Differential progression of neonatal diethylstilbestrol-induced disruption of the hamster testis and seminal vesicle

Reproductive Toxicology 21 (2006) 225–240

Differential progression of neonatal diethylstilbestrol-induced disruption of the hamster testis and seminal vesicle William J. Hendry III a,∗ , Benjamin P. Weaver a , Teran R. Naccarato a , Shafiq A. Khan b a b

Department of Biological Sciences, Wichita State University, 1845 Fairmount, Wichita, KS 67260-0026, United States Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA 30314, United States Received 12 May 2005; received in revised form 10 September 2005; accepted 26 September 2005 Available online 24 January 2006

Abstract The synthetic estrogen diethylstilbestrol (DES) is now recognized as the prototypical endocrine disruptor. Using a hamster experimental system, we performed a detailed temporal assessment of how neonatal DES-induced disruption progresses in the testis compared to the seminal vesicle. Both morphological and Western blot analyses confirmed that neonatal DES exposure alters androgen responsiveness in the male hamster reproductive tract. We also determined that the disruption phenomenon in the male hamster is manifest much earlier in the seminal vesicle than in the testis and that testis disruption often occurs differently between the pair of organs in a given animal. In the neonatally DES-exposed seminal vesicle, histopathological effects included: (1) general atrophy, (2) lack of exocrine products, (3) epithelial dysplasia, (4) altered organization of stromal cells and extracellular matrix, and (5) striking infiltration with polymorphonuclear leukocytes. Also, the morphological disruption phenomenon in the seminal vesicle was accompanied by a range of up-regulation and down-regulation responses in the whole organ levels of various proteins. © 2005 Elsevier Inc. All rights reserved. Keywords: Endocrine disruption; Diethylstilbestrol (DES); Testis; Seminal vesicle; Male reproductive system

1. Introduction Diethylstilbestrol (DES), synthesized in 1938, was the first orally active estrogen [1]. Beginning in 1947, it was widely prescribed for pregnant women in the mistaken belief that it would protect against miscarriage [2]. That practice ceased in 1971 with the first report of reproductive tract anomalies and cancer in the offspring of DES-treated mothers [3,4]. During the over 2 decades of DES usage in the United States alone, it is estimated that at least four million pregnant women and their fetuses were exposed to the potent estrogen [5]. The scope of this unfortunate phenomenon attracted considerable biomedical and legal attention and became commonly known as the “DES syndrome”. Historically, most epidemiological and experimental investigations of the DES syndrome have focused on females [6]. However, the sons of DES-treated mothers also suffered reproductive tract perturbations including infertility, cryptorchidism, epididymal cysts, and retained M¨ullerian ducts [7]. This set of

∗

Corresponding author. Tel.: +1 316 978 6086; fax: +1 316 978 3772. E-mail address: [email protected] (W.J. Hendry III).

0890-6238/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2005.09.014

perturbations in DES-exposed sons has been called a developmental estrogenization syndrome [7]. The extent and severity of the phenomenon in both sexes have established DES as the prototypical endocrine disruptor. Various rodent species have been used to investigate the progression and mechanism of the DES syndrome [8–11]. The species we have used is the hamster and it proved to be a very convenient and sensitive experimental system to probe the phenomenon of perinatal endocrine disruption induced by DES and by other agents [12]. After using the hamster system to focus on disruptive effects in the female reproductive tract, especially the uterus, more recently we have used it to study neonatal DES-induced disruption in the male reproductive tract [13,14]. Those studies suggested that the temporal and functional nature of the disruption phenomenon was different in two male reproductive tract organs, the testis and the seminal vesicle. To more stringently test that hypothesis, we performed a weekly evaluation of the initiation and progression of neonatal DES-induced histopathology in the two organs. That analysis showed that the seminal vesicle exhibited much earlier signs of DES-induced disruption. To gain further insight into the mechanism of the disruption phenomenon, we conducted a proteomic

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screening process. It showed that the morphological disruption phenomenon in the seminal vesicle was accompanied by a range of up-regulation and down-regulation responses in the whole organ levels of various proteins. 2. Materials and methods

vanadate, 1% SDS), received 4.5 volumes of 2× electrophoresis sample buffer (125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% ␤-mercaptoethanol), mixed, and boiled for 3 min. In other words, both procedures yielded groups of extracts that were matched or normalized based on tissue equivalents rather than total protein content. According to evaluation of Coomassie-stained gels and blots probed with the same antibody (anti-Ecadherin), electrophoretic resolution, and immunoblotting performance was comparable with the two types of extracts (data not shown).

2.1. Animals 2.5. Western blot analysis Procedures for neonatal DES treatment and tissue harvesting were as described previously [13,14]. In brief, timed-pregnant Syrian golden hamsters (Mesocricetus auratus) from Harlan Sprague–Dawley Inc. (Indianapolis, IN) were caged singly under a 14-h light/10-h dark photoperiod with food and water ad libitum. The food was a 2:1 mixture of #5001 rodent diet and #5015 mouse diet from LabDiet (PMI Nutrition Int. LLC, Brentwood, MO). According to the manufacturer, the total isoflavone (diadzein, genistein, glycitein) content of that diet mixture is 426 mg/kg diet. Within 6 h of birth (day 0), the size of each litter was adjusted by retaining all of its male members and enough of its female members to yield a total of eight pups per litter. The surplus females were killed by CO2 asphyxiation and the retained females were later used for other studies. All retained animals received a single s.c. injection of 50 ␮l of corn oil vehicle either alone (control) or containing 100 ␮g of DES (∼33 mg/kg body weight). As acknowledged previously [12,15,16], that dose level is high but not unreasonable considering that DES ingestion by pregnant women was as much as 150 mg daily and 18.2 g in total during their pregnancy [17]. However, it should also be acknowledged that such a single and relatively high dose of DES administered to newborn hamsters does not exactly model the clinical DES syndrome where exposure was to many but relatively lower doses in utero over months. At weekly time points from 1 to 13 weeks of age after DES treatment, males were anesthetized with CO2 , weighed, and then killed either by cervical dislocation when they were so small (weeks 1 and 2) that only tissue collection was practical, or by decapitation (weeks 3–13) when they were large enough that a sufficient volume of trunk blood could also be collected and then processed for serum separation.

2.2. Tissue harvesting and processing All testes and most seminal vesicles were immersed in commercial fixative (Z-Fix® from ANATECH Ltd., Battle Creek, MI) at room temperature. At 24-h intervals, tissues were reimmersed in fresh fixative and then in 70% ethanol. Before histological processing (paraffin embedding and sectioning for standard hematoxylin and eosin staining), all fixed tissues were lightly blotted and then weighed so that normalized values (mg individual testis or seminal vesicle weight/g whole body weight) could be calculated. For Western blot analyses (see below), unfixed seminal vesicles were repeatedly pierced with a needle and then blotted between paper towels to remove internal secretory material, rinsed in chilled PBS, reblotted, and cryostored at −80 ◦ C. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and were performed in an AAALAC-approved facility.

2.3. Testosterone radioimmunoassay Serum testosterone levels were determined by an immunoassay procedure as described previously [18]. The anti-testosterone antiserum [19] was provided by Dr. G. Niswender (Fort Collins, CO).

2.4. Preparation of total protein extracts from seminal vesicles Frozen seminal vesicles from pubertal (week 6) animals were quickly weighed and used to prepare total protein extracts by two methods. In one method, tissues received 9 volumes (ml/g) of sample buffer (final conditions = 0.1 M dithiothreitol, 2% SDS, 0.08 M Tris base (pH 6.8), 10% glycerol, and 1% saturated bromophenol blue solution), were homogenized (3 × 5 s) with a Tekmar Model TR-19 Tissuemizer at a power setting of 70, and finally boiled for 3 min. In the other method, tissues were homogenized (as above) in 4.5 volumes of lysis buffer (10 mM Tris, pH 7.4, 1.0 mM sodium ortho-

Sample aliquots (50 ␮l/lane) were run under denaturing conditions on 5–15% acrylamide gradient gels. To visualize the overall pattern of resolved proteins, gels were rocked at room temperature first in gel fixer (10% glacial acetic acid (GAA), 50% methanol, and 50% H2 O) twice for 15 min and secondly in Coomassie Brilliant Blue (CBB) solution (0.05% CBB R250, 50% methanol, 10% GAA, and 40% H2 O) for 30 min. The gels were then rocked at room temperature in destain solution (5% methanol, 7% GAA, and 88% H2 O) until background staining was removed and protein bands were distinct. To prevent cracking during drying, gels were rocked at room temperature in two changes of gel dry solution (35% ethanol, 2% glycerol, and 63% H2 O) for 15 min intervals. The gels were then sandwiched between two wet cellophane sheets and dried. For immunoblotting, gels were electrotransferred to a nitrocellulose membrane using a Genie apparatus (Idea Scientific Co., Minneapolis, MN) and a buffer containing 192 mM glycine, 25 mM Tris base, 15% methanol, and 0.02% SDS. To visualize the positions of standard proteins and assess efficiency of protein transfer out of the gel plus adsorption to the membrane, the relevant membrane lane was removed and then rocked at room temperature for 30 min in 5 ml SDS-PAGE fixer containing 0.05% CBB. The remainder of the membrane was immunoprobed as follows. First, non-specific protein binding sites were blocked by incubating the membrane for 30 min at room temperature in PTM (Dulbecco phosphate buffered saline, 1% Tween 20, and 5% nonfat dry milk, pH 7.5). After this and all the following immunodetection steps, the membrane was washed five times with PBS/Tween (Dulbecco phosphate buffered saline containing 0.05% Tween 20, pH 7.5). The membrane was then incubated with primary antibody (see Table 1 for descriptions of antibody provider, species source, monoclonal or polyclonal type, and dilution or concentration used) in PTM for 2 h at room temperature or overnight at 4 ◦ C. Next, the membrane was incubated for 1 h at room temperature in PTM containing 3.75 ␮g/ml of appropriate species-specific anti-IgG antibody:biotin conjugate (Vector Laboratories, Burlingame, CA). The next step used an avidin–biotin-peroxidase complex reagent (Vectastain® ABC) prepared in PTM according to manufacturer instructions (Vector Laboratories). The membrane was incubated in the ABC reagent for 30 min at room temperature. The membrane was then exposed to a commercial substrate solution (SigmaFastTM from Sigma, St. Louis, MO) containing 0.7 mg/ml 3,3 -diaminobenzidine, 0.17 mg/ml urea/hydrogen peroxide, and 0.06 M Tris buffer. A positive enzymatic reaction in this system generates deposition of an insoluble, dark-brown product. Specificity of the signal bands was verified by the omission of first antibody and/or its substitution by normal mouse, rabbit, or goat IgG. Densitometric scans of the blots were analyzed using Quantity One® quantitation software (Version 4.1) from Bio-Rad Laboratories (Chicago, IL).

2.6. Microscopy All microscopy was done on a Nikon Eclipse E800 using Infinity Optics and Plan Apo objectives. Images were captured using a Magna Fire digital camera (Optronics, Goleta, CA) and AnalySIS® digital image analysis software (Soft Imaging System Corp., Lakewood, CO).

2.7. Statistics All statistics were determined using the Statistical Analysis System (SAS) PC Version 8.01. For most quantitative observations, Student’s t-tests were performed to determine if significant differences existed at the indicated time points between mean values for control versus DES-exposed groups. Also for the normalized weight of individual testes, we performed a homogeneity-of-variance

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Table 1 Summary of Western blot analyses and antibody informationa Proteinb

CON dens.d

DES dens.d

Amplified in breast cancer-1 (AIB1) 175 Myles Brown; Dana-Farber Cancer Institute, Boston, MA; rabbit pAb; 1:1000 [66]

175 ± 19

135 ± 16

Androgen receptor (AR) 108 (C-19) Santa Cruz Biotechnology; Santa Cruz, CA; rabbit pAb; 1:500

241 ± 24

142 ± 6

×

Cables 66 Lawrence Zukerberg; Massachusetts General Hospital; Boston, MA; rabbit pAb; 1:400 [21,22]

853 ± 46

249 ± 12

×

2201 ± 30 872 ± 22 3073 ± 18

649 ± 69 1178 ± 65 1826 ± 130

× × ×

401 ± 25

354 ± 13

1202 ± 68 502 ± 30 (1704 ± 85)

1194 ± 52 379 ± 29 (1573 ± 44)

E-cadherin

kDac

122 36

All isoforms (Clone 36) BD Transduction Laboratories; San Diego, CA; mouse mAb; 1:2500 Caspase-3 (CPP32) 36 Joseph L. Goldstein; UT Southwestern Medical Center, Dallas, TX; rabbit pAb; 1:1000 [67] ␤-Catenin

101 85

(all isoforms) (H-102) Santa Cruz Biotechnology; Santa Cruz, CA; rabbit pAb; 1:500 p120-Catenin 116 (H-90) Santa Cruz Biotechnology; Santa Cruz, CA; rabbit pAb; 1:500

760 ± 39

584 ± 131

Erythroblastosis B2 (c-erbB2) 176, 165 (Ab-1) Lab Vision/NeoMarkers; Fremont, CA; rabbit pAb; 1:500

355 ± 36

193 ± 9

Fibroblast growth factor, basic (bFGF) 21 (Clone 3) BD Transduction Laboratories; San Diego, CA; mouse mAb; 1:1000

322 ± 51

551 ± 51

Focal adhesion kinase (FAK) 111 317 ± 29 (4:47) William G. Cance; University of Florida College of Medicine, Gainesville, FL; mouse mAb; 1:100 [68]

p < 0.05e

×

×

230 ± 13

Insulin receptor substrate-1 (IRS-1) 148 (Clone 6) BD Transduction Laboratories; San Diego, CA; mouse mAb; 1:1000

144 ± 37

80 ± 11

Lactoferrin (LTF) 69 (Anti-mouse) Christina Teng; NIEHS, Research Triangle Park, NC; rabbit pAb; 1:5000 [69]

231 ± 25

1035 ± 87

2024 ± 133

1567 ± 247

376 ± 13

721 ± 79

×

Nuclear factor-kappa B, p65 (NF␬B) 66 (C-20) Santa Cruz Biotechnology; Santa Cruz, CA; rabbit pAb; 1:500

1063 ± 103

1451 ± 87

×

p63 70 (Ab-1) Lab Vision/NeoMarkers; Fremont, CA; mouse mAb; 0.5 ␮g/ml

72 ± 10

196 ± 26

×

Proliferating cell nuclear antigen (PCNA) 37 (K110) Biomeda Corporation; Foster City, CA; mouse mAb; 1:500

497 ± 18

624 ± 87

Tenascin 207 113 ± 23 (HxB-2873) Harold P. Erickson; Duke University Medical Center; Durham, NC; rabbit pAb; 1:1000 [70]

135 ± 31

Mammalian homolog of yeast transcriptional 140 Corepressor, Sin3p (mSin3A) (Clone 2) BD Transduction Laboratories; San Diego, CA; mouse mAb; 1:250 Nuclear factor-kappa B, p50 (NF-␬B) 47 StressGen Biotechnologies; Victoria, BC, Canada; rabbit pAb; 0.5 ␮g/ml

×

Transcription intermediary factor-1␤ (TIF1␤) 112 884 ± 19 967 ± 33 (1Tb-1A9) Pierre Chambon; CNRS/INSERM/ULP/College de France, Illkirch-Cedex, Strasbourg, France; mouse mAb; 1:500 [62] Uteroglobin (UG) 26 (Anti-human) Anil Mukherjee; NIH/NICHD, Bethesda, MD; rabbit pAb; 1:1000 [71] Vimentin 60 (Ab-2) Lab Vision/NeoMarkers; Fremont, CA; mouse mAb; 1:1000

824 ± 89

488 ± 79

1474 ± 209

1738 ± 148

×

Animals were injected on the day of birth with vehicle either alone (control, CON) or containing 100 ␮g of DES. Total protein extracts from the seminal vesicles of early adult animals (6 weeks of age) underwent Western blot analysis with the indicated antibodies. Specific immunodetected protein band levels were measured by densitometric analysis. a Antibody information includes identifying details in parentheses, donator or commercial source, whether it was a mouse monoclonal (mAb) or rabbit polyclonal (pAb) antibody, either the dilution or IgG concentration at which it was used, and for donated antibodies, a reference citing use of that antibody. b Name of the target protein and its common abbreviation. c Size of the target protein in kilodaltons. d Signal band density expressed as mean ± standard error, n = 3. e Significant difference between the extracts from control vs. DES-exposed organs according to Student’s t-test.

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test to determine if significant differences existed at the indicated time points for weight variance within the control versus DES-exposed groups (includes both within individual animal and within group variation). Both Student’s t-tests and homogeneity-of-variance tests that yielded differences with p values ≤0.05 were considered significant.

3. Results 3.1. Circulating testosterone levels The effect of neonatal DES treatment on serum testosterone levels in immature and adult hamsters is shown in Fig. 1. From weeks 3 to 6, levels were not significantly different between the control and DES-exposed groups. During that period, levels were low from weeks 3 to 5 (prepubertal stage) and then increased sharply between weeks 5 and 6 (pubertal stage). Thereafter (adult stage), levels in control animals were stable except for the elevated value measured at week 9. In contrast, levels in DES-exposed animals continued to rise after week 6 so that they were significantly higher than those in control animals at weeks 7 and 8. Then levels in DES-exposed animals fell so that they were lower than those in control animals at week 9. Finally, levels in the DES-exposed animals matched those in the control animals from weeks 10 to 13. These data indicate that the pubertal rise in testicular testosterone production begins similarly in both groups. However, a subsequent surge in testosterone production occurs sooner and is more prolonged and pronounced in the neonatally DES-exposed group. 3.2. Body and gross organ effects According to weekly determinations of body weight (Fig. 2A), the earlier and more prolonged hyperandrogenic environment induced by neonatal DES treatment affected neither the prepubertal growth rate nor the maximal body mass attained by adult male hamsters. Normalizing individual testis weight to body weight (Fig. 2B) showed that, in both the control and neonatally DES-exposed groups, gonad growth rate surpassed

Fig. 1. Effect of neonatal DES treatment on serum testosterone levels in male hamsters. At the indicated weeks of age, animals were killed, trunk blood was collected, and serum testosterone levels (mean ± S.E., n = 3) were determined by RIA. Asterisks indicate means for the DES-exposed groups that were significantly different (p < 0.05) from the control (CON) groups at the indicated time points.

Fig. 2. Effect of neonatal DES treatment on body weight and normalized weight of the testes and seminal vesicle in the hamster. At the indicated weeks of age, animals were weighed (panel A; mean ± S.E., n = 3), killed, and then tissues were removed, fixed, blotted, weighed, and the weights were expressed relative to animal body weight for the testes (panel B; mean ± S.E., n = 6) and seminal vesicles (panel C; mean ± S.E., n = 3). Asterisks indicate means for the DES-exposed groups that were significantly different (p < 0.05) from the control (CON) groups at the indicated time points.

whole animal growth rate beginning well before puberty and continuing until around weeks 7 or 8 in adult animals. The effect of neonatal DES exposure on mean values of normalized testis weight was transitory and variable. For instance, mean values were significantly decreased on week 5 but were significantly increased on weeks 4, 6, and 7 in DES-exposed animals compared to controls. Thereafter, mean values for normalized testis weight stabilized at levels that were not significantly different between the two groups. Otherwise in the testis, we verified a previous anecdotal observation [13,14]. That is, dramatic differences often developed in size and morphology between right and left testes as animals matured. For instance, a homogeneity-of-variance test determined that testis weight variance was significantly greater in the DES-exposed group compared to the control group at weeks 10–13 (p < 0.008 at all four time points) and the actual

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Table 2 Normalized testis weight data for time points at which variance was significantly greater in the DES-exposed group Age (weeks)

Testis weight/body weight (mg/g)a CON

10 11 12 13

17.8/18.8 (1.0) 16.5/17.2 (0.7) 17.2/18.3 (1.1) 22.1/21.0 (1.1)

DES 18.1/18.7 (0.6) 22.1/22.6 (0.5) 18.8/18.4 (0.4) 21.2/21.4 (0.2)

17.6/18.3 (0.7) 20.4/20.9 (0.5) 20.7/20.4 (0.3) 23.3/22.2 (1.1)

17.7/26.7 (9.0) 11.5/11.8 (0.3) 21.5/34.4 (12.9) 21.5/22.7 (1.2)

18.4/24.9 (6.5) 20.3/34.9 (14.6) 18.2/18.4 (0.2) 13.1/23.6 (10.5)

8.5/23.9 (15.4) 5.6/17.9 (12.3) 18.1/22.8 (4.7) 6.8/20.6 (14.6)

a For each of the three of animals in the control (CON) and neonatally DES-exposed treatment groups at the indicated ages in weeks, the pair of testes were removed, fixed, blotted, individually weighed (mg) and each was expressed relative to the animal’s body weight (g). The difference in normalized weight (variance) between each pair of testes is shown in parentheses.

normalized testis weight data for those time points is presented in Table 2. Photographic examples of the observed spectrum of gonad differences are shown in Fig. 3. The upper panels show the extent of enhanced size of testis pairs at puberty (week 6) in DES-exposed animals compared to controls. In contrast, the

lower right panel shows the extent to which one of a pair of testes often became smaller and degenerate in outward appearance as the DES-exposed animals aged (week 13). We have not found any evidence that this phenomenon favors the left or the right testis.

Fig. 3. Effect of neonatal DES treatment on gross morphology of the testes and seminal vesicle in the hamster. Shown are representative examples of testes and seminal vesicles that were harvested at 6 or 13 weeks of age from control (CON) or DES-exposed animals, fixed, and then photographed. Note: The irregular emergences from the left-hand testes in each of the two left-hand panels are due to extrusion of seminiferous tubules through nicks in the outer tunica albuginea that sometimes occurred during the dissection and fixation procedures.

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A very different pattern of effects was observed for the seminal vesicle (Fig. 2C). At the earliest time point (week 1), normalized seminal vesicle weight was increased significantly in neonatally DES-exposed animals compared to control animals but then that trend was quickly reversed. While relative growth of the seminal vesicle continued throughout the study period in control animals, it languished at a stable, low level in DES-exposed animals. In fact, mean values for normalized seminal vesicle weight were significantly less in DES-exposed compared to control animals from weeks 4 to 13. The extent of seminal vesicle atrophy in DES-exposed hamsters at both the pubertal (week 6) and late adult time points (week 13) in this study is shown photographically in Fig. 3. Also note that seminal vesicle atrophy persisted even during the hyperandrogenic environment present at weeks 7 and 8 (Fig. 1) in DES-exposed animals. These data provide morphological support for the hypothesis that neonatally DES-induced disruption in the male hamster is manifest much earlier in the seminal vesicle than in the testis. 3.3. Testis histology This study confirmed earlier observations [13,14] that neonatally DES-exposed testes developed normally during puberty with the initiation and completion of the first wave of spermatogenesis and normal serum testosterone levels but then displayed progressive disruption of spermatogenesis during adult life. For instance, in this study the presence of mature sperm was first detected at week 5 in both the control and the neonatally DESexposed group of animals (not shown) and then the earliest evidence of neonatal DES-induced disruption of spermatoge-

nesis was observed at week 9 (Fig. 4). Also as noted above at the gross level, disruption at the histological level was manifest in only one of the pair of testes from the particular animal represented in the bottom two panels of Fig. 4. From this age onward, disrupted seminiferous tubule histology was observed in either one or both of the testes from neonatally DES-treated animals (data not shown). 3.4. Seminal vesicle histology These analyses further confirm that neonatal DES-induced disruption in the male hamster initiates and progresses much differently in the seminal vesicle than in the testis. At an early prepubertal age (week 2), low magnification images (Fig. 5, top panels) show that overall dimensions of the organ and number of developing acinar elements were similar in control and DESexposed animals. However, intermediate magnification images (Fig. 5, middle panels) begin to reveal differences in cellular organization of the stromal and epithelial tissue compartments comprising the acinar structures in control versus DES-exposed organs. The higher magnification pictures (Fig. 5, bottom panels) show further details of the DES treatment-dependent differences. For instance: (1) the distribution and thus staining pattern of extracellular matrix in the sub-epithelial stromal compartment was different between control and DES-exposed organs; (2) acinar epithelial cells were low cuboidal in control organs but exhibited a degree of pseudostratification in the DES-exposed organs; (3) cells with small and darkly staining nuclei appeared to be infiltrating from the stroma across the luminal epithelium into some of the acinar lumina.

Fig. 4. Effect of neonatal DES treatment on seminiferous tubule histology in the testes from 9-week-old hamsters. The upper panel is an example of the normal histology that accompanies active spermatogenesis in the adult testes of control (CON) animals. The lower two panels display a representative example of the divergent situation often observed between pairs of adult testes in neonatally DES-treated hamsters. All panels include size bars of 500 ␮m.

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Fig. 5. Effect of neonatal DES treatment on seminal vesicle histology in 2-week-old hamsters. Shown are representative examples of the histology observed at an early prepubertal age (week 2) in control (CON, left panels) and DES-exposed (right panels) hamsters. Photomicrographs were taken at low (2×, upper panels), intermediate (10×, middle panels), and higher (40×, lower panels) magnification and include respective size bars of 1 mm, 200 ␮m, and 50 ␮m in the lower right corners of the panels. Indicated in the lower two panels are the epithelial (E) and stromal (S) tissue compartments.

At puberty (week 6), neonatal DES-induced disruption of seminal vesicle histology was evident at all magnification levels (Fig. 6). In control animals (left panels of Fig. 6), acini often contained secretory material and were lined by an extensively folded epithelium. In contrast, the DES-exposed seminal vesicles were devoid of normal secretory material. Instead, acini were often filled with masses of cells (double asterisks in the top right panel of Fig. 6) that apparently infiltrated from the stromal compartment across the lining epithelium and then could be observed at varying stages of degeneration (lower inset in the middle, right panel of Fig. 6). Other acinar structures in the DES-exposed organs were associated with regions of abnormal sub-epithelial stroma (single asterisks in the right panels of Fig. 6) that contained scant extracellular matrix but instead were heavily populated by cells with very dark-staining and mostly ovoid nuclei (upper inset in the middle, right panel of Fig. 6). The higher magnification micrographs show that the epithelium lining acini in control organs was low columnar with mostly basally located nuclei (lower left panel of Fig. 6), whereas that in DES-exposed organs was much taller and had a very chaotic,

pseudostratified (metaplasia) organization (lower right panel of Fig. 6). The latter panel also shows further evidence of cells with small and darkly staining nuclei that appear to be infiltrating from the stroma across the pseudostratified luminal epithelium. At a late adult stage (week 13), disruption of seminal vesicle histology was still evident at all magnification levels (Fig. 7). In control animals at this age (left panels of Fig. 7), seminal vesicle histology was the same as that described for the seminal vesicles in 6-week old, control animals (Fig. 6). In DES-exposed animals (right panels of Fig. 7), the seminal vesicle remained devoid of normal secretory material and, again, some acini were filled with masses of infiltrated and degenerating cells (double asterisks in the top plus middle right panels and lower inset in the middle right panel of Fig. 6). Other acinar structures in the DES-exposed organs appeared to be filled with epithelial cells plus cells with small and darkly stained nuclei (single asterisks in the top plus middle right panels and upper inset in the middle right panel of Fig. 6). The higher magnification micrographs (lower panels of Fig. 7) indicate a situation similar to that observed in the seminal vesicles from 6-week-old animals (Fig. 6). In particular, that

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Fig. 6. Effect of neonatal DES treatment on seminal vesicle histology in 6-week-old hamsters. Shown are representative examples of the histology observed at puberty (week 6) in control (CON, left panels) and DES-exposed (right panels) hamsters. Photomicrographs were taken at low (2×, upper panels), intermediate (10×, middle panels), and higher (40×, lower panels) magnification and include respective size bars of 1 mm, 200 ␮m, and 50 ␮m in the lower right corners of the panels. Indicated in the left panels are masses of secretory (se) material in acinar lumina. Indicated in the lower two panels are the epithelial (E) sand stromal (S) tissue compartments. Single asterisks and the upper inset in the right, middle panel indicate regions of abnormal sub-epithelial stroma. Double asterisks and the lower inset in the right, middle panel indicate acini distended with infiltrated cells. Size bars in the highest magnification (100×) insets indicate 20 ␮m.

scenario included: (1) an epithelium lining the acini in control organs that was low columnar with mostly basally located nuclei (lower left panel of Fig. 7), whereas that in DES-exposed organs was much taller and metaplastic (lower right panel of Fig. 7) and (2) further evidence of cells with small and darkly staining nuclei that appear to be infiltrating from the stroma across the metaplastic luminal epithelium in the DES-exposed organs (lower right panel of Fig. 7). Some of the characteristics described above for each of the three age points are displayed at maximum magnification in Fig. 8. The capped bar in each panel indicates height of the epithelium lining acinar lumina. They show that those elements were taller in the DES-exposed organs (right panels) than in the control organs (left panels) at all the ages studied. These highest magnification micrographs also demonstrate the degree of pseudostratification and metaplasia that emerges prior to puberty and then progresses through adulthood in the acinar epithelium of DES-exposed organs. Lastly, in the right-hand panels are indicated examples of the cells with small, dark-stained, and

polylobular nuclei both within the epithelial compartment (white arrows) and infiltrated into the acinar lumina (black arrows). According to the judgment of independent experts asked to evaluate the histology figures shown here, the infiltrating cells can be generally classified as polymorphonuclear leukocytes. 3.5. Protein expression levels in the seminal vesicle For Western blot analyses, we used total protein extracts of seminal vesicles from pubertal (week 6) animals because at that time: (1) circulating androgen levels were substantial but not significantly different between the control and DES-exposed animals (Fig. 1); (2) neonatal DES-induced atrophy was significant (Figs. 2 and 3); (3) histological disruption was dramatic (Fig. 6) and exhibited some of the same characteristics that were observed at the late adult (week 13) stage (Figs. 7 and 8). Note that the organs used for these analyses were drained of accumulated secretory material before proteins were extracted. Thus, the extract aliquots loaded per gel lane truly represented

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Fig. 7. Effect of neonatal DES treatment on seminal vesicle histology in 13-week-old hamsters. Shown are representative examples of the histology observed at a late adult age (week 13) in control (CON, left panels) and DES-exposed (right panels) hamsters. Photomicrographs were taken at low (2×, upper panels), intermediate (10×, middle panels), and higher (40×, lower panels) magnification and include respective size bars of 1 mm, 200 ␮m, and 50 ␮m in the lower right corners of the panels. Indicated in two of the left panels are masses of secretory (se) material in acinar lumina. Indicated in the lower two panels are the epithelial (E) and stromal (S) tissue compartments. Single asterisks and the upper inset in the right, middle panel indicate an acinus engorged with epithelial cells and infiltrated cells. Double asterisks and the lower inset in the right, middle panel indicate acini distended with infiltrated cells. Size bars in the highest magnification (100×) insets indicate 20 ␮m.

tissue equivalents because they were not biased by the copious secretory material within control organs compared to the complete lack of such material within the DES-exposed organs. Furthermore, the drained tissue weights were still significantly different between the two groups (control = 122 ± 8 mg, DESexposed = 38 ± 4 mg, mean ± S.E., n = 6, p < 0.0001). The choice of antibodies tested was influenced by our ongoing studies of neonatal DES-induced disruption of protein expression in various regions of the female hamster reproductive tract and on clues from the literature about gene products implicated in the development and regulated function of male reproductive tract tissues. Of the 31 antibodies tested, Table 1 lists: (1) those from the indicated sources and of the indicated type (species, polyclonal or monoclonal) that generated detectable and specific signal bands on an immunoblot; (2) molecular weight of the target band according to migration position relative to that of standard proteins; (3) densitometric quantitation of the bands in the triplicate sample extracts from control versus

DES-exposed seminal vesicles; and (4) whether differences in the mean densitometric values between the control versus DESexposed groups for each protein were statistically significant. For all the immunopositive target proteins listed in Table 1, the relative differences in mean band intensity between the extracts from control versus DES-exposed seminal vesicles are shown in Fig. 9. Evidence that tissue protein loading was equivalent among the electrophoretically resolved sample lanes prior to Western blotting is shown in the left panel of Fig. 10. With the exception of the two major protein bands marked by asterisks at around 78 and 22 kDa in that panel, the pattern and density of all other stained proteins were quite similar in all six sample lanes. The right panel of Fig. 10 shows actual immunoblot results for: (1) all those proteins that were present at significantly different levels in extracts of seminal vesicles from control versus DESexposed animals (Table 1 and Fig. 9) and (2) three proteins that were present at similar levels in the seminal vesicle extracts from control and DES-exposed animals.

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Fig. 8. Highest magnification views of seminal vesicle disruption in the neonatally DES-exposed hamster. Shown are representative examples of the histology observed in the seminal vesicles harvested from control (CON, left panels) and DES-exposed (right panels) animals at early prepubertal (week 2), pubertal (week 6), and late adult (week 13) ages. Photomicrographs were taken at highest magnification (100×) and include size bars of 20 ␮m in the lower right corners of the panels. Also indicated are: (1) the epithelial (E) and stromal (S) tissue compartments; (2) infiltrating cells with dark-staining, multilobular nuclei (→) in both the acinar lumen and epithelial compartment of DES-exposed organs (right panels); (3) thickness of the epithelium (white bars with caps) lining acinar lumina.

Of the 21 proteins that were detected with the antibodies tested in this study, levels of 11 were not significantly different in extracts from the control compared to the DES-exposed seminal vesicles. Three examples of such non-affected proteins with regulatory (proliferating cell nuclear antigen (PCNA), transcription intermediary factor-1␤ (TIF1␤)) and structural (vimentin) function are shown in Fig. 10. Those immunoblotting results plus the total protein staining patterns (left panel of Fig. 10) confirm that neonatal DES-exposure did not derange the overall protein makeup of the seminal vesicle. On the other hand, neonatal DES-exposure did affect the total organ level of a wide range of specific proteins. The affected proteins, described below, included several cytoplasmic and membrane-associated regulatory factors and two are exocrine secretory products. As expected, the normal hamster seminal vesicle expresses the androgen receptor (AR) protein that is a member of the large superfamily of nuclear receptors/transcription factors [20]. While a previous study did not detect a significant change in AR expression at the RNA level in the seminal vesicle from neonatally DES-exposed hamsters [13], here we did find that its expression at the protein level was down-regulated by ∼41%

in the pubertal seminal vesicle from neonatally DES-exposed animals. Cables is a cyclin-dependent kinase binding protein that plays a role in proliferation and/or differentiation, particularly in epithelia [21] and its loss has been associated with the development of endometrial hyperplasia and endometrial cancer [22]. Thus, we were somewhat surprised that levels of the protein in the generally atrophic seminal vesicle present in DES-exposed animals was down-regulated (∼71%) rather than up-regulated. Also complicating the issue is the evidence that expression of the gene can be hormonally regulated, but by the female sex steroids progesterone (up-regulation) and estrogen (downregulation) [22]. Cadherins are transmembrane glycoproteins that mediate calcium-dependent cell–cell adhesion and E-cadherin is found in the adherins junctions near the apical surface of polarized epithelial cells [23]. Cellular stress induces caspase-mediated proteolytic cleavage of E-cadherin and produces a major fragment of about 30 kDa in size [24]. The antibody we used recognized the full-length protein plus a smaller isoform that was similar in size to the cleavage product described in the

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Fig. 9. Effect of neonatal DES treatment on relative levels of immunodetected proteins in the seminal vesicle of pubertal hamsters. Total protein extracts from control (CON) and DES-exposed seminal vesicles of 6-week-old hamsters underwent Western blot and densitometric analysis. Shown are the values for mean band density from DES-exposed tissues/mean band density from control tissues (DES/CON) as listed in Table 1. Asterisks indicate instances where differences between the means for each group were statistically significant as indicated in Table 1.

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Keller and Nigam report [24]. In the pubertal seminal vesicle from neonatally DES-exposed animals, the full-length protein was down-regulated (∼71%) and the smaller isoform was upregulated (∼35%). Consequently, the summed levels of the two isoforms were down-regulated (∼59%). The family of catenin proteins are involved in both cell–cell adhesion and regulation of specific gene expression [25,26]. The family member known as ␤-catenin was first identified as a structural adaptor linking cadherins to the actin cytoskeleton in cell–cell adhesion but is now also known as a transcription cofactor with the T cell factor/lymphoid enhancer factor (TCF/LEF) in the Wnt pathway that regulates cell proliferation and differentiation [27]. The antibody we used recognized two isoforms of the protein. Levels of the larger form were not different between the control and DES-exposed organs but they were higher than that of the smaller form in both groups of organs. Although levels of the smaller form were down-regulated (∼25%) in the DES-exposed organs, the summed levels of the two isoforms were not. The protein product of the c-erbB-2 oncogene, also known as HER-2 or neu, belongs to the HER family of receptor tyrosine kinases and it was first associated with breast carcinoma [28]. In extracts from the control organs, the antibody we used recognized a doublet of proteins with levels of the larger form

Fig. 10. Effect of neonatal DES treatment on total protein pattern and specific immunodetected proteins in the seminal vesicle of pubertal hamsters. Total protein extracts from control (CON) and DES-exposed seminal vesicles of 6-week-old animals were resolved by denaturing gel electrophoresis (left panel) and analyzed by Western blotting (right panel). Indicated on the left side of the left-hand panel are the migration positions of standard proteins with the indicated molecular weights (kDa) and the positions of two stained bands (asterisks) that were obviously darker in the seminal vesicle extracts from control animals than from DES-exposed animals. For the right-hand panel, multiple immunodetected isoforms of a given protein are identified by their molecular weight (kDa) in parentheses.

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(176 kDa) being somewhat higher than that of the smaller form (165 kDa). The summed levels of both forms were clearly downregulated (∼46%) in the DES-exposed organs. Lactoferrin is an iron-binding glycoprotein synthesized by neutrophils and exocrine glands and it plays an important role in innate defense mechanisms against bacteria, fungi, and viruses [29]. Furthermore, expression of the lactoferrin gene is responsive to steroid hormone and growth factor regulation in both male and female reproductive tract tissues [30]. Consistent with observations in mice treated prenatally with DES [31–33], levels of lactoferrin were highly up-regulated (∼350%) in the neonatally DES-exposed hamster seminal vesicle. The lactoferrin signal bands shown in Fig. 10 most likely represent an intracellular rather than secreted state of the protein since both groups of organs were drained of secretory contents prior to extract preparation and, as determined by the weekly histological assessment, DES-exposed seminal vesicles were devoid of secretory material at all ages. The nuclear factor-␬B (NF-␬B) protein family contains five transcription factors (p65 [RelA], RelB, c-Rel, p50 [NF-␬B1], and p52 [NF-␬B2]) [34]. They play an essential role in several aspects of human health including the development of innate and adaptive immunity and their dysregulation is associated with many disease states [35]. We found that neonatal DES exposure enhanced the expression of two family members in the pubertal seminal vesicle. The p50 protein was up-regulated by ∼92% and the p65 protein was up-regulated by 37%. The p63 protein is encoded by a homolog of the p53 tumor suppressor gene and it plays important roles in normal epithelial commitment, maintenance, and differentiation [36] plus tumorigenesis [37]. The levels of this protein (just beneath the non-specific signal band always detected at the same intensity in all six lanes) were also up-regulated (∼72%) in the DES-exposed organs. Uteroglobin was the first mammalian protein found to be progesterone regulated and, after its original observation in the uterus, it was detected in other organs including the seminal vesicle [38]. Here we demonstrate that the hamster seminal vesicle also produces the protein but its level was down-regulated (∼59%) in the DES-exposed organs. According to the same argument presented for lactoferrin above, the uteroglobin signal bands shown in Fig. 10 probably represent an intracellular rather than secreted state of the protein. 4. Discussion To more completely assess the process of neonatal DESinduced disruption of the male hamster reproductive tract, we began by performing weekly evaluations (from early prepubertal to late adult stages) of circulating testosterone levels plus gross morphology and histology of the testis and seminal vesicle. The results of those evaluations support two important observations made in previous studies [13,14]: (1) in neonatally DES-exposed hamster testes, spermatogenesis initiates normally around week 5 but then fails as animals mature; (2) neonatal DES exposure impairs the action of androgens on target organs in male hamsters. Further histological and immunoblot analyses revealed

new details about the striking histopathological process that develops in the neonatally DES-exposed seminal vesicle and the accompanying pattern of disrupted proteomics. 4.1. Circulating testosterone levels Serum testosterone assays were performed to determine if and to what extent neonatal DES treatment influenced testicular steroidogenesis and thus the onset of puberty and then the maintenance of an adult endocrine environment. In control animals, the developmental pattern and levels of circulating testosterone were consistent with those reported in an earlier study of male hamsters from week 1 to just short of week 9 of life [39]. In both cases, levels began to rise between weeks 5 and 6. Thereafter, levels plateaued at the 2–4 ng/ml range with the exception of an isolated spike at slightly different time points in the two studies: (1) ∼5 ng/ml at week 7 in the Vomachka and Greenwald study [39] and (2) ∼8 ng/ml at week 9 in this study. The developmental pattern we observed in control animals was also consistent with that described in another study [40] where plasma testosterone levels in male hamsters were measured weekly from weeks 3 to 9 of life but were expressed in relative rather than absolute terms. For instance, levels in that study began to rise between weeks 5 and 6, plateaued between weeks 7 and 8, and then almost doubled between weeks 8 and 9. Consistent with all three studies is the conclusion that, from the standpoint of gonadal androgen production, normal male hamsters attain puberty around week 6 of life. The significance of the postpubertal and transient peak of circulating testosterone measured in our study and by others [39,40] is unclear since it was not accompanied by a rise in the weight of seminal vesicles harvested from normal animals in either our study or in the study by Miller et al. [40]. The steroid assays also confirmed that neonatal DES exposure does not significantly alter circulating testosterone levels in pubertal (week 6) or late adult (week 13) male hamsters. However, between those time points, DES-exposed animals experienced an earlier, more pronounced, and more prolonged androgen surge. One way to interpret those observations is that neonatal DES treatment did not alter the age at which male hamsters entered puberty but it did alter subsequent steps of that process. In any case, the significance in DES-exposed animals of the earlier, more pronounced, and more prolonged surge in circulating testosterone is also unclear since it too was not accompanied by a surge in the weight of seminal vesicles harvested from the DES-exposed animals. Lastly, the steroid assays demonstrate that although neonatal DES-exposure can lead to failure of spermatogenesis as hamsters age [13,14], it does not block pubertal initiation nor does it lead to later failure of testicular androgen production. 4.2. Testis morphology/histology Consistent with previous studies [13,14], this study showed that neonatal DES exposure did not affect the body weight of male hamsters at any age, nor did it affect the onset of puberty according to the appearance of mature sperm (week 5), but it did affect other aspects of testicular development. For instance,

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the present study recorded both enhanced (week 4) and reduced (week 5) testis weight in DES-exposed animals. Perhaps pertinent to this topic is the anecdotal observation in previous reports [13,14] and verified statistically in this report that testis weight variance in older animals became significantly greater in the DES-exposed compared to the control organs. As illustrated in the bottom right panel of Fig. 3, the variance was sometimes due to the unilateral presence of a smaller but cleanly isolated testis that was degenerate in its gross morphology. In other cases encountered in this study (not shown) and noted in previous studies [13,14], one of a pair of DES-exposed testes in older animals had become a very deformed mass. Such masses often contained apparently purulent material and were adherent either to abdominal structures (cryptorchidism) or to the parietal layer of the tunica vaginalis lining the interior of the scrotum and inguinal canal. When the histology of these grossly abnormal structures was evaluated previously [13,14] and in this study (not shown), seminiferous tubules were completely replaced by a uniform population of cells with a fibroblastic morphology. The difficulty in cleanly dissecting out such abnormal and adherent masses likely contributed to the large variance in individual testis weights determined from the older, DES-exposed animals. These phenomena also stymied our attempts at testis proteomics screening because extracts prepared from such abnormal tissues did yield evidence of deranged overall protein makeup and thus replicate immunoblotting could not be reliably performed and evaluated as was possible for the seminal vesicle extracts.

tal DES-induced disruption of male reproductive tract structures [31,32,47–52]. The phenomenon of intense cell infiltration activity has been documented in the estrogen-stimulated adult uterus [12,43,46] and in the adult seminal vesicle (this report) of neonatally DES-treated hamsters. According to ultrastructural information [12,43], at least some of the infiltrating leukocytes in the neonatally DES-exposed and then estrogen-stimulated adult hamster uterus are eosinophils. However, immunohistochemical evaluation by new collaborators with expertise in eosinophil biology indicate that the majority of infiltrating cells in the neonatally DES-exposed hamster seminal vesicle are not eosinophils (personal communication from the laboratory of Dr. James J. Lee, Mayo Clinic, Scottsdale, AZ). Consequently, the identity and relative makeup of the population of infiltrating cells remains to be determined. Other possible histopathological correlations between the DES-exposed hamster uterus and seminal vesicle deserve comment. For instance, the histopathological lesions reported in the endometrial epithelial compartment of the neonatal DESexposed hamster uterus [12] involve disruption of both cell proliferation and apoptosis dynamics [15,45,46,53] and similar responses have been reported in the male reproductive tract of hamsters and rats following adult DES exposure [54–56]. Whether such altered dynamics are also involved in the neonatally DES-disrupted hamster seminal vesicle is the subject of planned studies.

4.3. Seminal vesicle morphology/histology

4.4. Protein expression in the seminal vesicle

At both the gross morphology and histological levels, neonatal DES-induced disruption commenced much earlier in the seminal vesicle than in the testis. A striking aspect of that disruption phenomenon was the total lack of exocrine function in the neonatally DES-exposed seminal vesicle. Normal exocrine function in the mammalian seminal vesicle is considered a classic androgen-dependent activity [41], and its loss was also reported in neonatally DES-exposed rats [42]. Perhaps part of the explanation for the difference in commencement of the disruption phenomenon is that the seminal vesicle is normally androgen responsive at an earlier stage of development than is the testis. We also noted that some of the histopathological details observed in the seminal vesicle of neonatally DES-treated male hamsters resemble those described in the uterus of neonatally DES-treated female hamsters [12,15,43–46]. Those shared details include: (1) emerging evidence of luminal epithelial pseudostratification and transepithelial cell infiltration in early prepubertal animals, (2) altered stromal cell and extracellular matrix organization in prepubertal and adult animals, (3) extreme hypertrophy and progression of pseudostratification/dysplasia of the luminal epithelium in adult animals, and (4) dramatic levels of transepithelial cell infiltration in adult animals. Interestingly, expression of these disruption end-points in the uterus represents a phenomenon of neonatal DES-induced disruption in estrogen responsiveness [12,15,45,46] and others have reported that altered estrogen responsiveness may play a role in perina-

Since the hamster genome has not yet been sequenced, convenient DNA microarrays are not available for high-throughput analysis of the relative expression of multiple genes in hamster tissues. Instead, we used the established method of Western blot analysis to individually assess the relative expression level of various genes at the protein (proteomic) level. The spectrum of screened targets included cell membrane and intracellular proteins with regulatory functions, exocrine products, paracrine/autocrine growth factors, intracellular structural proteins, and extracellular matrix proteins. We were somewhat surprised that levels of proliferating cell nuclear antigen (PCNA) were unaffected because, along with the Ki-67 antigen, immunohistological detection of intranuclear PCNA is considered a marker of proliferating cells in a wide variety of tissues [57] but neonatal DES exposure induced a general condition of atrophy in the pubertal seminal vesicle. However, newer studies have revealed PCNA’s ability to interact with multiple cellular partners that are involved in various metabolic pathways including Okazaki fragment processing, DNA repair, translesion DNA synthesis, DNA methylation, chromatin remodeling, and cell cycle regulation [58]. Furthermore, the ability of an antibody to detect antigen may vary under the conditions of immunoblotting versus immunohistological analysis. Another non-affected protein, TIF1␤, is also known as KAP-1 or KRIP-1 and was originally identified as a co-repressor for the large family of KRAB domain-containing zinc-finger proteins [59–61]. We first became interested in this

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protein because of its reported involvement in the maintenance of spermatogenesis in the rodent [62] but, perhaps due to the proteomic screening complications discussed above, we have been unable to show that its expression level in the hamster testis is affected by neonatal DES exposure. In addition to those regulatory proteins, also unaffected were the levels of vimentin which is the major intermediate filament protein present in mesenchymal or stromal cells [63]. On the other hand, the dramatic differences observed in gross morphology and histology of the control versus DES-exposed seminal vesicle at puberty were accompanied by significant differences in the organ extract levels of several other proteins. As mentioned above, a basic observation reported in previous studies [13,14] and confirmed by this study is that neonatal DES exposure alters androgen responsiveness in the reproductive tract of male hamsters. Although one of the previous studies found that expression of the AR gene at the RNA level was not significantly altered [13], this study found that it was downregulated at the protein level in the pubertal seminal vesicle of neonatally DES-exposed hamsters. This suggests that neonatal DES exposure disrupts AR gene expression at the posttranscriptional level. The mechanism of this effect and whether it represents a cause or a consequence of altered androgen responsiveness need to be investigated. For instance, the connection between dynamic processing of steroid receptor proteins and steroid hormone action has been suggested for some time and evidence now exists that such processing involves ubiquitination and proteasome function [64]. Another member of the disrupted protein expression list prompts an additional consideration of altered hormone responsiveness. In the pubertal seminal vesicle from neonatally DESexposed hamsters, we detected dramatic up-regulation of the lactoferrin protein. A similar observation in the hamster uterus was considered confirmatory evidence that neonatal DES exposure directly and permanently disrupts estrogen responsiveness in that organ [12,15]. Furthermore, enhanced expression of the lactoferrin gene at the RNA and protein level in the male and female reproductive tracts following perinatal DES exposure also occurs in the mouse [7,30–33]. Indeed, such phenomena in the male have been described as examples of molecular feminization [33] and/or developmental estrogenization [7] and perinatal DES-induced changes in estrogen receptor expression have been reported in the seminal vesicle of other rodents [32,47,48,65]. Also, the observed down-regulation of Cables protein levels in the neonatally DES-exposed seminal vesicle is consistent with a scenario of enhanced sensitivity to estrogens. However, using two different antibodies that do detect estrogen receptor-specific signal bands by Western blot analysis of hamster uterine extracts, we detected no such signal bands from either the control or DES-exposed seminal vesicle extracts generated for this study (data not shown). 4.5. Summary and conclusions This study confirms that neonatal DES exposure alters androgen responsiveness in some regions of the male hamster reproductive tract. It also determined that the disruption phenomenon

in the male hamster is manifest much earlier in the seminal vesicle than in the testis and that testis disruption often occurs differently between the pair of organs in a given animal. In the neonatally DES-exposed seminal vesicle, histopathological effects included: (1) general atrophy, (2) lack of exocrine products, (3) epithelial dysplasia, (4) altered organization of stromal cells and extracellular matrix, and (5) striking cell infiltration with polymorphonuclear leukocytes. Not surprisingly, the morphological disruption phenomenon in the seminal vesicle was accompanied by a range of up-regulation and down-regulation responses in the whole organ levels of various proteins. The tissue-specific nature of those responses and whether they also represent alterations in estrogen responsiveness remain to be determined. In any case, these results suggest that the hamster seminal vesicle is a very sensitive target of perinatal endocrine disruption action. Further exploitation of both the male and female aspects of this experimental system should include not only lower doses of the established endocrine disruptor DES, but also environmentally relevant doses of other suspected endocrine disruptor agents. Acknowledgments We thank Dr. Karen L. Brown-Sullivan for valuable guidance in performing statistical analyses and Ana Cecilia Millena for expert technical assistance and RIA performance. This work was supported by United States Public Health Service grants ES 10232, G12 RR03062 and B.P. Weaver was supported by a STAR Trainee scholarship funded through NIH grant number P20 RR016475 from the BRIN/INBRE program of the National Center for Research Resources. Portions of this work appeared in the theses submitted by T.R. Naccarato and B.P. Weaver in partial fulfillment of the MS degree in Biological Sciences and at the 36th Annual Meeting of the Society for the Study of Reproduction, 2003. References [1] Dodds EC, Goldberg L, Lawson W, Robinson R. Estrogenic activity of certain synthetic compounds. Nature 1938;141:247–8. [2] Swan SH. Intrauterine exposure to diethylstilbestrol: long-term effects in humans. Apmis 2000;108(12):793–804. [3] Herbst AL, Ulfelder H, Poskanzer DC. Adenocarcinoma of the vagina: association of maternal stilbestrol therapy with tumor appearance in young women. New Engl J Med 1971;284:878–81. [4] Herbst AL, Ulfelder H, Poskanzer DC. Perinatal carcinogenesis. Natl Cancer Inst Monograph 1971;51:25–35. [5] Mittendorf R. Teratogen update: carcinogenesis and teratogenesis associated with exposure to diethylstilbestrol (DES) in utero. Teratology 1995;51:435–45. [6] Metzler M. The metabolism of diethylstilbestrol. CRC Crit Rev Biochem 1981;10(3):171–212. [7] McLachlan JA, Newbold RR, Burow ME, Li SF. From malformations to molecular mechanisms in the male: three decades of research on endocrine disrupters. Apmis 2001;109(4):263–72. [8] Bern HA. Diethylstilbestrol (DES) syndrome: present status of animal and human studies. In: Li JJ, Nandi S, editors. Hormonal carcinogenesis. New York: Springer-Verlag; 1991. p. 1–8. [9] Greco TL, Duello TM, Gorski J. Estrogen receptors, estradiol, and diethylstilbestrol in early development: the mouse as a model for

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