Increased Endogenous Estrogen Synthesis Leads to the Sequential Induction of Prostatic Inflammation (Prostatitis) and Prostatic Pre-Malignancy
2009; Elsevier BV; Volume: 175; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2009.081107
ISSN1525-2191
AutoresStuart J. Ellem, Hong Wang, Matti Poutanen, Gail P. Risbridger,
Tópico(s)Reproductive Physiology in Livestock
ResumoProstatitis causes substantial morbidity to men, through associated urinary symptoms, sexual dysfunction, and pelvic pain; however, 90% to 95% of cases have an unknown etiology. Inflammation is associated with the development of carcinoma, and, therefore, it is imperative to identify and study the causes of prostatitis to improve our understanding of this disease and its role in prostate cancer. As estrogens cause prostatic inflammation, here we characterize the murine prostatic phenotype induced by elevated endogenous estrogens due to aromatase overexpression (AROM+). Early-life development of the AROM+ prostate was normal; however, progressive changes culminated in chronic inflammation and pre-malignancy. The AROM+ prostate was smaller at puberty compared with wild-type controls. Mast cell numbers were significantly increased at puberty and preceded chronic inflammation, which emerged by 40 weeks of age and was characterized by increased mast cell, macrophage, neutrophil, and T-lymphocyte numbers. The expression of key inflammatory mediators was also significantly altered, and premalignant prostatic intraepithelial neoplasia lesions emerged by 52 weeks of age. Taken together, these data link estrogens to prostatitis and premalignancy in the prostate, further implicating a role for estrogen in prostate cancer. These data also establish the AROM+ mouse as a novel, non-bacterial model for the study of prostatitis. Prostatitis causes substantial morbidity to men, through associated urinary symptoms, sexual dysfunction, and pelvic pain; however, 90% to 95% of cases have an unknown etiology. Inflammation is associated with the development of carcinoma, and, therefore, it is imperative to identify and study the causes of prostatitis to improve our understanding of this disease and its role in prostate cancer. As estrogens cause prostatic inflammation, here we characterize the murine prostatic phenotype induced by elevated endogenous estrogens due to aromatase overexpression (AROM+). Early-life development of the AROM+ prostate was normal; however, progressive changes culminated in chronic inflammation and pre-malignancy. The AROM+ prostate was smaller at puberty compared with wild-type controls. Mast cell numbers were significantly increased at puberty and preceded chronic inflammation, which emerged by 40 weeks of age and was characterized by increased mast cell, macrophage, neutrophil, and T-lymphocyte numbers. The expression of key inflammatory mediators was also significantly altered, and premalignant prostatic intraepithelial neoplasia lesions emerged by 52 weeks of age. Taken together, these data link estrogens to prostatitis and premalignancy in the prostate, further implicating a role for estrogen in prostate cancer. These data also establish the AROM+ mouse as a novel, non-bacterial model for the study of prostatitis. Prostatitis, an exceedingly common condition in the male population worldwide, has an incidence of ∼9% and a prevalence of ∼14%, and, unlike benign prostatic hyperplasia and prostate cancer (PCa), which are predominantly diseases of older men, prostatitis afflicts men of all ages. It is also the most common outpatient condition seen in men under 50 years of age and accounts for more clinical visits than either PCa and/or benign prostatic hyperplasia.1Vykhovanets EV Resnick MI MacLennan GT Gupta S Experimental rodent models of prostatitis: limitations and potential.Prostate Cancer Prostatic Dis. 2007; 10: 15-29Crossref PubMed Scopus (85) Google Scholar, 2Collins MM Stafford RS O'Leary MP Barry MJ How common is prostatitis? A national survey of physician visits.J Urol. 1998; 159: 1224-1228Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar At present our knowledge of prostatitis is lacking, with 90% to 95% of cases having an unknown etiology. This represents a significant problem, particularly as prostatitis has also been implicated in the development of PCa.3De Marzo AM Marchi VL Epstein JI Nelson WG Proliferative inflammatory atrophy of the prostate: implications for prostatic carcinogenesis.Am J Pathol. 1999; 155: 1985-1992Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar, 4Palapattu GS Sutcliffe S Bastian PJ Platz EA De Marzo AM Isaacs WB Nelson WG Prostate carcinogenesis and inflammation: emerging insights.Carcinogenesis. 2005; 26: 1170-1181Crossref PubMed Scopus (306) Google Scholar, 5Balkwill F Mantovani A Inflammation and cancer: Back to Virchow?.Lancet. 2001; 357: 539-545Abstract Full Text Full Text PDF PubMed Scopus (6054) Google Scholar Given the prevalence of prostatitis and this putative link to PCa, it is vitally important to identify the unknown causes of prostatitis. Several causes of prostatitis have been postulated, including hormonal variation or exposure, infection due to sexually transmitted disease,6Strickler HD Goedert JJ Sexual behavior and evidence for an infectious cause of prostate cancer.Epidemiol Rev. 2001; 23: 144-151Crossref PubMed Scopus (75) Google Scholar dietary factors, and physical trauma from urine reflux.7Kirby RS Lowe D Bultitude MI Shuttleworth KE Intra-prostatic urinary reflux: an aetiological factor in abacterial prostatitis.Br J Urol. 1982; 54: 729-731Crossref PubMed Scopus (195) Google Scholar However, of particular interest is an apparent link between estrogen and prostatic inflammation,8Naslund MJ Strandberg JD Coffey DS The role of androgens and estrogens in the pathogenesis of experimental nonbacterial prostatitis.J Urol. 1988; 140: 1049-1053PubMed Google Scholar which has emerged mainly from the administration of pharmacological levels of exogenous estrogen to rodents.9Harris MT Feldberg RS Lau KM Lazarus NH Cochrane DE Expression of proinflammatory genes during estrogen-induced inflammation of the rat prostate.Prostate. 2000; 44: 19-25Crossref PubMed Scopus (83) Google Scholar Additional data obtained from further rodent studies show that the prostate gland is particularly sensitive to estrogenic exposure during development in fetal and neonatal life; transient estrogen exposure before puberty results in inflammation later in life, well after the exposure has occurred.10Bianco JJ McPherson SJ Wang H Prins GS Risbridger GP Transient neonatal estrogen exposure to estrogen-deficient mice (aromatase knockout) reduces prostate weight and induces inflammation in late life.Am J Pathol. 2006; 168: 1869-1878Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 11Prins GS Huang L Birch L Pu Y The role of estrogens in normal and abnormal development of the prostate gland.Ann NY Acad Sci. 2006; 1089: 1-13Crossref PubMed Scopus (103) Google Scholar Several decades of research from various laboratories, including our own, has demonstrated that this action is mediated by the estrogen receptor (ER) α subtype and involves a cascade of events that permanently and irreversibly alter gene expression patterns in the gland.12Prins GS Birch L Couse JF Choi I Katzenellenbogen B Korach KS Estrogen imprinting of the developing prostate gland is mediated through stromal estrogen receptor alpha: studies with alphaERKO and betaERKO mice.Cancer Res. 2001; 61: 6089-6097PubMed Google Scholar These data indicate that exposure to estrogen causes prostatic inflammation and directly links estrogen and prostatitis. Studies have shown that chronic inflammation predisposes individuals to various types of cancer; indeed, underlying infection and inflammatory responses have been linked to 15% to 20% of all human cancers.5Balkwill F Mantovani A Inflammation and cancer: Back to Virchow?.Lancet. 2001; 357: 539-545Abstract Full Text Full Text PDF PubMed Scopus (6054) Google Scholar, 13Coussens LM Werb Z Inflammation and cancer.Nature. 2002; 420: 860-867Crossref PubMed Scopus (11277) Google Scholar, 14Kuper H Adami HO Trichopoulos D Infections as a major preventable cause of human cancer.J Intern Med. 2000; 248: 171-183Crossref PubMed Scopus (375) Google Scholar This is also believed to be true for the prostate, and there is an emerging and growing body of evidence implicating inflammation in the etiology of PCa similar to other organs such as the liver and stomach.3De Marzo AM Marchi VL Epstein JI Nelson WG Proliferative inflammatory atrophy of the prostate: implications for prostatic carcinogenesis.Am J Pathol. 1999; 155: 1985-1992Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar, 4Palapattu GS Sutcliffe S Bastian PJ Platz EA De Marzo AM Isaacs WB Nelson WG Prostate carcinogenesis and inflammation: emerging insights.Carcinogenesis. 2005; 26: 1170-1181Crossref PubMed Scopus (306) Google Scholar, 5Balkwill F Mantovani A Inflammation and cancer: Back to Virchow?.Lancet. 2001; 357: 539-545Abstract Full Text Full Text PDF PubMed Scopus (6054) Google Scholar Epidemiological evidence also indicates that men with a history of prostatitis have an increased risk for PCa.15Dennis LK Dawson DV Meta-analysis of measures of sexual activity and prostate cancer.Epidemiology. 2002; 13: 72-79Crossref PubMed Scopus (207) Google Scholar Additionally, lesions characterized by proliferating epithelial cells and activated inflammatory cells (proliferative inflammatory atrophy) are frequently observed in juxtaposition to premalignant prostatic lesions (prostatic intraepithelial neoplasia; PIN) and PCa.3De Marzo AM Marchi VL Epstein JI Nelson WG Proliferative inflammatory atrophy of the prostate: implications for prostatic carcinogenesis.Am J Pathol. 1999; 155: 1985-1992Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar To study the effects of estrogen on the prostate, previous studies have typically relied on the addition of exogenous estrogens at either low or pharmacological doses. This, however, introduces a number of complicating factors. Low dose effects can be difficult to discern, while pharmacological doses may not mimic normal physiological responses. In addition, this methodology also precludes the ability to examine the effects of estrogen and the progression of disease throughout development and adult life. Consequently, the development of new experimental animal models of prostatitis is essential to determine whether inflammation is linked to development of PCa. This need was highlighted and stressed in the report from the Bar Harbor Consensus meeting.16Shappell SB Thomas GV Roberts RL Herbert R Ittmann MM Rubin MA Humphrey PA Sundberg JP Rozengurt N Barrios R Ward JM Cardiff RD Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee.Cancer Res. 2004; 64: 2270-2305Crossref PubMed Scopus (486) Google Scholar Although two models of prostatitis have recently been developed, they are of limited utility: one is bacterial17Elkahwaji JE Zhong W Hopkins WJ Bushman W Chronic bacterial infection and inflammation incite reactive hyperplasia in a mouse model of chronic prostatitis.Prostate. 2007; 67: 14-21Crossref PubMed Scopus (75) Google Scholar while the other is rat-based and requires the administration of exogenous hormones.18Bernoulli J Yatkin E Talvitie EM Santti R Streng T Urodynamic changes in a noble rat model for nonbacterial prostatic inflammation.Prostate. 2007; 67: 888-899Crossref PubMed Scopus (24) Google Scholar As a result, both of these models preclude the ability to cross-breed with other types of genetically manipulated mice to delineate and study specific mechanisms. Consequently the imperative remains to develop new mouse models for the study of prostatitis. The aromatase overexpressing (AROM+) transgenic mouse provides a novel model to examine the effect of altered aromatase activity, and therefore estrogen levels and action, in the prostate within physiological hormonal environment. Estrogen levels in these mice are significantly elevated and are associated with a concomitant decrease in androgens. It has been previously reported that the prostates of these mice are rudimentary due to the suppression of androgens.19Li X Nokkala E Yan W Streng T Saarinen N Warri A Huhtaniemi I Santti R Makela S Poutanen M Altered structure and function of reproductive organs in transgenic male mice overexpressing human aromatase.Endocrinology. 2001; 142: 2435-2442Crossref PubMed Scopus (130) Google Scholar In this study, we show for the first time that the AROM+ mouse develops chronic prostatitis by 40 weeks of age. This inflammation is characterized by an elevation in mast cell numbers from puberty and ultimately leads to chronic inflammation and the development of PIN lesions. This demonstrates a continuum, with estrogen-inducing inflammation, which subsequently results in the onset of pre-malignancy. AROM+ mice exhibit ubiquitous human aromatase expression, driven by the human ubiquitin C promoter.19Li X Nokkala E Yan W Streng T Saarinen N Warri A Huhtaniemi I Santti R Makela S Poutanen M Altered structure and function of reproductive organs in transgenic male mice overexpressing human aromatase.Endocrinology. 2001; 142: 2435-2442Crossref PubMed Scopus (130) Google Scholar These mice are of FVB/N genetic background and were maintained under controlled conditions (lights on 0700 to 1900; temperature 20 °C to 24 °C), with access to mouse feed (Glen Forrest Stockfeeders, Glen Forrest, Australia) and water ad libitium. Litters of AROM+ mice were generated by breeding transgenic females with wild-type FVB/N males to produce AROM+ and wild-type offspring. Anterior, ventral, dorsal and lateral prostate lobes (AP, VP, DL, and LP, respectively) and seminal vesicles (SV) were collected from AROM+ and wild-type animals for analysis at day 0 (d0; postnatal) and day 3 (d3; postnatal), then weekly from 3 to 52 weeks of age. For consistency and unless otherwise noted, all data presented were generated using VP tissues. SCID mice were obtained from Central Animal Services, Monash University (Clayton, Australia) and housed as detailed for the AROM+ animals. All animal studies were conducted in accordance with the National Health and Medical Research Council and Monash University animal ethics regulations and guidelines. Analysis of branching morphogenesis in neonatal prostate tissues was conducted using combined confocal microscopy and computerized image analysis as previously described.20Almahbobi G Hedwards S Fricout G Jeulin D Bertram JF Risbridger GP Computer-based detection of neonatal changes to branching morphogenesis reveals different mechanisms of and predicts prostate enlargement in mice haplo-insufficient for bone morphogenetic protein 4.J Pathol. 2005; 206: 52-61Crossref PubMed Scopus (9) Google Scholar Briefly, VP or AP were dissected from the urogenital tracts of neonatal day 0 and day 3 wild-type and AROM+ mice and prepared for wholemount immunostaining of epithelial cell cytokeratins. Six parameters of branching were recorded and analyzed; specifically, prostate ductal length, volume, number of main ducts, branches, branch points, and tips. Immunohistochemistry was performed using a DAKO autostainer (DAKO, Carpentaria, CA) as previously described.21McPherson S Wang H Jones M Pedersen J Iismaa T Wreford N Simpson E Risbridger G Elevated androgens and prolactin in aromatase deficient (ArKO) mice cause enlargement but not malignancy of the prostate gland.Endocrinology. 2001; 142: 2458-2467Crossref PubMed Scopus (132) Google Scholar, 22Bianco JJ Handelsman DJ Pedersen JS Risbridger GP Direct response of the murine prostate gland and seminal vesicles to estradiol.Endocrinology. 2002; 143: 4922-4933Crossref PubMed Scopus (86) Google Scholar Briefly, following fixation the prostate tissues or recombinants were processed and embedded in paraffin wax and serially sectioned at 5 μm for histological and/or stereological analysis. After dewaxing, the sections were rehydrated antigen retrieval (0.01 M/L citrate buffer at pH 6.0 or 0.05 M/L Tris/0.01% EDTA at pH 9.0) was performed. Peroxidase block (Dako) was used to inactivate endogenous peroxidase activity and nonspecific binding was blocked using CAS block (Zymed, San Francisco, CA) followed by incubation with primary antibody for 60 to 120 minutes at room temperature. Primary antibody binding was detected using a biotinylated secondary antibody (Zymed) followed by incubation with an avidin-biotin peroxidase kit (ABC Elite; Vector Laboratories, Burlingame, CA). Antibody localization was ultimately visualized using the chromogen diaminobenzidine tetrachloride (Dako). Finally, sections were counterstained with Mayer’s hematoxylin, gradually dehydrated with alcohol, cleared with Histolene, and covered with dansylated polymyxin B mounting solution and a coverslip. The localization and expression of the androgen receptor (AR), ERα, ERβ, smooth muscle α-actin, proliferating cell nuclear antigen and high molecular weight cytokeratin (CKH) were determined using specific antibodies for AR (N-20, Santa Cruz Biotechnology, CA), ERα (Dako, a-actin, Sigma Aldrich, St. Louis, MO), proliferating cell nuclear antigen (Dako), CKH (Dako), and ERβ (Novocastra Laboratories Ltd., Newcastle, UK). The presence and localization of specific leukocytes within the tissues was also examined using immunohistochemistry. The localization of neutrophils, T lymphocytes, B lymphocytes, and macrophages was determined using antibodies specific for Ly6g (BD Pharmingen, San Diego, CA), CD3 (Santa Cruz Biotechnology, CA), CD45R (BD Pharmingen), and F480 (Abcam, Cambridge, UK), respectively. Mast cells contain granules composed of heparin, sulfated glycosaminoglycan and histamine. Consequently, acidified toluidine blue will stain these cells metachromatically, making them readily distinguishable from the surrounding tissue; the mast cells will stain a red-purple (metachromatic staining) and the surrounding tissue will be blue (orthochromatic staining). Briefly, 5-μm paraffin-embedded sections were deparaffinized and re-hydrated. The sections were then stained in a solution of 0.1% Toluidine Blue O (Sigma Aldrich), 7% alcohol, and 1% NaCl at pH 2.3 for 3 minutes. The sections were then washed in distilled water, rapidly dehydrated in 95% and 100% alcohol washes, mounted in dansylated polymyxin B, and coverslipped for analysis. All assessments were performed using a Bioprecision2 Microscope Stage (Ludl, NY), 99A400 Focus drive (Ludl), MAC5000 Controller (Ludl), and ND-281 Encoder (Heidenhain, IL) coupled to an Olympus BX-51 microscope (Olympus, Tokyo, Japan). The images were captured using a PixeLink PL-623C digital camera (PixeLink, Ottawa, ON, Canada) coupled to a computer. The newCAST component (version 2.14; Visiopharm, Hørsholm, Denmark) of the Visiopharm Integrator System (version 2.16.1.0; Visiopharm) was used to generate a set of counting frames and a point grid (grid properties were assessed individually for each marker). Uniform systematic random sampling was then used to quantitate the number of positive cells on toluidine blue or immunohistochemical stained sections, as previously described.21McPherson S Wang H Jones M Pedersen J Iismaa T Wreford N Simpson E Risbridger G Elevated androgens and prolactin in aromatase deficient (ArKO) mice cause enlargement but not malignancy of the prostate gland.Endocrinology. 2001; 142: 2458-2467Crossref PubMed Scopus (132) Google Scholar, 22Bianco JJ Handelsman DJ Pedersen JS Risbridger GP Direct response of the murine prostate gland and seminal vesicles to estradiol.Endocrinology. 2002; 143: 4922-4933Crossref PubMed Scopus (86) Google Scholar, 23Cancilla B Jarred RA Wang H Mellor SL Cunha GR Risbridger GP Regulation of prostate branching morphogenesis by activin a and follistatin.Dev Biol. 2001; 237: 145-158Crossref PubMed Scopus (68) Google Scholar In brief, the sections were examined under ×40 magnification; they were mapped to define tissue boundaries and were sampled at predetermined intervals along the x- and y- axes using a single point grid-counting frame. Positive cells were then scored and totaled for each respective gland, the final number being expressed as the number of positive cells per unit area. Serum levels of testosterone and estradiol were determined using the DSL-4000–coated tube radioimmunoassay and the DSL-4800 ultra-sensitive double antibody radioimmunoassay, respectively, as per the manufacturer’s instructions (Diagnostic System Laboratories Inc., Webster, TX). Values for the samples were derived via interpolation using standards provided with the kit. The expression of 84 inflammatory cytokines and chemokines was examined using the Mouse Inflammatory Cytokines and Receptors RT2 Profiler PCR Array (SuperArray Bioscience Corporation, Frederick, MD). Quantitative real-time PCR array was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Forster City, CA). VPs were dissected from AROM+ and wild-type mice at 40 weeks of age when chronic inflammation was evident. Total RNA was first extracted using TRIzol reagent, per the manufacturer’s instructions (Invitrogen, Carlsbad, CA), then further cleaned using the RT2 qPCR-Grade RNA isolation kit (SuperArray). RNA integrity was checked using Experion RNA StdSens Analysis kits and the Experion Automated Electrophoresis System (Biorad, Hercules, CA). cDNA was synthesized from the purified RNA using the RT2 First Strand Kit (SuperArray) and samples were initially run on a RT2 RNA QC PCR Array (SuperArray) to check housekeeping gene expression levels, RT efficiency and for genomic DNA carry-over. PCR array analysis was performed according to the manufacturer protocol with the RT2 Real-Time SYBR Green PCR Master Mix (SuperArray). mRNA expression for each gene was normalized using the expression of HPRT as a control housekeeping gene and compared with the data obtained with RNA from the control wild-type samples according the 2−ΔΔCT method.24Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C[T]) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (124899) Google Scholar Results were considered significant when relative mRNA expression was at least twofold higher or lower than that of the wild-type control mice and the statistical P value was <0.05. The results were also subsequently confirmed by quantitative real-time PCR on individual RNA samples using the QuantiTect SYBR Green PCR Kit (Qiagen Inc., Mississauga, Canada). To examine aromatase expression in the wild-type mouse VP compared with the AROM+ VP, relative quantitative PCR was performed as previously described.25Ellem SJ Schmitt JF Pedersen JS Frydenberg M Risbridger GP Local aromatase expression in human prostate is altered in malignancy.J Clin Endocrinol Metab. 2004; 89: 2434-2441Crossref PubMed Scopus (146) Google Scholar Briefly, RNA was extracted from the VPs of wild-type and AROM+ mice using TRIzol (Invitrogen), DNase treated and cDNA generated. Real time PCR was subsequently performed using the Roche LightCycler per manufacturer’s instructions (Roche Diagnostics, Basel, Switzerland). cDNA was normalized to 18S expression and relative aromatase levels in the different tissues were subsequently determined. In total, samples from 5 AROM+ and four wild-type animals were examined, with all samples run in triplicate. Tissue recombinants were prepared as previously described.26Risbridger G Wang H Young P Kurita T Wang YZ Lubahn D Gustafsson JA Cunha G Wong YZ Evidence that epithelial and mesenchymal estrogen receptor-alpha mediates effects of estrogen on prostatic epithelium.Dev Biol. 2001; 229: 432-442Crossref PubMed Scopus (150) Google Scholar, 27Cunha GR Donjacour A Stromal-epithelial interactions in normal and abnormal prostatic development.Prog Clin Biol Res. 1987; 239: 251-272PubMed Google Scholar, 28Cunha GR Young P Higgins SJ Cooke PS Neonatal seminal vesicle mesenchyme induces a new morphological and functional phenotype in the epithelia of adult ureter and ductus deferens.Development. 1991; 111: 145-158PubMed Google Scholar, 29Day KC McCabe MT Zhao X Wang Y Davis JN Phillips J Von Geldern M Ried T KuKuruga MA Cunha GR Hayward SW Day ML Rescue of embryonic epithelium reveals that the homozygous deletion of the retinoblastoma gene confers growth factor independence and immortality but does not influence epithelial differentiation or tissue morphogenesis.J Biol Chem. 2002; 277: 44475-44484Crossref PubMed Scopus (27) Google Scholar SVs were removed from day 0 wild-type or AROM+ mice and digested with 1% trypsin (Life Technologies, Gaithersburg, MD) in calcium- and magnesium-free Hanks’ balanced salt solution for 60 minutes at 4°C. Seminal vesicle mesenchyme was then separated from epithelium using a Graefe knife and fine forceps. APs were removed from adult male wild-type or AROM+ mice, and ductal tips of ∼1 mm in length were excised. The tissue recombinants were constructed in vitro with wild-type or AROM+ epithelia (-E) and stroma (-S); specifically wild-type-E/wild-type-S or AROM+-E/AROM+-S. The assembled recombinant tissues were cultured in a humidified incubator at 37°C with 5% CO2 for 36 hours before being grafted under the renal capsules of intact male immune-deficient SCID mice. Grafts were harvested after 4 weeks of growth in vivo and fixed in Bouins for analysis. A minimum of five grafts were prepared for each group. Data were analyzed to determine normality and significant differences were determined by t-test with a significance threshold used at a level of 5% (P < 0.05). Analyses were conducted using Prism 5.00 software (GraphPad Software Inc., San Diego, CA). Data are expressed as mean ± SEM, unless otherwise noted. Analysis and characterization of the urological phenotype in the AROM+ mouse revealed that these animals develop a number of prostate and genitourinary pathologies. Particularly apparent was the development of chronic inflammation in the ventral prostates (VP’s) of 40-week-old AROM+ mice, which was characterized by a number of distinct lesions with varied cellular infiltrate. This included mononuclear lymphocyte-like cells and granulocytes throughout the stroma, as well as extensive infiltrate within the lumen, comprised of macrophages, plasma cells, neutrophils, and some cellular debris (Figure 1, B and C). No evidence of inflammation was apparent in the prostates of age-match wild-type littermate controls (Figure 1A). Following the emergence of the inflammation, the AROM+ mice were also found to develop PIN lesions that were reminiscent of PIN in the human (Figure 1D). The AROM+ mice also developed scrotal hernias, leading to protrusions of abdominal fat and intestine into the scrotum (Figure 1E). This pathology increased in incidence and severity with age, affecting in excess of 60% of AROM+ males by 35 weeks of age (Figure 1F). In severe cases, the majority of the intestinal tract was found to have protruded through into the scrotum as a result. Although severe hernias such as these may ultimately cause inflammation due to drainage defects of blood vessels and possible bacterial infections, this is not the cause of the inflammation in the AROM+ mice: the chronic inflammation observed is seen in animals that develop hernias, as well as in those that do not. The AROM+ mouse has ubiquitous, constitutively activated expression of aromatase,19Li X Nokkala E Yan W Streng T Saarinen N Warri A Huhtaniemi I Santti R Makela S Poutanen M Altered structure and function of reproductive organs in transgenic male mice overexpressing human aromatase.Endocrinology. 2001; 142: 2435-2442Crossref PubMed Scopus (130) Google Scholar including significant overexpression of aromatase within the prostate itself (Figure 2, A and B). Consequently, these animals should exhibit elevated levels of systemic estrogens with a concomitant decrease in androgen levels. Measurement of serum hormone concentrations in the AROM+ mice confirmed this, and, when compared with the wild-type littermate controls, showed that these mice have significantly decreased levels of serum testosterone at all ages, except in the 45- to 59-week age group (Table 1), and significantly increased levels of serum estrogens at all ages (Table 2).Table 1Reduced Serum Testosterone Levels in AROM+ MiceTestosterone (ng/ml)Age (weeks)Wild-typeAROM+Significance58.54 ± 5.850.10 ± 0.06P = 0.0141157.44 ± 9.480.66 ± 0.29P = 0.021626–367.50 ± 10.780.94 ± 0.69P = 0.021445–595.84 ± 8.183.07 ± 4.54NSCirculating serum testosterone levels in AROM+ mice were determined by RIA and were significantly reduced in AROM+ mice versus wild-type controls across all age groups, except at 45 to 59 weeks of age (n ≥10 for each age group). Open table in a new tab Table 2Serum Estradiol Levels Are Increased in AROM+ MiceEstradiol (pg/ml)SignificanceAge (weeks)Wild-typeAROM+515.61 ± 8.8249.86 ± 18.5P < 0.04431526.44 ± 12.26143.9 ± 82.67P < 0.000326–3642.61 ± 20.67165.3 ± 92.68P < 0.000245–5938.08 ± 10.91302.6 ± 301.5P < 0.0089Circulating serum estradiol levels in AROM+ mice were determined by RIA. Estradiol levels were found to be significantly increased in AROM+ mice when compared with wild-type controls across all age groups (n ≥10 for each age group). Open table in a new tab Circulating serum testosterone levels in AROM+ mice were determined by RIA and were significantly reduced in AROM+ mice versus wild-type controls across all age groups, except at 45 to 59 weeks of age (n ≥10 for each age group). Circulating serum estradiol levels in AROM+ mice were determined by RIA. Estradiol levels were found to be significantly increased in AROM+ mice when compared with wild-type controls across all age groups (n ≥10 for each age group). Closer analysis of the hormone levels in individual animals was made by expressing the absolute hormone levels as a ra
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