External genitalia of the rat: normal development and the histogenesis of 5 alpha-reductase inhibitor-induced abnormalities

The normal histogenesis of the rat genital tubercle and the effect of exposure in utero to the 5 alpha-reductase inhibitor finasteride (L-652,931; MK-0906; Proscar) on that process were studied. In normal males and females, the genital tubercle was first seen on Day 14.25 of gestation. It contained a urethral plate which extended from the cloaca (and after Day 15.25, from the urogenital sinus) to the tip of the tubercle. On Day 18.25 the glans lamellae, which would separate the glans penis or the clitoris from the prepuce, began to develop in both sexes. Also on Day 18.25 a dense, midline plate of mesenchymal cells was first evident between the urogenital sinus and the rectum in normal males. This plate acted as a wedge, first increasing the separation between the rectum and the urogenital sinus, and subsequently separating the urethral plate from the surface epithelium in the genital tubercle. As a result, by Day 21.25 the urethra in males followed an "S"-shaped course, extending from the pelvis through the center of the glans penis to an orifice near the tip of the genital tubercle. In females, in which a mesenchymal plate did not develop, the urethral orifice remained at the base of the tubercle, and the clitoris contained the remnants of the urethral plate, extending as an open groove from the urethral orifice to the tip of the tubercle. Finasteride did not affect development of the genital tubercle in females. However, in males exposed to finasteride in utero, there was variable failure of the mesenchymal wedge to develop. As a result, the urethral plate remained in contact with the surface epithelium and eventually opened to form a groove on the ventral surface of the glans penis (hypospadias). Also, the persistence of the urethral plate along the ventral midline in finasteride-treated male fetuses and its subsequent opening as a groove interfered with development of the glans lamellae, causing displacement of the frenulum distally on the glans penis and the development of a cleft in the prepuce.     

Cell lineage analysis demonstrates an endodermal origin of the distal urethra and perineum

Congenital malformations of anorectal and genitourinary (collectively, anogenital) organs occur at a high frequency in humans, however the lineage of cells that gives rise to anogenital organs remains poorly understood. The penile urethra has been reported to develop from two cell populations, with the proximal urethra developing from endoderm and the distal urethra forming from an apical ectodermal invagination, however this has never been tested by direct analysis of cell lineage. During gut development, endodermal cells express Sonic hedgehog (Shh), which is required for normal patterning of digestive and genitourinary organs. We have taken advantage of the properties of Shh expression to genetically label and follow the fate of posterior gut endoderm during anogenital development. We report that the entire urethra, including the distal (glandar) region, is derived from endoderm. Cloacal endoderm also gives rise to the epithelial linings of the bladder, rectum and anterior region of the anus. Surprisingly, the lineage map also revealed an endodermal origin of the perineum, which is the first demonstration that endoderm differentiates into skin. In addition, we fate mapped genital tubercle ectoderm and show that it makes no detectable contribution to the urethra. In males, formation of the urethral tube involves septation of the urethral plate by continued growth of the urorectal septum. Analysis of cell lineage following disruption of androgen signaling revealed that the urethral plate of flutamide-treated males does not undergo this septation event. Instead, urethral plate cells persist to the ventral margin of the tubercle, mimicking the pattern seen in females. Based on these spatial and temporal fate maps, we present a new model for anogenital development and suggest that disruptions at specific developmental time points can account for the association between anorectal and genitourinary defects.     

Expression of the androgen receptor and 5α-reductase type 2 in the developing human fetal penis and urethra

Normal penile development is dependent on testosterone, its conversion via steroid 5 alpha-reductase type 2 to dihydrotestosterone, and a functional androgen receptor (AR). The goal of this study was to investigate the distribution of AR and 5 alpha-reductase type 2 in the developing human fetal external genitalia with special emphasis on urethra formation. Twenty fetal genital specimens from normal human males (12-20 weeks gestation) were sectioned serially and stained by avidin-biotinylated peroxidase complex method with antigen retrieval. Stained sections throughout male genital development documented the expression of AR and 5 alpha-reductase type 2 in the phallus. Between 12 and 14 weeks of gestation, AR was localized to epithelial cells of the urethral plate in the glans, the tubular urethra of the penile shaft, and stromal tissue surrounding the urethral epithelium. In the fetal penis between 16 and 20 weeks gestation, the density of AR expression was greatest in urethral epithelial cells versus the surrounding stromal tissues. There was a characteristic pattern of AR expression in the glandular urethral epithelium between 16 and 20 weeks gestation. AR expression was greater along the ventral aspect of the glandular urethra than along the dorsal aspect of the urethral epithelium. The expression of 5 alpha-reductase type 2 was localized to the stroma surrounding the urethra, especially along the urethral seam area in the ventral portion of the remodeling urethra. These anatomical studies support the hypothesis that androgens are essential for the formation of the ventral portion of the urethra and that abnormalities in either the AR or 5 alpha-reductase type 2 can explain the occurrence of hypospadias.     

Cellular basis of urothelial squamous metaplasia: roles of lineage heterogeneity and cell replacement

Although the epithelial lining of much of the mammalian urinary tract is known simply as the urothelium, this epithelium can be divided into at least three lineages of renal pelvis/ureter, bladder/trigone, and proximal urethra based on their embryonic origin, uroplakin content, keratin expression pattern, in vitro growth potential, and propensity to keratinize during vitamin A deficiency. Moreover, these cells remain phenotypically distinct even after they have been serially passaged under identical culture conditions, thus ruling out local mesenchymal influence as the sole cause of their in vivo differences. During vitamin A deficiency, mouse urothelium form multiple keratinized foci in proximal urethra probably originating from scattered K14-positive basal cells, and the keratinized epithelium expands horizontally to replace the surrounding normal urothelium. These data suggest that the urothelium consists of multiple cell lineages, that trigone urothelium is closely related to the urothelium covering the rest of the bladder, and that lineage heterogeneity coupled with cell migration/replacement form the cellular basis for urothelial squamous metaplasia.     

Loss of Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia

In humans and mice, mutations in Hoxa13 cause malformation of limb and genitourinary (GU) regions. In males, one of the most common GU malformations associated with loss of Hoxa13 function is hypospadia, a condition defined by the poor growth and closure of the urethra and glans penis. By examining early signaling in the developing mouse genital tubercle, we show that Hoxa13 is essential for normal expression of Fgf8 and Bmp7 in the urethral plate epithelium. In Hoxa13(GFP)-mutant mice, hypospadias occur as a result of the combined loss of Fgf8 and Bmp7 expression in the urethral plate epithelium, as well as the ectopic expression of noggin (Nog) in the flanking mesenchyme. In vitro supplementation with Fgf8 restored proliferation in homozygous mutants to wild-type levels, suggesting that Fgf8 is sufficient to direct early proliferation of the developing genital tubercle. However, the closure defects of the distal urethra and glans can be attributed to a loss of apoptosis in the urethra, which is consistent with reduced Bmp7 expression in this region. Mice mutant for Hoxa13 also exhibit changes in androgen receptor expression, providing a developmental link between Hoxa13-associated hypospadias and those produced by antagonists to androgen signaling. Finally, a novel role for Hoxa13 in the vascularization of the glans penis is also identified.     

Postnatal development of vagina, clitoris and urethral glands of the golden hamster

We have made a histological study of the postnatal development of the clitoris, preputial glands, urethral glands and vagina of the golden hamster. The 'phallic groove' of the clitoris is closed at 10 days of life, then the urethra has a cuboidal stratified, a stratified squamous and a stratified keratinized epithelium. The preputial glands are composed of branched saccular glands. These glands develop, with few changes during their maturation period. Formation of the urethral glands begins at 5 days and the alveoli are fully developed at puberty. The hamster vagina has two origins; the upper part is Müllerian, the caudal part is sinusal. The wall of the Müllerian vagina has a cylindrical epithelium at birth, which becomes 'double epithelium' at puberty and thereafter changes cyclically in connection with the estrous cycle. The sinusal vagina is solid at birth, its lumen being formed in the first 10 days of life and its wall having a cuboidal stratified epithelium. At 15 days it becomes a stratified keratinized epithelium, which will later line the vaginal pouch. At the 5th day, an ectodermic invagination (stratified keratinized epithelium) is observed in the zone of the future introitus. At the time of vaginal opening this zone forms the distal segment.     

Close correlation between the distribution of tenascin and that of actin filaments in the mouse urethral mesenchyme during active morphogenesis.

The distribution of tenascin, an extracellular matrix glycoprotein, and that of actin filaments were studied in the developing urethra of mouse embryos by antitenascin immunofluorescent and rhodamine-phalloidin staining. Tenascin appeared transiently in the urethral mesenchyme at the site of active morphogenesis in which the urethral epithelium separated from the surface epithelia of the glans and prepuce, being tubular, and the bilateral mesenchymes lining the preexisting urethral epithelium were seamed together in the ventral side of the tubular urethra immediately after the epithelial separation. The spatially and temporally restricted distribution of tenascin corresponded well to that of mesenchymal cells which possessed many actin filaments. These observations suggest that tenascin is involved in the cytoskeletal organization of mesenchymal cells in the active phase of morphogenesis.     

Auditory epithelial migration. III. Development of the stratified squamous epithelium of the tympanic membrane and external canal in the mouse

The development of the stratified squamous epithelium of the tympanic membrane and external auditory canal was studied in serial sections of 124 mouse ears aged from 11 gestational days to 100 days. A fold developed from the edge of the fundus of the primary canal. It possessed two regions: firstly the meatal plate, which produced the pars tensa-covering epithelium (zone 2) and most of the deep ear canal epithelium (zone 3), and secondly the fundal extension plate, which grew from that part of the fundus not forming the meatal plate. The fundal extension plate gave rise to the pars flaccida-covering epithelium (zone 1) and also to the adjacent deep canal epithelium (zone 3). A difference from human development was that zone 3 in the mouse, in both the meatal plate- and the fundal extension plate-derived areas, formed adnexal structures. In the early development of the meatal plate, zone 3, at its tip, was swollen and actively mitotic and extended always for a short distance on to the zone 2 side. Zone 2, first perceived two days after zone 3, became progressively attenuated, and by the fourth day after its formation was a single thin layer. It is suggested that the proximal part of zone 3, situated in the mature ear around the periphery of the tympanic membrane, is a generation center for unidirectional outward flux of epithelium which terminates in the mouse at the first adnexal structure. It may cause the whole of zone 2 to move in the same direction by negative contact inhibition.     

Cosinor demonstration of circatrigintan (circavigintan) biorhythm in cell component volume of female urethral ejaculate

In urethral expulsions of a multipara, aged 37 years, induced by digital stimulation over a time span of almost one and a half 26-day menstrual cycle, the volume of desquamated cellular component changed, according to two nearly identical versions of cosinor, with the period of 22-27 days, with the acrophase between 17th and 23rd day of menstrual cycle and with the amplitude between 0.5 and 1.2 ml/day. The issue testifies to periodic quantitative changes in squamous cells of vaginal type in female urethra, large paraurethral ducts and trigone of the bladder during menstrual cycle, with maximal desquamation during its secretory phase. A link on stop function of urethral epithelium and thus on female miction problems is suggested.     

Differentiation of epithelial cells in the urinary tract

Uroplakins, cytokeratins and the apical plasma membrane were studied in the epithelia of mouse urinary tract. In the simple epithelium covering the inner medulla of the renal pelvis, no uroplakins or cytokeratin 20 were detected and cells had microvilli on their apical surface. The epithelium covering the inner band of the outer medulla became pseudostratified, with the upper layer consisting of large cells with stalks connecting them to the basal lamina. Uroplakins and cytokeratin 20 were not expressed in these cells. However, some superficial cells appeared without connections to the basal lamina; these cells expressed uroplakins Ia, Ib, II and III and cytokeratin 20, they contained sparse small uroplakin-positive cytoplasmic vesicles and their apical surface showed both microvilli and ridges. Cytokeratin 20 was seen as dots in the cytoplasm. This epithelium therefore showed partial urothelial differentiation. The epithelium covering the outer band of the outer medulla gradually changed from a two-layered to a three-layered urothelium with typical umbrella cells that contained all four uroplakins. Cytokeratin 20 was organized into a complex network. The epithelium possessed an asymmetric unit membrane at the apical cell surface and fusiform vesicles. Umbrella cells were also observed in the ureter and urinary bladder. In males and females, the urothelium ended in the bladder neck and was continued by a non-keratinized stratified epithelium in the urethra in which no urothelial cell differentiation markers were detected. We thus show here the expression, distribution and organization of specific proteins associated with the various cell types in the urinary tract epithelium.W. Mello Jr. thanks FAPESP, São Paulo, Brazil for financial support.     

Neurotransmission and viscoelasticity in the ovine fetal bladder after in utero bladder outflow obstruction

Fetal bladder outflow obstruction, predominantly caused by posterior urethral valves, results in significant urinary tract pathology; these lesions are the commonest cause of end-stage renal failure in children, and up to 50% continue to suffer from persistent postnatal bladder dysfunction. To investigate the physiological development of the fetal bladder and the response to urinary flow impairment, we performed partial urethral obstruction and complete urachal ligation in the midgestation fetal sheep for 30 days. By electrical and pharmacological stimulation of bladder strips, we found that muscarinic, purinergic, and nitrergic mechanisms exist in the developing fetal bladder at this gestation. After bladder outflow obstruction, the fetal bladder became hypocontractile, producing less force after nerve-mediated and muscarinic stimulation with suggested denervation, and also exhibited greater atropine resistance. Furthermore, fetal bladder urothelium exerted a negative inotropic effect, partly nitric oxide mediated, that was not present after obstruction. Increased compliance, reduced elasticity, and viscoelasticity were observed in the obstructed fetal bladder, but the proportion of work performed by the elastic component (a physical parameter of extracellular matrix) remained the same. In addition to denervation, hypocontractility may result from a reduction in the elastic modulus that may prevent any extramuscular components from sustaining force produced by detrusor smooth muscle.     

Urethral seam formation and hypospadias

Knowledge of the formation of the normal male urethra may elucidate the etiology of hypospadias. We describe urethral formation in the mouse, show the similarities and relevance to human urethral development, and introduce the concept of the epithelial seam formation and remodeling during urethral formation. Three mechanisms may account for epithelial seam formation: (1) epithelial-mesenchymal transformation similar to that described in the fusion of the palatal shelves, (2) apoptosis, and/or (3) tissue remodeling via cellular migration. Urethral development in the embryonic mouse (14-21 days of gestation) was compared with urethral formation in embryonic human specimens (8-16 weeks of gestation) by using histology, immunohistochemistry, and three-dimensional reconstruction. The urethra forms by fusion of the epithelial edges of the urethral folds, giving a midline epithelial seam. The epithelial seam is remodeled via cellular migration into a centrally located urethra and ventrally displaced remnant of epithelial cells. The epithelial seam is remodeled by narrowing approximately at its midpoint, with subsequent epithelial migration into the urethra or penile skin. The epithelial cells are replaced by mesenchymal cells. This remodeling seam displays a narrow band (approximately 30 microns wide) of apoptotic activity corresponding to the mesenchymal cells and not to epithelial cells. No evidence was seen of the co-expression of cytokeratin and mesenchymal markers (actin or vimentin). Urethral seam formation occurs in both the mouse and the human. Our data in the mouse support the hypothesis that seam transformation occurs via cellular migration and not by epithelial mesenchymal transformation or epithelial apoptosis. We postulate that disruption of epithelial fusion remodeling, and cellular migration leads to hypospadias.     

Normal development of the male anterior urethra

A histological study was performed on serially sectioned human and mouse embryos to study the influences of programmed cell death (PCD) during morphogenesis for clarifying the existing controversies on the morphology and basic processes involved in the embryonic development of the male anterior urethra. The following new insights into the development of the anterior urethra could be established. The formation of the urethra starts with the early adhesion of the arms of the genital tubercle. In this way an epithelial plate is formed, located in the ventral midline, that is in continuity with the cloacal membrane. Male sex differentiation takes place following rupture of this cloacal membrane through programmed cell death. Fusion of the urogenital swellings with primary luminization gives rise to the penile urethra, whereas the glandular part of the urethra is formed through secondary luminization of the epithelial cord that is formed during fusion of the arms of the genital tubercle, i.e., the glans. In both fusion processes, apoptosis plays a key role. The consequence of fusion of the urogenital swellings is that their mesodermal cores unite on the ventral aspect of the penile urethra, where they differentiate into the integumental structures. The prepuce starts to develop as a fold of ectoderm with a mesodermal core after complete fusion of the entire urethra. Finally, the scrotum was found to develop through merging of the labioscrotal swellings and not by fusion.     

Cytokeratin polypeptide patterns of different epithelia of the human male urogenital tract: immunofluorescence and gel electrophoretic studies

Intermediate filament proteins of normal epithelia of the human and the bovine male urogenital tract and of certain human renal and bladder carcinomas have been studied by immunofluorescence microscopy and by two-dimensional gel electrophoresis of cytoskeletal fractions from microdissected tissue samples. The patterns of expression of cytokeratin polypeptides differ in the various epithelia. Filaments of a cytokeratin nature have been identified in all true epithelial cells of the male urogenital tract, including renal tubules and rete testis. Simple epithelia of renal tubules and collecting ducts of kidney, as well as rete testis, express only cytokeratin polypeptides nos. 7, 8, 18, and 19. In contrast, the transitional epithelia of renal pelvis, ureter, bladder, and proximal urethra contain, in addition to those polypeptides, cytokeratin no. 13 and small amounts of nos. 4 and 5. Most epithelia lining the human male reproductive tract, including those in the epididymis, ductus deferens, prostate gland, and seminal vesicle, synthesize cytokeratin no. 5 in addition to cytokeratins nos. 7, 8, 18, and 19 (cytokeratin no. 7 had not been detected in the prostate gland). Cytokeratin no. 17 has also been identified, but in very low amounts, in seminal vesicle and epididymis. The cytokeratin patterns of the urethra correspond to the gradual transition of the pseudostratified epithelium of the pars spongiosa (cytokeratins nos. 4, 5, 6, 13, 14, 15, and 19) to the stratified squamous epithelium of the fossa navicularis (cytokeratins nos. 5, 6, 10/11, 13, 15, and 19, and minor amounts of nos. 1 and 14). The noncornified stratified squamous epithelium of the glans penis synthesizes cytokeratin nos. 1, 5, 6, 10/11, 13, 14, 15, and 19. In immunofluorescence microscopy, selective cytokeratin antibodies reveal differential staining of different groups or layers of cells in several epithelia that may relate to the specific expression of cytokeratin polypeptides. Human renal cell carcinomas show a simple cytokeratin pattern consisting of cytokeratins nos. 8, 18, and 19, whereas transitional cell carcinomas of the bladder reveal additional cytokeratins such as nos. 5, 7, 13, and 17 in various proportions. The results shows that the wide spectrum of histological differentiation of the diverse epithelia present in the male urogenital tract is accompanied by pronounced changes in the expression of cytokeratin polypeptides and suggest that tumors from different regions of the urogenital tract may be distinguished by their cytokeratin complements.     

Molecular analysis of external genitalia formation: the role of fibroblast growth factor (Fgf) genes during genital tubercle formation

The molecular mechanisms underlying the development of the external genitalia in mammals have been very little examined. Recent gene knockout studies have suggested that the developmental processes of its anlage, the genital tubercle (GT), have much in common with those of limb buds. The Fgf genes have been postulated as regulating several downstream genes during organogenesis. Fgf8 was expressed in the distal urethral plate epithelium of the genital tubercle (GT) together with other markers such as the Msx1, Fgf10, Hoxd13 and Bmp4 expressed in the mesenchyme. To analyze the role of the FGF system during GT formation, an in vitro organ culture system was utilized. It is suggested that the distal urethral plate epithelium of GT, the Fgf8-expressing region, regulates the outgrowth of GT. Ectopic application of FGF8 beads to the murine GT induced mesenchymal gene expression, and also promoted the outgrowth of the GT. Experiments utilizing anti-FGF neutralizing antibody suggested a growth-promoting role for FGF protein(s) in GT outgrowth. In contrast, despite its vital role during limb-bud formation, Fgf10 appears not to be primarily essential for initial outgrowth of GT, as extrapolated from Fgf10(-/-) GTs. However, the abnormal external genitalia development of Fgf10(-/-) perinatal mice suggested the importance of Fgf10 in the development of the glans penis and the glans clitoridis. These results suggest that the FGF system is a key element in orchestrating GT development.     

Options for histological study of the structure and ultrastructure of human urinary bladder epithelium

The urothelium lines all urinary passages, with exception of the distal portions of the urethra. For the first time the structure of the human bladder was described by Leonardo Da Vinci in 15^th century, however, the exact ultrastructure and function of the bladder’s epithelium have not been fully understood. The aim of our study was to investigate the structure of normal human urinary bladder epithelium with methods of classical histology, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). We obtained biopsies from non-tumor areas from the human urinary bladder of tumor-bearing patients during transurethral resections of these tumours in general or spinal anaesthesia. Totally we investigated biopsies from 20 patients, 16 males and 4 females. The mean age of this group of patients was averaged 66.5 years. The urothelium is comprised of three cell types including polyhedral basal cells, piriform intermediate cells, and superficial umbrella cells. In human urinary bladder epithelium we found a direct connection between intermediate cells and the basement membrane. These thin cytoplasmic projections are detectable not only on slides for light microscopy (semi-thin sections), but also in transmission electron-micrographs. In semi-thin sections we found also direct connections between superficial umbrella cells and basement membrane. These connections we were not able to verify via transmission electron-microscopy. Nevertheless our results show that the human urinary bladder urothelium is a special type of pseudostratified epithelium and each cell has a thin cytoplasmic projection with a direct contact with basement membrane.     

Structure and postnatal transformation of the intermediate epithelium lining the mouse nasopalatine duct

In the epithelium lining the nasopalatine duct of the infant mouse, a transitional zone between the stratified squamous epithelium and the ciliated columnar one can be observed. The epithelium lining the transitional zone shows gradations ranging from the stratified squamous through the stratified cuboidal to the ciliated stratified low-columnar type, and gradually transforms into the stratified squamous epithelium with advancing ages. In the adult mouse, the nasopalatine duct is lined with the stratified squamous epithelium throughout up to the vicinity of the nasal cavity, and changes abruptly into the ciliated columnar epithelium lining the nasal cavity. It is suggested that the epithelium lining the transitional zone is identical with the 'intermediate epithelium' in the mouse nasopharynx and epiglottis.     

The normal development of the anorectum in the pig

The development of the anorectum was studied in forty-four embryos and foetuses of pig varying in length from 9 mm total length to 210 mm crownrump length and in three newborn pigs. The presence of some features during critical stages in the development of the cloaca in the pig such as an epithelial mass protruding into the dorsal cloaca near its intestinal orifice and distinct differences in the type of epithelium in different regions of the cloacal system greatly facilitated the study of the developmental process. Thus it could be established that a change in position of the dorsal cloaca and adjacent structures such as the distal part of the gut and the urorectal septum via the dorsal part of the cloacal plate towards the tailgroove is of major importance for the partition of the cloaca into a separate intestinal and urogenital division. A subsequent disintegration of the dorsal part of the cloacal plate results in two separate openings for both the systems at the same time. Disintegration of the ventral part of the cloacal plate leads only to a further widening of the external opening of the urogenital system. In the cloacal system three distinct zones were discerned, a dorsal and ventral cloacal and a cloacal plate region. From the dorsal cloacal epithelium the whole anorectal segment between the intestinal mucosa and the anal epidermis develops. The epithelium of ventral cloacal origin seems to disappear completely . Cloacal plate epithelium forms the epithelial lining of distal parts of the urogenital system. The penile urethra in the male is formed by a ventralward movement of the urogenital opening by the growing perineum and not by fusion of genital folds.     

Immunomorphological study of the distribution of prekeratin with a molecular weight of 49 kilodaltons in various epithelial cells in rats

Cryostat sections of various tissues of rat were stained using an indirect immunofluorescent method with monoclonal antibody against individual prekeratin with the molecular mass of 49 kilodalton (PK-49). Connective tissue endothelial cells, neurons, glia, haematopoetic tissue and smooth muscles were completely negative in this test. 46 morphological variants of epithelial structures were investigated. PK-49 was absent from all the stratified epithelia (epidermis, hair folliculi, oesophagus) but was expressed in virtually all simple epithelia of endodermal origin (exceptions: squamous lung alveolar epithelium and germinative epithelium of testis). There were negative (kidney tubules) as well as positive (bladder, mammary, glands) cell elements among mesodermal and ectodermal simple epithelia. High specificity of individual PK in respect to morphological variants of epithelia points out to the important role played by prekeratin-type intermediate filaments in morphogenesis.