Language：English   Publishing type：Research paper (scientific journal)  

Juvenile hormone (JH) are a family of multifunctional hormones regulating larval development, molting, metamorphosis, reproduction, and phenotypic plasticity in arthropods. Based on its importance in arthropod life histories, many insect growth regulators (IGRs) mimicking JH have been designed to control harmful insects in agriculture and aquaculture. These JH analogs (JHAs) may also pose hazards to nontarget species by causing unexpected endocrine-disrupting (ED) effects such as molting and metamorphosis defects, larval lethality, and disruption of the sexual identity. This critical review summarizes the current knowledge of the JH-mediated effects in the freshwater cladoceran crustaceans such as Daphnia species on JHA-triggered endocrine disruptive outputs to establish a systematic understanding of JHA effects. Based on the current knowledge, adverse outcome pathways (AOPs) addressing the JHA-mediated ED effects in cladoceran leading to male offspring production and subsequent population decline were developed. The weight of evidence (WoE) of AOPs was assessed according to established guidelines. The review and AOP development aim to present the current scientific understanding of the JH pathway and provide a robust reference for the development of tiered testing strategies and new risk assessment approaches for JHAs in future ecotoxicological research and regulatory processes.

DOI： 10.1016/j.aquatox.2021.106058

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Under the Organisation for Economic Co-operation and Development (OECD), the Ministry of the Environment of Japan (MOE) added Japanese medaka (Oryzias latipes) to the test guideline fish short-term reproduction assay (FSTRA) developed by the United States Environmental Protection Agency (US EPA) using fathead minnow (Pimephales promelas). The FSTRA was designed to detect endocrine disrupting effects of chemicals interacting with the hypothalamic-pituitary-gonadal axis (HPG axis) such as agonists or antagonists on the estrogen receptor (Esr) and/or the androgen receptor (AR) and steroidogenesis inhibitors. We conducted the FSTRA with Japanese medaka, in accordance with OECD test guideline number 229 (TG229), for 16 chemicals including four Esr agonists, two Esr antagonists, three AR agonists, two AR antagonists, two steroidogenesis inhibitors, two progesterone receptor agonists, and a negative substance, and evaluated the usability and the validity of the FSTRA (TG229) protocol. In addition, in vitro reporter gene assays (RGAs) using Esr1 and ARβ of Japanese medaka were performed for the 16 chemicals, to support the interpretation of the in vivo effects observed in the FSTRA. In the present study, all the test chemicals, except an antiandrogenic chemical and a weak Esr agonist, significantly reduced the reproductive status of the test fish, that is, fecundity or fertility, at concentrations where no overt toxicity was observed. Moreover, vitellogenin (VTG) induction in males and formation of secondary sex characteristics (SSC), papillary processes on the anal fin, in females was sensitive endpoints to Esr and AR agonistic effects, respectively, and might be indicators of the effect concentrations in long-term exposure. Overall, it is suggested that the in vivo FSTRA supported by in vitro RGA data can adequately detect effects on the test fish, O. latipes, and probably identify the mode of action (MOA) of the chemicals tested.

DOI： 10.1002/jat.4104

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Mechanisms underlying sex determination and differentiation in animals are known to encompass a diverse array of molecular clues. Recent innovations in high-throughput sequencing and mass spectrometry technologies have been widely applied in non-model organisms without reference genomes. Crustaceans are no exception. They are particularly diverse among the Arthropoda and contain a wide variety of commercially important fishery species such as shrimps, lobsters and crabs (Order Decapoda), and keystone species of aquatic ecosystems such as water fleas (Order Branchiopoda). In terms of decapod sex determination and differentiation, previous approaches have attempted to elucidate their molecular components, to establish mono-sex breeding technology. Here, we overview reports describing the physiological functions of sex hormones regulating masculinization and feminization, and gene discovery by transcriptomics in decapod species. Moreover, this review summarizes the recent progresses of studies on the juvenile hormone-driven sex determination system of the branchiopod genus Daphnia, and then compares sex determination and endocrine systems between decapods and branchiopods. This review provides not only substantial insights for aquaculture research, but also the opportunity to re-organize the current and future trends of this field.

DOI： 10.3390/genes12020305

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The G protein-coupled estrogen receptor 1 (Gper1) is a membrane-bound estrogen receptor that mediates non-genomic action of estrogens. A Gper1-mediating pathway has been implicated in reproductive activities in fish, including oocyte growth, but Gper1 has been characterized in only a very limited number of fish species. In this study, we cloned and characterized two genes encoding medaka (Oryzias latipes) Gper1s, namely, Gper1a and Gper1b, and phylogenic and synteny analyses suggest that these genes originate through a teleost-specific whole genome duplication event. We found that Gper1a induced phosphorylation of mitogen-activated protein kinase (MAPK) in 293T cells transfected with medaka Gper1s on exposure to the natural estrogen, 17β-estradiol (E2) and a synthetic Gper1 agonist (G-1), and treatment with both E2 and G-1 also decreased the rate of spontaneous maturation in medaka oocytes. These findings show that the processes for oocyte growth and maturation are sensitive to estrogens and are possibly mediated through Gper1a in medaka. We also show that 17α-ethinylestradiol (EE2), one of the most potent estrogenic endocrine-disrupting chemicals, and bisphenol A (BPA, a weak environmental estrogen) augmented phosphorylation of MAPK through medaka Gper1s in 293T cells. Interestingly, however, treatment with EE2 or BPA did not attenuate maturation of medaka oocytes. Our findings support that Gper1-mediated effects on oocytes are conserved among fish species, but effects of estrogenic endocrine-disrupting chemicals on oocytes acting through Gper1 may be divergent among fish species.

DOI： 10.1002/jat.4130

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Freshwater zooplankton Daphnia magna has been widely used in ecotoxicology studies. During the last 20 years, it has been demonstrated that the topical application of juvenile hormone (JH) or JH analogs to mother daphnids induce male offspring production. Based on this finding, an in vivo screening validation method for chemicals with JH agonistic effect has developed. Although this screening system successfully identified a number of JH-like chemicals, molecular mechanisms underlying the male sex-determining process remain largely unknown. To address this issue, we established a reliable male- or female-producing system using Daphnia pulex WTN6 strain by changing the rearing photoperiod. Taking advantage of this rearing system, we successfully found several factors involving male sex determination such as ionotropic glutamate receptors, protein kinase C and pantothenate. Here, we used two D. magna strains that can also control the production of female or male offspring by photoperiod differences as model species for ecotoxicology studies. We demonstrated that either treatment of antagonist of ionotropic glutamate receptors or inhibitor of protein kinase C strongly suppressed male offspring production even under male-producing conditions. Moreover, we revealed that male sex-determining processes are likely diverged between D. magna and D. pulex based on the current experiment. This study provides a fine experimental method for in vivo screening not only JH agonists but also JH antagonists. Moreover, using daphnids with photoperiod-dependent sex determination manner will hugely contribute to understanding the mode-of-action of JH in daphnids.

DOI： 10.1002/jat.4035

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Juvenile hormone (JH) is an important endocrine factor regulating many biological activities in arthropods. In daphnids, methoprene-tolerant (Met) belongs to a basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family protein which has recently been confirmed as a JH receptor and can bind and be activated by JHs and JH agonists. Although the activation of the JH signaling pathway causes many physiological effects, the molecular basis for the structural feature and ligand binding properties of Daphnia Met are not fully understood. To study the ligand preference in terms of structural features of Daphnia Met, we built in silico homology models of the PAS-B domain of Daphnia Mets from cladoceran crustaceans, Daphnia pulex and D. magna. Structural comparison of two Daphnia Met PAS-B domain models revealed that the volume in the main cavity of D. magna Met was larger than that of D. pulex Met. Compared with insect Met, Daphnia Met had a less hydrophobic cavity due to polar residues in the core-binding site. Molecular docking simulations of JH and its analogs with Daphnia Met indicated that the interaction energies were correlated with each of the experimental values of in vivo JH activities based on male induction and in vitro Met-mediated transactivation potencies. Furthermore, in silico site-directed mutagenesis supported experimental findings that Thr292 in D. pulex Met and Thr296 in D. magna Met substitution to valine contribute to JH selectivity and differential species response. This study demonstrates that in silico simulations of Daphnia Met and its ligands may be a tool for predicting the ligand profile and cross species sensitivity.

DOI： 10.1021/acs.chemrestox.0c00179

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In the mouse ovary, interactions between oocytes and somatic cells are essential for folliculogenesis and subsequent follicle development. The polyovular follicle (PF), which contains more than two oocytes in a follicle, can be induced in the neonatal mouse ovary when interactions between oocytes and somatic cells are disrupted by agents such as the potent synthetic estrogen diethylstilbestrol (DES) acting through estrogen receptor (ER) β. Hedgehog signaling is known to regulate granulosa cell proliferation, thecal cell differentiation, and follicle growth. To investigate the role of hedgehog signaling in the early folliculogenesis and in PF induction by DES, neonatal mouse ovaries were cultured with or without 10 μM cyclopamine (CPA), an inhibitor of hedgehog signaling, and grafted under the kidney capsule of adult ovariectomized host mice. The number and the incidence of PFs were significantly increased in organ-cultured ovaries post-grafting. Expression of procollagen type IV, alpha 1 (Col4a1) in organ-cultured ovaries was significantly reduced by CPA, but not by DES. The expression of two hedgehog ligands, Desert hedgehog (Dhh) and Indian hedgehog (Ihh), and a target gene, Hedgehog interacting protein (Hhip), was significantly increased by DES both in WT and ERβ KO mice. Therefore, we infer that DES can affect expression of those genes through ERα but not via suppression of hedgehog signaling. Thus, PFs are induced by DES or CPA, but the induction mechanism is different. Our results revealed an important role of hedgehog signaling in basement membrane remodeling during folliculogenesis even before thecal cell differentiation.

DOI： 10.1007/s00441-020-03222-9

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.21873/invivo.11578

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Institute of Anticancer Research  

Background/Aim: Neonatal diethylstilbestrol (DES) treatment induces polyovular follicles (PFs), which contain more than two oocytes in a follicle, through estrogen receptor (ER) β, not ERα. 2,3-Bis(4-hydroxyphenyl)-propionitrile (DPN) is a specific ERβ agonist
the effects of neonatal DPN exposure on PF induction and gene expression in the mouse ovary were examined. Materials and Methods: Histological analysis and real-time reverse transcription-polymerase chain reaction were performed. Results: The PF incidence was significantly high in the ovary of neonatally DPN-exposed mice compared to that in oil-exposed mice. The gene expression of growth differentiation factor 9 (Gdf9), Mullerian-inhibiting substance, steroidogenic factor 1 (Sf1) and steroidogenic acute regulatory protein (Star) in the ovary was significantly increased in the mice neonatally exposed to 40 μg DPN compared to oil-treated mice. Conclusion: Since SF1 is an important transcription factor of several genes involved in ovarian function, up-regulation of Sf1 expression by neonatal exposure to DPN, through ERβ, might affect expression of Gdf9, Mis and Star, resulting in increased PFs in mouse ovary.

DOI： 10.21873/invivo.11199

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Language：English   Publishing type：Part of collection (book)   Publisher：Elsevier  

The vagina (meaning sheath in Latin) is an important part of the female reproductive tract or reproductive organs located in the pelvis. It is a thin-walled fibromuscular tube (approximately 8-10 cm in length) connecting the uterine cervix internally and the vestibule externally. A stratified, squamous and nonkeratinized epithelium is placed on a dense connective tissue that contains a vascular venous plexus, and act as an erectile tissue. In human and mice, during embryogenesis, the vagina develops from the Müllerian duct. The epithelium of the Müllerian vagina migrates towards the posterior, and then vaginal epithelial cell differentiation is stimulated by paracrine factors from the surrounding mesenchyme. 17Β-Estradiol and estrogen receptor a are also important for epithelial cell proliferation and squamous cell differentiation.

DOI： 10.1016/B978-0-12-801238-3.64407-0

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DOI： 10.21873/invivo.11391

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Photoperiodism is a biological seasonal timing system utilized to regulate development and reproduction in organisms. The freshwater micro-crustacean Daphnia pulex displays environmental sex determination, the precise physiological mechanisms of which are largely unknown due to the lack of an experimental system to induce female or male offspring production by alterations of the rearing environment. We recently found that D. pulex, WTN6 strain, produces female or male offspring in response to long-day or short-day conditions, respectively. Taking advantage of this system, here we report the photoperiodic response curve for male offspring production, showing 12 hours as natural critical daylength (50% incidence of male-producing mothers), and that male offspring inducibility is highly sensitive to photoperiodic alterations. By using monochromatic light emitting diode (LED) devices, we found that the effective wavelength is red-light (627 nm), which stably induces male offspring production. This suggests that the red-light photoreceptor may be decisive in the primary step of sex determination process in this strain. Our findings provide the first insights into photoperiodism and red-light as key factors in triggering male offspring production in daphnids.

DOI： 10.2108/zs170005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Estrogens play fundamental roles in regulating reproductive activities and they act through estrogen receptor (ESR) in all vertebrates. Most vertebrates have two ESR subtypes (ESR1 and ESR2), whereas teleost fish have at least three (Esr1, Esr2a and Esr2b). Intricate functionalization has been suggested among the Esr subtypes, but to date, distinct roles of Esr have been characterized in only a limited number of species. Study of loss-of-function in animal models is a powerful tool for application to understanding vertebrate reproductive biology. In the current study, we established esr1 knockout (KO) medaka using a TALEN approach and examined the effects of Esr1 ablation. Unexpectedly, esr1 KO medaka did not show any significant defects in their gonadal development or in their sexual characteristics. Neither male or female esr1 KO medaka exhibited any significant changes in sexual differentiation or reproductive activity compared with wild type controls. Interestingly, however, estrogen-induced vitellogenin gene expression, an estrogen-responsive biomarker in fish, was limited in the liver of esr1 KO males. Our findings, in contrast to mammals, indicate that Esr1 is dispensable for normal development and reproduction in medaka. We thus provide an evidence for estrogen receptor functionalization between mammals and fish. Our findings will also benefit interpretation of studies into the toxicological effects of estrogenic chemicals in fish.

DOI： 10.1111/dgd.12386

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Study Objective: To evaluate the effect of fibroblast growth factor (FGF) on epithelialization of neovagina in patients with Mayer-Rokitansky- Kuster-Hauser syndrome who underwent vaginoplasty.
Design: Observational study.
Setting: University hospital.
Participants: Seven patients with Mayer-Rokitansky-Kuster-Hauser syndrome.
Interventions: Cytological examination was done on vaginal smear samples from the site of completed epithelialization, and tissue was collected from the epithelialized part for histological evaluation. Immunostaining for estrogen receptor a, and keratin 13 and 14, and reverse transcription polymerase chain reaction (RT-PCR) analysis of the FGF receptor (FGFR) 1-4 were performed in samples from case 2 three times (ie, during the surgery, during the period of vaginal creation, and at 3 months and 6 months after the surgery).
Main Outcome Measures: The primary outcome was the FGF effects on the epithelialization speed and FGFR expression in the neovagina. The second was the role of FGF in the mechanism of vaginal epithelial cell proliferation.
Results: The histological structure of the neovagina was consistent with that of normal vagina. RT-PCR analysis revealed that FGFR was expressed in the control vaginas and neovaginas. Among the FGFR subtypes, FGFR-4 was overexpressed during the process of epithelialization and its level decreased after completion of creation of the new vagina.
Conclusion: The epithelium of the neovagina was morphologically similar to that of normal vagina. It is suggested that FGF plays the role as a growth factor.

DOI： 10.1016/j.jpag.2015.12.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

The freshwater zooplankton Daphnia magna has been extensively employed in chemical toxicity tests such as OECD Test Guidelines 202 and 211. Previously, it has been demonstrated that the treatment of juvenile hormones (JHs) or their analogues to female daphnids can induce male offspring production. Based on this finding, a rapid screening method for detection of chemicals with JH-activity was recently developed using adult D. magna. This screening system determines whether a chemical has JH-activity by investigating the male offspring inducibility. Although this is an efficient high-throughput short-term screening system, much remains to be discovered about JH-responsive pathways in the ovary, and whether different JH-activators act via the same mechanism. JH-responsive genes in the ovary including developing oocytes are still largely undescribed. Here, we conducted comparative microarray analyses using ovaries from Daphnia magna treated with fenoxycarb (Fx; artificial JH agonist) or methyl farnesoate (MF; a putative innate JH in daphnids) to elucidate responses to JH agonists in the ovary, including developing oocytes, at a JH-sensitive period for male sex determination. We demonstrate that induction of hemoglobin genes is a well-conserved response to JH even in the ovary, and a potential adverse effect of JH agonist is suppression of vitellogenin gene expression, that might cause reduction of offspring number. This is the first report demonstrating different transcriptomics profiles from MF and an artificial JH agonist in D. magna ovary, improving understanding the tissue-specific mode-of-action of JH. Copyright © 2016 John Wiley & Sons, Ltd.

DOI： 10.1002/jat.3368

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Sex determination of Daphnia pulex is decided by environmental conditions. We established a suitable experimental system for this study using D. pulex WTN6 strain, in which the sex of the offspring can be controlled by photoperiod. Long-day conditions induced females and short-day conditions induced males. Using this system, we previously found that methy farnesoate (MF), which is a putative innate juvenile hormone molecule in daphnids, is necessary for male sex determination and that protein kinase C (PKC) is a candidate factor of male sex determiner. In this study, we demonstrated that a PKC inhibitor [bisindolylmaleimide IV (BIM)] application strongly suppressed male offspring induction in the short-day condition. Moreover, co-treatment of BIM with MF revealed that PKC signaling acts upstream of MF signaling for male sex determination. This is the first experimental evidence that PKC is involved in the male sex determination process associated with methyl farnesoate signaling in daphnid species.

DOI： 10.1242/bio.021857

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Stable and sustainable spermatogenesis is supported by the strict regulation of self-renewal and differentiation of spermatogonial stem cells (SSC), which are a rare population of undifferentiated spermatogonia. It has been revealed that some signaling factors regulate the self-renewal of SSC; however, the molecular mechanism of SSC maintenance is still not completely understood. Notch signaling is an evolutionarily conserved juxtacrine signaling that plays important roles in the cell fate determination of various tissue stem cells. Recently, analyses of loss-and gain-of-function suggested that Notch signaling was necessary for normal spermatogenesis. However, the expression of Notch signal components in spermatogonia is still unclear. Here, we analyzed the distribution of NOTCH3-expressing spermatogonia and the target genes. Double immunostaining with differentiation markers revealed that NOTCH3 was expressed in some undifferentiated and differentiated spermatogonia in mouse testes. To define the target gene of Notch3 signaling in spermatogonia, we analyzed the mRNA expression pattern of Hes and Hey family genes during testis development. Hes1 abundance was decreased during testis development, suggesting that spermatogonia may express Hes1. Immunohistochemical analysis showed that HES1 was expressed in prepubertal spermatogonia, whereas it was expressed predominantly in adult Sertoli cells and weakly in adult spermatogonia. Furthermore, NOTCH3-HES1 double-positive spermatogonia were in pup and adult testes. These results suggest that Notch3 signaling in spermatogonia could promote Hes1 expression. (C) 2017 S. Karger AG, Basel

DOI： 10.1159/000481772

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The Mullerian duct develops into the oviduct, uterus, and vagina, all of which are quite distinct in their morphology and function. The epithelial fate of these female reproductive organs in developing mice is determined by factors secreted from the stroma; however, how stromal differentiation occurs in the female reproductive organs derived from the Mullerian duct is still unclear. In the present study, roles of retinoic acid (RA) signaling in developing female reproductive tracts were investigated. Retinol dehydrogenase 10 (RDH10) and aldehyde dehydrogenase family 1 subfamily A2 (ALDH1A2) mRNAs and proteins and transactivation activity of endogenous RA were found in the stroma of proximal Mullerian ducts and gradually decreased from the proximal to caudal regions in fetal mice. In organ-cultured Mullerian ducts, retinaldehyde or RA treatment induced uterine epithelial differentiation, defined as a layer of columnar epithelial cells negative for oviductal and vaginal epithelial markers. In contrast, inhibition of RA receptor (RAR) signaling induced vaginal epithelial differentiation, characterized as vaginal epithelial marker genes-positive stratified epithelium. Grafting experiments of the organ-cultured Mullerian duct revealed irreversible epithelial fate determination. Although RAR did not directly bind to the homeobox A10 (Hoxa10) promoter region, RA-RAR signaling stimulated Hoxa10 expression. Thus, RA-RAR signaling in the Mullerian duct determines the fate of stroma to form the future uterus and vagina.

DOI： 10.1073/pnas.1608808113

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

In the fetal mouse ovary, oocytes are connected by an intercellular bridge and form germ cell cysts. Folliculogenesis begins after birth. To study the role of Notch signaling in folliculogenesis, double-immunohistochemical localization of laminin and Ki-67 was performed in mouse ovaries from embryonic day 17.5 (E17.5) to postnatal day 4 (P4). Most cysts and follicles contained Ki-67-negative cells; however, a few Ki-67-positive cells were present in cysts from E17.5 through P4, indicating that a small number of pre-granulosa cells continue to proliferate during folliculogenesis. To examine the effects of an inhibitor of Notch signaling (DAPT) and a synthetic estrogen (diethylstilbestrol [DES]) on folliculogenesis, an organ-culture system was established. The numbers of cysts, primordial follicles (PrFs) and primary follicles were unchanged by DES, whereas the total number of PrFs and of PrFs with Ki-67-negative cells was reduced by DAPT. In organ-cultured neonatal ovaries, only DAPT treatment increased degenerating cells defined as oocytes. On the contrary, the number of polyovular follicles (PFs) and the PF incidence were significantly increased in ovaries organ-cultured with DES at day 20 post-grafting. In organ-cultured fetal and neonatal ovaries, DAPT reduced Notch 3 and Hey2 mRNAs, whereas DES increased Hey2 mRNA. These results suggest that Notch signaling in fetal ovaries is involved with PrF assembly by the regulation of oocyte survival rather than by cell proliferation. In PF induction, as a result of the disruption of interactions between oocytes and pre-granulosa cells, DES and Notch signaling act independently.

DOI： 10.1007/s00441-016-2371-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The regulation of Sertoli cells by some hormones and signaling factors is important for normal spermatogenesis. Notch signaling is considered to be necessary for normal spermatogenesis in mouse. In this study, we revealed two new facts about Sertoli cells by western blotting experiments on different types of primary cells and microdissected tubules. The first is that Sertoli cells express the Jagged1 ligand in mice testes. The second is that the expression level of Jagged1 oscillates in the seminiferous epithelial cycle. Therefore, we inferred that Jagged1 in Sertoli cells contributes to the Notch signaling involved in spermatogenesis. Furthermore, we examined the regulation of Jagged1 expression and found that Jagged1 expression was suppressed by cAMP signaling and was promoted by TNF-alpha signaling in Sertoli cells. When cAMP and TNF-alpha were simultaneously added to Sertoli cells, Jagged1 expression was suppressed. Therefore, cAMP signaling dominates Jagged1 expression over TNF-alpha signaling. These results suggest that cAMP signaling may cause the periodicity of Jagged1 expression in the seminiferous epithelial cycle, and controlling Jagged1 expression by adding TNF-alpha or cAMP may contribute to normal spermatogenesis in vitro. (c) 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

DOI： 10.1016/j.reth.2016.02.005

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

Estrogen receptor alpha (ER alpha) plays a pivotal role in the mouse uterine and vaginal epithelial cell proliferation stimulated by estrogen, whereas ER beta inhibits cell proliferation. ER beta 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 ER beta, and its roles in cell proliferation in neonatal uteri and vaginae of ER beta knockout (beta ERKO) mice. ER beta 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 beta ERKO mice, cell proliferation was greater than that in wild-type animals and in uterine epithelia of 90- and 365-day-old beta ERKO mice. In addition, p27 protein, known as a cyclin-dependent kinase inhibitor, was decreased in the uteri of 90- and 365-day-old beta ERKO 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 ER beta is important in the persistent inhibition of epithelial cell proliferation with accumulation of p27 protein in the mouse uterus. Thus, suppression of ER beta function in the uterine epithelium during the neonatal period may be responsible for a risk for proliferative disease in adults.

DOI： 10.1210/en.2015-1012

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS LTD  

Gonadotrophs in the anterior pituitary secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Neonatal diethylstilbestrol (neoDES) treatment affects reproductive function of male and female mice, but the effect of this treatment on the development as well as direct effects on pituitary gonadotrophs have not been ascertained. We investigated LH-secreting gonadotropes and the expression of genes involved in the synthesis and secretion of gonadotropins in the anterior pituitary of neoDES mice using immunohistochemistry and real-time reverse transcription-polymerase chain reaction (RT-PCR). The percentage of LH-secreting gonadotropes in 90-day-old female mice treated neonatally with an oil vehicle (neoOil) was significantly lower than in 30-day-old neoOil females but not in males, indicating a significant reduction after reproductive maturation in females. The percentage of LH-secreting gonadotropes in the medial area of 90-day-old neoDES females was significantly lower than that of 90-day-old neoOil females, ovariectomized neoOil females, and neoOil and neoDES males. The expression of the LH beta (Lhb) subunit in the anterior pituitary of 90-day-old neoDES females was similar to that in neoOil females, but it was significantly lower than that observed in 90-day-old males. Ovariectomy increased the expression of the alpha subunit of glycoprotein hormones, FSH beta (Fshb) subunit and Lhb subunit both in neoOil and neoDES females, suggesting that the anterior pituitary of neoDES female mice is regulated by ovarian hormones via negative feedback. In organ-cultured, anterior pituitaries exposed to DES exhibited no change in the number of LH-secreting gonadotropes but did reduced gene expression. These results suggest that LH-secreting gonadotropes in the female mice are not only directly affected by neoDES but also are influenced by the masculinization of the hypothalamus. That is, neonatal DES exposure can masculinize or defeminize the hypothalamus of female mice. However, the regulation of the pituitary gonadotropins by the hypothalamus could be different from that in intact male mice.

DOI： 10.1177/1535370213519722

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT INST ANTICANCER RESEARCH  

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.

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHANN AMBROSIUS BARTH VERLAG MEDIZINVERLAGE HEIDELBERG GMBH  

In the ovary of neonatally DES-treated mice, lipid droplets accumulation was observed in the hypertrophied interstitial tissues. Our previous results demonstrated that the impaired steroidogenesis in the ovary of neonatally DES-treated mice was caused by altered gonadotropins levels, and resulted in the hypertrophy of ovarian interstitial cells. We speculated that lipid droplets in the ovary mainly consisted of cholesterol. This study was aimed to examine the effects of neonatal DES on cholesterol homeostasis in the ovary. The serum and ovarian total cholesterol concentrations in 3-month-old neonatally DES-treated mice were significantly higher than those in the neonatally oil-treated mice, but triglyceride concentrations were not altered. In the ovary of neonatally DES-treated mice, expression of Hmgcr, a rate-limiting enzyme in de novo cholesterol biosynthesis, was reduced but expression of Ldlr and Scarb1, involved in cholesterol uptake, was not changed. These results suggest that cholesterol uptake is not altered in the ovary of 3-month-old neonatally DES-treated mice. However, the expression of Acat1, the microsomal acyl coenzyme A cholesterol acyltransferase which is involved in cholesterol esterification and storing was increased compared with that in the ovary of neonatally oil-treated mice. Since ovarian steroidogenesis in neonatally DES-treated mice was impaired, synthesized and/or obtained cholesterol from the blood may not be used sufficiently. Thus, in the ovary of neonatally DES-treated mice, cholesterol is esterified by ACAT1 and stored in the interstitial cells.

DOI： 10.1055/s-0033-1333780

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC VET SCI  

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 a 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.

DOI： 10.1292/jvms.12-0182

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT INST ANTICANCER RESEARCH  

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 17 beta-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 Writ] 1, 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, Hey!, Dill and p.53 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.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

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 a 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. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.tox.2012.02.010

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Both the uterus and vagina develop from the Mullerian 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.

DOI： 10.1007/s00441-012-1350-7

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Neonatally diethylstilbestrol (DES) treatment causes hypertrophy-hyperplasia in the interstitial tissue of mouse ovaries. To understand the induction mechanism of the hypertrophy, mRNA expression involved in steroidogenesis in the ovary of neonatally DES-treated mice was examined. The expression of StAR and Cyp11a1 was significantly reduced while Cyp19 and Sf-1 were stimulated in the ovary of neonatally DES-treated 3-month-old mice. Expression of those genes was not different between DES- and oil-treated mice after the gonadotropins treatment. Lhb in the pituitary of 3-month-old neonatally DES-treated mice was significantly decreased. Finally, ovaries from DES-treated mice transplanted to neonatally oil-treated hosts had developing follicles at several stages and corpora lutea, whereas grafted ovaries from neonatally oil-treated mice in 3-month-old neonatally DES-treated hosts showed lipid accumulation in the interstitial tissue. Thus, hypertrophy and accumulation of lipid droplets in interstitial cells of neonatally DES-treated mice is caused by impaired steroidogenesis due to the alterations of gonadotropins levels. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.reprotox.2011.10.013

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

The uterus and upper3/5 of the vagina originate from the Mullerian 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 Mullerian 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. (C) 2011 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.diff.2011.03.005

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

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 beta A-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 beta A-subunit) in the stroma stimulates epithelial cell proliferation in the neonatal vagina. DES treatment decreases the expression of the beta A-subunit and activin receptor IIB but increases the expression of the beta B-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.

DOI： 10.1007/s00441-011-1161-2

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC EXPERIMENTAL BIOLOGY MEDICINE  

Estrogen and insulin-like growth factor-I (IGF-I) stimulate prolactin (PRL) production, release and proliferation of PRL-producing cells (PRL cells) in the anterior pituitary. PRL cells in adult estrogen receptor alpha (ER alpha) knockout (alpha ERKO) mice and IGF-I knockout (IGF-IKO) mice are decreased considerably in number. To investigate a correlation between 17 beta-estradiol (E2) and IGF-I on PAL production, IGF-I wild-type (WT) or IGF-IKO mice were ovariectomized at day 8 and the number of PRL cells was examined at days 20 and 60. Although PRL cell number at day 20 and WT or IGF-IKO mice ovariectomized at day 8 was similar to that in intact WT or IGF-IKO mice, PAL cells in adult WT or IGF-IKO mice ovariectomized at day 8 were significantly decreased as compared with those in intact WT or IGF-IKO mice. Therefore, estrogen is essential for PRL cell differentiation between days 20 and 60, regardless of IGF-I. While PRL cells in WT ovariectomized mice increased from days 20 to 60, those in IGF-IKO ovariectomized mice did not increase, suggesting that IGF-I modified PAL cell differentiation after day 20. ICI 182,780 (anti-estrogen) treatment canceled an increase of PRL cells in 30-day-old ovariectomized WT mice, indicating that the presence of ER alpha is important. The number of PAL cells in alpha ERKO mice was similar to that in WT mice at day 20; however, PRL cells in alpha ERKO mice at day 60 were not increased in number from day 20, supporting the idea that estrogen is essential for PAL cell differentiation after day 20. Finally, the percentage of PRL cells in IGF-IKO mice was decreased as compared with that in WT mice at day 20; therefore, IGF-I affects PAL cells before day 20. In conclusion, PRL cell differentiation is differently regulated by E2 and IGF-I depending on the age.

DOI： 10.1258/ebm.2010.009396

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Estradiol (E2) stimulates not only secretion of prolactin (PRL) and proliferation of PRL-producing cells (PRL cells) in the anterior pituitary, but also the expression of growth factors. In insulin-like growth factor-I (IGF-I) knockout (KO) mice, the number of PRL cells is decreased and administration of IGF-I does not increase either the number of PRL cells or plasma PRL levels, indicating that IGF-I plays a pivotal role in PRL cells. The effect of E2 on PRL cells in KO mice was investigated by immunohistochemistry and real-time RT-PCR. The number of PRL cells in KO mice was significantly lower than in the wild-type (WT) control mice. E2 increased the PRL mRNA in WT and KO mice; however, an increase of PRL mRNA in KO was less than that in WT. In addition, no vasoactive intestinal peptide (VIP)-immunoreactive cells were found in KO mice, therefore IGF-I is essential for VIP expression. To investigate the roles of IGF-I on PRL cells in the postnatal development, double-immunostaining with PRL and BrdU was performed in WT and KO mice from days 5-20. The percentages of PRL cells and BrdU-labeled cells in the anterior pituitary of KO mice were lower than in WT mice. Thus, IGF-I may be responsible for proliferation and differentiation of PRL cells in this postnatal period. Differentiation and the proliferation of PRL cells are controlled by IGF-I during the postnatal development, and IGF may be a mediator of E2 action through VIP induction in PRL cells of adults.

DOI： 10.1007/s00441-010-0937-0

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOSCIENTIFICA LTD  

IGF1 knockout (IGF1KO) mice show a reduced number of prolactin (PRL) producing cells (PRL cells); however, the role of IGF1 in PRL cell proliferation and differentiation in immature Mice is Unclear. In this study, ontogenic changes in the percentages of PRL cells, GH producing cells (GH cells), and 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in the anterior pituitary of male IGF1KO mice during the postnatal period were investigated. The percentage of PRL cells in IGF1KO mice was significantly lower at day 20 compared with that in wild-type (WT) mice, while GH cells in IGF1KO mice were significantly increased from day 10. From days 5 to 20, the percentage of BrdU-labeled cells in WT and IGF1KO mice was similar. PRL cells and GH cells are thought to originate from the same progenitor cells, therefore, PRL cells in IGF1KO mice are not able to differentiate because progenitor cells have already committed to be GH cells. However, IGF1, 17 beta-estradiol (E-2), epidermal growth factor (EGF), Or IGF1 plus E-2 treatments increased the PRL cell number in the pituitaries in vitro of 10-day-old WT and IGF1KO mice. This fact suggests that these factors are involved in PRL cell proliferation and differentiation. In addition, the increase of PRL cells in IGF1KO mice stimulated by E, or EGF was less than that of WT mice. Thus, IGF1 plays a crucial role in PRL cell proliferation and differentiation in mouse pituitaries by regulating the differentiation of progenitor cells and mediating the actions of E-2 and EGF. Journal of Endocrinology (2009) 203, 231-240

DOI： 10.1677/JOE-09-0232

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

In mice, neonatal exposure to a synthetic estrogen, diethylstilbestrol (DES), induces polyovular follicles, which contain two or more oocytes per ovarian follicle. We reported previously that the estrogen receptor beta (ESR2) mediates DES signaling in polyovular follicle induction. However, the specific mechanism of polyovular follicle induction has not yet been clarified. Folliculogenesis in rodents begins soon after birth, accompanied by programmed oocyte death and germ cell loss. In this study, the effects of DES on oocyte death and on mRNA expression of genes thought to be involved in polyovular follicle induction were analyzed during a crucial period of folliculogenesis in the ovary of C57BL/6J, Fas(lpr/lpr) (lacking cell death receptor, FAS), and Esr2 knockout (Esr2 KO) mice. Neonatal DES exposure reduced programmed oocyte death in C57BL/6J mice; however, this reduction was not observed in Esr2 KO mice. In control Fas(lpr/lpr) mice, the oocyte apoptotic index was significantly lower than that in the control C57BL/6J mice. However, the polyovular follicle incidence in control 20-day-old Fas(lpr/lpr) mice was similar to that in the control C57BL/6J mice. Moreover, DES exposure changed mRNA expression of inhibin-alpha (Inha) in 2-day-old C57BL/6J mice. These results suggest that inhibition of oocyte death by DES through ESR2 may be one of the triggers for polyovular follicle induction. The FAS system is also involved in neonatal oocyte death; however, reduction of oocyte death is not sufficient for polyovular follicle induction. The combination of increased Inha mRNA and reduction of oocyte death in the ovaries of mice by DES through ESR2 might be correlated with polyovular follicle induction.

DOI： 10.1095/biolreprod.108.070599

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Natural and synthetic estrogens, including diethylstilbestrol (DES), given during the critical period of newborn life induce abnormalities in ovaries of mice. Induction of polyovular follicles (PFs) containing two or more oocytes in a follicle is one example. In this study, the involvement of estrogen receptor subtypes ER alpha and ER beta in induction of PFs by neonatal treatment with DES was analyzed by using ER alpha knockout (alpha ERKO) and ER beta knockout (alpha ERKO) mice. Ovaries of mice injected with 3 mu g DES for 5 days from the day of birth were examined histologically from 10 to 60 days of age, and the expression of genes involved in folliculogenesis was analyzed by real-time quantitative PCR. The PF incidence (percent of PFs per 100 follicles greater than 50 mu m in diameter) in the ovary of alpha ERKO mice treated with DES was not different from that in the DES-treated wild-type mice. However, neonatal DES treatment did not increase the PF incidence in beta ERKO mice, suggesting that PFs were induced by DES through ER beta but not ER alpha. The expression of bone morphogenetic protein 15, growth differentiation factor 9, inhibin-alpha, Mullerian inhibiting substance, and other genes in the ovaries of DES-treated beta ERKO mice was not different from that in the ovaries of DES-treated wild-type mice. These results indicate that ER beta but not ER alpha is essential for DES to induce PFs in mice.

DOI： 10.2108/zsj.26.704

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Previous results show that diethylstilbestrol (DES) causes polyovular follicles through estrogen receptor (ER)beta and increases the number of follicles, suggesting that DES might affect follicular growth and development. Effects of neonatal DES exposure on follicle development were precisely examined in the ovaries of C57BL/6J and ER beta knockout (beta ERKO) mice. In the DES-exposed C57BL/6J mice, both primary follicle (PmF) progression from primordial follicles at 5 days of age and secondary follicle (SF) progression from PmFs at 10 days of age were delayed as compared with those in the oil-exposed controls. These results indicate that DES may suppress follicle development in neonatal mouse ovaries. DES exposure also decreased the number of follicles in 5-day-old C57BL/6J, WT and beta ERKO mice, suggesting that DES inhibits follicle formation and development through ER alpha in the neonatal mouse ovaries. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.reprotox.2008.10.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

Insulin-like growth factor-1 (IGF-1) is a positive regulatorin proliferation and differentiation of skeletal muscle cells, while myostatin (MSTN) is a member of transforming growth factor beta superfamily that acts as a negative regulator of skeletal muscle mass. The present study was performed to detail whether a correlation exists between MSTN and IGF-1 in skeletal muscle of IGF-1 knockout mice (IGF-1(-/-)) and their wild type (WT; i.e., IGF-1(+/+)) littermates. The body weight of IGF-1(-/-) animals was 32% that of WT littermates. The fiber cross-sectional areas (CSA) and number of fibers in M. rectus femoris of IGF-1(-/-) animals were 49 and 59% those of WT animals, respectively. Thus, muscle hypoplasia of IGF-1(-/-) undoubtedly was confirmed. Myostatin mRNA levels and protein levels were similar between M. gastrocnemius of IGF-1(-/-) and WT animals. Myostatin immunoreactivity was similarly localized in muscle fibers of both IGF-1(-/-) and WT M. rectus femoris. The mRNA levels of MyoD family (Myf5, MyoD, MRF4, myogenin) were differentially expressed in IGF-1(-/-) M. gastrocnemius, in which the mRNA expression of MRF4 and myogenin was significantly lower, whereas there were no changes in the mRNA expression of Myf5 and MyoD. These findings first describe that myostatin expression is not influenced by intrinsic failure of IGF-1, although MRF4 and myogenin are downregulated. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.cellbi.2007.05.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC VET SCI  

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 mu g/kg 17 beta-estradiol E-2 using a microarray method in order to identify primary E-2-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 I (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 E-2-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.

DOI： 10.1292/jvms.69.725

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC EXPERIMENTAL BIOLOGY MEDICINE  

In utero exposure to diethylstilbestrol (DES) induces various abnormalities in the Mullerian duct of the mouse. In order to understand the underlying molecular mechanisms associated with DES-induced abnormalities of the Mullerian duct, gene expression was examined on Gestation Day (GD) 19 in mouse fetuses exposed to DES (67 mu g/kg body weight) from GDs 10 to 18. Microarray analysis revealed that 387, 387, and 225 genes were upregulated and 177, 172, and 75 genes were down-regulated by DES in the oviduct, uterus, and vagina, respectively. DES exposure in utero commonly upregulated 72 genes and down-regulated 15 genes in these three organs. The present study demonstrated that organ-specific gene expression patterns in the mouse Mullerian duct were altered by in utero DES exposure. DES-induced changes in expression of genes such as Dkk2, Nkd2, and sFRP1 as well as changes in genes of the Hox, Wnt, and Eph families in the female mouse fetal reproductive tract could be the basis for various abnormalities in reproductive tracts following exposure to this estrogenic drug.

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT INST ANTICANCER RESEARCH  

Reproductive organs in female mice are susceptible to exposure to estrogenic substances especially during development. In the present study, C57BL/6J female mice were exposed to the synthetic estrogens ethinylestradiol (EE2) or diethylstilbestrol (DES), 10-100 or 6.7-67 mu g/kg bw, respectively, in utero from day 10 to 18 of pregnancy, and their effects were analyzed at 30 and 40 days of age. Both EE2 and DES reduced the survival rate of fetuses and newborns in a dose-dependent manner. Polyovular follicles (PF) were found in the ovaries of all groups at 30 days of age including oil-injected controls. However, the incidence of PF was significantly higher in the 50 mu g/kg EE2- and 33.3 mu g/kg DES-exposed mice than the control. In vaginal epithelia of the in utero EE2 exposed, ovariectomized mice, stratification and cornification were encountered even 10 days after ovariectomy. Especially, vaginae in the ovariectomized mice given high dose of EE2 or DES in utero showed ovary-independent proliferation of the epithelium. Thus, it is clear that prenatal exposure to EE2 or DES induces reproductive abnormalities, including PF, ovary-independent vaginal epithelial stratification and cornification.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC EXPERIMENTAL BIOLOGY MEDICINE  

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 mu g 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 alpha, p21, 14-3-3 sigma, small proline-rich protein 2f (Sprr2f), and Krupple-like factor 4 (KIN), 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.

DOI： 10.1177/153537020623100518

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC EXPERIMENTAL BIOLOGY MEDICINE  

Effects of 17 beta-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.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

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 mug 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-a (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. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.reprotox.2004.05.004

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/S0890-6238(03)00011-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Pharmaceutical Society of Japan  

Placental transfer of bisphenol-A (BPA) was studied in mice and Japanese monkeys (Macaca fuscata). BPA was found in maternal and fetal sera, liver, brain, uteri, testes and placenta as early as 30 min after a single subcutaneous (s.c.) injection to 17 days of pregnancy in mice. BPA was also found in fetal liver, kidney, and brain of Japanese monkeys 1 hr after a single s.c. injection to 150 days of pregnancy. These results clearly indicate that the maternal placental barrier can not protect the fetus from the consequences of BPA exposure in these species.

DOI： 10.1248/jhs.48.579

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Use of soy-based infant formulas and soy/isoflavone supplements has aroused concern because of potential estrogenic effects of the soy isoflavones genistein and daidzein. Here we show that s.c. genistein injections in ovariectomized adult mice produced dose-responsive decreases in thymic weight of up to 80%. Genistein's thymic effects occurred through both estrogen receptor (ER) and non-ER-mediated mechanisms, as the genistein effects on thymus were only partially blocked by the ER antagonist ICI 182,780. Genistein decreased thymocyte numbers up to 86% and doubled apoptosis, indicating that the mechanism of the genistein effect on loss of thymocytes is caused in part by increased apoptosis. Genistein injection caused decreases in relative percentages of thymic CD4(+)CD8(-) and double-positive CD4(+)CD8(+) thymocytes, providing evidence that genistein may affect early thymocyte maturation and the maturation of the CD4(+)CD8(-) helper T cell lineage. Decreases in the relative percentages of CD4(+)CD8(-) thymocytes were accompanied by decreases in relative percentages of splenic CD4(+)CD8(-) cells and a systemic lymphocytopenia. In addition, genistein produced suppression of humoral immunity. Genistein injected at 8 mg/kg per day produced serum genistein levels comparable to those reported in soy-fed human infants, and this dose caused significant thymic and immune changes in mice. Critically, dietary genistein at concentrations that produced serum genistein levels substantially less than those in soy-fed infants produced marked thymic atrophy. These results raise the possibility that serum genistein concentrations found in soy-fed infants may be capable of producing thymic and immune abnormalities, as suggested by previous reports of immune impairments in soy-fed human infants.

DOI： 10.1073/pnas.102650199

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/S0890-6238(02)00005-9

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/ar.10033

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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DOI： 10.1016/S0303-7207(02)00211-3

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1677/jme.0.0280087

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC ENDOCRINOLOGY  

To evaluate mechanisms of cell proliferation in the fetal female rat reproductive tract, diethylstilbestrol (DES) effects on cell division and estrogen receptor (ER), epidermal growth factor (EGF) and EGF receptor (EGF-R) expressions were determined from gestational day (GD) 15.5 to 21.5. Reproductive tracts were evaluated within three regions along the Mullerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. In fetuses from non-treated dams, epithelial and mesenchymal proliferation, as evaluated by 5-bromo-2'-deoxyuridine incorporation, was decreased with development in all regions of the Mullerian duct. EGF levels were determined by immunohistochemistry. Mullerian epithelial EGF immunoreactivity was intense in the proximal and middle regions on GDs 15.5 and 17.5. EGF staining remained intense only in the proximal epithelia by GD 19.5 and was weak in the caudal epithelium, but substantially reduced throughout epithelia in all regions by GD 21.5. Thus, decreased cell proliferation correlated with decreased EGF expression in the developing Mullerian duct. DES (100 mug/kg body,weight) was injected from GD 15 to 19 and female fetuses were collected on GD 19.5. DES increased Mullerian duct cell proliferation in the proximal epithelium and mesenchyme but decreased it in the caudal epithelium compared with oil-treated controls. No proliferative DES effect was observed in any cell type in the middle region. Mullerian duct EGF immunoreactivity was suppressed by DES compared with oil. Competitive PT-PCP, indicated DES also decreased mRNAs for EGF, EP beta1 and ER beta2, but not EP alpha and EGF-R. These results indicate EGF may be an important regulatory factor of Mullerian duct cell proliferation, and that DES may alter cell proliferation by disrupting normal EGF, ER beta1 and ER beta2 expression in the developing female rat reproductive tract.

DOI： 10.1677/joe.0.1700539

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DOI： 10.1093/toxsci/57.2.302

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Deciduoma induced by mechanical stimulation in pseudopregnant mice is similar to the decidua in normal pregnancy and it undergoes regression after a certain period. Therefore, we examined cell death in deciduomas which were induced by artifical stimulation. To analyze the regression mechanism of artificially induced deciduoma, DNA fragmentation, in situ 3'-DNA nick end labeling, and RT-PCR were performed on day 6 to 14 of pseudopregnancy DNA fragmentation appeared on day 8 and it increased to day 10 of pseudopregnancy in the traumatized uterine horn. A large number of apoptotic cells were found on day 10 in the periphery of deciduoma at the antimesometrial side. Deciduoma underwent degeneration on day 11 of pseudopregnancy. Expression of tumor necrosis factor-alpha (TNF-alpha) mRNA was high on days 8 and 10, then decreased, whereas the expression increased again on day 14. TNF-alpha protein was expressed from day 8 to day 12, showing a peak expression on day 10 when deciduoma reached maximum weight. Serum progesterone level was high in the traumatized pseudopregnant mice on day 6, then it gradually decreased. Life span of deciduoma was prolonged 4 days more by daily injection of progesterone. A reduction in serum progesterone coincides with TNF-alpha increase, resulting in an increase of apoptotic deciduomal cells at the regression period, and that the life span of deciduoma is prolonged by additive supply of progesterone. Anat Rec 254:205-213, 1999. (C) 1999 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0185(19990201)254:2<205::AID-AR6>3.0.CO;2-K

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

In the rodent uterus, the metrial gland develops during midpregnancy and undergoes regression prior to parturation. The involution of the gland is reported to be accompanied by the loss of gland cells due to their death in situ. Cell death has been classified by using morphological criteria into two types: necrosis and apoptosis. To study the mechanism involved in the peripartum regression of the rat metrial gland, we examined the mode of cell death in the gland during the last week of gestation. We identified apoptotic cells in the regressing metrial gland by using DNA fragmentation, in situ DNA 3'-end labeling, and electron microscopy. Expression of progesterone receptor (PR) and estrogen receptor (ER) was also demonstrated by immunohistochemistry in the gland. The mean weight of metrial gland nodes decreased after day 18 of pregnancy. The apoptotic granulated metrial gland (GMG) cells that were detected by using the in situ DNA 3'-end labeling method were observed on day 16 of pregnancy, and they increased in number after day 20 of pregnancy. Intense fragmentation of DNA was also found from day 20 to day 22 of pregnancy. Electron microscopy demonstrated apoptotic GMG cells in the regressing metrial glands, confirming the results of the labeling studies. Immunohistochemical study revealed that expression of PR and ER, which were localized mainly in fibroblast-like stromal cells but not in GMG cells, was almost unchanged during late pregnancy. Apoptotic cell death is the major mode of rat metrial gland cell death in the peripartum loss of metrial gland cells. Anat. Rec. 252:369-377, 1998. (C) 1998 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0185(199811)252:3<369::AID-AR4>3.0.CO;2-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

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 coil-elation between cell death and cell proliferation in rat uterine and vaginal epithelial cells during the estrous cycle. (C) 1997 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0185(199705)248:1<76::AID-AR9>3.0.CO;2-D

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

Pregnancy-dependent mammary tumors (PDMT) of GR/A mice and transplantable PDMT (TPDMT-4 line) in DDD mice, are exceptionally stable in hormone dependence, continue to grow until parturition and regress soon after delivery. In order to study the regression mechanism of PDMT and TPDMT-4, morphological and biochemical changes were examined in the tumors removed on day 18 (TPDMT-4) or day 20 (PDMT) of pregnancy, and on the expected parturient and the following postpartum days. DP;A fragmentation occurred from day 18 (TPDMT-4) or day 20 (PDMT) of pregnancy to the day after parturition. Apoptotic cells were demonstrated by an in situ 3'-end labeling method, and the plateau of the number of apoptotic cells was observed on the parturient day in PDMT and on the day after parturition in TPDMT-4. Reverse transcriptase polymerase chain reaction showed that expression of Fas was slightly increased but that of bcl-2 was decreased during the process of involution of TPDMT-4 and PDMT. These results suggest that both an increase in expression of Fas and decrease in expression of bcl-2 are involved in the apoptosis of pregnancy-dependent mammary tumor cells after parturition.

DOI： 10.1016/S0304-3835(96)04469-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Background: Tamoxifen (Tx) is known as an antiestrogen because of its competitive inhibition of estrogen binding to estrogen receptor (ER), and it is used as an estrogen antagonist in the human breast. However, Tx is known to have estrogen agonist activity in the human fetal reproductive tracts and vaginal epithelium and endometrium of postmenopausal women as has been known in the mouse uterus. Therefore, we examined estrogenic potency of Tx on the uterus and vagina in newborn mice and adult ovariectomized mice.
Methods: Using immunohistochemistry and in situ hybridization, we studied changes in expression of ER protein and ER mRNA in the uterus and vagina of C57BL/Tw mice exposed neonatally to 100 mu g Tx and 0.03-3 mu g diethylstilbestrol (DES), and changes in expression of ER mRNA in the ovariectomized adult mice given injections of 100 mu g Tx and 3 mu g DES.
Results: Nuclei of the epithelial and stromal cells in the vagina and of the stromal cells in the uterus showed strong ER immunostaining on the day of birth (= day 0), whereas nuclei of the epithelial cells in the uterus exhibited the ER immunostaining by day 5. In uterine epithelial cells, however, ER was induced by DES, 17 beta-estradiol, testosterone or Tx 24 h after a single injection on day 0, but not by the injection of 5 alpha-dihydrotestosterone, progesterone, or epidermal growth factor. ER in uterine epithelial cells was detected even 12 h after a single injection of 3 mu g DES on day 0. ER mRNA expression of uterine and vaginal epithelial cells of newborn mice increased 4 h after a single injection of 3 mu g DES. ER mRNA expression of uterine and vaginal stromal cells in neonatal mice increased 4 h after a single injection of 100 mu g Tx. In uterine epithelial and stromal cells and vaginal epithelial cells of ovariectomized adult mice, ER mRNA expression increased 12 h after a single injection of 3 mu g DES and 100 mu g Tx.
Conclusions: The present study indicates that Tx acts as ER inducer in the uterus and vagina of neonatal and ovariectomized adult mice. However, responsiveness of reproductive tracts to Tx is different between newborn and adult mice. (C) 1996 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0185(199603)244:3<374::AID-AR9>3.0.CO;2-Y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

The ontogenic expression of progesterone and estrogen receptors (PR and ER) and effect of estrogen on these receptors were investigated immunohistochemically in rat uterus from the day of birth (=0 day) to 30 days of age. Uterine epithelial and stromal cells showed a negative PR immunoreaction at 0 day. The PR in the epithelial cell nuclei appeared by 5 days, while the stromal cells showed a negative PR reaction until 12 days. The staining of the stromal cells appeared from 12 to 15 days. In both the epithelial and stromal cells, the initiation of the PR appearance was not affected by ovariectomy performed at 0 day or 5 days prior to the appearance of PR in the epithelial and stromal cells. Estrogen injections from 0 day failed to initiate the appearance of PR in the epithelial cells, regardless of doses of estradiol-17 beta (0.1, 1 and 10 mu g daily), but induced PR in the stromal cells. The staining of ER appeared at 5 days in the epithelial cells and at 1 day in the stromal cells, respectively. ER appeared after 2-3 daily injections of estrogen from 0 day depending upon the doses. These results suggest that steroid hormones secreted from neonatal ovary do not play any important role in ontogenic expression of PR during the postnatal uterine maturation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Netherlands  

Mammary gland involution after cessation of milk production is associated with extensive loss of secretory epithelial cells. In order to study the mechanism of mammary gland involution, litters were removed on day 20 of lactation and morphological and biochemical changes were examined in GR/A mice and Iprcg mice lacking functional Fas. DNA fragmentation occurred 24 h after weaning both in GR/A mice and Iprcg mice indicating that apoptotic cell death occurs during involution of mammary glands. In situ 3′-end labelling method revealed apoptotic cells in epithelial cells lining alveolar lumens and in cells shed into the alveolar lumen. The number of apoptotic cells plateaued on day 2 of weaning in mammary glands of GR/A mice. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that expression of bcl-2, Fas ligand, keratinocyte growth factor (KGF) and transforming growth factor-β1 (TGF-β1) increased on day 1 of weaning in the process of involution of mammary glands in GR/A mice. In mammary glands of Iprcg mice, RT-PCR showed that expression of bcl-2, Fas ligand, KGF, tumour necrosis factor-α(TNF-α) and TGF-βs increased in the process of involution. These results suggest that the Fas ligand, TGF-β1 and TNF-α are involved in the involution of mammary glands after weaning. TNF-α and anti-Fas antibody directly killed more than 80% of mammary cells from p53 knockout mice in vitro within 24 h in the presence of actinomycin D, supporting the hypothesis that Fas and/or TNF-α are involved in the induction of apoptosis of mouse mammary glands. © 1996 Rapid Science Publishers.

DOI： 10.1007/BF01321103

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHANN AMBROSIUS BARTH VERLAG  

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), cjun, c-fos and c-myc mRNAs by 17 beta-estradiol (E(2)) 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 E(2) 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 E(2) 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 E(2). 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 postpuberal E(2) and may be related to ovary-independent persistent changes in the genital tract.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Estrogen receptor (ER) and ER mRNA expression and cell division induced by neonatal diethylstilbestrol (DES) exposure in male C57BL/Tw mouse genital organs were studied using immunohistochemistry, in situ hybridization, and cell counting at metaphase. A single injection of 3 mu g DES on the day of birth induced ER in both epithelial (E) and stromal (S) cells of the epididymis and the seminal vesicles, in E cells of the ductus deferens, and in S cells of the ductus efferens and the anterior prostate 24 h after the injection. After 5 daily injections of DES, ER was induced in 5 cells of the ventral prostate at 5 days of age. The staining intensity of ER increased in E cells of the epididymis and the ductus efferens at 5 days. A single injection of DES induced ER mRNA in both E and S cells of the ductus efferens, the caput epididymis, and the ductus deferens 4 to 12 h after injection of the newborn mice. A single injection of DES stimulated cell division of both E and S cells in the ductus deferens and the epididymis; cell division of S cells of the ductus efferens, the anterior prostate, and the urethra was stimulated 24 h after the injection. After 5 daily injections of DES, cell division was stimulated significantly in E and S cells of the epididymis, the ductus deferens, and the ventral prostate, and in S tells of the seminal vesicle at 5 days. At 30 days of age, mitotic rates were significantly higher in E cells of the seminal vesicles and the epididymides of mice given 5 daily injections of DES. A single or 5 daily neonatal injections of 10 mu g testosterone (T) or 5 alpha-dihydrotestosterone (DHT) stimulated cell division at 1 and 5 days without inducing ER except in the caput epididymis and the ductus deferens; ER was induced in each of these organs by T but not by DHT. These results suggest that the ER induced by neonatal injections of DES is of functional significance, as cell division in male genital organs was also stimulated by DES exposure.

DOI： 10.1016/0890-6238(94)90021-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Developmental changes in mouse placentae from the 6th to the 18th day of pregnancy were studied in vivo and in vitro. Placental volume increased from the 6th to the 18th day of pregnancy; however, the total number of cells per placenta reached a plateau on the 14th day. Decidual cells were predominant in the placenta on the 6th day. Placentae obtained from the 10th to the 18th day contained decidual cells, trophoblastic (labyrinth and spongiotrophoblast) cells, and trophoblast giant cells. Decidual cells increased in number from the 6th to the 10th but decreased on the 14th day, whereas trophoblastic cells increased linearly until the 14th day. Two types of placental cells were distinguished in vitro: small fibroblast-like cells and large flattened cells containing 2-3 nuclei. The large cells reacted to anti-desmin antibody, indicating their decidual character. The small cells reacting to anti-keratin antibody appeared to be trophoblastic cells. Decidual cells from all days of gestation were nonproliferative, regressing with time in culture. 17Beta-estradiol (E, 10(-9) and 10(-8) M), progesterone (P, 10(-10), 10(-9), and 10(-8) M), and a combination of E and P (10(-9) M each) stimulated proliferation of the trophoblastic cells only from the 6th and the 10th days. Keoxifene (2 x 10(-7) M), but not tamoxifen, significantly inhibited the E-induced proliferation of the trophoblastic cells. Both mouse and human epidermal growth factor (EGF, 10 and 100 ng/ml) and human transforming growth factor alpha (TGFalpha, 10 ng/ml) stimulated the proliferation of trophoblastic cells from the 6th and the 10th days but not from the 14th or the 18th day. The present results indicate that E, P, EGF, and TGFalpha were effective in stimulating the proliferation in vitro of placental trophoblastic cells from early pregnancy, but were ineffective in stimulating the proliferation of decidual cells from any day of gestation and that of trophoblastic cells from the 14th and the 18th days.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

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. The uterine cells of ovariectomized rats given hormone treatment to promote a deciduogenic reaction showed PR expression similar to that in the pregnant females. These results indicate that PR expression in the uterus and vagina is regulated by ovarian steroids during the estrous cycle and early pregnancy.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The pelves of male and female C57BL/Tw mice given five daily injections of 100 mug tamoxifen, 50 mug dihydrotestosterone (DHT), or 2 mug diethylstilbestrol (DES) from the day of birth were examined morphometrically and histomorphometrically. Total areas of the pelvis, ilium, ischium, and pubis were significantly smaller in neonatally tamoxifen-treated mice than in the controls. There was no significant difference in length of the ischium between tamoxifen-treated and control mice of both sexes. However, lengths of ilium and pubis, and widths of ilium, pubis, and ischium in tamoxifen-treated male and female mice were significantly smaller than in the respective controls. In contrast, neonatal treatment with DHT or DES did not affect the shape of the pelvis of either sex. In the neonatally tamoxifen-treated females, the number of osteoblasts and osteoclasts per 200 mum trabecular surface length and per 10,000 mum2 subperiosteal area of pubic bone section was smaller than in the controls. Inhibition of ossification persisted in the junction of the pubis and ischium of pelves treated with tamoxifen in vitro. These results suggest that neonatally administered tamoxifen mainly retards the growth of the ilium and pubis in mice by changing the activities of osteoclasts and osteoblasts, and that tamoxifen acts directly on the neonatal mouse pubis to inhibit its ossification.

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Language：English   Publishing type：Research paper (scientific journal)  

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 2μg 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 μg 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.

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Language：English   Publishing type：Research paper (scientific journal)  

Development and mitotic activity of metrial gland in rat uterus were investigated focusing on the point of implantation during mid-pregnancy. In decidua basalis, the granulated metrial gland (GMG) cells slightly increased in number during days 8-12 of pregnancy and then decreased, while the cells appearing in the mesometrial triangle on day 9 continued to increase until day 16, forming the metrial gland in situ. By contrast, the mitotic change in the mesometrial triangle was similar to that in the other 2 regions, decidua basalis and the inner layer of myometrium. In all 3 regions, mitotic rate rose drastically on day 9, and then gradually decreased until day 16. However, most of the mitotic cells lacked the PAS-positive cytoplasmic granules in any regions. Mitotic activity in GMG cells was very low and almost unchanged throughout the gland development. These results suggest that the rapid rise of mitotic rate is due to the active mitosis of PAS-negative cells, presumably GMG precursor cells, existing widely in the implantation point of rat uterus during the early development of the gland. Immunocytochemical study revealed that progesterone receptors were localized in decidual and muscle cells and fibroblasts, but not in GMG cells during the development of the gland. The present study suggests that the metrial gland cells are proliferated, differentiated and maintained by the progesterone- sensitive stromal cells, such as decidual cells and/or fibroblasts.

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publisher：AMER SOC ZOOLOGISTS  

The developing organism is particularly sensitive to exposure to estrogenic chemicals during a critical period in the induction of longterm changes in female reproductive organs, and persistent molecular alterations induced by the perinatal estrogenic agents. The perinatal mouse model can be utilized as an Indicator of possible longterm consequences of exposure to exogenous estrogenic compounds including environmental endocrine disrupters. Attention should be paid to abnormalities in female genital organs exposed to estrogenic endocrine disrupters during fetal and early postnatal development in mammals including humans.

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Language：Japanese   Publisher：東京化学同人  

CiNii Books

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Language：Japanese   Publisher：横浜市立大学学術研究会  

CiNii Books

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Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00034198/

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/1998243256

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CiNii Books

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Language：Japanese   Publisher：日本産科婦人科学会  

CiNii Books

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=425059

Language：English   Publisher：MEDIMOND S R L  

In order to establish a model system to study effects of chemicals in the environment on aquatic animals, we examined the effects of a natural estrogen, 17 beta-estradiol (E-2), on early development of Fundulus heteroclitus. Embryos of F. heteroclitus were reared in seawater containing 10(-10), 10(-8) and 10(-6) M E-2 throughout the experiment. Hatching and survival rates decreased in a dose-dependent manner. Body weight, head and body lengths were significantly smaller than in controls through the experiment. In fry treated with E2, more than 85% of fry showed malformations, ossification was not completed. Sex ratio of control fry was 57% male and 43% female, whereas fry treated with 10(-8) M E2 was 100% female.

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Language：English   Publisher：MEDIMOND S R L  

The authors outline the particular sensitivity of the developing organism to exposure to estrogenic agents in the induction of longterm changes in female reproductive organs, and persistent molecular alterations induced by the perinatal estrogenic agents. The neonatal mouse model can be utilized as an indicator of possible longterm consequences of exposure to exogenous estrogenic compounds including environmental endocrine disrupters. More attention should be paid to abnormalities in female genital organs exposed to estrogenic endocrine disrupters during fetal and early postnatal development in mammals including humans.

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Language：Japanese   Publisher：JAPAN SOCIETY FOR COMPARATIVE ENDOCRINOLOGY  

DOI： 10.5983/nl2001jsce.22.82_4

CiNii Books

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Language：English  

CiNii Books

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Language：English   Publisher：BLACKWELL SCIENCE INC  

Binding of epidermal growth factor (EGF) to membrane preparations of vagina, uterus, ovary, oviduct, and liver was examined in mice treated neonatally with diethylstilbestrol (DES) and compared with that in untreated mice. Binding in the vagina (12.5 +/- 0.73 fmol/mg protein) was somewhat higher than in the uterus (8.0 +/- 0.34 fmol/mg protein). Level of specific binding was of the order: liver (18.4 +/- 1.09 and 16.0 +/- 1.53 fmol/mg protein) &gt; vagina (12.5 +/- 0.73 and 8.2 +/- 0.57 fmol/mg protein) &gt; uterus (8.0 +/- 0.34 and 6.8 +/- 0.56 fmol/mg protein) &gt; ovary (6.8 +/- 0.36 and 8.0 +/- 1.05 fmol/mg protein) &gt; oviduct (2.1 +/- 0.32 and 1.7 +/- 0.05 fmol/mg protein) in control and neonatally DES-exposed mice, respectively. Thus, neonatal DES exposure significantly lowered the binding site level only in the vagina, without modifying the binding affinity (K-d = 5.4 x 10(-9) M in controls vs 4.6 x 10(-9) M in DES exposed mice). Reduction of EGF receptor level in the vagina correlates with ovary-independent persistent proliferation and keratinization of the vagina induced by neonatal DES exposure. EGF receptors were immunohistochemically demonstrated in epithelial cells of vagina, uterus, and oviduct and in stromal cells in uterus and oviduct using a polyclonal antibody to human EGF receptor protein.

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution)  

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Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Presentation type：Oral presentation (general)  

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Presentation type：Poster presentation  

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