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To evaluate ontogenetic expression and localization of estrogen receptor (ER) alpha and beta in fetal female rat reproductive tract, competitive RT-PCR and immunohistochemistry were performed. Expression levels for Mullerian ERalpha, ERbeta1 and ERbeta2 mRNAs were determined by competitive RT-PCR. ERalpha expression on gestational day (GD) 15 x 5 increased 4 x 4-fold by GD 21 x 5, whereas both ERbeta1 and ERbeta2 gene expression were maintained at lower constant levels compared with ERalpha during development. ER immunolocalization was evaluated within three regions along the Mullerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. Nuclear ERalpha was localized predominantly in proximal Mullerian epithelium, and middle and caudal Mullerian mesenchyme on GDs 15 x 5-21 x 5. Staining intensity for ERalpha increased with development in all regions. However, ERbeta immunoreactivity was not detected in any region during prenatal life after separate staining with three different polyclonal anti-rat ERbeta antibodies. These findings provide fundamental information critical for clarifying the species-specific physiological roles of ER subtypes during fetal development and for investigating the tissue-specific mechanisms underlying the prenatal response to estrogen and estrogen receptor agonists.
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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 ERalpha, ERbeta, total PR (PR-A+B), PR-B and AR mRNA expressions. All receptors except for ERbeta 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, ERalpha 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 ERbeta staining. AR was detected in stromal and muscle cells throughout the oviduct region, and in epithelial cells of the inf/AMP. Taken together, ERalpha, 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 beta-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.
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In order to understand early events caused by estrogen in vivo, temporal uterine gene expression profiles at early stages were examined using DNA microarray analysis. Ovariectomized mice were exposed to 17beta-estradiol and the temporal mRNA expression changes of ten thousand various genes were analyzed. Clustering analysis revealed that there are at least two phases of gene activation during the period up to six hours. One involved immediate-early genes, which included certain transcription factors and growth factors as well as oncogenes. The other involved early-late genes, which included genes related to RNA and protein synthesis. In clusters of down-regulated genes, transcription factors, proteases, apoptosis and cell cycle genes were found. These hormone-inducible genes were not induced in estrogen receptor (ER) alpha knockout mice. Although expression of ERbeta is known in the uterus, these findings indicate the importance of ERalpha in the changes in gene expression in the uterus.
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Estrogen regulates proliferation and differentiation of epithelial cells in the mammalian oviduct, but pathways for cell-specific differentiation are not well understood. In the epithelial cells of the developing rat oviduct, we found estrogen receptor (ER) alpha is expressed at birth and persists in all cells through neonatal day (ND) 7 when ciliated cells appear. To determine a specific function of ER and foxj1, a transcription factor known to have fundamental roles in ciliogenesis in the lung, in differentiation of the ciliated epithelial cells, we treated newborn rats from ND 0 to 5 with estradiol-17beta (E2) with and without a selective ER antagonist. E2 enhanced the number of proliferating cells and accelerated the process of epithelial cell differentiation resulting in ciliogenesis by ND 5, and co-treatment with an ER antagonist inhibited these changes. Foxj1 was expressed only in the infundibulum and ampulla (INF/AMP). That expression preceded the appearance of cilia and was induced by E2. Cilia were absent in oviducts of foxj1-deficient mice, indicating that foxj1 plays a critical role in oviductal ciliogenesis. However, we found the presence of cilia in the ERalpha-deficient mouse oviduct. The widespread expression of ERalpha in oviductal epithelium, but restriction of cilia to the INF/AMP regions, and importantly, the presence of cilia in the ERalpha-deficient mice, suggested ER signaling is not essential for ciliated epithelial cell differentiation. These observations demonstrate that, although E2 stimulates the differentiation process of ciliated epithelial cells, foxj1 is directly required for epithelial cell ciliogenesis of the neonatal oviduct.
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Developmental exposure to a synthetic estrogen, diethylstilbestrol (DES), induces carcinogenesis in human and laboratory animals. In mice, neonatal DES treatment induces persistent proliferation and keratinization of the vaginal epithelium, even in the absence of the ovaries, resulting in cancerous lesions later in life. To understand the mechanisms underlying this persistent cell proliferation and differentiation, we characterized the gene expression patterns in the neonatally DES-exposed mouse vagina using DNA microarray and real-time quantitative RT-PCR. We found that genes related to cellular signaling, which are candidates for mediating the persistent proliferation and differentiation, were altered, and genes related to the immune system were decreased in the neonatally DES-exposed mouse vagina. We also noted high expression of interleukin-1 (IL-1)-related genes accompanied by phosphorylation of JNK1. In addition, expression IGF-I and its binding proteins was modulated and led to phosphorylation of IGF-I receptor and Akt, which is one of the downstream factors of IGF-I signaling. This led us to characterize the expression as well as the phosphorylation status of IL-1 and IGF-I signaling pathway components which may activate the phosphorylation cascade in the vagina of mice exposed neonatally to DES. These findings give insight into persistent activation in the vagina of mice exposed neonatally to DES.
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Alkylphenols perturb the endocrine system and are considered to have weak estrogenic activities. Although it is known that nonylphenol can bind weakly to the estrogen receptor, it is unclear whether all reported effects of nonylphenol are attributable to its estrogen receptor-binding activity. In order to examine whether alkylphenols have similar effects to the natural hormone, estradiol, we used a mouse model to examine the effects of nonylphenol on gene expression and compared it with estradiol. DNA microarray analysis revealed that, in the uterus, most of the genes activated by this alkylphenol at a high dose (50 mg/kg) were also activated by estradiol. At lower doses, nonylphenol (0.5 mg/kg and 5 mg/kg) had little effect on the genes that were activated by estradiol. Thus, we concluded that the effects of nonylphenol at a high dose (50 mg/kg) were very similar to estradiol in uterine tissue. Moreover, since evaluation of estrogenic activity by gene expression levels was comparable with the uterotrophic assay, it indicated that analysis of gene expression profiles can predict the estrogenic activities of chemicals. In contrast to the similar effects of nonylphenol and estradiol observed in the uterus, in the liver, gene expression was more markedly affected by nonylphenol than by estradiol. This indicated that, in the liver, nonylphenol could activate another set of genes that are distinct from estrogen-responsive genes. These results indicated that nonylphenol has very similar effects to estradiol on gene expression in uterine but not in liver tissue, indicating that tissue-specific effects should be considered in order to elucidate the distinct effects of alkylphenols.
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collége de France, 67404 Illkirch, Strasbourg, France
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Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collége de France, 67404 Illkirch, Strasbourg, France
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Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collége de France, 67404 Illkirch, Strasbourg, France
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Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collége de France, 67404 Illkirch, Strasbourg, France
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Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collége de France, 67404 Illkirch, Strasbourg, France
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Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collége de France, 67404 Illkirch, Strasbourg, France
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Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collége de France, 67404 Illkirch, Strasbourg, France
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Recent studies have revealed that hundreds of genes in the uterus are activated by estrogen. Their expression profiles differ over time and doses and it is not clear whether all these genes are directly regulated by estrogen via the estrogen receptor. To select the genes that may be regulated by estrogen, we treated mice with several doses of estrogen and searched for those genes whose dose–response expression pattern mirrored the uterine growth pattern. Among those genes, we found that the dose-dependent expression of the adrenomedullin (ADM) gene correlated well with the uterotrophic effect of estrogen. ADM expression is induced early after estrogen administration and is restricted to the endometrial stroma. The spatiotemporal gene expression pattern of ADM was similar to that of receptor-modifying protein 3 (RAMP3). RAMP3 is known to modify calcitonin gene-related receptor (CRLR) so that it can then serve as an ADM receptor. Chromatin immunoprecipitation assays indicated that the estrogen receptor binds directly to the ADM promoter region and RAMP3 intron after estrogen administration. It was also shown that neither the ADM nor RAMP3 gene could be activated in estrogen receptor-α-null mouse. Although uterine ADM expression has been reported to occur in the myometrium, our observations indicate that estrogen-induced ADM is also expressed in the uterine stroma and that such variable, spatiotemporally regulated ADM expression contributes to a wider range of biological effects than previously expected.
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Administration of physiological and non-physiological estrogens during pregnancy or after birth is known to have adverse effects on the development of the reproductive tract and other organs. Although it is believed that both estrogens have similar effects on gene expression, this view has not been tested systematically. To compare the effects of physiological (estradiol; E2) and non-physiological (diethylstilbestrol; DES) estrogens, we used DNA microarray analysis to examine the uterine gene expression patterns induced by the two estrogens. Although E2 and DES induced many genes to respond in the same way, different groups of genes showed varying levels of maximal activities to each estrogen, resulting in different dose-response patterns. Thus, each estrogen has a distinct effect on uterine gene expression. The genes were classified into clusters according to their dose-responses to the two estrogens. Of the eight clusters, only two correlated well with the uterotropic effect of different doses of E2. One of these clusters contained genes that were upregulated by E2, which included genes encoding several stress proteins and transcription factors. The other cluster contained genes that were downregulated by E2, including genes related to metabolism, transcription and detoxification processes. The expression of these genes in estrogen receptor-deficient mice was not affected by E2 treatment, indicating that these genes are affected by the E2-bound estrogen receptor. Thus, of the many genes that are affected by estrogen, it was suggested that only a small number are directly involved in the uterotropic effects of estrogen treatment.
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The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many organisms. TCDD exposure is known to be associated with abnormal development, hepatotoxicity and endocrine effects. It has also been reported to have antiestrogenic activity in addition to estrogenic activity. In order to clarify the effects of TCDD in the uterus, we evaluated the patterns of gene expression after TCDD and estradiol administration. Of the 10 000 arrayed genes, only a few were affected by both estradiol and TCDD. Although the subset of genes that responded to estrogen was also activated by TCDD, the response to TCDD was more limited than that observed in response to estradiol. Therefore, according to our analysis of gene expression patterns, TCDD had partial and weak estrogenic activity in the uterus.
