Genital Tubercle Research Articles

7850 Developmental ZNHIT3 expression in mice embryos in Mayer-Rokitansky-Küster-Hauser (MRKH) Syndrome related organs

Abstract Disclosure: D. Kira: None. B. Eroglu, MS, PhD: None. Z. Hawkins: None. L.P. Chorich: None. S. Brakta: None. L.C. Layman: None. Introduction: MRKH is a severe form of the Mullerian duct (MD) anomaly where the upper 2/3 of the vagina and the uterus fail to develop. Individuals with MRKH still have normally functioning ovaries and normal chromosomes. MRKH may be isolated Mullerian aplasia (type 1) or associated with renal, skeletal, cardiac, and/or auditory dysfunction (type 2). The etiology of MRKH syndrome remains unknown for most probands, although a genetic contribution is likely. Most evidence suggests autosomal dominant inheritance with incomplete penetrance. WNT4 and HNF1B pathogenic variants have been identified in some MRKH individuals. Recently, our lab identified heterozygous (HTZ) ZNHIT3 loss of function variants in persons with MRKH, Both variants predict functional changes in the ZNHIT3 C-terminus and impair protein expression in vitro. Previously, other investigators found biallelic variants in the N-terminus of ZNHIT3 in individuals with PEHO syndrome, a severe autosomal recessive neurodevelopmental disorder characterized by extreme cerebellar atrophy. We are currently characterizing a novel Znhit3 knockout (KO) mouse model, in which homozygous KO mice exhibit embryonic lethality. We have demonstrated ZNHIT3 RNA expression in the human uterus, heart, kidney, and skeleton, but developmental expression of ZNHIT3 in wild type (WT) animals has not been studied. In the WT mouse embryo, the kidney forms between E8.0 and E11, while the MDs form through invagination of epithelium ∼E11.5 and extend along the Wolffian duct until reaching the cloaca at ∼E13.5. Hypothesis: We hypothesize that ZNHIT3 is involved in MD development and expressed in the urogenital system and MRKH-related organs during embryonic development of WT mice. Method: Timed mating of WT mice produced embryos for analysis. Embryos were dissected out at E11.5, 12.5 and 13.5, fixed in 4% PFA, embedded in paraffin, and sectioned sagittally at 5mm thickness. H&E staining was done to identify sections showing kidney, genital tubercle, skeleton and cerebellum. Immunohistochemistry was also done using a ZNHIT3 antirabbit antibody and DAPI as nuclear counter stain. Imaging was performed using the Keyence fluorescence microscope (BZ-X800) at 10x and 40x magnification. Results: At all 3 time points, ZNHIT3 protein expression was observed in the developing urogenital organs including kidneys and bipotential gonads, as well as in vertebral bones and cerebellum. Heart protein expression studies are ongoing. Conclusion: ZNHIT3 protein expression is seen as early as E11.5 and is present in MRKH-related organs, suggesting it may play a role in the development of urogenital and MRKH-related organs. These findings provide the basis to compare expression in the Znhit3 KO mouse, a mouse model for MRKH. Presentation: 6/2/2024

Prenatal exposure to di-n-butyl phthalate promotes RIPK1-regulated necroptosis of uroepithelial cells and induces hypospadias through the epithelial-mesenchymal transition process in newborn male rats

Maternal exposure to di-n-butyl phthalate (DBP) has been linked to the induction of hypospadias; however, the underlying mechanism remains unclear. Necroptosis is reported to be implicated in developmental malformations. This study aimed to investigate the underlying mechanism of necroptosis in the development of hypospadias. DBP was dissolved in corn oil, and pregnant rats were administered a precisely measured dose of DBP (750mg/kg/day) via gastric intubation from gestation day 14 to 18. Control rats received only corn oil. The day of birth was considered postnatal day (PND) 1. Male hypospadias rats were identified on PND 7. Genital tubercle tissues were collected and stored at −80°C for subsequent PCR analysis, cryopreserved in liquid nitrogen for western blot, or fixed in formalin for immunohistochemistry (IHC) staining. IHC staining and western blot analysis revealed increased expression of RIPK1 and necroptosis markers in genital tubercle (GT) tissue compared to the control group. Additionally, higher levels of EMT and impaired androgen receptor expression were observed in GT tissue. Exposure to increased DBP concentrations in rat primary uroepithelial cells (PUCs) led to elevated ROS production. Necroptosis markers and EMT expression levels were upregulated in PUCs following DBP incubation. Notably, treatment with DBP combined with necrostatin-1, a necroptosis inhibitor, reduced the expression of EMT markers and ROS production compared to DBP treatment alone. In vitro studies further revealed that DBP-induced necroptosis promoted the degradation of E-cadherin through the ubiquitin-proteasome pathway in PUCs. Our findings suggest that maternal exposure to DBP promotes necroptosis in uroepithelial cells by elevating ROS level and EMT status. Thus, necroptosis may play an essential role in the development of hypospadias.

The Predictive Accuracy of Anogenital Distance and Genital Tubercle Angle for First-Trimester Fetal Sex Determination.

Early identification of fetal gender is crucial for managing gender-linked genetic disorders. This study aimed to evaluate the predictive performance of anogenital distance (AGD) and genital tubercle angle (GTA) for fetal sex determination during the first trimester. A multicenter retrospective cohort study was conducted on 312 fetal cases between 11 and 13 + 6 weeks of gestation from two tertiary hospitals. AGD and GTA measurements were taken from midsagittal plane images using ultrasound, with intra- and inter-reader reproducibility assessed. Binomial logistic regression and ROC curve analysis were employed to determine the diagnostic performance and optimal cutoff points. AGD had a mean of 7.16 mm in male fetuses and 4.42 mm in female fetuses, with a sensitivity of 88.8%, specificity of 94.4%, and an area under the ROC curve (AUC) of 0.931 (95% CI: 0.899-0.962) using 5.74 mm as a cutoff point. For GTA, the mean was 35.90 degrees in males and 21.57 degrees in females, with a sensitivity of 92%, specificity of 84.7%, and an AUC of 0.932 (95% CI: 0.904-0.961) using 28.32 degrees as a cutoff point. The reproducibility results were excellent for AGD (intra-operator ICC = 0.938, inter-operator ICC = 0.871) and moderate for GTA (intra-operator ICC = 0.895, inter-operator ICC = 0.695). The findings suggest that AGD and GTA are reliable markers for early fetal sex determination, with AGD showing higher reproducibility. The findings highlight the feasibility and accuracy of these non-invasive sonographic markers and their potential usefulness in guiding timely interventions and enhancing the management of gender-linked genetic conditions.

Transvaginal three-dimensional ultrasound assessment of embryonic genital tubercle at 8-10+6weeks of gestation.

To assess embryonic genital tubercle using transvaginal three-dimensional (3D) ultrasound at 8-10+6weeks of gestation. One-hundred and two transvaginal 3D ultrasound scans were performed for first-trimester dating at 8-10+6weeks of gestation. The genital tubercle angle (GTA) and genital tubercle length (GTL) were measured with a mid-sagittal view of the embryo using the 3D ultrasound multiplanar mode. Intra- and inter-observer agreements regarding GTA and GTL were also assessed with Bland-Altman plots and intra- and inter-correlation coefficients. There were no significant differences in GTA between male and female embryos at 8, 9, 10weeks, or 8-10+6weeks of gestation, respectively. There were also no significant differences in GTL between male and female embryos at 8, 9, 10weeks, or 8-10+6weeks of gestation, respectively. However, GTL increased linearly with advancing gestation (r=0.8276, p<0.00001). Mean GTL (SD) values at 8, 9, and 10weeks were 0.833 mm (0.274), 1.623 mm (0.262), and 2.152 mm (0.420), respectively (p<0.001). Intra- and inter-reproducibilities of GTA and GTL were excellent. The intra- and inter-correlation coefficients of GTA and GTL were 0.964 and 0.995, and 0.996 and 0.9933, respectively. The genital tubercle could be identified using transvaginal 3D ultrasound at 8-10+6weeks of gestation. However, sex differentiation could not be performed at this age. The genital tubercle linearly developed with advancing gestation during the mid-first trimester of pregnancy.

Rodent anogenital distance recommendations.

Measurement of rat anogenital distance (AGD) dates to at least 1912. Increased interest in endocrine disrupting chemicals and the use of AGD as a biomarker for fetal androgen effects have increased the number of studies with this endpoint in recent decades. A literature review revealed different landmarks, methods of measurement, and methods to adjust for body weight differences. AGD is often reported to hundredths of millimeters and as such, deserves precision in all these aspects. This paper presents recommendations for the measurement and analysis of rodent AGD. Literature and regulatory guidance documents that mentioned or measured rodent AGD were reviewed. Four adjustment methods were evaluated using available online data from three rat studies each with two generations of offspring. Tabulation of studies reveals that species/stocks and time of data collection, but more importantly anatomical landmarks and methods of measurement have produced a variety of results which are difficult to compare. Not all studies have adjusted for test article effects on body weight (and thus size). The four adjustment methods were fairly comparable. Recommendations are as follows. A microscopic method should be used to measure AGD of late rodent fetuses and early postnatal pups. The caudal edge of the genital tubercle and the cranial edge of the anus are clear and identifiable landmarks. The simplest adjustment is to divide individual AGDs by the cube root of animals' body weight. These recommendations will help ensure data consistency and accuracy, and facilitate meaningful comparisons across laboratories and chemical classes.

Phenotypic variability and management of patients with mosaic monosomy X and Y chromosome material: a case series

Backgroundwe aim to discuss the origin and the differences of the phenotypic features and the management care of rare form of disorder of sex development due to Mosaic monosomy X and Y chromosome materiel.MethodsWe report our experience with patients harboring mosaic monosomy X and Y chromosome material diagnosed by blood cells karyotypes and cared for in our department from 2005 to 2022.ResultsWe have included five infants in our study. The current average age was 8 years. In four cases, the diagnosis was still after born and it was at the age of 15 years in one case. Physical examination revealed a variable degree of virilization, ranging from a normal male phallus with unilateral ectopic gonad to ambiguous with a genital tubercle and bilateral not palpable gonads in four cases and normal female external genitalia in patient 5. Karyotype found 45, X/46, XY mosaicism in patient 1 and 2 and 45, X/46, X, der (Y) mosaicism in patient 3, 4 and 5. Three cases were assigned to male gender and two cases were assigned to female. After radiologic and histologic exploration, four patients had been explored by laparoscopy to perform gonadectomy in two cases and Mullerian derivative resection in the other. Urethroplasty was done in two cases of posterior hypospadias. Gender identity was concordant with the sex of assignment at birth in only 3 cases.ConclusionBecause of the phenotypic heterogeneity of this sexual disorders and the variability of its management care, then the decision should rely on a multidisciplinary team approach.

Prenatal post-implantation development of collared peccaries (Pecari tajacu Linnaeus, 1758).

Given the importance of information on intrauterine development in diagnosing anomalies in the gestational development of the species for the development of assisted reproduction technologies as well as understanding the autonomy and responsiveness of the newborn, the aim of the present study was to describe the external morphology of collared peccary conceptuses. For this study, two conceptuses were used per gestational age of 25-120 days post-copulation (dpc) and neonates with 145 dpc, totalling 22 animals. Females were euthanised, and embryos/foetuses were examined, measured, and photographed. During the first third of the gestational period (25-50 dpc, n = 8), a marked body curvature, brain vesicles, somites, internal organs, placid lens, auricular protrusion and limb buds are noted. In the second third of the gestational period (51-100 dpc, n = 10), foetuses lose their body curvature, displaying greater anatomical definition, including skeletal, external ears, nostrils, eyelids and tactile hair formation and cranial suture closure. In addition, dorsal scent gland and genital tubercle differentiation were visualized at 50 days post-copulation. In the third of the gestational period (101-145 dpc, n = 4), the organs become completely formed, alongside skin darkening, eyelid opening, dental eruption, dorsal odorous gland development, sexual organ externalization, and fanero attachment development. These data allowed for the construction of a prenatal growth curve, providing comparative anatomy information for ungulates and further contributing towards rational reproductive management and reproductive biotechnologies for this species.

Neurovascular anatomy of the developing human fetal penis and clitoris.

The human penile and clitoral development begins from a morphologically indifferent genital tubercle. Under the influence of androgen, the genital tubercle forms the penis by forming a tubular urethra within the penile shaft. Without the effect of the androgen, the genital tubercle differentiates into the clitoris, and a lack of formation of the urethra within the clitoris is observed. Even though there are similarities during the development of the glans penis and glans clitoris, the complex canalization occurring along the penile shaft eventually leads to a morphological difference between the penis and clitoris. Based on the morphological differences, the main goal of this study was to define the vascular and neuronal anatomy of the developing penis and clitoris between 8 and 12 weeks of gestation using laser scanning confocal microscopy. Our results demonstrated there is a co-expression of CD31, which is an endothelial cell marker, and PGP9.5, which is a neuronal marker in the penis where the fusion is actively occurring at the ventral shaft. We also identified a unique anatomical structure for the first time, the clitoral ridge, which is a fetal structure running along the clitoral shaft in the vestibular groove. Contrary to previous anatomical findings which indicate that the neurovascular distribution in the developing penis and clitoris is similar, in this study, laser scanning confocal microscopy enabled us to demonstrate finer differences in the neurovascular anatomy between the penis and clitoris.

Rab25 is involved in hypospadias via the β1 integrin/EGFR pathway

BackgroundHypospadias 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. MethodsThe 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. ResultsAn expanded distal cleft and V-shaped urethral opening were observed in GD 18.5 Rab25−/− mice. We demonstrated that Rab25 mediated hypospadias through the β1 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. ConclusionThese findings suggest that Rab25 plays an essential role in hypospadias by activation of β1 integrin/EGFR pathway, and Rab25 is a critical mediator of urethral seam formation in GD18.5 male fetal mice.

P-51 Association of Bardet-Biedl syndrome with differences of sex development and pituitary hypoplasia

Abstract Introduction Bardet-Biedl syndrome (BBS) is a rare autosomal recessive genetic disorder involving hypogonadism manifested by cryptorchidism, micropenis and/or delayed puberty Clinical Case We report the case of a 13-year-old child, who was consulted for obesity and whose physical examination showed major signs of SBB: global obesity, polydactyly, learning difficulties and visual disorders. Genital examination revealed a 1 cm genital tubercle with a single orifice and two genital swelling without palpable gonads. Biological investigations revealed hypogonadotropic hypogonadism, corticotropic insufficiency, low AMH with a non- stimulable HCG test and a 46XY male karyotype. Pelvic MRI did not show any gonads or internal genitalia but noted ectopy and malrotation of the right kidney. The diagnosis of a testicular regression syndrome is very likely in this case. At the fundus photography: presence of bilateral retinal dystrophy with high myopia. At the hypothalamohypophyseal level: a strong pituitary hypoplasia was objectified. Conclusion BBS is a rare autosomal recessive multisystem ciliopathy. The incidence of BBS is 1 in 150,000–160,000 in Europe and North America. BBS is caused by a dysfunction of the gene coding for proteins that are implicated in the function of the primary cilia and in important signaling pathways. The core features of the syndrome include retinal degeneration, obesity, polydactyly, cognitive impairment, renal anomalies and urogenital malformations. To date, pathogenic variants in 26 genes have been shown to be involved in the molecular basis of this rare ciliopathy. To our knowledge, only one study conducted on 11 children with BBS found among them 7 cases of pituitary abnormalities, 4 of which showed hypoplasia. While no association with a testicular regression syndrome has been described. This makes our patient the first described association of these 3 pathologies.

Classic genetic and hormonal switches during fetal sex development and beyond.

Critical genetic and hormonal switches characterize fetal sex development in humans. They are decisive for gonadal sex determination and subsequent differentiation of the genital and somatic sex phenotype. Only at the first glace these switches seem to behave like the dual 0 and 1 system in computer sciences and lead invariably to either typically male or female phenotypes. More recent data indicate that this model is insufficient. In addition, in case of distinct mutations, many of these switches may act variably, causing a functional continuum of alterations of gene functions and -dosages, enzymatic activities, sex hormone levels, and sex hormone sensitivity, giving rise to a broad clinical spectrum of biological differences of sex development (DSD) and potentially diversity of genital and somatic sex phenotypes. The gonadal anlage is initially a bipotential organ that can develop either into a testis or an ovary. Sex-determining region Y (SRY) is the most important upstream switch of gonadal sex determination inducing SOX9 further downstream, leading to testicular Sertoli cell differentiation and the repression of ovarian pathways. If SRY is absent (virtually "switched off"), e. g., in 46,XX females, RSPO1, WNT4, FOXL2, and other factors repress the male pathway and promote ovarian development. Testosterone and its more potent derivative, dihydrotestosterone (DHT) as well as AMH, are the most important upstream hormonal switches in phenotypic sex differentiation. Masculinization of the genitalia, i. e., external genital midline fusion forming the scrotum, growth of the genital tubercle, and Wolffian duct development, occurs in response to testosterone synthesized by steroidogenic cells in the testis. Müllerian ducts will not develop into a uterus and fallopian tubes in males due to Anti-Müllerian-Hormone (AMH) produced by the Sertoli cells. The functionality of these two hormone-dependent switches is ensured by their corresponding receptors, the intracellular androgen receptor (AR) and the transmembrane AMH type II receptor. The absence of high testosterone and high AMH is crucial for anatomically female genital development during fetal life. Recent technological advances, including single-cell and spatial transcriptomics, will likely shed more light on the nature of these molecular switches.

An improved multicellular human organoid model for the study of chemical effects on palatal fusion.

Tissue fusion is a mechanism involved in the development of the heart, iris, genital tubercle, neural tube, and palate during embryogenesis. Failed fusion of the palatal shelves could result in cleft palate (CP), a common birth defect. Organotypic models constructed of human cells offer an opportunity to investigate developmental processes in the human. Previously, our laboratory developed an organoid model of the human palate that contains human mesenchyme and epithelial progenitor cells to study the effects of chemicals on fusion. Here, we developed an organoid model more representative of the embryonic palate that includes three cell types: mesenchyme, endothelial, and epithelial cells. We measured fusion by a decrease in epithelial cells at the contact point between the organoids and compared the effects of CP teratogens on fusion and toxicity in the previous and current organoid models. We further tested additional suspect teratogens in our new model. We found that the three-cell-type model is more sensitive to fusion inhibition by valproic acid and inhibitors of FGF, BMP, and TGFβRI/II. In this new model, we tested other suspect CP teratogens and found that nocodazole, topiramate, and Y27632 inhibit fusion at concentrations that do not induce toxicity. This sensitive human three-cell-type organotypic model accurately evaluates chemicals for cleft palate teratogenicity.

First trimester identification of fetal sex by ultrasound.

The hypothesis was fetal sex determination by ultrasound at 11-14weeks' gestation has sufficient accuracy to be clinically relevant. Fetal sex assessment by transabdominal ultrasound was performed in 567 fetuses at 11-14weeks' gestation (CRL: 45-84mm). A mid-sagittal view of the genital region was obtained. The angle of the genital tubercle to a horizontal line through the lumbosacral skin surface was measured. The fetus was assigned male sex if the angle was > 30°, and female sex if the genital tubercle was parallel or convergent (< 10°). At an intermediate angle of 10-30°, the sex was not assigned. The results were divided into three categories based on gestational age: 11 + 2 to 12 + 1, 12 + 2 to 13 + 1, and 13 + 2 to 14 + 1weeks' gestation. To establish its accuracy, the first trimester fetal sex determination was compared to fetal sex determined on a mid-second trimester ultrasound. Sex assignment was successful in 534/683 (78%) of the cases. The overall accuracy of fetal sex assignment across all gestational ages studied was 94.4%. It was 88.3%, 94.7%, and 98.6% at 11 + 2 to 12 + 1, 12 + 2 to 13 + 1, and 13 + 2 to 14 + 1weeks' gestation, respectively. Prenatal sex assignment at the time of first trimester ultrasound screening has a high accuracy rate. The accuracy improved with increasing gestational age, which suggests that if clinically important decisions, such as chorionic villus sampling, are to be made based on fetal sex, they should be delayed until the latter part of the first trimester.

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-β 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-β pathway. This study provided a novel direction for studying the occurrence of hypospadias and may provide a hypospadias predictive marker in the future.

Isolated aphallia: a case report and review of literature

BackgroundAphallia is a rarest of rare congenital anomaly the incidence being 1 in 10 to 30 million live births. Seen at birth, it leaves the parents and family disturbed. Immediate counselling though done; it may take time for the parents to come to a decision.Case presentationA newborn was brought for genital examination and since the penis was not present and the scrotum with testes were normal, aphallia was suspected. At 1 month after all the necessary investigations, isolated aphallia without any other congenital anomalies was confirmed. The options offered were gender reassignment before the age of 18 months or staged phallo and urethroplasty. Parents are so far undecided.ConclusionsAphallia results from failure of the genital tubercle to develop, leading to the absence of all the penile components. Diagnosis of aphallia includes the absence of the phallus, male karyotype and normally developed scrotum with normal testicles. Usually, this anomaly is associated with multiple other anomalies involving the urinary, gastrointestinal and musculoskeletal systems. Management of this condition is challenging and requires a multidisciplinary approach. In addition to managing the associated anomalies, the options to treat aphallia will depend on the type, severity of associated anomalies, family background and socioeconomic status of the family. Psychological counselling along with surgery (in the form of gender reassignment or phalloplasty) are the mainstays of treatment. The literature of this anomaly and the treatment is reviewed.

The role of p63 in embryonic external genitalia outgrowth in mice.

Embryonic external genitalia (genital tubercle [GT]) protrude from the cloaca and outgrow as cloacal development progresses. Individual gene functions and knockout phenotypes in GT development have been extensively analyzed; however, the interactions between these genes are not fully understood. In this study, we investigated the role of p63, focusing on its interaction with the Shh-Wnt/Ctnnb1-Fgf8 pathway, a signaling network that is known to play a role in GT outgrowth. p63 was expressed in the epithelial tissues of the GT at E11.5, and the distal tip of the GT predominantly expressed the ΔNp63α isoform. The GTs in p63 knockout embryos had normal Shh expression, but CTNNB1 protein and Fgf8 gene expression in the distal urethral epithelium was decreased or lost. Constitutive expression of CTNNB1 in p63-null embryos restored Fgf8 expression, accompanied by small bud structure development; however, such bud structures could not be maintained by E13.5, at which point mutant GTs exhibited severe abnormalities showing a split shape with a hemorrhagic cloaca. Therefore, p63 is a key component of the signaling pathway that triggers Fgf8 expression in the distal urethral epithelium and contributes to GT outgrowth by ensuring the structural integrity of the cloacal epithelia. Altogether, we propose that p63 plays an essential role in the signaling network for the development of external genitalia.

Development of an in-vitro high-throughput screening system to identify modulators of genitalia development.

Sexually dimorphic outgrowth and differentiation of the embryonic genital tubercles (GTs) give rise to the penis in males and the clitoris in females. Defects in androgen production or in response to androgen signaling can lead to various congenital penile anomalies in both mice and humans. Due to lack of a high-throughput screening system, identification of crucial regulators of GT sexual differentiation has been slow. To overcome this research barrier, we isolated embryonic GT mesenchymal (GTme) cells to model genitalia growth and differentiation in vitro. Using either a mechanical or fluorescence-activated cell sorting-assisted purification method, GTme cells were isolated and assayed for their proliferation using a microscopy and image analysis system, on a single cell level over time. Male and female GTme cells inherently exhibit different cellular dynamics, consistent with their in-vivo behaviors. This system allows for the rapid quantitative analyses of numerous drug treatments, and enables the discovery of potential genetic modulators of GT morphogenesis on a large scale. Using this system, we completed a 438-compound library screen and identified 82 kinase inhibitor hits. In mice, in-utero exposure to one such candidate kinase inhibitor, Cediranib, resulted in embryos with severe genitalia defects, especially in males. Gene silencing by RNAi was optimized in this system, laying the foundation for future larger-scale genetic screenings. These findings demonstrate the power of this novel high-throughput system to rapidly and successfully identify modulators of genitalia growth and differentiation, expanding the toolbox for the study of functional genomics and environmental factors.

A genome-wide association study with tissue transcriptomics identifies genetic drivers for classic bladder exstrophy

Classic bladder exstrophy represents the most severe end of all human congenital anomalies of the kidney and urinary tract and is associated with bladder cancer susceptibility. Previous genetic studies identified one locus to be involved in classic bladder exstrophy, but were limited to a restrict number of cohort. Here we show the largest classic bladder exstrophy genome-wide association analysis to date where we identify eight genome-wide significant loci, seven of which are novel. In these regions reside ten coding and four non-coding genes. Among the coding genes is EFNA1, strongly expressed in mouse embryonic genital tubercle, urethra, and primitive bladder. Re-sequence of EFNA1 in the investigated classic bladder exstrophy cohort of our study displays an enrichment of rare protein altering variants. We show that all coding genes are expressed and/or significantly regulated in both mouse and human embryonic developmental bladder stages. Furthermore, nine of the coding genes residing in the regions of genome-wide significance are differentially expressed in bladder cancers. Our data suggest genetic drivers for classic bladder exstrophy, as well as a possible role for these drivers to relevant bladder cancer susceptibility.

Distal vaginal atresia: a report of a rare type found a late-term fetus and its histological comparison with the normal pelvis.

Solitary distal vaginal atresia is generally caused by a transverse septum or an imperforate hymen. We found a novel type of distal vaginal atresia in a late-term fetus (gestational age approximately 28 weeks) in our histology collection. This fetus had a vaginal vestibule that was closed and covered by a thick subcutaneous tissue beneath the perineal skin in the immediately inferior or superficial side of the imperforate hymen. The uterus, uterine tube, anus, and anal canal had normal development. The urethral rhabdosphincters were well-developed and had a normal topographical relationship with the vagina, but the urethrovaginal sphincter was absent. Thus, vaginal descent seemed to occur normally and form the vestibule. However, the external orifice of the urethra consisted of a highly folded duct with hypertrophied squamous epithelium. Notably, the corpus cavernosum and crus of the clitoris had poor development and were embedded in the subcutaneous tissue, distant from the vestibule. Normally, the cloacal membrane shifts from the bottom of the urogenital sinus to the inferior aspect of the thick and elongated genital tubercle after establishment of the urorectal septum. Therefore, we speculate there was a failure in the transposition of the cloacal membrane caused by decreased elongation of the genital tubercle. The histology of this anomaly strongly suggested that the hymen does not represent a part of the cloacal membrane, but is instead a product that appears during the late recanalization of the distal vagina after vaginal descent. The transverse septum was also likely to form during this recanalization.

Sexual dimorphism through androgen signaling; from external genitalia to muscles.

Sexual dimorphisms can be seen in many organisms with some exhibiting subtle differences while some can be very evident. The difference between male and female can be seen on the morphological level such as discrepancies in body mass, presence of body hair in distinct places, or through the presence of specific reproductive structures. It is known that the development of the reproductive structures is governed by hormone signaling, most commonly explained through the actions of androgen signaling. The developmental program of the male and female external genitalia involves a common anlage, the genital tubercle or GT, that later on develop into a penis and clitoris, respectively. Androgen signaling involvement can be seen in the different tissues in the GT that express Androgen receptor and the different genes that are regulated by androgen in the mesenchyme and endoderm component of the GT. Muscles are also known to be responsive to androgen signaling with male and female muscles exhibiting different capabilities. However, the occurrence of sexual dimorphism in muscle development is unclear. In this minireview, a summary on the role of androgen in the sexually dimorphic development of the genital tubercle was provided. This was used as a framework on analyzing the different mechanism employed by androgen signaling to regulate the sexual dimorphism in muscle development.