Localization of estrogen and androgen receptors in male reproductive tissues of mice and rats.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 279A:768 –778 (2004) Localization of Estrogen and Androgen Receptors in Male Reproductive Tissues of Mice and Rats SHUJI YAMASHITA* Electron Microscope Laboratory, School of Medicine, Keio University, Tokyo, Japan ABSTRACT Using immunohistochemical methods, we studied the cell-type- and species-speciﬁc expressions of estrogen receptor (ER) isoforms (ER␣ and ER␤) and androgen receptors (ARs) in the male reproductive tract and accessory sex glands of mature mice and rats. ER␣ and ER␤ showed cell-type- and species-speciﬁc distributions, respectively. In contrast, AR was localized in the epithelial and stroma cells of all tissues examined in this study, in both species. In mice, the epithelial cells of the ductuli efferentes showed a strong ER␣-immunoreaction, and those of the caput epididymis, coagulating glands, and prostate also exhibited a positive reaction. Stroma cells, except in the ductuli efferentes, showed a positive ER␣immunostaining. In rats, ER␣ was detected in very few cell types: the epithelial cells of the ductuli efferentes showed a strong reaction, and the stroma cells of the ampullary and urethral glands exhibited a weak reaction. ER␤ was localized in the epithelial cells of the prostate in mice, while the reaction was faint or negative in both the epithelial and stroma cells of other tissues. In rats, the ER␤-immunoreaction was strongest in the epithelial cells of the ventral prostate. The epithelial cells of the corpus and cauda epididymis, ductus deferens, and urethral glands, and the stroma cells of the urethral glands were also positively ER␤immunostained. Almost the same AR distribution pattern was observed in both species. In particular, strong AR-immunostaining was present in the epithelial cells of the caput and corpus epididymis, seminal vesicle, and ventral prostate. These results indicate that species and tissues differences should be taken into careful consideration in assessing the physiological and pharmacological effects of sex steroids (particularly estrogens) on the reproductive tissues of male rodents. © 2004 Wiley-Liss, Inc. Key words: estrogen receptor ␣; estrogen receptor ␤; androgen receptor; male reproductive tissue Androgens are essential for the normal development and functional maintenance of male reproductive organs. The actions of sex hormones are mediated through their nuclear receptors. Hormone-occupied receptors bind directly to their corresponding hormone responsive elements of target genes, and subsequently recruit transcriptional cofactors to modulate the transcription (Evans, 1988; Beato and Sanchez-Pacheco, 1996; Moras and Gronemeyer, 1998). Androgen receptors (ARs) have been demonstrated in most cell types of male reproductive tissues (Schleicher et al., 1985; Sar et al., 1990; Zhou et al., 2002). Two isoforms of estrogen receptors (ERs)—ER␣ and ER␤— have also been localized in several male reproductive tissues (Iguchi et al., 1991; Kuiper et al., 1997; Pelletier, 2000; Atanassova et al., 2001; Zhou et al., 2002). Although the roles of estrogens in male reproductive organs are still poorly understood, estrogens are widely be© 2004 WILEY-LISS, INC. lieved to participate in normal and abnormal processes of male physiology through ERs (Iguchi, 1992; Carreau et al., 1999; Hess et al., 2001). Recent studies using ER␣knockout mice have demonstrated the presence of ER␣ in Grant sponsor: Ministry of Education, Science and Culture, Japan; Grant number: 11670030; Grant sponsor: Gijuku Academic Development Fund. *Correspondence to: Shuji Yamashita, Electron Microscope Laboratory, School of Medicine, Keio University, 35-Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Fax: 81-3-3353-3290. E-mail: email@example.com Received 8 June 2003; Accepted 23 November 2003 DOI 10.1002/ar.a.20061 Published online 7 July 2004 in Wiley InterScience (www.interscience.wiley.com). ER␣, ER␤ AND AR IN MALE MICE AND RATS the ductuli efferentes to be essential for luminal ﬂuid absorption from the testis (Hess et al., 1997; Lee et al., 2000). In addition, it is well known that exogenous estrogen administration during the perinatal period elicits morphological and functional changes in both the female and male reproductive organs of immature and mature rodents (McLachlan et al., 1975; Newbold et al., 1985; Iguchi, 1992). ER␣ and ER␤ appear to have unique and sometimes opposite roles (Paech et al., 1997; Hall and McDonnell, 1999; Liu et al., 2002). ER␤ reportedly acts as a negative regulator in epithelial proliferation in the uterus and prostate (Weihua et al., 2000, 2001). Both isoforms of ERs and AR can also modulate transcription indirectly through contact with various transcription factors, such as activator protein-1 (AP-1) (Paech et al., 1997; Sato et al., 1997). Kushner et al. (2000) demonstrated that ER␣ and ER␤ have differential interactions with AP-1, depending on the ligands used. In reproductive tissues, sex steroids induce cell-typespeciﬁc responses. Several synthetic sex hormones and antihormones also act in a tissue- and species-speciﬁc manner (Campen et al., 1985; Rutqvist et al., 1995). These differential responses to the ligands may be due to the concentrations of ER isoforms and AR, transcriptional cofactors, and other transcription factors (including AP-1) in tissues and cells. In addition, Cunha and colleagues (Bigsby and Cunha, 1986; Cooke et al., 1997; Kurita et al., 2001) demonstrated that the presence of ERs and AR in stroma cells is important for responses to sex steroids in the epithelial cells of female and male reproductive tissues. Mice and rats are the animals most extensively used to study the effects of sex steroids and the mechanisms of hormone actions, and hormone target tissues from both species are usually assumed to provide similar responses to hormonal stimulation. However, systematic and comparative examinations concerning the distribution of ER isoforms and AR have not been carried out in the male reproductive tissues of mice and rats. Therefore, in the present study, were studied cell-type- and species-speciﬁc expressions of AR, ER␣, and ER␤ in the reproductive tracts and accessory sex glands of mature male mice and rats, using immunohistochemical techniques. MATERIALS AND METHODS Reagents Anti-ER␣ rabbit antibody (MC-20, sc-542), anti-AR rabbit antibody (C-19, sc-815), and their immunizing peptides (sc-542P and sc-815P) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ER␤ rabbit antibody (PA1-310B) and the neutralizing peptide (PEP-007) were obtained from Afﬁnity Bioreagents (Golden, CO). Peroxidase-linked anti-rabbit IgG F(ab)⬘2 fragment (from donkey), HRP-F(ab)⬘2, was purchased from Amersham Japan (Tokyo, Japan). The blocking reagent was from Boehringer Mannheim (Mannheim, Germany), and the block ace was from Dainippon Parmaceutical (Osaka, Japan). A protease inhibitor cocktail (complete) and a protein quantiﬁcation kit were obtained from Roche Diagnostics (Tokyo, Japan) and Dojindo Molecular Technologies (Gaithersburg, MD), respectively. Molecular-weight markers (precision plus protein standards) were obtained from BioRad Laboratories (Hercules, CA), and the ELC Western blotting detection kit was from Amersham Japan. 769 Animals and Tissue Preparation Male CD-1 mice (8 weeks old), male Wistar rats (8 weeks old), and female CD-1 mice (3 weeks old) were obtained from Clea Japan (Tokyo, Japan). The uteri and ovaries from 3-week-old mice were used as a positive control and a standard for the immunohistochemistry and Western blot analysis of ER␣ and ER␤. In addition, we used 8-week-old male mice that were injected with 17␤-estradiol (E2) or vehicle, and killed 1 hr after the injection, to determine a suitable ﬁxing procedure for ERs, since we previously observed that hormone-unoccupied ER␣ is easily extracted from frozen sections during ﬁxation (Yamashita and Korach, 1988). Tissues were mounted in OTC compound. They were then frozen in dry ice-cooled acetone for the immunohistochemistry, or on a dry ice block for Western blotting, and were stored at – 80°C. Six male mice, six male rats, and four female mice were used for the immunohistochemistry. Tissues for the Western blotting were collected from six male mice, six female mice, and four male rats. Immunohistochemistry Frozen sections (7 m thick) were ﬁxed with 4% paraformaldehyde dissolved in 0.15 M phosphate buffer (pH 7.4) for 20 min at room temperature (Yamashita, 2002). The ﬁxed sections were washed with phosphate-buffered saline (PBS), 10 mM phosphate buffer, and pH 7.4 containing 0.85% NaCl, and were then treated with 0.2% glycine in PBS for 30 min. After the sections were treated with the blocking solution (1% bovine serum albumin and 1% blocking reagent dissolved in PBS) for 60 min, they were incubated with each antibody overnight at 4°C. AntiER␣ antibody was diluted 500-fold, and other antibodies were diluted 200-fold. All primary antibodies and HRPF(ab)⬘2 were diluted with the blocking solution. Primary antibodies that were preabsorbed with their immunizing peptides were employed for the control immunostaining. The diluted antibodies (1 ml) were incubated with 5 l of each peptide solution overnight at 4°C. The sections were then incubated with HRP-F(ab)⬘2 diluted 50-fold for 60 min at room temperature. Immunoreaction was visualized with an imidazol-DAB solution. Western Blot Analysis The frozen tissues were homogenized with a glassTeﬂon homogenizer in ice-cold 50 mM Tris-HCl buffer (pH 7.4) containing 0.4 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, and the protease inhibitor cocktail, and centrifuged at 14,000 ⫻ g for 10 min at 4°C. The protein concentration of the supernatant was measured with the protein quantiﬁcation kit, and the supernatant was aliquoted and stored at – 80°C. Each sample was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with a 10% gel, and the precision plus protein standards were used as molecular-weight markers. The separated polypeptides were then transblotted onto a PVDF membrane. The membrane was treated with the block ace for 2 hr at room temperature, and then with the ﬁrst antibodies overnight at 4°C with a gentle shaking (anti-ER␣ antibody, 1:2000; anti-ER␤ antibody, 1:500; anti-AR antibody, 1:400). The membrane was subsequently incubated with HRP-F(ab⬘)2, 5,000-fold dilution, for 1 hr at room temperature. All of the antibodies were diluted with the blocking solution employed for the immunohistochemistry. The enzyme activity of HRP was detected with the use of an ELC 770 YAMASHITA Western blotting detection kit and a Lumivision Imager (HSII; Aisin Seiki, Aichi, Japan). stronger ER␤-immunoreaction than mice in tissues in which ER␤ was detected. RESULTS Immunohistochemical Procedures and Speciﬁcities of Antibodies Reproductive tract. The epithelium of the ductuli efferentes exhibited the strongest ER␣-immunoreaction in the male reproductive tissues of both species (Fig. 3a and b); however, no clear ER␤-immunostaining was present in the epithelium (Fig. 4a). In the mouse epididymal duct, ER␣-immunoreaction was strongest in the epithelial cells of the caput (Fig. 3a). However, in the initial segment, ER␣-immunostaining of the principal cells was faint, although apical cells and narrow cells exhibited moderate staining (Fig. 3a). ER␣immunoreaction in epithelial cells gradually diminished from the corpus to the cauda, and only clear cells showed a positive reaction in the distal portions (Fig. 3c). Stroma cells showed a positive ER␣-immunoreactivity throughout the epididymal duct, with the cauda exhibiting the strongest reaction. In the rat epididymis, ER␣-immunoreaction was negative in both epithelial and stromal cells (Fig. 3b and e). The epididymal epithelium of mice showed a faint ER␤-immunoreaction, whereas those of the corpus and cauda epididymis were weakly immunostained for ER␤ in rats (Fig. 4a and b). In the ductus deferens, ER␣-immunoreaction was positive in the stroma cells, but not in the epithelium, of both species (Fig. 3d and f), while a faint to weak ER␤-immunoreaction was seen in both cell types (Fig. 4c and d). We examined various ﬁxing protocols and antigen-retrieval methods to obtain a suitable immunohistochemical procedure. As the concentration of phosphate buffer-dissolving formaldehyde was increased from 0.05 M to 0.2 M, the extraction of the ERs was suppressed, and hormonetreated and -untreated tissues began to show a similar ER-immunoreaction intensity accompanied by a reduction in immunostaining. Therefore, in this study, we ﬁxed frozen sections with 4% formaldehyde in 0.15 M phosphate buffer at room temperature to reduce the extraction of hormone-unoccupied ERs, and to obtain a relatively high immunoreaction sensitivity. Antigen retrieval procedures by heating were ineffective for the immunostaining of ER␣, ER␤, and AR. When the frozen sections ﬁxed with formaldehyde were soaked in boiling solutions or microwaved in solutions at pH 3.5, 6.0, or 9.0, the immunoreactions of these receptors were completely lost or reduced. ER␣, ER␤, and AR proteins were exclusively localized in the nuclei in the standard tissues (Fig. 1a, c, and e) and all tissues examined in this study. Uterine gland epithelia from 3-week-old mice were used as a standard for ER␣immunostaining and for scoring the intensity of staining (Fig. 1a). Granulosa cells of 3-week-old mouse ovary served as a standard for ER␤-immunostaining intensity (Fig. 1c), and epithelial cells of seminal vesicles in 8-weekold rats were used as a standard for AR-immunostaining (Fig. 1e). In every specimen, immunostaining by antibodies preabsorbed with their respective immunizing peptides almost completely disappeared (Fig. 1b, d, and f). The speciﬁcities of the antibodies were further conﬁrmed by Western blot analyses. Anti-ER␣ antibody recognized the main 67-kDa band (which corresponds to the molecular weight of ER␣ protein) and minor 56- and 50-kDa bands (which may be degradation products of ER␣ (Korach et al., 1988)) in extracts from a 3-week-old mouse uterus (Fig. 2A, lane 1). These bands were recognized in the mouse ductuli efferentes (lane 2), epididymis (lane 3), and ventral prostate (lane 4). In the rat tissues, 67- and 50-kDa bands and a few very faint bands were detected in the ductuli efferentes (lane 5), but not in the epididymis (lane 6) or ventral prostate (lane 7). ER␣-antibody pretreated with the antigenic peptide showed a faint reaction with 67-kDa polypeptide only in the mouse uterus. On the Western blots, anti-ER␤ antibody recognized a single band in the ovary with an apparent molecular weight of 54 kDa (which corresponds to that of ER␤ protein (Kuiper et al., 1996)), whereas preabsorbed antibody with the immunizing peptide provided a negative reaction (Fig. 2B, lane 1). Polypeptides, 140 and 105, 54 and 35 kDa, from the mouse caput epididymis reacted with anti-AR antibody (Fig. 2C, lane 1). The molecular weight of AR is reported to be about 110 kDa (Weihua et al., 2002). Male reproductive tissues showed a differential molecular weight of positive bands, although 54 kDa was a major band (data not shown). Antibody treated with the immunizing peptide showed a negative immunoreaction (lane 2). Localization of ERs ER␣ was localized in many reproductive tissues in mice, but in few cell types in rats. In contrast, rats showed a Accessory glands. In mice, epithelial cells contained relatively high amounts of ER␣ proteins in the coagulating glands (Fig. 3i) and a low level of ER␣ protein in the ventral and dorsal prostate (Fig. 3g). A faint to weak ER␣-immunoreaction was seen in the stroma cells of seminal vesicles, coagulating glands, prostate glands, and urethral glands (Fig. 3g and i). Stromal cells of the ampullary gland exhibited a moderate reaction. In rats, ER␣-immunoreaction was barely recognizable in the epithelial cells of the accessory glands (Fig. 3h and j). Stroma cells in the proximal portion of the ampullary and urethral glands showed a positive ER␣-immunoreaction. ER␤-immunoreaction was present in the epithelial cells of the prostate in both species, with the ventral prostate showing a stronger reaction than the dorsal prostate (Fig. 4e– g and i). In the urethral glands, ER␤-immunoreaction was seen in the epithelial and stromal cells of both mice and rats (Fig. 4h and j). Localization of AR The epithelial cells in rats exhibited nearly the same or a stronger intensity of AR-immunostaining in male reproductive tissues compared to those in mice, except in the coagulating and ampullary glands. Reproductive tract. AR-immunoreaction was seen in the epithelial cells and stroma cells throughout the male reproductive tract in mice and rats. The ductuli efferentes showed faint to weak AR-immunoreaction in both species. The caput and corpus epididymis showed a strong AR-immunoreaction in the epithelial cells (Fig. 5a and b). In the initial segment of the caput epididymis, the apical cells showed a slightly weaker AR-immunoreactivity than the principal and clear cells (Fig. 5a and b). The reaction slightly decreased in the distal portions of the reproductive tract ER␣, ER␤ AND AR IN MALE MICE AND RATS 771 Fig. 1. Speciﬁcities of antibodies and localization of ER isoforms and AR in the tissues used as standards. a: The uterus of a 3-week-old CD-1 mouse was used as a standard tissue for ER␣-immunostaining. Arrows indicate glandular epithelium showing intense staining (⫹⫹⫹⫹). b: The antibody pretreated with the immunizing peptide was used as the control immunostaining. ER␤ localized in the ovaries of a 3-week-old mouse was employed for a standard of ER␤-immunoreaction in the male reproductive tissues. The sections were treated with anti-ER␤ antibody (c), or the antibody preabsorbed with the immunizing peptide (d). Strong ER␤immunostaining (⫹⫹⫹) corresponds to the reaction in the granulosa cells. e: Seminal vesicles of 8-week-old Wistar rats were used as a standard tissue for AR-immunoreaction; immunostaining in the epithelium was scored as strong (⫹⫹⫹). f: Anti-AR antibody preabsorbed with the antigenic peptide was employed for the control immunostaining. Bar ⫽ 50 m. (i.e., the cauda epididymis and ductus deferens; Fig. 5c and d). The AR-immunoreaction in the stroma cells was nearly homogeneous throughout the reproductive tracts of both species. The epithelia of the ventral prostate showed a stronger reaction than those of the dorsal prostate, and the lobespeciﬁc expression of AR was signiﬁcant in mice (Fig. 5e– g and i). The epithelial cells of the coagulating, ampullary, and urethral glands also exhibited a positive AR-immunoreaction (Fig. 5h and j). AR-immunostaining in the stroma cells was almost constant in the accessory glands of both species. Accessory glands. A strong AR-immunoreaction was present in the epithelial cells of the seminal vesicles and ventral prostate in both species (Figs. 1e, and 5e and f). 772 YAMASHITA Fig. 2. Western blot analysis of receptor proteins. A: ER␣ expression in the reproductive tissues of 8-week-old male mice and rats, and the uterus of a 3-week-old mice used as a standard. Gels were loaded with 100 g protein per lane: mouse uterus (lane 1), ductuli efferentes (lane 2), caput epididymis removing (or eliminating) the initial segment (lane 3), ventral prostate (lane 4), rat ductuli efferentes (lane 5), caput epididymis removing (or eliminating) the initial segment (lane 6), and ventral prostate (lane 7). Blots were stained with ER␣ antibody (bottom blot) and the antibody preabsorbed with the immunizing peptide (upper blot), respectively. B: ER␤ in the mouse ovary. Proteins (200 g) were subjected to SDS-PAGE. ER␤ was detected with anti-ER␤ antibody (lane 1) and antibody preincubated with the immunizing peptide (lane 2). C: AR in the mouse caput epididymis removing (or eliminating) the initial segment. Proteins (125 g) were separated by SDS-PAGE. AR was detected with anti-AR antibody (lane 1) and the antibody absorbed with the antigenic peptide (lane 2). Arrows show the positions of the standard proteins for molecular weight. Arrowheads indicate bands detected with the respective antibodies in the mouse uterus (A), ovary (B), and epididymis (C). Table 1 summarizes the localizations of the ER isoforms and ARs in the male reproductive tissues of mature mice and rats. DISCUSSION Western blot and immunohistochemical studies demonstrated that the antibodies used in this study are speciﬁc for each receptor protein, since the antibodies preabsorbed with their respective immunizing peptides exhibited no immunostaining in the tissue sections or blots (although a faint reaction with ER␣ antibody suggestive of an incomplete absorption was seen in the mouse uterus). On the Western blot, anti-ER␣ antibody reacted with 67-kDa ER␣ protein in the mouse ductuli efferentes, epididymis, ventral prostate, and rat ductuli efferentes, but not in the rat epididymis or ventral prostate. These results are in good agreement with those obtained by immunohistochemistry. The predominant 54-kDa band recognized with anti-AR antibody in the mouse epididymis may be a proteolytic fragment of a 105-kDa receptor. Several polypeptides that are assumed to be degradation products of intact receptor proteins were recognized with antibodies in the blots, when the male reproductive tissues were analyzed with antibodies to AR and ER␤. The molecular weights and amounts of the polypeptides varied among tissues, which may be due to differences in activities and the kinds of proteases contained in the tissues. AR proteins were localized in both the epithelial and stromal cells of all of the male reproductive tissues examined in the present study, which is essentially in accordance with results obtained previously by immunohistochemistry and steroid autoradiography (Schleicher et al., 1985; Sar et al., 1990; Prins and Birch, 1993; Pelletier et al., 2000; Zhu et al., 2000; Zhou et al., 2002). On the other hand, ER␣ and ER␤ were expressed in cell-type- and species-speciﬁc manners. Previous studies (Lubahn et al., 1993; Eddy et al., 1996; Krege et al., 1998) observed no signiﬁcant morphological changes in the reproductive tissues of young male ER␣ and ER␤ knockout mice. In addition, Fisher et al. (1998) reported that knockout mice for aromatase had no severe lesions in male reproductive organs, and were capable of breeding. These results indicate that androgens and ARs are essential for the development and normal functioning of male reproductive tissues, and that ERs apparently act as modulators of androgen action. This study demonstrates that ER␣ is highly expressed in the epithelium of the ductuli efferentes in mice and rats (Fig. 3a and b). ER␣ and ER␤ are undetectable in the rete testis of both species (S. Yamashita, unpublished observation). ER␣ has also been localized primarily in the epithelium of the ductuli efferentes in several species, including mice and rats (West and Brenner, 1990; Iguchi et al., 1991; Fisher et al., 1997; Goyal et al., 1997; Hess et al., 1997; Zhu et al., 2000; Zhou et al., 2002). These ﬁndings support previous results obtained using ER␣ knockout mice, which indicated that ER␣ is essential for the ﬂuidreabsorbing function of the ductli efferentes (Hess et al., 1997; Lee et al., 2000); however, the mechanism of ﬂuidreabsorption through the ER␣ system is still unclear. Zhou et al. (2002) demonstrated strong and nearly homogeneous ER␤-immunostaining throughout the epithelial cells of the male reproductive tract in mice. Atanassova et al. (2001), employing the same antibody used by Zhou et al. (2002), demonstrated similar ER␤ localization in Wistar rats. However, the present study demonstrates an ER␤ distribution pattern for both species that is quite different from those previous results, although the cell-type-speciﬁc distribution patterns of ER␣ and AR are in good agreement with those obtained by Zhou et al. (2002) in mice. The antibody used by Zhou et al. (2002) may have a high titer for ER␤ proteins, and thus may be able to detect a low concentration of ER␤. However, ER␤ mRNA levels analyzed by Northern blot were shown to be much lower in the epididymis and ductus deferens than in the ovaries of mice and rats (Couse et al., 1997; Kuiper et al., 1997; Jefferson et al., 2000), and steroid autoradiography demonstrated nuclear labeling of [3H]E2 in the ep- ER␣, ER␤ AND AR IN MALE MICE AND RATS Fig. 3. Localization of ER␣ in male reproductive tissues. Tissues from 8-week-old rodents were used for immunohistochemistry of ER␣. ER␣ was localized in the mouse ductuli efferentes and caput epididymis (a), cauda epididymis (c), ductus deferens (d), ventral prostate (g), and coagulating gland (i), and in the rat ductuli efferentes and caput epidid- 773 ymis (b), caput epididymis (e), ducts deferens (f), ventral prostate (h), and coagulating gland (j). Arrows (c) indicate clear cells. DE, ductuli efferentes; Ei, initial segment of caput epididymis; Ec, caput epididymis. Bar ⫽ 50 m. 774 YAMASHITA Fig. 4. Immunohistochemistry of ER␤ in male reproductive tissues. ER␤ proteins were localized in the mouse ductuli efferentes and caput epididymis (a), ductus deferens (c), ventral prostate (e), dorsal prostate (g), and urethral gland (h). In rats, ER␤ was immunostained in the corpus epididymis (b), ducts deferens (d), ventral prostate (f), dorsal prostate (i), and urethral gland (j). DE, ductuli efferentes; Ei, initial segment of caput epididymis; Ec, caput epididymis. Bar ⫽ 50 m. ER␣, ER␤ AND AR IN MALE MICE AND RATS Fig. 5. Expression of AR proteins in male reproductive tissues. ARimmunostaining is shown in the mouse ductuli efferentes and caput epididymis (a), ducts deferens (c), ventral prostate (e), dorsal prostate (g), and coagulating gland (h), and in the rat caput epididymis (b), ductus 775 deferens (d), ventral prostate (f), dorsal prostate (i), and coagulating gland (j). DE, ductuli efferentes; Ei, initial segment of caput epididymis; Ec, caput epididymis. Bar ⫽ 50 m. 776 YAMASHITA TABLE 1. ER␣, ER␤ and AR expression in the reproductive tissues of mice and rats* ER␣ Tissues Ductuli efferentes Epithelium Stroma Epididymis Caput epididymis Epithelium Stroma Corpus epididymids Epithelium Stroma Cauda epididymis Epithelium Stroma Ductus deferens Epithelium Stroma Seminal vesicles Epithelium Stroma Ampullary glands Epithelium Stroma Coagulating glands Epithelium Stroma Prostate Ventral prostate Epithelium Stroma Dorsal prostate Epithelium Stroma Urethral glands Epithelium Stroma ER␤ AR Mouse Rat Mouse Rat Mouse Rat ⫹⫹⫹⫹ – ⫹⫹⫹ – – – – – ⫾⬃⫹ ⫾ ⫹ ⫾ ⫾⬃⫹⫹a ⫾⬃⫹ – – ⫾ – ⫾ – ⫹⫹ ⫾ ⫹⫹⬃⫹⫹⫹ ⫾⬃⫹ ⫾⬃⫹ ⫾⬃⫹ – – ⫾ – ⫾⬃⫹ – ⫹⫹ ⫹ ⫹⫹⫹ ⫹ ⫾b ⫹⬃⫹⫹ – – ⫾ ⫾ ⫾⬃⫹ – ⫹ ⫾⬃⫹ ⫹⫹ ⫹ – ⫹⫹ – ⫹ ⫾ ⫾ ⫹ ⫾ ⫹ ⫹ ⫹⫹ ⫹⬃⫹⫹ ⫾ ⫾ – – – – ⫾ – ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹ – ⫹⫹ – ⫾⬃⫹⫹c – – – – ⫹⫹ ⫹ ⫹⬃⫹⫹ ⫹ ⫹⬃⫹⫹ ⫹ – – ⫾ – ⫾ – ⫹ ⫹ ⫾⬃⫹ ⫹ ⫹ ⫾ – – ⫹ ⫾ ⫹⫹ – ⫹⫹⬃⫹⫹⫹ ⫹ ⫹⫹⬃⫹⫹⫹ ⫹ ⫹ ⫾ – – ⫾ ⫾ ⫾⬃⫹ – ⫹ ⫹ ⫹⫹ ⫹ – ⫹ – ⫹ ⫾ ⫾ ⫹ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹ *The intensity of ER␣-immunostaining was scored using uterine sections from 3-week-old mice (Fig. 1a): ⫹⫹⫹⫹ (intense), corresponds to the staining in the glandular epithelium; ⫹⫹⫹ (strong), ⫹⫹ (moderate); ⫹ (weak); ⫾ (faint), – (negative). The ovaries of 3-week-old mice served as a standard for ER␤-immunostaining intensity (Fig. 1c): ⫹⫹⫹ (strong); corresponds to the ER␤-immunostaining in the granulosa cells of growing follicles. AR-immunostaining was also described as ranging from strong (⫹⫹⫹) to negative (–): ⫹⫹⫹; corresponds to staining in the epithelial cells of seminal vesicles in rats (Fig. 1e). a Immunoreaction was moderate in the principal cells of caput epididymis, while reaction was faint in the principal cells and weak to moderate in apical and narrow cells in the initial segment. b Clear cells exhibited moderate staining but principal cells showed negative reaction. c Stromal cells were moderately immunostained in the proximal portion and faintly stained in the distal portion. ithelial cells of the corpus and cauda epididymis and the ductus deferens to be very low or absent (Schleicher et al., 1984). The ER␤-immunostaining pattern in the present study supports the results obtained by Northern blot analyses and steroid autoradiography in mice and rats. The rodent prostate has been used as a model system to study the mechanism of hormone actions and hormone-dependent carcinogenesis in the male reproductive tissues (Prins and Birch, 1993; Paris et al., 1994; Yeh et al., 1998). In rodents, lobe-speciﬁc responses to androgens and estrogens have been reported (Prins and Birch, 1993; Banerjee et al., 2001; Risbridger et al., 2001a). The coagulating glands (anterior prostate) ap- pear to be most sensitive to estrogens (as in the case of squamous metaplasia induced by the administration of exogenous estrogen into mature mice (Risbridger et al., 2001a)). In the young CD-1 mice, the epithelial cells of the coagulating gland exhibited the strongest ER␣-immunostaining (Fig. 3i). Iguchi et al. (1991) reported a similar ER␣ distribution in mature C57BL mice. These results suggest that responses to exogenous estrogens depend on the concentration of ER␣, and support the conclusion that ER␣ is the predominant ER isoform in ER␣ knockout mice for mediating estrogen actions, including estrogen-induced squamous metaplasia in the mouse prostate (Risbridger et al., 2001b). ER␣, ER␤ AND AR IN MALE MICE AND RATS It has been reported that both testosterone (T) and estrogen are required for the development of benign prostatic hyperplasia and prostate cancer in both rodents and humans (Wang and Wong, 1998; Cunha et al., 2003; Steiner and Raghow, 2003). Cunha et al. (2003) demonstrated that the presence of AR and ER␣ is essential in hormonal carcinogenesis elicited by treatment with T plus E2 in rodents, based on experiments employing ER␣ and ER␤ knockout mice. The contribution of ER isoforms has not yet been elucidated in the human prostate (Linja et al., 2003; Steiner and Raghow, 2003). However, several reports have suggested that a lower expression of ER␤, which may act as a negative regulator for the proliferation of prostate epithelia, is related to benign prostate hyperplasia and cancer in humans (Horvath et al., 2001; Leav et al., 2001; Fixemer et al., 2003; Tsurusaki et al., 2003). In conclusion, AR proteins were localized in both the epithelial and stromal cells of all of the male reproductive tissues from 8-week-old mice and rats examined in this study. However, ER␣ and ER␤ showed cell-type- and species-speciﬁc distributions. Recently, Harris et al. (2002) reported that the ligand-binding proﬁles of ER␣ and ER␤ are species-dependent. Furthermore, distributions of coactivators and corepressors of nuclear receptors may differ among species and strains of rodents. 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