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• W2016235551 abstract "Exposure of newborn male mice to estrogens is associated with age-related changes in prostate size and induction of epithelial hyperplasia and dysplasia. Whether these changes directly result from systemic estrogen administration or indirect effects of estrogens on systemic testosterone levels is unclear. We have addressed this question using aromatase-knockout (ArKO) mice that are estrogen-deficient during their lifespan but have elevated androgen levels and develop prostate enlargement and hyperplasia (McPherson SJ, Wang H, Jones ME, Pedersen J, Iismaa TP, Wreford N, Simpson ER, Risbridger GP: Endocrinology 2001, 142:2458–2467). Circulating testosterone and dihydrotestosterone levels were significantly decreased by neonatal diethylstilbestrol treatment, remained suppressed in adult wild-type mice, but rapidly returned to control levels in ArKO animals. However, adult prostate weight and luminal size were reduced in both wild-type and ArKO animals. Because both wild-type and ArKO mice developed epithelial hyperplasia and inflammation following neonatal diethylstilbestrol treatment, this validates that estrogens directly cause prostatic inflammation and epithelial hyperplasia. Furthermore, because ArKO mice are estrogen-deficient, this study demonstrates the sensitivity of the neonatal period to estrogen exposure and the long range and permanent nature of the prostatic responses that occur. Finally, this study establishes the ArKO mouse model of estrogen deficiency as a unique approach to study the effects of estrogens, estrogenic factors, and endocrine disruptors on prostate development. Exposure of newborn male mice to estrogens is associated with age-related changes in prostate size and induction of epithelial hyperplasia and dysplasia. Whether these changes directly result from systemic estrogen administration or indirect effects of estrogens on systemic testosterone levels is unclear. We have addressed this question using aromatase-knockout (ArKO) mice that are estrogen-deficient during their lifespan but have elevated androgen levels and develop prostate enlargement and hyperplasia (McPherson SJ, Wang H, Jones ME, Pedersen J, Iismaa TP, Wreford N, Simpson ER, Risbridger GP: Endocrinology 2001, 142:2458–2467). Circulating testosterone and dihydrotestosterone levels were significantly decreased by neonatal diethylstilbestrol treatment, remained suppressed in adult wild-type mice, but rapidly returned to control levels in ArKO animals. However, adult prostate weight and luminal size were reduced in both wild-type and ArKO animals. Because both wild-type and ArKO mice developed epithelial hyperplasia and inflammation following neonatal diethylstilbestrol treatment, this validates that estrogens directly cause prostatic inflammation and epithelial hyperplasia. Furthermore, because ArKO mice are estrogen-deficient, this study demonstrates the sensitivity of the neonatal period to estrogen exposure and the long range and permanent nature of the prostatic responses that occur. Finally, this study establishes the ArKO mouse model of estrogen deficiency as a unique approach to study the effects of estrogens, estrogenic factors, and endocrine disruptors on prostate development. The prostate gland is an androgen-dependent organ. Androgen ablation by castration results in involution of the prostate and activation of apoptosis, and these effects can be reversed following the restoration of androgens.1Kyprianou N Isaacs JT Activation of programmed cell death in the rat ventral prostate after castration.Endocrinology. 1988; 122: 552-562Crossref PubMed Scopus (633) Google Scholar, 2Isaacs JT Antagonistic effect of androgen on prostatic cell death.Prostate. 1984; 5: 545-557Crossref PubMed Scopus (290) Google Scholar Circulating testosterone entering the prostate can be metabolized to 5α-dihydrotestosterone (DHT) by local 5α-reductase activity.3Anderson KM Liao S Selective retention of dihydrotestosterone by prostatic nuclei.Nature. 1968; 219: 277-279Crossref PubMed Scopus (435) Google Scholar, 4Bruchovsky N Wilson JD The conversion of testosterone to 5-alpha-androstan-17-beta-ol-3-one by rat prostate in vivo and in vitro.J Biol Chem. 1968; 243: 2012-2021Abstract Full Text PDF PubMed Google Scholar The effects of testosterone and DHT are mediated by activation of the intracellular androgen receptor.Although primarily influenced by androgens, the prostate is also an estrogen-target organ. The biosynthesis of estrogens occurs via metabolism of an androgenic substrate, catalyzed by an enzyme complex known as aromatase.5Goto J Fishman J Participation of a nonenzymatic transformation in the biosynthesis of estrogens from androgens.Science. 1977; 195: 80-81Crossref PubMed Scopus (87) Google Scholar Detection of aromatase expression in the prostate is evidence for local estrogen synthesis,6Tsugaya M Harada N Tozawa K Yamada Y Hayashi Y Tanaka S Maruyama K Kohri K Aromatase mRNA levels in benign prostatic hyperplasia and prostate cancer.Int J Urol. 1996; 3: 292-296Crossref PubMed Scopus (39) Google Scholar, 7Stone NN Laudone VP Fair WR Fishman J Aromatization of androstenedione to estrogen by benign prostatic hyperplasia, prostate cancer and expressed prostatic secretions.Urol Res. 1987; 15: 165-167Crossref PubMed Scopus (15) Google Scholar, 8Negri-Cesi P Colciago A Poletti A Motta M 5α-Reductase isozymes and aromatase are differentially expressed and active in the androgen-independent human prostate cancer cell lines DU145 and PC3.Prostate. 1999; 41: 224-232Crossref PubMed Scopus (44) Google Scholar, 9Matzkin H Soloway MS Immunohistochemical evidence of the existence and localization of aromatase in human prostatic tissue.Prostate. 1992; 21: 309-314Crossref PubMed Scopus (57) Google Scholar, 10Kaburagi Y Marino MB Kirdani RY Greco JP Karr JP Sandberg AA The possibility of aromatization of androgen in human prostate.J Steroid Biochem. 1987; 26: 739-742Crossref PubMed Scopus (37) Google Scholar, 11Hiramatsu M Maehara I Ozaki M Harada N Orikasa S Sasano H Aromatase in hyperplasia and carcinoma of the human prostate.Prostate. 1997; 31: 118-124Crossref PubMed Scopus (69) Google Scholar, 12Harada N Utsumi T Takagi Y Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and promoters, and switching of tissue-specific exons 1 in carcinogenesis.Proc Natl Acad Sci USA. 1993; 90: 11312-11316Crossref PubMed Scopus (348) Google Scholar, 13Ellem 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 and the identification of estrogen receptor (ER) subtypes ERα and ERβ14Kuiper GG Enmark E Pelto-Huikko M Nilsson S Gustafsson JA Cloning of a novel estrogen receptor expressed in rat prostate and ovary.Proc Natl Acad Sci USA. 1996; 93: 5925-5930Crossref PubMed Scopus (4194) Google Scholar, 15Prins GS Birch L Neonatal estrogen exposure up-regulates estrogen receptor expression in the developing and adult rat prostate lobes.Endocrinology. 1997; 138: 1801-1809Crossref PubMed Scopus (142) Google Scholar confirms that estrogen signaling pathways exist in the prostate.Direct effects of estrogens are difficult to determine because the effects of exogenous estrogen administration are centrally mediated and disrupt the normal endocrine environment, eliciting negative feedback inhibition of endogenous gonadotropin production and depression of testicular androgen biosynthesis.16Huhtaniemi IT Warren DD Catt KJ Comparison of oestrogen and GnRH agonist analogue-induced inhibition of the pituitary-testicular function in rat.Acta Endocrinol (Copenh). 1983; 103: 163-171PubMed Google Scholar, 17Karr JP Wajsman Z Kirdani RY Murphy GP Sandberg AA Effects of diethylstilbestrol and estramustine phosphate on serum sex hormone binding globulin and testosterone levels in prostate cancer patients.J Urol. 1980; 124: 232-236PubMed Google Scholar However, we recently studied direct estrogen actions in the prostate using the hypogonadal mouse (hpg) model, deficient in gonadotropin and sex steroid synthesis.18Bianco 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 (83) Google Scholar The effects of unopposed estradiol exposure were proliferative and included significant enlargement of prostate lobes. Aberrant growth was observed, including proliferation of stromal fibroblasts and epithelial basal cells accompanied by a disruption and reduction to smooth muscle and secretory epithelial cells. A prostatic inflammatory response was also identified in this model as has been previously shown using other models.18Bianco 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 (83) Google Scholar Conversely, the effects of unopposed androgens on the prostate were also proliferative, as evident in the aromatase-knockout (ArKO) mouse model, which is estrogen-deficient.19Fisher CR Graves KH Parlow AF Simpson ER Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene.Proc Natl Acad Sci USA. 1998; 95: 6965-6970Crossref PubMed Scopus (758) Google Scholar As a consequence of failure to metabolize androgens to estrogens, the ArKO mouse exhibited elevated peripheral and intraprostatic androgen levels as well as increased androgen receptor immunoexpression.20McPherson SJ Wang H Jones ME Pedersen J Iismaa TP Wreford N Simpson ER Risbridger GP Elevated androgens and prolactin in aromatase-deficient mice cause enlargement, but not malignancy, of the prostate gland.Endocrinology. 2001; 142: 2458-2467Crossref PubMed Scopus (132) Google Scholar Prostate lobes were enlarged and showed uniform volumetric expansion of stromal, epithelial, and luminal compartments.1Kyprianou N Isaacs JT Activation of programmed cell death in the rat ventral prostate after castration.Endocrinology. 1988; 122: 552-562Crossref PubMed Scopus (633) Google Scholar These data therefore provide evidence that both androgens and estrogens are proliferative in the prostate but in different ways. Coordinated growth and proliferation occur in response to androgens, whereas estrogens lead to uncoordinated and aberrant patterns of proliferation.The brief exposure of newborn male rats to pharmacological doses of estrogens produces multiple changes to prostate growth, morphology, and function, including alterations to hormonal sensitivity in later life.21Prins GS Woodham C Lepinske M Birch L Effects of neonatal estrogen exposure on prostatic secretory genes and their correlation with androgen receptor expression in the separate prostate lobes of the adult rat.Endocrinology. 1993; 132: 2387-2398Crossref PubMed Scopus (63) Google Scholar, 22Prins GS Neonatal estrogen exposure induces lobe-specific alterations in adult rat prostate androgen receptor expression.Endocrinology. 1992; 130: 3703-3714Crossref PubMed Scopus (76) Google Scholar, 23Rajfer J Coffey DS Sex steroid imprinting of the immature prostate. Long-term effects.Invest Urol. 1978; 16: 186-190PubMed Google Scholar, 24Naslund MJ Coffey DS The differential effects of neonatal androgen, estrogen and progesterone on adult rat prostate growth.J Urol. 1986; 136: 1136-1140Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 25Naslund 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 This process is referred to as neonatal imprinting or developmental estrogenization. Exposure to estrogens during early life reduces androgen sensitivity in later life24Naslund MJ Coffey DS The differential effects of neonatal androgen, estrogen and progesterone on adult rat prostate growth.J Urol. 1986; 136: 1136-1140Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 26Rajfer J Coffey DS Effects of neonatal steroids on male sex tissues.Invest Urol. 1979; 17: 3-8PubMed Google Scholar as a result of down-regulation and accelerated degradation of prostatic androgen receptor.21Prins GS Woodham C Lepinske M Birch L Effects of neonatal estrogen exposure on prostatic secretory genes and their correlation with androgen receptor expression in the separate prostate lobes of the adult rat.Endocrinology. 1993; 132: 2387-2398Crossref PubMed Scopus (63) Google Scholar, 22Prins GS Neonatal estrogen exposure induces lobe-specific alterations in adult rat prostate androgen receptor expression.Endocrinology. 1992; 130: 3703-3714Crossref PubMed Scopus (76) Google Scholar, 27Prins GS Birch L The developmental pattern of androgen receptor expression in rat prostate lobes is altered after neonatal exposure to estrogen.Endocrinology. 1995; 136: 1303-1314Crossref PubMed Google Scholar, 28Woodham C Birch L Prins GS Neonatal estrogen down-regulates prostatic androgen receptor through a proteosome-mediated protein degradation pathway.Endocrinology. 2003; 144: 4841-4850Crossref PubMed Scopus (67) Google Scholar The subsequent effects on the prostate include manifestation of atypical pathological changes with age, including inflammation, epithelial hyperplasia, and the emergence of dysplastic lesions.22Prins GS Neonatal estrogen exposure induces lobe-specific alterations in adult rat prostate androgen receptor expression.Endocrinology. 1992; 130: 3703-3714Crossref PubMed Scopus (76) Google Scholar, 29Santti R Newbold RR Pylkkanen MS McLachlan JA Developmental estrogenization and prostatic neoplasia.Prostate. 1994; 24: 67-78Crossref PubMed Scopus (131) Google Scholar, 30Prins 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, 31Pylkkanen L Santti R Newbold R McLachlan JA Regional differences in the prostate of the neonatally estrogenized mouse.Prostate. 1991; 18: 117-129Crossref PubMed Scopus (76) Google Scholar, 32Pylkkanen L Makela S Valve E Harkonen P Toikkanen S Santti R Prostatic dysplasia associated with increased expression of c-myc in neonatally estrogenized mice.J Urol. 1993; 149: 1593-1601PubMed Google Scholar, 33Gilleran JP Putz O DeJong M DeJong S Birch L Pu Y Huang L Prins GS The role of prolactin in the prostatic inflammatory response to neonatal estrogen.Endocrinology. 2003; 144: 2046-2054Crossref PubMed Scopus (48) Google Scholar These changes are of major importance, because specific inflammatory pathologies may be positively associated with benign and malignant changes in the prostate.34De 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 (721) Google Scholar, 35Putzi MJ De Marzo AM Morphologic transitions between proliferative inflammatory atrophy and high-grade prostatic intraepithelial neoplasia.Urology. 2000; 56: 828-832Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 36van Leenders GJ Gage WR Hicks JL van Balken B Aalders TW Schalken JA De Marzo AM Intermediate cells in human prostate epithelium are enriched in proliferative inflammatory atrophy.Am J Pathol. 2003; 162: 1529-1537Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Dysplastic epithelial lesions, otherwise referred to as prostatic intraepithelial neoplasia (PIN), precede the emergence of prostatic carcinoma.37McNeal JE Bostwick DG Intraductal dysplasia: a premalignant lesion of the prostate.Hum Pathol. 1986; 17: 64-71Abstract Full Text PDF PubMed Scopus (479) Google Scholar, 38McNeal JE Significance of duct-acinar dysplasia in prostatic carcinogenesis.Urology. 1989; 34: 9-15PubMed Google Scholar, 39McNeal JE Villers A Redwine EA Freiha FS Stamey TA Microcarcinoma in the prostate: its association with duct-acinar dysplasia.Hum Pathol. 1991; 22: 644-652Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 40Bostwick DG Brawer MK Prostatic intra-epithelial neoplasia and early invasion in prostate cancer.Cancer. 1987; 59: 788-794Crossref PubMed Scopus (452) Google Scholar, 41Brawer MK Prostatic intraepithelial neoplasia: a premalignant lesion.Hum Pathol. 1992; 23: 242-248Abstract Full Text PDF PubMed Scopus (90) Google Scholar The role of estrogens in mediating these changes is not well understood, because androgen production and action are suppressed, and it is difficult to distinguish between androgenic and estrogenic effects. However, it was shown using ER-deficient mouse models that the ERα subtype mediates acute and chronic pathological responses to developmental estrogenization, whereas ERβ is not required for these changes to occur.30Prins 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 Furthermore, neonatal exposure to the androgen receptor antagonist flutamide was shown not to mimic any of the changes elicited by early estrogen exposure,22Prins GS Neonatal estrogen exposure induces lobe-specific alterations in adult rat prostate androgen receptor expression.Endocrinology. 1992; 130: 3703-3714Crossref PubMed Scopus (76) Google Scholar supporting a direct effect of estrogen action on the prostate during estrogenization. To examine the long range effects of developmental estrogenization and assess the direct and indirect responses of the prostate gland, the estrogen-deficient (ArKO mouse) model was used.Materials and MethodsAnimalsThe ArKO mouse colony was originally bred on a C57BL/6J background, generated by targeted disruption of the cyp19 gene as previously described.19Fisher CR Graves KH Parlow AF Simpson ER Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene.Proc Natl Acad Sci USA. 1998; 95: 6965-6970Crossref PubMed Scopus (758) Google Scholar All mice were maintained under controlled conditions (lights on 7:00 AM to 7:00 PM; temperature 20 to 4°C), with free access to mouse feed and water. Soy-free mouse chow (Glen Forrest Stockfeeders, Glen Forrest, Australia) was provided for all animals in this study, because regular mouse chow has been shown to contain isoflavones.42Robertson KM O'Donnell L Simpson ER Jones ME The phenotype of the aromatase knockout mouse reveals dietary phytoestrogens impact significantly on testis function.Endocrinology. 2002; 143: 2913-2921Crossref PubMed Scopus (84) Google Scholar All studies were conducted with approval from the animal ethics committee of Monash University in accordance with guidelines of the National Health and Medical Research Council.The mice used in this study were generated by breeding male and female mice heterozygous for the cyp19 gene, for the production of homozygous aromatase +/+ or −/− offspring. Pregnant mice were monitored daily for the delivery of pups, and the day of birth was designated day 0. Pups were sexed, and the genotyping of tail DNA from male offspring was determined by PCR analysis. Pups were allocated randomly to one of two treatment groups, administered either 2 μg of diethylstilbestrol (DES; Sigma Chemical Co., St. Louis, MO) in peanut oil (Sigma Chemical Co.) or peanut oil alone on postnatal days 1–5 via injection at the nape of the neck. For each group five to eight animals were used.Monthly body weights were determined, and serum samples were collected by tail bleeding into capillary blood collection tubes (Microvette CB300KE; Sarstedt, Nümbrecht, Germany) from 50 days (∼7 weeks) of age. At 90 and 180 days of age, animals were weighed followed by sacrifice via cervical dislocation, and terminal serum samples were collected and stored at −20°C. To prevent variations in hormone concentrations as a consequence of diurnal hormonal secretion, sera were collected during a controlled period (1000 to 1400 hours). Testes, seminal vesicles (SV) and anterior (AP), ventral (VP), dorsal (DP), and lateral (LP) prostate lobes were excised and dissected free of extraneous fat. Organs were weighed and immersion-fixed in Bouin's fixative before processing. Following dehydration, organs were embedded in paraffin wax and serially sectioned (5 μm).HistopathologyFollowing serial sectioning of tissues, sections were sampled at 100-μm intervals (every 20th section) for AP and at 50-μm intervals (every 10th section) for remaining prostate lobes (VP, DP, and LP). Sections were stained with Mayer's hematoxylin and eosin before unbiased blind histological examination by a pathologist.Hormone AssaysTestosterone levels in plasma were assessed via organic extraction with a 3:2 mixture of hexane and ethyl acetate. Procedural recoveries were calculated with a parallel sample with tritiated testosterone added. The organic fraction was then dried overnight and reconstituted with a 1% gelatin phosphate-buffered saline buffer. The reconstituted aliquots were processed in a radioimmune precipitation assay with a specific antibody raised to testosterone (T3-125; Endocrine Sciences Laboratories, Calabasas, CA) and a liquid chromatography-purified tritiated testosterone. After 16-hour incubation at 4°C, free and bound hormones were separated with dextran T70-coated charcoal. The testosterone standard was calibrated against a World Health Organization testosterone preparation (coefficient of variation 3.1–7.5%). DHT was extracted, dried, and reconstituted as per the testosterone assay. The aliquots were then oxidized by exposure to 0.5% potassium permanganate for 30 minutes. Oxidation was terminated via a second organic extraction. The dried organic extract was reconstituted and assayed using antibody C0457 (Bioquest, North Ryde, Australia) and a liquid chromatography-purified tritiated DHT tracer. Procedural recoveries were calculated as per testosterone but with a DHT tracer (coefficient of variation 3.8–4.6%).Stereological AnalysisAll assessments were performed using a BX-51 microscope (Olympus Corp., Tokyo, Japan). The images were captured by a JVC TK-C1380 color video camera (Victor Co., Tokyo, Japan) coupled to an IBM computer and projected directly onto a video screen using an Integral Flashpoint 3Dx frame grabber video adaptor (Integral Technologies Inc., Indianapolis, IN). CAST software (version 2.1.4; Olympus Corp.) was used to generate a set of counting frames and a point grid (grid properties were assessed individually for each organ and treatment group; sampling was conducted at predetermined intervals along x- and y-axes). Fields were selected by a systematic uniform random sampling scheme, and volumes of tissue compartments were determined based on protocols modified from those previously used in the testis43Meachem SJ McLachlan RI de Kretser DM Robertson DM Wreford NG Neonatal exposure of rats to recombinant follicle stimulating hormone increases adult Sertoli and spermatogenic cell numbers.Biol Reprod. 1996; 54: 36-44Crossref PubMed Scopus (142) Google Scholar and prostate.20McPherson SJ Wang H Jones ME Pedersen J Iismaa TP Wreford N Simpson ER Risbridger GP Elevated androgens and prolactin in aromatase-deficient mice cause enlargement, but not malignancy, of the prostate gland.Endocrinology. 2001; 142: 2458-2467Crossref PubMed Scopus (132) Google Scholar, 44Singh J Zhu Q Handelsman DJ Stereological evaluation of mouse prostate development.J Androl. 1999; 20: 251-258PubMed Google ScholarHematoxylin and eosin-stained serial sections 50 μm apart were examined under ×40 magnification. VP tissues were classified into three compartments: stroma, epithelium, and lumen. The relative volume of stroma, epithelium, and lumen per organ was determined by the sum of the number of points that landed on each compartment divided by the sum of the number of points contacting the entire organ. At least 100 counts per tissue compartment were obtained. The absolute volume of each tissue compartment was determined by multiplying the organ weight and the relative volume.ImmunohistochemistryProliferating cells were identified by immunostaining for proliferating cell nuclear antigen (PCNA; DAKO, Carpentaria, CA). Immunohistochemistry was performed by using a DAKO autostainer. Briefly, individual organs were sectioned longitudinally (5 μm) to reveal the proximo-distal orientation and deparaffinized, rehydrated, and treated with peroxidase block (DAKO) for 10 minutes. Antigen retrieval (0.01 mol/L citrate buffer, pH 6.0, as per the manufacturer's specifications) was used before inactivation of endogenous peroxidase, and nonspecific binding was blocked using CAS block (Zymed, San Francisco, CA), followed by incubation with primary antibody for 30 minutes at room temperature. Primary antibody binding was detected using biotinylated rabbit anti-mouse IgG2A antibody (Zymed) followed by incubation with an avidin-biotin peroxidase kit (ABC Elite; Vector Laboratories, Burlingame, CA) for 15 minutes (PCNA). Antibody localization was visualized using diaminobenzidine tetrachloride as a chromogen. Finally, sections were counterstained with Mayer's hematoxylin, gradually dehydrated with alcohol, cleared with Histolene, and covered with a coverslip and with DPX mounting solution.PCNA QuantitationA semiquantitative approach was used to determine the percentage of cells (stromal and epithelial) showing positive immunostaining for PCNA. Briefly, blocks of prostate tissues (four to five animals per group) were selected randomly and sectioned longitudinally to reveal the proximo-distal orientation; sections from each animal included paired lobes. Tissues from all groups were processed in the same immunohistochemical assay to eliminate interassay variation. Following PCNA immunostaining, 5-μm sections were subjected to systematic sampling commencing at a random point using an unbiased counting frame generated using CAST software (version 2.1.4; Olympus Corp.). Using ×40 magnification, stromal and epithelial cells were classified as PCNA-positive or -negative. Cell numbers were combined (minimum of 600 cells counted per organ), and percentages of PCNA-positive epithelial and stromal cells were determined.Statistical AnalysisData were compared among genotypes (wild-type (WT) and ArKO), treatment groups (oil and DES), and ages (90 and 180 days). 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), or by one-way analysis of variance analysis followed by the posthoc Tukey multiple comparison test. The analyses were conducted using Prism 4.00 software (GraphPad Software Inc., San Diego, CA). Data are expressed as mean ± SE.ResultsNeonatal DES Treatment Reduces Serum Androgen Levels in ArKO Mice but Not Below Normal Levels in Untreated WT ControlsCirculating testosterone concentrations were significantly elevated in ArKO control versus WT control mice aged 50 days as previously reported (Figure 1A; P < 0.05). DES treatment to WT mice significantly reduced testosterone and DHT concentrations at ages 50 and 90 days (Figure 1, A and B; P < 0.05). DES treatment to ArKO mice reduced testosterone and DHT levels in mice 50 days old, although they were no different from WT controls (Figure 1, A and B; P < 0.05); no changes were identified after this time. Following DES treatment, the testosterone and DHT levels in ArKO mice were higher than or no different from (but never lower than) WT controls treated with DES (Figure 1, A and B; P = 0.08 and 0.08, respectively).Body and Organ WeightsBody, testicular, and seminal vesicle weights were measured as indirect indicators of androgen status in DES-treated WT and ArKO mice. No changes to body weights were identified between WT and ArKO control animals, and DES treatment did not significantly alter body weights in WT or ArKO groups (data not shown). Although androgen levels were elevated in ArKO mice, the testis weights were not significantly different to WT; however, neonatal DES treatment significantly reduced testis weights of both WT and ArKO animals at 90 and 180 days of age (Figure 2A; P < 0.01), consistent with the initial decrease in androgen levels between 50 or 90 days of age (see Figure 1). Similarly, SV organ weights were significantly elevated (P < 0.05) in ArKO control versus WT control animals at 90 (P < 0.01) and 180 days (Figure 2B; P < 0.05). DES treatment significantly reduced WT and ArKO SV weights at both ages (P < 0.05 and P < 0.001), although by 180 days ArKO SVs had again become heavier than in WT littermates (162.9 ± 44.9 vs. 75.8 ± 40.07 mg).Figure 2Effects of neonatal DES on nonprostatic reproductive organ weights. A: Testis weights of WT and ArKO control animals were not significantly different, but neonatal DES treatment significantly reduced testis weights of WT and ArKO animals at 90 and 180 days of age (P < 0.01). B: Seminal vesicle weights were significantly elevated (P < 0.05) in ArKO control versus WT control animals at 90 (P < 0.01) and 180 days (P < 0.05). DES treatment significantly reduced WT and ArKO SV weights at 90 (P < 0.05) and 180 days (P < 0.001). Different superscripts indicate significant comparisons.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The weights of the lobes of the prostate gland (VP, AP, LP, and DP) were determined as shown in Table 1. A" @default.
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• W2016235551 title "Transient Neonatal Estrogen Exposure to Estrogen-Deficient Mice (Aromatase Knockout) Reduces Prostate Weight and Induces Inflammation in Late Life" @default.
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• W2016235551 doi "https://doi.org/10.2353/ajpath.2006.050623" @default.
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