301 Expression of estrogen, and androgen receptors in the oviduct of developing, cycling and pre-implantation rats

A Okada1,2, Y Ohta2,3, S Inoue3,4,5, H Hiroi6, M Muramatsu5 and T Iguchi3,7 1Safety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd, Itabashi, Tokyo 174–8511, Japan 2Laboratory of Animal Science, Department of Veterinary Science, Faculty of Agriculture, Tottori University, Tottori 680–8553, Japan 3CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332–0012, Japan 4Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113–8655, Japan 5Research Center for Genomic Medicine, Saitama Medical School, Saitama 350–1241, Japan 6Department of Obstetrics and Gynecology, Graduate School of Medicine, University of Tokyo, Tokyo 113–8655, Japan 7Center for Integrative Bioscience, Okazaki National Research Institutes, and Department of Molecular Biomechanics, School of Life Science, Graduate University for Advanced Studies, Okazaki, Aichi 444–8585, Japan

(Requests for offprints should be addressed to T Iguchi; Email: [email protected])

Abstract

To determine expression and localization of receptors for estrogen (ER), progesterone (PR) and androgen (AR), detailed immunohistochemical evaluations were performed in the Sprague–Dawley rat oviduct during pre- and neonatal development, estrous cycle and pre-implantation period. In addition, real-time RT-PCR studies were conducted to evaluate changes in ERα,ERβ, total PR (PR-A+B), PR-B and AR mRNA expressions. All receptors except for ERβ were detected in epithelial, and stromal or mesenchymal cells of the fetal and neonatal oviduct, and increased with development. During the estrous cycle and early pregnancy, ERα and PR-A+B were expressed in epithelial, stromal and muscle cells throughout the oviduct region, and showed changes in expression predominantly in the isthmus. Only a few epithelial cells in the infundibulum (INF) and ampulla (AMP) showed ERβ staining. AR was detected in stromal and muscle cells throughout the oviduct region, and in epithelial cells of the INF/AMP. Taken together, ERα, PR-A+B and AR were detected in the epithelium of the INF/AMP region, but all of these receptors were expressed in a distinct subset of epithelial cells which were negative for β-tubulin IV, a ciliated epithelial cell marker. These results contribute to a better understanding of the respective roles of ERs, PRs and AR in the rat oviduct. Journal of Molecular Endocrinology (2003) 30, 301–315

Introduction -B (PR-B) respectively, belonging to the superfamily of ligand-inducible The mammalian oviduct, or fallopian tube, is an transcription factors (Mangelsdorf et al. 1995). organ known as the female reproductive tract that Evaluation of the expression and localization of has a fundamental role in gamete transport, these receptors is key in clarifying the mechanisms fertilization and subsequent early embryo develop- of estrogen and P4 actions on cell proliferation, ment. Functions of the oviduct, as well as those of cytodifferentiation and functional differentiation of uterus and vagina, are believed to be regulated by the reproductive tissues. In the uterus and vagina, two ovarian sex steroid hormones, estrogen and ERs and PRs expressions have been well progesterone (P4) (Jansen 1984, Harper 1994). In documented. The use of ER or PR gene knockout most tissues, estrogen and P4 actions are mediated (KO) mouse tissue has strongly suggested the by  (ER) and estrogen receptor importance of epithelial–stromal tissue/cell inter-  (ER), and P4 receptor-A (PR-A) and P4 action in cell proliferation, cytodifferentiation and

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functional differentiation of uterine and vaginal been demonstrated. Weihua et al. (2002) have cells (Cooke et al. 1997, 1998, Buchanan et al. 1998, recently reported the essential role of AR in the Kurita et al. 2000a,b, 2001). However, few data estrogen-induced uterine epithelial cell proliferation regarding the expression and regulation of these of rats, and indicated that stromal AR amplified the receptors in the oviducts of mammals, except for ER signal by induction of insulin-like growth humans and nonhuman primates, have been factor-I, which is known to be produced in stromal accumulated (Brenner & Slayden 1994). Neither cells and induces epithelial cell proliferation in a has there been evaluation of the role of ERs and paracrine fashion. Moreover, direct inhibitory PRs using ER or PR KO mice. The reason for the effects of the ER/AR heterodimer on both ER lack of information about rodent oviductal ERs and and AR transactivational properties have been PRs may be the difficulty of examination because reported (Panet-Raymond et al. 2000). Accordingly, of its smaller size and more complex morphological identification of regional and cellular AR localiz- features compared with the uterus, which has no ation may allow a better understanding of not only regional differences and only a single epithelial cell the role of AR, but also the mechanism of estrogen type. In contrast, the oviduct has a coiled structure action in the rat oviduct. and is composed of four different regions: the In this report, changes in mRNA expression and infundibulum (INF), ampulla (AMP), isthmus (IST) protein localization of ER,ER, total PRs and uterotubal junction (UTJ). Depending on the (PR-A+B), PR-B and AR were determined during oviductal region, there are at least two types of the pre- and neonatal development, estrous cycle, epithelial cells: ciliated epithelial cells and noncili- and pre-implantation period in the rat oviduct ated or secretory epithelial cells. Therefore, cell- using real-time RT-PCR and immunohistochem- and region-specificity of ER,ER, PR-A and istry. In addition, to identify the epithelial cell types PR-B expressions should be determined for better expressing steroid hormone receptors, -tubulin understanding of the molecular and cellular IV was used as a ciliated epithelial cell marker mechanisms of estrogen and P4 actions in the rat (Renthal et al. 1993) in double immunohisto- oviduct. Although some previous reports have chemical studies. revealed ER and ER expressions in the rat oviduct (Saunders et al. 1997, Sar & Welsch 1999, Mowa & Iwanaga 2000a,b, Wang et al. 2000), Materials and methods details of cell- and region-dependency still have not been determined or are varied between the reports. Animals Regarding oviductal PR expression, Li (1994) Male and female Sprague–Dawley rats obtained reported neonatal ontogeny in the mouse, but no from Charles River Japan, Inc. (Kanagawa, Japan) reports of rat ontogeny and isoform expression, or were used (13 weeks of age). Animals were housed in cycling and pre-implantation mice and rats were individually in stainless-steel cages with controlled found. temperature (232 C) and relative humidity Androgens have uterotrophic effects in intact and (5510%), and a 13 h light:11 h darkness cycle ovariectomized immature female rats (Armstrong (0800–2100). Pellet food (CRF-1; Oriental Yeast et al. 1976), and testicular feminized female Co., Ltd, Tokyo, Japan) and municipal tap water (Tfm/Tfm) mice showed impaired reproductive were freely available. The day on which sperm was performance (Lyon & Glenister 1980), suggesting found in the vaginal smear was designated as the importance of androgens in females as well as gestation day (GD) 0 or prenatal day (PD) 0, and males. Androgens are known to exert their effects the day when neonates were born was designated via (AR), which is another as neonatal day (ND) 0. The stage of the regular member of the nuclear receptor superfamily 4-day estrous cycle was specified by the vaginal (Mangelsdorf et al. 1995), and AR mRNA smear examination using a light microscope every expression has been reported in endometrium, morning. For each total RNA and tissue prep- endometrial glands and myometrium of the rat aration, ten oviducts were removed from five uterus (Hirai et al. 1994). However, the molecular ether-anesthetized rats in each group (PD 15 and mechanism of androgen action and the role of 19 from female fetuses, ND 0, 3, 5, 7, 10, 15 and AR in the female reproductive tract have not yet 20 from female neonates, metestrus, diestrus,

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Downloaded from Bioscientifica.com at 09/28/2021 07:29:16AM via free access ERs, PRs and AR in the rat oviduct · A OKADA and others 303 proestrus and estrus from cycling rats, and GD 0, 1, standard scale prepared from rat genomic DNA 2, 3 and 4 from pregnant rats). All animals were (Clontech Laboratories, Inc., Palo Alto, CA, USA). maintained in accordance with the Institutional The expression level of each target gene was Guidelines for Care and Use of Laboratory calculated by standardizing the target gene copy Animals. number with the GAPDH copy number in a sample. Purity and specificity of all products were confirmed by omitting the reverse transcriptase, Total RNA preparation and real-time RT-PCR and by single melting temperature, appropriate size Procedures for total RNA preparation and real- and their sequence. Analysis of results is based time RT-PCR were described previously (Okada on duplicate samples from four independent et al. 2002b). Template total RNA (500 ng) treated experiments. with DNase I was reverse-transcribed by using SuperScript II RNase H reverse transcriptase Antibodies (Invitrogen Corp., Carlsbad, CA, USA) with  oligo(dT)12–18 primer for 50 min at 42 C and then A mouse monoclonal antibody against ER (6F11; chilled on ice. An aliquot of generated cDNA was Novocastra Laboratories Ltd, Newcastle upon amplified with a pair of primers (ER, forward Tyne, UK) was used at a dilution of 1:50. A rabbit 5CTGACAATCGACGCCAGAA3 and reverse polyclonal anti-rat ER antiserum against a syn- 5CAGCCTTCACAGGACCAGAC3;ER, for- thesized peptide (CSSTEDSKNKESQNLQSQ) ward 5CTTGCCCACTTGGAAACATC3 and corresponding to the C-terminal amino acid reverse 5CCAAAGGTTGATTTTATGGCC3; residues 467–485 of rat ER protein was generated PR-A+B, forward 5CTTTGTTTCCTCTGCAA and affinity-purified as described previously (Hiroi AAATTG3 and reverse 5GTATACACGTAAG et al. 1999, Okada et al. 2002a), and was used at a GCTTTCAGAAGG3; PR-B, forward 5CAGAC dilution of 1:100. A pre-diluted mouse monoclonal CAACCTGCAACCAGAA3 and reverse 5AGT antibody against PR-A+B (10A9) was obtained CCTCACCAAAACCCTGGG3; and AR, for- from Immunotech (Marseille Cedex, France). The ward 5ACCCTCCCATGGCACATTTT3 and epitope of the PR antibody is located on the reverse 5TTGGTTGGCACACAGCACAG3) C-terminus of PR, which is a common domain derived from rat mRNA sequences (GenBank between A and B isoforms. A rabbit polyclonal Accession No. Y00102, U57439, L16922, M20133 antiserum against AR (N-20; Santa Cruz Biotech- and U06637 respectively). Primers for PR-A+B, nology, Inc., Santa Cruz, CA, USA) and a mouse and PR-B were designed to detect a sequence in monoclonal antibody against -tubulin IV the 3-UTR common to the A and B isoforms, (ONS1A6; BioGenex, San Ramon, CA, USA) were and in the 5-UTR unique to the B isoform respect- used at a dilution of 1:200 and 1:250 respectively. ively. Glyceraldehyde-3-phosphate-dehydrogenase Binding specificity of all antibodies has been (GAPDH) was likewise amplified as an internal previously established (Banerjee et al. 1992, Fisher control (forward 5TCTACCCACGGCAAGTT et al. 1997, Hiroi et al. 1999, Okada et al. 2002b, CAAT3 and reverse 5ACCCCATTTGATGTT Pelletier et al. 2000, Weihua et al. 2002). AGCGG3; GenBank M17701). Quantitative real- time PCR was carried out in an ABI Prism 7700 Tissue preparation and immunohistochemistry Sequence Detector (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master Oviducts were fixed with 4% paraformaldehyde in Mix reagent (Applied Biosystems) as the detector. 0·1 M phosphate buffer overnight at 4 C. Sections PCR cycle parameters were 94 C for 15 s, 60 C cut in paraffin at 4 µm were deparaffinized and for 30 s and 72 C for 60 s, followed by a hold rehydrated. Antigen retrieval was performed by temperature for 10 min at 95 C. The threshold autoclaving at 121 C for 15 min in 10 mM citrate parameter was set as the cycle at which each buffer (pH 6·0) for ER, PR-A+B and AR, or at fluorescent signal was first detected above back- 121 C for 10 min in 0·8 M urea for ER. Sections ground, and the number of template copies present were then rinsed in distilled water and treated with at the start of the reaction was determined at 0·3% hydrogen peroxide in methanol for 30 min at exponential increase by comparison with a room temperature (RT). After rinsing in 0·01% www.endocrinology.org Journal of Molecular Endocrinology (2003) 30, 301–315

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Triton X-100 in PBS (PBT), sections were blocked in normal sheep serum (Dako Corp., Carpinteria, CA, USA) for 30 min at RT, and then incubated overnight at 4 C with the anti-ER antibody or the anti-AR antibody, for 48 h at 4 C with the anti-ER antibody, or for 2 h at RT with the anti-PR-A+B antibody. Following treatment with each primary antibody, sections were rinsed in PBT and treated with Simple Stain Rat PO (Nichirei, Tokyo, Japan) for 30 min at RT. After a final PBT wash, sections were treated with 0·01% 3,3-diaminobenzidine tetrahydrochloride (Dojindo Laboratories, Kumamoto, Japan) in 0·05 M Tris– HCl at pH 7·6 including 0·068% imidazole (Sigma, St Louis, MO, USA) and 0·02% hydrogen peroxide for 5 min at RT. For double immunohistochemistry, sections Figure 1 Comparison of ERα,ERβ, PR-A+B, PR-B and stained for ER, PR-A+B or AR as described AR mRNA expressions by real-time RT-PCR between above were rinsed in PBT and blocked in normal the oviduct, uterus and prostate. Data are represented as means±S.D. Analysis of results is based on duplicate sheep serum, followed by incubation with the samples each from three pooled total RNAs (three anti--tubulin IV antibody overnight at 4 C. animals for each pooled sample). **P<0·01 and Sections were rinsed in PBT and treated with *P<0·05 vs oviduct. EnVision/AP (Dako) for 30 min at RT. After rinsing in PBT, they were treated with fuchsin multiple comparison test was carried out for (Dako) including levamisole (Dako) for 5 min changes in mRNA expression of ER,ER, at RT. PR-A+B, PR-B and AR in the real-time RT-PCR All sections, except those for ER staining, were study (Fig. 2). Data are represented as meansS.D. lightly counter-stained with hematoxylin (Dako and considered significantly different at P<0·05. A/S, Glostrup, Denmark). Normal mouse IgG (Dako A/S) and normal rabbit immunoglobulin fraction (Dako A/S) were used as negative controls Results in place of primary antibodies for ER, PR-A+B, -tubulin IV, and ER and AR stainings Expressions of ER,ER, PR-A+B, PR-B and respectively, showing no specific immunoreactivity. AR mRNAs in the rat oviduct Quantitative real-time RT-PCR was employed to evaluate changes in expressions of ERs, PRs and Immunohistochemical evaluation and AR mRNAs in the rat oviduct. First, to confirm statistical analysis the primers’ specificity, expression levels for ER, Sections were examined and photographed using ER, PR-A+B, PR-B and AR mRNAs were a light-microscope (BX60; Olympus Optical Co., determined in the diestrous uterus and prostate, as Ltd, Tokyo, Japan) attached to a digital camera positive control tissues, in addition to the diestrous (DP50; Olympus). Staining intensity was graded as oviduct of the rats (Fig. 1). Expressions of ER, negative, slight, weak, moderate or marked for PR-A+B and PR-B were much lower in the ER,ER, PR-A+B and AR immunohistochem- prostate than in the oviduct, while those of ER istries. At least seven specimens from each of five and AR were inversely highly in the prostate. The animals were examined for all investigations. percentage for PR-B against PR-A+B was 33·8% Student’s t-test or Welch’s t-test were performed in the uterus, being in agreement with previous in cases of equal variance or unequal variance reports (Ilenchuk & Walters 1987). Also, similar respectively, after ANOVA between oviduct, and expression patterns for ER,ER, PRs and AR in uterus or prostate for comparison of gene the rat prostate have been previously reported (Lau expressions between tissues (Fig. 1). Duncan’s et al. 1998).

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In fetal rat oviduct, ER mRNA was expressed of immunoexpressions for ER and PR-A+B in the at least from PD 15, and increased by PD 19. After rat oviduct was determined, and summarized in birth, oviductal ER mRNA continued to increase Table 1. Pre- and neonatal oviducts showed simple until ND 3, and was maintained at a high constant tubal structure from PD 15 to ND 5, and level through to ND 20 (Fig. 2A). Expressions of subsequently differentiated morphologically into ER mRNA during the regular 4-day estrous cycle the INF, AMP, IST and UTJ after ND 7. In the and early pregnancy are also presented. ER level prenatal oviduct, immunohistochemical staining for was significantly lower at estrus than at other stages ER exhibited nuclear signals rated as slight and (P<0·05). In the pre-implantation oviduct, ER weak in epithelial cells, and negative and slight in was low on GD 0, which were similar to those at mesenchymal cells on GD 15 and 19 respectively. estrus, and increased significantly from GD 1 to 3. In the neonatal oviduct, epithelial ER was In the pregnant oviduct, the peak of ER increase exhibited as weak and moderate stainings at birth was noted on GD 2 (P<0·01), while, ER mRNA (ND 0) and from ND 3 to 20 respectively (Fig. 4, was detected in a low and constant manner as left panels). However, some ER-negative cells compared with ER throughout the development were present in the epithelium of the INF/AMP and physiological changes examined (Fig. 2A). region after ND 10. Stromal cells showed ER For PRs, two distinct primer pairs were used stainings at weak, moderate and marked levels at to determine isoform-specific change in the rat ND 0, from ND 3 to 10, and ND 15 and 20 oviduct, one detected A and B isoforms equally and respectively, and no marked difference in ER the other was specific for B isoform. Oviductal staining was noted among regions in the oviduct. PR-A+B and PR-B were equally expressed at low Moderate ER staining was also found in muscle levels from PD 15 to ND 3, and both increased cells of the IST/UTJ region from ND 7 to 20. gradually from PD 15 to ND 5 (Fig. 2B and C). PR-A+B immunoexpression was evident in epi- PR-A+B then increased markedly until ND 20, but thelial cells at slight levels on PD 15 and 19; PR-B expression continued to increase moderately however, it was not present in mesenchymal cells from ND 7 to ND 20, resulting in a decrease in on either prenatal day (Table 1). Slight epithelial the percentage for PR-B to one-tenth to a quarter PR-A+B expression continued to ND 5 in the against PR-A+B. During the estrous cycle, undifferentiated oviduct, and to ND 20 in the PR-A+B mRNA level was significantly lower at INF/AMP region (Fig. 4, middle panels). However, estrus than at other stages (P<0·05). However, some epithelial cells showed negative or moderate PR-B was not significantly changed throughout the PR-A+B signals in the differentiated INF/AMP estrous cycle. In the pre-implantation oviduct, both region from ND 7 to 20. In contrast, epithelial PR-A+B and PR-B expressions were low on GD 0, PR-A+B staining was intense in the differentiated and increased from GD 1 to 3. Moreover, the IST/UTJ region, and showed moderate and percentage for PR-B against PR-A+B on GD 0 marked signals on ND 7 and 10, and ND 15 and (21·5%) increased to 46·6% on GD 2 (Fig. 2C). 20 respectively. PR-A+B was also detected in AR mRNA was also expressed in the rat oviduct, stromal cells after ND 3, but absent in the and increased gradually with development of the IST/UTJ region on ND 7 and 10. Muscle cells pre- and neonatal rats. However, no change was showed slight and moderate PR-A+B stainings in exhibited in the cycling and pre-implantation rats the IST/UTJ region on ND 10, and ND 15 and 20 (Fig. 2A). respectively (Table 1).

Immunohistochemical expressions of ER and Immunohistochemical expressions of ER and PR-A+B in the prenatal and neonatal rat PR-A+B in the cycling and pre-implantation oviducts oviducts In the uterus, as positive control tissue, ER and ER and PR immunoreactivities were detected in PR-A+B were detected in nuclei of luminal and nuclei of epithelial, stromal and muscle cells of all glandular epithelia, and stroma and muscle layer regions in the diestrous oviduct (Fig. 5). Although (Fig. 3) as reported (Ohta et al. 1993, Wang et al. most of the stromal cells (INF, AMP, IST and UTJ) 2000). With these monoclonal antibodies, ontogeny and muscle cells (IST and UTJ) were positive for www.endocrinology.org Journal of Molecular Endocrinology (2003) 30, 301–315

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Figure 2 Changes in expressions of ERα,ERβ and AR (A), and PR-A+B and PR-B (B) in the oviduct of the prenatal, neonatal, cycling and pre-implantation rats. Target mRNA expression levels were evaluated by real-time RT-PCR and standardized to GAPDH mRNA expression. Analysis in duplicate was repeated four times in each experiment. Data are represented as means±S.D. from three independent experiments. **P<0·01 and *P<0·05 vs PD 15, diestrus and GD 0 in developing, cycling and pre-implantation oviduct respectively. Change in the percentage for PR-B against PR-A+B is shown (C).

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Gestational increases in ER and PR-A+B staining intensities were observed in the IST/UTJ cells (Fig. 6).

Immunohistochemical expression of oviductal ER For immunohistochemistry of ER, Weihua et al. (2002) recently reported an improved method to detect the ER signal in the rat uterus by using urea in place of citrate buffer as the antigen retrieval buffer, and detected apparent nuclear ER stainings in rat uterine epithelial and stromal cells. Effectiveness of the antigen retrieval with urea has also been reported for stainings of ER, PR, AR and other intranuclear antigens in paraffin sections (Taylor et al. 1994). Therefore, we employed this method for ER immunohistochem- istry, and could detect ER immunoreactivity in nuclei of uterine luminal and glandular epithelial, and peripheral stromal cells in addition to granulosa cells of the ovary, which served as Figure 3 Immunohistochemistry for ERα, PR-A+B and controls (Fig. 7). To confirm our findings of ER AR in the uterus and prostate of adult rats. ERα and protein expression in the rat tissues, we employed PR-A+B stainings were seen in nuclei of luminal two commercially available rabbit polyclonal epithelium (Le) and stroma (St) of the uterus. Nuclear staining for AR was detected in glandular epithelium (Ge) antisera to rat ER as reported (Okada et al. of the prostate. ERα and PR-A+B, and AR stainings were 2002a): PA1–310 (Affinity Bioreagents, Inc., abolished by incubation with normal mouse and rabbit Golden, CO, USA) and 06–629 (Upstate Biotech- IgG respectively (Negative). Bar: 50 µm. nology, Lake Placid, NY, USA) against amino acid residues 467–485 and 54–71 of rat ER respect-  both ER and PR-A+B, the number of positive ively. With both ER antisera, similar results to epithelial cells varied depending on the oviduct those with our antibody were obtained in the ovary and uterus by immunohistochemistry using either region. Despite positive stainings for ER and PR- ff A+B in all epithelial cells of the IST/UTJ, positive citrate bu er or urea (data not shown).  stainings were less in those of the INF/AMP (Fig. 5). In the diestrous oviduct, however, few ER - Staining intensity of ER was marked in all cell positive cells were found in epithelial cells of the  types of all regions. PR-A+B staining intensity was INF/AMP with our (Fig. 7) and two other ER  also marked in stromal and muscle cells of all re- antibodies (data not shown). No ER was detected gions, and in epithelial cells of the IST/UTJ, in stromal cells of the INF/AMP, or in any cells of  whereas it was weak in epithelial cells of the INF/ the IST/UTJ of the diestrous oviduct. ER AMP. PR-A+B staining in epithelial cells was immunoreactivity was indistinguishable in the pre- greater in the IST than in the UTJ (Fig. 5). and neonatal oviduct (Table 1), and showed no Although changes in the staining intensity of remarkable change during the estrous cycle and ER and PR-A+B in all regions were weak during pre-implantation period in the adult oviduct (data the estrous cycle and early pregnancy, epithelial, not shown). stromal and muscle cells in the IST/UTJ showed apparent changes, the intensity being higher at Immunohistochemical expression of diestrus and metestrus than at proestrus and estrus oviductal AR (Fig. 6). In the pre-implantation oviduct, immuno- In the adult rat prostate, positive AR was detected localization of ER and PR-A+B exhibited a in nuclei of glandular epithelial cells (Fig. 3) pattern similar to that in the cycling oviducts. as previously reported (Husmann et al. 1990). www.endocrinology.org Journal of Molecular Endocrinology (2003) 30, 301–315

Downloaded from Bioscientifica.com at 09/28/2021 07:29:16AM via free access 308 ora fMlclrEndocrinology Molecular of Journal OKADA A n tes· others and R,PsadA ntertoviduct rat the in AR and PRs ERs,

Table 1 Ontogenetic immunolocalization of ER,ER, PR-A+B and AR in the prenatal and neonatal female rat oviduct (2003) PD ND

30, 15190357 10 15 20 301–315 Cell type INF/AMP IST/UTJ INF/AMP IST/UTJ INF/AMP IST/UTJ INF/AMP IST/UTJ

ER Epi±++++++++++++a ++ ++a ++ ++a ++ Str−±+++++++++++++++++++++++++ Mus UDUDUDUDUDUD ++ UD ++ UD ++ UD ++ ER Epi−−−−−− − − − − − − − Str−−−−−− − − − − − − − Mus UDUDUDUDUDUD − UD − UD − UD − PRsEpi±±±±±±b ++ ±b ++ ±b +++ ±b +++ Str−−−±±± − + − + ± + + Mus UDUDUDUDUDUD − UD ± UD ++ UD ++ AREpi−−−±±± ± ± ± ± ± ± ± Str−−−±±± ± ± ± ± ± ± ± Mus UDUDUDUDUDUD ± UD ± UD + UD + Downloaded fromBioscientifica.com at09/28/202107:29:16AM PD: prenatal days, ND: neonatal days, Epi: epithelial cells, Str: stromal cells, Mus: muscle cells. INF: infundibulum, AMP: ampulla, IST: isthmus, UTJ: uterotubal junction. +++: marked, ++: moderate, +: weak, ±: slight, −: negative, UD: undistinguishable. aSome epithelial cells showed negative stainings. bSome epithelial cells showed negative or moderate stainings. www.endocrinology.org via freeaccess ERs, PRs and AR in the rat oviduct · A OKADA and others 309

However, in the developing oviduct, no AR tissues of humans and laboratory rodents, expres- immunoreactivity was detected in Müllerian duct sion, localization and function of these receptors in cells on PD 15 and 19. Both epithelial and stromal the oviduct remain unclear. This study demon- ARs were first detected at a low level on ND 3, and strates that ER,ER, PR-A, PR-B and AR are maintained at a similar level until ND 20 in all expressed in the rat oviduct throughout physiologi- differentiated regions (Table 1, and Fig. 4, right cal conditions in a cell- and region-dependent panels). In the IST and UTJ, muscle AR was manner. A lack of these receptors in ciliated observed at slight and weak levels on ND 7 and 10, epithelial cells of the rat oviductal epithelium was and ND 15 and 20 respectively. noted. In the diestrous oviduct, some AR stainings were In the real-time RT-PCR study, expressions of detected at weak levels in epithelial cells of the ER, PR-A+B and PR-B mRNA were detected to INF/AMP (Fig. 5, right panels). In contrast to ER be regulated in the fetal, neonatal, cycling and and PR expressions in all epithelial cells, most pregnant rat oviduct, suggesting that these recep- epithelial cells did not show AR immunoreactivity tors may play roles in the physiological functions of in the IST/UTJ. Stainings for stromal and muscle the rat oviduct. Oviductal ER expression was ARs were weak or moderate throughout the reported to be low in rats (Saunders et al. 1997, oviduct region. However, no remarkable change Mowa & Iwanaga 2000a,b) and mice (Couse et al. was observed in the expression and localization of 1997), while Sar & Welsch (1999) and Jefferson AR during the estrous cycle and early pregnancy. et al. (2000) failed to detect ER immunopositive cells in oviduct of rats and mice respectively. In the present study, although ER mRNA was detected Immuno-co-localization of epithelial ER, in the rat oviduct by RT-PCR, it was present only PR-A+B and AR with -tubulin IV at low constant levels throughout physiological To determine surface epithelial cell types expressing conditions. Immunoreactivity for ER was limited ER, PRs and AR, double immunohistochemical to a few nonciliated epithelial cells in the staining for each receptor with -tubulin IV, a cili- INF/AMP. Thus, abundant ER may be a major ated epithelial cell marker (Renthal et al. 1993) was ER subtype and play an essential role in performed. In the INF/AMP, the mucosa formed development and function of the rat oviduct. On numerous elaborately branched folds and their the other hand, two PR isoforms, PR-A and PR-B, lumina were labyrinthine systems of narrow spaces are produced from a single gene by transcription between the branching folds covered with the at two distinct promoters (Kastner et al. 1990), and epithelium. The epithelia were basically simple col- the ratio of isoforms varies depending on the umnar and consisted of two kinds of cells, ciliated reproductive tissues during development (Shyamala and nonciliated epithelial cells. They sometimes et al. 1990) and estrous cycle (Mangal et al. 1997). In appeared pseudostratified in the oblique sections. the rat oviduct, the present study demonstrated a The ciliated cells were positive for -tubulin IV and definite variation in the mRNA expression of PR contained round pale nuclei in the apical or middle isoforms evaluated by the ratio of PR-B to total region of the cells, while the nonciliated cells were PR during development and different hormonal negative for -tubulin IV and had dark nuclei oval to circumstances as adults. At present, although little slender in the basal region. Each expression of ER, evidence has been available to define the physio- PR-A+B and AR was restrictedly observed in non- logical significance of P4 action via PR-A and/or ciliated epithelial cells, but not in ciliated epithelial PR-B, it seems highly probable that differential cells of the INF/AMP (Fig. 8). In contrast, almost all expression of two isoforms in the oviduct is epithelial cells were negative for -tubulin IV in the fundamental for the cell growth, differentiation and UTJ/IST, but positive for ER and PR-A+B. functions in response to P4. PR-A is reported to be able to act as a transcriptional inhibitor of PR-B when both proteins are co-expressed (Vegeto et al. Discussion 1993). In the developing oviduct, ER, PR-A+B and Despite extensive research on expression of sex AR were expressed in both epithelial and steroid hormone receptors in female reproductive mesenchymal or stromal cells, and the staining www.endocrinology.org Journal of Molecular Endocrinology (2003) 30, 301–315

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Figure 4 Immunohistochemical localization of ERα, PR-A+B and AR in the developing rat oviduct. ERα (left), PR-A+B (middle) or AR (right) was detected in nuclei of epithelial, stromal and muscle cells of the neonatal rat oviduct. After ND 7, morphological differentiation of the neonatal oviduct occurred. See Table 1 for detail of changes in expressions. All immunoreactivity was abolished by incubation with normal mouse or rabbit IgG as controls (data not shown). Bar: 50 µm.

intensity and mRNA level increased with the reported in their electron-microscopic study that growth of neonates. During this early postnatal differentiation of ciliated cells, which is believed to period when increases in these receptors were be elicited by the initiation of endogenous estrogen observed, immunoreactivity for -tubulin IV in the (17-estradiol; E2) production, occurred on ND 5 rat oviduct appeared between ND 5 and ND 7 in the mouse oviduct. Furthermore, neonatal (data not shown). Komatsu & Fujita (1978) have estrogen administration accelerated cilia formation

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Figure 5 Immunohistochemical localization of ERα, PR-A+B and AR in the diestrous rat oviduct. In the INF and AMP, ERα (left), PR-A+B (middle) and AR (right) were detected in nuclei of epithelial and stromal cells. However, fewer epithelial cells showed immunoreactivity for ERα, PR-A+B and AR. In the IST and UTJ, ERα, PR-A+B and AR were detected in nuclei of epithelial, stromal and muscle cells, except for epithelial AR. Arrows represent high magnification of AR-positive cells, as shown in the inset in AR-AMP. All immunoreactivity was abolished by incubation with normal mouse or rabbit IgG as controls (data not shown). Bar: 100 µm.

in the mouse oviduct (Eroschenko 1982). The documented (Herbst & Bern 1981, Newbold et al. teratogenic and carcinogenic effects of perinatal 1983, Iguchi 1992), and ER KO mice showed exposure to diethylstilbestrol (DES), a synthetic resistance to those adverse effects of DES (Couse estrogen, on the mouse and rat oviduct are well et al. 2001). Therefore, the increase in oviductal www.endocrinology.org Journal of Molecular Endocrinology (2003) 30, 301–315

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ER may have a critical role in the neonatal proliferation and cytodifferentiation of oviductal cells in response to endogenous and exogenous estrogens. If estrogen regulates epithelial ciliary formation directly via epithelial ER in the neonatal oviduct, this potential mechanism may be different from that in ER-negative epithelial cells of the adult oviduct discussed below. In contrast, although the physiological role of PRs and AR has not been clearly demonstrated in the development of the rat oviduct, both epithelial and stromal cells could be targets for endogenous progestins and androgens, and exogenous PRs and AR modula- tors. Especially, because epithelial cells of neonatal IST markedly expressed PRs, they could be a Figure 6 Change in PR-A+B immunoexpression in the IST during the estrous cycle and early pregnancy. critical target for progestin action. Immunohistochemistry for PR-A+B exhibited marked Oviductal cilia are believed to have the critical staining in epithelial, stromal and muscle cells in the IST role in ovum transport through oviduct to uterus in at diestrous stage (Diestrus). However, they decreased cycling and pregnant rats (Halbert et al. 1989). in intensity to moderate at estrus stage (Estrus). In Estrogen accelerated ovum transport in pregnant pregnant rats, PR-A+B staining was observed at moderate levels in epithelial, stromal and muscle cells in rats, and concomitant P4 treatment blocked the the GD 0 oviduct, similar to the estrus oviduct (data not estrogen-induced events (Banik & Pincus 1964, shown), and increased to marked levels on GD 2. Fuentealba et al. 1988). In the cycling and preg- PR-A+B immunoreactivity was abolished by incubation nant rats, ovariectomy or hypophysectomy, with normal mouse IgG as negative control (Negative). Bar: 100 µm.

Figure 7 Immunohistochemical localization of ERβ in the ovary (Ovary), uterus and oviduct. Marked staining was found in ovarian granulosa cells. In the uterus, ERβ staining was detected at moderate and slight levels in luminal and glandular epithelia, and peripheral stroma respectively (Uterus). Also, few epithelial cells in the AMP showed positive staining for ERβ (arrows), but no positive cell was noted in the IST. ERβ immunoreactivity was abolished by incubation with normal rabbit immunoglobulin fraction in the ovary and AMP (Negative). Bar: 200 µm for the ovary, 100 µm for the uterus, AMP and IST.

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et al. 1997, 1998, Buchanan et al. 1998, Kurita et al. 2000a,b, 2001), there is likely to be a similar situation in the oviduct. In contrast, change of cell phenotype from secretory to ciliated cells with estrogen treatment was demonstrated in primary culture of human oviductal epithelial cells (Comer et al. 1998). Moreover, Reeder & Shirley (1999) suggested the possibility of transformation from ciliated cells to secretory cells following loss of cilia in an electron microscopic study. Further elucidation of this matter is necessary. Although effects of androgens on the oviduct still remain obscure, AR was predominantly detected in nonciliated epithelial cells in the INF/AMP, and stromal and muscle cells in the rat oviduct. Figure 8 Double immunohistochemical localization of ff ERα, PR-A+B or AR, with β-tubulin IV in the AMP of the Recently, a direct inhibitory e ect of ER/AR diestrous oviduct. Staining of ERα, PR-A+B or AR heterodimer on both ER and AR transactivational (brown), and β-tubulin IV (red) were not found to be properties has been reported (Panet-Raymond et al. co-localized in the same surface epithelial cells. 2000). Although, in the rat oviduct, co-localization α Arrowheads indicate ER -, PR-A+B- or AR-positive, but of AR and ER was not determined, both AR and β-tubulin IV-negative, cells. Arrows indicate ERα-, PR-A+B- or AR-negative, but β-tubulin IV-positive, cells. ER were expressed in stromal, muscle and noncili- Immunoreactivity was abolished by incubation with ated epithelial cells. Interaction of AR and ER, normal mouse and rabbit IgG (Negative). Bar: 20 µm. therefore, may possibly occur in the rat oviduct as well as in the uterus, if AR and ER co-localize in the identical cell. and treatment with the aromatase inhibitor In conclusion, the cell- and region-dependent 4-hydroxyandrostenedione, caused delayed ovum ER,ER, PRs and AR expressions were transport due to reduction of estrogen production immunohistochemically determined in the rat (Wu et al. 1971, Forcelledo & Croxatto 1986, 1988). oviduct throughout various physiological condi- In contrast, treatment with the PR antagonist tions. This study could be helpful for understanding RU486 caused accelerated ovum transport in rats the molecular and cellular mechanisms underlying (Fuentealba et al. 1987). Thus, estrogen and P4 may estrogen, progestin and androgen actions on the rat have roles in ovum transport by regulating oviduct. oviductal ciliogenesis in rats. In the present study,  however, a lack of ER and PRs in ciliated Acknowledgements epithelial cells was shown by double immunostain- ing with -tubulin IV, suggesting no possibility of The authors gratefully acknowledge Emeritus direct regulation of ciliogenesis of ciliated cells by Professor Noboru Takasugi, Yokohama City estrogen and P4. We hypothesized, therefore, two University for valuable advice and critical reading possible mechanisms of ciliogenesis regulation by of the manuscript. The authors also thank estrogen and P4 in the rat oviduct: (i) interaction members of the Molecular Medicine Laboratories, between epithelial and stromal cells via intermedi- and the Safety Research Laboratories, ate molecules which are produced by ER- and Yamanouchi Pharmaceutical Co., Ltd for their PRs-positive stromal cells, and (ii) cytodifferenti- helpful support and comments. This work was ation from ER- and PRs-positive nonciliated supported in part by a Grant-in-Aid for Scientific (progenitor?) epithelial cells to ciliated epithelial Research on Priority Areas (A) from the Ministry cells with ciliation and subsequent loss of ER and of Education, Culture, Sports, Science and PRs. Since a fundamental role of epithelial–stromal Technology of Japan, the Environmental Research tissue/cell interaction was clearly demonstrated in Grant from the Ministry of Environment, and the estrogen action on cell proliferation and epithelial Health Sciences Research Grant from the Ministry PR regulation in mouse uterus and vagina (Cooke of Health, Labor and Welfare, Japan. www.endocrinology.org Journal of Molecular Endocrinology (2003) 30, 301–315

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References Halbert SA, Becker DR & Szal SE 1989 Ovum transport in the rat oviductal ampulla in the absence of muscle contractility. Biology of ff Reproduction 40 1131–1136. Armstrong DT, Moon YS & Leung PC 1976 Uterotrophic e ects Harper MJK 1994 Gamete and zygote transport. In The Physiology  of testosterone and 5 -dihydrotestosterone in intact and of Reproduction, edn 2, pp 123–187. Eds E Knobil & JD Neill. Biology of Reproduction ovariectomized immature female rats. 15 New York: Raven Press. 107–114. Herbst AL & Bern HA 1981 Developmental Effects of Diethylstilbestrol Banerjee A, Roach MC, TrckaP&LuduenaRF1992Preparation (DES) in Pregnancy, pp 203. New York: Thieme-Stratton. of a monoclonal antibody specific for the class IV isotype of Hirai M, Hirata S, Osada T, HagiharaK&KatoJ1994 Androgen -tubulin. Purification and assembly of  ,  and  II III IV receptor mRNA in the rat ovary and uterus. Journal of Steroid tubulin dimers from bovine brain. Journal of Biological Chemistry 267 Biochemistry and Molecular Biology 49 1–7. 5625–5630. Hiroi H, Inoue S, Watanabe T, Goto W, Orimo A, Momoeda M, Banik UK & Pincus G 1964 Estrogens and transport of ova in the Tsutsumi O, TaketaniY&Muramatsu M 1999 Differential rat. Proceedings of the Society for Experimental Biology and Medicine 116 immunolocalization of estrogen receptor  and  in rat ovary and 1032–1034. uterus. Journal of Molecular Endocrinology 22 37–44. Brenner RM & Slayden OD 1994 Cyclic changes in the primate Husmann DA, Wilson CM, McPhaul MJ, Tilley WD & Wilson JD oviduct and endometrium. In The Physiology of Reproduction, 1990 Antipeptide antibodies to two distinct regions of the edn 2, pp 541–569. Eds E Knobil & JD Neill. New York: Raven androgen receptor localize the receptor protein to the nuclei of Press. target cells in the rat and human prostate. Endocrinology 126 Buchanan DL, Kurita T, Taylor JA, Lubahn DB, Cunha GR & 2359–2368. Cooke PS 1998 Role of stromal and epithelial estrogen receptors ff in vaginal epithelial proliferation, stratification, and cornification. Iguchi T 1992 Cellular e ects of early exposure to sex hormones and antihormones. International Review of Cytology 1–57. Endocrinology 139 4345–4352. 139 Comer MT, Leese HJ & Southgate J 1998 Induction of a Ilenchuk TT & Walters MR 1987 Rat uterine 3 ffi differentiated ciliated cell phenotype in primary cultures of analyzed by [ H]R5020 photoa nity labeling: evidence that the A Fallopian tube epithelium. Human Reproduction 13 3114–3120. and B subunits are not equimolar. Endocrinology 120 1449–1456. Cooke PS, Buchanan DL, Young P, Setiawan T, Brody J, Korach Jansen RPS 1984 Endocrine response in the fallopian tube. Endocrine KS, Taylor J, Lubahn DB & Cunha GR 1997 Stromal estrogen Reviews 5 525–551. ff receptors mediate mitogenic effects of estradiol on uterine Je erson WN, Couse JF, Banks EP, Korach KS & Newbold RR epithelium. PNAS 94 6535–6540. 2000 Expression of estrogen receptor  is developmentally Cooke PS, Buchanan DL, Lubahn DB & Cunha GR 1998 regulated in reproductive tissues of male and female mice. Biology Mechanism of estrogen action: lessons from the estrogen of Reproduction 62 310–317. receptor- knockout mouse. Biology of Reproduction 59 470–475. Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer H Couse JF, Lindzey J, Grandien K, Gustafsson JÅ & Korach KS & Chambon P 1990 Two distinct estrogen-regulated promoters 1997 Tissue distribution and quantitative analysis of estrogen generate transcripts encoding the two functionally different receptor- (ER) and estrogen receptor- (ER) messenger human progesterone receptor forms A and B. EMBO Journal 9 ribonucleic acid in the wild-type and ER-knockout mouse. 1603–1614. Endocrinology 138 4613–4621. Komatsu M & Fujita H 1978 Electron-microscopic studies on the Couse JF, Dixon D, Yates M, Moore AB, Ma L, Maas R & development and aging of the oviduct epithelium of mice. Anatomy Korach KS 2001 Estrogen receptor- knockout mice exhibit and Embryology 152 243–259. resistance to the developmental effects of neonatal Kurita T, Lee K, Cooke PS, Taylor JA, Lubahn DB & Cunha GR diethylstilbestrol exposure on the female reproductive tract. 2000a Paracrine regulation of epithelial progesterone receptor by Developmental Biology 238 224–238. estradiol in the mouse female reproductive tract. Biology of Eroschenko VP 1982 Surface changes in oviduct, uterus and vaginal Reproduction 62 821–830. cells of neonatal mice after estradiol-17 and the insecticide Kurita T, Lee K, Cooke PS, Lydon JP & Cunha GR 2000b chlordecone (Kepone) treatment: a scanning electron microscopic Paracrine regulation of epithelial progesterone receptor and study. Biology of Reproduction 26 707–720. lactoferrin by progesterone in the mouse uterus. Biology of Fisher JS, Millar MR, Majdic G, Saunders PTK, Fraser HM & Reproduction 62 831–838. Sharpe RM 1997 Immunolocalisation of oestrogen receptor- Kurita T, Cooke PS & Cunha GR 2001 Epithelial-stromal tissue within the testis and excurrent ducts of the rat and marmoset interaction in paramesonephric (Müllerian) epithelial monkey from perinatal life to adulthood. Journal of Endocrinology differentiation. Developmental Biology 240 194–211. 153 485–495. LauKM,LeavI&HoSM1998 Rat estrogen receptor- and -, Forcelledo ML & Croxatto HB 1986 Ovum transport in cyclic rats and progesterone receptor mRNA expression in various prostatic rendered hypo-oestrogenic by treatment with 4-hydroxy-4- lobes and microdissected normal and dysplastic epithelial tissues of androstene-3,17-dione. Journal of Reproduction and Fertility 76 the noble rats. Endocrinology 139 424–427. 325–330. Li S 1994 Relationship between cellular DNA synthesis, PCNA Forcelledo ML & Croxatto HB 1988 Effects of 4-hydroxyandro- expression and sex status in the stenedione and exogenous testosterone on blood concentrations of developing mouse ovary, uterus and oviduct. Histochemistry 102 oestradiol and oviductal embryo transport in the rat. Journal of 405–413. Endocrinology 118 93–100. Lyon MF & Glenister PH 1980 Reduced reproductive performance Fuentealba B, NietoM&Croxatto HB 1987 Ovum transport in in androgen-resistant Tfm/Tfm female mice. Proceedings of the Royal pregnant rats is little affected by RU486 and exogenous Society of London Series B. Biological Sciences 208 1–12. progesterone as compared with cycling rats. Biology of Reproduction Mangal RK, Wiehle RD, Poindexter AN III & Weigel 1997 37 768–774. Differential expression of uterine progesterone receptor forms A Fuentealba B, NietoM&Croxatto HB 1988 Progesterone and B during menstrual cycle. Journal of Steroid Biochemistry and abbreviates the nuclear retention of estrogen receptor in the rat Molecular Biology 63 195–202. oviduct and counteracts estrogen action on egg transport. Biology of Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Reproduction 38 63–69. Umesono K, Blumberg B, Kastner P, Mark M, Chambon P &

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Downloaded from Bioscientifica.com at 09/28/2021 07:29:16AM via free access ERs, PRs and AR in the rat oviduct · A OKADA and others 315

Evans RM 1995 The nuclear receptor superfamily: the second Sar M & Welsch F 1999 Differential expression of estrogen decade. Cell 83 835–839. receptor- and estrogen receptor- in the rat ovary. Endocrinology Mowa CN & Iwanaga T 2000a Differential distribution of oestrogen 140 963–971. receptor- and - mRNAs in the female reproductive organ of Saunders PTK, Maguire SM, GaughanJ&Millar MR 1997 rats as revealed by in situ hybridization. Journal of Endocrinology 165 Expression of oestrogen receptor beta (ER) in multiple rat tissues 59–66. visualised by immunohistochemistry. Journal of Endocrinology 154 Mowa CN & Iwanaga T 2000b Developmental changes of the R13–R16. oestrogen receptor- and - mRNAs in the female reproductive Shyamala G, Schneider W & Schott D 1990 Developmental organ of the rat – an analysis by in situ hybridization. Journal of regulation of murine mammary progesterone receptor gene Endocrinology 167 363–369. expression. Endocrinology 126 2882–2889. Newbold RR, Bullock BC & McLachlan JA 1983 Exposure to Taylor CR, Shi SR, Chaiwun B, Young L, Imam SA & Cote RJ diethylstilbestrol during pregnancy permanently alters the ovary 1994 Strategies for improving the immunohistochemical staining and oviduct. Biology of Reproduction 28 735–744. of various intranuclear prognostic markers in formalin-paraffin Ohta Y, Sato T & Iguchi T 1993 Immunocytochemical localization sections: androgen receptor, estrogen receptor, progesterone of progesterone receptor in the reproductive tract of adult female receptor, protein, proliferating cell nuclear antigen, and Ki-67 rats. Biology of Reproduction 48 205–213. antigen revealed by antigen retrieval techniques. Human Pathology Okada A, Ohta Y, Buchanan DL, Sato T, Inoue S, Hiroi H, 25 263–270. a Muramatsu M & Iguchi T 2002 Changes in ontogenetic Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O’Malley BW expression of and not of estrogen receptor & McDonnell DP 1993 Human Journal of Molecular beta in the female rat reproductive tract. form is a cell- and promoter-specific repressor of human Endocrinology 87–97. 28 progesterone receptor B function. Molecular Endocrinology 7 b ff Okada A, Ohta Y, Buchanan DL, Sato T & Iguchi T 2002 E ect 1244–1255. of estrogens on ontogenetic expression of progesterone receptor in Wang H, ErikssonH&Sahlin L 2000 Estrogen receptors  and  in the fetal female rat reproductive tract. Molecular and Cellular the female reproductive tract of the rat during the estrous cycle. Endocrinology 195 55–64. Biology of Reproduction 1331–1340. Panet-Raymond V, Gottlieb B, Beitel LK, PinskyL&Trifiro MA 63 2000 Interactions between androgen and estrogen receptors and Weihua Z, Ekman J, Almkvist Å, Saji S, Wang L, Warner M & the effects on their transactivational properties. Molecular and Gustafsson JÅ 2002 Involvement of androgen receptor in Cellular Endocrinology 167 139–150. 17-estradiol-induced cell proliferation in rat uterus. Biology of Pelletier G, LabrieC&LabrieF2000Localization of oestrogen Reproduction 67 616–623. receptor , oestrogen receptor  and androgen receptors in the Wu JT, Dickmann Z & Johnson DC 1971 Effects of ovariectomy or rat reproductive organs. Journal of Endocrinology 165 359–370. hypophysectomy on day one of pregnancy on development Reeder RL & Shirley B 1999 Deciliation in the ampulla of the rat and transport of fertilized rat eggs. Journal of Endocrinology 49 oviduct and effects of estrogen on the process. Journal of 507–513. Experimental Zoology 283 71–80. Renthal R, Schneider BG, Miller MM & Ludueña RF 1993 IV is the major -tubulin isotype in bovine cilia. Cell Motility and the Received in final form 6 March 2003 Cytoskeleton 25 19–29. Accepted 8 March 2003

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