Biomedical subjects

Emily Willingham

Publications and source records attributed to Emily Willingham.

9 recordsLinked to original sources

PubMed2006

Activating transcription factor 3 is estrogen-responsive in utero and upregulated during sexual differentiation.

BACKGROUND/AIMS: Synthetic estrogens induce hypospadias, an anomaly of genital tubercle/urethral development. Activating transcription factor 3 (ATF3), which is estrogen-responsive in vitro, is upregulated in hypospadiac human tissue. We used a mouse model of steroid-dependent genital tubercle development to elucidate the ontogeny of ATF3 expression and the developmental response of ATF3 in vivo to estrogen exposure. METHODS: We used quantitative RT-PCR to assess ontogenic expression of ATF3 and its response to estrogen treatment in utero. Immunohistochemistry was used to localize the protein. RESULTS: Quantitative RT-PCR showed that ATF3 mRNA is upregulated in all estrogen-exposed fetal genital tubercles compared to controls (p = 0.024), including specifically in males exposed in utero (p = 0.049). Additionally, its expression increases significantly during the period of sexual differentiation in both sexes and significantly correlates with female development (p = 0.004), a phenomenon that appears to be attributable to higher levels at birth in females. The protein localizes in the nucleus, as expected. CONCLUSIONS: ATF3 is estrogen-responsive in vivo. The response of ATF3 to estrogenic stimulation in utero at an earlier stage may contribute to urethral abnormalities observed in estrogen-exposed male fetuses, although it is likely not the only gene involved, which supports the general understanding that hypospadias is subject to multifactorial influences. ATF3 may therefore be an appropriate gene for further investigations in an endocrine context.

Activating Transcription Factor 3↗

PubMed2006

Embryonic exposure to the fungicide vinclozolin causes virilization of females and alteration of progesterone receptor expression in vivo: an experimental study in mice.

BACKGROUND: Vinclozolin is a fungicide that has been reported to have anti-androgenic effects in rats. We have found that in utero exposure to natural or synthetic progesterones can induce hypospadias in mice, and that the synthetic progesterone medroxyprogesterone acetate (MPA) feminizes male and virilizes female genital tubercles. In the current work, we selected a relatively low dose of vinclozolin to examine its in utero effects on the development of the genital tubercle, both at the morphological and molecular levels. METHODS: We gave pregnant dams vinclozolin by oral gavage from gestational days 13 through 17. We assessed the fetal genital tubercles from exposed fetuses at E19 to determine location of the urethral opening. After determination of gonadal sex, either genital tubercles were harvested for mRNA quantitation, or urethras were injected with a plastic resin for casting. We analyzed quantified mRNA levels between treated and untreated animals for mRNA levels of estrogen receptors alpha and beta, progesterone receptor, and androgen receptor using nonparametric tests or ANOVA. To determine effects on urethral length (males have long urethras compared to females), we measured the lengths of the casts and performed ANOVA analysis on these data. RESULTS: Our morphological results indicated that vinclozolin has morphological effects similar to those of MPA, feminizing males (hypospadias) and masculinizing females (longer urethras). Because these results reflected our MPA results, we investigated the effects of in utero vinclozolin exposure on the mRNA expression levels of androgen, estrogen alpha and beta, and progesterone receptors. At the molecular level, vinclozolin down-regulated estrogen receptor alpha mRNA in females and up-regulated progesterone receptor mRNA. Vinclozolin-exposed males exhibited up-regulated estrogen receptor alpha and progesterone receptor mRNA, effects we have also seen with exposure to the synthetic estrogen, ethinyl estradiol. CONCLUSION: The results suggest that vinclozolin virilizes females and directly or indirectly affects progesterone receptor expression. It also affects estrogen receptor expression in a sex-based manner. We found no in vivo effect of vinclozolin on androgen receptor expression. We propose that vinclozolin, which has been designated an anti-androgen, may also exert its effects by involving additional steroid-signaling pathways.

Analysis of Variance↗

PubMed2006

Steroid receptors and mammalian penile development: an unexpected role for progesterone receptor?

PURPOSE: We investigated the role of steroid receptors in normal and abnormal genital tubercle development in males and females. We hypothesized that progesterone receptor expression might be involved in abnormal development in both sexes. MATERIALS AND METHODS: We examined the effects of medroxyprogesterone acetate on steroid receptor mRNA expression and assessed the involvement of androgen receptor in the action of medroxyprogesterone acetate on genital tubercle development using androgen receptor deficient (Tfm) mice. RESULTS: Quantitative reverse transcriptase polymerase chain reaction and morphological results demonstrated a pattern of virilized females and feminized males in medroxyprogesterone acetate exposed embryos. Progesterone receptor was the only steroid receptor examined that did not differ between medroxyprogesterone acetate treated males and vehicle treated females. At the morphological level in utero exposure to medroxyprogesterone acetate from gestational days 12 to 17 feminized male genital tubercles, producing a more proximal urethral opening. Female fetuses exposed for the same period exhibited virilized genitalia, with a more distal urethral opening. We also exposed Tfm mice to medroxyprogesterone acetate to assess the role of androgen receptor in the activity of medroxyprogesterone acetate. These medroxyprogesterone acetate exposed mice did not differ morphologically from vehicle treated Tfm mice, indicating that medroxyprogesterone acetate requires androgen receptor to elicit genital tubercle abnormalities. CONCLUSIONS: The increase of progesterone receptor mRNA expression in males and the decrease in females as a result of exposure to medroxyprogesterone acetate, which also causes urethral abnormalities in both sexes, suggests a previously unidentified role for progesterone receptor, possibly interacting with androgen receptor, in anomalous genital tubercle development.

Animals↗

PubMed2006

Loratadine exerts estrogen-like effects and disrupts penile development in the mouse.

PURPOSE: Hypospadias is a developmental anomaly of the penis and urethra that can be steroid mediated. It is characterized by a urethral opening occurring below the normal location at the tip of the penis. The link between loratadine, the active ingredient in a common over-the-counter antihistamine, and hypospadias, the most common congenital abnormality, has been the subject of controversy. We examined the effect of in utero exposure to an over-the-counter loratadine syrup on urethral development, and expression of androgen and estrogen receptors. MATERIALS AND METHODS: We orally gavaged pregnant dams with the equivalent of a daily dose of loratadine syrup, with 3 times that dose or with a corn oil gavage control from GD 12 through GD 17. Using gross and histological assessment and 3D reconstruction, we looked for urethral abnormalities in fetal GTs at E 19. We also used real-time quantitative PCR to characterize the expression levels of steroid receptor mRNA in the GT at E 19, a critical stage for completion of urethral and penile development in this species. RESULTS: Loratadine syrup disrupted normal urethral development in the mouse, based on gross morphology and histological assessment, and also disrupted steroid receptor expression, producing an expression profile similar to that resulting from in utero exposure to ethinyl estradiol. CONCLUSIONS: In utero exposure to over-the-counter loratadine syrup can result in hypospadias in this model, and creates changes in the steroid receptor mRNA expression profile similar to those elicited by a synthetic estrogen.

Animals↗

PubMed2006

Ontogeny of androgen receptor and disruption of its mRNA expression by exogenous estrogens during morphogenesis of the genital tubercle.

PURPOSE: The ontogeny of androgen receptor expression in male and female mouse genital tubercles, and the effects of in utero ethinyl estradiol exposure on androgen receptor mRNA expression in the hypospadias model were studied. MATERIALS AND METHODS: Androgen receptor mRNA expression was measured in mouse genital tubercles from fetuses and pups collected on gestational days 12, 14, 16 and 18, and from newborns using immunohistochemistry and real-time quantitative polymerase chain reaction. Pregnant dams were exposed to ethinyl estradiol or corn oil as controls from gestational days 12 to 17. Genital tubercles of gestational day 19 fetuses were then examined by further quantitative polymerase chain reaction analysis after identification of the seam area using a dissecting microscope to diagnose hypospadias in males. RESULTS: Androgen receptor protein was detected in genital tubercles by gestational day 14. Androgen receptor mRNA expression increased gradually in each sex during normal development. However, female genital tubercles expressed a higher level of androgen receptor mRNA throughout development compared to male genital tubercles (p <0.0001). In utero ethinyl estradiol exposure led to a 5.4 and 4.5-fold increase in androgen receptor mRNA in the genital tubercles of female and male embryos (p = 0.004 and 0.001, respectively). Hypospadiac male genital tubercles showed increased androgen receptor mRNA expression compared to control males (p = 0.006). Levels in hypospadiac males did not differ from those in control females but they were less than those in ethinyl estradiol treated females (p >0.05 and 0.01, respectively). CONCLUSIONS: Androgen receptor protein is expressed abundantly in male and female genital tubercles. Androgen receptor mRNA levels are higher in female than in male genital tubercles through development and they increase in response to in utero ethinyl estradiol exposure with ethinyl estradiol treated females having the highest levels of expression, followed by ethinyl estradiol treated hypospadiac males. We infer that higher estrogen in genital tubercles results in a physiological response of increased androgen receptor mRNA expression. We found no direct association between changes in androgen receptor mRNA expression and the presence or absence of hypospadias in males, suggesting that alterations in the expression of proteins other than or in addition to androgen receptor result in anomalous urethral development. This finding supports the idea that the etiology of hypospadias is multifactorial in origin.

Animals↗

PubMed2006

Serum response factor, its cofactors, and epithelial-mesenchymal signaling in urinary bladder smooth muscle formation.

Little is known about the mechanism of bladder smooth muscle differentiation. We hypothesize that epithelial-mesenchymal signaling induces the expression of smooth muscle proteins in bladder mesenchyme resulting in smooth muscle differentiation. We confirmed that smooth muscle differentiation in the mouse urinary bladder occurs first at gestational day 14 (E14) based upon immunohistochemical localization of smooth muscle alpha-actin (SMAA). To investigate murine bladder smooth muscle differentiation and epithlelial-mesenchymal signaling in the developing bladder, we analyzed gene expression profiles of intact embryonic murine bladders and separated epithelial and mesenchymal components at embryonic days E13, E14, E15, E16, and postnatal day 1 (P1). Using cDNA microarray, we identified regulators of vascular smooth muscle differentiation in bladder mesenchyme, including serum response factor (SRF) and its cofactors, ELK1 and SRF accessory protein (SAP)1, as well as two SRF-associated pathways, angiotension receptor II and transforming growth factor- beta2. Immunohistochemistry showed diffuse expression of SRF in the bladder at E12 with localization of expression to the peripheral mesenchyme at E13 and E14. Our results suggest that bladder smooth muscle differentiation may share a similar gene expression program as occurs during vascular smooth muscle differentiation. The unique structure of the urinary bladder makes it an ideal model for studies of smooth muscle differentiation and epithelial-mesenchymal signaling.

Animals↗

PubMed2006

Gene expression profiles in mouse urethral development.

OBJECTIVE: To analyse the gene expression profiles of the mouse genital tubercle (GT) during urethral tube development at embryonic (E) days E14, E15, E16 and E17, as the aetiology of hypospadias, one of the most common congenital anomalies, remains unknown. MATERIALS AND METHODS: During GT development the urethral folds fuse to form an epithelial seam; subsequently, the epithelial seam disappears, resulting in the normal tubular urethra. Abnormalities in urethral seam formation and remodelling might explain hypospadias, and elucidating the molecular developmental mechanisms underlying normal penile development might provide the basis for understanding hypospadias. Total RNA was isolated from the genital tubercle at embryonic days E14, E15, E16, and E17. Together with reference RNA, sample RNA was labelled with Cy-3 and Cy-5 respectively and hybridized to a 16 000-mouse gene array that included the Incyte GEM2.1 and NIA 7k sets. Candidate genes were analysed by immunohistochemistry and real-time polymerase chain reaction. RESULTS: Using cDNA microarrays, we identified the up-regulation of genes involved in the transforming growth factor (TGF)-beta and Wnt-Frizzled pathways, and of thrombospondin (TSP) 4, a member of a cell-migration molecule family, all candidates for involvement in urethral tube formation. Immunohistochemistry showed TGFbeta1, TGFbeta receptor III, and Frizzled1 were expressed exclusively in E14-E17 urethral epithelium. TSP4 was expressed in the mesenchymal basal layer underlying E17 GT skin epidermis. CONCLUSIONS: Many signalling pathways are involved in late GT development, and cell migration molecules might have an important role in urethral tube formation.

Animals↗

PubMed2005

Activating transcription factor 3 is up-regulated in patients with hypospadias.

Hypospadias is a congenital anomaly of the genitalia characterized by abnormalities of the urethra and foreskin, with the urethral meatus located in an abnormal position anywhere from the distal ventral penile shaft to the perineum. Because the incidence of hypospadias is approximately 1/200-1/300 live male births, it is one of the most common congenital malformations, but its etiology is largely uncharacterized. Genomic analysis of hypospadic tissue indicated a potential role for activating transcription factor 3 (ATF3) in the development of this anomaly. ATF3 may be involved in homeostasis, wound healing, cell adhesion, or apoptosis, and normally it is expressed at a steady-state in quiescent cells. Additionally, it has been shown to be an estrogen-responsive gene, and the etiology of hypospadias may be related to in utero exposure to estrogenic or anti-androgenic compounds. We examined the expression of ATF3 in tissues from 28 children with hypospadias compared with 20 normal penile skin tissue samples from elective circumcision. Eighty-six percent of the hypospadias samples were immunohistochemically positive, compared with 13% of normal tissue samples. Seventy-five percent of hypospadias samples were positive from in situ hybridization, compared with 1% of circumcision samples. Our results indicate that ATF3 is up-regulated in the penile skin tissues of boys with hypospadias, suggesting a role for this transcription factor in the development of this abnormality. Because the etiology of hypospadias may include exposure to estrogenic compounds, the responsiveness of ATF3 to estrogen is also discussed.

Activating Transcription Factor 3↗

PubMed2004

Endocrine-disrupting compounds and mixtures: unexpected dose-response.

The current study examined the effects on the red-eared slider turtle of extremely low doses of three endocrine-disrupting compounds (EDCs)--trans-Nonachlor, chlordane, and p,p'-DDE-singly and in mixtures. Previous studies using the red-eared slider turtle have proven its value as an organism for obtaining information about the effects of endocrine-disrupting compounds. The sex of the turtle, easily manipulated by exposure during embryogenesis, continues to be a marker of effects. When red-eared slider turtle embryos incubating at a temperature that normally produces a male-biased sex ratio are exposed to these compounds singly, the sex ratio of the resulting hatchlings shifts significantly to a female bias. The current work offers further evidence that the red-eared slider turtle provides a way to quantify the effects of mixtures and has the potential as a model for evaluating additivity and synergy. The results provide information about how very low doses (parts per billion) of these compounds behave in mixtures. The dose ranges were 0.125-0.5 ng/egg for trans-Nonachlor and chlordane and 7-28 ng/egg for p,p'-DDE. Results suggest the possibility that two of the compounds may exert effects in mixtures via complementary pathways; when applied singly, the effects of chlordane and p,p'-DDE were inversely related to dose, but in mixtures, which were essentially a greater dose of EDC, the compounds had an increased effect. In all cases, mixtures resulted in 100% females.

Animals↗