Search

Background/aim: The mouse vagina exhibits stratified squamous epithelium, which is comprised of multiple cell layers. We previously showed that erbB signaling, induced by epithelial estrogen receptor 1 (ESR1), is required for the initial differentiation of the epithelium. However, the downstream effector that mediates terminal differentiation in the apical layers remains elusive. The contribution of fibroblast growth factor (FGF) to vaginal epithelial cell differentiation was investigated., Materials and Methods: Vaginas from wild-type or epithelium-specific Esr1 conditional knockout (cKO) mice were analyzed using immunohistochemistry and quantitative real-time RT-PCR., Results: Of the FGF ligands examined, Fgf22 mRNA was significantly induced following estrogen treatment. Furthermore, FGF downstream signaling, phosphorylated FRS2 and ERK1/2 were exclusively expressed in the apical layers of the vaginal epithelium. No changes in such expression were observed in the Esr1 cKO mice., Conclusion: FGF-ERK/MAPK pathway may be a main inducer of terminal differentiation in the mouse vaginal epithelium., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Estrogen-mediated actions in female reproductive organs are tightly regulated, mainly through estrogen receptor 1 (ESR1). The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen and provides a unique model for analyzing the homeostasis of stratified squamous epithelia. To address the role of ESR1-mediated tissue events during homeostasis, we analyzed mice with a vaginal epithelium-specific knockout of Esr1 driven by keratin 5-Cre (K5-Esr1KO). We show here that loss of epithelial ESR1 in the vagina resulted in aberrant epithelial cell proliferation in the suprabasal cell layers and led to failure of keratinized differentiation. Gene expression analysis showed that several known estrogen target genes, including erbB growth factor ligands, were not induced by estrogen in the K5-Esr1KO mouse vagina. Organ culture experiments revealed that the addition of erbB growth factor ligands, such as amphiregulin, could activate keratinized differentiation in the absence of epithelial ESR1. Thus, epithelial ESR1 integrates estrogen and growth factor signaling to mediate regulation of cell proliferation in squamous differentiation, and our results provide new insights into estrogen-mediated homeostasis in female reproductive organs.

Estrogen receptor α (ERα) plays a pivotal role in the mouse uterine and vaginal epithelial cell proliferation stimulated by estrogen, whereas ERβ inhibits cell proliferation. ERβ mRNA is expressed in neonatal uteri and vaginae; however, its functions in neonatal tissues have not been ascertained. In this study, we investigated the ontogenic mRNA expression and localization of ERβ, and its roles in cell proliferation in neonatal uteri and vaginae of ERβ knockout (βERKO) mice. ERβ mRNA and protein were abundant in the uterine and vaginal epithelia of 2-day-old mice and decreased with age. In uterine and vaginal epithelia of 2-day-old βERKO mice, cell proliferation was greater than that in wild-type animals and in uterine epithelia of 90- and 365-day-old βERKO mice. In addition, p27 protein, known as a cyclin-dependent kinase inhibitor, was decreased in the uteri of 90- and 365-day-old βERKO mice. Inhibition of neonatal ERs by ICI 182780 (an ER antagonist) treatment stimulated cell proliferation and decreased p27 protein in the uterine luminal epithelium of 90-day-old mice but not in the vaginal epithelium. These results suggest that neonatal ERβ is important in the persistent inhibition of epithelial cell proliferation with accumulation of p27 protein in the mouse uterus. Thus, suppression of ERβ function in the uterine epithelium during the neonatal period may be responsible for a risk for proliferative disease in adults.

Numerous studies support a role of phosphatase and tensin homolog deleted from chromosome 10 (Pten) as a tumor suppressor gene that controls epithelial cell homeostasis to prevent tumor formation. Mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen and provides a unique model for analyzing homeostasis of stratified squamous epithelia. We analyzed vaginal epithelium-specific Pten conditional knockout (CKO) mice to provide new insights into Pten/phosphoinositide-3-kinase (PI3K)/Akt function. The vaginal epithelium of ovariectomized (OVX) mice (control) was composed of 1-2 layers of cuboidal cells, whereas OVX CKO mice exhibited epithelial hyperplasia in the suprabasal cells with increased cell mass and mucin production. This is possibly due to misactivation of mammalian target of rapamycin and mitogen-activated protein kinase. Intriguingly, estrogen administration to OVX Pten CKO mice induced stratification and keratinized differentiation in the vaginal epithelium, as in estrogen-treated controls. We found that Pten is exclusively expressed in the suprabasal cells in the absence of estrogens, whereas estrogen administration induced Pten expression in the basal cells. This suggests that Pten acts to prevent excessive cell proliferation as in the case of other squamous tissues. Thus, Pten exhibits a dual role on the control of vaginal homeostasis, depending on whether estrogens are present or absent. Our results provide new insights into how Pten functions in tissue homeostasis.

Background: In neonate mice exposed to diethylstilbestrol (neoDES), vaginal epithelium shows persistent proliferation and stratification even after ovariectomy. Tissue recombination studies suggest that neonatally-estrogenized vaginal stroma can induce vaginal epithelial hyperplasia depending on the stromal age. This study examined the proliferative effect of the vaginal stroma from 8-day-old mice treated with DES on the vaginal epithelia of 8-day-old and adult mice., Materials and Methods: Vaginal epithelium and stroma from 8-day-old and adult mice was recombined, and grafted to ovariectomized host mice., Results: The 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in the epithelium and the number of epithelial cell layers were not significantly different between epithelia from 8-day-old and adult mice when combined with stroma from 8-day-old control mice. BrdU-labeled cells in the vaginal epithelia from both age groups combined with the stroma from 8-day-old neoDES mice exhibited higher values. The epithelium from neoDES adult mice had a lower percentage of BrdU-labeled cells., Conclusion: The stroma from 8-day-old neoDES mice induces epithelial cell proliferation, but lower stromal cell proliferation.

Female reproductive organs show organ-specific morphological changes during estrous cycles. Perinatal exposure to natural and synthetic estrogens including diethylstilbestrol (DES) or estrogenic chemicals induces estrogen-independent persistent proliferation of vaginal epithelium in mice. To understand the underlying mechanism of the estrogen-independent persistent vaginal changes induced by perinatal DES exposure, we examined global gene expressions in the vaginae of ovariectomized adult mice exposed neonatally to DES using a microarray. The cell cycle-related gene, p21, a cyclin-dependent kinase inhibitor, showed upregulation in the vagina, and p21 protein was localized in the basal layer of the vaginal epithelium in mice exposed neonatally to DES and an estrogen receptor α agonist, propyl pyrazole triol (PPT). The expressions of Notch receptors and Notch ligands were unchanged; however, the mRNAs of hairy-related basic helix-loop-helix (bHLH) transcription factor genes, Hes1, Hey1 and Heyl were persistently downregulated in the vagina, indicating estrogen-independent epithelial cell proliferation in mice exposed neonatally to DES and PPT.

Estrogen regulates morphological changes in reproductive organs, such as the vagina and uterus, during the estrous cycles in mice. Estrogen depletion by ovariectomy in adults results in atrophy accompanied by apoptosis in vaginal and uterine cells, while estrogen treatment following ovariectomy elicits cell proliferation in both organs. Sequential changes in mRNA expression of wingless-related MMTV integration site (Wnt) and Notch signaling genes were analyzed in the vagina and uterus of ovariectomized adult mice after a single injection of 17β-estradiol to provide understanding over the molecular basis of differences in response to estrogen in these organs. We found estrogen-dependent up-regulation of Wnt4, Wnt5a and p21 and down-regulation of Wnt11, hairy/enhancer-of-split related with YRPW motif-1 (Hey1) and delta-like 4 (Dll4) in the vagina, and up-regulation of Wnt4, Wnt5a, Hey1, Heyl, Dll1, p21 and p53 and down-regulation of Wnt11, Hey2 and Dll4 in the uterus. The expression of Wnt4, Hey1, Hey2, Heyl, Dll1 and p53 showed different patterns after the estrogen injection. Expression patterns for Wnt5a, Wnt11, Dll4 and p21 in the vagina and uterus were similar, suggesting that these genes are involved in the proliferation of cells in both those organs in mice.

Proliferation and differentiation of cells in female reproductive organs, the oviduct, uterus and vagina, are regulated by endogenous estrogen. In utero exposure to a synthetic estrogen, diethylstilbestrol (DES), induces vaginal clear-cell adenocarcinoma in humans. In mice, perinatal exposure to DES results in abnormalities such as polyovular follicles, uterine circular muscle disorganization and persistent vaginal epithelial cell proliferation. We reported the persistent gene expression change such as interleukin-1 (IL-1) related genes, insulin-like growth factor-I (IGF-I) and its downstream signaling in the mouse vagina exposed neonatally to DES. In this study, we found persistent up-regulation of Wnt4 and persistent down-regulation of Wnt11 in the vagina of mice exposed neonatally to DES and estrogen receptor α specific ligand. Also Wnt4 expression in vagina is correlated to the stratification of epithelial cells with the superficial keratinization of vagina, but not epithelial cell stratification only., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Both the uterus and vagina develop from the Müllerian duct but are quite distinct in morphology and function. To investigate factors controlling epithelial differentiation and cell proliferation in neonatal uterus and vagina, we focused on Hedgehog (HH) signaling. In neonatal mice, Sonic hh (Shh) was localized in the vaginal epithelium and Indian hh (Ihh) was slightly expressed in the uterus and vagina, whereas all Glioma-associated oncogene homolog (Gli) genes were mainly expressed in the stroma. The expression of target genes of HH signaling was high in the neonatal vagina and in the uterus, it increased with growth. Thus, in neonatal mice, Shh in the vaginal epithelium and Ihh in the uterus and vagina activated HH signaling in the stroma. Tissue recombinants showed that vaginal Shh expression was inhibited by the vaginal stroma and uterine Ihh expression was stimulated by the uterine stroma. Addition of a HH signaling inhibitor decreased epithelial cell proliferation in organ-cultured uterus and vagina and increased stromal cell proliferation in organ-cultured uterus. However, it did not affect epithelial differentiation or the expression of growth factors in organ-cultured uterus and vagina. Thus, activated HH signaling stimulates epithelial cell proliferation in neonatal uterus and vagina but inhibits stromal cell proliferation in neonatal uterus.

The uterus and upper 3/5 of the vagina originate from the Müllerian duct; however, these organs show quite distinct characteristics in morphology and function. To investigate factors controlling vaginal epithelial cell differentiation from a single layer of pseudostratified epithelium to a multi-layered stratified epithelium with keratin, we focused on fibroblast growth factors (Fgfs). Transformation related protein 63 (Trp63) expression, a marker of stratified epithelium, increased in the Müllerian vaginal epithelial cells from days 0 to 5, and keratin 14 (Krt14) was expressed from day 5, suggesting that Trp63-negative vaginal epithelial cells can differentiate into Trp63-positive cells after birth. Fgf7 and Fgf10 were localized in the vaginal stroma but their receptor, Fgf receptor 2IIIb (Fgfr2IIIb), was localized in the vaginal epithelium. Both Fgf9 and its receptor, Fgfr2IIIc, were localized in the vaginal epithelium. Vaginae cultured with FGF10 or anti-FGF9 antibody showed stratified epithelium with an intense Krt14 expression; however, an inhibitor of phosphorylation of mitogen-activated protein kinase 1/3 (MAPK1/3) canceled the effect of FGF10 and anti-FGF9 antibody. Thus, Fgf10 stimulates the differentiation of pseudostratified epithelial cells into stratified cells via MAPK1/3 pathway, and Fgf9 inhibits this differentiation in the neonatal mouse vagina., (Copyright © 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Perinatal exposure to a synthetic estrogen, diethylstilbestrol (DES), causes cervicovaginal adenosis and permanent hyperplastic cornified vaginal epithelium with keratinization in mice. To investigate the mechanisms of the induction of vaginal abnormalities by DES, we have focused on activin A signaling. We have found that the βA-subunit mRNA is mainly expressed in the neonatal vaginal stroma, whereas activin A receptor type IB is localized in the neonatal vaginal epithelium. SMAD2, the intracellular signaling protein, is phosphorylated in the neonatal vagina. Cell proliferation in the vaginal epithelium grown in vitro is reduced by DES treatment or by activin signaling suppression through inhibin treatment. Thus, activin A (a homodimer of the βA-subunit) in the stroma stimulates epithelial cell proliferation in the neonatal vagina. DES treatment decreases the expression of the βA-subunit and activin receptor IIB but increases the expression of the βB-subunit and inhibin receptor. Neonatal DES treatment inhibits the phosphorylation of SMAD2 in the vaginal epithelium, indicating the inhibition of activin A signaling in the vaginal epithelium by neonatal DES treatment. Treatment with DES or inhibin, a native antagonist of activin, induces adenosis-like structures and keratinization in the vagina grown in vitro. These data suggest that the suppression of activin A signaling by DES is involved in the induction of cervicovaginal adenosis and keratinization in the neonatal mouse vaginal epithelium.

Development of the reproductive organs can be strongly affected by the hormonal environment. In the mouse, exposure to estrogens and androgens during the critical developmental period induces estrogen-independent cell proliferation and differentiation in the adult vaginal epithelium, which often results in cancerous lesions later in life. In the present study, we assessed the contributions of estrogen receptor 1 (alpha) (ESR1) to the developmental effects of the nonaromatizable androgen 5alpha-dihydrotestosterone (DHT) on female mouse vagina and external genitalia. The vagina of Esr1(-/-) mice treated neonatally with DHT showed atrophic epithelium, whereas the vaginal epithelium of Esr1(+/+) mice was stratified and keratinized even after ovariectomy. In addition, neonatal treatment with DHT led to persistent phosphorylation of ESR1 in the vaginae of 60-day-old ovariectomized mice. We infer from these data that ESR1 is obligatory for the induction and maintenance of persistent vaginal epithelial changes induced by neonatal administration of DHT. Neonatal DHT treatment also induced hypospadias in both Esr1(-/-) and Esr1(+/+) mice. In contrast, DHT-induced formation of an os penis-like large bone in the clitoris was found in Esr1(-/-) mice but not in Esr1(+/-) or Esr1(+/+) mice. These results shed light on mechanisms of the induction of developmental effects elicited by sex steroid hormones on the developing animals.

Female reproductive organs are mainly regulated by estrogen and progesterone. Specifically, the uterus, vagina and mammary gland show organ-specific mitosis and morphological changes during proliferative events, such as estrous cycle, gestation and lactation. The mechanism underlying these organ-specific estrogen-dependent events is still unknown. We examined, therefore, global gene expression in the mature uterus, vagina and mammary gland of ovariectomized adult mice 6 hr after an injection of 5 microg/kg 17beta-estradiol (E2) using a microarray method in order to identify primary E2-responsive genes. Half of the E2 up-regulated genes in the uterus were similar to those in the vagina. E2 up-regulated the expression of Insulin-like growth factor 1 (Igf-1) genes in the uterus and vagina. In the vagina, E2 up-regulated the expression of IGF binding proteins (Igfbp2 and Igfbp5). In the mammary gland, unlike the uterus and vagina, no gene showed altered expression 6 hr after the E2 exposure. These results suggest that expression of Igf-1 and morphogenesis genes is regulated by E2 in an organ-specific manner, and it is supported by the results of BrdU labeling showing E2-induced mitosis in the uterus and vagina except the mammary gland. The differences in organ specificity in response to E2 may be attributed by differences in gene expression regulated by E2 in female reproductive organs. The candidate estrogen-responsive genes in the uterus and vagina identified by profiling provide an important foundation understanding functional mechanisms of estrogen regulating morphogenesis and maintenance of each reproductive organ.

Estrogens regulate the proliferation and differentiation of mouse vaginal epithelial cells. We examined the temporal and spatial expression of DDV10, a novel C-type lectin during stratification and cornification of the vaginal epithelium. DDV10 was expressed in vagina but not uterus in ovariectomized mice treated with 17beta-estradiol (E2). In mouse stomach, the expression of DDV10 was detected in pars proventricularis but not in pars glandularis. Furthermore, the DDV10 gene was found to possess two transcripts, a long form (DDV10) and a short form (OCILrP1, osteoclast inhibitory lectin-related protein 1). DDV10 mRNA but not OCILrP1 mRNA was expressed in the stratified and cornified epithelial tissues. DDV10 mRNA was first detected between 12 and 18 h after E2 treatment in the vaginal epithelium, and was detected in the vagina of the neonatally diethylstilbestrol (DES)-treated mouse. Recently, a unified name was registered in GenBank (C-type lectin domain family 2, member g; Clec2 g). Taken together, these data suggest that DDV10 is the long form of Clec2 g (Clec2g-L), and DDV10/Clec2g-L may play a role in the stratification and/or cornification of epithelial cells during differentiation.

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 mug 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 Gadd45alpha, 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.

Effects of 17beta-estradiol (E2) on uterine and vaginal epithelial cell proliferation could be mediated by stromal cell-derived paracrine factors. To study the epithelial-stromal interactions in mice, an in vitro model of uterine and vaginal stromal cells of immature mice is essential. Therefore, we established a primary culture model of stromal cells both from uterus and vagina and examined the effect of E2 on proliferation of cultured stromal cells. We found that E2 stimulated proliferation of stromal cells from both organs in vitro, showing an increase in the number of cells and the percentage of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells. Interestingly, vaginal stromal cells responded to lower E2 than uterine stromal cells in proliferation (10(-12) M vs. 10(-8) M) and BrdU labeling (10(-14) - 10(-10) M vs. 10(-10) - 10(-6) M). To examine the effect of E2 in vivo, cells were grafted into the subrenal capsule of the host mice and grown for 2 weeks. The BrdU labeling in cultured stromal cells was increased by E2 in vivo. To examine the effect of cultured stromal cells on epithelial cell proliferation, uterine and vaginal epithelium of adult mice were separated, recombined with the cultured stromal cells, and grafted under the renal capsule of hosts for 3 weeks. Epithelial cells recombined with cultured stromal cells showed simple columnar morphology in uterine grafts and stratified and keratinized morphology in vaginal grafts under the influence of the hormonal environment of the hosts. The BrdU labeling in epithelial cells was increased by E2, suggesting that cultured stromal cells can stimulate epithelial cell proliferation. In conclusion, we established a primary culture model of uterine and vaginal stromal cells, which can be mitogenically stimulated by E2 in vitro and in vivo after being grafted under the renal capsule. This culture system will be useful for investigating the underlying molecular mechanisms of uterine and vaginal epithelial-stromal interactions.

Neonatal treatment of female mice with natural and synthetic estrogens including diethylstilbestrol (DES) results in persistent proliferation and cornification of vaginal epithelium. In order to study the mechanism of persistent proliferation of vaginal epithelium, histological and biochemical changes were examined in the vagina of C57BL female mice exposed neonatally to 3 microg DES for 5 days. In intact control adult mice, ovariectomy induced apoptotic cell death in vaginal epithelial cells detected by in situ 3'-DNA nick end labeling method accompanied by low DNA synthesis detected by incorporation of bromodeoxyuridine. In neonatally DES-exposed adult mice, however, ovariectomy did not induce reduction of DNA synthesis and showed only a slight increase in apoptotic cells of vaginal epithelium. In neonatally DES-exposed mouse vagina, semi-quantitative reverse transcription polymerase chain reaction revealed a continuous higher expression of mRNAs encoding epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). These results indicate that neonatal DES exposure causes the increase in expression of EGF and TGF-alpha mRNA, possibly resulting in the induction of persistent proliferation and cornification of vaginal epithelium in mice.

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.

Growth factors and estrogen receptor (ER) signaling cooperate to play essential roles in cell proliferation, differentiation and tumor progression in mouse reproductive organs. Treatment of neonatal mice with diethylstilbestrol (DES) induces an estrogen-independent persistent proliferation and cornification of the vaginal epithelium, which results in cancerous lesions later in life. However, the mechanisms of the estrogen-dependent and -independent pathways essentially remain unknown. We characterized the expression of epidermal growth factor (EGF)-like growth factors (EGF, transforming growth factor alpha (TGF-alpha), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC), amphiregulin (APR), epiregulin (EPR) and neuregulin (NRG) 1) and erbB receptors (EGF receptor (EGFR), erbB2/neu, erbB3 and erbB4) in the vaginae of mice treated either neonatally (0-4 day) or as adults (55-59 day) with estrogens. EGFR and erbB2 were activated in the vaginal epithelium of mice by estrogen treatment. This activation was also encountered in vaginae from neonatally DES-exposed mice, along with the expression of EGF, TGF-alpha, HB-EGF, BTC, APR, EPR and NRG1. Immunohistochemical analysis indicated that erbB2 was primarily expressed in vaginal epithelium. Finally, we found that serine 118 and 167 located in the AF-1 domain of ERalpha were phosphorylated in these vaginae. AG825, AG1478 or ICI 182,780 administration blocked proliferation of vaginal epithelium induced by neonatal DES exposure. Thus, signal transduction via EGFR and erbB2 could be related to the estrogen-induced vaginal changes and persistent erbBs phosphorylation and sustained expression of EGF-like growth factors, leading to ERalpha activation that may result in cancerous lesions in vaginae from neonatally DES-exposed mice later in life.

Estrogens regulate the proliferation and differentiation of mouse vaginal epithelial cells. However, the molecular mechanisms underlying estrogen-induced changes have not been elucidated. The goal of this study was to identify estrogen-responsive genes related to the proliferation and differentiation of mouse vaginal epithelial cells. We used differential display to reveal specific genes regulated by estrogens and identified a transcript that was designated DDV10. DDV10 encodes a membrane protein with a C-type lectin domain in the carboxyl-terminal region; thus, we inferred that it belongs to the C-type lectin family. We analyzed the temporal and spatial expression of DDV10 using RT-PCR, quantitative real-time RT-PCR, and in situ hybridization. Ovariectomy decreased DDV10 mRNA levels, whereas 17beta-estradiol treatment increased expression of DDV10 mRNA in vaginas of ovariectomized mice. DDV10 mRNA was first detected between 20 and 30 d after birth and was found in eye, tongue, stomach, and stratified and cornified vaginal epithelial cells, but not in stromal cells or uterus. DDV10 transcripts were not detected in vaginas of estrogen receptor alpha knockout mice. Taken together, these data suggest that DDV10 encodes a novel, 17beta-estradiol-regulated, C-type lectin in the mouse vagina. DDV10 may play a role in the stratification and/or cornification of epithelial cells during differentiation.

Withdrawal of sex hormones by gonadectomy results in rapid involution of mouse reproductive organs. To study the regression mechanism in the uterus and vagina after ovariectomy, histologic and biochemical changes were examined. Apoptotic cells were detected by in situ 3'-DNA nick end labeling method and electron microscopy, while the number of cells showing incorporation of bromo-deoxyuridine (BrdU) decreased in the uterus and vagina after ovariectomy. DNA fragmentation in the uterus was observed even at estrus and the degree of fragmentation increased after ovariectomy. DNA fragmentation in the vagina occurred 1-5 days after ovariectomy. Semi-quantitative RT-PCR revealed that expression of Fas-ligand and tumor necrosis factor-alpha (TNF-alpha) mRNA in the uterus and vagina was increased by ovariectomy. These results suggest that apoptotic cell death is induced by ovariectomy through the mediation of both Fas and TNF-alpha in the mouse uterus and vagina; however, uterine and vaginal cells in CBA lpr(cg)/lpr(cg) mice lacking functional Fas showed apoptosis, indicating that Fas is not the sole regulator of apoptosis in female reproductive organs in mice.

Perinatal treatment of female mice with natural or synthetic estrogens including diethylstilbestrol (DES) results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium. However, the molecular mechanisms of the estrogen-independent changes have not been elucidated. To analyze the mechanism of estrogen-independent cell proliferation and cornification of the vaginal epithelium, we used differential display and determined specific genes expressed in neonatally DES-treated vagina. A candidate clone that designated DDV5 was identical to the serine protease, neuropsin that is reportedly expressed in the mouse central nervous system. We then analyzed the expression pattern of DDV5/neuropsin using Northern blot analysis. We found: (1). DDV5/neuropsin mRNA is expressed in vaginae from neonatally DES-treated ovariectomized mice but not in vaginae from ovariectomized control mice, (2). its expression is not detected in uteri from neonatally DES-treated mice, (3). DDV5/neuropsin is expressed in vaginae from normal intact mice during estrus. Furthermore, we found that DDV5/neuropsin mRNA rapidly decreased in vaginae after ovariectomy. DDV5/neuropsin was detected in vaginae from ovariectomized mice 48 h after estrogen treatment. These results suggest that DDV5/neuropsin is expressed in estrogen-stimulated mouse vagina, and its gene expression is regulated by estrogen. Neonatal DES exposure affects transcriptional control of DDV5/neuropsin in the mouse vagina, which results in persistent expression of DDV5/neuropsin even after ovariectomy, thus, DDV5/neuropsin may play a role in estrogen-independent persistent proliferation and cornification of the vaginal epithelium. Using in situ hybridization method, we found DDV5/neuropsin mRNA localized in epithelial cells but not stromal cells in vaginae. This is the first report on the gene expression of a serine-protease neuropsin in the mouse vagina, and as a marker of the estrogen-independent persistent proliferation and cornification of the vaginal epithelium.

Background: Rodent uterus and vagina show marked histological changes during the estrous cycle. Apoptotic cell death has been demonstrated in hamster and rat uterine epithelium during the estrous cycle by electron microscopy: numerous epithelial cells undergo apoptosis at estrus. We examined cell death and cell proliferation in rat uterus and vagina during estrous cycle., Methods: To examine the rate of proliferation in uterine and vaginal cells at each estrous stage, the numbers of cells at metaphase were counted separately in epithelial and stromal cells. We identified the apoptotic cells in uterus and vagina at each estrous stage by using DNA fragmentation, in situ DNA 3'-end labeling, and electron microscopy., Results: Mitotic rates in uterine luminal and glandular epithelial cells were low at metestrus and estrus, respectively. Intense fragmentation was found in the uterus at metestrus and in the vagina at proestrus and metestrus. In uterine luminal and glandular epithelial cells, apoptotic index showed peaks at metestrus and estrus, respectively. In vaginal epithelial cells, many apoptotic cells were encountered in the superficial layer at proestrus, which may contribute to keratinization. In the middle and basal layer of vaginal epithelial cells, apoptotic index was high at metestrus, when mitotic rate was low. Electron microscopy confirmed the results of the labeling studies., Conclusions: Apoptotic cell death was encountered in the uterus and vagina during estrous cycle in rats. There is an inverse correlation between cell death and cell proliferation in rat uterine and vaginal epithelial cells during the estrous cycle.

Neonatal exposure to a synthetic estrogen, diethylstilbestrol (DES), induces the ovary-independent persistent proliferation of vaginal epithelium in mice. Mouse vagina at the estrous stage in the normal estrous cycle shows 10 to 15 layers of epithelium with superficial keratinized layers, and ovariectomy induces a decrease of these epithelial cell layers and lymphocyte infiltration. Thus, cell proliferation and regression of vaginal epithelium are ovary dependent. Neonatally DES-treated mouse vagina showed the same phenotype as normal mouse vagina at the estrous stage, but ovariectomy did not induce a decrease of epithelial cell layers or a lymphocyte infiltration, and there was persistent proliferation of vaginal epithelium even after ovariectomy. In addition, apoptotic cell death characterized by oligonucleosomal DNA fragmentation, Fas expression, and Bcl-2 downregulation were induced after ovariectomy in normal mouse vagina, but not in DES-treated mouse vagina. These results suggest that neonatal DES-exposure in mice prevents vaginal Fas-mediated apoptosis following the downregulation of Bcl-2, and these abnormalities in expression are involved in persistent proliferation of the vaginal epithelium.

Normal mouse vaginae and uteri regress following ovariectomy, whereas the vagina of mice given five daily injections of 3 micrograms diethylstilbestrol (DES) from the day of birth exhibit ovary (estrogen)-independent persistent stratification and cornification of the epithelium. Zymography indicated expression of four proteinases in both vaginae and uteri of normal mice after ovariectomy. Two proteinases detected in gelatin-containing gel and two others in casein-containing gel proved to be metalloproteinases and serineproteinases, respectively. The two metalloproteinases were identified as gelatinases A and B. Only gelatinase B was intensified 1 d after ovariectomy; however, all four proteinases showed an increase in expression 3 d after ovariectomy. In the uterus, the two gelatinases showed increased expression after ovariectomy. Progelatinase B and serineproteinase II were expressed in the vagina of normal mice at estrus; ovariectomy intensified expression and activation of gelatinases and serineproteinases II in the vagina. Vaginae of mice treated neonatally with DES exhibited a weak expression of proteinases. Ovariectomy changed neither the histology nor the expression of proteinases in these DES-exposed vaginae. Expression of gelatinases was inhibited by estrogen; progesterone stimulated expression and activation of gelatinase B. Serineproteinases found in the vagina and uterus of ovariectomized mice were also inhibited by estrogen but neither was affected by progesterone. These results suggest that gelatinase B and both gelatinases participate in vaginal and uterine regression, respectively, following ovariectomy. Estrogen negatively regulates expression of gelatinases and serineproteinases in the vagina, and of gelatinase A and serineproteinase II in the uterus.

Perinatal treatment of female mice with natural and synthetic estrogens including diethylstilbestrol (DES) results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium. The dynamics of the induction of estrogen receptor (ER), c-jun, c-fos and c-myc mRNAs by 17 beta-estradiol (E2) was examined in the uterus and vagina of neonatally DES-exposed and -unexposed ovariectomized adult mice. In the uterus of neonatally DES-unexposed ovariectomized mice, the expression of ER mRNA increased within 1 h after E2 administration and declined by 12 h thereafter. ER mRNA in the vagina decreased within 1 h after the stimulation and recovered by 12 h thereafter. In the uterus, c-jun and c-fos mRNAs increased in concentration within 1 h after E2 administration, showing a peak 3 h after the stimulation; they decreased with time thereafter. In the vagina, the concentration of c-jun and c-fos mRNAs increased rapidly, reaching a peak within 1 h after the stimulation. However, the expression of c-myc in uterus and vagina was not changed by postpuberal E2. These results suggest that estrogen regulation of ER and proto-oncogene mRNAs in the vagina differs from those in the uterus. In the neonatally DES-exposed ovariectomized adult mice, uterine ER mRNA expression levels were significantly higher than in the unexposed ovariectomized controls; however, vaginal levels were drastically lower than in the controls. Expression of c-jun and c-fos mRNAs was greater in both the uterus (3- and 6-fold, respectively) and the vagina (18- and 4-fold) of neonatally DES-exposed mice than in controls. The ER mRNA and the increased levels of c-fos and c-jun mRNAs in both uterus and vagina of neonatally DES-exposed ovariectomized mice were not further altered by post-puberal E2 and may be related to ovary-independent persistent changes in the genital tract.

Proliferation of the vagina and uterus in rodents is stimulated by ovarian estrogen, and ovariectomy induces regression of these tracts. In mouse, reproductive tracts, organ weights, vaginal epithelial thickness, the number of epithelial cell layers, and uterine epithelial cell height were significantly reduced 1 to 20 days after ovariectomy when compared with those of intact, mice taken at the estrous stage. No significant differences in those parameters were found between ovariectomized mice implanted with estradiol and intact estrous mice. Vaginal epithelium at estrus was composed of 10-15 layers of epithelial cells with superficial keratinized layers, however, the vaginal epithelium began to regress one day after ovariectomy. Infiltration of leukocytes was encountered in the vaginal epithelium 2-3 days after ovariectomy. Protein expression in the regressing vagina and uterus after ovariectomy was examined by two-dimensional polyacrylamide gel electrophoresis. After ovariectomy, new proteins appeared in the regressing vagina, but not in the regressing uterus. Expressions of 7 and 6 proteins in the vagina and uterus, respectively, reached peak levels 2-3 days after ovariectomy. The expression of ovariectomy-specific proteins and infiltration of leukocytes in vaginal epithelium was inhibited by the injection of cycloheximide, an inhibitor of protein synthesis. These results suggest that some proteins participate in the regression of mouse reproductive tracts after estrogen withdrawal induced by ovariectomy.

Neonatal treatment of female mice with diethystilbestrol (DES) is known to induce ovary-independent persistent proliferation and cornification of vaginal epithelium. This irreversibly changed vaginal epithelium persistently expressed higher levels of c-jun and c-fos mRNAs, which was not altered by postpubertal estrogen. Sexual dimorphism was encountered in mouse pelvis and anococcygeus muscle. Postpubertal estrogen changed the shape of the pelvis to the female type and postpubertal androgen changed it to the male type. Neonatal exposure to DES and to the antiestrogen tamoxifen altered the developmental pattern of the pelvis, which contained lower concentrations of calcium and phosphorus than controls. The size of anococcygeus muscle was increased by postpubertal androgen but decreased by postpubertal estrogen. However, neonatal estrogen (DES) exposure permanently enlarged the anococcygeus muscle. Thus, neonatal treatment of mice with estrogen and antiestrogen results in irreversible changes in nonreproductive as well as reproductive structures.

Neonatal treatment of female mice with a synthetic estrogen, diethylstilbestrol (DES), results in adenosis, ovary-independent epithelial proliferation and cornification, and downgrowths in the vagina. Protein synthesis was examined in the vagina of 45-day-old, ovariectomized C57BL/Tw mice which had been given 5 daily injections of 2 micrograms DES or the oil vehicle alone from the day of birth, and in those of age-matched, ovariectomized mice given 3 daily injections of 0.1 microgram DES from 42 days of age. [35S]Methionine-labeled proteins of the vagina were analyzed by two-dimensional polyacrylamide gel electrophoresis. Major changes were observed in 11 proteins of the vagina in both neonatally and postnatally DES-exposed groups. Six of the 11 proteins were increased in expression, but one of the remaining proteins was decreased. In the group of neonatally DES-exposed mice alone, expressions of 4 proteins (MW. 128, 90, 46 and 44 kDa) were markedly increased. These results indicate that neonatal exposure of mice to DES induced ovary-independent, persistent alteration in the protein synthesis of the vagina.

The distribution of the progesterone receptor (PR) was investigated immunocytochemically in female reproductive tracts of rats during the estrous cycle and early pregnancy through use of an anti-PR monoclonal antibody. PR was localized predominantly in the nuclei of epithelial, stromal, and muscle cells in the uterus and vagina during the estrous cycle. In the uterus, the nuclei of epithelial cells were stained intensively at diestrus, while the PR staining of the stromal cells was more intense at proestrus than at any other stage of the cycle. PR expression during the cycle in muscle cells of the myometrium was similar to that in the endometrial stromal cells. In the vagina, however, PR expression during the cycle was approximately the same among epithelial, stromal, and muscle cells, the nuclei of which were stained deeply at proestrus. Ovariectomy at various stages of the cycle altered the PR expression appearing in the uterus and vagina during the cycle. In ovariectomized rats, estrogen increased the PR immunoreaction of various types of cells examined in the uterus and vagina except for the uterine epithelial cells. The reaction of these uterine epithelial cells was decreased by estrogen but was increased by progesterone given after estrogen; however, progesterone given alone reduced the reaction. In the epithelial and stromal cells of the uterus, intensity of the staining was increased after mating, reaching maximum on Day 3 of pregnancy, and then decreased on Day 4 (day of implantation), while in epithelial and stromal cells of the vagina the staining remained weak during early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Postpubertal progesterone injections slightly inhibited the proliferation and cornification of the vaginal epithelium induced by neonatal injections of diethylstilbestrol (DES). In vaginae of neonatally DES-exposed mice (DES mice), 9 protein expressions (PEX) appeared newly; 13 PEX decreased; 5 PEX increased compared to those in the controls. In vaginae of DES mice, PEX were altered by postpubertal injections of progesterone (DES-P); 3 PEX disappeared; 11 PEX were reversed to those in the controls. Progesterone injections impaired the proliferation of the vaginal epithelium, reversing 11 PEX compared to those in DES mice. In uteri of DES-P mice, 3 PEX increased, 2 PEX decreased, 2 PEX appeared and 1 PEX disappeared compared to those in DES mice. In conclusion, it was shown that postpubertal injections of progesterone alter the protein expression in the vagina and uterus of DES mice.

Postpubertal injections of a synthetic estrogen, diethylstilbestrol (-DES), and an anti-estrogen, tamoxifen (-Tx), stimulate proliferation of vaginal and uterine epithelial cells of ovariectomized (OVX) adult mice. In vaginae of two groups of OVX mice treated neonatally with DES (DES-) and oil vehicle alone (Oil-), postpubertal injections of DES altered all and 25 out of 37 protein expressions examined, except for keratin polypeptides, respectively. Twenty-one of the DES- and Tx-altered protein expressions showed the same behavior, suggesting that Tx acts as an estrogen agonist on these proteins. Both neonatally Oil- and DES-treated OVX mice given postpubertal injections of DES (Oil-DES and DES-DES) and Tx (Oil-Tx and DES-Tx) showed similar histological changes in the vagina. Epithelial proliferation and superficial cornification were observed in vaginae of five groups of Oil-DES, Oil-Tx, DES-DES, DES-Tx and DES-Oil (given oil postpubertally) OVX mice. In these groups, 13 proteins showed the same behavior, implying that these proteins are related to proliferation and cornification of mouse vagina. The histology of the uterus in DES-Oil OVX mice closely resembled that in Oil-Oil OVX mice; in protein expressions of the uterus, 5 proteins in DES-DES and 7 proteins in DES-Tx OVX mice were different from those in Oil-DES and Oil-Tx OVX mice, respectively. These findings indicate that neonatal DES treatment alters the behavior of the vagina and uterus in response to postpubertally administered DES and Tx.

Neonatal treatment of female mice with diethylstilbestrol (DES) results in genital tract abnormalities including ovary-independent vaginal proliferation and cornification. Protein profiles were examined in vagina and uterus from 45-day-old, ovariectomized C57BL/Tw mice which had been given 5 daily injections of 2 micrograms DES or oil vehicle alone from the day of birth, and in those from 45-day-old, ovariectomized mice given 3 daily injections of 0.1 microgram DES from 42 days of age. Proteins extracted were analyzed by two-dimensional polyacrylamide gel electrophoresis. In the vagina, expression of 37 non-keratin proteins was altered by postpubertal injections of DES as compared with the controls. In the neonatally DES-exposed vagina, expression of 26 of 37 proteins was altered as compared with controls. In the uterus, expression of 22 proteins was altered in the postpubertally DES-exposed group as compared with that in the control; however, the protein expression pattern of the neonatally DES-exposed group closely resembled that of the control except for one protein (no. 23, pI = 6.1, MW = 39.5 kDa) which was specifically increased in the neonatally DES-exposed group. By immunoblot analysis, 6 keratin polypeptides (49.5, 50, 52, 53, 57 and 58 kDa) were identified in vaginae of ovariectomized mice exposed neonatally and postpubertally to DES and of the controls. These results indicate that neonatal DES exposure induces organ specific alterations in the synthesis of proteins in mouse vagina and uterus.

Neonatal treatment with diethylstilbestrol (DES) induces ovary-independent vaginal epithelial changes in mice. The response of vaginal epithelial cells from intact prepuberal BALB/cCrgl mice treated neonatally with 2 micrograms of DES for 5 days to growth-stimulatory and -inhibitory factors was studied using a serum-free collagen gel culture system that sustains the growth of normal vaginal epithelial cells. Cells from control and DES-exposed mice at 21 days of age showed about a 5-fold increase in number during 10 days in a serum-free medium supplemented with transferrin, bovine serum albumin fraction V, insulin, and epidermal growth factor. Epidermal growth factor and insulin stimulated dose-related proliferation of vaginal epithelial cells from both control and DES-exposed mice; however, cells from DES-exposed mice showed a reduced growth response to epidermal growth factor and an increased growth response to insulin, compared with control cells. Insulin-like growth factor I (1-100 ng/ml) tested in the absence of insulin failed to stimulate cell growth. Transforming growth factor-beta (0.05-5 ng/ml) consistently inhibited cell growth in a dose-dependent manner.

Pregnant ICR/JCL mice were given 4 daily subcutaneous injections of 0.2-2000 micrograms diethylstilbestrol (DES) starting on day 15 of gestation. Offspring of mothers given DES were killed at 1-10 days of age and examined for nodules of enlarged polygonal cells under the epithelium of the Müllerian (upper) vagina. Some offspring were ovariectomised at 30 days and killed at 120 days. The nodules which appeared in the prenatally DES-exposed mice (2-2000 micrograms/day) at 3-7 days were not connected with the epithelium of the sinus vagina and reacted positively to an antibody to epidermal growth factor. Nodule formation may prove to be prodromic of later ovary-independent vaginal changes in the DES-exposed mice. Epithelial stratification (2-2000 micrograms/day) and downgrowths and/or pegs (20-2000 micrograms/day) occurred in vaginae of ovariectomized mice exposed prenatally to DES; however, adenosis-like lesions occurred only in the offspring of mothers given the highest prenatal injections of 2000 micrograms DES. Wolffian remnants and hypospadias (2-2000 micrograms/day) were also encountered in the DES-exposed mice. Ovary-independent stratification of the uterine epithelium (20-2000 micrograms/day) and disorganization of the circular musculature (2-2000 micrograms/day) were also observed in the DES-exposed mice. None of these changes was found in ovariectomised 0.2 micrograms DES-exposed and control mice.

Female mice of the C57 Black/Tw strain were injected daily with 100 microng testosterone, 50 microng testosterone propionate (TP), 100 microng 5 alpha-dihydrotestosterone (DHT) or 50 microng 5 alpha-dihydrotestosterone propionate (DHTP), for 10 days from the day of birth. Two other groups of female mice were given neonatal injections with 20 microng estradiol-17 beta and 100 microng progesterone for 10 days, respectively. All mice were ovariectomized at 60 days of age and killed at 90 days. In 100% of neonatally estrogenized or androgenized, ovariectomized mice, the cranial part of the vagina was lined with stratified epithelium with either cornification or parakeratosis or mucification. Stratification only or stratification with superficial squamous metaplasia or cornification took place in the uterine epithelia of 18% of the TP-treated, 75% of the DHT-treated and 50% of the DHTP-treated, ovariectomized mice. In contrast, neonatally estrogenized, ovariectomized mice did not show the estrogen-independent, persistent uterine changes. Neonatal progesterone treatment failed to induce the permanent changes in the vaginal and uterine epithelia.

Normal mouse vaginal epithelial cells isolated from ovariectomized ca. 35-d-old BALB/cCrgl mice were grown in primary culture using collagen gel matrix and a serum-free medium composed of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 (D:H) medium supplemented with insulin (IN), epidermal growth factor (EGF), cholera toxin, transferrin, and bovine serum albumin V (BSA). Three-dimensional cellular outgrowths occurred inside the collagen gel matrix. The contribution of each factor to cell growth was examined by individual addition to the basic D:H medium and by individual deletion from the complete serum-free medium. When added individually, only IN promoted growth. Deletion of IN from the complete serum-free medium markedly diminished growth; deletion of EGF or BSA slightly diminished growth. When horse, fetal bovine, or chicken serum was added to the basal D:H medium, only with increasing doses of horse serum was there enhanced cell growth. The effect of 17 beta-estradiol and diethylstilbestrol on the growth of cells was also tested, using a suboptimal medium of D:H supplemented with BSA and IN, or a minimal medium supplemented with IN alone. During the 8-d time period, addition of estrogen did not enhance cell growth in either medium. To date, we have been unable to demonstrate a mitogenic effect of estrogen; rather a dose-dependent inhibition of proliferation is seen.

Neonatal injections of 20 micrograms 17 beta-estradiol (E2) induced persistent proliferation and cornification of the vaginal epithelium in adult ovariectomized C57 Black/Tw mice. However, permanent vaginal changes were prevented by various retinoids when, simultaneously with E2 treatment, the animals were given injections of 100 micrograms daily dose of retinol, retinol acetate, retinal or of 200 micrograms daily dose of retinol palmitate (RoP). Neonatal injections of a 100 micrograms daily dose of RoP had no preventive effect on the occurrence of E2-induced permanent vaginal changes. This finding suggests that the preventive effect of RoP is weaker than that of other retinoids showing approximately the same degree of prevention. Combined treatment with E2 plus retinoic acid (even a small dose of 20 micrograms) had such a toxic effect on newborn mice that they died within 7 days after birth, while the animals given neonatal injections of 20 micrograms retinoic acid alone survived until the termination of the experiment.

In the vaginal epithelium of mice given 5 daily injections of 100 microgram 5 alpha-dihydrotestosterone from the day of birth, highly electron-dense cells with numerous cytoplasmic processes appeared in the basal layer within 35 days after the last injection. These cells were similar to the B cells reported in earlier works in neonatally estrogen-treated mice undergoing ovary-independent, permanent proliferation and cornification. The present electron-microscopic study suggests that the permanently proliferating, highly electron-dense cells are derived from electronlucent cells (clear cells) appearing in the basal layer of the vaginal epithelium 15-20 days after neonatal androgen treatment.

Female mice of the C57 Black/Tw strain given 5 daily injections with 100 microng testosterone (T) or 5 alpha-dihydrotestosterone (DHT) from the day of birth showed estrogen-independent persistent proliferation and cornification of the vaginal epithelium in adulthood. The vaginal epithelium of the mice was essentially similar to that of the controls in histological structure during or shortly after neonatal injections of the androgens. In T- and DHT-mice aged over 20 days, however, a marked proliferation with or without superficial cornification took place in the epithelium lining the proximal and middle parts of the vagina (Müllerian vagina), while neither proliferation nor cornification occurred in the epithelium of the distal vagina (urogenital sinus vagina). On the second day of postnatal life in mice given a single injection with T on the day of birth, the mitotic activity in the epithelium of the middle vagina was heightened, but it dropped to the control level on the third day and remained low until 20 days. By contrast, the mitotic rates in the epithelium of the rest of the vagina in T-mice and of all parts of the vagina in DHT-mice were approximately the same as in the controls until 20 or 30 days. The mitotic rates in the epithelium of the Müllerian vagina were markedly elevated in T-mice at 20 days of age and DHT-mice at 30 days, and thereafter remained almost unchanged until 60 days of age. These results were different from the findings in mice given neonatal injections with the dose of estradiol-17 beta (E) capable of estrogen-independent vaginal cornification (Iguchi et al., 1976). The present finding seem to indicate that the mechanism involved in the induction of estrogen-independent vaginal changes by neonatal administration of androgen (T, DHT) is different from that following neonatal treatment with estrogen (E), although androgen and estrogen act directly on the vaginal epithelium of neonates.

Estrogen-independent, persistent proliferation and cornification of the vaginal epithelium occurred in ovariectomized adult mice which had received neonatal injections of 50 microgram 5alpha-dihydrotestosterone (DHT). The occurrence of the permanent vaginal changes was prevented by injections of 200 IU vitamin A acetate (VA) given simultaneously with DHT. In 45% of the DHT plus VA-treated, ovariectomized mice, clear cells with pale cytoplasm appeared in the basal layer of the degenerating epithelium, while they came out in the epithelium of 13% of the ovariectomized mice receiving neonatal injections of DHT alone. For suppressing the permanent vaginal changes, including high mitotic rate, more than 1 month was required after the combined treatment. However, the occurrence of the permanent changes was not blocked by VA given after the DHT treatment. Permanent proliferation and squamous metaplasia took place in the uterine epithelium of the DHT-treated mice. Neonatal injections of VA also prevented the occurrence of the permanent uterine changes when given simultaneously with DHT but not when given after the androgen treatment.

Sustained growth in primary culture of vaginal epithelial cells from ovariectomized adult BALB/cCrgl mice embedded within or seeded on collagen gel matrix was achieved in a serum-free medium composed of Ham's F-12 medium/Dulbecco's modified Eagle's medium, 1:1 (vol/vol), supplemented with insulin, bovine serum albumin fraction V, epidermal growth factor, cholera toxin, and transferrin. Three-dimensional growth of vaginal epithelial cells occurred inside the collagen gel matrix. Cell numbers increased 4- to 8-fold in collagen gel and about 4-fold on collagen gel after 9-10 days in culture. The effect of 17 beta-estradiol (0.00018-180 nM in gel or 0.018-180 nM on gel) and diethylstilbestrol (DES; 0.0186-186 nM in gel) on the growth of vaginal epithelial cells was examined. The addition of estrogen did not enhance the growth of vaginal epithelial cells during this time period either in the complete medium or in a suboptimal medium. Cultures on floating collagen gels in the serum-free medium are composed of 1-3 cell layers with superficial cornification. Estrogen does not appear to be a direct mitogen for vaginal epithelial cells, at least in this system.

Adenosis-like lesions (ADL) were found in the upper vagina including the cervical region of ICR mice as early as 1 day after 5 daily injections of 200 micrograms tamoxifen (Tx) starting on the day of birth (200-micrograms Tx mice). ADL developed progressively from 5 to 30 days of age. Involution of the musculature and suppression of the gland genesis occurred in the uterus of 5- to 30-day-old, 200-micrograms Tx mice. The uterine abnormality resulted in a loss of type III collagen and laminin, and in an increase in fibronectin and type I collagen in the mesenchymal stroma of 15-day-old, 200-microgram Tx mice. Vaginal ADL and uterine myometrial involution were also encountered in 35-day-old mice given neonatal injections of 2-200 micrograms Tx, 200 micrograms clomiphene (Clm) and 200 micrograms nafoxidine (Naf), respectively. These changes were never observed in control and 0.2-microgram Tx mice. Ovariectomy performed at 10 days resulted in a reduction in the weight of uteri and the extent of ADL occupancy in Tx, Clm and Naf mice. Five daily injections of 0.1 microgram 17 beta-estradiol (E2) beginning at 30 days of age increased uterine weight and ADL occupancy extent in Tx (2-200 micrograms), Clm and Naf mice. The present findings suggest, therefore, that the antiestrogens act as an estrogen agonist to mouse uterus and vagina and that the induced ADL develops progressively depending on ovarian estrogen.

Adenosis occurred in transplanted C57BL and BALB/c mice Müllerian-derived reproductive tract regions, cervix, and/or fornix (FX), and middle vagina but never in the urogenital sinus-derived portion of the vagina, after a 1-mo exposure to endogenous ovarian hormones or exogenous estradiol (E2). Grafts in ovariectomized hosts did not exhibit adenosis, confirming its dependence on estrogen. C57BL FX and midvaginal transplants from 1-, 3-, and 5-day-old donors but not from 7- or 10-day-old donors developed adenosis, indicating a critical period before day 6. Prolonged E2 exposure (to 2 mo) decreased the adenosis incidence observed in the C57BL FX group but not in midvaginal transplants. Progesterone added during the second half of transplantation to continuing exogenous E2 prevented this reduction in the FX group; however, adenosis incidence in the similarly treated middle vagina group was less than that observed after 1 or 2 mo of E2 treatment alone. Progesterone present throughout the 2-mo transplantation period did not significantly affect adenosis incidence induced by 2-mo exposure of midvaginal or FX grafts to E2 alone. Changes suggestive of squamous cell carcinoma were found in a few BALB/c midvaginal grafts after E2 exposure for 1 mo and in some C57BL midvaginal and FX grafts after E2 and progesterone exposure for 2 mo.

In adult mice, neonatal injections of estrogen induce persistent, estrogen-independent cornification of the vaginal epithelium. Since the occurrence of nodules composed of affected epithelial cells (B cells) is a prodromic phenomenon of the irreversible vaginal alterations, the relationship between the occurrence of B-cell nodules and the time of DNA synthesis in the vaginal epithelial cells was examined by means of the techniques of serial mapping and autoradiography. B-cell nodules appeared in the epithelium of mullerian vagina 3-5 days after the initiation of neonatal estrogen treatment, without spatial continuity to cuboidal epithelial cells of the sinus vagina. The time of DNA synthesis was different in B cells from that in both columnar mullerian and cuboidal sinus cells, suggesting that the mother cells of B cells might be a population of still unidentified, undifferentiated cells.

Female mice of the C57 Black/Tw strain were injected daily with 100 microgram testosterone, 50 microgram testosterone propionate (TP), 100 microgram 5alpha-dihydrotestosterone (DHT) or 50 microgram 5alpha-dihydrotestosterone propionate (DHTP), for 10 days from the day of birth. Two other groups of female mice were given neonatal injections with 20 microgram estradiol-17beta and 100 microgram progesterone for 10 days, respectively. All mice were ovariectomized at 60 days of age and killed at 90 days. In 100% of neonatally estrogenized or androgenized, ovariectomized mice, the cranial part of the vagina was lined with stratified epithelium with either cornification or parakeratosis or mucification. Stratification only or stratification with superficial squamous metaplasia or cornification took place in the uterine epithelia of 18% of the TP-treated, 75% of the DHT-treated and 50% of the DHTP-treated, ovariectomized mice. In contrast, neonatally estrogenized, ovariectomized mice did not show the estrogen-independent, persistent uterine changes. Neonatal progesterone treatment failed to induce the permanent changes in the vaginal and uterine epithelia.

Normal mouse vaginal epithelial cells isolated from ovariectomized ca. 40-day-old BALB/cCrg1 mice were purified by Percoll density gradient centrifugation and grown in primary culture using a collagen gel matrix and a serum-free complete medium. During the 9-day culture period, a 6-fold increase in cell number was observed. Addition of estrogen to the medium did not enhance epithelial cell proliferation. In fact, all doses of estrogen (180 fM to 18 nM) were inhibitory, resulting in only a 3- to 4-fold increase in cell number by day 9 of culture. Continuous exposure to estradiol (1.8 nM) for 9 days in the serum-free complete medium resulted in a decrease in cytosolic estrogen receptors with associated nuclear accumulation of estrogen receptors. A corresponding increase of cytosolic progestin receptors was also observed, indicating that no qualitative modification of the estrogen receptor system had occurred. Thus, despite its effectiveness in specific product synthesis (progestin receptors), estrogen does not stimulate proliferation of vaginal cells in this culture system, but rather inhibits epithelial cell proliferation.

Female mice of the C57BL/Tw strain were injected daily with 1 microgram estradiol-17 beta (E2), 1 or t microgram testosterone (T) and 5 alpha-dihydrotestosterone (5 alpha-DHT), the dose being increased varying from 5 to 100 microgram for 5 days from the day of birth. These mice were ovariectomized at 90 days of age and kept for 50 days until sacrifice. Permanent-anovulatory ovaries were found in 30-40% of mice treated neonatally with large doses of 5 alpha-DHT. All of the E2-treated and 42% of the 5 microgram T-treated mice showed permanent anovulation in the ovaries at 90 days of age, while none of the 1 microgram T-treated mice had such anovulatory ovaries. Estrogen-independent, permanent vaginal changes occurred in mice receiving injections of 5 microgram T or 5-100 microgram 5 alpha-DHT, but not in those given the injections of 1 microgram T.