Urine-derived exosomes and their role in modulating uroepithelial cells to prevent hypospadias.

PURPOSE: Urethral hypospadias, a common congenital malformation in males, is closely linked with disruptions in uroepithelial cell (UEC) processes. Evidence exists reporting that urine-derived exosomes (Urine-Exos) enhance UEC proliferation and regeneration, suggesting a potential role in preventing hypospadias. However, the specific influence of Urine-Exos on urethral hypospadias and the molecular mechanisms involved are not fully understood. This study focuses on investigating the capability of Urine-Exos to mitigate urethral hypospadias and aims to uncover the underlying molecular mechanisms. METHODS: Bioinformatics analysis was performed to identify key gene targets in Urine-Exos potentially involved in hypospadias. Subsequent in vitro and in vivo experiments were conducted to validate the regulatory effects of Urine-Exos on hypospadias. RESULTS: Bioinformatics screening revealed syndecan-1 (SDC1) as a potential pivotal gene for the prevention of hypospadias. In vitro experiments demonstrated that Urine-Exos enhanced the proliferation and migration of UECs by transferring SDC1 and inhibiting cell apoptosis. Notably, Urine-Exos upregulated ß-catenin expression through SDC1 transfer, further promoting UEC proliferation and migration. These findings were confirmed in a congenital hypospadias rat model induced by di(2-ethylhexyl) phthalate (DEHP). CONCLUSION: This study reveals the therapeutic potential of Urine-Exos in hypospadias, mediated by the SDC1/ß-catenin axis. Urine-Exos promote UEC proliferation and migration, thereby inhibiting the progression of hypospadias. These findings offer new insights and potential therapeutic targets for the management of congenital malformations.

Rab25 is involved in hypospadias via the ß1 integrin/EGFR pathway.

BACKGROUND: Hypospadias is a common congenital abnormality of the penile. Abnormal regulation of critical genes involved in urethral development leads to hypospadias. We used the Rab25-/- mice and foreskin fibroblasts transfected with lentivirus in vitro and in vivo to investigate the role of Rab25 in hypospadias. METHODS: The expression levels of various molecules in tissue samples and foreskin fibroblasts were confirmed using molecular biology methods (western blotting, PCR, immunohistochemistry, etc.). A scanning electron microscope (SEM) was used to visualize the external morphology of genital tubercles (GTs) of gestation day (GD) 18.5 male wild-type (WT) and Rab25-/- mice. RESULTS: An expanded distal cleft and V-shaped urethral opening were observed in GD 18.5 Rab25-/- mice. We demonstrated that Rab25 mediated hypospadias through the ß1 integrin/EGFR pathway. In addition, silencing Rab25 inhibited cell proliferation and migration and promoted apoptosis in the foreskin fibroblasts; Ki-67- and TUNEL-positive cells were mainly concentrated near the urethral seam. CONCLUSION: These findings suggest that Rab25 plays an essential role in hypospadias by activation of ß1 integrin/EGFR pathway, and Rab25 is a critical mediator of urethral seam formation in GD18.5 male fetal mice.

Maternal exposure to dibutyl phthalate (DBP) impairs angiogenesis and AR signalling pathway through suppression of TGFB1I1 in hypospadias offspring.

Early exposure to dibutyl phthalate (DBP) can cause hypospadias in newborn foetuses. However, the underlying molecular mechanism is not well defined. Aberrant angiogenesis is associated with various dysplasias including urogenital deficits. In vivo and in vitro angiogenesis assays showed reduced angiogenesis in the hypospadias group and DBP exposed group. RNA-sequencing analysis of DBP-treated HUVECs revealed decreased expression of transforming growth factor beta 1-induced transcript 1 (TGFB1I1) and a significantly enriched angiogenesis-associated pathway. Further experiments revealed that decreased TGFB1I1 expression was associated with disrupted tube formation and migration, which resulted in decreased angiogenesis. Functional assays revealed that the overexpression of TGFB1I1 promoted tube formation and migration of HUVECs in the DBP-treated group. Moreover, we showed that the transcription factor AR was regulated by TGFB1I1 through inhibiting its translocation from the cytoplasm to the nucleus. Together, our results identified TGFB1I1 as a component of aberrant angiogenesis in hypospadias rats and its interaction with AR might be a potential target for hypospadias development.

Di-n-butyl phthalate induces oversecretion of vascular endothelium-derived NAP-2 and promotes epithelial-mesenchymal transition of urothelial cells in newborn hypospadias rats.

Di-n-butyl phthalate (DBP) is a plasticizer commonly used in industrial production and is present in our daily life. It has been confirmed that DBP causes genitourinary malformations, especially hypospadias. However, the research of hypospadias mainly focusses on the genital tubercle in previous studies. In this study, we found DBP could affect the exocrine function of the vascular endothelium which disturb the development of genital nodules and induced hypospadias. We used cytokine array to find that vascular endothelium-derived NAP-2 may be a major abnormal secreted cytokine with biological functions. The transcriptomic sequencing analysis showed that abnormal activation of the RhoA/ROCK signaling pathway was the main reason for increased NAP-2 secretion. The expression levels of epithelial-mesenchymal transition (EMT) biomarkers and NAP-2 in hypospadias animal models were detected with Immunohistochemistry, Western blot, Immunofluorescence, and ELISA methods. The expression levels of NAP-2, RhoA/ROCK signaling pathway related proteins, reactive oxygen species (ROS) levels in HUVEC cells, EMT biomarkers and migration capacity of urothelial cells cocultured with HUVEC were measured with ELISA, flow cytometry, Western blot or Transwell assay for further cell experiments. The results showed that DBP leaded to NAP-2 oversecretion from vascular endothelium mainly rely on the activation of RhoA/ROCK signaling pathway and ROS accumulation. The RhoA/ROCK inhibitor fasudil could partially decrease ROS production, and both fasudil and N-acetyl-L-cysteine (NAC) could decrease NAP-2 secretion. Meanwhile, the oversecretion of NAP-2 from HUVEC in coculture system promoted EMT and migration capacity of urothelial cells, and TGF-ß inhibitor LY219761 could block the aberrant activation of EMT process. Therefore, it could be concluded that DBP increase NAP-2 secretion from vascular endothelium by RhoA/ROCK/ROS pathway, and further promote EMT in urothelial cells through TGF-ß pathway. This study provided a novel direction for studying the occurrence of hypospadias and may provide a hypospadias predictive marker in the future.

Role of epigenetics in the etiology of hypospadias through penile foreskin DNA methylation alterations.

Abnormal penile foreskin development in hypospadias is the most frequent genital malformation in male children, which has increased dramatically in recent decades. A number of environmental factors have been shown to be associated with hypospadias development. The current study investigated the role of epigenetics in the etiology of hypospadias and compared mild (distal), moderate (mid shaft), and severe (proximal) hypospadias. Penile foreskin samples were collected from hypospadias and non-hypospadias individuals to identify alterations in DNA methylation associated with hypospadias. Dramatic numbers of differential DNA methylation regions (DMRs) were observed in the mild hypospadias, with reduced numbers in moderate and low numbers in severe hypospadias. Atresia (cell loss) of the principal foreskin fibroblast is suspected to be a component of the disease etiology. A genome-wide (> 95%) epigenetic analysis was used and the genomic features of the DMRs identified. The DMR associated genes identified a number of novel hypospadias associated genes and pathways, as well as genes and networks known to be involved in hypospadias etiology. Observations demonstrate altered DNA methylation sites in penile foreskin is a component of hypospadias etiology. In addition, a potential role of environmental epigenetics and epigenetic inheritance in hypospadias disease etiology is suggested.

Quantification of the Androgen and Estrogen Receptors in the Penile Tissues of Hypospadias in Comparison with Normal Children.

INTRODUCTION: Hypospadias is a common congenital abnormality typified by a proximally placed ectopic urethral meatus along the ventral surface of the penis. Androgen receptor (AR) and estrogen receptor (ER) expression in the hypospadias tissues may be altered in hypospadias. METHODOLOGY: We evaluated by immunohistochemistry the AR and ER expression in 75 tissues from hypospadias repair, and compared this expression to that of tissue from 75 patients undergoing circumcision. We also compared the intensity of AR and ER expression between different severities of hypospadias. RESULTS: AR quantitative grading score decreased with severity of hypospadias, while the ER score increased as the hypospadias worsened, which was statistically significant (p-value <0.05). CONCLUSION: The penile tissue AR expression is decreased and ER expression is increased with increasing severity of hypospadias.

Erectile dysfunction in hypospadiac male adult rats induced by maternal exposure to di-n-butyl phthalate.

For the treatment of hypospadias, a significant number of studies focus on penile reconstruction. However, scant attention is given to sexual behavior of hypospadiac patients and underlying mechanisms. A rat model of hypospadias was constructed by maternal di-n-butyl phthalate (DBP) exposure (800 mg/kg/day by gavage during gestational days 14-18). Ten-week-old male rats with hypospadias undertook significantly decreased penis/body weight ratio, reduced testis/body weight ratio, lower serum testosterone level and thinner myelin sheath thickness of cavernosum nerves. Meanwhile, erectile dysfunction (ED) was found in hypospadiac rats, which showed significant increases in transforming growth factor-ß1 (TGF-ß1) protein expression and decreases in the expression of alpha smooth muscle actin (α-SMA) protein, neuronal and endothelial nitric oxide synthase protein (nNOS and eNOS). In addition, phosphorylated protein kinase B/protein kinase B (pAkt/Akt) ratios were remarkably lower, but the Bcl-2-associated X protein (Bax)/Bcl-2 ratios, caspase-3 protein expression, nuclear factor erythroid 2-related factor 2/ Kelch-like ECH-associated protein 1 (Nrf2/Keap-1) ratios, NAD(P)H dehydrogenase quinone 1(NQO1) protein expression and heme oxygenase-1 (HO-1) protein expression were higher in the hypospadias groups than the control group. Notably, ED is comorbid with hypospadias in cases. Penile fibrosis, testosterone deficiency, and endothelial dysfunction lead to ED in hypospadias induced by DBP eventually, which might be explained by activating Akt/Bad/Bax/caspase-3 pathway, Nrf2/Keap-1 pathway and suppressing NOS/cGMP pathway in penis.

Homozygosity for a novel INHA mutation in two male siblings with hypospadias, primary hypogonadism, and high-normal testicular volume.

Background: The human INHA gene encodes the inhibin subunit alpha protein, which is common to both inhibin A and B. The functional importance of inhibins in male sex development, sexual function, and reproduction remain largely unknown. Objective: We report for the first time two male siblings with homozygous INHAmutations. Methods: The medical files were examined for clinical, biochemical, and imaging data. Genetic analysis was performed using next-generation and Sanger sequencing methods. Results: Two brothers complained of gynecomastia, testicular pain, and had a history of hypospadias. Biochemistry revealed low serum testosterone, high gonadotropin and anti-Mullerian hormone, and very low/undetectable inhibin concentrations, where available. Both patients had azoospermia in the spermiogram. We have identified a homozygous 2 bp deletion (c.208_209delAG, R70Gfs*3) variant, which leads to a truncated INHA protein in both patients, and confirmed heterozygosity in the parents. The external genital development, pubertal onset and progression, reproductive functions, serum gonadotropins, and sex hormones of mother and father, who were heterozygous carriers of the identified mutation, were normal. Conclusion: Homozygosity for INHA mutations causes decreased prenatal and postnatal testosterone production and infertility in males, while the heterozygous female and male carriers of INHA mutations do not have any abnormality in sex development and reproduction.

Expression of mRNA vascular endothelial growth factor in hypospadias patients.

BACKGROUND: Hypospadias is a relatively common genital anomaly in humans, usually followed by inelastic dartos that causes penile chordee. Vascular endothelial growth factor (VEGF) is strongly linked to the viscoelasticity of tissues and their elastic phase. This study aimed to evaluate VEGF expressions in (1) fascia dartos between hypospadias and controls and (2) chordee severity. METHODS: This prospective cohort study involved 65 specimens from patients with hypospadias and ten specimens from controls. The samples were analyzed by quantitative real-time polymerase chain reaction (qPCR) for VEGF expression. RESULTS: The expressions of VEGF were not different between proximal and distal hypospadias patients and controls (fold change: distal - 0.25; fold change: proximal - 0.2; p = 0.664). The scaled expressions related to chordee severity were mild - 0.1; moderate 0.1; severe - 0.25 (p = 0.660). CONCLUSIONS: VEGF expressions might not affect the severity of hypospadias and chordee, implying the pathogenesis is complex involving many growth factors. Further study with a larger sample size is necessary to clarify and confirm our findings.

Urethral Tissue Reconstruction Using the Acellular Dermal Matrix Patch Modified with Collagen-Binding VEGF in Beagle Urethral Injury Models.

OBJECTIVES: Urethral tissue reconstruction for hypospadias is challenging for urologists. In this study, bovine acellular dermal matrix (ADM) patch loading with collagen-binding vascular endothelial growth factor (CBD-VEGF) was used to repair the urethral injury in beagles. METHODS: The safety and effectiveness of the scaffold implantation were carefully evaluated by comparing among the urethral injury control group, ADM implantation group, and ADM modified with CBD-VEGF implantation group during 6 months. Urodynamic examination, urethral angiography, and pathological examination were performed to evaluate the recovery of urethral tissue. RESULTS: Stricture, urethral diverticulum, and increased urethral closure pressure were observed in the control group. Fistula was observed in one animal in the ADM group. By contrast, no related complications or other adverse situations were observed in animals treated with ADM patch modified with CBD-VEGF. The average urethra diameter was significantly smaller in the control animals than in scaffold implantation groups. Pathological examination revealed more distribution of proliferative blood vessels in the animals treated with ADM modified with CBD-VEGF. CONCLUSIONS: Overall, ADM patches modified with CBD-VEGF demonstrated an optimized tissue repair performance in a way to increase tissue angiogenesis and maintain urethral function without inducing severe inflammation and scar formation.

Evaluation of surgical procedures of mouse urethra by visualization and the formation of fistula.

Visualization of the surgically operated tissues is vital to improve surgical model animals including mouse. Urological surgeries for urethra include series of fine manipulations to treat the increasing number of birth defects such as hypospadias. Hence visualization of the urethral status is vital. Inappropriate urethral surgical procedure often leads to the incomplete wound healing and subsequent formation of urethro-cutaneous fistula or urethral stricture. Application of indocyanine green mediated visualization of the urethra was first performed in the current study. Indocyanine green revealed the bladder but not the urethral status in mouse. Antegrade injection of contrast agent into the bladder enabled to detect the urethral status in vivo. The visualization of the leakage of contrast agent from the operated region was shown as the state of urethral fistula in the current hypospadias mouse model and urethral stricture was also revealed. A second trial for contrast agent was performed after the initial operation and a tendency of accelerated urethral stricture was observed. Thus, assessment of post-surgical conditions of urogenital tissues can be improved by the current analyses on the urethral status.

KCTD1 mutants in scalp­ear­nipple syndrome and AP­2α P59A in Char syndrome reciprocally abrogate their interactions, but can regulate Wnt/ß­catenin signaling.

Potassium­channel tetramerization-domain-containing 1 (KCTD1) mutations are reported to result in scalp­ear­nipple syndrome. These mutations occur in the conserved broad­complex, tramtrack and bric a brac domain, which is associated with inhibited transcriptional activity. However, the mechanisms of KCTD1 mutants have not previously been elucidated; thus, the present study aimed to investigate whether KCTD1 mutants affect their interaction with transcription factor AP­2α and their regulation of the Wnt pathway. Results from the present study demonstrated that none of the ten KCTD1 mutants had an inhibitory effect on the transcriptional activity of AP­2α. Co­immunoprecipitation assays demonstrated that certain mutants exhibited changeable localization compared with the nuclear localization of wild­type KCTD1, but no KCTD1 mutant interacted with AP­2α. Almost all KCTD1 mutants, except KCTD1 A30E and H33Q, exhibited differential inhibitory effects on regulating TOPFLASH luciferase reporter activity. In addition, the interaction region of KCTD1 to the PY motif (amino acids 59­62) in AP­2α was identified. KCTD1 exhibited no suppressive effects on the transcriptional activity of the AP­2α P59A mutant, resulting in Char syndrome, a genetic disorder characterized by a distinctive facial appearance, heart defect and hand abnormalities, by altered protein cellular localization that abolished protein interactions. However, the P59A, P60A, P61R and 4A AP­2α mutants inhibited TOPFLASH reporter activity. Moreover, AP­2α and KCTD1 inhibited ß­catenin expression levels and SW480 cell viability. The present study thus identified a putative mechanism of disease­related KCTD1 mutants and AP­2α mutants by disrupting their interaction with the wildtype proteins AP­2α and KCTD1 and influencing the regulation of the Wnt/ß­catenin pathway.

Expression of androgen receptor and estrogen receptor-beta in foreskin tissue of hypospadiac and healthy boys. / Expresión de receptores androgénicos y estrogénicos beta en la piel prepucial de hipospadias y niños sanos.

OBJECTIVES: This prospective study aimed to investigate the expression of the androgen receptor(AR) and the estrogen receptor-ß (ER-ß) in foreskint issues in boys with and without distal hypospadias. METHODS: Thirty boys with distal hypospadias were evaluated. Fifteen boys who under went elective circumcision over a period of 18 months served as the control group. The presence of AR and ER-ß in foresk in tissues was investigated immunohistochemically. RESULTS: The percentages of AR in epithelial cells were18.9 ± 27.3% in the hypospadias group and 3.3 ±5.3% in the circumcision group, and the difference betweent he groups was significant (p=0.034). Of the stromal cells, 19.5 ± 26.8% in the hypospadias group and2.6 ± 4.4% in the circumcision group were positive lystained for AR (p=0.004). In the hypospadias group,significantly higher stromal cell percentage of ER-ß was found compared to that in the control group (24± 24.5% and 1.3 ± 1.1%, respectively, p<0.001). Moreover, epithelial cell percentage of ER-ß was higher in the hypospadias group than that in the control group,and the respective values were 6.8 ± 10.1% and 0.9± 1.3% (p<0.0001)CONCLUSION: The percent of AR and ER-ß expression were higher in hypospadias-affected foresk in than in the normal foreskin. Whether the normal function of these receptors reveals, there is a need for more detailed studies.

OBJETIVO: Este estudio prospectivo que pretende investigar la expresión del receptor androgénicoy estrogénico en la piel prepucial en niños con y sin hipospadias distal.MÉTODOS: Treinta niños con hipospadias distal fueron evaluados. 15 niños recibieron una circuncisión electiva en un periodo de 18 meses y sirvieron de grupo control.La presencia de RA y RE-ß en la piel prepucial se investigo por immunohistoquimica. RESULTADOS: El porcentaje de expresión del receptor androgenico en células epiteliales fue de 18,9+/-27,3% en el grupo hipospadias y 3,3+/- 5,3% en el grupo de circuncisión. La diferencia entre ambos grupos fue significativo (p=0,034). En las células estromales,19,5+/- 26,8% en el grupo hipospadias y 2,6+/-4,4% en el grupo circuncisión fueron positivos para el RA (p=0,004). En el grupo de hipospadias, un porcentaje mas elevdo de expresión de RE-b ß se evidencio en comparación al grupo control (24+/-24,5%y 1,3+/-1,1, respectivamente, pel porcentaje de células epiteliales con RE-ß fue superior en el grupo hipospadias que en el grupo control; los valores respectivos fueron 6,8+/-10,1% y 0,9+/-1,3%(p<0,0001).CONCLUSIÓN: En este estudio se sugiere que la expresiónde RA y RE fueron superiores en el grupo conhipospadias que en piel prepucial normal. Se requierenmas estudios para determinar el significado de esta expresión.

Expression of androgen receptor and estrogen receptor-beta in foreskin tissue of hypospadiac and healthy boys / Expresión de receptores androgénicos y estrogénicos beta en la piel prepucial de hipospadias y niños sanos

OBJECTIVES: This prospective study aimed to investigate the expression of the androgen receptor(AR) and the estrogen receptor-β (ER-β) in foreskint issues in boys with and without distal hypospadias. METHODS: Thirty boys with distal hypospadias were evaluated. Fifteen boys who under went elective circumcision over a period of 18 months served as the control group. The presence of AR and ER-Beta in foresk in tissues was investigated immunohistochemically. RESULTS: The percentages of AR in epithelial cells were18.9 ± 27.3% in the hypospadias group and 3.3 ±5.3% in the circumcision group, and the difference betweent he groups was significant (p = 0.034). Of the stromal cells, 19.5 ± 26.8% in the hypospadias group and2.6 ± 4.4% in the circumcision group were positive lystained for AR (p = 0.004). In the hypospadias group,significantly higher stromal cell percentage of ER-β was found compared to that in the control group (24 ± 24.5% and 1.3 ± 1.1%, respectively, p < 0.001). Moreover, epithelial cell percentage of ER-β was higher in the hypospadias group than that in the control group,and the respective values were 6.8 ± 10.1% and 0.9 ± 1.3% (p < 0.0001). CONCLUSION: The percent of AR and ER-Beta expression were higher in hypospadias-affected foresk in than in the normal foreskin. Whether the normal function of these receptors reveals, there is a need for more detailed studies

OBJETIVO: Este estudio prospectivo que pretende investigar la expresión del receptor androgénicoy estrogénico en la piel prepucial en niños con y sin hipospadias distal. MÉTODOS: Treinta niños con hipospadias distal fueron evaluados. 15 niños recibieron una circuncisión electiva en un periodo de 18 meses y sirvieron de grupo control.La presencia de RA y RE-Beta en la piel prepucial se investigó por immunohistoquimica. RESULTADOS: El porcentaje de expresión del receptor androgenico en células epiteliales fue de 18,9 +/- 27,3% en el grupo hipospadias y 3,3 +/- 5,3% en el grupo de circuncisión. La diferencia entre ambos grupos fue significativo (p = 0,034). En las células estromales,19,5 +/- 26,8% en el grupo hipospadias y 2,6+/-4,4% en el grupo circuncisión fueron positivos para el RA (p = 0,004). En el grupo de hipospadias, un porcentaje más elevdo de expresión de RE-b β se evidencio en comparación al grupo control (24 +/- 24,5%y 1,3 +/- 1,1, respectivamente, pel porcentaje de células epiteliales con RE-Beta fue superior en el grupo hipospadias que en el grupo control; los valores respectivos fueron 6,8 +/- 10,1% y 0,9 +/- 1,3%(p < 0,0001). CONCLUSIÓN: En este estudio se sugiere que la expresiónde RA y RE fueron superiores en el grupo conhipospadias que en piel prepucial normal. Se requierenmas estudios para determinar el significado de esta expresión

Di-n-butyl phthalate (DBP) reduces epithelial-mesenchymal transition via IP3R in hypospadias during maternal exposure.

OBJECTIVE: This study focused on the oxidative stress effect of di-n-butyl phthalate (DBP) on development of the urinary system. METHODS: We examined the mRNA expression of genital tubercle (GT) in control and DBP induced hypospadias group by Affymetrix Rat 230 2.0 Array. Real-time PCR and Western Blot were used to detect the protein and mRNA expression levels of inositol-1,4,5-triphate-receptor (IP3R) and epithelial-mesenchymal-transition (EMT)-related molecular markers, such as E-cadherin, ß-Catenin, Snail, N-cadherin, in the GT of hypospadiac male rats and controls. The results of array were further confirmed in vitro. The changes of intracellular calcium concentration in urethral epithelial cells were detected by Fluo-3-AM before and after DBP treatment. The levels of reactive oxygen species (ROS) in urethral epithelial cells were measured by DCFH-DA with different concentrations of DBP (0, 1, 10, 100 µmol/L) treatment. RESULTS: The mRNA expression profiles of GT in control and DBP induced hypospadias group showed high expression of IP3R and the abnormalities of EMT. Compared to the control group, the expression levels of IP3R, E-cadherin and ß-Catenin increased at both the protein and mRNA levels. However the expression levels of Snail and N-cadherin decreased. The intracellular calcium concentration increased significantly after DBP treatment. The effect of DBP on urethral epithelial cells was linked to the generation of oxidative stress. CONCLUSION: DBP can influence the development of GT through its oxidative stress effect, which significantly increases the concentration of calcium and inhibits EMT in urethral epithelial cells, and block the fusion process of urethral groove, causing the occurrence of hypospadias. This study provides a new understanding of DBP's molecular mechanisms on hypospadias and may lead to new treatment strategies for the disease.

TGF-ß1 relieves epithelial-mesenchymal transition reduction in hypospadias induced by DEHP in rats.

BACKGROUNDS: To investigate the potential mechanism of hypospadias induced by DEHP in rats to reveal the preventative effect of TGF-ß1 in hypospadias induced by DEHP via the reduction of EMT. METHODS: Time-mated Sprague-Dawley rats underwent cesarean section, and the penises of male pups were collected after exposure to corn oil or DEHP to establish a rat model of hypospadias and to further study the molecular mechanisms of hypospadias in vivo. In addition, the penises were cultured and treated with MEHP or MEHP+TGF-ß1 in vitro. Subsequently, histomorphology and elements in TGF-ß/Smad signaling pathway changes were evaluated using scanning electron microscopy, immunofluorescence, polymerase chain reaction, and western blot. RESULTS: The development of rat penis and urethral seam fusion were delayed after the treatment with DEHP in vivo or MEHP in vitro compared with the Control group. Moreover, TGF-ß1, Smad2/Smad3, and the mesenchymal biomarkers, including α-SMA, N-cadherin, and Vimentin, were decreased. However, the epithelial biomarkers, including E-cadherin, ZO-1, ß-catenin, and occludin, were increased. In addition, TGF-ß1 could relieve all of the above changes. CONCLUSION: Gestational DEHP exposure could lead to hypospadias by reducing urethral EMT. Moreover, TGF-ß1 could prevent it by regenerating EMT through activating the TGF-ß/Smad signal pathway.

FGF8, FGF10 and FGF receptor 2 in foreskin of children with hypospadias: an analysis of immunohistochemical expression patterns and gene transcription.

INTRODUCTION: Fibroblast growth factors (FGFs) play a crucial role in early embryogenesis of the genital tubercle and are involved in the development of hypospadias, affecting both endo- and ectodermally derived tissues. It was hypothesized that expression of FGFs could be qualitatively or quantitatively altered in skin of children with hypospadias. OBJECTIVE: The objective of the study was to investigate expression patterns and transcription levels of FGF8, FGF10, and FGF Receptor 2 (FGFR2) in patients with hypospadias compared to normal controls. PATIENTS AND METHODS: Skin samples from the ventro-lateral aspect of the foreskin of 32 patients with hypospadias (17 distal and 15 proximal, mean age 25 months) and 10 normal foreskin samples (mean age 77 months) were analyzed by immunohistochemistry. Staining, localization, and distribution of positive cells in epidermis and dermis were categorized independently by two researchers. Complementary DNA (cDNA) samples prepared from messenger RNA (mRNA) isolates of the same samples were analyzed by quantitative polymerase chain reaction (qPCR), comparing expressions of FGF8, FGF10, and FGFR2 with loading controls. RESULTS: Patients with hypospadias consistently showed aberrant immunohistochemical staining patterns for FGF8/FGF10/FGFR2 in epidermis and dermis compared to patients without penile malformation (p < 0.01 for all markers). qPCR displayed no difference in expression levels on mRNA level (FGFR2 p = 0.44, FGF8 p = 0.77, and FGF10 p = 0.17) comparing normal foreskin with foreskin from patients with hypospadias. Figure. DISCUSSION: The results point at an impact of FGF signaling during embryological development of hypospadias on skin, as an ectodermally derived tissue. Similar to the urethral development, this might be a result of mesothelial-epithelial interactions. The differing expression patterns in immunohistochemistry are not matched by a quantitative difference in marker expression on the mRNA level, putatively caused by post-translational modifications or alterations of the downstream pathway. FGFs, particularly FGF10 and FGFR2, are critically involved in wound healing. CONCLUSIONS: There are significant differences in localization and distribution of FGF8, FGF10, and FGFR2 in comparisons of normal foreskin to foreskin of patients with hypospadias, whereas there is no difference in the quantitative expression of these markers on the mRNA level. This confirms the notion that penile skin is affected as well by the embryological aberrations during the embryogenesis of hypospadias.

Di-n-butyl phthalate induced autophagy of uroepithelial cells via inhibition of hedgehog signaling in newborn male hypospadias rats.

Maternal exposure to di-n-butyl phthalate (DBP) induces hypospadias via regulation of autophagy in uroepithelial cells. Here, we use gene express analysis to explore the underlying molecular mechanisms. Pregnant rats received DBP orally at a dose of 750 mg/kg/day during gestational days 14-18. Gene expression analysis showed an increased expression of the hedgehog interacting protein (HhIP) gene. In DBP-induced hypospadiac male offspring, immunohistochemistry (IHC) staining and Western blot analysis confirmed increased expression of the HhIP protein and inhibited hedgehog signaling. in vitro experiments suggest the involvement of the reactive oxygen species (ROS)-HhIP-Gli1-autophagy axis in DBP-treated primary rat urethral epithelial cells. Taken together, our findings show that prenatal exposure to DBP induces abnormal hedgehog signaling and autophagy in uroepithelial cells that may play important roles in the development of hypospadias.

A critical role for estrogen signaling in penis development.

Hypospadias, a developmental defect of the penis, is one of the most common congenital malformations in humans. Its incidence has rapidly increased over recent decades, and this has been largely attributed to our increased exposure to endocrine-disrupting chemicals. Penis development is primarily an androgen-driven process; however, estrogen and xenoestrogens are known to affect penis development in both humans and mice. Here, we investigated the role of estrogen in the developing penis. Using a novel penis culture system, we showed that exogenous estrogen directly targets the developing penis in utero to cause hypospadias. In addition, we also uncovered an unexpected endogenous role for estrogen in normal postnatal penis development and showed that a loss of estrogen signaling results in a mild hypospadias phenotype, the most common manifestation of this disease in humans. Our findings demonstrated that both androgen and estrogen signaling are intrinsically required for normal urethral closure. These findings confirmed that penis development is not an entirely androgen-driven process but one in which endogenous estrogen signaling also plays a critical role.-Govers, L. C., Phillips, T. R., Mattiske, D. M., Rashoo, N., Black, J. R., Sinclair, A., Baskin, L. S., Risbridger, G. P., Pask, A. J. A critical role for estrogen signaling in penis development.

The role of genetic variation in DGKK on moderate and severe hypospadias.

BACKGROUND: Recent genome-wide association studies of hypospadias have implicated the role of genetic variants in or near the diacylglycerol kinase kappa (DGKK) gene. However, these variants are largely identified among samples of mild and moderate hypospadias cases. Therefore, we evaluated previously identified DGKK variants among second- and third-degree hypospadias cases and controls recruited in Arkansas, a state characterized by a high birth prevalence of hypospadias. METHODS: Second- and third-degree hypospadias non-Hispanic white cases (n = 36 and n = 9, respectively) and controls (n = 45) were recruited at Arkansas Children's Hospital. Preputial tissue was collected on cases and controls between 2013 and 2017. Cases and controls were genotyped using the Illumina Infinium Global Screening Array. We used logistic regression models to assess the association of genotyped and imputed genetic variants mapped to the DGKK region with second- and third-degree hypospadias. RESULTS: All families self-reported as non-Hispanic white and genetic principal component analyses did not demonstrate evidence of population stratification. Five DGKK variants previously reported as associated with hypospadias were identified in the genotype data. None of the variants were associated with second- or third-degree hypospadias (range of odds ratios = 0.7-0.9, all p > .05). CONCLUSIONS: In our analyses, genetic variation in DGKK does not play a role in the development of moderate and severe hypospadias. Our findings provide support to the etiologic heterogeneity of hypospadias by all classifications of severity.