Evaluation of the potential role of diethylstilbestrol on the induction of endometriosis in a rat model - An alternative approach.

Endometriosis is known to be a gynaecological condition characterised by persistent inflammation and abnormal development of endometrial stroma and glands. Researchers require a rodent model to analyse the disease environment. Animal models are the best option for investigating the etiology and effective treatment of debilitating illnesses in women since rodents, like humans, menstruate. In order to develop the model system, diethylstilbestrol (DES) was examined for its ability to induce endometriosis in rats by investigating its effect on the estrus cycle, hormones, and key markers. The results demonstrated that animals given DES had an erratic estrus cycle and aberrant hormone levels. Histomorphology revealed the development of an endometriosis environment with degenerative epithelium and enlarged glandular cells after DES induction. The higher levels of estrogen, progesterone, and MCP-1 were shown in the endometriosis induced animals. Endometriosis-induced groups had decreased levels of HOXA10 and HOXA11 and increased levels of VEGF and COX-2. Finally, the DES demonstrated endometriosis induction efficacy, implying that it might be a viable replacement for endometriosis induction.

New frontiers of developmental endocrinology opened by researchers connecting irreversible effects of sex hormones on developing organs.

In the early 1960's, at Professor Bern's laboratory, University of California, Berkeley) in the US, Takasugi discovered ovary-independent, persistent vaginal changes in mice exposed neonatally to estrogen, which resulted in vaginal cancer later in life. Reproductive abnormalities in rodents were reported as a result of perinatal exposure to various estrogenic chemicals. Ten years later, vaginal cancers were reported in young women exposed in utero to the synthetic estrogen diethylstilbestrol (DES) and this has been called the "DES syndrome". The developing organism is particularly sensitive to developmental exposure to estrogens inducing long-term changes in various organs including the reproductive organs. The molecular mechanism underlying the persistent vaginal changes induced by perinatal estrogen exposure was partly demonstrated. Persistent phosphorylation and sustained expression of EGF-like growth factors, lead to estrogen receptor α (ESR1) activation, and then persistent vaginal epithelial cell proliferation. Agents which are weakly estrogenic by postnatal criteria may have major developmental effects, especially during a critical perinatal period. The present review outlines various studies conducted by four generations of investigators all under the influence of Prof. Bern. The studies include reports of persistent changes induced by neonatal androgen exposure, analyses of estrogen responsive genes, factors determining epithelial differentiation in the Müllerian duct, ESR and growth factor signaling, and polyovular follicles in mammals. This review is then expanded to the studies on the effects of environmental estrogens on wildlife and endocrine disruption in Daphnids.

Molecular mechanisms of estrogen action in female genital tract development.

The development of the female reproductive tract can be divided into three parts consisting of Müllerian duct organogenesis, pre-sexual maturation organ development, and post-sexual maturation hormonal regulation. In primates, Müllerian duct organogenesis proceeds in an estrogen independent fashion based on transcriptional pathways that are suppressed in males by the presence of AMH and SRY. However, clinical experience indicates that exposure to xenoestrogens such as diethylstilbestrol (DES) during critical periods including late organogenesis and pre-sexual maturational development can have substantial effects on uterine morphology, and confer increased risk of disease states later in life. Recent evidence has demonstrated that these effects are in part due to epigenetic regulation of gene expression, both in the form of aberrant CpG methylation, and accompanying histone modifications. While xenoestrogens and selective estrogen receptor modulators (SERMS) both can induce non-canonical binding confirmations in estrogen receptors, the primate specific fetal estrogens Estriol and Estetrol may act in a similar fashion to alter gene expression through tissue specific epigenetic modulation.

DNA methylation and transcriptome aberrations mediated by ERα in mouse seminal vesicles following developmental DES exposure.

Early transient developmental exposure to an endocrine active compound, diethylstilbestrol (DES), a synthetic estrogen, causes late-stage effects in the reproductive tract of adult mice. Estrogen receptor alpha (ERα) plays a role in mediating these developmental effects. However, the developmental mechanism is not well known in male tissues. Here, we present genome-wide transcriptome and DNA methylation profiling of the seminal vesicles (SVs) during normal development and after DES exposure. ERα mediates aberrations of the mRNA transcriptome in SVs of adult mice following neonatal DES exposure. This developmental exposure impacts differential diseases between male (SVs) and female (uterus) tissues when mice reach adulthood due to most DES-altered genes that appear to be tissue specific during mouse development. Certain estrogen-responsive gene changes in SVs are cell-type specific. DNA methylation dynamically changes during development in the SVs of wild-type (WT) and ERα-knockout (αERKO) mice, which increases both the loss and gain of differentially methylated regions (DMRs). There are more gains of DMRs in αERKO compared with WT. Interestingly, the methylation changes between the two genotypes are in different genomic loci. Additionally, the expression levels of a subset of DES-altered genes are associated with their DNA methylation status following developmental DES exposure. Taken together, these findings provide an important basis for understanding the molecular and cellular mechanism of endocrine-disrupting chemicals (EDCs), such as DES, during development in the male mouse tissues. This unique evidence contributes to our understanding of developmental actions of EDCs in human health.

Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

Widespread enhancer activation via ERα mediates estrogen response in vivo during uterine development.

Little is known regarding how steroid hormone exposures impact the epigenetic landscape in a living organism. Here, we took a global approach to understanding how exposure to the estrogenic chemical, diethylstilbestrol (DES), affects the neonatal mouse uterine epigenome. Integration of RNA- and ChIP-sequencing data demonstrated that ∼80% of DES-altered genes had higher H3K4me1/H3K27ac signal in close proximity. Active enhancers, of which ∼3% were super-enhancers, had a high density of estrogen receptor alpha (ERα) binding sites and were correlated with alterations in nearby gene expression. Conditional uterine deletion of ERα, but not the pioneer transcription factors FOXA2 or FOXO1, prevented the majority of DES-mediated changes in gene expression and H3K27ac signal at target enhancers. An ERα dependent super-enhancer was located at the Padi gene locus and a topological connection to the Padi1 TSS was documented using 3C-PCR. Chromosome looping at this site was independent of ERα and DES exposure, indicating that the interaction is established prior to ligand signaling. However, enrichment of H3K27ac and transcriptional activation at this locus was both DES and ERα-dependent. These data suggest that DES alters uterine development and consequently adult reproductive function by modifying the enhancer landscape at ERα binding sites near estrogen-regulated genes.

Puberty visualized: sexual maturation in the transparent Casper zebrafish.

Transparent Casper zebrafish allow studies of vertebrate sexual maturation and gonad development in vivo. Casper gonad dynamics can be observed longitudinally over time and non-invasively. Gonad maturation and reproduction are complex processes subject to disruption by endocrine-disrupting chemicals (EDCs), such as diethylstilbestrol (DES). DES was used as a 'proof of principle' to ascertain the usefulness of the Casper model to determine EDC effects on gonad maturation. Puberty onset in control juvenile Casper zebrafish (N = 43) averaged 13.2 weeks post fertilization (WPF) for females and included increased vent size, while in males puberty occurred at 11.7 WPF along with maintenance of small vents. DES treatment for 6 days in early juveniles (N = 20) induced an average delay in puberty of 5 weeks in females and 10 weeks in males. DES induced loss of breeding tubercles and vent enlargement in post-pubescent males. Puberty in control fish was correlated with an average body length of 1.7 cm for males and 1.8 cm for females. Increased testes opacity, small vent and breeding tubercles denoted male puberty. Puberty in females was defined as ovarian follicle diameters reaching 400 µm with increasingly opaque follicles and by an increased vent size. These results are like those for wild-type zebrafish and indicate that the Casper model is a useful system for studying gonad dynamics in vivo. Future use of transgenic reporter lines in Casper will allow new avenues of investigation into the reproductive biology of this vertebrate model.

Wnt pathway: A mechanism worth considering in endocrine disrupting chemical action.

Endocrine disrupting chemicals (EDCs) are defined as exogenous substances that can alter the development and functioning of the endocrine system. The Wnt signaling pathway is an evolutionarily conserved pathway consisting of proteins that transmit cell-to-cell receptors through cell surface receptors, regulating important aspects of cell migration, polarity, neural formation, and organogenesis, which determines the fate of the cell during embryonic development. Although the effects of EDCs have been studied in terms of many molecular mechanisms; because of its critical role in embryogenesis, the Wnt pathway is of special interest in EDC exposure. This review provides information about the effects of EDC exposure on the Wnt/ß-catenin pathway focusing on studies on bisphenol A, di-(2-ethylhexyl) phthalate, diethylstilbestrol, cadmium, and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Histone deacetylase inhibitors suppress transdifferentiation of gonadotrophs to prolactin cells and proliferation of prolactin cells induced by diethylstilbestrol in male mouse pituitary.

Diethylstilbestrol (DES), an estrogen agonist, increases prolactin (PRL) cells through transdifferentiation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells to PRL cells as well as proliferation of PRL cells in adult male mouse pituitary. Since hyperacetylation of histone H3 is implicated in the regulation of activation of various genes, we examined the effect of DES on the state of histone H3 acetylation. DES significantly reduced the immunohistochemical signal for acetylated histone H3 at lysine 9 (H3K9ac) in PRL, LH and FSH cells, but not for H3K18ac or H3K23ac. DES-treated mice were injected intraperitoneally with HDAC inhibitors (HDACi), sodium phenylbutyrate (NaPB) or valproic acid (VPA), to mimic the acetylation level of histone H3. As expected, HDACi treatment restored the level of H3K9ac expression in these cells, and also inhibited DES-induced increase in PRL cells. Furthermore, NaPB and VPA also abrogated the effects of DES on the population density of both LH and FSH cells. Similarly, the numbers of proliferating and apoptotic cells in the pituitary in NaPB- or VPA-treated mice were comparable to those of the control mice. Considered together, these results indicated that the acetylation level of histone H3 plays an important role in DES-induced transdifferentiation of LH to PRL cells as well as proliferation of PRL cells.

The Effect of Structural Diversity on Ligand Specificity and Resulting Signaling Differences of Estrogen Receptor α.

Numerous chemicals have been reported to exert estrogen-like endocrine disrupting effects via a receptor binding mechanism that directly interacts with the ligand binding domain of estrogen receptor α (ERα). However, not only their binding affinities to ERα but also their interference in specific cell and tissue functions are clearly different. In this regard, significant regulation differences among three representative estrogenic chemicals (diethylstilbestrol (DES), bisphenol A (BPA), and diarylpropionitrile (DPN)), well-known ERα agonists with very similar structures, have been observed. Molecular dynamics simulation is used to explore the underlying mechanism of different regulation effects induced by the similar estrogen-like chemicals. The DES-induced 12 Å motion of the H9-H10 loop markedly expands the negative electrostatic potential surface of the AF-2 domain, which is consistent with the over-regulation effect of the agonist. In comparison, the 3 Å motion induced by BPA and DPN corresponds to the low-regulation effect of the chemicals. Cross-correlation analysis indicates that the different ERα motions and resulting surface feature of AF-2 domain are brought by the distinguished binding modes of the agonists. Moreover, only hydrophobic DES with estrogen-like size and flexibility has a high binding affinity of -23.47 kcal/mol binding free energy. Both the hydrophilic group in DPN and the small molecular size of BPA dramatically decrease the agonist binding ability, and their binding free energies are only -12.43 kcal/mol and -11.82 kcal/mol, respectively. Our study demonstrates that similar chemicals interact differently with ERα and induce different allosteric effects, which explains the observed regulation diversity.

Long non-coding RNA MIAT is estrogen-responsive and promotes estrogen-induced proliferation in ER-positive breast cancer cells.

Estrogen drives the development and progression of estrogen receptor (ER)-positive breast cancer. However, the detailed mechanism underlying ER-driven carcinogenesis remains unclear despite extensive studies. Previously reports indicated higher expression of long non-coding RNA (lncRNA) myocardial infarction associated transcript (MIAT) in ER-positive breast cancer tissues than in ER-negative tissues. However, the functional relevance of MIAT in ER-positive breast cancer tumorigenesis was poorly understood. Here, we investigated the role of lncRNA MIAT in ER-positive breast cancer cells. MIAT was over-expressed in ER-positive breast cancer tissues and ER-positive breast cancer cell line MCF-7. Activating estrogen signaling by diethylstilbestrol (DES) led to a dose- and time-dependent up-regulation of MIAT in MCF-7 cells that was dependent on ERα, as evidenced by ERα silencing and pharmacological inhibition using ER antagonist ICI 182780. Silencing MIAT decreased DES-induced MCF-7 cell proliferation while overexpressing MIAT increased MCF-7 cell proliferation. Further mechanistic study identified that MIAT was critical for G1 to S phase cell cycle transition. Taken together, these results suggest that lncRNA MIAT is an estrogen-inducible lncRNA and a key regulator in ER-positive breast cancer cell growth. MIAT could serve as a potential biomarker and promising therapeutic target for ER-positive breast cancer.

Diethylstilbestrol regulates mouse gubernaculum testis cell proliferation via PLC-Ca2+ -CREB pathway.

Recent evidence suggested a positive correlation between environmental estrogens (EEs) and high incidence of abnormalities in male urogenital system, but the mechanism remains unclear. Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen that disrupts the morphology and proliferation of gubernaculum testis cells, but the underlying mechanism is unclear. In this study, mouse gubernaculum testis cells were pretreated with phospholipase C (PLC) inhibitor U-73122 and then treated with DES. The results demonstrated that U-73122 impaired DES-evoked intracellular Ca2+ mobilization in gubernaculum testis cells and inhibited DES-induced proliferation of gubernaculum testis cells. Mechanistically, we found that U-73122 inhibited DES-induced activation of cAMP-response element binding protein (CREB) in gubernaculum testis cells. In conclusion, these data suggest that the effects of DES on mouse gubernaculum testis cells are mediated by PLC-Ca2+ -CREB pathway. SIGNIFICANCE OF THE STUDY: Environmental estrogens remain a serious threat to male reproductive health, and it is important to understand the mechanism by which EEs affect the male productive system. Here we explore potential mechanisms how the proliferation and contractility of gubernaculum testis cells are regulated by diethylstilbestrol. Our findings provide the first evidence that PLC-Ca2+ -CREB signalling pathway mediates the nongenomic effects of diethylstilbestrol on gubernaculum testis cells. These findings provide new insight into the role of diethylstilbestrol in the aetiology of male reproductive dysfunction and will help develop better approaches for the prevention and therapy of male reproductive malformation.

Response of xenografts of developing human female reproductive tracts to the synthetic estrogen, diethylstilbestrol.

Human female fetal reproductive tracts 9.5-22 weeks of gestation were grown for 1 month in ovariectomized athymic adult female mouse hosts that were either untreated or treated continuously with diethylstilbestrol (DES) via subcutaneous pellet. Normal morphogenesis and normal patterns of differentiation marker expression (KRT6, KRT7, KRT8, KRT10, KRT14, KRT19, ESR1, PGR, TP63, RUNX1, ISL1, HOXA11 and α-ACT2) were observed in xenografts grown in untreated hosts and mimicked observations of previously reported (Cunha et al., 2017) non-grafted specimens of comparable age. DES elicited several notable morphological affects: (a) induction of endometrial/cervical glands, (b) increased plication (folding) of tubal epithelium, (c) stratified squamous maturation of vaginal epithelium and (d) vaginal adenosis. DES also induced ESR1 in epithelia of the uterine corpus, cervix and globally induced PGR in most cells of the developing human female reproductive tract. Keratin expression (KRT6, KRT7, KRT8, KRT14 and KRT19) was minimally affected by DES. Simple columnar adenotic epithelium was devoid of TP63 and RUNX1, while DES-induced mature vaginal epithelium was positive for both transcription factors. Another striking effect of DES was observed in grafts of human uterine tube, in which DES perturbed smooth muscle patterning. These results define for the first time IHC protein markers of DES action on the developing human reproductive tract, which provide bio-endpoints of estrogen-induced teratogenesis in the developing human female reproductive tract for future testing of estrogenic endocrine disruptors.

Prediction of the combined effects of multiple estrogenic chemicals on MCF-7 human breast cancer cells and a preliminary molecular exploration of the estrogenic proliferative effects and related gene expression.

The environmental risks of environmental estrogens (EEs) are often assessed via the same mode of action in the concentration addition (CA) model, neglecting the complex combined mechanisms at the genetic level. In this study, the cell proliferation effects of estrone, 17α-ethinylestradiol, 17ß-estradiol, estriol, diethylstilbestrol, estradiol valerate, bisphenol A, 4-tert-octylphenol and 4-nonylphenol were determined individually using the CCK-8 method, and the proliferation effects of a multicomponent mixture of estrogenic chemicals mixed at equipotent concentrations using a fixed-ratio design were studied using estrogen-sensitive MCF-7 cells. Furthermore, transcription factors related to cell proliferation were analyzed using RT-PCR assays to explore the potential molecular mechanisms related to the estrogenic proliferative effects. The results showed that the estrogenic chemicals act together in an additive mode, and the combined proliferative effects could be predicted more accurately by the response addition model than the CA model with regard to their adverse outcomes. Furthermore, different signaling pathways were involved depending on the different mixtures. The RT-PCR analyses showed that different estrogens have distinct avidities and preferences for different estrogen receptors at the gene level. Furthermore, the results indicated that estrogenic mixtures increased ERα, PIK3CA, GPER, and PTEN levels and reduced Akt1 level to display combined estrogenicity. These findings indicated that the potential combined environmental risks were greater than those found in some specific assessment procedures based on a similar mode of action due to the diversity of environmental pollutions and their multiple unknown modes of action. Thus, more efforts are needed for mode-of-action-driven analyses at the molecular level. Furthermore, to more accurately predict and assess the individual responses in vivo from the cellular effects in vitro, more parameters and correction factors should be taken into consideration in the addition model.

Diethylstilbestrol activates CatSper and disturbs progesterone actions in human spermatozoa.

STUDY QUESTION: Is diethylstilbestrol (DES), a prototypical endocrine-disrupting chemical (EDC), able to induce physiological changes in human spermatozoa and affect progesterone actions? SUMMARY ANSWER: DES promoted Ca2+ flux into human spermatozoa by activating the cation channel of sperm (CatSper) and suppressed progesterone-induced Ca2+ signaling, tyrosine phosphorylation and sperm functions. WHAT IS KNOWN ALREADY: DES significantly impairs the male reproductive system both in fetal and postnatal exposure. Although various EDCs affect human spermatozoa in a non-genomic manner, the effect of DES on human spermatozoa remains unknown. STUDY DESIGN, SIZE, DURATION: Sperm samples from normozoospermic donors were exposed in vitro to a range of DES concentrations with or without progesterone at 37°C in a 5% CO2 incubator to mimic the putative exposure to this toxicant in seminal plasma and the female reproductive tract fluids. The incubation time varied according to the experimental protocols. All experiments were repeated at least five times using different individual sperm samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human sperm intracellular calcium concentrations ([Ca2+]i) were monitored with a multimode plate reader following sperm loading with Ca2+ indicator Fluo-4 AM, and the whole-cell patch-clamp technique was performed to record CatSper and alkalinization-activated sperm K+ channel (KSper) currents. Sperm viability and motility parameters were assessed by an eosin-nigrosin staining kit and a computer-assisted semen analysis system, respectively. The ability of sperm to penetrate into viscous media was examined by penetration into 1% methylcellulose. The sperm acrosome reaction was measured using chlortetracycline staining. The level of tyrosine phosphorylation was determined by western blot assay. MAIN RESULTS AND THE ROLE OF CHANCE: DES exposure rapidly increased human sperm [Ca2+]i dose dependently and even at an environmentally relevant concentration (100 pM). The elevation of [Ca2+]i was derived from extracellular Ca2+ influx and mainly mediated by CatSper. Although DES did not affect sperm viability, motility, penetration into viscous media, tyrosine phosphorylation or the acrosome reaction, it suppressed progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation. Consequently, DES (1-100 µM) significantly inhibited progesterone-induced human sperm penetration into viscous media and acrosome reaction. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although DES has been shown to disturb progesterone actions on human spermatozoa, this study was performed in vitro, and caution must be taken when extrapolating the results in practical applications. WIDER IMPLICATIONS OF THE FINDINGS: The present study revealed that DES interfered with progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation, ultimately inhibited progesterone-induced human sperm functions and, thereby, might impair sperm fertility. The non-genomic manner in which DES disturbs progesterone actions may be a potential mechanism for some estrogenic endocrine disruptors to affect human sperm function. STUDY FUNDING/COMPETING INTERESTS: National Natural Science Foundation of China (No. 31400996); Natural Science Foundation of Jiangxi, China (No. 20161BAB204167 and No. 20142BAB215050); open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07) to T. Luo; National Natural Science Foundation of China (No. 81300539) to L.P. Zheng. The authors have no conflicts of interest to declare.

[Impacts of DES on the expressions of related genes in the gubernaculums testis of newborn mice].

OBJECTIVE: To investigate the influence of diethylstilbestrol (DES) on the mRNA expressions of the androgen receptor (AR), estrogen receptor α (ERα), proliferating cell nuclear antigen (PCNA), and actin alpha 1 (ACTα1) in the gubernaculums testis of newborn mice and explore their action mechanisms. METHODS: A total of 140 male Kunming mice were randomly divided into a blank control, a dimethyl sulfoxide (DMSO) control, and 5 experimental groups to be treated subcutaneously with normal saline, DMSO, and DES at 0.02, 0.1, 0.5, 10 and 50 µg per kg of the body weight per day, respectively, at gestation days 9－17. On the first day after birth, the animals were sacrificed and the gubernaculums testis collected for detection of the mRNA expressions of AR, ERα, PCNA and ACTα1 by RT-PCR. RESULTS: Compared with the DMSO control, the experimental groups, particularly the DES 10 and 50 µg groups, showed significant increases in the mRNA expression of ERα (RE2 = 0.825, P <0.05), but remarkable decreases in those of AR, PCNA and ACTα1 (RA2 = 0.713, RP2 = 0.946, RT2 = 0.960, P <0.01), all in a dose-dependent manner. CONCLUSIONS: The AR, ERα, PCNA, and ACTα1 mRNA are expressed in the gubernaculum testis of normal newborn mice, and their expression levels may be influenced by intervention with different concentrations of DES during the gestation. Exogenous estrogens may affect the proliferation and contraction of gubernaculum testis cells and consequently the normal development of the testis or even the whole male reproductive system by influencing the metabolism of ER and/or AR.

Role of Notch signaling in granulosa cell proliferation and polyovular follicle induction during folliculogenesis in mouse ovary.

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.

Diethylstilbestrol Regulates the Expression of LGR8 in Mouse Gubernaculum Testis Cells.

BACKGROUND Hormonal effects on the gubernaculum can affect testicular descent. Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen that disrupts the outgrowth of gubernaculums, leading to testis maldescent. However, the underlying mechanisms remain elusive. MATERIAL AND METHODS The gubernaculum were removed from 3-day-old mice and cultured. The subcultured cells were randomly divided into a normal control group and experimental groups. The DES groups were administered 10 µg/ml, 1 µg/ml, 0.1 µg/ml, 0.01 µg/ml of diethylstilbestrol dissolved in dimethyl sulfoxide (DMSO) respectively. The cell morphology was observed under an inverted microscope, and leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8) was localized by immunofluorescence. The expressions of LGR8 gene and protein in gubernaculum cells were quantified by RT-PCR and Flow Cytometer respectively. RESULTS DES treatment converted cells from a normal fibroblast-like morphology into a more refractile, spindle-shaped morphology or irregular elliptical shapes along with cytoplasmic shrinkage. LGR8 was expressed in the cytoplasmic membrane, DES dose-dependently downregulated LGR8 expression at low doses (≤1.0 µg/ml), but upregulated LGR8 at high doses (10 µg/ml) at both the mRNA and protein levels. CONCLUSIONS These results suggest that DES causes testicular maldescent by altering the LGR8 pathway in mouse gubernaculum testis cells.

Exposure to diethylstilbestrol during pregnancy modulates microRNA expression profile in mothers and fetuses reflecting oncogenic and immunological changes.

Prenatal exposure to diethylstilbestrol (DES) is known to cause an increased susceptibility to a wide array of clinical disorders in humans. Previous studies from our laboratory demonstrated that prenatal exposure to DES induces thymic atrophy and apoptosis in the thymus. In the current study, we investigated if such effects on the thymus result from alterations in the expression of microRNA (miR). To that end, pregnant C57BL/6 mice who were exposed to DES and miR profiles in thymocytes of both the mother and fetuses on postnatal day 3 (gestation day 17) were studied. Of the 609 mouse miRs examined, we noted 59 altered miRs that were common for both mothers and fetuses, whereas 107 altered miRs were specific to mothers only and 101 altered miRs were specific to fetuses only. Upon further analyses in the fetuses, we observed that DES-mediated changes in miR expression may regulate genes involved in important functions, such as apoptosis, autophagy, toxicity, and cancer. Of the miRs that showed decreased expression following DES treatment, miR-18b and miR-23a were found to possess complementary sequences and binding affinity for 3' untranslated regions of the Fas ligand (FasL) and Fas, respectively. Transfection studies confirmed that DES-mediated downregulation of miR-18b and miR-23a led to increased FasL and Fas expression. These data demonstrated that prenatal DES exposure can cause alterations in miRs, leading to changes in the gene expression, specifically, miR-mediated increased expression in FasL and Fas causing apoptosis and thymic atrophy.

Expression Analysis of DGKK during External Genitalia Formation.

PURPOSE: Genetic variants in diacylglycerol kinase κ (DGKK) have been strongly associated with risk of hypospadias. We investigated the expression pattern of Dgkk during development of mouse external genitalia to better understand its function and mechanism in the etiology of hypospadias. MATERIALS AND METHODS: We performed Dgkk expression analysis via indirect immunofluorescence in histological sections of CD-1 mouse embryonic and postnatal male, female and diethylsilbestrol treated external genitalia. Histological findings were supplemented with DGKK expression analysis using quantitative real-time polymerase chain reaction assays. RESULTS: In mouse external genitalia Dgkk was expressed in the membrane and cytoplasm of differentiating squamous epithelial cells of urethral plate/groove and epidermis but not in the undifferentiated epithelial cells of preputial lamina or basal layer of urethral groove epithelium. CD-1 gestation day 18 male mouse genital tubercle treated with oil or diethylstilbestrol showed similar patterns of Dgkk expression despite many morphological differences, including formation of preputial cleft observed in diethylsilbestrol treated mice. CONCLUSIONS: Dgkk appears to be a marker or mediator of squamous cell differentiation during development of mouse external genitalia. However, no association exists between Dgkk expression and formation of preputial cleft in the genital tubercle of diethylsilbestrol treated mice, suggesting that these 2 events may follow independent pathways in mice. Further studies are needed to elucidate the role of DGKK in hypospadias.