The Journal of Toxicological Sciences, Review 75 Vol.30, No.2, 75-89, 2005

SEX STEROID HORMONE RECEPTORS IN THE DEVELOPING FEMALE REPRODUCTIVE TRACT OF LABORATORY RODENTS

Akinobu OKADA1*, Tomomi SATO2,YasuhikoOHTA3, 4 andTaisenIGUCHI4, 5

1Safety Research Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 1-1-8 Azusawa, Itabashi-ku, Tokyo 174-8511, Japan 2Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan 3Department of Veterinary Science, Faculty of Agriculture, Tottori University, 4-104 Koyama-Minami, Tottori 680-8553, Japan 4CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi 332-0012, Japan 5Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Science, and Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan

(Received January 7, 2005; Accepted February 8, 2005)

ABSTRACT — Many chemicals released into the environment potentially disrupt the endocrine system in wildlife and humans. Some of these chemicals exhibit estrogenic activity by binding to the estrogen receptors. The developing organism is particularly sensitive to estrogenic chemicals during the critical period in which the induction of long-term changes and persistent molecular alterations in female repro- ductive tracts occur. Perinatal mouse and rat models can be utilized as indicators for determining the con- sequences of exposure to exogenous estrogenic agents, including possible xenoestrogens or environmental endocrine disruptors. Estrogen receptors (ER) and estrogen responsive , therefore, need to be iden- tified in order to understand the molecular basis of estrogenic actions. Recent identifications of ER sub- types and isoforms make understanding target organ responses to these estrogenic chemicals even more difficult. Indeed, many reports suggest that these chemicals do affect the reproductive and developmental processes of female laboratory rodents that had been perinatally exposed, and that interactions between sex steroid hormone receptors occur. Much information concerning the expression of sex steroid receptors in rodents has been reported concerning the normal development of the Müllerian duct. Thus, accumulated information on the expression of ER subtypes and isoforms as well as that of progesterone and androgen receptors in laboratory rodents is herein reviewed, in addition to the presentation of our own data.

KEY WORDS: Estrogen , , , Reproductive tracts, Mülle- rian duct, Endocrine disruption

INTRODUCTION turb the action of estrogen and many of them are known to possess -binding activity (Danzo, Recently, there has been considerable concern 1997). This fact brings to mind historical work by about the potential endocrine-disrupting effects of Herbst et al. done in the early seventies (1971) when a chemicals released into the environment in wildlife and close correlation between the occurrence of vaginal humans (Colborn and Clement, 1992). Chemicals that clear cell adenocarcinoma in young women and early have estrogenic activity are termed xenoestrogens or intrauterine exposure to diethylstilbestrol (DES), a syn- endocrine disruptors. They are thought to mimic or dis- thetic estrogen, was demonstrated (Herbst and Bern,

Correspondence: Akinobu OKADA * Present address: Drug Safety Research Laboratories, Astellas Pharma Inc., 1-6, Kashima 2-chome, Yodogawa-ku, Osaka 532-8514, Japan. Vo l . 3 0 N o . 2 76

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1981). Similarly, during prenatal and neonatal develop- understanding of the molecular and cellular mecha- ment of the paramesonephric or Müllerian duct, estro- nisms underlying effects of sex steroid hormones dur- gen exposure has been found to induce a variety of ing the development of female reproductive tracts, this abnormalities in the Müllerian derivatives such as article will review recent progress in the subtype and uncoiling of the oviduct and uterine hyperplasia in isoform expression of ER in laboratory rodents, as well addition to neoplasia, vaginal hyperplasia and ovary- as those of PR and androgen receptor (AR). independent cornification with an abnormal cell prolif- PR is one of the well-studied estrogen-regulated eration rate in mice and rats (Dunn and Green, 1963; genes that contain estrogen response elements (ERE) Takasugi and Bern, 1964; Iguchi and Takasugi, 1987; (Kastner et al., 1990; Kraus et al., 1994). Previous Newbold et al., 1983; Iguchi, 1992; Sato et al., 1994, reports demonstrated that progesterone-binding sites 2004; Okada et al., 2001). Laboratory rodents exposed and PR expression were regulated by the estrogens in perinatally to estrogens, therefore, provide a model for female reproductive tracts (Ennis and Stumpf, 1988; the exploration of the consequences of xenoestrogen Ohta et al., 1996; Okada et al., 2002b). Kurita and co- exposure in humans, since rodent genital tract develop- workers (2000) showed mechanistic evidence of PR ment at birth is similar to that of the human fetus at the regulation by estradiol (E2) in mouse uterine and vagi- end of the first trimester. nal epithelia in vivo in tissue recombinants made with Recent developments in molecular and cellular and stroma from wild-type and/or αERKO biology techniques have identified many subtypes and mice. They suggested paracrine regulation via stromal isoforms of sex steroid hormone receptors and the ERα and direct regulation via epithelial ERα in mouse complex interaction between them. The specific uterus and vagina, respectively. Androgens also have importance of subtypes and isoforms of estrogen uterotrophic effects in intact and ovariectomized receptor (ER) and progesterone receptor (PR) in repro- immature female rats (Armstrong et al., 1976), and tes- ductive and non-reproductive tissues was proposed in ticular feminized male (Tfm/Tfm) mice show impaired ER or PR knockout mice (Lubahn et al., 1993; Krege et reproductive performance (Lyon and Glenister, 1980), al., 1998; Couse and Korach, 1999; Couse et al., 1999; both of which suggest the importance of androgens in Conneely and Lydon, 2000; Dupont et al., 2000; female reproduction (Kowalski et al., 2004). Weihua et Conneely et al., 2003). However, reproductive tract al. (2002) have recently reported the essential role of development is normal during the prenatal and neona- AR in estrogen-induced uterine epithelial cell prolifer- tal stages in the knockout mice, suggesting that ER and ation in rats, and they indicated that stromal AR ampli- PR signals are not necessary for the morphogenesis of fied the ERα signal by induction of insulin-like growth the female reproductive tract. Nevertheless, as factor I (IGF-I), which is known to be produced in stro- described above, perinatal administration of estrogens mal cells and induce epithelial cell proliferation in a exerts teratogenic and carcinogenic effects on female paracrine fashion. Moreover, direct inhibitory effects rodent reproductive tracts. Findings in estrogen recep- of the ERα/AR heterodimer on both ERα and AR tor α knockout (αERKO) mice and in transgenic mice transactivational properties have been reported (Panet- which overexpress ERα lead to the speculation that the Raymond et al., 2000). Thus, identification of regional teratogenic and carcinogenic effects of estrogens are and cellular AR and PR localization may allow a better mediated through ERα (Couse et al., 1997, 2001). understanding not only of the role of AR and PR, but There have been accumulating reports regarding the also the mechanism of estrogen action in female repro- expression pattern of sex steroid hormone receptors in ductive tracts. the developing female reproductive tracts in mice and rats. This information allows us to understand which A brief description of the development of the female cells and tissues are targets for physiological and exog- reproductive tract enous estrogens. The prenatal and neonatal ontogenies In mammals, both the paramesonephric or Mülle- of sex steroid hormone receptors in female reproduc- rian duct and the mesonephric or Wolffian duct are sit- tive tracts have been reported (Pasqualini and Sumida, uated within the genital ridge. There are two ducts 1986; Greco et al., 1993). However, experiments have present during fetal development. The Müllerian duct been performed using radio-labeled ligands or immu- develops in the cranial to caudal direction along with nohistochemistry with the antibody, both of which are the Wolffian duct during gonad formation (Dohr and non-specific to the receptor subtypes or isoforms Tarmann, 1984; Byskov and Høyer, 1994). After gonad (Murakami et al., 1990). Thus, in order to have a better differentiation, androgens produced by Leydig cells

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Sex steroid receptors in the female reproductive tract. induce Wolffian duct development, and Müllerian al., 1997) and humans (Mosselman et al., 1996), show- inhibiting substance (MIS) produced by Sertoli cells ing high homology between the species. E2 bound induces Müllerian duct degeneration in male fetuses. ERα with a higher affinity than ERβ and increased In female fetuses, Müllerian duct development and ERα transcriptional activity at the EREs in a reporter Wolffian duct degeneration occur due to a lack of assay (Kuiper et al., 1997; Pettersson et al.,2000).In androgens and MIS after gonadal differentiation (Josso addition to these differences in transcriptional activity, and Picard, 1986). Thus, male reproductive organs ERα and ERβ can have entirely opposite transcrip- such as the epididymis, vas deferens, and seminal ves- tional effects at activating -1 (AP-1) sites, icles arise from the Wolffian duct, and female repro- depending on the ligands (Paech et al., 1997). More- ductive organs such as the oviduct, uterus, and upper over, the role of ERβ as a negative regulator of ERα vagina arise from the Müllerian duct. has been suggested in mouse uterus (Weihua et al., The Müllerian duct is comprised of epithelial 2000). Five isoforms of ERβ mRNA, ERβ1(ERβ), cells surrounded by mesenchymal cells. The pattern of ERβ2, ERβ1-δ3, ERβ2-δ3, and ERβ1-δ4, have been cell proliferation of the Müllerian duct has been dem- reported in various rat tissues as products of alternative onstrated in prenatal, immature, and adult rodents splicing (Maruyama et al., 1998; Petersen et al., 1998; (McCormack and Glasser, 1980; Quarmby and Price et al., 2000). Only ERβ1andERβ2, which has an Korach, 1984; Li, 1994; Okada et al., 2001). In tissue additional 18 amino acids in the ligand binding domain recombination experiments, epithelial-mesenchymal of ERβ1, have the ability to bind both ligand and ERE. interactions are critically important for the proper dif- However, ERβ2 has an apparently lower binding affin- ferentiation of the Müllerian duct derivatives (Kurita et ity for E2 as compared to ERβ1(Petersenet al., 1998). al., 2001). At birth, Müllerian epithelium is negative Therefore, ERβ2 can act as a dominant negative regu- for uterine and vaginal epithelial markers, and uterine latory partner during heterodimerization with ERα or and vaginal expression patterns are induced in ERβ1. This action has been demonstrated during rat neonatal Müllerian epithelium by the respective mes- mammary gland development (Saji et al., 2001). enchymes. Kurita et al. (2001) also showed that func- Two PR isoforms, PR-A and PR-B, are produced tional differentiation of uterine and vaginal epithelia from a single gene by transcription at two distinct pro- required organ-specific mesenchymal/stromal factors, moter sites (Horwitz and Alexander, 1983; Kastner et but mesenchymal/stromal signals regulating prolifera- al., 1990; Savouret et al., 1990). The expression of two tion of epithelial cells in the uterus and vagina protein isoforms from the one gene is conserved in sev- appeared to be nonspecific. Since it is well known that eral species, including mice, rats, and humans (Lessey the functional differentiation of uterine and vaginal et al., 1983; Schott et al., 1991; Kraus et al., 1993). epithelia is regulated by E2 and progesterone (P4), The human PR-A isoform differs from the PR-B in that mesenchymal/stromal signals for regulating functional it lacks 164 amino acids on its N terminus. The ratio of epithelial differentiation may be controlled through isoforms varies depending on the reproductive tissues mesenchymal/stromal ERs and PRs. Thus, temporal during development (Shyamala et al., 1990) and the expressions of these receptors may be helpful for estrous cycle (Mangal et al., 1997). PR-A- and PR-B- understanding Müllerian duct development. deficient mice suggest the different actions of each PR isoform in vivo (Lubahn et al., 1993; Krege et al., Subtypes and isoforms of estrogen and progester- 1998; Couse and Korach, 1999; Couse et al., 1999; one receptors Conneely and Lydon, 2000; Dupont et al., 2000; Estrogen, progesterone, and androgen actions are Conneely et al., 2003). mediated by ERs, PRs, and AR, respectively, which belong to the superfamily of ligand- ONTOGENIC EXPRESSION OF inducible transcription factors (Mangelsdorf et al., ESTROGEN RECEPTORS 1995). The steroid -ligand complex binds target DNA at hormone response elements During prenatal development of the Müllerian duct (HREs) to transcribe various downstream genes such Immunohistochemistry of ERα and ERβ showed as and genes encoding growth factors. In differential expression of the two ER subtypes in the rats, two subtypes of ERs, classical ERα (Koike et al., ovaries and uterus of adult rats (Shughrue et al., 1998; 1987) and novel ERβ (Kuiper et al., 1996) have been Hiroi et al., 1999; Sar and Welsch, 1999; Wang et al., identified. ERβ was also cloned in mice (Tremblay et 1999; Mowa and Iwanaga, 2000a; Pelletier et al.,

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2000). In female rodents, expression of ERα was 15.5 dpc, mesenchymal ERα staining could be detected in both the epithelium and stroma of the detected, but epithelial staining was only slight. Epi- uterus and vagina (Sato et al., 1992, 1996; Ohta et al., thelial and mesenchymal ERα levels within the oviduct 1993, 1996; Graham and Clarke, 1997; Mowa and region increased gradually from 15.5 dpc to 19.5 dpc. Iwanaga, 2000a; Shughrue et al., 1998; Wang et al., Uterine and vaginal ERα levels continually increased 1999). ERβ is reportedly expressed at a lower level within the mesenchyme throughout gestational devel- than ERα in the uterus and vagina of adult mice and opment and reached marked intensity levels by 21.5 rats (Mowa and Iwanaga, 2000a; Shughrue et al., 1998; dpc. However, epithelial ERα staining was slight or Wang et al., 1999). ER is expressed in the Müllerian absent from the uterine and vaginal regions throughout duct of fetal mice (Greco et al., 1991; Wu et al., 1992; gestation until 21.5 dpc. Although this study evaluated Greco et al., 1993; Lemmen et al., 1999; Jefferson et ERα expression in the upper vagina, which originated al., 2000; Nielsen et al., 2000) and rats (Mowa and from the Müllerian duct, earlier ERα expression at 16 Iwanaga, 2000b). Greco and co-workers (1991) dpc was detected in the epithelium of the lower vagina, reported that ER immunoreactivity was observed in which was derived from the urogenital sinus in mice epithelial cells of female mouse reproductive tract at (Kurita et al., 2001). These observations clearly dem- 15 days post-coitus (dpc), but was occasionally onstrate that ERα is expressed region- and cell type- observed at 17 dpc and on the day of birth. The results dependently during the prenatal development of the of immunohistochemistry with a monoclonal antibody Müllerian duct. The patterns of ERα localization sug- specific to ERα in mice (Nielsen et al., 2000), and in gest potential tissue-specific mechanisms by which situ hybridization with a radio-labeled probe specific estrogenic chemicals may influence Müllerian duct to ERα in rats (Mowa and Iwanaga, 2000b) demon- cell growth and differentiation. strate that ERα localization varies with the region of No expression of ERβ1(ERβ) was detected in the Müllerian duct involved. Nielsen et al. (2000) the mouse uterus at 16 dpc in the ribonuclease protec- detected a faint immunopositive signal in the nuclei of tion assay (RPA) (Jefferson et al., 2000). Two in situ the surrounding cells of the Müllerian duct as early as hybridization studies revealed similar negative results 11.5 dpc in mice. Epithelial ERα was first observed in in the epithelium and mesenchyme of the Müllerian the proximal oviduct region at 13.5 dpc. At the mRNA duct throughout the prenatal period in rats and mice level, expression of ERα was exhibited in the oviductal (Lemmen et al., 1999; Mowa and Iwanaga, 2000b). epithelium and all mesenchyme at 17 dpc. It was However, in the RT-PCR study, rat Müllerian duct expressed in the uterine and vaginal epithelia on post- ERβ1andERβ2mRNAsweredetectedandfoundto natal days. The semi-quantitative reverse-transcription be expressed at constant levels from 15.5 dpc to 21.5 polymerase chain reaction (RT-PCR) study showed dpc (Okada et al., 2002a). Expression levels of ERβ1 that the ERα mRNA level in the Müllerian duct at 21.5 and ERβ2 mRNA were much lower than that of ERα. dpc was 4.4-fold higher than that at 15.5 dpc in rats Comparison of ERβ1andERβ2 expression levels indi- (Okada et al., 2002a). In immunohistochemical evalu- cated that ERβ2 mRNA expression is significantly ations, ERα protein showed a similar expression pat- greater than that of ERβ1 in the 19.5 dpc Müllerian tern to its mRNA (Table 1). In the oviduct region at duct. The tissue-specific expression ratio of ERβ1ver-

Table 1. Ontogenetic immunolocalization of ERα in the fetal female rat reproductive tracta. Tissue Cell type Prenatal days 15.5 17.5 19.5 21.5 Oviduct Epithelium ± + ++ ++ Mesenchyme − ± + + Uterus Epithelium − − − − Mesenchyme − ± + ++ Upper vagina Epithelium NF − − ± Mesenchyme NF ± + ++ a: Data from Okada et al. (2002a) ++: marked, +: moderate, ±: slight, −: negative, NF: Not formed.

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Sex steroid receptors in the female reproductive tract. sus ERβ2hasbeendescribedinvariousrattissues; or mice, respectively, at all. In a quantitative real-time approximately 1:1 in prostate, ovary, muscle, and pitu- RT-PCR study, oviductal ERα mRNA increased until itary, and 2- to 6-fold expression of ERβ1 compared to neonatal day (ND) 3, and was maintained at a high that of ERβ2 in tissues of the nervous system (Petersen constant level through ND 20. However, ERβ mRNA et al., 1998). Recently, the importance of ERβ2asa was detected in the oviduct in a low and constant man- negative regulator of ERα has been demonstrated in ner, as compared with ERα, throughout pre- and post- the rat mammary gland in which ERβ2levelsarecom- natal development (Okada et al., 2003). In the parable or higher than those for ERβ1(Sajiet al., immunohistochemical study of cell- and region-speci- 2001). Although both ERβ1andERβ2 levels are obvi- ficity (Table 2), epithelial ERα was weak at birth (ND ously higher than ERα in the mammary gland, both 0) and exhibited moderate stainings from ND 3 to 20. isoforms were extremely low in rat Müllerian duct However, some ERα negative cells were present in the compared to ERα, implying insufficient amounts of epithelium of the INF/AMP region after ND 10. Stro- ERβs acting as a negative regulator of ERα in the rat mal cells showed ERα staining at weak, moderate, and Müllerian duct. The physiological significance of marked levels at ND 0, from ND 3 to 10, and ND 15 ERβ2inratMüllerian duct is unknown, but different and 20, respectively, and no marked difference in ERα ratios of ERα/ERβ1/ERβ2 among tissues may suggest staining was noted between regions in the oviduct. tissue-specific regulation depending on the ER sub- Moderate ERα staining was also found in muscle cells types and isoforms. Taken together, these results indi- of the IST/UTJ region from ND 7 to 20. ERβ immu- cate that ERα is likely a dominant ER subtype during noreactivity was indistinguishable in the neonatal ovi- Müllerian duct development. Furthermore, region-spe- duct. Thus, abundant ERα may be a major ER subtype cific ERα expression firmly indicates that functional and play an essential role in the development and func- differentiation within the Müllerian duct occurs before tion of the neonatal rat oviduct. morphological differentiation during the neonatal As described above, in the developing rat ovi- period, and that regional targets for chemicals that may duct, ERα was expressed in both epithelial and stromal act via or influence ER-mediated mechanisms are spe- cells, and the staining intensity and mRNA level cific during late gestational Müllerian duct differentia- increased with the growth of the neonates. With the tion. increases in these receptors during the early postnatal period, immunoreactivity for β-tubulin IV, a cilia During neonatal development of the oviduct marker protein, in the rat oviduct appeared between The mammalian oviduct, or fallopian tube, is part ND 5 and ND 7. Komatsu and Fujita (1978) have of the female reproductive tract that has a fundamental reported in their electron-microscopy study that the role in gamete transport, fertilization, and subsequent differentiation of ciliated cells, which is believed to be early embryo development (Jansen, 1984). During elicited by the initiation of endogenous estrogen pro- neonatal development, the oviduct forms a coiled duction, occurs in the mouse oviduct on ND 5. Further- structure and is composed of four different regions: the more, neonatal E2 administration accelerated cilia infundibulum (INF), ampulla (AMP), isthmus (IST) formation in the mouse and rat oviduct (Eroschenko, and uterotubal junction (UTJ). Depending on the ovi- 1982; Okada et al., 2004a, 2004b). Although the stim- ductal region, there are at least two types of epithelial ulation of ER signaling accelerates the differentiation cells: ciliated epithelial cells and nonciliated or secre- process of ciliated epithelial cells, the presence of cilia tory epithelial cells. Growth and development of these in the αERKO mouse oviduct suggests that it is not cell types are under regulation by the sex steroid hor- fundamentally required for this event in the ERα-neg- mones E2 and P4 (Abe and Oikawa, 1993). ERα and ative ciliated epithelial cells of the adult oviduct ERβ expression in the rat oviduct has been reported (Okada et al., 2004a). Double immunohistochemical (Saunders et al., 1997; Sar and Welsch, 1999; Mowa staining for ERα with β-tubulin IV was reported in the and Iwanaga, 2000a, 2000b; Wang et al., 2000), but the determination of surface epithelial cell types express- predominant ERα expression was shown in the neona- ing ERα (Okada et al., 2003). The ciliated cells were tal oviduct. Oviductal ERβ expression was low in rats positive for β-tubulin IV, while the nonciliated (secre- (Saunders et al. 1997, Mowa and Iwanaga, 2000a, tory) cells were negative for it. ERα was selectively 2000b) and mice (Couse et al., 1997), while Sar and observed in nonciliated epithelial cells, but not in cili- Welsch (1999) and Jefferson et al. (2000) failed to ated epithelial cells of the INF/AMP. In contrast, detect ERβ immunopositive cells in the oviducts of rats almost all epithelial cells in the UTJ/IST were negative

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A. OKADA et al. − − − + ± ± + ++ ++ ++ +++ +++ IST/UTJ b c − − + ± ± ± 20 ID ID ID ID ++ +++ INF/AMP − − − ± ± ± + ++ ++ ++ +++ +++ . a IST/UTJ b c − − + ± ± ± 15 ID ID ID ID ++ +++ INF/AMP − − − − ± ± ± ± ++ ++ ++ ++ IST/UTJ b c − − + ± ± ± 10 ID ID ID ID ++ ++ INF/AMP − − − − − ± ± ± ++ ++ ++ ++ IST/UTJ c 7 − − ± ± ± ± ID ID ID ID ++ ++ INF/AMP 5 − − ± ± ± ± ID ID ID ID ++ ++ , PR-A+B, and AR in the prenatal neonatal female rat oviduct β 3 − − ± ± ± ± ID ID ID ID ++ ++ , ER α 0 + + − − ± − − − : negative, ID: indistinguishable. : negative, ID ID ID ID Neonatal days − : Some epithelial cells stained negative or moderately. : Some epithelial cells stained negative c : slight, + ± − − ± − − − ± 19 ID ID ID ID : weak, + ± − − − ± − − − Prenatal days 15 ID ID ID ID (2003) et al. : moderate, Epi Str Mus Epi Str Mus Epi Str Mus Epi Str Mus ++ Cell type Ontogenetic immunolocalization of ER β α : marked, : marked, AR PRs ER ER : Some epithelial cells stained negative. : Some epithelial cells stained negative. : Data from Okada Epi: epithelial cells, Str: stromal Mus: muscle cells. isthmus, UTJ: uterotubal junction. AMP: ampulla, IST: INF: infundibulum, +++ Table 2. Table a b

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Sex steroid receptors in the female reproductive tract. for β-tubulin IV, but positive for ERα. ONTOGENIC EXPRESSION OF PROGESTERONE RECEPTORS During neonatal development of the uterus and vagina During prenatal development of the Müllerian duct Region-specific expression of ER was previously In adult rodents, expression of PR has been reported in the female reproductive tracts of neonatal reported in both the epithelium and the stroma of the mice.Onthedayofbirth,ERwasexpressedinepithe- uterus and vagina (Ohta et al., 1993, 1996; Graham and lial cells of the oviduct, cervix and vagina, but not in Clarke, 1997; Mowa and Iwanaga, 2000a; Shughrue et the uterus. In contrast, stromal cells expressed ER in all al., 1998; Wang et al., 1999). Developmental changes of these organs, including the uterus (Yamashita et al., in PR have been reported in the uterus of guinea pigs 1989; Sato et al., 1992, 1996; Ohta et al., 1993, 1996; and rats using binding assay with radio-labeled ligand Li, 1994; Fishman et al., 1996). The onset of uterine (Pasqualini and Nguyen, 1980; Nguyen et al., 1988), epithelial ER immunostaining was ND 4 in mice showing a developmental increase in P4 binding sites. (Yamashita et al., 1989) and ND 5 in rats (Table 3, In the RT-PCR study, PR mRNA expression was Ohta et al., 1996). ER mRNA was observed starting on detected from 15.5 dpc and increased gradually by ND7inrats(Fishmanet al., 1996). However, strain 21.5 dpc in the rat fetus (Okada et al., 2002b). Signifi- differences in the ontogenetic localization of ER were cant increases were exhibited on 21.5 dpc compared to reported in uterine epithelial cells of neonatal BALB/c 15.5 dpc and 17.5 dpc. Localization of PR protein was andCD-1mice(Bigsbyet al., 1990). Mowa and assessed during rat development by immunohis- Iwanaga (2000b) evaluated ERα expression in the neo- tochemistry using a monoclonal antibody against both natal female rat reproductive tract using in situ hybrid- PR-A and PR-B (PR-A+B) (Table 4). PR-A+B immu- ization. No ERα mRNA signals were found in the nostaining was localized within the nuclei of the Mül- uterine, cervical or vaginal epithelia until ND 4-6. The lerian epithelium. Although the vagina had not yet first appearance of ERα mRNA signals in uterine and been formed by 15.5 dpc, slight epithelial PR staining vaginal epithelia was recognized by ND 4 and 6, could be detected in the oviduct and uterus by this respectively. In the vagina, the strongest and most dis- time. Weak staining of Müllerian epithelial PR tinct signals for ERα mRNA were localized in the appeared in the upper vaginal region by 17.5 dpc. The basal layer of the epithelium and diminished in intensity of Müllerian epithelial PR staining increased strength towards the lumen. Uterine glandular epithe- gradually from 15.5 dpc until 19.5 dpc within all three lium expressed signals for ERα mRNA by ND 14. The of the regions. Staining levels became constant by 19.5 cervix was the only portion of the developing repro- dpc and moderate staining appeared through 21.5 dpc. ductive tract lacking epithelial ERα. Jefferson et al. In contrast, mesenchymal PR staining was faint in all (2000) also revealed postnatal ERα and ERβ expres- regions of the Müllerian duct throughout gestational sion in the female mouse reproductive tract using RPA development (Okada et al., 2002b). Kurita et al. (2001) and immunohistochemistry. The results of RPA also reported negative immunostaining for PR-A+Bin revealed that the expression of ERα mRNA in the mesenchymal cells of the uterus, upper vagina, and uterus was found at all stages of neonatal development, lower vagina during prenatal development in mice. In but no ERβ mRNA appeared until ND 26. Moreover, in order to investigate the differential expression of PR immunohistochemistry, ERα was detected in stromal isoform mRNAs in the oviduct, two distinct primer cells of the uterus throughout the postnatal period and pairs were used; one detected A and B isoforms equally in epithelial cells as early as ND 5. Similarly, ERα and the other was specific for B isoform (Okada et al., immunostaining was detected on ND 5 in mouse uter- 2003). Oviductal PR-A+B and PR-B were equally ine epithelium, and at birth in both the Müllerian and expressed at low levels from 15 dpc until birth. sinus vagina (Kurita et al., 2001). Signals for ERβ mRNA were only weakly expressed in the reproductive During neonatal development of the oviduct tract during the rat postnatal period (Mowa and Neonatal development of oviductal epithelial Iwanaga, 2000b), and there was no expression of ERβ cells is regulated by E2 and P4 (Abe and Oikawa, protein in the mouse uterus (Jefferson et al., 2000). 1993). Neonatal PR-A+B ontogeny in oviduct has been reported in mice (Li, 1994) and rats (Okada et al., 2003). Li (1994) demonstrated that in mice, staining of the epithelial PR was weak to positive from ND 1 to 3,

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A. OKADA et al. 30 ++ ++ ++ ++ +++ +++ 25 ++ ++ ++ ++ ++ +++ + 20 ++ ++ ++ ++ +++ + / 15 ++ ++ ++ ++ − +++ + / − 12 ++ ++ ++ ++ − . a + + − − 10 ++ ++ ++ ++ 7 − − / / ++ ++ + + + + / / 5 − − ++ ++ − − 4 − − − ID ID ++ 3 − − − ID ID ++ , and PR-A+B in the neonatal female rat uterus α 2 − − − ID ID ++ : negative, ID: indistinguishable. : negative, ++ 1 − − − − / ID ID + : weak, + 0 − − − − ID ID Neonatal days . (1996) et al : moderate, Epi Str Mus Epi Str Mus ++ Cell type Ontogenetic immunolocalization of ER α : marked, : marked, PRs ER : Data from Ohta Table 3. Table a Epi: epithelial cells, Str: stromal Mus: muscle cells. +++

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Sex steroid receptors in the female reproductive tract. and moderately positive from ND 7, stromal and mus- B between cell types in the cycling mouse oviduct. PR- cular PR was evident from ND 1. In our rat study, PR A was the predominant isoform in the oviduct, immunoexpression was evident in epithelial cells at observed within both epithelial and stromal cells of the slight levels at birth, however, it was not present in AMP and IST regions. In contrast to PR-A expression, stromal cells (Table 2). Slight epithelial PR expression PR-B was detectable only in epithelial cells lining the continued until ND 5 in the undifferentiated oviduct, AMP and IST regions. and until ND 20 in the INF/AMP region. However, some epithelial cells showed a negative or moderate During neonatal development of the uterus and PR signal in the differentiated INF/AMP region from vagina ND 7 to 20. A double immunohistochemical study on Immunohistochemical uterine and vaginal PR PR-A+Bwithβ-tubulin IV revealed that PR-A+B is expression has been described in mice (Li, 1994; expressed specifically in ciliated epithelial cells, but Kurita et al., 2001) and rats (Ohta et al., 1993, 1996). not in nonciliated cells, the same as ERα (Okada et al., Li (1994) detected positive PR staining in uterine epi- 2003). In contrast, epithelial PR staining was intense in thelial cells at birth. However, other reports failed to the differentiated IST/UTJ region, and showed moder- detect PR at birth, but first detected it on ND 3 in mice ate and marked signals on ND 7 to 10, and ND 15 to and ND 5 in rats (Table 3, Ohta et al., 1996). Uterine 20, respectively. PR was also detected in stromal cells epithelial PR increased with subsequent postnatal after ND 3, but was absent from the IST/UTJ region on development in both mice and rats. Similarly, uterine ND 7 to 10. Muscle cells showed slight and moderate stromal and muscular cells expressed PR from birth in PR staining in the IST/UTJ region on ND 10, and from mice, but not in rats. Rat stromal and muscle PR was ND 15 to 20, respectively. Oviductal PR-A+B and PR- found in the uterus from ND 12 and ND 15, respec- B mRNAs were equally expressed at low levels from tively. Kurita et al. (2001) evaluated vaginal PR expres- birth to ND 3, and both increased gradually from 15 sion during postnatal development in mice, but dpctoND5.PR-A+B then increased markedly until epithelial immunostaining for PR was negative from ND 20, but PR-B expression continued to increase birth to ND 14 in both the Müllerian and sinus vagina. moderately from ND 7 to 20, resulting in a decrease in No report on isoform expression for uterine or vaginal the percentage of PR-B to one tenth vs. one quarter PR during postnatal development has been found. against PR-A+B(Okadaet al., 2003). At present, although little evidence has been available to define the ONTOGENIC EXPRESSION OF physiological significance of P4 action via PR-A and/ ANDROGEN RECEPTOR or PR-B, it seems highly probable that differential expression of the two isoforms in the oviduct is funda- In rat uterus, AR has been identified by both mental for cell growth, differentiation, and function in immunohistochemistry and in situ hybridization in epi- response to P4. PR-A is reportedly able to act as a tran- thelial, stromal, and smooth muscle cells (Hirai et al., scriptional inhibitor of PR-B when both are 1994). In human vagina, AR immunoreactivity was co-expressed (Vegeto et al., 1993). Gava et al. (2004) found in both epithelial and stromal cells (Hodgins et described the differential expression of PR-A and PR- al., 1998; Pelletier et al., 2000). AR was also expressed

Table 4. Ontogenetic immunolocalization of PR-A+B in the fetal female rat reproductive tracta. Tissue Cell type Prenatal days 15 17.5 19.5 21.5 Oviduct Epithelium ± ± + + Mesenchyme − − − − Uterus Epithelium ± ± + + Mesenchyme − − − − Upper vagina Epithelium NF ± + + Mesenchyme NF − − − a: Data from Okada et al. (2002b) +: moderate, ±: slight, −: negative, NF: Not formed.

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A. OKADA et al. in the female reproductive tracts of rat fetuses. type-specific expression patterns in the female repro- Although AR levels were similar at 17 dpc in the uro- ductive tract, demonstrating the importance of finding genital tubercle of male and female fetuses, the amount which are target organs and cells for each developmen- of AR decreased after 18 dpc in female urogenital tis- tal stage. In addition, we need to elucidate the molecu- sues (Bentvelsen et al., 1994). AR mRNA was also lar mechanisms of estrogenic chemicals. Microarray expressed in the rat oviduct, and increased gradually methodology will provide a powerful technique for with development of the pre- and neonatal rats (Okada identifying global gene expression changes during et al., 2003). Drews et al. (2001) investigated the development and for understanding the molecular immunohistochemical localization of AR in the Mülle- basis of the adverse effects of the estrogenic chemicals rian duct of fetuses at 17 dpc and 18 dpc. AR staining that can alter signaling systems (Watanabe et al., 2002, showed negative throughout the duct except for mesen- 2003, 2004; Wu et al., 2003; Hu et al., 2004). It is nec- chymal cells in the sinus region. In the developing ovi- essary to study targets of estrogenic chemicals with duct, no AR immunoreactivity was detected on 15 dpc consideration to the developmental stage and with an and 19 dpc (Table 2, Okada et al., 2003). Both epithe- understanding of the phenotypes that result from peri- lial and stromal ARs were first detected at a low level natal exposure, in addition to the changes in gene on ND 3, and maintained at similar level until ND 20 in expression obtained from microarray and other molec- all oviduct regions. In the IST and UTJ, muscle AR ular techniques. was observed at slight and weak levels from ND 7 to 10, and ND 15 to 20, respectively. AR was predomi- ACKNOWLEDGMENT nantly detected in nonciliated epithelial cells in the INF/AMP, and stromal and muscle cells in the rat ovi- This work was supported in part by a Grant-in- duct. Recently, a direct inhibitory effect of the ERα/ Aid for Scientific Research on Priority Areas (A) from AR heterodimer on both ERα and AR transactivational the Ministry of Education, Culture, Sports, Science, properties has been reported (Panet-Raymond et al., and Technology of Japan, a Research Grant from the 2000). Although, in the rat oviduct, co-localization of Ministry of Environment, and a Health Sciences AR and ERα has not been determined, both AR and Research Grant from the Ministry of Health, Labour, ERα were expressed in stromal, muscle, and noncili- andWelfare,Japan. ated epithelial cells. Interaction of AR and ERα, there- fore, may possibly occur in the rat oviduct as well as in REFERENCES the uterus. Abe, H. and Oikawa, T. (1993): Effects of estradiol FINALLY and progesterone on the cytodifferentiation of epithelial cells in the oviduct of the newborn Xenoestrogens or endocrine disruptors bind ER golden hamster. Anat. Rec., 235, 390-398. and may cause estrogenic effects in humans. In labora- Armstrong, D.T., Moon, Y.S. and Leung, P.C. (1976): tory rodents, perinatal exposure to estrogenic chemi- Uterotrophic effects of testosterone and 5α- cals causes a variety of abnormalities in the female dihydrotestosterone in intact and ovariecto- reproductive tract. Thus rats and mice have been used mized immature female rats. Biol. Reprod., 15, as a model system for the investigation of the perinatal 107-114. effects of estrogenic chemicals to human embryos and Bentvelsen, F.M., McPhaul, M.J., Wilson, J.D. and fetuses. Meanwhile, several subtypes and isoforms of George, F.W. (1994): The androgen receptor of sex steroid hormone receptors, including ER, have the urogenital tract of the fetal rat is regulated been identified, and understanding the interactions by androgen. Mol. Cell. Endocrinol., 105,21- between these subtypes and isoforms can shed light on 26. the molecular mechanism of hormone action. In order Bigsby, R.M., Aixin, L., Luo, K. and Cunha, G.R. to clarify the effect of estrogenic chemicals on the (1990): Strain differences in the ontogeny of reproductive tract, it is essential to understand in which estrogen receptors in murine uterine epithelium. organs, on which cell types, and during what stages of Endocrinology, 126, 2592-2596. development these chemicals can act. Accumulating Byskov, A.G. and Høyer, P.E. (1994): Embryology of evidence suggests that different receptor subtypes and mammalian gonads and duct. In The Physiology isoforms showed developmental stage-, organ- and cell of Reproduction, 2nd ed. (Knobil, E. and Neill,

Vo l . 3 0 N o . 2 85

Sex steroid receptors in the female reproductive tract.

J.D., eds.), pp. 487-540, Raven Press, New Anat., 120, 123-128. York. Drews, U., Sulak, O. and Oppitz, M. (2001): Immuno- Colborn, T. and Clement, C. (1992): Chemically- histochemical localisation of androgen receptor induced Alterations in Sexual and Functional during sex-specific morphogenesis in the fetal Development: The Wildlife/Human Connection. mouse. Histochem. Cell Biol., 116, 427-439. pp. 403, Princeton Sci Pub, Princeton, New Jer- Dunn, T.B. and Green, A.W. (1963): Cysts of the epid- sey. idymis, cancer of the cervix, granular cell myo- Conneely, O.M. and Lydon, J.P. (2000): Progesterone blastoma, and other lesions after estrogen receptors in reproduction: Functional impact of injection in newborn mice. J. Natl. Cancer Inst., the A and B isoforms. Steroids, 65, 571-577. 31, 425-455. Conneely, O.M., Mulac-Jericevic, B. and Lydon, J.P. Dupont, S., Krust, A., Gansmuller, A., Dierich, A., (2003): Progesterone-dependent regulation of Chambon, P. and Mark, M. (2000): Effect of female reproductive activity by two distinct single and compound knockouts of estrogen progesterone receptor isoforms. Steroids, 68, receptors α (ERα)andβ (ERβ) on mouse repro- 771-778. ductive phenotypes. Development, 127, 4277- Couse, J.F., Davis, V.L., Hanson, R.B., Jefferson, 4291. W.N., McLachlan, J.A., Bullock, B.C., New- Ennis, B.W. and Stumpf, W.E. (1988): Differential bold, R.R. and Korach, K.S. (1997): Accelerated induction of progestin-binding sites in uterine onset of uterine tumors in transgenic mice with cell types by estrogen and antiestrogen. Endo- aberrant expression of the estrogen receptor crinology, 123, 1747-1753. after neonatal exposure to diethylstilbestrol. Eroschenko, V.P. (1982): Surface changes in oviduct, Mol. Carcinog., 19, 236-242. uterus and vaginal cells of neonatal mice after Couse, J.F., Lindzey, J., Grandien, K., Gustafsson, J-Å. estradiol-17β and the insecticide chlordecone and Korach, K.S. (1997): Tissue distribution and (Kepone) treatment: A scanning electron micro- quantitative analysis of estrogen receptor-α scopic study. Biol. Reprod., 26, 707-720. (ERα) and estrogen receptor-β (ERβ) messen- Fishman,R.B.,Branham,W.S.,Streck,R.D.and ger ribonucleic acid in the wild-type and ERα- Sheehan, D.M. (1996): Ontogeny of estrogen knockout mouse. Endocrinology, 138, 4613- receptor messenger ribonucleic acid expression 4621. in the postnatal rat uterus. Biol. Reprod., 55, Couse, J.F. and Korach, K.S. (1999): Estrogen recep- 1221-1230. tor null mice: What have we learned and where Gava, N., Clarke, C.L., Byth, K., Arnett-Mansfield, will they lead us? Endocrine Rev., 20, 358-417. R.L. and DeFazio, A. (2004): Expression of Couse, J.F., Hewitt, S.C., Bunch, D.O., Sar, M., progesterone receptors A and B in the mouse Walker, V.R., Davis, B.J. and Korach, K.S. ovary during the estrous cycle. Endocrinology, (1999) Postnatal sex reversal of the ovaries in 145, 3487-3494. mice lacking estrogen receptors α and β.Sci- Graham, J.D. and Clarke, C.L. (1997): Physiological ence, 286, 2328-2331. action of progesterone in target tissues. Endo- Couse, J.F., Dixon, D., Yates, M., Moore, A.B., Ma, crine Rev., 18, 502-519. L., Maas, R. and Korach, K.S. (2001): Estrogen Greco, T.L., Furlow, J.D., Duello, T.M. and Gorski, J. receptor-α knockout mice exhibit resistance to (1991): Immunodetection of estrogen receptors the developmental effects of neonatal diethyl- in fetal and neonatal female mouse reproductive stilbestrol exposure on the female reproductive tracts. Endocrinology, 129, 1326-1332. tract. Dev. Biol., 238, 224-238. Greco, T.L., Duello, T.M. and Gorski, J. (1993): Estro- Danzo, B.J. (1997): Environmental xenobiotics may gen receptors, estradiol, and diethylstilbestrol in disrupt normal endocrine function by interfering early development: The mouse as a model for with the binding of physiological ligands to ste- the study of estrogen receptors and estrogen roid receptors and binding proteins. Environ- sensitivity in embryonic development of male mental. Health Perspectives, 105, 294-301. and female reproductive tracts. Endocrine Rev., Dohr, G. and Tarmann, T. (1984): Contacts between 14, 59-71. Wolffian and Müllerian cells at the tip of the Herbst, A.L., Ulfelder, H. and Poskanzer, D.C. (1971): outgrowing Müllerian duct in rat embryos. Acta Adenocarcinoma of the vagina. Association of

Vo l . 3 0 N o . 2 86

A. OKADA et al.

maternal stilbestrol therapy with tumor appear- Koike, S., Sakai, M. and Muramatsu, M. (1987): ance in young women. New Eng. J. Med., 284, Molecular cloning and characterization of rat 878-881. estrogen receptor cDNA. Nucleic Acids Res., Herbst, A.L. and Bern, H.A. (1981): Developmental 15, 2499-2513. Effects of Diethylstilbestrol (DES) in Preg- Komatsu, M. and Fujita, H. (1978): Electron-micro- nancy. 1981. pp.203. Thieme-Stratton, New scopic studies on the development and aging of York. the oviduct epithelium of mice. Anat. Embryol., Hirai,M.,Hirata,S.,Osada,T.,Hagihara,K.and 152, 243-259. Kato, J. (1994): Androgen receptor mRNA in Kowalski, A.A., Vale-Cruz, D.S., Simmen, F.A. and the rat ovary and uterus. J. Steroid Biochem. Simmen, R.C.M. (2004): Uterine androgen Mol. Biol., 49,1-7. receptors: Roles in estrogen-mediated gene Hiroi, H., Inoue, S., Watanabe, T., Goto, W., Orimo, expression and DNA synthesis. Biol. Reprod., A., Momoeda, M., Tsutsumi, O., Taketani, Y. 70, 1349-1357. and Muramatsu, M. (1999): Differential immu- Kraus, W.L., Montano, M.M. and Katzenellenbogen, nolocalization of estrogen receptor α and β in B.S. (1993): Cloning of the rat progesterone rat ovary and uterus. J. Mol. Endocrinol., 22, gene 5’-region and identification of two func- 37-44. tionally distinct promoters. Mol. Endocrinol., 7, Hodgins, M.B., Spike, R.C., Mackie, R.M. and 1603-1616. MacLean, A.B. (1998): An immunohistochemi- Kraus, W.L., Montano, M.M. and Katzenellenbogen, cal study of androgen, estrogen and progester- B.S. (1994): Identification of multiple, widely one receptors in the vulva and vagina. Br. J. spaced estrogen-responsive regions in the rat Obstet. Gynecol., 105, 216-222. progesterone receptor gene. Mol. Endocrinol., Horwitz, K.B. and Alexander, P.S. (1983): In situ pho- 8, 952-969. tolinked nuclear progesterone receptors of Krege, J.H., Hodgin, J.B., Couse, J.F., Enmark, E., human breast cancer cells: Subunit molecular Warner, M., Mahler, J.F., Sar, M., Korach, K.S., weights after transformation and translocation. Gustafsson, J-Å. and Smithies, O. (1998): Gen- Endocrinology, 113, 2195-2201. eration and reproductive phenotypes of mice Hu, J., Gray, C.A. and Spencer, T.E. (2004): Gene lacking estrogen receptor β.Proc.Natl.Acad. expression profiling of neonatal mouse uterine Sci. U.S.A., 95, 15677-15682. development. Biol. Reprod., 70, 1870-1876. Kuiper, G.G.J.M., Enmark, E., Pelto-Huikko, M., Iguchi, T. and Takasugi, N. (1987): Postnatal develop- Nilsson, S. and Gustafsson, J-Å. (1996): Clon- ment of uterine abnormalities in mice exposed ing of a novel estrogen receptor expressed in rat to DES in utero. Biol. Neonate, 52, 97-103. prostate and ovary. Proc. Natl. Acad. Sci. Iguchi, T. (1992): Cellular effects of early exposure to U.S.A., 93, 5925-5930. sex hormones and antihormones. Int. Rev. Kuiper, G.G.J.M., Carlsson, B., Grandien, K., Cytol., 139, 1-57. Enmark, E., Häggblad, J., Nilsson, S. and Jansen, R.P.S. (1984): Endocrine response in the fallo- Gustafsson, J-Å. (1997): Comparison of the pian tube. Endocrine Rev., 5, 525-551. ligand binding specificity and transcript tissue Jefferson, W.N., Couse, J.F., Banks, E.P., Korach, K.S. distribution of estrogen receptors α and β. and Newbold, R.R. (2000): Expression of estro- Endocrinology, 138, 863-870. gen receptor β is developmentally regulated in Kurita, T., Lee, K., Cooke, P.S., Taylor, J.A., Lubahn, reproductive tissues of male and female mice. D.B. and Cunha, G.R. (2000): Paracrine regula- Biol. Reprod., 62, 310-317. tion of epithelial progesterone receptor by estra- Josso, N. and Picard, J.Y. (1986): Anti-Müllerian hor- diol in the mouse female reproductive tract. mone. Physiol. Rev., 66, 1038-1090. Biol. Reprod., 62, 821-830. Kastner, P., Krust, A., Turcotte, B., Stropp, U., Tora, Kurita, T., Cooke, P.S. and Cunha, G.R. (2001): Epi- L., Gronemeyer, H. and Chambon, P. (1990): thelial-stromal tissue interaction in parameso- Two distinct estrogen-regulated promoters gen- nephric (Müllerian) epithelial differentiation. erate transcripts encoding the two functionally Dev. Biol., 240, 194-211. different human progesterone receptor forms A Lemmen, J.G., Broekhof, J.L.M., Kuiper, G.G.J.M., andB.EMBOJ.,9, 1603-1614. Gustafsson, J-Å., van der Saag, P.T. and van der

Vo l . 3 0 N o . 2 87

Sex steroid receptors in the female reproductive tract.

Burg, B. (1999): Expression of estrogen recep- in the female reproductive organ of rats as tor alpha and beta during mouse embryogenesis. revealed by in situ hybridization. J. Endocrinol., Mech. Dev., 81, 163-167. 165, 59-66. Lessey, B.A., Alexander, P.S. and Horwitz, K.B. Mowa, C.N. and Iwanaga, T. (2000b): Developmental (1983): The subunit structure of human breast changes of the oestrogen receptor-α and -β cancer progesterone receptors: Characterization mRNAs in the female reproductive organ of the by chromatography and photoaffinity labeling. rat - an analysis by in situ hybridization. J. Endocrinology, 112, 1267-1274. Endocrinol., 167, 363-369. Li, S. (1994): Relationship between cellular DNA syn- Murakami, R., Shughrue, P.J., Stumpf, W.E., Elger, W. thesis, PCNA expression and sex steroid hor- and Schulze, P.E. (1990): Distribution of mone receptor status in the developing mouse progestin-binding cells in estrogen-treated and ovary, uterus and oviduct. Histochemistry, 102, untreated neonatal mouse uterus and oviduct: 405-413. Autoradiographic study with [125I] progestin. Lubahn, D.B., Moyer, J.S., Golding, T.S., Couse, J.F., Histochemistry, 94, 155-159. Korach, K.S. and Smithies, O. (1993): Alter- Newbold, R.R., Tyrey, S., Haney, A.F. and McLachlan, ation of reproductive function but not prenatal J.A. (1983): Developmentally arrested oviduct: sexual development after insertional disruption A structural and functional defect in mice fol- of the mouse estrogen receptor gene. Proc. Natl. lowing prenatal exposure to diethylstilbestrol. Acad.Sci.U.S.A.,90, 11162-11166. Teratology, 27, 417-426. Lyon, M.F. and Glenister, P.H. (1980): Reduced repro- Nguyen, B.L., Hatier, R., Jeanvoine, G., Roux, M., ductive performance in androgen-resistant Tfm/ Grignon, G. and Pasqualini, J.R. (1988): Effect Tfm female mice. Proc. R. Soc. Lond. Ser. B- of estradiol on the progesterone receptor and on Biol. Sci., 208,1-12. morphological ultrastructures in the fetal and Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, newborn uterus and ovary of the rat. Acta Endo- P., Schütz, G., Umesono, K., Blumberg, B., crinol., 117, 249-259. Kastner, P., Mark, M., Chambon, P. and Evans, Nielsen, M., Björnsdóttir, S., Høyer, P.E. and Byskov, R.M. (1995): The nuclear receptor superfamily: A.G. (2000): Ontogeny of oestrogen receptor α The second decade. Cell, 83, 835-839. in gonads and sex ducts of fetal and newborn Mangal, R.K., Wiehle, R.D., Poindexter, A.N. III. and mice. J. Reprod. Fertil., 118, 195-204. Weigel, N.L. (1997): Differential expression of Ohta, Y., Sato, T. and Iguchi, T. (1993): Immunocy- uterine progesterone receptor forms A and B tochemical localization of progesterone receptor during menstrual cycle. J. Steroid Biochem. in the reproductive tract of adult female rats. Mol. Biol., 63, 195-202. Biol. Reprod., 48, 205-213. Maruyama, K., Endoh, H., Sasaki-Iwaoka, H., Kanou, Ohta, Y., Fukazawa, Y., Sato, T., Suzuki, A., H.,Shimaya,E.,Hashimoto,S.,Kato,S.and Nishimura, N. and Iguchi, T. (1996): Effect of Kawashima, H. (1998): A novel isoform of rat estrogen on ontogenic expression of progester- with 18 amino acid inser- one and estrogen receptors in rat uterus. Zool. tion in the ligand binding domain as a putative Sci., 13, 143-149. dominant negative regulator of estrogen action. Okada, A., Sato, T., Ohta, Y., Buchanan, D.L. and Biochem. Biophys. Res. Commun., 246, 142- Iguchi, T. (2001): Effect of diethylstilbestrol on 147. cell proliferation and expression of epidermal McCormack, S.A. and Glasser, S.R. (1980): Differen- growth factor in the developing female rat repro- tial response of individual uterine cell types ductive tract. J. Endocrinol., 170, 539-554. from immature rats treated with estradiol. Endo- Okada, A., Ohta, Y., Buchanan, D.L., Sato, T., Inoue, crinology, 106, 1634-1648. S., Hiroi, H., Muramatsu, M. and Iguchi, T. Mosselman, S., Polman, J. and Dijkema, R. (1996): (2002a): Changes in ontogenetic expression of ERβ: Identification and characterization of a and not of estrogen novel human estrogen receptor. FEBS Lett., receptor beta in the female rat reproductive 392, 49-53. tract. J. Mol. Endocrinol., 28, 87-97. Mowa, C.N. and Iwanaga, T. (2000a): Differential dis- Okada, A., Ohta, Y., Buchanan, D.L., Sato, T. and tribution of oestrogen receptor-α and -β mRNAs Iguchi, T. (2002b): Effect of estrogens on onto-

Vo l . 3 0 N o . 2 88

A. OKADA et al.

genetic expression of progesterone receptor in Price, R.H. Jr., Lorenzon, N. and Handa, R.J. (2000): the fetal female rat reproductive tract. Mol. Cell Differential expression of estrogen receptor beta Endocrinol., 195, 55-64. splice variants in rat brain: Identification and Okada, A., Ohta, Y., Inoue, S., Hiroi, H., Muramatsu, characterization of a novel variant missing exon M. and Iguchi, T. (2003): Expression of estro- 4. Mol. Brain Res., 80, 260-268. gen, progesterone and androgen receptors in the Quarmby, V.E. and Korach, K.S. (1984): The influ- oviduct of developing, cycling and pre-implan- ence of 17β-estradiol on patterns of cell division tation rats. J. Mol. Endocrinol., 30, 301-315. in the uterus. Endocrinology, 114, 694-702. Okada, A., Ohta, Y., Brody, S.L., Watanabe, H., Renthal, R., Schneider, B.G., Miller, M.M. and β β Chambon, P. and Iguchi, T. (2004a): Role of Ludueña, R.F. (1993): IV is the major -tubulin foxj1 and estrogen receptor alpha in ciliated epi- isotype in bovine cilia. Cell Motil. Cytoskele- thelial cell differentiation of the neonatal ovi- ton, 25, 19-29. duct. J. Mol. Endocrinol., 32, 615-625. Saji, S., Sakaguchi, H., Andersson, S., Warner, M. and Okada,A.,Ohta,Y.,Brody,S.L.andIguchi,T. Gustafsson, J-Å. (2001): Quantitative analysis (2004b): Epithelial c-jun and c-fos are tempo- of estrogen receptor proteins in rat mammary rally and spatially regulated by estradiol during gland. Endocrinology, 142, 3177-3186. neonatal rat oviduct differentiation. J. Endo- Sar, M. and Welsch, F. (1999): Differential expression crinol., 182, 219-227. of estrogen receptor-β and estrogen receptor-α Paech, K., Webb, P., Kuiper, G.G.J.M., Nilsson, S., in the rat ovary. Endocrinology, 140, 963-971. Gustafsson, J-Å., Kushner, P.J. and Scanlan, Sato, T., Okamura, H., Ohta, Y., Hayashi, S., Takasugi, T.S. (1997): Differential ligand activation of N. and Iguchi, T. (1992): Estrogen receptor estrogen receptors ERα and ERβ at AP1 sites. expression in the genital tract of female mice Science, 277, 1508-1510. treated neonatally with diethylstilbestrol. In Panet-Raymond, V., Gottlieb, B., Beitel, L.K., Pinsky, Vivo, 6, 151-156. L. and Trifiro, M.A. (2000): Interactions Sato, T., Chiba, A., Hayashi, S., Okamura, H., Ohta, between androgen and estrogen receptors and Y., Takasugi, N. and Iguchi, T. (1994): Induction the effects on their transactivational properties. of estrogen receptor and cell division in genital Mol. Cell. Endocrinol., 167, 139-150. tracts of male mice by neonatal exposure to Pasqualini, J.R. and Nguyen, B.L. (1980): Progester- diethylstilbestrol. Reprod. Toxicol., 8, 145-153. one receptors in the fetal uterus and ovary of the Sato, T., Ohta, Y., Okamura, H., Hayashi, S. and Igu- guinea pig: Evolution during fetal development chi, T. (1996): Estrogen receptor (ER) and its and induction and stimulation in estradiol- messenger ribonucleic acid expression in the primed animals. Endocrinology, 106, 1160- genital tract of female mice exposed neonatally 1165. to tamoxifen and diethylstilbestrol. Anat. Rec., Pasqualini, J.R. and Sumida, C. (1986): Ontogeny of 244, 374-385. steroid receptors in the reproductive system. Int. Sato, T., Fukazawa, Y., Ohta, Y. and Iguchi, T. (2004): Rev. Cytol., 101, 275-324. Involvement of growth factors in induction of Pelletier, G., Labrie, C. and Labrie, F. (2000): Local- persistent proliferation of vaginal epithelium of ization of oestrogen receptor α, oestrogen mice exposed neonatally to diethylstilbestrol. receptor β and androgen receptors in the rat Reprod. Toxicol., 19, 43-51. reproductive organs. J. Endocrinol., 165, 359- Saunders, P.T.K., Maguire, S.M., Gaughan, J. and 370. Millar, M.R. (1997): Expression of oestrogen Petersen, D.N., Tkalcevic, G.T., Koza-Taylor, P.H., receptor beta (ERβ) in multiple rat tissues visu- Turi, T.G. and Brown, T.A. (1998): Identifica- alized by immunohistochemistry. J. Endocrinol., β tion of estrogen receptor 2, a functional variant 154, R13-R16. of estrogen receptor β expressed in normal rat Savouret, J.F., Misrahi, M. and Milgrom, E. (1990): tissues. Endocrinology, 139, 1082-1092. Molecular action of progesterone. Int. J. Bio- Pettersson, K., Delaunay, F. and Gustafsson, J-Å. chem., 22, 579-594. (2000): Estrogen receptor β actsasadominant Schott, D.R., Shyamala, G., Schneider, W. and Parry, regulator of estrogen signaling. , 19, G. (1991): Molecular cloning, sequence analy- 4970-4978. ses, and expression of complementary DNA

Vo l . 3 0 N o . 2 89

Sex steroid receptors in the female reproductive tract.

encoding murine progesterone receptor. Bio- Genome-wide analysis of changes in early gene chemistry, 30, 7014-7020. expression induced by oestrogen. Genes Cells, Shughrue, P.J., Lane, M.V., Scrimo, P.J. and Merch- 7, 497-507. enthaler, I. (1998): Comparative distribution of Watanabe, H., Suzuki, A., Kobayashi, M., Lubahn, estrogen receptor-α (ER-α)andβ (ER-β) D.B., Handa, H. and Iguchi, T. (2003): Similar- mRNA in the rat pituitary, gonad, and reproduc- ity and differences in uterine gene expression tive tract. Steroids, 63, 498-504. patterns caused by treatment with physiological Shyamala, G., Schneider, W. and Schott, D. (1990): and non-physiological oestrogens. J. Mol. Endo- Developmental regulation of murine mammary crinol., 31, 487-497. progesterone receptor gene expression. Endocri- Watanabe, H., Suzuki, A., Goto, M., Lubahn, D.B., nology, 126, 2882-2889. Handa, H. and Iguchi, T. (2004): Tissue-specific Takasugi, N. and Bern, H.A. (1964): Tissue changes in estrogenic and non-estrogenic effects of a mice with persistent vaginal cornification by xenoestrogen, nonylphenol. J. Mol. Endocrinol., early postnatal treatment with estrogens. J. Natl. 33, 243-252. Cancer Inst., 33, 855-865. Weihua, Z., Saji, S., Mäkinen, S., Cheng, G., Jensen, Tremblay, G.B., Tremblay, A., Copeland, N.G., E.V., Warner, M. and Gustafsson, J-Å. (2000): Gilbert, D.J., Jenkins, N.A., Labrie, F. and Estrogen receptor (ER) β,amodulatorofERα Giguère, V. (1997): Cloning, chromosomal in the uterus. Proc. Natl. Acad. Sci. U.S.A., 97, localization, and functional analysis of the 5936-5941. murine estrogen receptor β.Mol.Endocrinol., Weihua, Z., Ekman, J., Almkvist, Å., Saji, S., Wang, 11, 53-365. L., Warner, M. and Gustafsson, J-Å. (2002): Vegeto, E., Shahbaz, M.M., Wen, D.X., Goldman, Involvement of androgen receptor in 17β-estra- M.E., O’Malley, B.W. and McDonnell, D.P. diol-induced cell proliferation in rat uterus. (1993): Human form is Biol. Reprod., 67, 616-623. a cell- and promoter-specific repressor of human Wu, T.C.J., Wang, L. and Wan, Y.J.Y. (1992): Expres- function. Mol. Endo- sion of estrogen receptor gene in mouse oocyte crinol., 7, 1244-1255. and during embryogenesis. Mol. Reprod. Dev., Wang, H., Masironi, B., Eriksson, H. and Sahlin, L. 33, 407-412. (1999): A comparative study of estrogen recep- Wu, X., Pang, S-T., Sahlin, L., Blanck, A., Norstedt, tors α and β in the rat uterus. Biol. Reprod., 61, G. and Flores-Morales, A. (2003): Gene expres- 955-964. sion profiling of the effects of castration and Wang, H., Eriksson, H. and Sahlin, L. (2000): Estro- estrogen treatment in the rat uterus. Biol. gen receptors α and β in the female reproductive Reprod., 69, 1308-1317. tract of the rat during the estrous cycle. Biol. Yamashita, S., Newbold, R.R., McLachlan, J.A. and Reprod., 63, 1331-1340. Korach, K.S. (1989): Developmental pattern of Watanabe, H., Suzuki, A., Mizutani, T., Khono, S., estrogen receptor expression in female mouse Lubahn, D.B., Handa, H. and Iguchi, T. (2002): genital tracts. Endocrinology, 125, 2888-2896.

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