III. the Formation of the Spongiose and Glandar Urethra

The British Association of Plastic Surgeons (2004) 57, 203–214

The development of the male genitourinary system: III. The formation of the spongiose and glandar urethra

P.J. Hynes*, J.P. Fraher

Department of Anatomy, University College, Cork, Ireland

Received 10 May 2002; accepted 21 August 2003

KEYWORDS Summary It is generally agreed that the urethral plate disintegrates, resulting in the Urethral groove; Urethral urethral groove. This is subsequently transformed into the urethra by fusion of the folds; Urethra; urethral folds, which ﬂank its sides. Recently, the existence of such a groove and folds Hypospadias has been denied and this challenge to the long accepted existence of such folds is signiﬁcant since hypospadias is considered to result from failure of their fusion. The present studies indicate that mesodermal fold formation and its subsequent subepithelial fusion across the midline plays an essential role in urethral tube formation. Disruption of this process readily explains common congenital abnormalities of the urethra. Q 2004 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.

According to a widely held view, the urethral plate from an ingrowth of ectoderm from the tip of the is the forerunner of the male urethra.1–5 It glans.2,3,5 subsequently disintegrates leaving a deep median By contrast, van der Putte13 – 15 and Kluth16 found groove on the undersurface of the genital tubercle. no evidence of a groove or of fusion of folds on the This, the urethral groove, is ﬂanked on both sides by undersurface of the genital tubercle. According to the cloacal (urethral) folds. These folds then fuse them, the urorectal septum, which divides the with one another ventralward from the level of the cloaca and forms the perineum, extends ventral- perineum and transform the groove into the ward by intrinsic growth, pushing the urogenital spongiose urethra.6–10 It has also been proposed opening distally to form the penile urethra. This that the entire glandar urethra is formed by fusion challenge to the generally held view is signiﬁcant, of the urethral folds as is that of the shaft.11 since the latter has provided the foundation on which According to Glenister12 and Altemus,10 the prox- were built hypotheses relating to the pathogenesis imal portion of the glandar urethra is formed by of urogenital anomalies including hypospadias. fusion of the urethral folds but its distal portion is derived from an infolding of the surface epithelium. Others consider that the glandar urethra results Materials and methods

*Corresponding author. Details of the material used, including ﬁxation, E-mail address: [email protected] embedding, sectioning, staining, image capture and

S0007-1226/$ - see front matter Q 2004 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2003.08.017 204 P.J. Hynes, J.P. Fraher processing, have been given previously.22 The same mesoderm to form the tip of the gt and the apical series of sections were used to examine the part of the glans (ap) (Fig. 1(a), (b) and (d)). Here it formation of the urethra. forms a ledge and bulges somewhat caudally on Computer assisted three-dimensional recon- each side, resulting in the shallow median lacunar structions were made, and the ﬁnal reconstructions groove (lg) on its caudal surface (Fig. 1(b) and (d)). were sectioned virtually, as before,22,23 so as to This is continuous proximally with the primitive view the median sagittal structures from a right urethral groove (pug), which overlies the urethral lateral perspective. The resulting images were plate (up) (Figs. 1(c) and (d) and 2(b) and (c)). The stored and measured as before. distal ends of the cloacal folds are pronounced and a shallow surface pit lies between them (Fig. 1(d)). Key to abbreviations in text: ap, apical part of the The surface ectoderm in the depths of this pit forms glans; c, corona; cf, cloacal fold; cm, cloacal the distal end (ventral face) of the urethral plate membrane; cs, coronal sulcus; ff, frenular fold; (Fig. 1(c)). gt, genital tubercle; hg, hindgut; lg, lacunar With the progressive apposition of the walls of groove; llf, lateral lacunar fold; mlf, medial lacunar the phallic cloaca (pc) (part II)23 the urethral plate fold; pc, phallic cloaca; pf, preputial fold; pl, (up) extends correspondingly further proximally preputial lamina; pm, preputial membrane; ps, from the level of the corona (c) at E13 (Fig. 1(a)) preputial swelling; pug, primitive urethral groove; to that of the mid-shaft by the end of E15 (Fig. rmgt, rostral mesoderm of genital tubercle; u, 3(b)). Subsequently, in reversal of this trend, the urethra; ud, urogenital duct; uf, urethral fold; up, phallic cloaca extends distally through the shaft urethral plate; urs, urorectal septum. part of the urethral plate so that, only the glandar part of the latter remains at the end of E16 (Fig. 4(b)). With this, median cleavage of the urethral plate has begun. Observations Two mesodermal preputial swellings (ps) develop, one on either side of the base of the Development of the genital tubercle genital tubercle (Fig. 1(d)). During E15, they become more prominent and each extends on to At embryonic day 13 (E13) the broad, shallow the rostral aspect of the shaft where they become coronal sulcus (cs) has developed and now dis- continuous with one another to form the primor- tinguishes the distal globular glandar part of the dium of the preputial fold (pf) (Fig. 3(c)). This does genital tubercle (gt) from its proximal shaft (Fig. not extend across the median aspect of the shaft, 1(a) and (d)). The rostral mesoderm of the genital caudally. It continues to extend distally along the tubercle (rmgt) has overgrown the cloacal fold (cf) shaft so that by E16 it is more closely related to the

Fig. 1 (a) Reconstructed mid-sagittal section of an E13 specimen. The RMGT extends distal to the urethral plate (up) to form the apical part of the glans (ap). (b,c) Light micrographs of transverse sections of an E13 specimen. Their levels are indicated in (a). On the under surface of the apical part of the glans the lacunar groove (lg) is evident (b) and is continuous proximally with the primitive urethral groove (pug) (c). (d) SEM of an E13 genital tubercle (caudal surface, tail removed). The coronal sulcus has appeared and it separates the glandar and shaft parts of the GT. The preputial swellings are now evident on either side of the shaft. Key to labels in Figures: ap, apical part of glans; c, corona; cf, cloacal fold; cm, cloacal membrane; cs, coronal sulcus; ff, frenular fold; hg, hindgut; lg, lacunar groove; llf, lateral lacunar fold; mlf, medial lacunar fold; pc, phallic cloaca; pf, preputial fold; pl, preputial lamina; pm, preputial membrane; ps, preputial swelling; pug, primitive urethral groove; rmgt, rostral mesoderm of genital tubercle; t, tail; u, urethra; ud, urogenital duct; uf, urethral fold; up, urethral plate; urs, urorectal septum; us, urogenital sinus. Note: in Figs. 1(a)–8(a), red, yellow and grey represent ectoderm, endoderm and mesoderm, respectively. The development of the male genitourinary system: III 205

Fig. 2 (a) Reconstructed mid-sagittal section of an E14 specimen. (b,c,d) Light micrographs of transverse sections of an E14 specimen. Their levels are indicated in (a). The urogenital duct lies at the base of shaft (a,d). Key to labels are given in caption of Fig. 1. glandar region of the phallus (Fig. 4(a) and (c)). By During E15 and E16, the perineal part of the the end of E16, the preputial fold has come to urorectal septum (urs) extends distally (Figs. 3(a) extend over the coronal sulcus (cs) to abut on the and (b) and 4(a) and (b)). The phallic cloaca (pc) glans (Fig. 4(b) and (d)) from which it is narrows proximally where its side walls, the cloacal separated by the ectodermal preputial lamina (pl) folds (cf), come closer to the midline in advance of except near the caudal midline where its mesoderm the growing perineum (Fig. 4(e) and (f)). Here, their is continuous with that of the cloacal folds mesodermal cells proliferate, become densely (Fig. 5(c)). packed and project medially to form the urethral folds (uf) (Fig. 4(f)). These come progressively Formation of the urethra of the shaft closer to the median plane proximally. They compress the cloacal walls so that their epithelia At E14 the urogenital duct (ud) (part I)22 lies at the are apposed to one another over a length of about base of the phallic shaft (Fig. 2(a)). It is bounded 30 mm(Fig. 4(g)). More proximally still they fuse posteriorly by the urorectal septum (urs) (perineal with one another across the midline (Fig. 4(h)). part), anteriorly by the ventral part of the cloacal Consequently, the superﬁcial and deep parts of the membrane (cm) and laterally by the superﬁcial urethral plate (up) (phallic cloaca) are separated parts of the cloacal folds (cf) (Fig. 2(d)). from one another. Lateral to the urethral folds

Fig. 3 (a,b) Reconstructed mid-sagittal sections of early (a) and late (b) E15 specimens. During E15 considerable distally directed growth of the perineum (urs) takes place with distal displacement of the urogenital duct. At late E15 the urethral plate extends to mid-shaft level. (c) SEM of a late E15 genital tubercle (caudal surface). The preputial swellings have become more prominent and have united rostrally to form the preputial fold (pf). Key to labels are given in caption of Fig. 1. 206 P.J. Hynes, J.P. Fraher

Fig. 4 (a,b) Reconstructed mid-sagittal sections of early (a) and late (b) E16 specimens. By late E16 the phallic cloaca has extended through the urethral plate to the level of the coronal sulcus. As such, median cleavage of the urethral plate has begun. (c,d). SEM of early (c) and late (d) E16 genital tubercles (caudal surfaces). The preputial fold now abuts the glans and the preputial lamina (pl) begins to form (d). (e–i) light micrographs of transverse sections of a late E16 specimen. Their levels are indicated in (b). The side walls of the phallic cloaca bulge medially (f) and the cloacal fold mesodermal cells condense to form the urethral folds (arrows) (g) which fuse proximally (h,i). Key to labels are given in caption of Fig. 1. The development of the male genitourinary system: III 207

Fig. 4 (continued )

Fig. 5 (a) Reconstructed mid-sagittal section and (b–e) light micrographs of transverse sections of E17 specimens. The levels of section are indicated in (a). The preputial fold covers the proximal glans (a). The medial (mlf) and lateral (llf) lacunar folds have developed from the apical part of the glans (b). Median cleavage of the urethral plate has extended to its glandar part (c). Frenular fold fusion is taking place at the base of the glans (d,e). Key to labels are given in caption of Fig. 1. 208 P.J. Hynes, J.P. Fraher

(Fig. 4(g)), and also proximal to the level of fusion Formation of the proximal glandar urethra (Fig. 4(i)), the mesodermal cells are less densely packed and are elongated transversely towards Median cleavage of the urethral plate (up) con- their respective urethral fold. tinues. It progresses distally through its glandar part The level of fusion of the urethral fold mesoderm during E17 (Fig. 5(a) and (c)) and is complete by shifts distally. At E15 it has reached the base of the E18, when it breaks through to the surface (Fig. 6(a) phallus (Fig. 3(a)); at E16 it is at the mid-phallus and (c)). The phallic cloaca thereby comes to have a level (Fig. 4(a)); and at the end of E16 it is in the new opening to the exterior at the base of the distal part of the shaft (Fig. 4(b)). With the distal apical part of the glans (ap) and the urethral plate progression of urethral (cloacal) fold mesodermal forms the deep and lateral walls of the proximal fusion, the phallic cloaca (pc) becomes increasingly part of the glandar urethra (u). separated from the surface (Fig. 4(b)). The preputial fold (pf) (Fig. 4(d)) continues to At each stage during the same period, as the grow distally. It covers the proximal glans by E17 cloacal membrane (cm) is traced proximally it (Fig. 5(a) and (c)), the base of the apical part of the broadens in conjunction with increasing separation glans (ap) by E18 (Fig. 6(b)), and the entire glans by of the most superficial parts of the cloacal folds (cf) postnatal day 1 (P1) (Fig. 9(a) and (b)). Its lateral from one another (Fig. 4(e–i)). Also its intercellular parts have extended round on to the caudal aspect spaces become more prominent and it finally of the glans, where their covering epithelia have undergoes cleavage in the midline most proximally. come into apposition with one another. The fused Together with growth of the perineum, this results epithelia comprise the preputial membrane (pm) in distal displacement of the urogenital duct (ud) (Figs. 5(a) and (c) and 6(a) and (c)). This comes to relative to the hindgut (hg) (Figs. 3(a) and (b) and bound the urogenital duct (ud) distally. It overlies 4(a) and (b)). This comes to lie in the distal half of the cleaved glandar urethral plate (up), with which the phallic shaft at the end of E16 (Fig. 4(b)). These it is in continuity deeply. processes lead to progressive shortening of the On the caudal aspect of the glans the preputial cloacal membrane so that only its distal shaft lamina (pl) is deficient close to the midline. Here portion remains at the end of E16 (Fig. 4(b)). the mesodermal cores of the preputial (pf) and

Fig. 6 (a) Reconstructed mid-sagittal section and (b,c,d) light micrographs of transverse sections of E18 specimens. The levels of section are indicated in (a). Median cleavage of the urethral plate is complete (c) and the phallic cloaca opens at the base of the apical part of the glans (a). Frenular fold fusion continues distally (d). Key to labels are given in caption of Fig. 1. The development of the male genitourinary system: III 209 urethral (cloacal) folds (cf) are continuous with one long (Fig. 7). By E17 the mesoderm of the apical another and form a common frenular fold (ff) on part of the glans (ap) has proliferated to form two either side of the urethral plate (up) (Fig. 5(c)). As ridges on its caudal paramedian aspect, the medial these are traced proximally they come progress- (mlf) and lateral (llf) lacunar folds (Fig. 5(b)). The ively closer to one another, compress the apposed former bounds the lacunar groove (lg) on its medial urethral plate epithelium over a length of 30 mm side. The latter lies on its lateral aspect. During (Fig. 5(d)) and fuse in the proximal part of the glans E18, these folds increase in prominence due to by E17 (Fig. 5(e)). Their behaviour closely mimics proliferation of their mesodermal cores (Fig. 6(b)). that of the urethral folds. They are also similar in The lacunar groove becomes correspondingly that their cells are densely packed. Laterally, the deeper. The epithelium (ectoderm) on the latter mesodermal cells are loosely packed and elongated is continuous proximally with that (endoderm) of transversely. With fusion of the frenular folds the the cleaved glandar urethral plate (up) (Fig. 6(c)). glandar part of the urethra (u) and the preputial The lacunar fold mesoderm is continuous proxi- frenulum begin to form simultaneously (Fig. 5(e)). mally with that of the urethral folds (uf) (Fig. 6(c)). At the site of this fusion the mesodermal cells The lacunar folds (mlf, llf) play a signiﬁcant part become less densely packed. in the formation of the distal part of the glandar The mesoderm of the perineum continues its urethra. As they are traced proximally, they distal extension during E17, associated with pro- become more prominent and incline medially at gressive distal fusion of the frenular folds (ff). their crests (Figs. 8(b) and (c) and 9(b) and (c)). The These are fused towards the base of the apical part free borders of the medial folds come into apposi- of the glans (ap) at E18 (Fig. 6(a) and (d)). tion with one another across the median plane The genital tubercle appears during E12 (part proximally in the apical part of the glans at E19 (Fig. 23 2) and is 750 mm long by E13 (Fig. 7). Its length 8(c)). Here they fuse, like tissue coming into increases steadily up to E16. It is 1350 mm long by continuity with like. The level of this fusion extends late E16. This increase in length is more due to progressively further distally as age advances. It is growth of the shaft than to growth of the glans, at the level of the middle of the apical part of the though both contribute (Fig. 7). It coincides with glans at P1 (Fig. 9(b)). At this stage too the urethral fold fusion and the formation of the mesoderm of the lateral folds has come into urethra (u) of the shaft (Figs. 3(a) and (b) and 4(a) continuity across the midline superﬁcial to that of and (b)). A rapid growth phase of the GT occurs the medial lacunar folds at the base of the apical during late E16 and E17 that is principally due to an part of the glans (Fig. 9(c)). As it does so, it also increase in the length of the glans (Fig. 7). This becomes continuous with the latter. period coincides with frenular fold fusion and the In advance of lacunar fold fusion the preputial formation of the proximal glandar urethra. The folds (pf) have come into continuity with one length of the GT changes little thereafter. another at progressively more distal levels of the glans and come to overlie the lacunar groove (lg) Formation of the distal glandar urethra (Figs. 8(b) and (c) and 9(b) and (c)). By P1 their caudal extremities are continuous with one another The apical part of the glans has increased slowly and as far distally as the tip of the apical part of the progressively in length up to E19, when it is 535 mm glans. More proximally the mesodermal cores of the preputial (pf) and lacunar folds (mlf, llf) are becoming continuous with one another on the caudal aspect of the glans (Fig. 9(c)) with the consequent formation of the preputial frenulum distally. With these changes the formation of the urethra (u) is completed and comes to lie deeply within the glans, surrounded by a thick coat of mesoderm (Fig. 9(c)).

Discussion

The present studies indicate that mesodermal fold formation and the subsequent subepithelial fusion Fig. 7 Key to labels are given in caption of Fig. 1. of the mesoderm of these folds across the midline 210 P.J. Hynes, J.P. Fraher

Fig. 8 (a) Reconstructed mid-sagittal section and (b,c) light micrographs of transverse sections of E19 specimens. The levels of section are indicated in (a). The lacunar folds have increased in size and incline medially as they are traced proximally (b,c). The medial lacunar folds (mlf) are fusing at the base of the apical part of the glans (c). The preputial fold (pf) now covers the entire glans (a). Fusion of its caudal free edges precedes that of the lacunar folds (b,c). Key to labels are given in caption of Fig. 1. play essential roles in urethral tube formation. of the urethra. This process begins proximally and Fusion proceeds as a distally moving wave along the extends progressively distally. long axis of the genital tubercle. Four pairs of folds From E14 to E16 fusion of the cloacal (urethral) are involved in forming the urethra (urethral, folds leads to the division of the phallic cloaca and frenular, preputial and lacunar). All act in a similar results in the formation of the shaft part of the manner during the formation of their corresponding urethra. Initially, this fusion involves the deeper portions of it. The mesodermal cells of each are mesoderm of the cloacal folds, that is, at the level densely packed and become more so as they are of urethral folds. As a result, this part of the urethra traced towards the level at which the fold becomes is lined with epithelium of endodermal origin. Also, continuous across the midline with its counterpart. the endodermal epithelium, lining the superﬁcial They appear to extend medially and compress the portions of the cloacal fold, is displaced on to the intervening epithelium immediately distal to their caudal median surface of the shaft. It is unclear level of fusion. The mesodermal cells are orien- from the present study if this epithelium differen- tated towards these folds. This suggests that they tiates so that it becomes ectodermal in phenotype may be migrating towards the midline. After or is replaced by adjacent ectoderm. Progressive merging, the fold cells become less densely packed, median cleavage of the ventral part of the cloacal are elongated transversely and come to be further membrane immediately precedes cloacal fold separated from one another by intercellular matrix. fusion. The coincident lateral movement of its These features strongly suggest that laterally adjacent superﬁcial cloacal fold mesoderm is placed mesoderm migrates medially and then thought to be responsible for this disruption.8,12 fuses across the median plane to form each part During E17 and E18, the deep mesoderm of the

Fig. 9 (a) Reconstructed mid-sagittal section and (b,c) light micrographs of transverse sections of P1 specimens. The levels of section are indicated in (a). At this stage the medial lacunar folds (mlf) are fusing at the middle of the apical part of the glans (b) whilst the lateral (llf) are fusing at its base (c). Key to labels are given in caption of Fig. 1. The development of the male genitourinary system: III 211 advancing preputial fold is continuous with the mesoderm to complete the frenulum. Their fusion superficial mesoderm of the cloacal folds and with results in the formation of the distal one third of the it forms a composite structure, the frenular fold. glandar urethra. This is lined only with ectoderm. Their subsequent fusion leads to the separation of Since the lacunar folds develop from the apical part the already cleaved urethral plate from the surface of the glans distal to the cloacal folds they differ and results in the formation of the proximal glandar from the latter although they are continuous urethra. This part is derived from urethral plate dorsally with them. No evidence was found of an (cloacal) epithelium and is, therefore, lined with ingrowth of surface ectoderm of the tip of the glans endoderm. Given that it is the superficial mesoderm to reach the distal extremity of the urethral plate, of the cloacal folds which comes to separate the such as has been reported by others.2,3,5 Nor was glandar part of the urethral plate from the surface any evidence found of inward growth or folding of ectoderm, it cannot be stated with certainty that ectoderm on the caudal surface of the glans during all surface ectoderm is excluded from its lining, but the formation of the lacunar groove such as was this seems likely to be the case. observed by Glenister in man.9,12 The ectoderm Concerning human development, Glenister’s9,12 covering the lacunar groove is in contact with and findings are consistent with the above. Thus, by level with the deeper endoderm of the distal fusion of the urethral folds the epithelium of the urethral plate from the beginning. These epithelia bulbar urethra is derived from the phallic cloaca come to form the lining of the proximal and distal whilst those of the spongy urethra and of the glandar urethra, respectively. proximal glandar urethra are derived from the Van der Putte (pig)13 – 15 and Kluth (rat)16 reject urethral plate. However, the findings in this study the traditional and generally accepted theory that that the urethral plate merely represents the the urethra is formed by fusion of folds. They were apposed walls of the phallic cloaca, combined unable to identify a urethral groove or urethral with the fact that the degree of this apposition folds. Accordingly, they proposed that the urethra may vary between species23 would suggest that the is formed as a result of ventralward growth of the entire urethra apart from its distal glandar part perineal mesoderm, i.e. longitudinally along the should be considered to arise from the phallic phallus. Their hypothesis is based on a study mainly cloaca. Also, both Glenister19,12 and Altemus10 have using sagittal sections and surface observations of shown that the urethral folds and the adjacent the developing phallus. In contrast to the above, caudal extremities of the preputial fold fuse the findings of the present study are consistent with simultaneously to form the caudal wall and the fold fusion transverse to the long axis of the frenulum of the proximal glandar urethra, respect- phallus. It shows that this takes place deep within ively. However, they described a deep urethral the phallus and so is better seen on transverse groove as extending along the caudal aspect of the sections, as used in this study, rather than sagittal. shaft to the base of glans subsequent to the The present study also shows that the cloacal folds formation of the urethra. Thus, it appears that are low and the cloacal groove (primitive urethral the development of the urethra in man may differ groove) is shallow. Neither of these features is from that in mouse only in that the ventral part of readily noticeable from surface examination, but the cloacal membrane becomes disrupted along its both are clearly evident on transverse sections. In entire length. In this way, in man, a deep addition, the present study shows that (cloacal) longitudinal groove (the definitive urethral groove) fold fusion takes place in the absence of an may come to exist before the urethral folds begin to extensive longitudinal groove along the undersur- fuse. Some of this appearance could be artefactual, face of developing phallus. This is because fusion is however. Postmortem changes and tissue proces- immediately preceded by disruption of the cloacal sing difficulties inherent in the acquisition of human membrane and the former progresses at a similar embryos may result in artificial disruption of the pace to that of the latter. The findings of van der delicate cloacal membrane. Putte13 – 15 and Kluth16 are compatible with the The distal glandar urethra is formed in two possibility that this is also the case in the pig stages. From E18 to P1 the edges of the preputial and rat. folds, which are continuous proximally with the Disruption of the process of mesodermal fold already fused frenular folds, fuse and overlie the formation and the subsequent subepithelial lacunar groove. Later (E19 to P1) the lacunar folds, mesodermal fusion across the midline readily which are also continuous proximally with the fused explains common congenital abnormalities of the frenular folds, fuse and separate the lacunar groove urethra. Thus, failure of folds formation or of its from the surface of the glans. As they do so they fusion with its counterpart will result in hypo- remain continuous superficially with the preputial spadias. This may occur with oestrogenic17 or 212 P.J. Hynes, J.P. Fraher inadequate androgenic3,24 stimulation or may occur increase in interstitial volume during fusion of as a result of end organ failure when these folds fail each fold type is associated with a corresponding to respond (possibly due to a deficiency in 5-alpha increase in growth rate of the related part of the reductase)3,25,26 despite normal testosterone genital tubercle. Moreover, in those cases in which levels. Such aetiology would also explain the more these mesodermal folds fail to fuse (hypospadias) complete or proximal types of hypospadias and in the length of the caudal aspect of the phallus is less particular those associated with a groove (definitive than that of its rostral aspect so that the phallus is urethral groove) passing distally from the hypospa- curved caudally (chordee).20,29 Both the failure of dic opening.18,26,27 This would suggest that in this an increase in mesodermal interstitial volume (that type of hypospadias the phallic cloaca, cloacal may be triggered by normal fold fusion) and the membrane and urethral plate develop normally persistence of the urethral plate could account for leading to the formation of such a groove on the this anomaly. disruption of the cloacal membrane. During the formation of the proximal glandar Van der Putte13 –15 has shown that defects of the urethra, fusion of the caudal extremities of the dorsal (proximal) part of the pig cloacal membrane preputial fold coincides with fusion of the urethral will result in imperforate anus and rectovesical folds. During formation of the distal glandar fistula. The present study indicates that such urethra, fusion of the caudal extremities of the defects may arise if the cloacal fold mesoderm is preputial fold precedes fusion of lacunar folds. This continuous across the midline from the beginning study proposes that formation of the entire urethra over any part of its length. When these defects results from waves of subepithelial migration of occur proximally, the process of urorectal septum mesoderm proximodistally. Thus, it follows that in formation by subepithelial mesodermal fold fusion order for the mesoderm of the caudal extremities becomes arrested or blocked by the median of preputial fold to become continuous, fusion of mesoderm of the dorsal part of the cloacal the urethral folds must progress normally at least as membrane. As a result the urorectal septum cannot far as the base of the glans. By doing so, and given displace its overlying endoderm onto the surface that the caudal extremities of the preputial fold are and rectourogenital epithelial continuity persists. If continuous with them at this level, their fusion defects occur more distally with a consequent provides a transmedian mesodermal bridge by discontinuity in the ventral part of the cloacal which fusion of the latter can begin and progress membrane, distal fusion of the urethral folds distally. Thus, in hypospadias where normal fusion beyond the level of abnormal continuity is pre- of the urethral folds to this level fails to take place, empted in a similar fashion and urethrocutaneous this mesodermal bridge is lacking. Consequently, epithelial continuity, through the persistent uro- the mesodermal cores of the caudal extremities of genital duct, presents as hypospadias. Such aetiol- the preputial fold cannot fuse and a hooded ogy might explain the types of hypospadias which prepuce is formed. Hypospadias at more distal have been described18,28 in association with a levels is associated with fusion of the urethral folds subcutaneous band of fibrous tissue and not a to the base of the glans at least. Therefore, in distal groove extending distally from the hypospadic glandar hypospadias normal preputial development opening. This band could be derived from the can take place. This is also consistent with the fused cloacal fold mesoderm of the defective findings in the present study that once begun, cloacal membrane. The findings of this study also preputial fold fusion can proceed independently of explain the presence of an accessory urethra, a urethral/lacunar fold fusion. common feature of these types of hypospadias19,29 These studies present a novel hypothesis for which extends distal to the hypospadic opening. On development of the entire urethra. This entails a the basis of the present hypothesis, this is likely to series of fusion events between a variety of folds. be a remnant of the phallic cloaca. Each of several pairs of folds behaves in a closely Following fusion of the mesodermal folds the similar way, entailing a distally moving wave of increase in their interstitial volume may play an medially directed mesodermal growth, leading to important role in the growth in length of the epithelial compression and displacement followed phallus. An increase in mesodermal interstitial by fold fusion. The mesoderm and epithelium space and turgor pressure, generated by the remain separate and distinct throughout and unlike progressive accumulation of extracellular matrix palatal fold fusion epithelial-mesenchymal trans- mucopolysaccharides synthesised by mesodermal formation21 does not take place. The waves of cells, has been shown to play an important role in fusion occur more or less sequentially to involve growth during embryogenesis.21 The findings in this most of the length of the phallus. The hypothesis study are in keeping with this, insofar as the based on the present studies provides a firm basis The development of the male genitourinary system: III 213 for explanations of a wide range of abnormalities. fusion. In man this cleavage may occur earlier in Failure of these processes leads to hypospadias and development and a deep longitudinal groove (the the related anomalies of chordee and hooded definitive urethral groove) may come to exist prepuce. Defects that occur earlier in development before urethral fold fusion begins. which distort normal cloacal morphology may also † Failure of formation of the various folds or of impede this process and may lead to a more their fusion with their counterparts will result in complex variety of congenital anomalies. Proximal hypospadias at varying levels along the penis. defects in the epithelial continuity of the cloacal † Hypospadias may also arise if a defect in the membrane could account for imperforate anus and cloacal membrane (due to a pre-existing con- urorectal fistula. More distally placed defects may tinuity of cloacal fold mesoderm across the account for the more complex forms of hypospadias midline) pre-empts fold fusion. in particular those associated with an accessory † Following mesodermal fusion an increase in its urethra and not with a urethral plate or groove. interstitial volume plays an important role in the growth in length of the phallus. In hypospadias, chordee results from the failure of an increase in this volume and the persistence of the urethral Conclusions plate.

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