corresponding lobes of wild-type controls (data not shown). Discrete focal areas of atypical glandular epithelium were observed in both DLP and VP lobes from around 12 wk of age. These lesions contained multiple intraluminal layers of atypical cells, showing tufting or papillary patterns. Cells were larger and more columnar and often displayed abundant cytoplasm with varying degrees of nuclear hyperchromasia and pleomorphism. The glandular dysplastic foci had several morphological characteristics in common with prostatic intraepithelial neoplasia (PIN) lesions previously reported in other genetically engineered mouse models (25), resembling the low-grade PIN I (Fig. 4IGo) and PIN II (Fig. 4JGo), of the recently proposed classification of PIN in genetically engineered mice by Park et al. (25). No high-grade PIN or prostate tumor formation was detected in Pb-PRL transgenic prostate. Ducts showing epithelial hyperplasia without atypical features were also regularly detected in all lobes of the adult Pb-PRL prostate (Fig. 4HGo), whereas similar localized epithelial hyperplasia was only sporadically seen in wild-type controls (data not shown).



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  		Figure 4. Prostate lobe histology. Representative histology from Pb-PRL (A, C, and E) and wild-type (B, D, and F) animals at 25 wk of age demonstrating stromal hyperplasia and ductal dilation in VP, DP, and LP lobes. G, Interstitial inflammatory reaction with mononuclear infiltration, mostly lymphocytes and macrophages (arrow), was frequently observed in VP and LP Pb-PRL prostate; VP lobe is shown. Ducts displaying nonatypical, glandular hyperplasia were also regularly observed in Pb-PRL prostate. H, Typical unilayered, columnar epithelium in adjacent ducts included; LP lobe is shown. I, Focal atypical epithelial cells seen in LP lobe epithelium of Pb-PRL transgenic mouse at 12 wk of age. Atypical cells are larger, more columnar than adjacent cells and display abundant pale cytoplasm. Inset shows affected duct at lower magnification. J, More pronounced epithelial cell atypia in DP of Pb-PRL transgenic mouse was observed at 25 wk of age. Multiple layers of atypical epithelial cells, partly demonstrating increasing nuclear pleomorphism and hyperchromasia. Normal epithelium in adjacent duct is included for comparison. Scale bars, A–F, 100 µm; G, H, J, 50 µm; I, 25 µm.

 
Increased stromal distribution of ER{alpha} and AR in Pb-PRL transgenic prostate
Immunohistochemical methods were applied to characterize the distribution of AR, ER{alpha}, and ERß in adult Pb-PRL prostate.

An increased distribution of ER{alpha} in stromal cells of adult Pb-PRL prostate was evident, with ER{alpha} nuclear staining detected in more than 25% of fibroblasts and smooth muscle cells of the interductal stroma in all lobe types (Fig. 5AGo). This was in contrast to wild-type controls in which only occasional cell nuclei of the scarcely populated stroma showed positive immunostaining for ER{alpha} (Fig. 5BGo).



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  		Figure 5. Localization of ER{alpha}, ERß, and AR in Pb-PRL transgenic prostate. Immunohistochemistry of paraffin-embedded prostate sections from 25-wk-old Pb-PRL (TG) and wild-type control (WT) mice. A and B, Stromal cells frequently demonstrated ER{alpha} immunoreactivity in Pb-PRL prostate, LP lobe shown (A), whereas only solitary cells showed positive nuclear staining in wild-type prostate (B). Weak background staining was seen in epithelial cell cytoplasm, and positive epithelial staining was detected in individual transgenic and wild-type mice (data not shown). C and D, Mouse uterus served as positive control tissue, strong immunoreactivity was seen in glandular epithelium (ge) and uterine stroma (s) (C), negative control section included (D). Ductal epithelial cells uniformly demonstrated strong nuclear ERß immunostaining in all lobes of both Pb-PRL and wild-type prostate, illustrated here in the DP (E and F) and VP (I and J) lobes, whereas no stromal immunoreactivity was noted. G, Pregnant ovary from rat was used as positive control tissue, and intense ERß immunoreactivity was uniformly observed in granulosa cells (gc). H, ERß-negative control section demonstrating only weak cytoplasmic background staining. K and M, Uniform AR immunoreactivity was seen in nuclei of epithelial cells in all lobes of Pb-PRL prostate (K), and frequent staining in Pb-PRL stromal cells was also demonstrated (M); LP lobe is shown. L, A majority of epithelial cells in wild-type controls also showed positive immunostaining, whereas stromal cells only infrequently displayed AR immunoreactivity (not shown). Scale bars, A, B, and M, 25 µm; C–L, 50 µm.

 
Limited epithelial ER{alpha} immunoreactivity was also noted in individual animals of both genotypes (data not shown). Mouse uterus was used as a positive control for the ER{alpha} antibody, demonstrating staining of uterine glandular epithelium and stroma (Fig. 5CGo). Control antibodies showed no immunoreactivity in either uterus (Fig. 5DGo) or prostate (data not shown). Epithelial staining for ERß was almost uniform (>90%) in all lobes of both Pb-PRL transgenic (Fig. 5Go, E and I) and wild-type controls (Fig. 5Go, F and J), whereas stromal cells did not display ERß staining in either Pb-PRL or wild-type controls. Ovary from a pregnant rat was used as positive control for the ERß antibody, demonstrating intense immunoreactivity specifically in the granulosa cells (Fig. 5GGo). Negative control sections from ovary were included (Fig. 5HGo).

In all lobes of the Pb-PRL transgenic prostate, epithelial nuclei showed uniform and strong AR staining (Fig. 5KGo). A majority of epithelial nuclei in wild-type controls also displayed immunoreactivity, although areas of more heterogenic staining were evident (Fig. 5LGo). Additionally, AR immunoreactivity was regularly seen in more than 25% of stromal cells in both DLP and VP lobes of Pb-PRL (Fig. 5MGo), whereas positive staining was only sporadically seen in stroma of wild-type controls (data not shown).

Increased stromal to epithelial ratio in Pb-PRL and Mt-PRL transgenic prostate
To further distinguish differences in prostate phenotype, analysis of relative tissue compartments was performed, and comparisons were made between Pb-PRL, Mt-PRL, and control animals aged 16–20 wk. Quantification was undertaken by measurement of tissue areas by manual tracing of epithelium, periductal stroma (including the duct basement membrane), interductal stroma, and lumen using calibrated image analysis software. Cell nuclei in these areas were also counted manually, and area density (nuclei/0.1 mm2) was calculated. Data are presented relative to matched wild-type controls (Table 2Go). The sensitivity of the morphometric technique is confirmed by its ability to clearly distinguish the distinctive morphology of the various prostate lobes in controls (Robertson, F., J. Harris, M. J. Naylor, S. R. Oakes, J. Kindblom, K. Dillner, H. Wennbo, J. Törnell, P. A. Kelly, J. Green, and C. J. Ormandy, submitted for publication). In Mt-PRL animals, the ducts were grossly distended; the luminal area increased (VP, 143%, P < 0.05; DP, 192%, P < 0.01; LP, 168%, P = 0.013, of control), with a flattened epithelium resulting in reduction of both epithelial area (VP, 46%; DP, 26%; and LP, 59% of control; P < 0.01) and epithelial cell density (Table 2Go). Area density was also significantly increased in the interductal stroma of Mt-PRL transgenic DP and LP lobes (Table 2Go), whereas the VP lobe stroma exhibited a nonsignificant increase (151% of control; P = 0.1746).


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  		Table 2. Area density (nuclei/0.1 mm2) in stromal and epithelial compartments of Mt-PRL and Pb-PRL transgenic prostate expressed relative to respective control group (100%)

 
Pb-PRL transgenic tissue component analysis likewise revealed a significant increase in cellular density of the interductal stroma compartment in VP, DP, and LP lobes (Table 2Go). In contrast, a significant reduction in epithelial area (50% of control; P < 0.05) and cellular density was evident only in the VP lobe (Table 2Go). Increased luminal area was also seen in the VP and DP lobes (VP, 167%; and DP, 143% of control; P < 0.05). Calculation of the stromal to epithelial cellular ratio (SER) thus showed a distinct shift in both transgenic models compared with normal mouse prostate. In wild-type controls, the lobe-specific SER varied between 1:2.5 and 1:10, whereas in all lobes of Mt-PRL and Pb-PRL, transgenic prostate stromal and epithelial cells were present in approximately equal numbers (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report the generation of a transgenic mouse with prostate-specific expression of the rPRL gene. The Pb-PRL transgenic males developed a significant enlargement of the prostate gland, characterized primarily by hyperplasia of the stromal compartment, distended ductal structures, and focal areas of glandular dysplasia. The stromal hyperplasia was associated with increases in stromal AR and ER{alpha}. In addition, we demonstrated a significant increase in ductal morphogenesis of both DLP and VP in Mt-PRL transgenic males, in contrast to the normal ductal structure observed in the Pb-PRL prostate.

The motive for generating a prostate-specific PRL transgenic was to enable us to study the in vivo effects of locally enhanced PRL action in the prostate gland without the possible systemic alterations resulting from a prolonged hyperprolactinemic state. Extrapituitary production of PRL has raised interest in the past few years, but secretory control and indeed relevance remains largely unknown. Although the contribution of local PRL production to circulating PRL levels is presumably low, it may be sufficient to exert significant activity on its local environment (1). In addition, serum PRL levels may not reflect prostatic PRL concentration, because some clinical reports have noted significantly elevated prostatic tissue PRL levels in patients with BPH (26). The recent detection of locally produced PRL in prostate epithelium has indicated a possible auto/paracrine action of PRL in prostate tissue (27). Earlier work has demonstrated PRL-induced delay in castration-induced prostatic regression (28). In line with this, PRL was recently shown to significantly inhibit apoptosis in androgen-deprived DP and LP rat prostate cultures, potentially acting as a survival factor for prostate epithelium (29).

In contrast to the Mt-PRL transgenic model (18), the Pb-PRL males did not display any elevations in serum testosterone levels. Our previous studies have demonstrated a lack of importance of elevated circulating androgens in the Mt-PRL adult prostate phenotype (19), and the distinct phenotype of the normoandrogenic Pb-PRL transgenic males support these findings. The detectable levels of transgenic rPRL in serum of adult Pb-PRL males were associated with the developing size increase of the proliferating prostate in the transgenic animals. This is indicated by the fact that no detectable levels of circulating rPRL were found in prepubertal animals, and restriction of transgenic expression to the prostate was maintained in the adult Pb-PRL males. We conclude from this that, due to the increase in Pb-PRL transgenic prostate size, the prostatic expression of rPRL gradually reaches levels leading to a limited but systemically detectable secretion. Adult serum PRL levels in the normal male mouse were characterized in the 1970s (30, 31). The serum rPRL levels detected in Pb-PRL are in the same range (5–10 ng/ml), however a direct comparison of transgenic rPRL and reported endogenous mPRL levels is difficult due to technical differences in standard preparations. A lack of significant systemic effects of detectable rPRL is nonetheless indicated by the unaffected circulating androgen levels. The unaltered expression level of liver PRLR in Pb-PRL males compared with controls further supports this conclusion. PRL ligand-induced up-regulation of PRLR expression in target tissues has been described (32), and significantly increased PRLR expression has previously been demonstrated in the liver of Mt-PRL transgenic males (33).

Development of glandular organs such as the prostate involves the process of branching morphogenesis. In rodents, the critical time period for ductal budding and the ensuing process of ductal growth and branching is reported to span from d 15 of gestation to approximately 4–5 wk postpartum (23, 34, 35). Furthermore, Sugimura et al. (23) have demonstrated that during the first 15 d after birth, over 75% of tips and branch-points of the adult gland are formed in the VP lobe, and a majority of ductal tips and branch-points are also formed in the DLP of the mouse. Interestingly, prostate lobe microdissections revealed a significantly increased ductal morphogenesis in Mt-PRL transgenic prostate, with approximately a doubling in branching points and ductal tips evident in the DP, LP, and VP lobes. Activation of the Mt-1 promoter during the early embryonic stage is well described, with abundant expression already by d 12 of gestation reported (36, 37). In contrast, expression of the androgen-dependent Pb-PRL transgene mRNA was not detected before 4 wk of age; expression was thus initiated after the neonatal period of ductal formation, and branching was essentially completed. Consequently, the Pb-PRL prostate exhibited no significant changes in mature ductal architecture when compared with controls. Marked ductal dilation and elongation was, however, evident in both transgenic models, and the formation of a dense fibrous and cellular interductal stroma appeared equally pronounced in Pb-PRL prostate. The disparity of ductal architecture in the PRL transgenic models can thus presumably be attributed to the temporal difference in transgene activation. The altered androgen status in Mt-PRL transgenic males may also have an impact on early ductal development in the prostate. This is indicated by a previous study in the VP of hypogonadal mice, demonstrating that a single neonatal dose of androgens can increase branching morphogenesis and lobe weight at adulthood (38). However, neonatal administration of androgens after castration in rat has not been shown to significantly alter final ductal architecture (39). Moreover, castration and androgen replacement studies performed by Donjacour and Cunha (39) have established that neonatal prostatic ductal morphogenesis is sensitive to, but does not require, chronic androgen stimulation. These findings demonstrate how PRL can, directly or indirectly through androgen stimulation, induce a significant increase in neonatal prostate morphogenesis.

The recent generation and characterization of the various estrogen-modulated mouse models [{alpha}ERKO, ßERKO, {alpha}ßERKO, and aromatase knockout mouse model (ArKO)] has provided new insights regarding the role of estrogens in prostate growth and development (40). The expression of both ER subtypes in human and rodent prostate is now well established, with expression of ER{alpha} described primarily in a subset of stromal cells and ERß restricted to the ductal epithelium (41, 42). We detected no distinguishable changes in distribution of ERß immunoreactivity, with uniform staining of ductal epithelium seen in both Pb-PRL and wild-type prostate and a lack of immunoreactive ERß noted in the stroma of both groups. In contrast, ER{alpha} was frequently detected in the Pb-PRL transgenic stroma of all lobes, whereas only sporadic stromal cells in wild-type controls demonstrated immunoreactivity. The limited presence of ER{alpha} detected in epithelial cells of both genotypes could indicate a degree of nonspecific ER immunoreactivity for the antibody, but epithelial ER{alpha} gene expression and immunoreactivity were previously reported (43, 44). PRL-induced up-regulation of ER expression has previously been described in several organs, including the liver, mammary gland, and corpus luteum (45, 46), and our results could indicate a PRL-mediated up-regulation of stromal ER{alpha} in the prostate. However, the relevance of a possible increase in stromal ER{alpha} content for the Pb-PRL prostate phenotype remains to be determined. Recent work has established that both initiation and progression of squamous metaplasia and neonatal imprinting after estrogen administration are mediated through stromal ER{alpha} (47, 48). A distinct phenotype of focal epithelial hyperplasia in the VP has been reported in aging mice lacking functional ERß (ßERKO; Refs. 49 and 50), whereas no apparent prostate pathology or enlargement has yet been reported in {alpha}ERKO or {alpha}ßERKO (40). These findings are indicative of an antiproliferative role for epithelial ERß and also suggest that an unbalanced stromal ER{alpha} action could contribute to the phenotype observed. The ArKO (51), lacking endogenous estrogen production due to a nonfunctional aromatase enzyme, also bears an interesting resemblance to the Pb-PRL transgenic prostate phenotype. In the ArKO mouse, the combined effects of estrogen absence and elevated androgen and PRL levels result in a moderate prostate enlargement with hyperplasia evident in all lobes and tissue compartments (52). In Pb-PRL prostate, an increase in stromal AR content was observed, as previously noted for the Mt-PRL (19). Furthermore, testosterone treatment of adult wild-type mice resulted in up-regulation of epithelial AR, but not stromal AR, and was not associated with any significant prostate hyperplasia (19). The importance of stromal AR in the prostate is well known, and mediation of some androgenic effects, such as ductal morphogenesis and epithelial growth, has been proposed not to require intraepithelial AR (53, 54). Our combined results from the Mt-PRL and Pb-PRL suggest that an increase in stromal rather than epithelial AR content is associated with the hyperplastic phenotype of the PRL transgenic prostate.

Comparative analysis of relative tissue areas and cellular area density confirmed the histological similarities of the transgenic models, with significant increases in stromal cellularity appearing most prominent. The stromal hyperplasia in both transgenic prostates resulted in a major increase in the SER. Interestingly, when calculating the ratio of stromal to epithelial hyperplasia in human BPH tissue, clinical reports have firmly established a dominance of the stromal component (55, 56, 57, 58). Furthermore, in symptomatic BPH patients, the SER has been reported significantly higher than in asymptomatic patients (56). The implications of our findings with regard to a clinical setting have yet to be determined. In addition, the presence of inflammatory response in LP and VP transgenic lobes corresponds well to previous findings demonstrating that prepubertal exposure to compounds that increase PRL secretion, including estradiol, results in LP inflammation in the adult rat (59). Furthermore, administration of a dopamine agonist (bromocriptin) reverses the induction of LP inflammation by estradiol, suggesting that PRL is necessary for the inflammatory effect. Our findings are thus further supportive of a role for PRL in the induction of prostate inflammation. The atypical epithelial lesions demonstrated in Pb-PRL prostate resemble the low-grade PIN described in the recently proposed classification of PIN in genetically engineered mice (25), thereby indicating a potential for proliferative disease. However, because we have not yet observed any progression to high-grade PIN or tumor formation in the Pb-PRL prostate, it must be considered that these lesions could represent a form of atypical hyperplasia without progressive potential.

Recent studies in PRLR-deficient mice (PRLR-/-) generated by Ormandy et al. (3) have shed further light on the role of PRL action in the prostate. Morphological studies revealed no alterations in branching morphology, but a subtle prostatic phenotype with partial loss of epithelial content specifically in the DP lobe was noted (Robertson, F., J. Harris, M. J. Naylor, S. R. Oakes, J. Kindblom, K. Dillner, H. Wennbo, J. Törnell, P. A. Kelly, J. Green, and C. J. Ormandy, submitted for publication). More interestingly, when crossed with transgenic mice that carry the SV40T oncogene under the C3 promoter (60), PRLR-/- males failed to develop any prostate tumors, and VP lobe PIN areas were significantly reduced (Robertson, F., J. Harris, M. J. Naylor, S. R. Oakes, J. Kindblom, K. Dillner, H. Wennbo, J. Törnell, P. A. Kelly, J. Green, and C. J. Ormandy, submitted for publication). These findings could indicate a role for PRL in prostate tumor formation. The results from the PRLR-deficient mice thus demonstrate a subtle essential role for PRL in normal prostate development and maintenance of normal tissue composition that contrast to the major effects of increased systemic or local PRL levels reported in our transgenic models.

In summary, we have demonstrated that prostate-specific expression of a PRL transgene results in abnormal growth of the prostate gland and that the phenotype is not associated with any alterations in circulating androgen levels. The Pb-PRL prostate is characterized primarily by stromal hyperplasia and ductal dilation, resulting in an increased stromal to epithelial ratio. The increased stromal component is of particular interest because it evidently resembles the situation in human BPH. Furthermore, our comparative studies of the Pb-PRL and Mt-PRL transgenic models demonstrate a dual ability of increased PRL activity to initiate abnormal growth in the normally developed adult gland and, directly or indirectly, to induce a significant increase in ductal morphogenesis of the neonate prostate.


   Acknowledgments
 
We thank Dr. R. Matusik (Vanderbilt University, Nashville, TN) for generously providing the probasin minimal promoter fragment and Mrs. B. Masironi and Mrs. M. Petersson for excellent technical assistance.


   Footnotes
 
This study was supported by grants from the Swedish Research Council (to L.S.), the Swedish Cancer Society (to J.T. and J.K.), Assar Gabrielssons Foundation (to K.D. and J.K.), Cancer Council of New South Wales and National Health and Medical Research Council of Australia (to C.J.O.) and the Gothenburg Medical Society (to J.K.).

Abbreviations: AR, Androgen receptor; ArKO, aromatase knockout mouse model; BPH, benign prostate hyperplasia; d, distilled; DLP, dorsolateral prostate; DP, dorsal prostate; ER, estrogen receptor; LP, lateral prostate; Mt-1, metallothionein I; Mt-PRL, PRL transgenic mouse; Pb, probasin; PIN, prostatic intraepithelial neoplasia; PRL, prolactin; PRLR, PRL receptor; rPRL, rat PRL; SER, stromal to epithelial cellular ratio; VP, ventral prostate.

Received December 23, 2002.

Accepted for publication February 7, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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