Urothelial sonic hedgehog signaling plays an important role in bladder smooth muscle formation.

During bladder development, primitive mesenchyme differentiates into smooth muscle (SM) under the influence of urothelium. The gene(s) responsible for this process have not been elucidated. We propose that the Sonic hedgehog (Shh) signaling pathway is critical in bladder SM formation. Herein, we examine the role of the Shh-signaling pathway during SM differentiation in the embryonic mouse bladder. Genes in the Shh pathway and SM expression in mouse embryonic (E) bladders (E12.5, 13.5, and 14.5) were examined by immunohistochemistry (IHC), in situ hybridization, and reverse transcription polymerase chain reaction (RT-PCR). To examine the effects of disrupting Shh signaling, bladder tissues were isolated at E12.5 and E14.5, that is, before and after bladder SM induction. The embryonic bladders were cultured on membranes floating on medium with and without 10 muM of cyclopamine, an Shh inhibitor. After 3 days, SM expression was examined by assessing the following: SM alpha-actin (SMAA), SM gamma-actin (SMGA), SM-myosin heavy chain (SM-MHC), Patched, GLI1, bone morphogenic protein 4 (BMP4), and proliferating cell nuclear antigen (PCNA) by IHC and RT-PCR. SM-related genes and proteins were not expressed in E12.5 mouse embryonic bladder before SM differentiation, but were expressed by E13.5 when SM differentiation was initiated. Shh was expressed in the urothelium in E12.5 bladders. Shh-related gene expression at E12.5 was significantly higher than at E14.5. In cyclopamine-exposed cultures of E12.5 tissue, SMAA, SMGA, GLI1, and BMP4 gene expression was significantly decreased compared with controls, but PCNA gene expression did not change. In cyclopamine-exposed E14.5 cultures, SMGA and SM-MHC gene expression did not change compared with controls. Using an in vitro embryonic bladder culture model, we were able to define the kinetics of SM- and Shh-related gene expression. Cyclopamine inhibited detrusor SM actin induction, but did not inhibit SM-MHC induction. SMAA and SMGA genes appear to be induced by Shh-signaling pathways, but the SM-MHC gene is not. Based on Shh expression by urothelium and the effects of Shh inhibition on bladder SM induction, we hypothesize that urothelial-derived Shh orchestrates induction of SM in the fetal mouse bladder.

Embryonic exposure to low-dose 17beta-estradiol decreases fetal mass sex specifically in male mice and does not cause hypospadias.

BACKGROUND: Endocrine-disrupting compounds are synthetic and natural compounds in the environment that can alter endocrine-governed developmental processes. Among these are the natural estrogens genistein, a plant isoflavone, and 17beta-estradiol (E2), which is present in dietary animal products, such as eggs and meat. In addition, natural and synthetic steroids are administered to beef cattle to promote growth, and low levels of the estrogens can persist in the beef. Most previous work using E2 has involved injection; however, oral administration results in a different suite of hormone products following first-pass metabolism in the liver. METHODS: Low doses of E2 were administered orally to pregnant dams to determine embryonic effects. As end points of effects, we examined whether embryonic exposure produced hypospadias, an endocrine-linked abnormality of the male genitalia, and we assessed fetal mass. RESULTS: Male fetuses from the two highest dosage groups were significantly smaller than their control male counterparts, and males from the highest dosage group were also significantly smaller than control females. Control males were significantly larger than all females, and there was no difference in mass among control and treated females. Additionally, the E2 dose was inversely correlated with mass overall. No effect of these doses of E2 on hypospadias was seen. CONCLUSIONS: These results indicate a sex-specific fetal effect of low-dose, orally administered E2, which appears to exert androgen-inhibiting effects on mass in males.

The effects of coal tar based pavement sealer on amphibian development and metamorphosis.

Coal tar based pavement sealers are applied regularly to parking lots and contain significant levels of polycyclic aromatic hydrocarbons (PAHs). Recently a connection between elevated levels of PAHs in streams and storm water runoff from parking lots has been identified. We tested the hypothesis that coal tar based pavement sealers could alter the survival, growth, and development of amphibians using a model species, Xenopus laevis. Ten fertilized individuals were placed singly into containers containing one of four treatment groups: control, low, medium, and high (respective nominal concentrations 0, 3, 30, and 300 ppm TPAH). All of the individuals in the high exposure group died by the sixth day of exposure. By day 14 there were significant patterns of stunted growth (p<0.0001) and slower development (p=0.006) in the medium and high exposure groups relative to the control and low treatment groups. When the experiment ended on day 52 the control and low-dose individuals had achieved more advanced developmental stages than the medium group (p=0.0007). These data indicate that these commonly used coal tar based pavement sealers may potentially affect the amphibian taxa living in areas that receive storm water runoff.

Turtle sex determination assay: mass balance and responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,3',4,4',5-pentachlorobiphenyl.

Polyhalogenated hydrocarbons have been implicated in the anomalous sexual differentiation of mammals and reptiles. Here, a temperature-sensitive turtle sex determination assay using the red-eared slider (Trachemys scripta elegans) was used to determine the estrogenic or antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (PCB-126). Neither TCDD nor PCB-126 showed a statistically significant difference in the resulting sex ratios (Fisher's exact test, p < 0.45). As a consequence of the dosing technique (eggshell spotting), the shell retained 90 and 96% of the dose for PCB- 126 and TCDD, respectively, similar to retention of estradiol- 17beta. However, the dosing allowed transfer of sufficient chemical to achieve tissue concentrations that were greater than most concentrations reported for environmentally incurred residues. Similar relative mass distributions of PCB-126 and TCDD were observed in albumin (14-20%), yolk (55-70%), and embryo (16-25%). Relative concentration distributions in the embryo approached those in the yolk, 37 to 40% and 40 to 52%, respectively, while relative concentrations in the albumin remained at 11 to 20%. Lipid-normalized TCDD and PCB-126 concentrations were 30- to 40-fold greater in the embryo than in the yolk. It is hypothesized that nonpassive partitioning processes may have occurred in the embryo.