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Pesticides suspected of being estrogenic--including arachlor, cisnonachlor, transnonachlor, DDE, and chlordane--were examined for their ability to override a male-producing incubation temperature and result in female hatchlings in the red-eared slider, a turtle with temperature-dependent sex determination. Compounds were assayed in the environmentally relevant concentrations detected in alligator eggs from Lake Apopka, Fla. singly, in concert with one another, and with estradiol. Compounds assayed alone and resulting in significant sex reversal were transnonachlor, cisnonachlor, arachlor, DDE, and chlordane. When applied together, the eight compounds assayed also resulted in significant sex reversal. However, when administered with estradiol, only one of the compounds, chlordane, caused sex reversal at significant levels.

Abstract 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 α and β, 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 α and β, and progesterone receptors. At the molecular level, vinclozolin down-regulated estrogen receptor α mRNA in females and up-regulated progesterone receptor mRNA. Vinclozolin-exposed males exhibited up-regulated estrogen receptor α 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.

Objectives: Air quality has been shown to impact the rates of fungal infection of the airway, causing diseases such as acute invasive fungal rhinosinusitis (AIFRS), particularly in immunocompromised patients. We theorize that patients with hematologic malignancies in units with aging air handling units (AHUs) have a higher attack rate of AIFRS., Methods: Retrospective chart review identified patients with hematologic malignancy and AIFRS in two distinct and equal time periods between 2013 and 2022, representing the presence of aging AHUs and new AHUs, respectively. Cubic feet per minute (CFM) air flows, AIFRS attack rates, and clinical data were compared between the two groups and statistical analyses performed., Results: The older AHUs produce air flow of 27,610 CFM and the newer AHUs produce air flow of 80,000 CFM. There were 18 patients with air supplied by older AHUs and 7 patients with air supplied by new AHUs who developed AIFRS. There was a significantly higher AIFRS attack rate for patients supplied by the older AHUs compared with patients supplied by newer AHUs (p = 0.033). The patients supplied by the older AHUs tended to be younger. The white blood cell counts, absolute neutrophil counts, and the mean time to diagnosis did not differ between the two groups., Conclusions: To our knowledge, this is the first study to examine AIFRS in immunocompromised patients' inpatient environment. Further research should explore whether higher CFM AHUs can decrease this disease among our most vulnerable patients., Level of Evidence: 3 Laryngoscope, 134:4466-4470, 2024., (© 2024 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives: The otolaryngology hospitalist (OH) model is an emerging paradigm for inpatient and acute patient care. This study presents encounter volume before and after the implementation of an OH service. Postimplementation trends are evaluated., Study Design: Retrospective administrative and clinical database review., Setting: Tertiary care university hospital., Methods: This review includes 2 distinct time frames (2008-2012, 2014-2018), representing periods before and after OH implementation. The number of billed patient encounters is compared between these 2 periods using the hospital data warehouse. Additional data is evaluated for the postimplementation period, using a clinical database. Encounter type, the reason for consultation, procedures, and requesting service/location are described., Results: After the OH implementation, there was a 451% increase in evaluation and management encounters submitted for billing. Since the OH model inception, there was an overall increase in encounters (849-910), procedures performed (319-345), and operative cases (46-54) per year. Each inpatient consultation request generates one or more procedures on average. Common reasons for consultation include sinonasal pathology (20.3%), dysphonia/dysphagia (17.5%), and airway evaluation (15%). Critical Care (24%), Emergency Medicine (21%), and Hospital Medicine (21%) requested most of the Otolaryngology consults. Most consults were seen on the inpatient medical/surgical floor (46%), with the ICU (27%) and the Emergency Department (22%) being the next most common locations., Conclusions: The OH model is an evolving paradigm that is viable and offers timely, specialized care for patients in a hospital or acute care setting., (© 2023 American Academy of Otolaryngology-Head and Neck Surgery Foundation.)

Atrazine is the most commonly detected pesticide contaminant of ground water, surface water, and precipitation. Atrazine is also an endocrine disruptor that, among other effects, alters male reproductive tissues when animals are exposed during development. Here, we apply the nine so-called "Hill criteria" (Strength, Consistency, Specificity, Temporality, Biological Gradient, Plausibility, Coherence, Experiment, and Analogy) for establishing cause-effect relationships to examine the evidence for atrazine as an endocrine disruptor that demasculinizes and feminizes the gonads of male vertebrates. We present experimental evidence that the effects of atrazine on male development are consistent across all vertebrate classes examined and we present a state of the art summary of the mechanisms by which atrazine acts as an endocrine disruptor to produce these effects. Atrazine demasculinizes male gonads producing testicular lesions associated with reduced germ cell numbers in teleost fish, amphibians, reptiles, and mammals, and induces partial and/or complete feminization in fish, amphibians, and reptiles. These effects are strong (statistically significant), consistent across vertebrate classes, and specific. Reductions in androgen levels and the induction of estrogen synthesis - demonstrated in fish, amphibians, reptiles, and mammals - represent plausible and coherent mechanisms that explain these effects. Biological gradients are observed in several of the cited studies, although threshold doses and patterns vary among species. Given that the effects on the male gonads described in all of these experimental studies occurred only after atrazine exposure, temporality is also met here. Thus the case for atrazine as an endocrine disruptor that demasculinizes and feminizes male vertebrates meets all nine of the "Hill criteria"., (Copyright © 2011. Published by Elsevier Ltd.)

Hypospadias is a penile developmental abnormality that may partly result from in utero exposure to estrogenic compounds. Expression of activating transcription factor 3 (ATF3) is elevated in human foreskin tissue from hypospadic patients, and in utero exposure to ethinyl estradiol (17-EE) causes ATF3 upregulation in a hypospadias mouse model. We investigated the effects of in vitro exposure to EE on ATF3 expression and promoter activity in human foreskin fibroblasts using immunocytochemistry, quantitative polymerase chain reaction (PCR), western blot, and the luciferase activity assay. Immunocytochemistry showed peak positive staining at 2 hours after 0.5 to 3 hours of EE treatment (0.1 microM). Western blot showed significantly increased ATF3 protein expression (P = 0.006) after EE exposure. ATF3 mRNA, as evaluated using reverse transcriptase PCR and TaqMan real-time PCR, also increased (P = 0.146). In addition, the luciferase activity assay showed that ATF3 promoter activity was significantly enhanced after 1 hour of EE exposure (P < 0.0001). Thus, EE upregulates ATF3 expression in fibroblasts in vitro, consistent with our previous results with human tissue and in vivo mouse models. ATF3 is involved in the TGF-beta epithelial-mesenchymal signaling pathway, and its involvement in hypospadias suggests that ATF3 plays a role in development of this anomaly as a result of exposure to estrogenic compounds. Its potential involvement in other manifestations of developmental endocrine disruption is worth investigating.

Purpose: Additives such as benzophenone-2 are commonly used in cosmetic products and food container plastics to filter out ultraviolet light. In pregnant women exposure may result in transplacental transfer of benzophenone-2 to fetuses. Benzophenone-2 is estrogenic in vitro and in the rat uterotropic assay. Estradiol causes hypospadias in mice and estrogen-like compounds are also postulated to cause hypospadias. We determined whether hypospadias would develop in male mice exposed to benzophenone-2 in utero and whether this outcome depended on estrogen receptor pathways., Materials and Methods: Timed pregnant C57BL/6 mice were administered benzophenone-2 (6.25 mg) or control vehicle by oral gavage from gestational days 12 through 17 and they were sacrificed on day 18. Fetuses were weighed and sexed, anogenital distance was measured and genital tubercles were harvested for paraffin sections or quantitative reverse transcriptase-polymerase chain reaction analysis of genes purportedly involved in genital tubercle development., Results: Eight of 57 benzophenone-2 treated male fetuses (14%) whose genital tubercles were examined histologically had hypospadias (p = 0.0064). Co-administration of benzophenone-2 with the estrogen receptor antagonist EM-800 resulted in normal genital tubercles, ie no hypospadias, in 26 of 26 mice. Likewise no EM-800 or control treated male genital tubercles showed hypospadias. Benzophenone-2 treated male mice had no changes in body mass adjusted anogenital distance relative to controls. Reverse transcriptase-polymerase chain reaction revealed that genital tubercles of benzophenone-2 treated male mice expressed higher levels of estrogen receptor-beta relative to male controls (p = 0.04)., Conclusions: These findings suggest that benzophenone-2 may cause hypospadias via signaling through the estrogen receptor. Further study of human benzophenone-2 exposure and its effects is needed to support this hypothesis.

Objectives: The phytoestrogen (plant estrogen) genistein, present in soy products, is of interest because in utero exposure to genistein can cause hypospadias in our mouse model and maternal consumption of soy is prevalent in human populations. Another compound of interest is the fungicide vinclozolin, which also causes hypospadias in the mouse and rat and can occur concurrently with genistein in the diet as a residue on exposed foods. A study in the United Kingdom found no relationship between a maternal organic vegetarian diet and hypospadias frequency, but women who consumed nonorganic vegetarian diets had a greater percentage of sons with hypospadias. Because nonorganic diets can include residues of pesticides such as vinclozolin, we sought to assess the interaction of realistic daily exposures to genistein and vinclozolin and their effects on the incidence of hypospadias., Methods: Pregnant mice were fed a soy-free diet and orally gavaged from gestational days 13 to 17 with 0.17 mg/kg/day of genistein, 10 mg/kg/day of vinclozolin, or genistein and vinclozolin together at the same doses, all in 100 microL of corn oil. The controls received the corn oil vehicle. The male fetuses were examined at gestational day 19 for hypospadias, both macroscopically and histologically., Results: We identified no hypospadias in the corn oil group. The incidence of hypospadias was 25% with genistein alone, 42% with vinclozolin alone, and 41% with genistein and vinclozolin together., Conclusions: These findings support the idea that exposure to these compounds during gestation could contribute to the development of hypospadias.

Purpose: We examined the ontogenic and sex specific expression of estrogen receptor-alpha and beta in mouse genital tubercles and assessed the effects of in utero estrogen exposure on these parameters., Materials and Methods: Expression of the 2 genes was detected in mouse genital tubercles from fetuses collected on gestational days 12, 14, 16 and 18, and from newborns using immunohistochemistry and quantitative polymerase chain reaction. Pregnant dams were exposed to ethinyl estradiol or corn oil as the control., Results: Estrogen receptor-alpha and beta proteins first appeared on gestational days 12 and 14, respectively. The 2 proteins were expressed in the urethral plate and mesenchyma. Staining intensity was more prominent in the mesenchyma for estrogen receptor-alpha and in the urethral plate for estrogen receptor-beta. Female genital tubercles expressed more estrogen receptor-alpha than male genital tubercles (p <0.01), while estrogen receptor-alpha expression increased gradually in the 2 sexes until birth. Estrogen receptor-beta expression did not differ between males and females, and it showed no notable variation during fetal life. Ethinyl estradiol led to a 2.1 and 3.8-fold increase in estrogen receptor-alpha expression in females and in males with hypospadias (p = 0.002 and 0.04, respectively). Estrogen receptor-beta expression did not change in response to ethinyl estradiol., Conclusions: This study provides in vivo evidence that estrogen receptor-alpha expression in the genital tubercles of each sex increases until parturition but estrogen receptor-beta expression does not, implying genital tubercle sensitivity to estrogen increases during fetal life. Exogenous administration of estrogens results in a response of increased expression of estrogen receptor-alpha but not of estrogen receptor-beta. These differential findings for estrogen receptor-alpha and beta imply that the 2 receptors may have different roles in normal or anomalous genital tubercle development.

The molecular events that lead to isolated hypospadias remain largely unknown, and the etiology of this common congenital anomaly seems to be multifactorial. We have explored the response of several candidate genes to environmental agents that cause hypospadias in a mouse model. Here, we provide an overview of current findings in relation to candidate genes and their response to environmental agents, including the results of genomic analyses of both mouse and human tissues. In addition to steroid-hormone receptors, one gene of specific interest is activating transcription factor 3 (ATF3). We hypothesize a potential mechanism of action for ATF3 and other identified genes, including TGF-B.

Purpose: An unexplained increase in the incidence of hypospadias has been reported, and yet to our knowledge the molecular events and their regulation leading to hypospadias remain unknown, although environmental compounds capable of endocrine activity are suspected. We screened on a global scale abnormalities in gene expression in human hypospadiac tissue compared to those in nonhypospadiac tissue. Additionally, microarray analysis of tissue from a pair of fraternal twins, including 1 with and 1 without hypospadias, served as a control for genetic variability. We hypothesized that gene expression would differ between hypospadiac vs nonhypospadiac tissue and fraternal twin data would show patterns similar to those of group data on hypospadiac and nonhypospadiac tissue., Materials and Methods: Microarray analysis was performed on tissue from patients with and without hypospadias, and from a pair of fraternal twins, including 1 with and 1 without hypospadias. Analysis incorporated the expression of 22,000 genes., Results: We found significant differences in gene expression, specifically with a group of genes, including CYR61, CTGF, ATF3 and GADD45beta, known to be responsive to estrogen or to interact with estrogen receptor., Conclusions: Our findings provide support for the hypothesis that endocrine active environmental compounds may contribute to the development of hypospadias. Additionally, regulation of these genes may have a role in formation of the urethra.

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.

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.

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.

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.

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.

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.

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.

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.

Red-eared slider turtles are genetically bipotential for sex determination. In this species, as in many other reptiles, incubation temperature of the egg determines gonadal sex. At higher incubation temperatures females are produced and increasing temperature appears to increase estrogen production in the embryonic brain. Treatment of eggs incubating at a male-producing temperature with exogenous estrogen causes ovaries to form. At a female-biased incubation temperature, prevention of estrogen biosynthesis or administration of nonaromatizable androgens results in the development of testes. In mammals, steroidogenic factor 1 (SF-1) regulates most genes required for estrogen biosynthesis, including aromatase. In both mammals and red-eared sliders, SF-1 is differentially expressed in males and females during gonadogenesis. We have examined both SF-1 gene expression and aromatase activity in embryos incubating at different temperatures and after manipulation to change the course of gonadal development. Our findings indicate a central role for SF-1 in enacting the effect of estrogen. Estrogen treatment directly or indirectly downregulates SF-1 and, ultimately, causes development of females. The inhibition of estrogen results in upregulation of SF-1 and male hatchlings. Thus, SF-1 may lie at the center of one molecular crossroad in male versus female differentiation of the red-eared slider., (Copyright 2001 Wiley-Liss, Inc.)

In the red-eared slider turtle, pesticides can alter expected sex outcomes, a major step in the inferred pathway of sex determination, and hatchling steroid physiology. Changes such as these can profoundly affect an organism's fitness. Other potential markers for effects on fitness include hatchling mass, hatchling use of maternal stores (residual yolk), and especially early hatchling growth rates. In the current study, red-eared slider turtles were exposed during embryogenesis to one of three compounds-chlordane, trans-Nonachlor, or p,p'-DDE-all of which affect sex determination in this species. Turtles were weighed at hatching, after a 28-d fasting period, and after 14 d of ad libitum feeding. All three compounds had some population-wide effects on changes in mass from time point to time point when compared to controls. From hatching to the end of the 28-d fast, turtles exposed in the egg to the mid-range doses of trans-Nonachlor and of p,p'-DDE lost mass and underwent a change in mass significantly different from controls. Additionally, turtles exposed to the two higher doses of trans-Nonachlor and the mid-range dose of chlordane grew significantly more than controls after 14 d of ad libitum feeding. These results point to a role for pesticides in endocrine disruption that extends beyond sex determination and sex development.

A variety of natural products and synthetic chemicals, known collectively as endocrine-disrupting compounds (EDCs), mimic or interfere with the mechanisms that govern vertebrate reproductive development and function. At present, research has focused on (i) the morphological and functional consequences of EDCs; (ii) identifying and determining the relative potencies of synthetic and steroidal compounds that have endocrine-disrupting effects; (iii) the mechanism of action of EDCs at the molecular level; and (iv) the recognition that in "real life," contamination usually reflects mixtures of EDCs. Future research must examine (i) the interactive nature of EDCs, particularly whether the threshold concept as developed in traditional toxicological research applies to these chemicals; (ii) when and how EDCs act at the physiological level, particularly how they may organize the neural substrates of reproductive physiology and behavior; (iii) the various effects these compounds have on different species, individuals, and even tissues; and (iv) how adaptations may evolve in natural populations with continued exposure to EDCs. Several predictions are offered that reflect these new perspectives. Specifically, (i) the threshold assumption will be found not to apply to EDCs because they mimic the actions of endogenous molecules (e.g., estrogen) critical to development; hence, the threshold is automatically exceeded with exposure. (ii) Behavior can compound and magnify the effects of EDCs over successive generations; that is, bioaccumulated EDCs inherited from the mother not only influence the morphological and physiological development of the offspring but also the offsprings' reproductive behavior as adults. This adult behavior, in turn, can have further consequences on the sexual development of their own young. (iii) The sensitivity of a species or an individual to a compound is related to species (individual)-typical concentrations of circulating gonadal steroid hormones. Related to this is the recent finding that alternate forms of the putative receptors are differentially distributed, thereby contributing to the different effects that have been observed. (iv) Except in extraordinary situations, populations often continue to exist in contaminated sites. One possible explanation for this observation that needs to be considered is that animals can rapidly adapt to the nature and level of contamination in their environment. It is unlikely that successive generations coincidentally become insensitive to gonadal steroid hormones fundamentally important as biological regulators of development and reproduction. Rather, adaptive alterations in the genes that encode steroid receptors may occur with chronic exposure to EDCs, allowing the sex hormone receptor to discriminate natural steroids from EDCs.

Gonadal sex in the red-eared slider turtle is determined by the incubation temperature that the embryo experiences during the mid-trimester of development. High temperatures result in female-biased sex ratios, and low temperatures produce male-biased sex ratios. The physiological equivalent of temperature appears to be a combination of the nature and abundance of steroidogenic enzymes and their products-including estradiol and its precursor, testosterone-and aromatase, the enzyme that converts testosterone to estradiol. Aromatase has been hypothesized to play a major role in the female developmental pathway in this species, and research in other species with temperature-dependent sex determination points to the brain as an organ that transduces the temperature signal into an aromatase response. In this study, we used a tritiated water assay to compare the pattern of estradiol biosynthesis at male- and female-producing temperatures in the brain and adrenal-kidney-gonad (AKG) through development. The pattern for both sexes in the AKG was one of increased activity after the temperature-sensitive period (TSP), but with no significant difference between sexes. In the brain, however, putative females exhibited a significantly higher level of aromatase activity than putative males at the beginning of the TSP, after which activity in both male and female brains decreased, dropping below detection in females before hatch. These results point to the brain as a site of aromatase response to temperature in this species, and they suggest that the product of aromatase activity, estradiol, may induce alterations in the neuroendocrine axis controlling gonadal sex steroid hormone production., (Copyright 2000 Academic Press.)

Incubation temperature determines gonadal sex in the red-eared slider turtle, Trachemys scripta. However, little is known about the long-term effects of incubation temperature on traits other than gonadal sex in this species. To investigate the hypothesis that incubation temperature (independent of gonadal sex) influences sex steroid levels after hatching, we incubated eggs of the red-eared slider turtle at three temperatures (26, 28.6, and 31 degrees C). We then measured plasma levels of dihydrotestosterone, estradiol, progesterone, and testosterone in 6-wk-old males from 26 degrees C and 28.6 degrees C eggs, and in 6-wk-old females from 28.6 degrees C and 31 degrees C eggs. We found that dihydrotestosterone levels were not influenced by incubation temperature or gonadal sex. However, progesterone levels were significantly higher in males from 26 degrees C eggs than in males from 28.6 degrees C eggs. In contrast, testosterone levels did not differ between males from 26 degrees C versus males from 28.6 degrees C eggs, but they were significantly higher in females from 28.6 degrees C than in females from 31 degrees C eggs. Progesterone and testosterone levels did not differ between males and females from 28.6 degrees C eggs. Temperature also influenced estradiol levels in both sexes, but the effects were enigmatic. We conclude that incubation temperature has lasting effects on sex steroid levels even after hatching.

Xenobiotics suspected of being estrogenic-the PCB aroclor 1242 and the pesticides toxaphene, dieldrin, p,p'-DDD, cis-Nonachlor, trans-Nonachlor, p,p'-DDE, and chlordane-were examined for their ability to override a male-producing incubation temperature and result in female hatchlings in the red-eared slider, a turtle with temperature-dependent sex determination. Compounds were assayed in the environmentally relevant concentrations detected in alligator eggs from Lake Apopka, Florida, singly, in concert with one another, and with estradiol. Compounds assayed alone and resulting in significant sex reversal were trans-Nonachlor, cis-Nonachlor, aroclor 1242, p,p'-DDE, and chlordane. When administered with estradiol, only one of the compounds, chlordane, caused sex reversal at significant levels. When applied together, however, the eight compounds assayed resulted in significant sex reversal., (Copyright 1999 Academic Press.)