Estrogen Receptor-α Mediates Diethylstilbestrol-Induced Feminization of the Seminal Vesicle in Male Mice

Background: Studies have shown that perinatal exposure to the synthetic estrogen diethylstilbestrol (DES) leads to feminization of the seminal vesicle (SV) in male mice, as illustrated by tissue hyperplasia, ectopic expression of the major estrogen-inducible uterine secretory protein lactoferrin (LF), and reduced expression of SV secretory protein IV (SVS IV). Objectives: The present study was designed to evaluate the role of the estrogen receptor (ER) in this action by using ER-knockout (ERKO) mice. Methods: Wild-type (WT), ERα-null (αERKO), and ERβ-null (βERKO) male mice were treated with either vehicle or DES (2 μg/day) on neonatal days 1–5. These mice were divided into two groups: In the first group, intact mice were sacrificed at 10 weeks of age; in the second group, mice were castrated at 10 weeks of age, allowed to recover for 10 days, treated with dihydrotestosterone (DHT) or placebo, and sacrificed 2 weeks later. Body weights and SV weights were recorded, and mRNA expression levels of Ltf (lactoferrin), Svs4, and androgen receptor (Ar) were assessed. Results: In DES-treated intact mice, SV weights were reduced in WT and βERKO mice but not in αERKO mice. DES-treated WT and βERKO males, but not αERKO males, exhibited ectopic expression of LF in the SV. DES treatment resulted in decreased SVS IV protein and mRNA expression in WT males, but no effect was seen in αERKO mice. In addition, DES-treated βERKO mice exhibited reduced Svs4 mRNA expression but maintained control levels of SVS IV protein. In DES-treated castrated mice, DHT implants restored SV weights to normal levels in αERKO mice but not in WT mice, suggesting full androgen responsiveness in αERKO mice. Conclusions: These data suggest that DES-induced SV toxicity and feminization are primarily mediated by ERα; however, some aspects of androgen response may require the action of ERβ.

volume 120 | number 4 | April 2012 • Environmental Health Perspectives Research Studies in laboratory animals have unequivo cally demonstrated that exposures to exogenous estrogens during certain periods of embryonic, fetal, and neo natal development lead to perma nent and detrimental changes in reproductive function, most notably structural malforma tions among the reproductive tissues, reduced fertility, and cancer Tomatis 1992, 1993). Diethylstilbestrol (DES), a highly potent, orally available synthetic estrogen, is perhaps the most studied among the class of exogenous estrogens since being strongly associated with vaginal adeno carcinoma and abnormalities of the uterus and cervix in young women who were exposed via pharmacologi cal use by their mothers during pregnancy (Herbst et al. 1971). Although DES was proscribed for use in women during pregnancy in 1973, these findings are the origin of the still cur rently held hypothesis that unintended expo sures to compounds with estrogenic activities, such as phyto estrogens (e.g., genistein and coumestrol), pesticides [e.g., DDT (dichloro diphenyl trichloroethane) and DDE (dichloro diphenyl dichloroethylene)], and plasticizers (e.g., bisphenol A), during embryonic through post natal development may be linked to certain reproductive and other abnormalities in humans.
Many of the pathologies associated with develop mental exposure to DES in humans can be replicated in outbred and inbred strains of laboratory mice (McLachlan et al. 1980;Newbold 2004). A few studies have focused on the health problems of men exposed to DES in utero (known as DES sons), and the results have been mixed. The most consistent finding indicates an increased risk for non cancerous epididymal cysts (Bibbo et al. 1977;Conley et al. 1983;Gill et al. 1979;Niculescu 1985;Wilcox et al. 1995); an increased risk of testicular cancer has not been ruled out but is not yet confirmed. The rele vancy of the present protocol to DES sons is supported by previous studies in mice showing that expo sure periods considered susceptible to per turbation by DES include exposure during gestation days (GDs) 9-16, on GD18 alone, and on post natal days (PNDs) 1-5 (Newbold et al. 2000). Outbred female mice treated with DES as neo nates develop a high incidence of uterine adeno carcinoma (Newbold et al. 1990); similarly treated male mice develop testicular cancer and abnormalities of the pros tate and seminal vesicles (SVs) (McLachlan 1977). Since reports of these findings were published almost three decades ago, the fetal and neo natal mouse has been used extensively to investigate the toxicology of estrogenic compounds on reproductive tract develop ment. Ostensibly, estrogen receptorα (ERα) gene (Esr1)knockout (αERKO) and ERβ (Esr2)knockout (βERKO) mice have added to our ability to investigate the mecha nisms by which exogenous estrogens exert their effects (Couse and Korach 1999). Conclusive studies have demonstrated that reproductive tracts of female αERKO mice are largely resistant to the develop mental effects of neo natal exposure to DES . These data indi cate a prominent role for ERα in mediating the toxicological effects of DES during repro ductive tract development in females.
Reproductive tissues of male mice appear to be especially sensitive to the toxicologi cal effects of DES (McLachlan et al. 1975;Prins 1992;Prins et al. 2001), and presum ably other exogenous estrogens, throughout develop ment. The development of the SV in the male reproductive tract involves three pro cesses (Shima et al. 1990): growth, epithelial branching morpho genesis, and epithelial cyto differentiation. In humans, SV development begins from the mesonephric (or Wolffian) duct at approximately 12 weeks of fetal age, and morpho genesis of the SV depends on fetal testicular androgens (Risbridger and Taylor 2006). At maturity, the SV consists of folded glandular epithelium with lumi nal secretory cells and a discontinuous layer of basal cells surrounded by a stromal layer of smooth muscle (Risbridger and Taylor 2006). In the mouse, the SV is present by embryonic day 16.5 (Lung and Cunha 1981) and develops from the meso nephric ducts. Morphogenesis begins on GD15, and during the first week of postnatal life the SV under goes intense morpho genesis that results in the complex folded structures charac teristic of the mature SV (Lung and Cunha 1981). ER is present in the mouse SV (Yamashita 2004) and appears during develop ment on PND6 (Cooke et al. 1991). Significant increases in cyto solic ER levels in the SV have been reported in mice following neo natal exposure to DES (Turner et al. 1989).
In addition to morphological changes of the male reproductive tract, studies have shown that developmental exposure to estrogens leads to femalelike patterns of gene expression in certain reproductive tissues during adulthood (Beckman et al. 1994;Newbold et al. 1989), including aberrantly high expression of progesterone receptor (Pgr), an estrogenregulated gene, in the stromal cells of the male reproductive tract (Williams et al. 2000(Williams et al. , 2001; ectopic expression of the major estrogeninducible uterine secretory protein, lactoferrin (LF; also called lacto transferrin); and loss of constitutive expression of the androgenregulated gene, SV secretory protein 4 (SVS IV), in the SVs (Beckman et al. 1994). To date, studies of DES exposure in ERnull male mice have focused largely on the prostate; these studies have demon strated that ERKO males are refractory to the effects of neo natal DES exposure, whereas βERKO males exhibit susceptibilities comparable to similarly treated wildtype (WT) mice (Prins et al. 2001). In the present study we evaluated the effects of neo natal DES exposure on the SV of αERKO and βERKO mice.

Animals and treatment.
All studies involving animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Research 2011) and approved by the National Institute of Environmental Health Sciences (NIEHS) Animal Care and Use Committee. Animals were treated humanely and with regard for alleviation of suffering. The genera tion of ERα and ERβnull mice is previously described (Krege et al. 1998;Lubahn et al. 1993). Mice were generated by breeding C57/BL6 mice hetero zygous for disruption of the ERα gene (Esr1) or the ERβ gene (Esr2) to produce homo zygous ERαnull (αERKO) or ERβnull (βERKO) mice, respectively, and WT litter mates.
Pregnant females were housed under controlled lighting (12hr light/dark cycle) and temperature conditions and were provided with NIH 31 laboratory mouse chow (Zeigler Brothers Inc., Gardners, PA) and fresh water ad libitum. On the day of parturition, considered day l of age, male offspring were pooled from multiple litters and randomly distributed among CD1 foster mothers at eight per female. All offspring then received a sub cutaneous injection of 2 μg (1-2 mg/kg/day) DES in corn oil or an equal volume of corn oil alone (vehicle) daily on PNDs 1-5. All offspring were weaned at 21 days of age and geno typed by polymerase chain reaction (PCR) on DNA extracted from tail biopsy using previously described methods (Couse et al. 2003). Mice in group 1 were killed at 10 weeks of age. Mice in group 2 were castrated at 10 weeks of age and allowed to recover for 10 days. Mice then received a sub dermal implant of a 1cm length of sealed Silastic tubing (1cm in length, 1.47 mm inner diameter, 1.95 mm outer diameter) packed with crystalline dihydro testosterone (DHT) or nothing (placebo) (implants were kindly provided by D. Handelsman, Sydney, Australia), and then were killed 2 weeks later (Lim et al. 2008). At necropsy, we recorded body weights, collected and heparinized whole blood from the inferior vena cava, and stored the plasma at -70°C until assayed. We collected the SV, trimmed off the coagulating gland, and recorded the wet weight. SVs were snapfrozen for RNA and protein analysis or fixed in paraformaldehyde solution for histological analysis. SV tissue sections (4 μm) were stained with hematoxylin and eosin.
Hormone serum assays. We evaluated serum estradiol and testosterone levels using Coat ACount radio immunoassay kits (Siemens Healthcare Diagnostics, Los Angeles, CA) and were assayed using an APEX automatic gamma counter (ICN Micromedic Systems Inc., Huntsville, AL).
RNA and protein isolation. Frozen SV tis sue from individual animals was pulverized, and the material was subdivided for either pro tein or RNA extraction. Total RNA was iso lated using the RNeasy isolation kit (Qiagen Inc., Valencia, CA) according to the manufac turer's protocol. Cytoplasmic and nuclear pro tein was extracted from frozen pulverized SV tissue using the NEPER Protein Extraction Kit (Pierce, Rockford, IL) according to the manufacturer's protocol.
Reverse-transcriptase polymerase chain reaction (RT-PCR). SV RNA (0.5 μg) was reverse transcribed using the SuperScript First Strand Synthesis Kit (Invitrogen, Carlsbad, CA) according to manufacturer's protocol. As a negative control, we used a sample con taining RNA but no reverse transcriptase. Realtime RTPCR was performed using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) and SYBR Green (Invitrogen). Primers were designed using Applied Biosystems Primer Express Software (version 2.0).
Realtime RTPCR was performed using 2.5 ng cDNA. Samples were analyzed in duplicate, and a negative control sample was included on each plate. For all samples, the cyclophilin gene [peptidylprolyl isomerase A (Ppia)] was used as an endogenous control for normalization. Expression ratios were calcu lated using the mathematical model described by Pfaffl (2001): where Ct is cycle threshold.
Western immunoblot analysis. Cytoplasmic SV protein (1 μg) was immobilized to nitro cellulose membrane using the BioRad Dot Blot apparatus (BioRad, Hercules, CA) according to the manufacturer's protocol. We performed the protein analysis using a single blot that was stripped and reprobed. Equal loading was determined using MEMCode total protein stain (Pierce) and then destained before Western blotting. Non specific peroxidases were eliminated using 3% hydrogen peroxide, and non specific sites were blocked with 10% bovine serum albumin in Trisbuffered saline, pH 7.4, plus 0.1% Tween20 (TBST). Blots were then incubated with primary anti bodies for 1 hr at room temperature. Antimouse SV secretory protein IV (SVS IV) rabbit polyclonal antibody, a gift from T. Teng (NIEHS), and rabbit antimouse LF polyclonal antibody, isolated as described previously (Jefferson et al. 1996), were used at 1:5,000 dilution. Blots were then incubated with secondary antibody, antirabbit horseradish peroxidase (Amersham, Piscataway, NJ), diluted 1:10,000 in TBST. Membranes were washed five times for 5 min each in TBST, and immuno reactive bands were visualized using WestDura Reagent (Pierce) following the manufacturer's instructions. Blots were exposed to film for 1 min, and images were captured by a camera (model c84845403G) using LabWorks s46 software (both from UVP BioImaging Systems, Upland, CA).
Statistical analysis. The data were analyzed using JMP software (version 7) and SAS soft ware (version 9.1), both from SAS Institute Inc. (Cary, NC). For body weight and SV wet weight, parametric tests were used to compare values. We evaluated realtime RTPCR data using analysis of variance (ANOVA) followed by Tukey's test. For estrogen and testosterone levels, statistical significance was determined using ttest, and groups were compared using non parametric MannWhitney tests. pValues < 0.05 were considered statistically significant.

DES Exposed αERKO male mice retain androgen responsiveness in SV.
The SVs of adult WT males exposed to DES as neo nates exhibited a 50% reduction in weight relative to untreated WT controls (Figure 1). This volume 120 | number 4 | April 2012 • Environmental Health Perspectives is consistent with previous reports (Prins et al. 2001) and indicates that DES treat ments were successful. DESexposed βERKO males exhibited a remarkably similar effect ( Figure 1). In contrast, DES exposure had no measurable effect on SV weights in αERKO males. Instead, both DEStreated and control αERKO males exhibited significantly larger SV weights compared with agematched WT and βERKO males (p < 0.05).
Neo natal DES exposure resulted in signifi cant histological alterations in the SV of WT and βERKO males but not αERKO males ( Figure 2). The SV of WT mice neo natally exposed to DES showed a significant increase in thickness of the smooth muscle layer, as well as the connective tissue that separates the epithelium from the smooth muscle layer ( Figure 2B). The single columnar epithelial structure is no longer present, replaced by increased glandular formation. However, these glands do not appear to be secretory in nature because they did not produce the secretions seen in the control SV ( Figure 2B). In addi tion, significant vasculature infiltration can be seen in the connective tissue layer, as well lymphocyte infiltration in the smooth muscle layer. DEStreated βERKO mice exhibited significant thickening and lymphocyte infil tration of the smooth muscle layer, similar to the histopathological effects observed in the WT mice. Despite potential hyper plasia of the epithelium, apparently some of the epithelium remained functional because epithelial cells still maintained a polarized columnar shape and produced some secretions ( Figure 2F).
In contrast, the welldocumented histologi cal identifiers of DES exposure in the SV exhibited by WT and βERKO males was not evident in the DEStreated αERKO males ( Figure 2D).
The expression of Svs4, a gene positively regulated by androgen in the mouse SV, was greatly reduced in the SVs of DESexposed WT males ( Figure 3A). This reduced Svs4 expression was further confirmed by the absence of detectable SVS IV protein as evaluated by Western blot ( Figure 3B). We observed a similar effect on Svs4 expression in DESexposed βERKO males, although levels of SVS IV protein were more variable in this geno type. Interestingly, βERKO mice retained SVS IV protein expression even though they had lower mRNA levels after DES exposure, unlike WT controls. These data suggest that some aspects of androgen response, such as maintenance of SVS IV protein, may require the action of ERβ and warrant further studies to evaluate a potential role for ERβ. In contrast, the SVs of DESexposed αERKO males exhibited normal Svs4 mRNA expression and SVS IV protein levels compared with controls.
Because maintenance of normal weights and Svs4 expression in mouse SV are depen dent on sufficient androgen stimulation, abnormally low SV weights and absence of Svs4 expression and SVS IV protein observed in DESexposed adult WT and βERKO males suggest that estrogen leads to a permanent   Figure 1). Given that AR expression patterns were normal, we turned our focus to the possibility that DES exposure led to a reduction in cir culating testosterone levels and hence reduced androgen signaling in the SV (Table 1). Surprisingly, we found that testosterone levels in DESexposed WT males were somewhat elevated, although not statistically signifi cant, compared with controls. In βERKO males, testosterone levels were normal in both treatment groups. Consistent with previ ous reports (Lindzey et al. 1998), untreated αERKO males exhibited significantly higher levels of circulating testosterone relative to WT and βERKO males (Table 1). Although neo natal DES exposure appeared to reduce testosterone levels in αERKO males, this dif ference was not significant compared with untreated ERαnull animals, and levels still remained 4 to 5fold higher than those of WT males. Estradiol levels were comparable among all genotypes and treatment groups [see Supplemental Material, The above data indicate that neo natal DES exposure causes the SV tissues to become refractory to androgen stimulation dur ing adulthood, despite exhibiting normal Ar expression patterns in a milieu of normal cir culating testosterone levels. Furthermore, the absence of this effect in DESexposed αERKO males strongly suggests that this effect is ERαmediated during development. It is conceivable, however, that the remarkably high testosterone levels in αERKO mice (Table 1) may mask the effects of DESinduced andro gen resistance, such as those observed in DES exposed WT males. To test this hypothesis, control and DESexposed WT and αERKO males were castrated at 10 weeks of age, pro vided a Silastic implant of placebo or DHT 10 days post surgery, and killed 2 weeks later. In this milieu, we assumed that circulating androgen levels among the genotypes would now be comparable, allowing for more defini tive evaluation of the role of ERα and the effects of neo natal DES exposure on the SV. As expected, castration led to an acute loss in SV weight in both WT and αERKO males regardless of neo natal treatment (Figure 4). Two weeks of DHT treatment totally restored SV weights in castrated control WT males but had no effect in DESexposed WT males. These data definitively demon strate that develop mental exposure to DES leads to androgen resistance in the adult SV. In con trast, DHT treatment fully restored castration induced losses in SV weights in both control and DESexposed αERKO males. These data indicate that αERKO males are largely resis tant to the effect of neo natal DES exposure on the SV, providing convincing evidence of the importance of ERα in mediating the toxico logical effects of DES.
ERα mediates DES-induced molecular feminization of SV. The LF gene (Ltf) is an estrogenresponsive gene in the uterus and that is not normally expressed at measurable levels in the SV. However, previous studies have shown ectopic LF expression in the SV after neo natal DES exposure in mice (Beckman et al. 1994;Newbold et al. 1989), indicating DES induced "feminization" of the tissue. As we expected, in the present study, Ltf expression and LF protein were undetectable in SVs of control adult males in the thre genotypes ( Figure 5). In contrast, Ltf expression and protein were easily detected in the SVs of DESexposed WT males. We also observed the same effect in DESexposed βERKO males, whereas Ltf expression in the SVs of αERKO males remained absent regardless of neo natal treatment, indicating that DESinduced feminization is ERα mediated.

Discussion
Our findings in this study demon strate the importance of ERα in mediating the toxicological effects of neo natal DES exposure in the male SV. In WT and βERKO males, DES exposure resulted in a reduction in SV weight, ectopic expression of Ltf mRNA with a concurrent decrease in Svs4 mRNA  . SV weight (wt) of male mice (WT and αERKO) treated neonatally on days 1-5 with vehicle (control) or 2 μg/day DES, castrated at 10 weeks of age, and exposed to DHT in Silastic tubing or Silastic tubing alone for an additional 2 weeks. Treatment groups: without (-) DHT: WT control, n = 11; WT DES, n = 6; αERKO control, n = 4; αERKO DES, n = 10; with (+) DHT: WT control + DHT, n = 8; WT DES + DHT, n = 6; αERKO control + DHT, n = 7; αERKO DES + DHT, n = 8).
*p < 0.05 compared with the corresponding control, by parametric tests.   (Marselos and Tomatis 1993;Prins 1992;Prins et al. 2001). In the present study, the hallmark histological alterations of DES exposure were evident in WT mice. However, the penetrance of these effects varied in the βERKO males, whereas αERKO males were resistant to the detrimental histological alterations. Existing evidence that the toxicological effects of develop mental exposure to DES in the female reproductive tract and the prostate of males are dependent on functional ERα at the time of exposure is quite convincing Couse and Korach 2004;Prins et al. 2001). Because many xeno estrogens, including DES, possess properties that can potentially disrupt cellu lar functions apart from their ability to act as estrogens, research toward a thorough understanding of their toxicology has been problematic. Results of investigations such as the present study using receptornull mice add to the growing evidence that the detrimental effects of DES are dependent on functional ERα and therefore are attributable to its hormonal properties and ability to disrupt endogenous estrogen signaling. These data definitively show that the developing male reproductive tract, although perhaps not dependent on estrogens or ER for normal development, expresses ER and is quite sensitive to the toxicological effects of exogenous estrogens when aberrantly exposed. Although the risk of DES exposure among humans, especially during development, has long been diminished (since the mid1970s), the toxicological effects of developmental exposure to DES are largely accepted as a harbinger of similar potentials among environmental and industrial estrogenic compounds, such as genistein and bisphenol A, to which humans are thought to be exposed. Perhaps the most prominent effect of developmental DES exposure in the reproduc tive tract of male mice is a permanent inability of the SVs to reach and maintain their normal size during adulthood. This hallmark effect of DES appears to be due to induction of a permanent resistance to androgen stimulation, on which growth of the SVs is so dependent, because it could not be rescued by a castration/ hormone replacement scheme where animals were provided pharmacological levels of andro gen for 2 weeks. Further evidence of androgen resistance in the SVs after DES exposure is the absence of androgeninduced Svs4 expression during adulthood. Somewhat similar morpho logical and biochemical indicators of androgen resistance are observed in the prostate of DES exposed mice. However, unlike in the prostate where this effect of DES appears to be due at least in part to a permanent reduction in AR expression (Prins 1992;Prins et al. 2001), we demonstrate here that DESinduced androgen resistance in the SVs occurs in the presence of normal AR expression patterns and circulat ing testosterone levels. Our findings are in agreement with other reports showing AR lev els are not significantly reduced in neo natally exposed SV (Turner et al. 1989). These data suggest that the androgen signaling pathway is vulnerable to develop mental exposure to DES at multiple points, both at the level of the AR, as in the prostate, and at points presumably downstream of the AR, as appears to be the case in the SV. This latter mechanism may involve altered expression of a coregulator that is critical to AR function.
The difference in apparent mechanisms by which DES exerts its effects in the prostate versus SV may be due to the tissues originat ing from a different embryonic anlagen, or the developmental stage of each at the time of exposure. The epithelium of the prostate is from the endodermal uro genital sinus, and the epithelium of the SV is from the Wolffian duct. The expression of AR is developmentally regulated, and expression occurs in different parts of the male reproductive tract tempo rally, suggesting some differences in develop mental programming (Cooke et al. 1991).
In addition to permanent androgen resis tance in the SV brought about by neo natal DES exposure, feminized patterns of gene expression also occur. Ltf expression is largely limited to the epithelial tissues of the female reproductive tract under stringent regu la tion by estradiol/ERα actions and is not normally expressed in the male reproductive tract. Previous studies, however, have shown that mice exposed prenatally to DES express signifi cantly high levels of Ltf in the SV (Beckman et al. 1994;Newbold et al. 1989;Pentecost et al. 1988). Here we demonstrate that this DESinduced feminization does not occur in the absence of ERα but remains in the absence of ERβ. These data demonstrate that ERα is fundamental to the molecular feminization of the SV. It remains unclear whether the observed feminization is also due to the onset of androgen resistance, leading to an environ ment of unopposed estradiol stimulation, or is attributable to a wholly separate disruption in normal signaling. Studies have shown that uteri of female mice exposed developmen tally to DES also have aberrantly high expres sion of Ltf in the absence of estrogen (Nelson et al. 1994;Newbold et al. 2007). This dys regulation has been associated with patterns of hypo methylation of CpG sites near the estrogen response element in the Ltf gene in uterine tissues of DESexposed mice (Li et al. 1997). The ectopic expression of Ltf in the SV is plausibly due to a similar mechanism, and the absence of this ectopic expression in mice lacking ERα suggests that this receptor is involved in the epi genetic changes associ ated with neo natal DES exposure. Further studies investigating epi genetic changes using αERKO mice will help elucidate molecular events that lead to permanent alterations in gene expression.

Conclusion
The data presented here demon strate that ERα plays a role in the develop mental effects resulting from DES toxicity in the SV. ERα is involved in the lack of androgen responsive ness determined by increases in SV weight after DHT treatment and by SVS IV protein and Svs4 gene expression, but this does not appear to be due to downregulation of the AR itself. Therefore, other factors that con trol androgen signaling must be affected. In addition, this study definitively shows that ERα is necessary for the molecular femini zation of the SV after neo natal exposure to DES, because we did not observe aberrant LF expression in αERKO mice.
Irrespective of the underlying mecha nisms, the toxicological effects of DES that lead to androgen resistance and feminization in the SV are dependent on functional ERα. Furthermore, this is clearly a toxicological effect of aberrant stimulation of ERα signaling in the SV during development, as unexposed αERKO males invariably exhibited overly wellmaintained SVs, thus indicating that nor mal development and function of the tissue are not dependent on functional ERα.