HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suzuki, A. Articles by Iguchi, T. Search for Related Content PubMed PubMed Citation Articles by Suzuki, A. Articles by Iguchi, T.

---

ORIGINAL RESEARCH ARTICLE

Global Gene Expression in Mouse Vaginae Exposed to Diethylstilbestrol at Different Ages

Atsuko Suzuki^*,,,,||, Hajime Watanabe^,,¶, Takeshi Mizutani^,, Tomomi Sato, Yasuhiko Ohta^*,,|| and Taisen Iguchi^,,¶,1

^* The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1, Yoshida, Yamaguchi, 753-8515, Japan; Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology, 4-1-8 Motomachi, Kawaguchi 332-0012, Japan; Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan; ^|| Laboratory of Experimental Animals, Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Koyama 680-8553, Japan; and ^¶ Department of Molecular Biomechanics, School of Life Science, Graduate University for Advanced Studies, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan

¹ To whom requests for reprints should be addressed at Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan. E-mail: taisen{at}nibb.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Estrogens regulate proliferation and differentiation of cells in target organs such as the female reproductive tract. In mature mice, estrogens stimulate cell proliferation, whereas ovariectomy results in atrophy of the female reproductive tract. In contrast, perinatal exposure to estrogens, including diethylstilbestrol (DES), induces persistent, ovary-independent vaginal stratification and cervico-vaginal tumors later in life. These effects are due to altered cell fate following DES exposure during a critical developmental period. The detailed mechanisms underlying the reversible and irreversible cell proliferation in vaginae induced by DES at different ages has not been clarified. Therefore, we examined differences in gene expression pattern using DNA microarray analysis in mouse vaginae 6 hrs after a single injection of 2 µg DES per gram of body weight, and proliferation of vaginal epithelial and stromal cells 24 hrs after the injection at postnatal days (PNDs) 0, 5, 20, and 70. After DES stimulation, vaginal epithelial and stromal cells showed cell proliferation at PNDs 20 and 70, and at PNDs 0 and 5, respectively. DNA microarray analysis exhibited 54 DES-induced genes and 9 DES-repressed genes in vaginae at PND 0, whereas more than 200 DES-induced genes were found in vaginae at PNDs 5 and 20, and 350 genes at PND 70. Clustering analysis of DES-induced genes in the vaginae at different ages revealed that genes induced by DES at PND 5 were closer to the adult type than that of PND 0. Genes related to keratinocyte differentiation, such as Gadd45, p21, 14–3–3 sigma, small proline-rich protein 2f (Sprr2f), and Krupple-like factor 4 (Klf4), were induced by DES. The number of DES-induced genes during the critical period, PND 0, was smaller than those found after the critical period. These results give insight toward understanding the molecular mechanisms underlying the critical period in mouse vaginae.

Key Words: microarray • gene expression • diethylstilbestrol • vagina • mouse

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Estrogens induce cell proliferation and differentiation, whereas estrogen depletion results in atrophy accompanied by apoptosis in adult female reproductive organs such as the uterus and vagina (1–3). Estrogen exposure during a critical period in early development induces persistent ovary-independent proliferation and keratinization in the vaginal epithelium during adulthood (4, 5). Diethylstilbestrol (DES), a synthetic estrogen used to prevent miscarriage during the 1940s to the early 1970s, induced vaginal clear cell carcinoma and uterine abnormalities in daughters of mothers exposed to DES during pregnancy (6). Similar abnormalities were reported in mice exposed to estrogens during a critical perinatal period (4, 5, 7). In female mice, various abnormalities such as polyovular follicles, oviductal tumors, uterine epithelial metaplasia, persistent vaginal stratification and keratinization, vaginal adenosis, and cervico-vaginal carcinomas, were induced by perinatal exposure to estrogens including DES (4, 5, 7–13). Vaginal epithelial proliferation persists even after ovariectomy in adult mice exposed to a sufficient dose of DES during the early neonatal period (4, 5).

During the normal estrous cycle, vaginal epithelial cell proliferation and keratinization occur at the estrous stage (1), whereas keratin 1 (K1) and progesterone receptor expressions were induced at the proestrous stage (14, 15). DES exposure during a critical developmental period results in alteration of the response to estrogen in the vagina, leading to a set of subsequent abnormalities. Epithelial cells failed to undergo apoptosis even after ovariectomy, and persistent expression of various genes was observed in the persistently proliferated vagina (15–17). Reduced expression of estrogen receptor (ER) mRNA and persistent expression of c-fos and c-jun mRNAs were observed in the vaginae of mice exposed to DES at the neonatal stage, even after ovariectomy (15). Persistent phosphorylation of erbB receptors, including epidermal growth factor (EGF) receptor, and sustained expression of EGF-like growth factors were found in the vaginae of mice neonatally exposed to DES (16). Neonatal exposure to a fibroblast growth factor family member, keratinocyte growth factor (KGF), resulted in persistent vaginal epithelial stratification (18). The induction of EGF by estrogens may play important roles in the proliferation of epithelial cells in the uterus and vagina (18–21).

We used DNA microarray to analyze gene expression in neonatally DES-exposed mouse vaginae and observed persistent expression of interleukin-1 (IL-1), IL-1 receptor, insulin-like growth factor-I (IGF-I) mRNAs, and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK), as well as phosphorylation of downstream genes (17).

The critical periods for the induction of abnormalities by estrogenic chemicals during mouse development varies by organ (22). Analyses of the molecular mechanisms underlying the critical sensitive window in each organ is essential for understanding the etiology of the persistent changes induced in the reproductive tracts. Therefore, we examined global expression in vaginae of early genes elicited by DES treatment at different ages in order to understand the differences in estrogen-responsive genes during and after the critical period, and in adulthood.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals.
C57BL/6J mice (CLEA, Tokyo, Japan) were used at postnatal days (PNDs) 0, 5, 20, and 70. Mice were maintained in a 12:12-hr light:dark cycle at 23–25°C. They were fed a commercial diet (CE-2; CLEA), and tap water was provided ad libitum. All experiments and animal husbandry protocols were approved by the animal care committee of the National Institutes of Natural Sciences.

Treatments.
DES (Sigma Chemical Co., St. Louis, MO) was dissolved in sesame oil. Unless otherwise stated, all materials were obtained from Wako Pure Chemical Industries, Osaka, Japan. The day of birth was designated as Day 0. For microarray experiments, mice at PND 0 (7–12 mice from three litters), PND 5 (7–12 mice from three litters), PND 20 (8 mice), and PND 70 (8 mice) were given a single subcutaneous (sc) injection of 2 µg DES per gram of body weight (bw) or oil vehicle alone. Mice at PND 70 were ovariectomized at 56 days of age. Vaginae from DES-exposed and control mice were collected for DNA microarray analysis and quantitative real time–polymerase chain reaction (QRT-PCR). In order to identify early genes induced by DES, tissues were dissected 6 hrs after the injection as described previously (23–25).

In addition, five mice each were given a single sc injection of 2 µg DES/gram bw or oil vehicle alone for the bromodeoxyuridine (BrdU) experiment.

DNA Microarray Analysis.
Total RNA from vaginae was extracted using TRIZOL (Invitrogen, Tokyo, Japan) and purified using an RNeasy mini kit (Qiagen, Tokyo, Japan). Quality and quantity of total RNA were confirmed by the Agilent 2100 bioanalyzer (Agilent, Tokyo, Japan). cRNA probes were prepared from the purified RNA using an Affymetrix cRNA probe kit (Affymetrix, Inc., Santa Clara, CA) according to the manufacturer’s protocol. All preparations met the recommended criteria of Affymetrix for use on their expression array. The amplified cRNA was hybridized to high-density oligonucleotide arrays (Mouse U74A; Affymetrix, Inc.) containing approximately 12,500 genes, and the scanned data were analyzed with GeneChip software (Affymetrix, Inc.) and processed as described previously (23). To confirm the estrogen-related changes in gene expression revealed by DNA microarray analysis, we independently repeated the same experiment at least twice. The expression data were analyzed with GeneSpring software (Agilent, Palo Alto, CA) as described previously (26).

For the clustering analysis, genes activated more than 2-fold by DES were selected, and similarities between experiments and expression levels were measured by standard correlation using the GeneSpring program as described (23–26). Gene expression change was estimated by the value of control PND 0 as one. Putative target genes were validated by QRT-PCR.

QRT-PCR.
Total RNA was purified as described above. cDNA was synthesized from purified total RNA with Superscript II RT (–) (Invitrogen) with random primers at 42°C for 60 mins. PCR reactions were performed in the PE Prism 5700 sequence detector (PE Biosystems, Tokyo, Japan) using SYBR-Green PCR core reagents (PE Biosystems) in the presence of appropriate primers according to the manufacturer’s instructions. PCR amplification was performed in triplicate under the following conditions: 2 mins at 50°C, 10 mins at 95°C, followed by a total of 40 cycles of 15 secs at 95°C and 1 min at 60°C.

Gene expression levels were normalized to the expression levels of ribosomal protein L8 mRNA (U67771), and gel electrophoresis and melting curve analyses were performed to confirm correct amplicon size and the absence of nonspecific bands. The primers were chosen to amplify short PCR products of less than 100 base pairs and their sequences are listed in Table 1.

Immunostaining of BrdU.
Five mice each given a single sc injection of 2 µg DES were killed 24 hrs after the injection. Two hours before dissection, 0.05 mg/g BrdU (Sigma, Tokyo, Japan) was injected intraperitoneally. Tissues fixed with neutral-buffered 10% formalin were embedded in paraffin. Sections cut at 8 µm were incubated with 0.3% H₂O₂ in methanol for 30 mins at room temperature (RT) to block endogenous peroxidase activity. They were washed with 0.5% Tween-20 in phosphate-buffered saline (PBS), incubated with 2 N HCl in water for 20 mins at RT. They were washed with 0.5% Tween-20 in PBS, incubated with 1% bovine serum (BSA) for 20 mins at RT, and with anti-BrdU antibody (Roche, Mannheim, Germany) at a dilution of 1:15 in 1% BSA at 4°C overnight. Washing with 0.5% Tween-20 in PBS, sections were incubated with mixture of 3,3'-diaminobenzidine tetrahydrochloride (DAB) and H₂O₂. Counterstain was conducted with methyl green. The number of BrdU-positive cells in 300 epithelial cells in the middle part of vagina and that in 500 stromal cells were recorded. Proliferation rate (%) was estimated as a percentage of BrdU-positive cells in epithelial cells and stromal cells, separately.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gene Expression in the Vaginae of Mice Treated with DES at Different Ages.
The number of detected genes in the mouse vagina from oil-injected controls was not very different among animals examined at different ages: newborn (PND 0) = 4988 genes, PND 5 = 4937 genes, PND 20 = 4881 genes, and PND 70 = 4903 genes. We selected genes showing at least a 2-fold change in expression in response to DES treatment for further analysis (listed at http://www.nibb.ac.jp/bioenv1/suzuki/). The number of genes induced or repressed by DES was modest at PND 0, but showed a sharp increase with age (Fig. 1). DES exposure induced 54, 208, 202, and 326 genes and repressed 9, 66, 117, and 152 genes in vaginae at PNDs 0, 5, 20, and 70, respectively (Fig. 1). The number of genes (208) induced by DES at PND 5 was similar to that of PND 20 (202). Ninety-two of 208 (44%) genes induced by DES at PND 5 were also induced at PND 20 by DES. Fifty-eight (28%) PND 5–specific genes were induced by DES (Fig. 1). Four genes (including two enhanced sequence tags [ESTs]) were specific to PND 0 (Table 2 and Fig. 1).

View larger version (24K): [in this window] [in a new window]   Figure 1. (a) The number of induced genes in vaginae 6 hrs after a single injection of 2 µg DES/g bw at PNDs 0, 5, 20, and 70. Mice at PND 70 were ovariectomized 2 weeks before. The number of DES-induced genes was small at PND 0, but it increased drastically at PND 5. (b) The number of repressed genes in vaginae 6 hrs after a single injection of 2 µg DES/g bw at PNDs 0, 5, 20, and 70. The numbers of DES-repressed genes increased linearly with age. Each pattern in the bar indicates genes commonly induced (a) or repressed (b) genes in respective ages. Days indicate ages given a single injection of DES.

Clustering Patterns and Category of DES-Regulated Genes in the Vaginae of Mice at Different Ages.
The 729 genes showing more than a 2-fold change following a single injection of DES at PNDs 0, 5, 20, and 70 were used for clustering analysis. These genes can be grouped as PND 0, PND 5, and PND 70 in controls, and as PND 0 and PND 5–70 in DES-exposed vaginae (Fig. 2) because the clustering patterns of genes in DES-exposed vaginae at PND 5 was more similar to those of PND 20 and PND 70 than that of PND 0 (Fig. 2).

View larger version (60K): [in this window] [in a new window]   Figure 2. Clustering analysis of DES-responsive genes in mouse vaginae selected in the present study. Mice at PNDs 0, 5, 20, and 70 were stimulated by a single injection of DES. Each color bar indicates the expression level of one gene: red, induction; green, repression; yellow, average expression in eight groups; gray, not detected. Genes showing more than a 2-fold change in expression 6 hrs after a single injection of 2 µg DES/g bw at all ages were used for clustering analysis. Seven-hundred seventy-nine genes were selected. Control mice exhibited separated trees between the neonatal period (PNDs 0 and 5) and adulthood (PNDs 20 and 70). Branching of clustered genes in DES-exposed vaginae at PND 5 was closer to that of PND 0.

View larger version (21K): [in this window] [in a new window]   Figure 3. Changes in gene expression of cell cycle and keratinocyte differentiation regulators were confirmed by QRT-PCR. Fold change of gene expression by QRT-PCR was estimated based on the value of each gene at PND 0 as one. Results are the mean and SEM of three experiments. Each experiment was performed in triplicate. P < 0.05 vs. age-matched controls.

View larger version (18K): [in this window] [in a new window]   Figure 4. Percentage of BrdU-positive cells in vaginal epithelial cells and stromal cells at different ages. Mean and SEM. Note reverse of vaginal cell proliferation in epithelium and stroma after PND 5. *P < 0.05 vs. age-matched controls.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study, global gene expression in vaginae was analyzed at different ages using DNA microarray analysis. We demonstrated age differences in vaginal responses to estrogen in the induction of gene expression from PND 0 to PND 70. ER staining was found in vaginal stromal cells in CD-1 mice at PND 0 (37), and in both vaginal epithelial and stromal cells, but in only stromal cells in uterine epithelial cells in C57BL/Tw mice at PND 0 (38). Epithelial expression of ER in the reproductive tracts occurs later in C57BL/6 mice than in CD-1 mice (39). Developmental effects of estrogens, including DES on neonatal mouse vaginae, are mediated through stromal ER (40). In the present study, we used C57BL/6 mice. Thus, neonatal mouse vaginae seem to be responsive to estrogen at PND 0. However, in the present study, the number of DES-induced genes in vaginae at PND 0 was smaller compared with those in PNDs 5, 20, and 70 mice. Moreover, vaginal epithelia in mice at PNDs 5, 20, and 70 were proliferated by DES, but not at PND 0 in the present study (Fig. 4). The PND 0 mouse vagina is still under development even without estrogenic stimulation (4, 41). Thus, in terms of gene expression, the vagina at PND 0 is less sensitive to estrogenic stimulation than it is at later stages. It has been shown that the response to DES is different between the Müllerian and urogenital sinus vaginal epithelium; proliferation in the vaginal fornix (Müllerian vagina) is inhibited by neonatal DES and the cell morphology is altered (7). Further studies examining differences in gene expression in Müllerian and urogenital sinus vagina in response to DES are essential.

Clustering analysis of estrogen-responsive genes in DES-exposed mouse vaginae showed that they could be broadly categorized into two types; a neonate type (PND 0) and an adult type (PNDs 5, 20, and 70). Vaginal stromal cells showed proliferative response to DES only at PNDs 0 and 5, but not at PNDs 20 and 70. In contrast, vaginal epithelial cells showed proliferative response to DES at PND 20 and PND 70 in the present study. The critical window for induction of estrogen-independent persistent changes in vaginae is within 3–5 days after birth (22). The underlying mechanism of the differences in responsiveness between vaginal epithelial cells and stromal cells at different ages needs to be analyzed in the near future to understand the molecular basis of the critical window. In mouse vaginae, proliferative response to DES in epithelial cells and stromal cells were reversed after PND 5, which may indicate the critical window of the mouse vagina.

Estrogens induce expression of genes related to cell cycle regulators, chromatin remodeling, IGF-I signaling, apoptosis, and keratin expression in mouse uteri (23–25, 42). Increased expression of mRNAs of cell cycle regulators were reported after 17ß-estradiol treatment in the uteri of adult ovariectomized mice (42). In the present study, these cell cycle regulators except for cyclin G1 and E1, were induced in adult vaginae; thus vaginae responded to estrogen similar to uteri at the gene expression level.

From PND 5 onward, DES induced the following cell regulatory genes: p21, which delays S phase progression (43); Gadd45, which acts at the G2/M checkpoint; and 14–3–3 sigma, which inhibits activation of cyclin B (32, 43, 44). Thus, induction of these cell cycle regulators at the mitotic phase checkpoint 6 hrs after DES stimulation may play a role in DNA synthesis required for vaginal cell proliferation.

Induction of keratinocyte differentiation regulators, such as Sprr1a, Sprr2a, and keratin complexes, was reported in estrogen-exposed uteri (45). Sprr family genes are expressed in all squamous cells, such as epithelial cells of skin, vagina, and digestive tract (46, 47). Sprr2a, Sprr2b, and Sprr2f are expressed in uteri and vaginae (48). Sprr2f is expressed most intensely in uteri and vaginae (48). Sprr2f and Keratin complex 2 (K2) mRNAs were elevated in vaginae 6 hrs after DES exposure from PND 5 to PND 70. Genes related to epithelial cell differentiation responded to DES earlier than genes related to proliferation at PND 5. The increase in expression of Sprr2f and K2 genes at PND 20 and PND 70 is probably related to proliferation of vaginal epithelial cells. Sprr2a and Sprr2b may be correlated with keratinization in vaginal epithelial cells.

Klf4 is a transcription factor of Sprr2 (47, 49) and plays a role in keratinocyte differentiation (30). DES induced Klf4 expression in vaginae even at PND 0 in the present study. The inductions of Klf4 and Sprr2a expressions in DES-exposed uteri at PND 5 were reported previously (45). In vaginae, Klf4 is an early estrogen-responsive gene and a candidate for persistent vaginal stratification by perinatal DES exposure.

14–3–3 Sigma also regulates the cell cycle by inhibiting G2/M progression-dependent p53 (44) and is induced in squamous cell carcinoma of the urinary bladder (50). Expression of 14–3–3 sigma was found in DES-stimulated mouse vaginae at PND 5 in the present study and also in neonatally DES-exposed vaginae (17), suggesting that this gene will be a candidate for further study in the persistent vaginal stratification and keratinization induced by perinatal estrogen exposure.

In conclusion, vaginal epithelial cells and stromal cells showed proliferation after a single injection of DES at PNDs 20 and 70, and at PNDs 0 and 5, respectively. The number of genes induced 6 hrs after DES exposure in mouse vaginae at PND 0 was lower than those induced at PNDs 5, 20, and 70. The DES-induced gene expression pattern in vaginae at PND 5 was closer to the adult type. Several cell cycle regulators such as Gadd45, G1 to S phase transition 1, and p21; and keratinocyte differentiation factors, 14–3–3 sigma and Sprr2f, were induced by DES in vaginae from PND 5 to adult. Thus, microarray analysis revealed that the gene expression pattern in vaginae during the critical period was different from that after the critical period. Further studies are essential to examine the time course of gene expression to discover late genes induced in mouse vaginae by DES exposure at different ages.

	Acknowledgments

 
We are grateful to Dr. Raphael Guzman, Department of Molecular Cell Biology, University of California at Berkeley and Cancer Research Laboratory, for his critical reading of this manuscript.

	Footnotes

 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; by a Health Sciences Research grant from the Ministry of Health, Labour and Welfare; and by a research grant from the Ministry of Environment, Japan.

Received for publication January 12, 2006. Accepted for publication February 14, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Evans GS, Gibson DF, Roberts SA, Hind TM, Potten CS. Proliferative changes in the genital tissue of female mice during the oestrous cycle. Cell Tissue Kinet 23:619–635, 1990.[Medline]
2. Suzuki A, Enari M, Abe Y, Ohta Y, Iguchi T. Effect of ovariectomy on histological change and protein expression in female mouse reproductive tracts. In Vivo 10:103–110, 1996.[Medline]
3. Sato T, Fukazawa Y, Kojima H, Ohta Y, Iguchi T. Multiple mechanisms are involved in apoptotic cell death in the mouse uterus and vagina after ovariectomy. Reprod Toxicol 17:289–297, 2003.[Medline]
4. Takasugi N, Bern HA, Deome KB. Persistent vaginal cornification in mice. Science 138:438–439, 1962.[Abstract/Free Full Text]
5. Takasugi N, Bern HA. Tissue changes in mice with persistent vaginal cornification induced by early postnatal treatment with estrogen. J Natl Cancer Inst 33:855–865, 1964.[Medline]
6. Herbst AL, Ulfelder H, Poskanzer DC. Adenocarcinoma of the vagina. Association of maternal stilbestrol therapy with tumor appearance in young women. N Engl J Med 284:878–881, 1971.[Medline]
7. Forsberg JG. The development of atypical epithelium in the mouse uterine cervix and vaginal fornix after neonatal oestradiol treatment. Br J Exp Pathol 50:187–195, 1969.[Medline]
8. Dunn TB, Green AW. Cysts of the epididymis, cancer of the cervix, granular cell myoblastoma, and other lesions after estrogen injection in newborn mice. J Natl Cancer Inst 31:425–455, 1963.[Medline]
9. Newbold RR, McLachlan JA. Vaginal adenosis and adenocarcinoma in mice exposed prenatally or neonatally to diethylstilbestrol. Cancer Res 42:2003–2011, 1982.[Abstract/Free Full Text]
10. Iguchi T, Takasugi N, Bern HA, Mills KT. Frequent occurrence of polyovular follicles in ovaries of mice exposed neonatally to diethylstilbestrol. Teratology 34:29–35, 1986.[Medline]
11. Iguchi T. Cellular effects of early exposure to sex hormones and antihormones. Int Rev Cytol 139:1–57, 1992.[Medline]
12. Newbold RR, Bullock BC, McLachlan JA. Progressive proliferative changes in the oviduct of mice following developmental exposure to diethylstilbestrol. Teratog Carcinog Mutagen 5:473–480, 1985.[Medline]
13. Suzuki A, Sugihara A, Uchida K, Sato T, Ohta Y, Katsu Y, Watanabe H, Iguchi T. Developmental effects of perinatal exposure to bisphenol-A and diethylstilbestrol on reproductive organs in female mice. Reprod Toxicol 16:107–116, 2002.[Medline]
14. Ohta Y, Sato T, Iguchi T. Immunocytochemical localization of progesterone receptor in the reproductive tract of adult female rats. Biol Reprod 48:205–213, 1993.[Abstract]
15. Kamiya K, Sato T, Nishimura N, Goto Y, Kano K, Iguchi T. Expression of estrogen receptor and proto-oncogene messenger ribonucleic acids in reproductive tracts of neonatally diethylstilbestrol-exposed female mice with or without post-puberal estrogen administration. Exp Clin Endocrinol Diabetes 104:111–122, 1996.[Medline]
16. Miyagawa S, Suzuki A, Katsu Y, Kobayashi M, Goto M, Handa H, Watanabe H, Iguchi T. Persistent gene expression in mouse vagina exposed neonatally to diethylstilbestrol. J Mol Endocrinol 32:663–677, 2004.[Abstract]
17. Miyagawa S, Katsu Y, Watanabe H, Iguchi T. Estrogen-independent activation of erbBs signaling and estrogen receptor alpha in the mouse vagina exposed neonatally to diethylstilbestrol. Oncogene 23:340–349, 2004.[Medline]
18. Hom YK, Young P, Thomson AA, Cunha GR. Keratinocyte growth factor injected into female mouse neonates stimulates uterine and vaginal epithelial growth. Endocrinology 139:3772–3779, 1998.[Abstract/Free Full Text]
19. Hom YK, Young P, Wiesen JF, Miettinen PJ, Derynck R, Werb Z, Cunha GR. Uterine and vaginal organ growth requires epidermal growth factor receptor signaling from stroma. Endocrinology 139:913–921, 1998.[Abstract/Free Full Text]
20. Nelson KG, Takahashi T, Bossert NL, Walmer DK, McLachlan JA. Epidermal growth factor replaces estrogen in the stimulation of female genital-tract growth and differentiation. Proc Natl Acad Sci U S A 88: 21–25, 1991.[Abstract/Free Full Text]
21. Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS. Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci U S A 89:4658–4662, 1992.[Abstract/Free Full Text]
22. Iguchi T, Watanabe H, Katsu Y, Mizutani T, Miyagawa S, Suzuki A, Kohno S, Sone K, Kato H. Developmental toxicity of estrogenic chemicals on rodents and other species. Congenit Anom (Kyoto) 42: 94–105, 2002.[Medline]
23. Watanabe H, Suzuki A, Mizutani T, Khono S, Lubahn DB, Handa H, Iguchi T. Genome-wide analysis of changes in early gene expression induced by oestrogen. Genes Cells 7:497–507, 2002.[Abstract]
24. Watanabe H, Suzuki A, Kobayashi M, Takahashi E, Itamoto M, Lubahn DB, Handa H, Iguchi T. Analysis of temporal changes in the expression of estrogen-regulated genes in the uterus. J Mol Endocrinol 30:347–358, 2003.[Abstract]
25. Watanabe H, Suzuki A, Kobayashi M, Lubahn DB, Handa H, Iguchi T. Similarities and differences in uterine gene expression patterns caused by treatment with physiological and non-physiological estrogens. J Mol Endocrinol 31:487–497, 2003.[Abstract]
26. Watanabe H, Suzuki A, Goto M, Lubahn DB, Handa H, Iguchi T. Tissue-specific estrogenic and non-estrogenic effects of a xenoestrogen, nonylphenol. J Mol Endocrinol 33:243–252, 2004.[Abstract]
27. Shah JV, Cleveland DW. Waiting for anaphase: Mad2 and the spindle assembly checkpoint. Cell 103:997–1000, 2000.[Medline]
28. Guerin M, Sheng ZM, Andrieu N, Riou G. Strong association between c-myb and oestrogen-receptor expression in human breast cancer. Oncogene 5:131–135, 1990.[Medline]
29. Foster KW, Frost AR, McKie-Bell P, Lin CY, Engler JA, Grizzle WE, Ruppert JM. Increase of GKLF messenger RNA and protein expression during progression of breast cancer. Cancer Res 60:6488–6495, 2000.[Abstract/Free Full Text]
30. Chen X, Whitney EM, Gao SY, Yang VW. Transcriptional profiling of Kruppel-like factor 4 reveals a function in cell cycle regulation and epithelial differentiation. J Mol Biol 326:665–677, 2003.[Medline]
31. Ziegler DM. Recent studies on the structure and function of multisubstrate flavin-containing monooxygenases. Annu Rev Pharmacol Toxicol 33:179–199, 1993.[Medline]
32. Fan W, Richter G, Cereseto A, Beadling C, Smith KA. Cytokine response gene 6 induces p21 and regulates both cell growth and arrest. Oncogene 18:6573–6582, 1999.[Medline]
33. Wang XW, Zhan Q, Coursen JD, Khan MA, Kontny HU, Yu L, Hollander MC, O’Connor PM, Fornace AJ Jr, Harris CC. GADD45 induction of a G2/M cell cycle checkpoint. Proc Natl Acad Sci U S A 96:3706–3711, 1999.[Abstract/Free Full Text]
34. Masui F, Matsuda M, Mori T. Vitamin A prevents the irreversible proliferation of vaginal epithelium induced by neonatal injection of keratinocyte growth factor in mice. Cell Tissue Res 311:251–258, 2003.[Medline]
35. Sato T, Fukazawa Y, Ohta Y, Iguchi T. Involvement of growth factors in induction of persistent proliferation of vaginal epithelium of mice exposed neonatally to diethylstilbestrol. Reprod Toxicol 19:43–51, 2004.[Medline]
36. Nelson KG, Sakai Y, Eitzman B, Steed T, McLachlan J. Exposure to diethylstilbestrol during a critical developmental period of the mouse reproductive tract leads to persistent induction of two estrogen-regulated genes. Cell Growth Differ 5:595–606, 1994.[Abstract]
37. Yamashita S, Newbold RR, McLachlan JA, Korach KS. Developmental pattern of estrogen receptor expression in female mouse genital tracts. Endocrinology 125:2888–2896, 1989.[Abstract/Free Full Text]
38. Sato T, Okamura H, Ohta Y, Hayashi S, Takamatsu Y, Takasugi N, Iguchi T. Estrogen receptor expression in the genital tract of female mice treated neonatally with diethylstilbestrol. In Vivo 6:151–156, 1992.[Medline]
39. Bigsby RM, Li AX, Lou K, Cunha GR. Strain differences in the ontogeny of estrogen receptors in murine uterine epithelium. Endocrinology 126:2592–2596, 1990.[Abstract/Free Full Text]
40. Cunha GR, Cooke PS, Kurita T. Role of stromal-epithelial interactions in hormonal responses. Arch Histol Cytol 67:417–434, 2004.[Medline]
41. Iguchi T, Ohta Y, Takasugi N. Mitotic activity of vaginal epithelial cells following neonatal injections of different doses of estrogen in mice. Dev Growth Differ 18:69–78, 1976.
42. Hewitt SC, Deroo BJ, Hansen K, Collins J, Grissom S, Afshari CA, Korach KS. Estrogen receptor-dependent genomic responses in the uterus mirror the biphasic physiological response to estrogen. Mol Endocrinol 17:2070–2083, 2003.[Abstract/Free Full Text]
43. Sherr CJ. G1 phase progression: cycling on cue. Cell 79:551–555, 1994.[Medline]
44. Hermeking H, Lengauer C, Polyak K, He TC, Zhang L, Thiagalingam S, Kinzler KW, Vogelstein B. 14–3–3 sigma is a p53-regulated inhibitor of G2/M progression. Mol Cell 1:3–11, 1997.[Medline]
45. Huang WW, Yin Y, Bi Q, Chiang TC, Garner N, Vuoristo J, McLachlan JA, Ma L. Developmental diethylstilbestrol exposure alters genetic pathways of uterine cytodifferentiation. Mol Endocrinol 19: 669–682, 2005.[Abstract/Free Full Text]
46. Song HJ, Poy G, Darwiche N, Lichti U, Kuroki T, Steinert PM, Kartasova T. Mouse Sprr2 genes: a clustered family of genes showing differential expression in epithelial tissues. Genomics 55:28–42, 1999.[Medline]
47. Patel S, Kartasova T, Segre JA. Mouse Sprr locus: a tandem array of coordinately regulated genes. Mamm Genome 14:140–148, 2003.[Medline]
48. Tesfaigzi J, Carlson DM. Expression, regulation, and function of the SPR family of proteins. A review. Cell Biochem Biophys 30:243–265, 1999.
49. Segre JA, Bauer C, Fuchs E. Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet 22:356–360, 1999.[Medline]
50. Moreira JM, Gromov P, Celis JE. Expression of the tumor suppressor protein 14–3–3 sigma is down-regulated in invasive transitional cell carcinomas of the urinary bladder undergoing epithelial-to-mesenchymal transition. Mol Cell Proteomics 3:410–419, 2004.[Abstract/Free Full Text]

This article has been cited by other articles:

				K.L. Greathouse, J.D. Cook, K. Lin, B.J. Davis, T.D. Berry, T.G. Bredfeldt, and C.L. Walker Identification of Uterine Leiomyoma Genes Developmentally Reprogrammed by Neonatal Exposure to Diethylstilbestrol Reproductive Sciences, October 1, 2008; 15(8): 765 - 778. [Abstract] [PDF]

				A. Suzuki, H. Urushitani, T. Sato, T. Kobayashi, H. Watanabe, Y. Ohta, and T. Iguchi Gene Expression Change in the Mullerian Duct of the Mouse Fetus Exposed to Diethylstilbestrol In Utero Experimental Biology and Medicine, April 1, 2007; 232(4): 503 - 514. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suzuki, A. Articles by Iguchi, T. Search for Related Content PubMed PubMed Citation Articles by Suzuki, A. Articles by Iguchi, T.