ABSTRACT
PURPOSE: We developed an organotypic genital tubercle culture system in vitro and used it to investigate the direct effects of the hyperestrogenic state on fetal mouse penile and urethral development. MATERIALS AND METHODS: Genital tubercles were dissected from embryonic day 14.5 C57B/L6 male mouse fetuses and cultured using an air-liquid interface on a microporous membrane support soaked in synthetic medium. Cultures were separated into 4 groups. Groups 1 to 3 were supplied with 10 nM dihydrotestosterone, estradiol and 10 nM dihydrotestosterone plus estradiol, respectively. Group 4 was cultured in hormone-free medium. After 36 to 72-hour culture morphological, histological, proliferation, apoptosis, androgen signaling and activating transcription factor 3 analyses were done. RESULTS: The physiological concentration of 10 nM dihydrotestosterone was essential for genital tubercle growth in vitro. Androgen induced growth and urethral development were significantly suppressed by high dose estrogen. Concurrently we observed increased apoptosis and decreased proliferation in the mesenchyma. Androgen signaling was disrupted and activating transcription factor 3, a factor related to hypospadias genesis, was up-regulated. CONCLUSIONS: High dose estrogen suppressed male genital tubercle development in vitro. The organotypic genital tubercle culture system in vitro consisting of urethral epithelial and mesenchymal cells can recapitulate the hormonal sensitivity of fetal penile and urethral development. This method is potentially useful for studying the effects of various factors, particularly endocrine disruptors.
Subject(s)
Estrogens/pharmacology , Genitalia, Male/drug effects , Genitalia, Male/embryology , Penis/drug effects , Penis/embryology , Urethra/drug effects , Urethra/embryology , Animals , Male , Mice , Organ Culture TechniquesABSTRACT
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.
Subject(s)
Abnormalities, Drug-Induced/etiology , Diet, Vegetarian/adverse effects , Endocrine Disruptors/toxicity , Fetus/drug effects , Food Contamination , Fungicides, Industrial/toxicity , Genistein/toxicity , Hypospadias/chemically induced , Oxazoles/toxicity , Pesticide Residues/toxicity , Phytoestrogens/toxicity , Abnormalities, Drug-Induced/embryology , Animals , Drug Interactions , Endocrine Disruptors/administration & dosage , Female , Fungicides, Industrial/administration & dosage , Genistein/administration & dosage , Gestational Age , Hypospadias/embryology , Male , Mice , Models, Animal , Oxazoles/administration & dosage , Phytoestrogens/administration & dosage , PregnancyABSTRACT
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.
Subject(s)
Activating Transcription Factor 3/genetics , Antigens, Differentiation/genetics , Hypospadias/genetics , Immediate-Early Proteins/genetics , Intercellular Signaling Peptides and Proteins/genetics , RNA/genetics , Up-Regulation , Activating Transcription Factor 3/metabolism , Antigens, Differentiation/metabolism , Cluster Analysis , Connective Tissue Growth Factor , Cysteine-Rich Protein 61 , Estrogens/metabolism , Foreskin/metabolism , Foreskin/pathology , Genetic Predisposition to Disease , Humans , Hypospadias/metabolism , Hypospadias/pathology , Immediate-Early Proteins/metabolism , Infant , Intercellular Signaling Peptides and Proteins/metabolism , Male , Microarray Analysis/methods , Receptors, Estrogen/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
AIM: To identify proteins that are differentially expressed in cells derived from normal and diseased tunica albuginea (TA) as related to Peyronie's disease (PD). METHODS: Cells with characteristics of fibroblasts were isolated from two tissue sources. Those from the plaque of patients with PD were designated as PT cells, and those from the normally-appearing TA of the same patients were designated as NT cells. Messenger RNAs of these cells were analyzed by real-time polymerase chain reaction (RT-PCR) for the expression of monocyte chemoattractant protein 1 (MCP-1). Crude protein lysates were analyzed by surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS) with IMAC30-Cu, CM10, and H50 chips. Each lysate was then separated into six fractions, which were further analyzed by SELDI-MS. RESULTS: RT- PCR analysis showed that PT cells expressed higher levels of MCP-1 than their counterpart NT cells. SELDI-MS analysis showed that the crude protein lysates of all four cell strains produced similar and reproducible protein profiles on IMAC30-Cu and CM10 chips. Additional SELDI-MS analyses with the fractionated lysates detected three proteins of 11.6 kDa, 14.5 kDa, 22.6 kDa that were upregulated in PT cells and two proteins of 6.3 kDa and 46.9 kDa that were downregulated in PT cells. CONCLUSION: MCP-1, which is often involved in tissue fibrosis, was expressed at higher levels in PT than that in NT cells. Five potential biomarkers for PD were identified by SELDI-MS analysis.
