Single cell transcriptomic analysis of external genitalia reveals complex and sexually dimorphic cell populations in the early genital tubercle.

External genital organs are among the most recognizable sexually dimorphic characters. The penis and clitoris develop from the embryonic genital tubercle, an outgrowth at the anterior margin of the cloaca that undergoes an extensive period of development in male and female embryos prior to the onset of sexual differentiation. In mice, differentiation into the penis and clitoris begins around embryonic day (E)15.5. Current knowledge of cell types that comprise the genital tubercle is limited to a few studies that have fate mapped derivatives of endoderm, mesoderm, and ectoderm. Here we use single cell transcriptomics to characterize the cell populations in the genital tubercles of male and female mouse embryos at E14.5, approximately 24 â€‹h before the onset of sexual differentiation, and we present the first comprehensive atlas of single-cell gene expression during external genital development. Clustering analyses and annotation using marker genes shows 19 distinct cell populations in E14.5 genital tubercles. Mapping of cell clusters to anatomical locations using in situ gene expression patterns revealed granularity of cellular specializations and positional identities. Although E14.5 precedes sexually dimorphic morphogenesis of the genital tubercle, comparative analysis of males and females identified sexual dimorphisms at the single cell level, including male-specific cell clusters with transcriptional signatures of smooth muscle and bone progenitors, both of which are known to be sexually dimorphic in adult genitalia, as well as immune cells. These results provide a new resource for classification of external genital cell types based on gene expression profiles and reveal sex-specific cellular specializations in the early genital tubercle.

Androgen and estrogen receptor expression in the developing human penis and clitoris.

To better understand how the human fetal penis and clitoris grows and remodels, we undertook an investigation to define active areas of cellular proliferation and programmed cell death spatially and temporally during development of human fetal external genitalia from the indifferent stage (8 weeks) to 18 weeks of gestation. Fifty normal human fetal penile and clitoral specimens were examined using macroscopic imaging, scanning electron microscopy and immunohistochemical localization for the cellular proliferation and apoptotic markers, Ki67 and Caspase-3. A number of hot spots of cellular proliferation characterized by Ki67 localization are present in the penis and clitoris especially early in development, most notably in the corporal body, glans, remodeling glanular urethra, the urethral plate, the roof of the urethral groove and the fully formed penile urethra. The 12-fold increase in penile length over 10 weeks of growth from 8 to 18 weeks of gestation based on Ki67 labelling appears to be driven by cellular proliferation in the corporal body and glans. Throughout all ages in both the developing penis and clitoris Ki67 labeling was consistently elevated in the ventral epidermis and ventral mesenchyme relative to the dorsal counterparts. This finding is consistent with the intense morphogenetic activity/remodeling in the ventral half of the genital tubercle in both sexes involving formation of the urethral/vestibular plates, canalization of the urethral/vestibular plates and fusion of the urethral folds to form the penile urethra. Areas of reduced or absent Ki67 staining include the urethral fold epithelium that fuses to form the penile tubular urethra. In contrast, the urethral fold mesenchyme is positive for Ki67. Apoptosis was rarely noted in the developing penis and clitoris; the only area of minimal Caspase-3 localization was in the epithelium of the ventral epithelial glanular channel remodeling.

Prenatal ultrasound diagnosis of chordee without hypospadias.

Chordee without hypospadias (CWH) is an extremely rare condition. Here, we describe a case of CWH diagnosed accurately on prenatal ultrasound at 25 weeks' gestation. Physical examination of the newborn confirmed the sonographic findings. To the best of our knowledge, prenatal ultrasound diagnosis of CWH has not been reported in the literature. We emphasize the importance of observing fetal micturition to rule out the possibility of CWH before the diagnosis of hypospadias is made on prenatal ultrasound.

Atrazine induces penis abnormalities including hypospadias in mice.

Use of the herbicide atrazine (ATR) is banned in the European Union; yet, it is still widely used in the USA and Australia. ATR is known to alter testosterone and oestrogen production and thus reproductive characteristics in numerous species. In this proof of concept study, we examined the effect of ATR exposure, at a supra-environmental dose (5 mg/kg bw/day), beginning on E9.5 in utero, prior to sexual differentiation of the reproductive tissues, until 26 weeks of age, on the development of the mouse penis. Notably, this is the first study to specifically investigate whether ATR can affect penis characteristics. We show that ATR exposure, beginning in utero, causes a shortening (demasculinisation) of penis structures and increases the incidence of hypospadias in mice. These data indicate the need for further studies of ATR on human reproductive development and fertility, especially considering its continued and widespread use.

Fetal anogenital distance using ultrasound.

OBJECTIVE: This study measured anogenital distance (AGD) during late second/early third trimester of pregnancy to confirm previous findings that AGD can be measured noninvasively in the fetus using ultrasound and further showed differences in reference ranges between populations. METHOD: Two hundred ten singleton pregnancies were recruited at the Rosie Hospital, Cambridge, UK. A 2D ultrasound was performed between 26 and 30 weeks of pregnancy. AGD was measured from the centre of the anus to the base of the scrotum in males and to the posterior convergence of the fourchette in females. RESULTS: A significant difference in AGD between males and females (P < .0001) was found, replicating previous results with a significant correlation between estimated fetal weight (EFW) and AGD in males only (P = .006). A comparison of AGD using reference data from an Israeli sample (n = 118) and our UK sample (n = 208) showed a significant difference (P < .0001) in both males and females, after controlling for gestational age (GA). CONCLUSION: Our results confirm that AGD measurement in utero using ultrasound is feasible. In addition, there are strong sex differences, consistent with previous suggestions that AGD is influenced by prenatal androgen exposure. AGD lengths differ between the UK and Israel; therefore, population-specific normative values may be required for accurate clinical assessments.

In utero effects of maternal phthalate exposure on male genital development.

BACKGROUND: Phthalates are used extensively in commercial and personal care products and maternal exposure is ubiquitous. Phthalates are anti-androgenic, but the potential effects of phthalates on male penile development have not been assessed in utero. OBJECTIVE: The study aims to investigate the association between early pregnancy phthalate exposure and fetal penile development, overall and by race. METHODS: Prospective cohort study of women with singleton pregnancies presenting for prenatal ultrasound between 18 and 22 weeks' gestation. Maternal urine samples were assayed for eight phthalate monoester metabolites. We used maternal phthalate levels at 18 to 22 weeks' gestation as predictors of fetal size using multiple linear regression models, adjusted for fetal gestational age, maternal age, race, smoking, and education. We incorporated a phthalate by race interaction into a second set of regression models. RESULTS: We detected statistically significant race interactions for continuous phthalates with penile width. Race interactions were also suggested for penile length and volume using tertiles of phthalates with point estimates generally positive for whites and negative for African Americans. CONCLUSION: Penile development is significantly influenced by race, and the impact of maternal phthalates on penile measurements also varies by race. Maternal phthalate exposure can adversely affect in utero penile growth and development, especially among African Americans.

Differential cell proliferation and cell death during the urethral groove formation in guinea pig model.

BACKGROUND: Urethral groove (UG) formation is an important step in penile formation. Because commonly used animal models do not have UG, the mechanisms of UG formation have never been discovered. We aim to discover the cellular mechanism of the UG formation using guinea pig model. METHODS: Histology was used to study the ontogeny of UG. BrdU immunofluorescence was used to label proliferating cells, cell death was determined using LysoTracker Red and TUNEL staining, and stereology was used for quantification. To reveal Shh mRNA expression patterns, in situ hybridization was performed in guinea pig genital tubercles (GTs) and ShhGFPcre-LacZ-reporter mice were used for comparison. RESULTS: Cell proliferation in the outer layers and programmed cell death in the inner layers of urethral epithelium played key roles during urethral canal movement from dorsal to ventral aspect and final opening to form UG. Shh mRNA expression domain shifted out to the ventral surface of GT from proximal throughout to distal in guinea pigs, but was excluded from the ventral surface epithelium in midshaft and distal of mouse GT. CONCLUSION: Differential cell proliferation and cell death in developing urethral epithelium lead to UG formation and Shh expression in ventral surface epithelium of GT may play an important role.

The intravesical phallus in patients with cloacal exstrophy: An embryologic conundrum.

BACKGROUND: Phalluses present inside the extrophied bladder of cloacal exstrophy (CE) newborns have been sporadically reported in the literature; this clinical entity has largely unknown origins and may represent an extremely rare anomaly of CE. OBJECTIVE: Along with nearly doubling the number of reported intravesical phalluses in the literature, this study aims to outline the common anatomic features and discuss the implications for theories of CE embryogenesis. STUDY DESIGN: The authors retrospectively identified patients with CE and a potential intravesical phallus between 1997 and 2017 at two high-volume centers. Information was obtained about karyotype, age at closure, neurologic and renal anomalies, diastasis, phallus anatomy, and phallus biopsy pathology. RESULTS: Six genotypic males met the inclusion criteria. Five of six had a diastasis greater than 6 cm. Four of six had paired corporal bodies in the intravesical phallus, one had a single corporal body, and one had a corporal-like structure. Five of six patients had a phallus located midline in the caudal aspect of the bladder; one was located midline in the bladder dome. Phallic biopsies were obtained in three of six patients. Two showed glanular and corporal tissue while the other showed vascular proliferation morphologically similar to that of erectile tissue. DISCUSSION: Previous reports suggested that a superior vesicle fissure configuration, fusion of the corporal bodies, and fused bladder plates were common findings with an intravesical phallus. With the addition of new cases, the only consistent variable between patients is a phallus located anywhere along the bladder plate that can comprise a corporal-like structure, a single corporal body, or fused corporal bodies. These findings have implications for several embryologic theories. Although this is a retrospective review with a limited number of patients, the condition is exceedingly infrequent making it only observable retrospectively over decades at high volume centers. CONCLUSIONS: The study outlined common anatomic features of the intravesical phallus in cloacal exstrophy and discussed the subsequent embryologic implications. In cloacal exstrophy newborns with presumed aphallia, meticulous inspection of the bladder plate and biopsy of any potential phallic structures can prevent resection of phallic tissue.

Contrasting mechanisms of penile urethral formation in mouse and human.

This paper addresses the developmental mechanisms of formation of the mouse and human penile urethra and the possibility that two disparate mechanisms are at play. It has been suggested that the entire penile urethra of the mouse forms via direct canalization of the endodermal urethral plate. While this mechanism surely accounts for development of the proximal portion of the mouse penile urethra, we suggest that the distal portion of the mouse penile urethra forms via a series of epithelial fusion events. Through review of the recent literature in combination with new data, it is unlikely that the entire mouse urethra is formed from the endodermal urethral plate due in part to the fact that from E14 onward the urethral plate is not present in the distal aspect of the genital tubercle. Formation of the distal portion of the mouse urethra receives substantial contribution from the preputial swellings that form the preputial-urethral groove and subsequently the preputial-urethral canal, the later of which is subdivided by a fusion event to form the distal portion of the mouse penile urethra. Examination of human penile development also reveals comparable dual morphogenetic mechanisms. However, in the case of human, direct canalization of the urethral plate occurs in the glans, while fusion events are involved in formation of the urethra within the penile shaft, a pattern exactly opposite to that of the mouse. The highest incidence of hypospadias in humans occurs at the junction of these two different developmental mechanisms. The relevance of the mouse as a model of human hypospadias is discussed.

Renal Subcapsular xenografing of human fetal external genital tissue - A new model for investigating urethral development.

In this paper, we introduce our novel renal subcapsular xenograft model for the study of human penile urethral and clitoral development. We grafted fifteen intact fetal penes and clitorides 8-11 weeks fetal age under the renal capsules of gonadectomized athymic mice. The mice were treated with a subcutaneous pellet of dihydrotestosterone (DHT), diethylstilbestrol (DES) or untreated with hormones. Xenografts were harvested after fourteen days of growth and analyzed via serial histologic sectioning and immunostaining for Ki-67, cytokeratins 6, 7 and 10, uroplakin and the androgen receptor. Non-grafted specimens of similar fetal age were sectioned and immunostained for the same antigenic markers. 14/15 (93.3%) grafts were successfully propagated and harvested. The developing urethral plate, urethral groove, tubular urethra, corporal bodies and preputial lamina were easily identifiable. These structures demonstrated robust cellularity, appropriate architecture and abundant Ki-67 expression. Expression patterns of cytokeratins 6, 7 and 10, uroplakin and the androgen receptor in xenografted specimens demonstrated characteristic male/female differences analogous to non-grafted specimens. DHT treatment reliably produced tubularization of nascent urethral and vestibular structures and male patterns of androgen receptor expression in grafts of both genetic sexes while estrogenic or hormonally absent conditions reliably resulted in a persistent open urethral/vestibular groove and female patterns of androgen receptor expression. This model's success enables further study into causal pathways by which endocrine-disrupting and endocrine-mimicking substances may directly cause disruption of normal human urethral development or hypospadias.

Di-n-butyl phthalate induced hypospadias relates to autophagy in genital tubercle via the PI3K/Akt/mTOR pathway.

OBJECTIVE: To explore the mechanisms of hypospadias induced by in utero exposure to din-butyl phthalate (DBP). METHODS: Timed-pregnant Sprague-Dawley rats were administered 750 mg/kg of DBP by gavage from GD (gestation days) 13 to GD 18, whereas control group received corn oil. Genital tubercles (GTs) and blood samples were collected from male fetuses on GD 19. The serum testosterone concentration, apoptosis activity, autophagosomes and their related proteins (light chain 3 (LC3-I, LC3-II) ), and sequestosomes (SQSTM1/p62) in the GTs were then measured. Protein expression of protein kinase B (Akt), Beclin 1, phosphorylated Akt (p-Akt), p-S6, and phosphorylated mammalian target of rapamycin (p-mTOR) in the GTs were analyzed by Western blotting. RESULTS: The incidence of hypospadias induced by DBP was 43.64% in male fetuses. The GT volume and GT volume/body weight of fetuses were significantly reduced in the hypospadias and the non-hypospadias groups. Apoptotic cell number was significantly decreased in the GTs of the hypospadias group, but unchanged in the non-hyposadias group. The ratio of LC3-II/LC3-I was higher in the GTs from DBP exposed fetuses compared to the control group. The ratio of LC3-II/LC3-I in the GTs was higher in the hypospadias group than in the non-hypospadias group. The number of autophagosomes was increased in the GTs of the hypospadias group. Protein expression of p-S6, p-mTOR, and p-Akt were significantly decreased in the GTs of hypospadiac rats. CONCLUSIONS: DBP-induced hypospadias might be associated with apoptosis and autophagy mediated by the PI3K/Akt/mTOR signaling pathway in the GT.

Developmental and Evolutionary Origins of the Amniote Phallus.

An intromittent phallus is used for sperm transfer in most amniote taxa; however, there is extensive variation in external genital morphology within and among the major amniote clades. Amniote phalluses vary in number (paired, single, or rudimentary), spermatic canal morphology (closed tube or open sulcus), and mode of transition between resting and tumescent states (inflation, rotation, eversion, or muscle relaxation). In a phylogenetic context, these varying adult anatomies preclude a clear interpretation for the evolutionary history of amniote external genitalia; as such, multiple hypotheses have been presented for the origin(s) of the amniote phallus. In combination with historic embryological studies, recent comparative developmental analyses have uncovered evidence that, despite extensive morphological variation in adult anatomy, embryonic patterning of the external genitalia is similar among amniotes and begins with emergence of paired swellings adjacent to the cloaca. External genital development in mammals, squamates (lizards, snakes, and amphisbaenians), Rhyncocephalians (tuataras), turtles, crocodilians (alligators, crocodiles, and gharials), and birds proceeds by iterative sequences of budding and fusion events, initiated by emergence of paired swellings adjacent to the embryonic cloaca. Conservation of the embryonic origins, morphogenetic processes, and molecular genetic mechanisms involved in external genital development across Amniota supports derivation from the common ancestor of amniotes, and suggests that lineage-specific divergence of later patterning events underlies the variation observed in extant adult amniote phallus morphology.

Mouse hypospadias: A critical examination and definition.

Hypospadias is a common malformation whose etiology is based upon perturbation of normal penile development. The mouse has been previously used as a model of hypospadias, despite an unacceptably wide range of definitions for this malformation. The current paper presents objective criteria and a definition of mouse hypospadias. Accordingly, diethylstilbestrol (DES) induced penile malformations were examined at 60 days postnatal (P60) in mice treated with DES over the age range of 12 days embryonic to 20 days postnatal (E12-P20). DES-induced hypospadias involves malformation of the urethral meatus, which is most severe in DES E12-P10, DES P0-P10 and DES P5-P15 groups, and less so or absent in the other treatment groups. A frenulum-like ventral tether between the penis and the prepuce was seen in the most severely affected DES-treated mice. Internal penile morphology was also altered in the DES E12-P10, DES P0-P10 and DES P5-P15 groups (with little effect in the other DES treatment groups). Thus, adverse effects of DES are a function of the period of DES treatment and most severe in the P0-P10 period. In "estrogen mutant mice" (NERKI, ßERKO, αERKO and AROM+) hypospadias was only seen in AROM+ male mice having genetically-engineered elevation is serum estrogen. Significantly, mouse hypospadias was only seen distally at and near the urethral meatus where epithelial fusion events are known to take place and never in the penile midshaft, where urethral formation occurs via an entirely different morphogenetic process.

Sonographic Assessment of the Fetal Penile Development.

The intrauterine diagnosis of micropenis is an important clue in the discernment of some syndromes and hormonal deficiencies. In this study, we tried to establish reference ranges for the fetal penile length and penile width. This prospective cross-sectional study included 179 healthy singleton male fetus pregnancies that were between 17 and 37 weeks of gestation. Of these pregnancies, the fetal penile length and width were measured using trans-abdominal ultrasound. The correlation coefficients of gestational age with penile measurements were calculated. We observed that as the gestational age increased both the penile length and width increased (p < .0001, correlation coefficients R(2) = 0.854 and R(2) = 0.883; respectively). Reference values of the penile length in the Turkish Population were similar to previously evaluated populations including English, American and Israeli populations. The penile width measurement is a convenient way to diagnose micropenis, but penile width measurement alone might miss some penile abnormalities including chordee and hypospadias.

Resurrecting embryos of the tuatara, Sphenodon punctatus, to resolve vertebrate phallus evolution.

The breadth of anatomical and functional diversity among amniote external genitalia has led to uncertainty about the evolutionary origins of the phallus. In several lineages, including the tuatara, Sphenodon punctatus, adults lack an intromittent phallus, raising the possibility that the amniote ancestor lacked external genitalia and reproduced using cloacal apposition. Accordingly, a phallus may have evolved multiple times in amniotes. However, similarities in development across amniote external genitalia suggest that the phallus may have a single evolutionary origin. To resolve the evolutionary history of amniote genitalia, we performed three-dimensional reconstruction of Victorian era tuatara embryos to look for embryological evidence of external genital initiation. Despite the absence of an intromittent phallus in adult tuataras, our observations show that tuatara embryos develop genital anlagen. This illustrates that there is a conserved developmental stage of external genital development among all amniotes and suggests a single evolutionary origin of amniote external genitalia.

Tissue-specific roles of Fgfr2 in development of the external genitalia.

Congenital anomalies frequently occur in organs that undergo tubulogenesis. Hypospadias is a urethral tube defect defined by mislocalized, oversized, or multiple openings of the penile urethra. Deletion of Fgfr2 or its ligand Fgf10 results in severe hypospadias in mice, in which the entire urethral plate is open along the ventral side of the penis. In the genital tubercle, the embryonic precursor of the penis and clitoris, Fgfr2 is expressed in two epithelial populations: the endodermally derived urethral epithelium and the ectodermally derived surface epithelium. Here, we investigate the tissue-specific roles of Fgfr2 in external genital development by generating conditional deletions of Fgfr2 in each of these cell types. Conditional deletion of Fgfr2 results in two distinct phenotypes: endodermal Fgfr2 deletion causes mild hypospadias and inhibits maturation of a complex urethral epithelium, whereas loss of ectodermal Fgfr2 results in severe hypospadias and absence of the ventral prepuce. Although these cell type-specific mutants exhibit distinctive genital anomalies, cellular analysis reveals that Fgfr2 regulates epithelial maturation and cell cycle progression in the urethral endoderm and in the surface ectoderm. The unexpected finding that ectodermal deletion of Fgfr2 results in the most severe hypospadias highlights a major role for Fgfr2 in the developing genital surface epithelium, where epithelial maturation is required for maintenance of a closed urethral tube. These results demonstrate that urethral tubulogenesis, prepuce morphogenesis, and sexually dimorphic patterning of the lower urethra are controlled by discrete regions of Fgfr2 activity.

The inconspicuous penis in children.

The term 'inconspicuous penis' refers to a group of anatomical abnormalities in which the penis looks smaller than is expected. Micropenis can be defined as 'true micropenis'--which results from a defect in the hypothalamic-pituitary-gonadal axis--and 'micropenis secondary to congenital anatomical anomalies of the surrounding and overlying structures'--also known as 'concealed penis'. The different forms of concealed penis include webbed penis, congenital megaprepuce and partially hidden penis caused by prepubic adiposity. This disorder can also have iatrogenic causes resulting from adhesions that are secondary to circumcision--this type of concealed penis is known as 'trapped penis'. However, in both groups, micropenis is defined as a stretched penile length that is at least 2.5 SD below the mean for the patient's age, but without any other penile defects. Patients with true micropenis can be managed with testosterone, which has demonstrated good penile elongation results in the long term. Surgery also has a pivotal role in reconstruction for elongating the penis and for correction of anatomical abnormalities in concealed penis.

Canalization of the urethral plate precedes fusion of the urethral folds during male penile urethral development: the double zipper hypothesis.

PURPOSE: We describe the "double zipper" mechanism of human male urethral formation, where the distal zipper opens the urethral groove through canalization of the urethral plate, and a second closing zipper follows behind and closes the urethral groove to form the tubular urethra. MATERIALS AND METHODS: Anonymous human fetal genital specimens were acquired and gender was determined by polymerase chain reaction of the Y chromosome. Specimens were processed for optical projection tomography, stained with E-cadherin, Ki67 and caspase 3, and imaged. RESULTS: Eight developing male fetal specimens from 6.5 to 16.5 weeks of gestation were analyzed by optical projection tomography, and an additional 5 specimens by serial sections. Phallus length ranged from 1.3 to 3.7 mm. The urethral plate canalized into a groove with 2 epithelial edges that subsequently fused. Ki67 staining was localized to the dorsal aspect of the urethral plate. In contrast, caspase 3 staining was not observed. The entire process was completed during a 10-week period. CONCLUSIONS: The human male urethra appears to form by 2 mechanisms, an initial "opening zipper" that facilitates distal canalization of the solid urethral plate to form the urethral groove, which involves a high rate of epithelial proliferation (apoptosis not observed), and a "closing zipper" facilitating fusion of the 2 epithelial surfaces of the urethral groove, and thus extending the penile urethra distally. Improved knowledge of the molecular mechanisms of these processes is critical to understanding mechanisms of abnormal urethral development, such as hypospadias.