Urethral Groove Research Articles

Growth, allometry, and characteristics of a sexually selected structure in wolverine (Gulo gulo (Linnaeus, 1758)), northern river otter (Lontra canadensis (Linnaeus, 1758)), and sea otter (Enhydra lutris (Linnaeus, 1758))

Allometric analyses of sexually selected structures have revealed many patterns of evolutionary and behavioural significance, for example, in weapons, ornaments, and genitalia. We investigated allometry of the baculum (penis bone) relative to body size in post-growth adults of three large mustelids: wolverine ( Gulo gulo (Linnaeus, 1758)), northern river otter ( Lontra canadensis (Linnaeus, 1758)), and sea otter ( Enhydra lutris (Linnaeus, 1758)). The baculum grew over a longer period than did body size. Correlations among bacular variables were positive in post-growth adults. No regression slopes expressed positive allometry (i.e., slope > 1 for linear variables). These trends point to the possibility that bacular size is adapted to the average size of the reproductive tract of sexually mature female northern river otters and possibly sea otters, and that pre-ejaculatory (“pre-copulatory”) selection is highest in those species. Bacular size varied more than skull or limb-bone size, and bacular shape also varied greatly. Species differed in size and complexity of the urethral groove and bacular apex, suggesting functional differences in intromission. Substantial variation in bacular shape resulted from healed fractures, especially in sea otter. Knowledge of copulatory behaviour, age of breeding, female reproductive anatomy, and genitalic interactions during intromission is needed for comprehensive understanding of bacular anatomy, allometry, and variation for these species.

Fossil bacula of five species of Borophaginae (Family: Canidae): Implications for their reproductive biology.

The baculum of mammals offers the opportunity to study the reproductive biology of extinct species given that it is a fossilizable part of the male genitalia and that its size and shape correlate with several aspects of the reproductive biology of extant mammals. Fossil bacula, however, are rare. Currently, bacula have been described from only two extinct species of canids, one from the subfamily Caninae and the other from the extinct subfamily Hesperocyoninae. Here, I describe the bacula of five extinct species of Borophaginae, each of which was found with other skeletal elements that have enabled identification to the species level. Two specimens (Aelurodon ferox and Aelurodon stirtoni) are largely complete, while the baculum from Carpocyon compressus is complete but still embedded in matrix that obscures some of its features. The bacula of Paratomarctus euthos and Desmocyon thomsoni are incomplete, but they provide useful information nonetheless. These borophagine bacula are similar to extant canines in being robust, having a urethral groove, and a simple distal end. These features suggest that the Borophaginae had long-lasting copulation and possibly spontaneous ovulation, similar to the extant canines. However, unlike the straight baculum of extant canines, borophagine bacula are ventrally curved (arched), which is also observed in the hesperocyonine baculum. The implication of this curvature for the reproductive biology of these animals remains unknown.

Urethral Plate Characteristics in Cases of Non-proximal Hypospadias May Not Be Associated With a Higher Risk of Complications When a Two-Stage Repair Is Applied.

PurposeTo investigate whether a two-stage repair of distal- and mid-shaft hypospadias (non-proximal hypospadias) could eliminate the risk factors resulting from adverse urethral plate characteristics and eventually reduce complication rates.MethodsWe retrospectively reviewed all cases of primary surgical repair of non-proximal hypospadias performed in our center between 2009 and 2018. In all cases where adverse urethral plate characteristics were found, such as meatal stenosis, a shallow urethral groove, a thick web of tissue between the native meatus and the urethral groove or in the presence of a very “thin,” skin-like distal urethra, a two-stage repair was routinely undertaken. In cases of native meatal stenosis, a meatotomy, and meatoplasty were performed. In cases of a very “thin” distal urethra we incised the skin proximally up to the point of a normal urethral fold and a meatoplasty was performed at that point. Hypospadias repair was then performed in a second operation, 3–6 months following the first procedure. Urethroplasty, both in cases of a single-stage repair and in cases of a two-stage repair, was always performed using the Thiersch-Duplay technique. Patients with a follow-up of less than 12 months were excluded from this study.ResultsOver a period of 10 years, 208 boys underwent primary surgical repair of non-proximal hypospadias. Eighty-nine of the 208 patients (42.8%) underwent single-stage hypospadias repair. Two-stage repair of the hypospadias was required in 119 (57.2%) of the patients. The overall complication rate was 3.4% in the group operated in a single stage and 7.6% in the group that required a two-stage repair (p = 0.09). The most frequent complication reported was urethrocutaneous fistula (p = 0.31), followed by meatal stenosis (p = 0.37), urethral stricture (p = 0.08) and wound dehiscence (p = 0.16). There was no significant difference between the complication rates of the two groups.ConclusionPatients with distal hypospadias and poor urethral plate characteristics repaired in a two-stage approach have comparable low-complications to those with favorable urethral plate characteristics repaired in a single-stage.

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

This study focused on the oxidative stress effect of di-n-butyl phthalate (DBP) on development of the urinary system. 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, β-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, 100μmol/L) treatment. 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 β-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. 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.

Hot spots in fetal human penile and clitoral development

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, respectively. 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.

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

Recent evidence suggest that estrogens along with androgens are necessary for male sexual differentiation, augmenting Jost’s original hypothesis. To better understand the mechanism of action of androgens and estrogens in human genital development, we undertook an investigation to define the precise location of the androgen receptor (AR), estrogen receptors alpha (ERα) and beta (ERβ) in the developing human penis and clitoris using scanning electron microscopy, optical projection tomography and immunohistochemical staining.AR, ERα and ERβ are all expressed to varying degrees and locations in the developing penis and clitoris. The location of the AR expression in close proximity to the urethral groove and in the area of formation of the penile and glanular urethra supports the role of androgens as necessary for normal male urethral fusion and development. The expression of ERα and ERβ to the epithelial cells of the preputial lamina and area of canalization and remodeling of the glanular urethra plate is consistent with the possible role of estrogens in normal development. Our data is also consistent with the hypothesis that disruption of the AR, ERα and ERβ can lead to under virilization of the penis and hypospadias by effecting fusion of the penile urethra and remodeling of the glanular urethra.

Development of the urinary system in guinea pig females (Cavia porcellus)

ABSTRACT: Guinea pigs are animal models widely used in research related to developmental biology. The objective of this work was to demonstrate the process of formation and differentiation of urinary organs in females of the species in the prenatal period. Four females were used at 25, 30, 45 and >65 DG (days of gestation). The animals were dissected, and then macroscopic and microscopic descriptions of the urinary organs were performed. At 25 DG metanephros were present in the urogenital crest into the abdominal cavity. Collecting ducts and glomerular precursor cells could be visualized. After this period, metanephros underwent microstructural modifications to form the kidneys at the end of the prenatal period. After 30 DG, the renal parenchyma already had a cortex, where the glomerulus and proximal convoluted tubules were present; and the medulla, where distal convoluted tubules, collecting ducts, and pelvis were present. The pelvis of each kidney was drained by the ureters. The ureters also underwent tissue differentiation to be differentiated (mucosa with transitional epithelium and lamina propria of connective tissue, muscular, and adventitia) at the end of the prenatal period. The urinary vesicle also underwent tissue changes to form the tunics similar to those found in the ureters, with emphasis on the greater volume of the muscular tunica and the lamina propria that constituted the submucosa in this organ. The pelvic urethra was evidenced by a mucosa lined by transitional epithelium, submucosa, muscular and adventitia. Finally, a partial clitoral urethra and a urethral meatus in the prepuce of the clitoris were also evidenced. The urethral channel began to form with the emergence of the urethral plate and the urethral groove at 30 DG and thereafter with the fusion of the urethral folds to form a partially channeled urethra in the clitoris. A urethral meatus was observed in the most distal portion of the clitoral tissue, formed by the fusion of the prepuce. It is concluded that the urinary organs of guinea pig have similar development to that described in domestic animals, except for the partial clitoral urethra and evident urethral meatus.

Insights Figure for "Differential cell proliferation and cell death during the urethral groove formation in guinea pig model".

Insights Figure for "Differential cell proliferation and cell death during the urethral groove formation in guinea pig model".

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

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. 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. 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. 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.

Human glans and preputial development

The urethra within the human penile shaft develops via (1) an “Opening Zipper” that facilitates distal canalization of the solid urethral plate to form a wide urethral groove and (2) a “Closing Zipper” that facilitates fusion of the epithelial surfaces of the urethral folds. Herein, we extend our knowledge by describing formation of the human urethra within the glans penis as well as development of the prepuce. Forty-eight normal human fetal penile specimens were examined using scanning electron microscopy and optical projection tomography. Serial histologic sections were evaluated for morphology and immunohistochemical localization for epithelial differentiation markers: Cytokeratins 6, 7, 10, FoxA1, uroplakin and the androgen receptor. As the closing zipper completes fusion of the urethral folds within the penile shaft to form a tubular urethra (~ 13 weeks), canalization of the urethral plate continues in proximal to distal fashion into the glans penis to directly form the urethra within the glans without forming an open urethral groove. Initially, the urethral plate is attached ventrally to the epidermis via an epithelial seam, which is remodeled and eliminated, thus establishing mesenchymal confluence ventral to the glanular urethra. The morphogenetic remodeling involves the strategic expression of cytokeratin 7, FoxA1 and uroplakin in endodermal epithelial cells as the tubular glanular urethra forms. The most ventral epithelial cells of the urethral plate are pinched off from the glanular urethra and are reabsorbed into the epidermis ultimately losing expression of their markers, a process undoubtedly regulated by androgens. The prepuce initially forms on the dorsal aspect of the glans at approximately 12 weeks of gestation. After sequential proximal to distal remodeling of the ventral urethral plate along the ventral aspect of glans, the prepuce of epidermal origin fuses in the ventral midline.

Development of the human penis and clitoris

The human penis and clitoris develop from the ambisexual genital tubercle. To compare and contrast the development of human penis and clitoris, we used macroscopic photography, optical projection tomography, light sheet microscopy, scanning electron microscopy, histology and immunohistochemistry. The human genital tubercle differentiates into a penis under the influence of androgens forming a tubular urethra that develops by canalization of the urethral plate to form a wide diamond-shaped urethral groove (opening zipper) whose edges (urethral folds) fuse in the midline (closing zipper). In contrast, in females, without the influence of androgens, the vestibular plate (homologue of the urethral plate) undergoes canalization to form a wide vestibular groove whose edges (vestibular folds) remain unfused, ultimately forming the labia minora defining the vaginal vestibule. The neurovascular anatomy is similar in both the developing human penis and clitoris and is the key to successful surgical reconstructions.

Distal urethral plate adhesions: New anatomical perspective in hypospadias

We found midline epithelial adhesions in the glandar urethral plate in patients with hypospadias. After dissolution, a blind epithelized channel becomes visualized inside of the plate pointing to immature embryonic luminization. In addition it reveals that the epithelized surface of the distal urethral plate is larger than previously considered. To determine the incidence and extent of these new anatomical details of urethral plate in hypospadias patients. We prospectively assessed the detailed anatomy of the urethral plate in 72 consecutive patients with hypospadias. We recorded the presence of adhesions in the middle of the glandar urethral groove that can be easily dissoluted (dissolution line - D-line). We recorded the plate width before and after D-line dissolution, the presence of the hidden blind channel at continuation of D-line (channel type-A) and of the visible blind channel between D-line and urethral hypospadiac meatus (type-B) (Figure). In 62 patients, where the urethral plate tubularization was considered (Duplay, TIP), septs between channels were opened in the midline and a final width of the plate was measured by rolling the plate around a tube. Midline adhesions (D-line) were found in all 72 patients. Mean length of D-line was 5.13±O.17mm. Mean plate width before dissolution was 5.9±0.15mm, and after dissolution 7.8±0.16mm. A blind channel of type A was detected in 22 patients (31%), type B in 24 (33%), type A and B in 16 (22%), and none in 10 patients (14%). Mean final plate width after D-line dissolution and opening of septs between channels in 62 patients with urethral plate tubularization was 8.7±0.15mm. The main contribution of our study is a new perspective of distal urethral plate anatomy that enables enlargement of the epithelized surface of the distal urethral plate by dissolution of the preexisting epithelized groove and opening of epithelized channels within the plate. To the best of our knowledge, this anatomical anomaly has not been described previously. The distal urethral plate of all hypospadias patients is partially "folded" in the midline by epithelial adhesions of different depth and extent that may be easily dissoluted. In half of the patients (53%) the "folded" part of the plate continues proximally as a blind channel inside the urethral plate (type A channel). Opening of these structures together with the well-known urethral plate pits (type B channel) helps augment the width and the overall epithelized surface of the distal urethral plate.

External Genital Development, Urethra Formation, and Hypospadias Induction in Guinea Pig: A Double Zipper Model for Human Urethral Development

To determine whether the guinea pig phallus would be an appropriate model of human penile development, we characterized the embryology and sexual differentiation of guinea pig external genitalia and attended to induce hypospadias in males and tubular urethra formation in females pharmacologically. The external genitalia of guinea pig were collected from genital swelling initiation to newborn stages, and scanning electronic microscopy and histology were performed to visualize the morphology and structure. Immunohistochemistry was used to determine the androgen receptor localization. Bicalutamide and methyltestosterone were given to pregnant dams to reveal the role and timing of androgen in guinea pig penile masculinization. Canalization and dorsal-to-ventral movement of the urethral canal develops the urethral groove in both sexes, and then the males perform distal-opening-proximal-closing to form tubular urethra. More nuclear-localized androgen receptor is found in proximal genital tubercles of males than in females at (E) 29. Antiandrogen treatment at E26-E30 can cause hypospadias, and methyltestosterone administration at E27-E31 can induce tubular urethra formation in females. Fetal development of the guinea pig phallus is homologous to that of humans. Although guinea pig has structures similar to mouse, the urethral groove and the tubular urethra formation are more similar to humans. Antiandrogen treatment causes hypospadias in males and additional androgen induces tubular urethra formation in females. Thus, guinea pig is an appropriate model for further study of cellular and molecular mechanisms involved in distal-opening-proximal-closing in tubular urethra formation and the evaluation of the pathophysiological processes of hypospadias.

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.

Tubularized parameatal based dartos-spongiosum flap urethroplasty in distal hypospadias with flat glans, shallow meatal groove, and severe glans tilt

Objective: To report the technique and results of single stage tubularized parameatal based dartos-spongiosum flap urethroplasty for distal hypospadias with flat glans, shallow meatal groove, and severe glans tilt. Materials and Methods: The study was conducted over a period of 2-year from October 2013 to October 2015, and 19 patients with distal hypospadias were included in the study with flat glans, poor urethral groove, and severe glans tilt, who otherwise would have been subjected to transection of urethral plate and a two-stage procedure. In this technique, bilateral flaps were fashioned along the preputial edge beginning from the hypospadiac meatus with intact dartos, keeping the base of the flaps attached to the bifurcated spongiosum. The urethral plate was transected. Glans was split wide open. Bilateral flaps joined at ventral midline creating new urethral plate which was tubularized with the creation of slit like wide meatus at the tip of glans. Neourethral coverage was provided with double dartos flap. Complications and aesthetic appearance on a score of 1-10 were outcome measures. Results: There was no urethral stricture, residual chordee, penile rotation, or asymmetrical penis. The average aesthetic score was 8.5. Meatus was at the tip of glans with adequate caliber in all patients. Two (10.5%) out of 19 patients developed subcoronal fistula that required surgical closure. Conclusion: Tubularized parameatal-based dartos-spongiosum flap urethroplasty is viable and easy to reproduce option for management of a specific group of patients with distal hypospadias (with flat glans, poor urethral plate, and glans tilt) and is associated with very low complication rate.

Adult Gli2+/-;Gli3Δ699/+ Male and Female Mice Display a Spectrum of Genital Malformation.

Disorders of sexual development (DSD) encompass a broad spectrum of urogenital malformations and are amongst the most common congenital birth defects. Although key genetic factors such as the hedgehog (Hh) family have been identified, a unifying postnatally viable model displaying the spectrum of male and female urogenital malformations has not yet been reported. Since human cases are diagnosed and treated at various stages postnatally, equivalent mouse models enabling analysis at similar stages are of significant interest. Additionally, all non-Hh based genetic models investigating DSD display normal females, leaving female urogenital development largely unknown. Here, we generated compound mutant mice, Gli2+/–;Gli3Δ699/+, which exhibit a spectrum of urogenital malformations in both males and females upon birth, and also carried them well into adulthood. Analysis of embryonic day (E)18.5 and adult mice revealed shortened anogenital distance (AGD), open ventral urethral groove, incomplete fusion of scrotal sac, abnormal penile size and structure, and incomplete testicular descent with hypoplasia in male mice, whereas female mutant mice displayed reduced AGD, urinary incontinence, and a number of uterine anomalies such as vaginal duplication. Male and female fertility was also investigated via breeding cages, and it was identified that male mice were infertile while females were unable to deliver despite becoming impregnated. We propose that Gli2+/–;Gli3Δ699/+ mice can serve as a genetic mouse model for common DSD such as cryptorchidism, hypospadias, and incomplete fusion of the scrotal sac in males, and a spectrum of uterine and vaginal abnormalities along with urinary incontinence in females, which could prove essential in revealing new insights into their equivalent diseases in humans.

Complex epithelial remodeling underlie the fusion event in early fetal development of the human penile urethra

We recently described a two-step process of urethral plate canalization and urethral fold fusion to form the human penile urethra. Canalization (“opening zipper”) opens the solid urethral plate into a groove, and fusion (“closing zipper”) closes the urethral groove to form the penile urethra. We hypothesize that failure of canalization and/or fusion during human urethral formation can lead to hypospadias. Herein, we use scanning electron microscopy (SEM) and analysis of transverse serial sections to better characterize development of the human fetal penile urethra as contrasted to the development of the human fetal clitoris.Eighteen 7–13 week human fetal external genitalia specimens were analyzed by SEM, and fifteen additional human fetal specimens were sectioned for histologic analysis. SEM images demonstrate canalization of the urethral/vestibular plate in the developing male and female external genitalia, respectively, followed by proximal to distal fusion of the urethral folds in males only. The fusion process during penile development occurs sequentially in multiple layers and through the interlacing of epidermal “cords”. Complex epithelial organization is also noted at the site of active canalization. The demarcation between the epidermis of the shaft and the glans becomes distinct during development, and the epithelial tag at the distal tip of the penile and clitoral glans regresses as development progresses.In summary, SEM analysis of human fetal specimens supports the two-zipper hypothesis of formation of the penile urethra. The opening zipper progresses from proximal to distal along the shaft of the penis and clitoris into the glans in identical fashion in both sexes. The closing zipper mechanism is active only in males and is not a single process but rather a series of layered fusion events, uniquely different from the simple fusion of two epithelial surfaces as occurs in formation of the palate and neural tube.

Hypospadias

Hypospadias is a hypoplasia of the tissues forming the ventral side of the penis responsible of an ectopic meatus of the urethra. This congenital anomaly results in a fusion defect of the two epithelial surfaces of the urethral groove between the 11th and the 18th weeks of development. The earlier this process arrests, the more the form is proximal and severe. This is the second genital malformation in boys with 1case per 250male births. Its origin is often multifactorial (genetic, endocrine, placental and environmental). Three anatomical forms exist: proximal, middle and distal (the most common). Additional exams (endocrine, genetic and morphological) are realized early, before surgery, in case of severe hypospadias, familial, associated with cryptorchidism, bifid scrotum, micropenis and/or skeletal, kidney, and/or heart abnormalities. It clarifies pubertal prognosis. The surgical management is made between 6months and 12months: it limits the functional and aesthetic impact of this malformation. Many surgical techniques are described. They all have in common the three operating time: penile straightening, urethroplasty, reconstruction of the ventral side of penis. They are based on direct sutures, local flaps pedicled, and grafts (skin or mucosa). The rate of postoperative complications is between 6and 30%. The two main complications are fistulae and stenoses. The psychological follow-up of these children is necessary to adulthood. Surgery of hypospadias remains a delicate surgery and must be performed by experienced surgeons.

Prenatal Diagnosis of Isolated Hypospadias by Using “Tulip Sign” with Two and Three-Dimensional Ultrasonography

Hypospadias is an anomaly of the male urogenital tract which occurs as a result of deficient closure of the urethral groove. Hypospadias is the most commonly seen urogenital anomaly in male neonates, but it is rarely diagnosed during prenatal period. Herein, a severe hypospadias case which was diagnosed by using 2D (two-dimensional) and 3D (three-dimensional) ultrasonography at 28 weeks of pregnancy with the aid of “tulip sign” finding was presented. 2D ultrasonography of 29 year old, primigravid woman at 28 weeks of gestation revealed an incurved, short penis with blunt tip located between the two scrotal folds and there was no additional anomaly. Findings were compatible with the “tulip sign” and case was diagnosed as isolated hypospadias. Isolated hypospadias was confirmed after delivery and surgical correction was planned. Prenatal diagnosis of hypospadias and relevant literature has been reviewed.

68 STEROIDOGENIC CONTROL OF INTRAUTERINE SEXUAL DIFFERENTIATION IN SPIX’S YELLOW-TOOTHED CAVY, GALEA SPIXII

The Spix’s yellow toothed (SYT) cavy is a species of rodent that lives in Caatinga vegetation of northeast Brazil. The SYT is utilised as a protein source by inhabitants of that region and has zootechnic potential. SYT cavies have been bred in captivity for studies related to reproductive biology. The presence of a penile clitoris trespassed by the urethra was described in the adult female. The aim of this research is describe sexual differentiation events in the male and female conceptus and relate molecular mechanisms of androgen and oestrogen metabolism of the placenta with the differentiation of external genitalia. Conceptuses after 15, 22, 30, 40, >50 (full term) days of gestation (DG; n = 9 males and 9 females at each gestational period) from 30 pregnant females, which were paired with 10 males, were used. After detection of copulation, the above described DG were awaited prior to the day of euthanasia. We performed anatomical description and light and scanning electron microscopy on the genital organs in the conceptus; concentration of testosterone (T) and oestradiol (E) by radioimmunoassay in the pregnant females; and described the immunoreactivity (by immunoblotting and immunohistochemistry) of enzymes: cytochrome P450 aromatase, cytochrome P450c17, 17-β-HSD, and 3-β-HSD in placentas of male and female conceptus. The genital organs emerge from a pair of undifferentiated gonads and a genital tubercle at 15 DG. These gonads differentiate into a pair of testicles or a pair of ovaries at 22 DG. The other internal genital organs arise from the urogenital sinus and Müllerian ducts in females or Wolffian ducts in males after 22 DG. The undifferentiated genital tubercle forms the penis in males with the urethra channeling at 30 DG. In females, the urethra channeling is complete after 40 DG with the closure of the urethral groove, and at >50 DG the clitoris has characteristics of a pseudo-penis. Those that are pregnant undergo an increase of E (from 5.456 ± 1.599 pg mL–1 at 22 DG to 107.186 ± 15.966 pg mL–1 at >50 DG) and T (from 122.444 ± 15.799 pg mL–1 at 22 DG to 718.555 ± 67.407 pg mL–1 at >50 DG) concentrations during the gestational period. The concentration of T was a minimum 5-fold higher than the E concentration at the moment of external sexual differentiation. The placentas from the male and female conceptus are immunoreactive for the enzyme 3-β-HSD producing progesterone throughout pregnancy, but these placentas are not immunoreactive for the oestrogen-producing enzyme cytochrome P450 aromatase throughout pregnancy. Conversely, the cytochrome P450c17 and 17-β-HSD enzymes are immunoreactive in the placentas of the male and female conceptus throughout pregnancy, producing androgen hormones. The absence of enzymes that produce oestrogen hormones in the placenta of SYT cavies indicate that testosterone from pregnant reaches the fetal circulation, and associated with the presence of enzymes that produce androgens, may act to increase androgen hormone concentration in the pregnant, which may act in the virilization process of the female external genitalia.