The Development of the Perineum in the Human: A Comprehensive Histological Study with a Special Reference to the Role of the Stromal Components (Advances in Anatomy, Embryology and Cell Biology)

Advances in Anatomy Embryology and Cell Biology

Vol. 177

Editors F. Beck, Melbourne B. Christ, Freiburg F. Clasc
, Madrid D. E. Haines, Jackson H.-W. Korf, Frankfurt W. Kummer, Gießen E. Marani, Leiden R. Putz, Mnchen Y. Sano, Kyoto T. H. Schiebler, Wrzburg K. Zilles, Dsseldorf

S.C.J. van der Putte

The Devlopment of the Perineum in the Human A Comprehensive Histological Study with a Special Reference to the Role of the Stromal Components

With 46 Figures

S. C. J. van der Putte Department of Pathology University Medical Centre Utrecht P.O. Box 85500, 3508 GA Utrecht, The Netherlands e-mail: [email protected]

Library of Congress Control Number: 2004102481 ISSN 0301-5556 ISBN 3-540-21039-3 Springer Berlin Heidelberg New York This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. Springer is a part of Springer Science+Business Media springeronline.com
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For Marijke

Acknowledgements

I would like to thank the technicians of the laboratories of histopathology and immunohistochemistry for their technical help and wish to acknowledge in particular the contribution of Mr. H. Sakkers, J.L. Hof, W.J.M. Hermsen, and J.A.S. van Ginkel. I am most grateful to Mr. D.F. van Wichen for his great help in preparing the illustrations and to Mrs. I.L. van Rooijen for her assistance in the preparation of the manuscript. I also wish to express my appreciation to my colleagues Prof. Dr. J. Huber, Drs. G. van Noort (Streeklaboratorium Pathologie, Enschede), Dr. P.G.J. Nikkels, and Dr. W.G.M. Spliet for providing the many specimens which were essential for this investigation. I gratefully acknowledge the generosity of the Department of Anatomy and Embryology of the University of Amsterdam (Prof. Dr. W. H. Lamers), Department of Anatomy and Embryology of the Leiden University Medical Centre (Prof. Dr. A.C. Gittenberger-de Groot), Department of Functional Anatomy of the University Medical Centre Utrecht (Dr. R.L.A.W. Bleys), and the Hubrecht Institute of Developmental Biology Utrecht (Prof. Dr. R.H.A. Plasterk) for giving me access to their collections of human embryos.

Contents

1

Introduction. . . . . . . . . . . . . . . . . . . . . . . .

1

2

Materials and Methods . . . . . . . . . . . . . . . . .

2

3 3.1 3.2 3.2.1

Development of the Sexually Indifferent Perineum Introduction . . . . . . . . . . . . . . . . . . . . . . . . Observations. . . . . . . . . . . . . . . . . . . . . . . . Precloacal Perineum (e.3–5 mm Total Length, 24–28 Days Fertilization Age, Carnegie Stage XI–XIII) . . . . . . . . . . . . . . . . Cloacal Perineum (e.5–15 mm CRL, 26–45 Days Fertilization Age, Carnegie Stages XIV–XVIII) . . Cloaca: Epithelial Structure . . . . . . . . . . . . . . Cloaca: Mesenchymal Structure . . . . . . . . . . . Vascular System. . . . . . . . . . . . . . . . . . . . . . Nervous System. . . . . . . . . . . . . . . . . . . . . . External Perineum . . . . . . . . . . . . . . . . . . . . Postcloacal Sexually Indifferent Perineum (e.15–32 mm CRL, 44–56 Days, Carnegie Stage XVIII–XXIII) . . . . . . . . . . . . . Urogenital Sinus . . . . . . . . . . . . . . . . . . . . . Anal Canal . . . . . . . . . . . . . . . . . . . . . . . . . Striated Perineal Musculature . . . . . . . . . . . . . Vascular System. . . . . . . . . . . . . . . . . . . . . . Nervous System. . . . . . . . . . . . . . . . . . . . . . External Perineum . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . Cloacal Eminence . . . . . . . . . . . . . . . . . . . . Cloaca and Allantois: Partition of Mesonephric and Ureteric Systems . . . . . . . . . . . . . . . . . . Division of the Cloaca. . . . . . . . . . . . . . . . . . Urogenital Sinus . . . . . . . . . . . . . . . . . . . . . Anal Canal . . . . . . . . . . . . . . . . . . . . . . . . . Perineal Striated Musculature . . . . . . . . . . . . . Vascular System. . . . . . . . . . . . . . . . . . . . . . Labioscrotal Swellings. . . . . . . . . . . . . . . . . .

3 3 4

3.2.2 3.2.2.1 3.2.2.2 3.2.2.3 3.2.2.4 3.2.2.5 3.2.3 3.2.3.1 3.2.3.2 3.2.3.3 3.2.3.4 3.2.3.5 3.2.3.6 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.3.7 3.3.8

4 5 5 11 13 14 15 17 17 25 28 28 30 30 31 32 32 34 36 40 41 42 43

X

4 4.1 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8 4.2.9 4.2.10 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.3.6 4.3.7 5 5.1 5.2 5.2.1 5.2.1.1 5.2.1.2 5.2.1.3 5.2.1.4 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6 5.2.7 5.2.8 5.2.9 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5

Development of the Female Perineum . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . Observations . . . . . . . . . . . . . . . . . . . . . . . Vagina . . . . . . . . . . . . . . . . . . . . . . . . . . . Transformation of the Urogenital Sinus into the Urethra and Vestibulum . . . . . . . . . . . . . . . . Urethra . . . . . . . . . . . . . . . . . . . . . . . . . . . Vestibulum . . . . . . . . . . . . . . . . . . . . . . . . Erectile Structures. . . . . . . . . . . . . . . . . . . . Fascial Tissues . . . . . . . . . . . . . . . . . . . . . . Labia Majora . . . . . . . . . . . . . . . . . . . . . . . Anal Canal . . . . . . . . . . . . . . . . . . . . . . . . Perineal Striated Musculature . . . . . . . . . . . . External Perineum . . . . . . . . . . . . . . . . . . . Discussion. . . . . . . . . . . . . . . . . . . . . . . . . Vagina . . . . . . . . . . . . . . . . . . . . . . . . . . . Transformation of the Urogenital Sinus into the Urethra and Vestibulum . . . . . . . . . . . . . . . . Urethra . . . . . . . . . . . . . . . . . . . . . . . . . . . Vestibulum . . . . . . . . . . . . . . . . . . . . . . . . Labia Majora . . . . . . . . . . . . . . . . . . . . . . . Anal Canal . . . . . . . . . . . . . . . . . . . . . . . . External Perineum . . . . . . . . . . . . . . . . . . . Development of the Male Perineum . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . Observations . . . . . . . . . . . . . . . . . . . . . . . Urethra . . . . . . . . . . . . . . . . . . . . . . . . . . . Prostatic Urethra . . . . . . . . . . . . . . . . . . . . Membranous Urethra . . . . . . . . . . . . . . . . . Spongy Urethra . . . . . . . . . . . . . . . . . . . . . Navicular Fossa . . . . . . . . . . . . . . . . . . . . . Erectile Structures. . . . . . . . . . . . . . . . . . . . Fascial Tissues . . . . . . . . . . . . . . . . . . . . . . Perineal Raphe, Septum, Body, and Fasciae . . . Scrotum . . . . . . . . . . . . . . . . . . . . . . . . . . Penis, Prepuce, Preputial Sac and Frenulum . . . Anal Canal . . . . . . . . . . . . . . . . . . . . . . . . Perineal Striated Musculature . . . . . . . . . . . . External Perineum . . . . . . . . . . . . . . . . . . . Discussion. . . . . . . . . . . . . . . . . . . . . . . . . Urethra . . . . . . . . . . . . . . . . . . . . . . . . . . . Erectile Structures. . . . . . . . . . . . . . . . . . . . Perineal Raphe, Septum, Body, and Fasciae . . . Penis, Prepuce, Preputial Sac, and Frenulum . . Scrotum . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . .

43 43 44 46

. . . . . . . . . . .

50 52 55 57 59 64 64 67 67 68 69

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

73 74 75 78 79 80 81 81 82 83 83 88 89 91 94 97 99 102 104 107 107 108 109 109 113 114 116 118

XI

6

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . 118

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

1 Introduction The developmental steps which lead to the formation of the human perineum seem firmly established (Arey 1965; Hamilton and Mossman1972; Moore and Persaud 1998; Wartenberg 1993; Sadler 1995; Larsen 1997). They form the base for the evaluation of the pathogenesis of a great variety of complicated and often serious malformations which occur in this region. This concept has, however, been challenged by the results of an investigation into the normal and abnormal development of the anorectum in pig (van der Putte and Neeteson 1983, 1984; van der Putte 1986). Observations revealed that at least in pig, a major element in current ideas about the early development of the perineum, namely the process by which the original simple cloaca is subdivided into a urogenital and anal part is incorrect, while additional observations strongly suggested that the same may be true for ideas about female and male sexual transformation. A preliminary investigation in human embryos gave similar indications (van der Putte 1986). The data supported earlier critical findings (Politzer 1931, 1932; Wijnen 1964; Ludwig 1965) which have apparently been ignored, possibly because they seemed to hinder the understanding of the pathogenesis of congenital malformations such as imperforate anus and hypospadias. The necessity to provide an embryological base for the explanation of these malformations has had a profound effect on prevalent theories about the development of the anogenital region and has even led to theoretical constructions which were apparently not based on observations in embryos (Bill and Johnson 1958; Duhamel et al. 1966; Stephens et al. 1988, 1996). The unexpected results from an investigation into hereditary congenital anorectal malformations of pig embryos have demonstrated the weakness of such interpretations and constructions (van der Putte and Neeteson 1984) and underlined the need for a new inquiry into the normal development of the area in human embryos. These results demonstrated that although data from malformations may offer extra information about the normal development of the area, great care has to be taken in using that information for the reconstruction of its normal development, which should be firmly based on the observation of the evolving microscopic anatomy of the region in the first place. However, in this respect original work on the development of the perineum gave a confusing picture of fragmentary and often conflicting information. It underlined the necessity that in case a new investigation was undertaken such a study should be comprehensive by addressing the whole developmental process, including not only the sexually indifferent period and female and male differentiation, but also an analysis of the until now almost completely neglected stromal tissues.

2

Materials and Methods

2 Materials and Methods The perineum was studied in 125 and fetuses ranging from 3 mm to 360 mm crown–rump length (CRL). Most of the embryos form part of collections (Departments of Anatomy and Embryology of the Universities of Amsterdam, Leiden, and Utrecht, and the Hubrecht Institute of Developmental Biology, Utrecht). The specimens had been prepared by conventional methods, namely fixing with 4% formalin or Bouins or Zenkers fixatives, embedding in paraffin, sectioning at 5–10 mm, and staining by hematoxylin and eosin or azophloxin. From fetuses between 50 and 150 mm CRL, pelvic-perineal specimens were prepared and serially sectioned. While some were mounted as complete series, most were mounted in step sections (1:5 to 1:10). The pelvic-perineal blocks from fetuses larger than 150 mm CRL were cut into thin slices prior to embedding, sectioning, and staining. The planes of sectioning were sagittal, transverse, and those tending to frontal and frontal to the surface of the perineum. The availability of a wide range of planes of sectioning proved to be essential in the analysis of still undifferentiated stromal elements of the perineum, which apart from their topography could be identified by differences in texture only. Male (M) and female (F) sex was determined on gonadal differentiation. The following embryos indicated by sex, measurements in millimeters CRL, and planes of sectioning (t, transverse; s, sagittal; f, frontal to the surface of the perineum) were studied: 3 t, 3 t, 4f, 5 t, 5 s, 6 t, 7 t, 9 t, 9.5 t, 10 t, 11.25 t, 12 t, 13 t, 13 t, 13 s, 14 t, 14 t, 14 t, 14 t, 14.5 t, 15 t, 15.25 t, 17 s, 18 t, M19 t, M19 t, M20 t, F21 t, M22 s, M22 t, F23 t and F23 f (twin), F25 t, M26 t, F28 t, M29 s, F30f, F32 t. The following female fetuses indicated by measurements in CRL and planes of sectioning were also studied: 32 s, 35 t, 37 f, 40 t, 41 s, 42 f, 45 s, 48 f, 50 t, 50 t, 52 f, 53 s, 55 s, 60 s, 66 f, 70 s, 70 t, 70 s, 85 s, 87 s, 93 s, 105 t, 110 s, 120 s, 125 f, 125 s, 130 t, 135 f, 140 f, 150 f, 160 s, 175 t, 182 f, 190 s, 210 s, 230 s, 230 f, 260 f, 320 f, 330 s, 360 s, as were the following male fetuses: 35 f, 36 t, 40 f, 40 t, 44 f, 45 t, 45 f, 49 s, 51 f, 58 f, 60 s, 60 s, 60 f, 60 s, 65 t, 65 f, 76 f, 80 f, 82 t, 85 s, 90 s, 90 f, 105 s, 110 t, 115 f, 135 f, 140 s and f (twin), 140 s, 150 f, 155 f, 155 t, 160 s and t (different parts), 160 s and t, 170 t, 190 t, 200 s, 205 s, 210 t, 210 t, 220 t, 225 f, 230 t, 230 f, 245 f, 270 t, 320 t, 330 t, 360 t and s, 360 s. Histochemical tests comprised periodic acidSchiff (PAS) stain for glycogen and mucoproteins, and Alcian blue for mucoproteins. Immunohistochemical tests have been applied to identify smooth muscle differentiation [anti-alpha-sma, Sigma (Zwijndrecht, The Netherlands)] low-molecular-weight keratins [anti-cytokeratins 07, Biogenex (San Ramon, CA, USA); Cam 5.2, Becton (San Jose, CA, USA)], and vimentin [anti-vimentin, Dako (Glastrup, Denmark)]. Throughout the text the developmental events are indicated by the CRL of the embryos. These measurements are preferred over age and staging because they form the

Introduction

3

only direct information available in almost all of the embryos and some of the fetuses and allow comparison between the present data and those of all major previous studies. It should be stressed that when added to the text these lengths indicate the earliest observation of a certain developmental event or a developmental period, and also that variation in development between embryos of the same age and length was considerable. The study comprises all that is currently designated as perineum, from skin to the pelvic diaphragm in the whole diamond-shaped region bordered by the pubic symphysis, ischial tuberosities, and os coccygis (Williams 1989; Moore and Dalley II 1996), and to which are added from an embryological perspective the urethra and muscular coat in the female, and the prostatic urethra and related muscular coat, prostate and colliculus seminalis with ejaculatory ducts and prostatic utricle in the male. Since the designation “perineum” is often used for the area between the anus and the vestibulum vaginae (“gynecological perineum”) in the female and for the area between the anus and the urethral orifice in the male, that part of the perineum is referred to as “midperineal region of the perineum” to avoid confusion.

3 Development of the Sexually Indifferent Perineum 3.1 Introduction Descriptions of the development of the sexually indifferent perineum tell how the widened caudal part of the hindgut becomes the cloaca after being contacted by the mesonephric ducts. This cloaca is thereafter divided into a urogenital part into which issue the allantois and mesonephric ducts and an anal part which receives the gut. Traditionally almost all attention of embryologists has been focused on this process of partition, most likely because disturbances in this process have been blamed for the relatively frequently occurring and sometimes life threatening anorectal anomalies. Prevalent views indicate the following sequence in this process: (a) partition of the cloaca by a descending coronal urorectal septum into a ventral urogenital part forming the bladder and urogenital sinus and an anal compartment forming the anal canal, (b) fusion between this septum and the cloacal membrane resulting in separate urogenital and anal membranes, and (c) breakdown of these membranes giving both divisions their own external opening. In a variant of this model, with a straightforward reference to the need to explain communications between the anorectum and the urogenital system in imperforate anus, the urorectal septum was thought to be formed by the combination of a deep descending septum fusing in the midline with two ridges extending from the lateral walls of the cloaca. In contrast, obser-

4

Development of the Sexually Indifferent Perineum

vations in pig and earlier critical findings suggested that the subdivision of the cloaca was the result of changes in the configuration of the anogenital region by differential growth and that a urorectal septum as a dividing structure does not exist. They also contradicted a process of fusion between the septum and the cloacal membrane and thereby the existence of separate urogenital and anal membranes. Ideas about other aspects of the formation of the perineum, too, are less undisputed than textbooks of embryology suggest. In original work, discussions have erupted now and then about such elements as the contribution of the allantois to the development of the urinary bladder; the partition of the mesonephric ducts and the ureters, their relationship to the cloaca and role in the formation of the trigone of the bladder. The definition of the urogenital sinus has remained rather vague, causing confusion about its precise limitation, its position in relation to the orifices of the mesonephric ducts and so-called vesico-urethral canal, and its subdivision into “pelvic” and “phallic” parts. Differences in opinion about the pathogenesis of malformations of the distal urethra such as hypospadias in the male has led to conflicting ideas about the role of the urogenital-sinus-related “urethral” plate. The development of the anal canal has created controversies about the existence of a separate anal membrane, the appearance of a secondary occlusion and the contribution by a proctodeum. Information concerning the development of the mesenchyme-derived elements of the perineum such as future erectile, fascial, vascular, nervous, and muscular structures is minimal. 3.2 Observations The development of the sexually indifferent perineum is described in three phases: (1) a precloacal period [embryos (e.)3–5 mm] from the involution of the primitive streak to the moment the tips of the caudalward-growing mesonephric ducts fuse with the lateral walls of the hindgut; (2) a cloacal period (e.5–15 mm) ending at the disintegration of the dorsal part of the cloacal membrane which results in the opening of the cloaca into the amniotic cavity; and (3) a postcloacal sexually indifferent period (e.15–32 mm) ending when the first histological features indicating the transformation of the perineum into a male composition can be identified. 3.2.1 Precloacal Perineum (e.3–5 mm Total Length, 24–28 Days Fertilization Age, Carnegie Stage XI–XIII) At this stage the caudal part of the embryo reveals a very simple basic configuration of an axial neural tube and notochord flanked by paraxial meso-

Observations

5

derm. Inside its lateral plate mesoderm the intra-embryonic coelom is formed cranially. In the region of the future perineum between the body stalk and the tail bud, a blind ending hind gut predominates. Cranially it communicates with the allantois, ending blindly inside the body stalk. Caudally the hind gut ends, together with the neural tube and notochord, in the tailbud. In a median zone and just caudal to the body stalk, a narrow direct contact between the columnar epithelium of the ventral wall of the hind gut and the cuboidal epithelia of the epiderm forms the anlage for the cloacal membrane. It is followed caudalward by the disappearing primitive streak from which mesoderm extends between all major structures of the area at the end of the period. At that later stage the distal part of the hind gut widens into a chamber, which receives the allantois ventrocranially and the gut dorsocranially, and continues into an increasingly longer and more tube-like blind ending tail gut inside the fast lengthening tail caudal to the cloacal membrane. The lining of this part of the hind gut consists of a dorsally low and ventrally high two-layered columnar epithelium which resembles that of the gut but is distinctly different from the epithelium of the allantois. The latter is a contrasting single layer of cuboidal cells with a faintly staining (“clear”) cytoplasm. A pair of ducts, extending caudalward lateral to the dense mesenchyme of the nephrogenic cord, form the mesonephric ducts and come into contact and subsequently fuse with the lateral walls of this part of the hind gut. This fusion marks the transition of that most distal part of the hindgut into the cloaca. 3.2.2 Cloacal Perineum (e.5–15 mm CRL, 26–45 Days Fertilization Age, Carnegie Stages XIV–XVIII) Development during this period is characterized by the transformation of the simple chamber-like early cloaca into a tube bending into a U-form simultaneously with the formation of a broad mantle of mesenchyme around the epithelial structure (Fig. 1a–d). Strong growth of both elements cause a marked increase in volume resulting in the appearance of a cloacal eminence occupying the area between the body stalk—umbilical cord from now on— tail and the anlage for the legs on both sides. 3.2.2.1 Cloaca: Epithelial Structure The epithelium of the cloaca is still characterized by a high columnar epithelium which is most likely pseudostratified in character (see footnote in Sect. 4.2.3) and distinctly different from the low epithelium of the allantois (Fig. 2).

6

Development of the Sexually Indifferent Perineum

Fig. 1a–f Schematic median sections through the perineum of embryos of 5 mm to 30 mm to demonstrate the configuration of the main median structures. In a an early chamber-like cloaca (1) shows the cloacal membrane (2), tailgut (3), orifice of a mesonephric duct (4), allantois (5), and gut (6). In b an indentation including coelom (7) develops cranially. In c the pericloacal mantle mesenchyme has increased, which results in an outward cloacal eminence, a bending and more tubular cloaca now revealing urogenital (1a) and anal (1b) compartments, with a short and narrowing communication (1c), and the transformation of the ventral part of the cloacal membrane into a plate. In d the cloacal eminence has become more prominent, especially in its ventral (urogenital) part. In the plate-like part of the cloacal membrane (2b), with its extension along the ventral wall of the cloaca, a histologically different glans plate (2a) has appeared. This contrasts with the dorsal cloacal membrane (2c), which becomes thinner and later disintegrates. Early structural differences in the mesenchyme indicate the primordial glans (8), corpora cavernosa (9), and primordial muscular layers of the deep urogenital compartment (10) and distal gut (11). In e the ventral part of the cloacal eminence has grown into the phallic structure. The dorsal part of the cloacal membrane has disintegrated (wide broken lines) creating a single opening to the amniotic cavity for both the urogenital and anal compartments and their original communication. This results, in effect, in a division of the cloaca into a urogenital sinus (1a) and anal canal (1b) as the open and very reduced communication (1c) becomes part of the surface. Mesonephric ducts (4) and ureters (12) have separated, transforming the allantois (5) into the urinary bladder by adding the intermediate area as the trigone; (13) is the puborectalis. In f the superficial part of the urogenital plate has split, widening the urogenital orifice. Paramesonephric ducts (14) have immigrated into the tuberculum M
lleri forming the mesonephric-paramesonephric complex. Smooth muscle layers differentiate around the deep urogenital sinus (10) and rectum (11). Stippled lines indicate the border between cloaca-derived ps.str.col. epithelium and the epidermis. The asterisk indicates the position of the primordium of the bulbourethral/greater vestibular gland; (15) external anal sphincter; (16) symphysis pubis. Original magnifications, 55 (a–d), 40 (e) and 30 (f)

Observations

7

The first alteration in the epithelial structure of the cloaca during this period is the regression and disappearance of the tail gut. This process is an early and rapid event (e.6–9 mm) noticeable by a reduction of mitotic activity and the presence of nuclear fragments indicative of a regulated cell death (apoptosis). The structure disappears completely although small isolated remnants may remain for a short while distally. At the same time, the simple early cloaca reveals an indentation on the cranial side that deepens and changes the shape of the cloaca into a U-form consisting of an increasingly more complicated ventral urogenital compartment and a dorsal anal compartment with a short narrowing communication between. The urogenital compartment shows an increasing distinction between a deep segment directly involved in the development of the mesonephric-ureteric structures and a superficial segment more strictly related to the cloacal eminence. The deep segment of the urogenital compartment forms a pair of lateral extensions (“horns” or “cornua”) where fusion between the cloaca and the mesonephric ducts had taken place near the entrance to the allantois (7– 9 mm; Figs. 2b, 3a) Initially such a cornu forms a narrow tube which con-

Fig. 2a, b Cloaca in embryos of 6 mm transverse section (tr.) (a) and 7 mm tr. (b). In a the cloaca (1) demonstrates its lining of characteristic ps.str.col. epithelium, relatively thin cloacal membrane (2) and early mantle mesenchyme (3). In b the striking contrast is shown between the high ps.str.col. epithelium of the cloaca (1) with cornua (arrows) and of the gut (4), and the low cuboidal epithelium of the allantois (5)

8

Development of the Sexually Indifferent Perineum

Fig. 3 Schematic drawings illustrating the development of the mesonephric ducts, ureters, and paramesonephric structure in embryos of 5 mm to 30 mm. They show in a the fusion of the caudalward growing mesonephric duct (1) and hindgut, later cloaca (2), in b the development of the ureteric diverticulum (3), in c the replacement of apoptotic mesonephric epithelium of the common outlet by cloacal epithelium (arrows), and in d and e the separation and parting of the mesonephric duct and lengthening ureter, leaving excrescences of mesonephric and ureteric epithelium (arrowheads). In f a frontal view shows parted mesonephric ducts (1) and ureters (3) with some epithelium (shaded) still present inside temporarily remaining lateral zones of cloacal epithelium (2), which flank a tongue of bladder epithelium (4) and outline the future trigone of the bladder. In g the solid ends of the paramesonephric ducts (5) have immigrated in direct contact with the epithelium of the mesonephric ducts, and form in h and i the mesonephric-paramesonephric complex consisting of expanding and fusing paramesonephric ducts, which push the associated mesonephric ducts lateralward and remain separated from the urogenital sinus by a column of dense mesenchyme (6) containing a nerve (arrow). Original magnification, 120

tinues into the mesonephric duct revealing a solid junction first and continuity between the lumina later. At the same time, the distal part of the mesonephric duct forms a medial ureteric diverticulum (Figs. 3b, 4a), which indents the caudal (metanephric) extremity of the nephrogenic cord. The diverticulum then shifts lateralward together with this metanephrogenic blastema and elongates with the increase in distance between the blastema and the cloaca to become the tubular ureter, which widens distally into the early renal pelvis. The epithelium of the segment of the mesonephric duct between this ureter and cloacal cornu shows extensive apoptosis (Fig. 4a) and is undermined and replaced by advancing cloacal epithelium (Fig. 3c, d; e.7 mm). As a result cloacal epithelium comes to directly border the different epithelia of the definitive mesonephric ducts and of the ureters, lifting them into excrescences around the new orifices (Figs. 3e, 4b). During this process the cloacal cornua and the adjacent parts of the cloaca and allantois greatly expand and receive the now separate mesonephric ducts and

Observations

9

Fig. 4a, b Relationship between cloaca, mesonephric duct and ureter in embryos of 7 mm t (a) and 13 mm t (b). In a the cloaca (1) with early cornu (arrowhead) meeting (at arrow) the distal part of the mesonephric duct (2), which widens into an early ureteric diverticulum (3) in contact with dense metanephric blastema (4). The cranial duct is out of the plane of section. Inset: apoptosis in the “common outlet” of the mesonephric duct (asterisk); arrow, border between cloaca and mesonephric duct. In b mesonephric duct (2) and ureter (3) have just separated, revealing their openings into the cornu of the cloaca (1), and still surrounded by excrescences of their epithelium (arrowheads), which are lifted by new, replacement epithelium of the sinus (arrow); 5, coelomic cavity

ureters dorsolaterally. This adds to a complicated epithelial configuration in this part of the cloaca, demonstrating a single layer of low cuboidal allantois epithelium cranially, high cuboidal to pseudostratified columnar (ps.str.col.) cloacal epithelium caudally, and just inside the cloacal domain the simple columnar epithelium of the mesonephric ducts and the more eosinophilic simple columnar epithelium of the ureters. At the same time the cornua give the deep segment of the urogenital sinus a crescentlike shape with the largest diameter in a transverse direction. It shows a relative decrease as compared with the rapid growth of the superficial part, which latter also differs by acquiring its largest diameter in the median plane. The rapid growth of the superficial segment of the urogenital compartment appears closely related to a disproportionately strong increase in its surrounding mesenchyme. This relationship is particularly well illustrated by the transformation of the ventral part of the original cloacal membrane into a solid epithelial plate (e.8 mm; Fig. 5a). This urogenital

10

Development of the Sexually Indifferent Perineum

Fig. 5a–d Cloaca in embryos of 10 mm t (a), 13 mm t (b), and 14 mm (c, d). In a the urogenital compartment shows wide cornua (1) and a cloacal membrane that becomes a solid plate (2), flanked by greatly increased mesenchyme forming cloacal labia (3). In b the ventral cloacal membrane has differentiated into the urogenital plate (2), revealing a tendency to splitting, and into the more densely stained solid glans plate (4), with epithelial tag differing from both the cloaca-related epithelium (2) internally and the epidermis (5) externally; abrupt transitions are indicated by arrows; (cf. Fig. 13b). In c the communication (6) between urogenital and anal compartments has widened and the membrane (2) has increased in width and thinned. 3, Cloacal labia; 7, gut; 8, mantle mesenchyme of the gut. In d a nearly rupturing membrane (2) still closes the anal compartment (9). Note the thin layer of mantle mesenchyme between the mantle (8) of the gut and the cloacal labia (3)

Observations

11

plate is largest ventrally where the cloacal eminence has its tip, and decreases dorsalward where it passes into the original membrane. It consists almost entirely of a cloacal type of epithelium with a thickness of four cell layers and reveals a tendency to focal dehiscence centrally (Fig. 5b). It is covered on the surface by an initially thin epidermal layer which thickens into an epithelial “tag” near the tip of the eminence. The formation of the tag is associated with the appearance of a new epithelial component between the cloacal and epidermal epithelia. This component distinguishes itself by a markedly eosinophylic cytoplasm and a strict apposition of the basal nuclei on the basement membrane. It is named glans plate as it is most closely related to the developing glans of the future phallic structure (Figs. 1d—f, 5b). The opposite side of this superficial part of the urogenital compartment shows a more rounded shape. The lumen is lined by a high ps.str.col. epithelium, the thickness of which decreases towards the plate-like ventral part of the cloacal membrane. While narrowing dorsalward it acquires an increasingly more superficial position before sharply bending inward as the tubular anal compartment, which gradually passes into the distal gut. This part of the cloacal membrane becomes wider and thinner (Fig. 5c, d) with the presence of nuclear fragments in the membrane and adjacent dorsal wall, indicating a process of regression by apoptosis. It is this part of the cloacal membrane that disintegrates at the end of the period creating a cloacal orifice into the amniotic cavity for both the urogenital compartment and anal compartment, and also for the short and superficial communication between the two compartments (Fig. 1e). In effect this means separate urogenital and anal orifices for the urogenital and anal compartments although still linked by a short cloacal groove on the surface. 3.2.2.2 Cloaca: Mesenchymal Structure It is most likely that the division of the cloaca is directly related to differential growth in the mantle of mesenchyme which develops around the epithelial structure and, in particular, around the superficial segment of the urogenital compartment. This mantle makes its appearance in the form of accumulations of mesenchyme around the cloaca (Fig. 5a) which extend around the distal ends of the mesonephric ducts and ureters (e.5–7 mm) but is far denser than the loose mesenchyme surrounding the allantois. During the following phase of rapid growth, a broad circumferential structure is formed around the urogenital compartment. It elongates this compartment in association with an outward expansion on the surface which creates most of the cloacal eminence. The mesenchyme extends as far as the inguinal folds and forms the tip of the eminence and the ridge-like

12

Development of the Sexually Indifferent Perineum

Fig. 6a–d Schematic median sections through the cloaca and its mantle of mesenchyme in embryos of 8 mm to 17 mm. Accentuation of the mantle demonstrates how the strong disproportionate growth of the mesenchyme around the urogenital compartment of the cloaca, as compared with the minor increase of mesenchyme related to the anal compartment and the intermediate communication, causes bending of the cloaca into a U-form tube, followed by its division into a urogenital sinus and anal canal. Original magnifications, 45 (a–c), 38 (d). See Fig. 1, also

cloacal labia1 flanking the cloacal membrane (Fig. 5). The cloacal eminence markedly decreases towards the anal compartment, which remains relatively short and practically holds its original position. As a result of the wide discrepancy between the strong-growing and protruding mantle around the urogenital compartment of the cloaca and the very moderate growth around the anal compartment, which stays at its original position in relation to the tail groove, the very short tubular communication between both becomes increasingly smaller and more superficial (Figs. 1d,e, 6). When the dorsal part of the cloacal membrane disintegrates, it forms no more than a short, narrow, and shallow external cloacal groove in the midline of the midperineal region between the urogenital and anal openings. Simultaneously, differences in density and structure appear in the mantle mesenchyme of the cloaca. They already indicate a number of different elements which will become better definable during the following developmental period (see Sect. 3.2.3). Dorsolateral to this mantle in sensu strictu develops a pair of postanal swellings with a conspicuous stromal pattern perpen1 These ridges flanking the cloacal membrane have been indicated by a confusing diversity of names such as genital, cloacal, anogenital, urogenital, urethral, and urethral-and-anal folds, which appears to be due to the altering status of the related structures. In the present study terms directly related to these structures are preferred with “folds” replaced by “labia,” which term better describes their specific position along the (future) orifices, does not suggest a non-existent process of folding, and is well in line the definitive “labia minora” for its most distinct derivation. In this structure, “cloacal labia” become “urogenital labia” ventrally (with the anal parts flattening and becoming incorporated into the wall of the superficial anal canal and orifice) after the division of the cloaca into a urogenital sinus and an anal canal. These urogenital labia become “labia minora” after the formation of the vestibulum in the female and regressing “urethral labia” in the male.

Observations

13

Fig. 7a, b Dorsolateral perineal complex in embryos of 10 mm t (a), and 12 mm t (b). In a the dorsolateral perineal complex (1) is shown between the tail (arrow) and the leg (arrowhead); 2, sacral plexus of spinal nerves; 3, external iliac vein; 4, autonomic nerve plexus. In b the dorsolateral perineal complex (1) shows its position by large future labial and muscular nerves (5); 4, autonomic nerve plexus; 6, cloacal labia; 7, anal compartment of the cloaca; arrowhead, leg

dicular to the epidermis and situated at the dorsal ends of the cloacal labia. Right and left dorsolateral perineal complexes also develop in relation to sacral somites and large spinal nerves and gradually extend ventralward (Figs. 7, 8a, b). This composition will become much more distinct during the following postcloacal period. 3.2.2.3 Vascular System The early vascular system still consists of vascular plexuses without a clear distinction between arterial and venous elements. At the very beginning it is based on a ventral plexus related to vessels around the allantois and a dorsal plexus related to some small contributions from the caudal aorta (future median sacral artery) and the postcardinal venous system (e.5–9 mm). The system becomes more extensive with the rapid and strong growth of the mantle mesenchyme (e.9–13 mm). It then reveals two arterial systems: (a) the median sacral system as a relatively large direct contribution from the caudal aorta which develops a pair of ventral branches supplying blood to plexuses on the left and right sides of the dorsal half of the cloaca including related parts

14

Development of the Sexually Indifferent Perineum

Fig. 8a–d Schematic drawings demonstrating the differentiation of the dorsolateral perineal complex derived from sacral somites into the perineal striated muscles and labioscrotal swellings in embryos of 13 mm to 30 mm. In a, a transverse section through the base of the tail (1) shows the dense left primordium of the external anal sphincter (2) and of the labioscrotal swellings (3) in line with some remaining somites (4). 5, Puborectalis; 6, dorsal wall of the rectum; 7, pudendal artery and its main branches; 8, external iliac vessels. In b, a sagittal section shows the position of the complex in relation to the dense mesenchyme (9) from which the future erectile tissue derives. In c, an oblique (dorsomedial to ventrolateral) section shows how the complex has differentiated into the ischiocavernosus (10), external urethral sphincter (11), bulbospongiosus (12), and external anal sphincter (2) muscles, and into the primordial dartos tissue of the labioscrotal swelling (13); 14, postanal swelling; 15 corpus cavernosum. In d, a frontal projection demonstrates the position of the muscles in relation to the crura of the corpora cavernosa (15), deep (16) and superficial (17) urogenital sinus, and anal canal (18). Original magnifications, 45 (a), 50 (b), 40 (c and d)

of the cloacal labia, and (b) the internal pudendal system which sprouts as two small bilateral branches from the internal iliac arteries (e.11 mm) and then develops (e.13 mm) its own branches toward the dorsolateral perineal complex and the ventral half of the cloaca (Fig. 9). 3.2.2.4 Nervous System The earliest nervous elements recognizable are a bilateral series of small concentrations of cells related to the early sympathic trunks (e.9 mm) dorsolateral to the gut and extending ventralward to the proximal parts of the ureters. After the appearance of fibers, the sympathic pelvic plexus is formed (e.11 mm). This plexus rapidly extends towards the deep part of the cloaca and allantois and becomes connected to the spinal nerves by the pelvic splanchnic nerves. During this period spinal nerves originating from the sacral stems grow into the perineum, initially reaching the nearby dorsolateral perineal complexes (e.11 mm). Thereafter, other nerves grow ventralward and ventrolateralward in close relation with the main vascular contributories.

Observations

15

Fig. 9a–c Schematic drawings demonstrating the development of the main arterial supply to the perineum in lateral projections in embryos of 13 mm and 30 mm. In a and b the original two systems are illustrated in a paramedian (a) and lateral (b) section with (a) a ventral branch of the caudal aorta (1) forming the median sacral system (2) that supplies the dorsal part of the cloacal eminence (cloaca indicated by broken lines), and (b) the lateral system originating from the internal iliac artery (3) forming the pudendal system (4, interrupted) and splitting into a ventral branch (5), supplying the ventral part of the eminence from which will derive the future erectile structures, and into a dorsolateral branch (6) supplying the dorsolateral perineal complex from which will derive the perineal striated muscles and the labioscrotal swellings. 7, Allantois-related venous plexus. In c, a more definitive configuration shows the reduction of the median sacral system (2), its anastomosis to the pudendal system forming the transverse artery of the perineum (8), and the predominance of the pudendal system revealing as its main elements the pudendal artery (3), giving off the inferior rectal artery (9) and labioscrotal arteries (10), the artery to a bulbus spongiosus/vestibularis (11), an artery in the ventral wall of the urogenital sinus (12), the deep artery (13) to the corpora cavernosa, and the dorsal artery to the glans (14). Not indicated are branches to the striated muscles. 7, Allantois-related venous plexus. Original magnifications, 55 (a and b), 25 (c)

3.2.2.5 External Perineum The external shape of the perineum transforms from a slightly convex but otherwise flat area (e.5–7 mm) into the increasingly more elevated cloacal eminence (Fig. 10a, b). This eminence becomes more or less pointed ventrally, where relatively large flanks develop. It slopes down dorsalward showing the cloacal plate in the bottom of a median slit flanked by the slightly elevated cloacal labia which end at each side in the postanal swellings near the tail groove. The ventral end of the cloacal plate is marked by an epithelial tag. Lateral to the labia form the flanks, a major part of the eminence. They become bordered laterally by the paired labioscrotal swellings which appear dorsolaterally at the end of the period. On the surface of the perineum the epidermis changes from a single layer of cuboidal cells into a double layer which characteristically shows a basal zone free of nuclei, and flat cells superficially. It covers the cloacal plate and

16

Development of the Sexually Indifferent Perineum

Fig. 10a–d Schematic drawings of the superficial stromal components in reconstructions of the external shape of embryos of 7 mm to 30 mm. They demonstrate in a and b the development of the cloacal eminence from a flat area around the oval cloacal membrane (1). Indicated are the mesenchyme of the glans (2), cloacal labia (3) along the cloacal membrane, and flanks (4), with the early labioscrotal swellings (5) in a dorsolateral position. 6, Tail; 7, postanal swellings. In c the ventral part of the eminence has become a phallic structure with large urogenital labia flanking the dorsally open urogenital orifice. The phallic structure is perpendicular to the labioscrotal swellings and overhangs a still inconspicuous anal area, with a small primary anal orifice, surrounded by labial stroma (8a); 7, postanal swellings; arrow, midperineal region with remnant of cloacal groove. In d the definitive anal opening (8b) has emerged after the addition of a newly formed superficial segment to the primary anal canal incorporating originally labial stroma. This drawing also demonstrates a basic composition in the stromal tissues, which has differentiated from the mesenchyme around the urogenital sinus, with fibrovascular (i.e., future erectile) tissues of the glans (2) and cloacal, later urogenital labia (3) embracing the sinus ventrally. Embracing it dorsally is stroma with a conspicuous ventralward orientation, i.e., future fascial tissues of the dorsal frenulum (9), superficial (5a) and deep (5b) dartos stroma, and superficial stroma of the shaft (10). Arrow, midperineal region with ventralward-extending perianal stroma. Original magnification, 25

forms the conspicuous tag-like thickening ventrally. During the last phase of the cloacal period an intermediate cell layer indicative of differentiation into an early stratified squamous (str.sq.) epithelium develops in the area of the glans and cloacal labia. The cloaca has thus been divided by the end of the cloacal period into a urogenital sinus and an anal canal separated by a bar. The most prominent ventral part of the cloacal eminence containing the urogenital sinus becomes the sexually indifferent phallic structure with the related ventral parts of the cloacal labia becoming the urogenital labia. The far less distinctive dorsal parts of the cloacal labia, related to the anal canal, flank the anal orifice.

Observations

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3.2.3 Postcloacal Sexually Indifferent Perineum (e.15–32 mm CRL, 44–56 Days, Carnegie Stage XVIII–XXIII) In the postcloacal sexually indifferent perineum, the basic architecture of the definitive perineum emerges. It consists of the urogenital sinus and its adnexal structures, the anal canal, striated perineal muscles, vascular and nervous systems, and the external perineum. It thus comprises all major elements which by differential growth will form either a male or female construction later. 3.2.3.1 Urogenital Sinus Major elements in the development of the urogenital sinus are: (a) its growing distinction from the allantois which forms most of the urinary bladder, and the related parting of the mesonephric ducts and ureters together with their orifices; (b) the formation of a specific deep urogenital sinus and the integration of mesonephric and paramesonephric systems; and (c) the development of a specific superficial urogenital sinus, including the formation of the phallic structure and most of the midperineal region between the urogenital and anal orifices. Formation of the Urogenital Sinus and Urinary Bladder; Parting of Mesonephric and Ureteric Orifices. The already existing marked differences in structure between the allantois and the cloaca become even more distinct. After the regression of the blind-ending part of the allantois inside the umbilical cord during the previous period, the remainder of the allantois becomes a wide urinary bladder lined by a now two-layered still conspicuously clear epithelium. Its wall consists of a loose radially oriented lamina propria tissue surrounded by a denser layer of primordial detrusor vesicae muscle (Fig. 11a). This layer develops into bundles of smooth muscle cells from inside the umbilicus (e.20 mm) where the regressing part of the allantois becomes the urachus, toward the urogenital sinus (e.30 mm). The urogenital sinus, in contrast, remains a narrow tube lined by a high three- to four-layered ps.str.col. epithelium surrounded by a broad layer of dense mesenchyme that differentiates into a dense lamina propria with a predominantly longitudinal orientation, and into an even denser primordial muscular layer (Fig. 11b). In the borderland between the urogenital sinus and bladder, those contrasting elements gradually merge, especially on the ventral side. Dorsally such a transition is interrupted by the partition of the mesonephric ducts and ureters together with their openings (e.18 mm), during which process the orifices of the mesonephric ducts become firmly established inside the

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Development of the Sexually Indifferent Perineum

Fig. 11a, b Bladder (a) and urogenital sinus (b) in an embryo of 30 mm t show the striking contrast in structure between the allantois-derived bladder (1), demonstrating its lining of low cuboidal, lightly stained allantois-type of epithelium, loosely structured lamina propria (2), and muscularis (3) at a level just cranial to the ureteric orifices; and (b) the cloaca-derived deep urogenital sinus (4) revealing a high ps.str.col. epithelium, dense lamina propria (5), and primordial muscularis (6) at a level just caudal to the orifices of the mesonephric ducts

deep urogenital sinus and those of the ureters inside the bladder (Fig. 1e). As all orifices are originally situated just inside the area lined by cloaca-derived epithelium, it is not surprising that initially the widening area between the orifices is lined by the urogenital sinus type of epithelium. Simultaneously with the lateralward and cranialward movement of the ureters, an initially small “tongue” of allantois-derived epithelium of the bladder extending caudalward in the midline becomes more prominent. It leaves thereby two bands of sinus epithelium between the ureteric and mesonephric orifices marking the boundaries of the developing trigone of the urinary bladder. Inside these bands are short intraepithelial caudalward extensions of the lumina of the ureters and short cranialward extensions of epithelium of the mesonephric ducts—temporarily visible vestiges of the parting process (Fig. 3f). At the end of the period the two bands become increasingly narrower and are replaced by the bladder type of epithelium near the orifices of the ureters. Some urogenital sinus epithelium remains, however, in a zone cranial to the mesonephric orifices.

Observations

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During the separation, the mesenchyme of the trigone becomes markedly denser than that in the adjacent areas of the bladder. This trigone-related tissue demonstrates at first a lateralward orientation which becomes increasingly more oblique in a ventralcaudalward direction as the trigone lengthens. This tissue extends to the developing ventral internal urethral sphincter musculature of the deep urogenital sinus and forms then a distinctive layer representing the primordial vesical sphincter just beneath the changing epithelium. Formation of the Deep Urogenital Sinus; Integration of Mesonephric and Paramesonephric Systems. The deep urogenital sinus greatly elongates during this period. The crescentic shape of its lumen is accentuated by the formation of a M
llerian tubercle dorsally and a median groove ventrally. The characteristic high three- to four-layered ps.str.col. epithelium becomes somewhat lower at the tubercle, but still remains markedly different from the single layer of mesonephric epithelium, which reveals a distinctive nucleus-free basal zone around the orifices. Just inside the tuberculum, each mesonephric duct becomes associated with an immigrating solid tip of a paramesonephric duct (e.25 mm; Fig. 3g). The latter had originated from the lining of the coelom in the cranial parts of both genital ridges (e.12 mm) and had grown caudalward with their solid tips progressing in direct contact with the epithelium of the mesonephric ducts. While progressing, these solid tips produce the paramesonephric ducts, which then separate from the mesonephric ducts, acquire a more lateral position in the genital ridges, but bend medialward after reaching the stroma of the dorsal wall of the urogenital sinus where the two ducts fuse (Figs. 3h, 12a). This fusion extends caudalward but does not involve the most distal portions of the solid tips which have halted inside the tuberculum, where they remain in direct contact with the medial sides of the mesonephric ducts near their orifices just before reaching the sinus epithelium between the orifices. The result is a highly characteristic mesonephric–paramesonephric complex of a median solid paramesonephric epithelium, which remains in direct contact with the mesonephric ducts laterally, but is still split distally and separated from the sinus epithelium by a column of dense mesenchyme (Figs. 3i, 12b). The broad mantle of mesenchyme of the deep urogenital sinus differentiates into an inner layer of dense lamina propria with a predominantly longitudinal orientation (e.20 mm). An outer layer revealing a transverse texture on the ventral side shows early smooth muscle cell differentiation adjacent to the superficial urogenital sinus and forms the earliest anlage for the internal urethral sphincter (e.25 mm). A bilateral concentration of dense tissue in that same area fuses ventrally and forms the anlage for the striated external urethral sphincter. In the broader layer of mesenchyme on the dorsal side and mainly caudal to the mesonephric orifices appear: (1) the dense smallcell stroma of the Mllerian tubercle surrounding the paramesonephric

20

Development of the Sexually Indifferent Perineum

Fig. 12a, b Development of the mesonephric–paramesonephric complex in embryos of 26 mm t (a) and 28 mm t(b). In a (upper illustration) paramesonephric ducts (1), still in contact with the mesonephric ducts (2), begin to fuse, with their split solid tips (asterisks; lower illustration) separated from the epithelium of the urogenital sinus (3) by a column of dense mesenchyme (arrow). In b the paramesonephric ducts (1) have fused; their solid ends (asterisks) remain in direct contact with the mesonephric ducts (2) and separated from the urogenital epithelium (between arrowheads) by the dense mesenchyme (arrow)

structure, and (2) a vague anlage for the vesical sphincter cranial to the orifices. Development of the Superficial Urogenital Sinus and Phallic Structure. The development of the superficial segment of the urogenital sinus is very different from the formation of the deep segment of the sinus both in its epithelial and its mesenchymal structure. The epithelial structure consists of a lower two- to three-layered ps.str.col. epithelium. It shows a further increase in its median diameter in proportion with the growing phallic structure. As a result, the urogenital plate acquires a lengthening extension along the ventral wall of the urogenital sinus (Fig. 13a), which extension remains when disintegration of the superficial part gradually widens the urogenital orifice (Fig. 1e, f). The superficial urogenital sinus also develops a left and right lateral groove and a dorsal groove (Fig. 13a). Superficial to the urogenital plate extension, the glans plate forms a distinct small area of solid, slightly more eosinophilic epithelium in the tip of the phallus underlying an epithelial tag (Fig. 13b).

Observations

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Fig. 13a, b Superficial part of the urogenital sinus in embryos of 23-mm frontal section (f a), and 20 mm t (b). In a the superficial part of the early urogenital sinus (1), still surrounded by a mantle of dense mesenchyme, demonstrates a dorsal (2) and early lateral grooves (3), and the extension of the urogenital plate (4) along the ventral wall. In b the glans plate (5) with tag differs from both the ventral urogenital plate extension (4) and the epidermis (6); abrupt transitions indicated by arrows

Thereafter, grooves and ridges become more distinct. They include a newly formed ventral ridge flanked by deep and partially still solid ventrolateral grooves, and situated deep to the ventral extension of the urogenital plate. (Figs. 14c, 15a). From each lateral groove grows a solid primordium of the bulbourethral/greater vestibular gland (e.25 mm). The broad superficial mantle of mesenchyme differentiates into a complicated composition of stromal tissues. Apart from a dense lamina propria with a strict longitudinal orientation on the dorsal and lateral sides of the sinus, two sets of tissues are identified that form the main structure: a ventral fibrovascular zone comprising the primordial erectile elements and embracing the urogenital sinus ventrally, and dorsal stromal tissues demonstrating a distinct tendency toward a ventral orientation and coming to embrace the urogenital sinus dorsally (Fig. 15a). The latter in particular develop in close relationship with the more peripherally developing striated musculature of the perineum and the labioscrotal swellings. In the fibrovascular zone appear the future erectile structures in sensu strictu, i.e., glans, corpora cavernosa, urogenital labia, and bulbi spongiosi/

22

Development of the Sexually Indifferent Perineum

Fig. 14 Schematic drawings of transverse (a–d) and paramedian (e) sections through 30mm embryos to demonstrate the fibrovascular zone, with erectile structures, that embraces the urogenital sinus ventrally (a–c), and future fascial elements that embrace it dorsally (d–e). In a the glans (1) embraces the glans plate (2). In b the distal part of the urogenital sinus (3) is embraced by the distal corpora cavernosa (4), linked by numerous vessels to the urogenital labia (5), from where spreads a layer of transversely oriented superficial shaft stroma (6). In c the middle part of the urogenital sinus is flanked by the bulbi spongiosi/vestibulares (7), which are closely linked to both the urogenital labia and the proximal corpora cavernosa (at some distance; direction indicated by arrows). 6, Superficial shaft stroma; 8, bulbospongiosus; 9, ventral extensions of the deep dorsal urogenital stroma; 10, dartos stroma. In the sinus (3) are shown the ventral ridge (11) and lateral grooves with the primordial bulbourethral/greater vestibular glands (asterisk); 12, ischiocavernosus. In d a transverse section through the midperineal region between the urogenital and anal orifices reveals the deep (9) and superficial (13) urogenital stroma, with the former extending over a short distance into the puborectalis (14) and bulbospongiosus (8) muscles, and receiving longitudinal bundles of the muscularis propria of the rectum (15). Arrow, external cloacal groove derivative. In e a paramedian section lateral to the urogenital sinus better demonstrates the deep dorsal urogenital stroma (9) as it bends ventralward superficial to the bulbi spongiosi/vestibulares (7) and bulbospongiosus (8); 15, muscularis propria of the rectum; 16, external urethral sphincter partially surrounding the lateral wall of the deep urogenital sinus (3); 17, external anal sphincter. Original magnification, 30

t

vestibulares, as more or less delineated elements, which remain, however, closely associated by numerous anastomosing vessels (Fig. 14a–c). The glans of the phallic structure remains a cap of dense small-cell tissue relatively poor in blood vessels. It forms a large ventral to lateral part of the phallic structure becoming increasingly thinner towards its periphery. It is

Fig. 15a–d Stromal components embracing the urogenital sinus dorsally in embryos of 32 mm t (a), 23 mm f (b), 30 mm t (c) and 32 mm t (d). In a dense tissue lateral to the urogenital sinus (1) are found a urogenital labium (2), early bulbus vestibularis (3), and bulbospongiosus with perineal nerves (4). 5, Lateral groove with a primordial greater vestibular gland (arrow); 6, Ventral ridge; 7, ventrolateral groove; arrowhead, border be-

Observations

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tween ps.str.col. sinus epithelium and str.sq. labium epithelium. (cf. Fig. 14c). In b superficial dorsal urogenital stroma (8), superficial stroma of the shaft (9), and dartos stroma (10) that embrace the urogenital sinus (1) dorsally demonstrate an pronounced ventralward orientation. In c dorsal to the sinus are shown the central and denser peripheral deep dorsal urogenital stroma (11), superficial dorsal urogenital stroma (8), and deep dartos stroma (10). 12, Puborectalis. In d a labioscrotal swelling consists of the deep component (10a) of primordial dartos stroma, with a parallel ventralward orientation, and of the superficial component (10b)

24

Development of the Sexually Indifferent Perineum

dorsally divided into two halves by the glans plate and envelops the most distal parts of the corpora cavernosa. The corpora cavernosa elongate while remaining undivided distally and split into the crura of the penis proximally. Their very dense small-cell tissue differentiates into a broad peripheral primordial tunica albuginea characterized by a transverse pattern leaving the central core “structureless.” Such demarcation is absent on the side of the urogenital sinus where dense tissue extends as far as the sinus epithelium and is flanked by conspicuous plexuses representing the anlage for future ventral urethral/vestibular arteries. The urogenital labia become prominent ridges which flank the urogenital orifice (Fig. 15a) but are in fact part of a much larger and deeper area with a high vascularity on both sides of the superficial urogenital sinus extending to the other erectile structures. This fibrovascular tissue forms a band with a parallel transverse orientation on the dorsal side of the urogenital orifice after the original continuity between the urogenital-sinus-related and analcanal-related parts of the cloacal labia has been interrupted in the midperineal region between the urogenital and anal orifices. The bulbi spongiosi/vestibulares are present, in embryos of about 25 mm, as a pair of vaguely delimited vascular oval-to-round structures on both sides of the deepest part of the superficial urogenital sinus (Fig. 15a). They are the deepest erectile structures to develop from the fibrovascular zone and are situated beneath the crura of the corpora cavernosa. The bulbi are bordered by the lamina propria medially, the bulbospongiosus muscle laterally, the early muscular coat around the deep urogenital sinus cranially, and the anlage for the bulbourethral/vestibular glands caudally. As for the rapidly proliferating mesenchyme which embraces the superficial urogenital sinus dorsally, this tissue as a whole reveals a markedly parallel orientation in its texture (Figs. 10d, 14d, e, 15b). That pattern persists during its differentiation into a number of future fascial components. In the earliest stage the orientation is almost transverse with the tissue spreading into the urogenital labia and into the right and left early labioscrotal swellings, which still occupy a rather dorsal position (e.15–20 mm). The orientation gradually alters into a ventrolateral to ventral orientation in relation with the development of those structures. Then differences in density and topography become more distinct and allow the recognition of four stromal components: a. Superficial dorsal urogenital stroma which derives from the mesenchyme dorsal to the urogenital sinus near the orifice (Figs. 15b, c, 14d). This stroma develops between the deeper, longitudinally oriented lamina propria and the orifice. It embraces this most superficial part of the urogenital sinus and spreads into the dorsal parts of the urogenital labia and more lateralward toward the surface of this midperineal region where it is flanked by

Observations

25

ventralward-bending deep dorsal urogenital stroma and primordial dartos stroma (Figs. 14d, 15b, c). b. Deep dorsal urogenital stroma which forms an ill-defined mass dorsal to the deeper part of the superficial urogenital sinus. This stroma demonstrates an orientation in its tissue which is in the deep part more or less parallel to the long axis of the urogenital sinus but more superficially bends ventralward while embracing the sinus to end lateral to the corpora cavernosa (Fig. 14e). It consists of a denser peripheral component which extends into the adjacent parts of the puborectalis and bulbospongiosus muscles, and of a less dense central part (Fig. 15c). c. Superficial shaft stroma which extends from the urogenital labia ventralward, thereby occupying the flanks of the phallic structure and ending on its ventral side (“dorsum”), and also spreading into the lateral sulci between the phallic structure and the labioscrotal swellings (Figs. 10d, 15b). d. Dartos stroma which consists of a deep component participating in the general tendency to parallel ventralward orientation of the dorsal mantle tissue, and a superficial component more ventrally that has no special orientation and is restricted to the labioscrotal swellings of which it constitutes most of the volume (Figs. 10d, 15b, c, d).

3.2.3.2 Anal Canal The anal canal forms a narrow tube lined by ps.str.col. epithelium. Initially it does not reveal features which discriminate it from the gut cranially (Fig. 16a). However, before long a subtle difference appears between the mucosa of that most distal part of the gut, i.e., rectum, and that of the cloacaderived portion, which then shows a less regular distribution of nuclei in the epithelium and a more longitudinal orientation in the texture of the lamina propria and forms the cloaca-derived anal canal (e.17 mm). External to the rapidly disappearing remnants of the cloacal membrane, the originally shallow groove between flattened dorsal parts of the cloacal labia deepens and forms a sort of depression; simultaneously the midperineal region increases in volume to constitute a ventral wall (Fig. 16b). This depression is lined by a relatively thick str.sq. epithelium similar to the epithelium which forms the inner lining of the rest of the cloacal labia. Following events demonstrate that these two segments deep and superficial to the now disintegrated cloacal membrane will form the structurally different cloaca-derived deep and labia-related superficial segments of the anal canal (Fig. 16c, d). The deep anal segment, with its cloaca-derived ps.str.col. epithelium with irregularly distributed nuclei and dense longitudinally oriented lamina propria, becomes increasingly more distinct from a rectum that shows a more regular ps.str.col. epithelium with an eosinophilic luminal zone, and also a more irregularly structured lamina propria. It then emerges that the border between the rectal and anal mucosa is situated well within the smooth mus-

26

Development of the Sexually Indifferent Perineum

Fig. 16a–d Schematic drawings illustrating the development of the anal canal in embryos of 14 mm to 30 mm. They demonstrate in a the anal compartment (1) of the cloaca at the time when the cloacal membrane (arrowhead), flanked by the cloacal labia (2), disintegrates, making that compartment the primary anal canal. In b a vague difference in epithelia indicates a border with the rectum (3), which transition is well within the primordial smooth muscle layer of the rectum (4), flanked by the puborectalis (5). The groove between the dorsal parts of the cloacal labia widens into a depression (6), which is bordered ventrally by the dorsalward bulging mantle (7) of the urogenital sinus (8), with a flattening cloacal groove (9), and is flanked by the original labial stroma (2) and the rapidly growing external anal sphincter (10). 11, Epidermis. In c and d the depression deepens and narrows, adding a new superficial anal segment (6) with a new anal orifice to the original primary anal canal, which now forms the deep anal segment (1). 3, Rectum. Arrowhead, narrowing within the deep segment associated with the distal ends of the muscularis of the rectum; arrows, early anal crypts. Original magnifications, 70 (a), 45 (b–d)

cle layer that has developed around the rectum (e.19 mm). The distal extremity of this muscularis propria bends inward and strikingly narrows the cloaca-derived deep segment of the anal canal at a short distance cranially to where the segment passes into the labia-related superficial anal segment (Fig. 16c). This narrow cloaca-derived part forms the “bottom” of a funnellike structure the wide part of which is constituted by the superficial anal segment and opens into the amniotic cavity. External to the investing muscularis propria of the rectum, the anal canal is flanked by the bilateral puborectalis muscles, which were already well established at the beginning of the period (Fig. 16b, c). The superficial anal segment undergoes a striking change in shape by the deepening and narrowing of the initially rather shallow depression in step with the growth of the external anal sphincter (Fig. 16c, d). It thereby acquires, external to the original orifice, a new anal orifice. That increasingly more tubular superficial segment is built up on its lateral sides by fibrovascular stromal tissue derived from the dorsal portions of the cloacal labia (Fig. 16c, d), which converges dorsally towards a bar of transversely oriented stromal tissue flanked temporarily by the postanal swellings. Ventrally the labial tissue flanks the regressing cloacal groove situated in the midline of a dorsalward bulging midperineal region. The superficial anal segment is

Observations

27

Fig. 17a, b Anal canal in an embryo of 23 mm t (a) and remnant of the cloacal groove in an embryo of 32 mm t (b). In a is illustrated the characteristic configuration of the anal canal with the deep cloaca-derived segment (1) lined by ps.str.col. epithelium, and a narrowed superficial labia-related segment (2) lined by a broad str.sq. epithelium. Arrowhead, border between the segments; arrow, border with the epidermis; asterisk, early anal column; 3, puborectalis; 4, external anal sphincter; 5, caudal extremity of muscularis propria of the rectum; 6, labial stroma. In b tufts of dark ps.str.col. cloaca-derived epithelium in the bottom of the cloacal groove, flanked by str.sq. epithelium on remnants of the cloacal labia (6), persist near the urogenital sinus (upper) and anal canal (lower). 7, Transversely oriented superficial dorsal urogenital stroma; 8, ventralward labia-derived stroma of the anal canal

lined by a broad str.sq. epithelium which is in conformity with its derivation from the cloacal labia. It is a noteworthy phenomenon that the rapidly thickening str.sq. epithelium in the deepest and narrowest part of the superficial anal segment narrows the preexisting lumen to such a degree that this passage is hard to discern but persists in all embryos scrutinized (Fig. 17a). Near the external margin of the “funnel” the broad labia-related str.sq. epithelium gradually passes into the epidermis, which can be distinguished by its marked nucleus-free basal zone, a more regular distribution of nuclei in horizontal rows and a decrease in thickness in a peripheral direction. Concentrations of dense tissue outside the superficial anal canal indicate the bilateral primordia of the external anal sphincter (Figs. 16b–d, 17a) They are linked by equally dense tissue rich in vessels dorsal and ventral to the canal. Differentiation starts in the original lateral parts (e.25 mm).

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Development of the Sexually Indifferent Perineum

At the end of the period (e.30 mm) the rectal epithelium often reveals undulation as a first sign of crypt formation. The dense longitudinally oriented lamina propria of the deep anal canal forms the first anal columns, and reveals also a distinct extension into the smooth muscle layer, i.e., the future internal anal sphincter. A broad ridge marked by the remnant of the cloacal groove on the ventral side of the superficial anal canal forms the first anal cushion in some embryos. 3.2.3.3 Striated Perineal Musculature Originating as bilateral primordia from the dorsolateral perineal complex, the future striated muscle tissue extends ventralward as far as the corpora cavernosa (Fig. 8). It then becomes divided into the following separate primordial muscles from ventral to dorsal: a. Ischiocavernosus located on the free surface of the early crura of the corpora cavernosa b. External urethral sphincter situated deep and medial to the ischiocavernosus and just ventral and lateral to the smooth muscle layer representing the differentiating internal urethral sphincter in its embrace of the ventral side of the deep urogenital sinus c. Bulbospongiosus situated dorsal to the ischiocavernosus and lateral to the bulbi spongiosa with dense tissue dorsally linking the two primordia to each other and to deep portions of the external anal sphincter d. External anal sphincter situated in line with and dorsal to the bulbospongiosus with the two halves linked by dense tissue ventral and dorsal to the superficial segment of the anal canal

Differentiation into striated muscle cells and a better demarcation are first recognized in the ischiocavernosus and bulbospongiosus (e.22 mm), followed by the external anal sphincter (e.25 mm). The muscle cells in the various muscles demonstrate a ventralward orientation. The primordial tissue of the external urethral sphincter spreads ventralward and cranialward forming a thin layer outside the ventral smooth musculature of the internal urethral sphincter. Myotubes can, however, not be recognized before the urethra is being structured during sexual differentiation (e.60 mm). 3.2.3.4 Vascular System During the first half of this period the right and left branches of the median sacral artery are still very prominent. They course lateral to the lower rectum and form a conspicuous ventral anastomosis at the level of the transition from rectum to anal canal, deep to the external anal sphincter. Branches

Observations

29

supply plexuses around the anal canal and in the dorsal wall of the urogenital sinus, including the dorsal parts of the urogenital labia. However, with the rapid decrease in caliber of the median sacral artery halfway through this period, the two arteries also decline, and although they can still be identified, their blood supply is largely taken over by the internal pudendal arteries (Fig. 9c). These internal pudendal vessels become the predominant system of the perineum. In association with the marked development of the perineum and the structures deriving from the dorsolateral perineal complex, and from the broad mantle around the urogenital sinus in particular, on each side a lateral and a ventral division is formed. The lateral division, which originally supplies the dorsolateral perineal complex, next develops branches to the perineal striated muscles and labioscrotal swelling. However, it also takes over most of the blood supply of the sacral system: (a) by a dorsal branch to the external anal sphincter, which then supplies the adjacent anal canal and perianal skin as the inferior rectal artery; (b) by branches linked to the anastomosing artery ventral to the transition from anal canal into rectum, which as the transverse artery of the perineum continues to supply the tissues between the urogenital sinus and anal canal and adjacent rectum; and (c) by branches to the labioscrotal swellings, which take over the supply to the dorsal parts of the urogenital labia and adjacent wall of the urogenital sinus. The ventral division, originally supplying the future erectile tissues in particular, develops branches on each side to the anlage for the bulbi spongiosi, to the tissues of the vascular zone between the corpora cavernosa and adjacent parts of the urogenital labia, as well as to the corpora cavernosa and glans. The veins often accompany the arteries, though in a more irregular pattern. Dorsally some communications remain with median sacral veins derived from the postcardinal system. Ventrally the original allantois-related plexus transforms into the deep dorsal vein of the penis/clitoris which will preserve wide communications with the venous plexuses around the base of the bladder and prostate. This configuration of arteries and veins represents in fact the definitive pattern that will undergo gradual alterations in conformity with the differential growth during the transformation into a female or male perineum. Lymph vessels have not yet entered the perineum. Large labyrinthine plexuses or sacci are still confined to the area around the main iliac veins. Sprouts from these structures will spread through the perineum much later (f.60–130 mm) as extensions of superficial inguinal plexuses and deep perivascular lymph channels.

30

Development of the Sexually Indifferent Perineum

3.2.3.5 Nervous System The pelvic autonomic nerve plexuses become very large as compared with the other structures of the perineum and are easily recognized by the many small concentrations of ganglion cells. They form bilateral masses extending from dorsolateral to the deep anal canal and adjacent rectum to the area lateral to the most caudal extension of the coelom and dorsolateral to the deepest part of the urogenital sinus and adjacent bladder, to end around the ureters. They send small nerves into the mantles around the anal canal and the urogenital sinus and bladder. The spinal nerves also become relatively large and conspicuous structures especially when compared with the closely linked developing striated muscles. Their pattern is largely similar to that of the arterial system. From the lumbosacral plexus form the bilateral pudendal nerves, each of which splits into a nerve to the ventral part of the urogenital sinus, including the future erectile tissues and skin, and into large bundles to both dorsolateral perineal complexes. These last form the right and left perineal nerves, each of which splits into inferior rectal nerves to the external anal sphincter, the anal canal, and perianal skin dorsally, and into nerves to the bulbospongiosus, external urethral sphincter, and ischiocavernosus muscles, to the dorsal parts of the urogenital sinus and of the urogenital labia and labioscrotal swellings. This construction of the nervous system will not change essentially during later female and male differentiation. 3.2.3.6 External Perineum The development of the characteristic external shape of the perineum mirrors the strong proliferation of stromal elements during the first half of this postcloacal sexually indifferent period (Figs. 1, 10). The combined rapid growth of fibrovascular tissues embracing the urogenital sinus ventrally and the more (myo)fibroblastic tissues embracing the sinus dorsally create a cylindrical phallic structure, with the future erectile elements forming most of its protruding parts and the future (myo)fibrous tissues predominating in its base. The latter combine with the labioscrotal swellings in the formation of a broadening midperineal region between the urogenital and anal orifices. The growth of this region makes the dorsal part of the phallic structure expand to such a degree that for some time it overhangs the anal orifice and flattens the original median cloacal groove between the two orifices (e.17– 24 mm). It thus obscures the anal orifice and the more so because that primary orifice at that time comes to lie in the bottom of a deepening depression, with stromal tissues on the lateral and dorsal sides also growing to such an extent that they form a new (secondary) anal opening. The area dor-

Discussion

31

sal to the anal orifice becomes flatter with the disappearance of the postanal swellings (e.23 mm). On the ventral side, the growth of the urogenital labia makes the slit-like urogenital orifice more conspicuous. The rapid expansion of the primordial dartos tissue from the dorsolateral perineal complexes at the dorsolateral base of the cloacal eminence ventralward brings the main volume of the labioscrotal swellings to the lateral sides of the eminence and thereby demarcates the phallic structure. During the second half of the period this phallic structure elongates further. A glans becomes more or less demarcated by a shallow groove in some embryos. The new anal orifice then appears dorsal to the phallic structure. It is surrounded by a wall built of fibrovascular tissue from the cloacal labia and the growing external anal sphincter underneath. The epithelium on the surface of the perineum shows a certain distinction, with a peripheral zone still covered by a usual type of two-layered epidermis with a conspicuous nucleus-free basal zone, and a central area covered by a much broader four- to six-layered str.sq. epithelium with nuclei at the basement membrane. The central region is directly related to structures derived from the primordial glans and cloacal labia, i.e., glans, urogenital labia, paramedian zones in the midperineal region between the urogenital and anal orifices initially flanking and later replacing remnants of the cloacal groove, and a narrow zone around the anal orifice. In the midline of this midperineal region the broad str.sq. epithelium may originally flank (remnants of) a narrow strip of ps.str.col. epithelium that had remained from the original communication between the urogenital and anal compartments of the cloaca prior to its transformation into the cloacal groove after the disintegration of the dorsal part of the cloacal membrane (Fig. 17b). Underneath are the superficial dorsal urogenital stroma at the side of the urogenital sinus and a ventralward extension of parallel ventralward-oriented stroma derived from the cloacal labia originally flanking the anal orifice. 3.3 Discussion The present study with its emphasis on the development of the early perineum as a whole, leads to a new concept which differs from current ideas and reveals major shortcomings in the descriptions in textbooks of embryology (Arey 1965; Hamilton and Mossman 1972; Moore and Persaud 1998; Wartenburg 1993; Sadler 1995; Larsen 1997). The present findings add new information about all major aspects of the development of the sexually indifferent perineum: the formation of the cloacal eminence; the relationship between the cloaca and allantois, including the partition of the mesonephric and ureteric systems; the division of the cloaca into the urogenital sinus and the anal canal; and the development of the urogenital sinus, including its relationship to the mesonephric and immigrating paramesonephric ductal sys-

32

Development of the Sexually Indifferent Perineum

tems, and of the anal canal, perineal striated musculature, labioscrotal swellings, and external perineum. 3.3.1 Cloacal Eminence The term “cloacal eminence” used by Retterer (1890) is reintroduced to indicate the swelling which develops during the cloacal period, occupies the whole area between the umbilical cord, the two hind buds, and the tail, and consists of the cloaca and its mantle of mesenchyme. It includes the anal region and later incorporates dorsolateral perineal complexes derived from the dermatomyotomes of sacral somites. The term therefore has a broader meaning than the terms “genital tubercle” and “cloacal tubercle,” as generally employed to name the not further defined prominence that is assumed to become the phallus first and the penis or clitoris later. The investigation shows that this cloacal eminence forms the anlage for the whole of the perineum. It is interesting to note that this concept fits well into the modern anatomical definition of the “perineum” as the diamondshape area superficial to the pelvic diaphragm and bordered by the pubic arch, ischial tuberosities, and coccyx (Williams 1989; Moore and Dalley II 1996), albeit that from an embryological point of view it should also include the female urethra and musculature, and the prostatic urethra and musculature, prostate, colliculus seminalis, and distal ejaculatory ducts in the male. The new concept concerning the cloacal eminence offers an embryological base for the structural unity of the perineum as is also illustrated by vascularization (internal pudendal vessels) and innervation (pudendal nerves). It should be noted that in embryology the term “perineum” is generally used for the much more limited area between the urogenital and anal orifices which is thought to derive from the so-called urorectal septum and persists in the female as the “gynecological perineum.” This area which lacks welldefined boundaries is referred to as “midperineal region between the urogenital/vestibular/urethral and anal orifices” in the present study to avoid confusion. 3.3.2 Cloaca and Allantois: Partition of Mesonephric and Ureteric Systems It is current opinion that the ventral (urogenital) part of the cloaca forms both the urethra and the urinary bladder (Arey 1965; Hamilton and Mossman 1972; Wartenberg 1993; Sadler 1995; Larsen 1997). This reflects interpretations from original work (Keibel 1896; Pohlman 1911; Chwalla 1927) which were mainly based on wax models and appear to be influenced by phylogenetic considerations. On histological grounds that conclusion is difficult to sustain, taking into account the striking differences in both epitheli-

Discussion

33

um and mantle tissue between the cloaca and the allantois from a very early stage onward, as is demonstrated in the present study. These striking differences remain evident in their respective derivations in female and male later. The observations indicate that the urinary bladder is derived from the allantois, with the exception of the trigone which is formed by a combination of elements from the allantois and cloaca as the result of the partition of the mesonephric ducts and ureters and their orifices. Such a conclusion is corroborated by the analysis of congenital malformations of both structures (Stephens et al. 1996). The first stage of that process of partition of the mesonephric ducts and ureters, which comprises the disappearance of the short common excretory duct and the establishment of separate mesonephric and ureteric openings into the ventral cloaca (later urogenital sinus), has been attributed to the absorption of the common excretory duct into the fast-expanding adjacent cornua of the cloaca, either by extension of the cloaca along the ducts followed by the disappearance of the intussusceptum (Frazer 1935), or by replacement of the original mesonephric epithelium by cloaca epithelium (Kempermann 1935; Gyllenstein 1949). The present observations indicate a process of apoptosis in the epithelium of the common excretory duct, which epithelium is replaced by cloaca epithelium that grows underneath, as suggested by Kempermann (1935), incorporating these undermined terminal ducts into the cornua of the cloaca and providing ducts and ureters with separate orificia. The second stage, which comprises the actual parting of the orifices leading to the formation of the trigone, has been considered the result of either the migration of the mesonephric ducts caudal- and medialward (Chwalla 1927), or of the increase in length of this part of the urogenital sinus, as the major factor (Frazer 1935; Gyllenstein 1949). The latter mechanism would cause the mesonephric ducts to form a loop caudalward with its inner part inside the sinus epithelium. This would then be followed by the breaking down of the front wall inside the sinus and progression of the orifice caudaland medialward, while at the same time (Frazer 1935) or later (Gyllenstein 1949) the orifices of the ureters are carried cranialward. They would thus make the trigone a sinus-derived structure. The idea of Brockis (1952) and Stephens et al. (1996) that the trigone and posterior part of the urethra derives from the mesonephric ducts is caused by misinterpretation of the wide cornua of the cloaca as representing dilated distal parts of the mesonephric ducts, a mistake most likely made because the clear differences in epithelium were not taken into account. The present observations of bilateral lengthening zones of sinus epithelium between the mesonephric and ureteric orifices and the increasingly more oblique orientation in the texture of the especially dense mesenchyme support this essential role of lengthening of the wall between the mesonephric and ureteric orifices, as suggested. However, the observations demon-

34

Development of the Sexually Indifferent Perineum

strate that the phenomenon of cranialward extending mesonephric epithelium which has been interpreted as “loops” by Gyllenstein (1949) is associated with a more or less similar phenomenon cranially in the form of short extensions of the ureters inside the lengthening zones of sinus epithelium. This combination of extensions offers insufficient proof of such a complicated process as loop formation. A simple explanation is favored in which shifting orifices during lengthening of the wall between the mesonephric ducts and ureters include not only the trailing epithelium but also the underlying mesenchyme. The analysis also demonstrates that it is too simple a construction to consider the trigone as a derivation of the urogenital sinus. It reveals that in the midline on the cranial side a tongue of allantois-derived epithelium of the bladder lengthens and before long replaces the original sinus epithelium near the orifices of the ureters. In addition, a direct relationship between the mesenchyme of the trigone and either the allantois or the urogenital sinus cannot be established with certainty. 3.3.3 Division of the Cloaca The notion that the cloaca consists not only of an epithelium-lined cavity but also of a broad mantle of mesenchyme is necessary in order to understand the mechanism underlying the subdivision of the cloaca into separate urogenital and anal parts. That subdivision has in the past caused considerable controversy between theories which evolved from fusion between lateral ridges (Rathke 1832; Retterer 1890), or a descending frontal septum (Tourneux 1888; Pohlman 1911; Chwalla 1927) to a combination of both (Stephens et al. 1996) possibly in the form of a horseshoe-like septum (De Vries and Friedland 1974). Thereby, all theories included a last stage during which the septum fuses with the still-intact cloacal membrane dividing that membrane into separate urogenital and anal membranes. It is evident from the literature that these constructions were often theoretical and intimately linked to the various variants of congenital malformations which were in need of an embryological base (Bill and Johnson 1958; Duhamel 1968; Stephens et al.1988, 1996). These theories based on fusion still monopolize the textbooks of embryology and still appear in original papers (Kromer 1999). Alternative opinions have hardly received attention. The latter comprise ideas about a simple expansion of mesenchyme between the two compartments of the cloaca (Politzer 1931), or about a decisive role for alterations in the shape of the caudal region of the embryo (Blechsmidt 1961; Wijnen 1964; Nievelstein 1998; Paidas et al. 2000). Fusion between a “septum” and the cloacal membrane has been doubted (Chwalla 1927; De Vries and Friedman 1974) or re-

Discussion

35

jected (Politzer1931; Ludwig 1965; van der Putte 1986; Nievelstein et al.1998; Paidas et al. 2000). The present investigation does not endorse the idea that the cloaca is divided by a descending “urorectal septum”. The concept of division by such a septum which is seemingly supported by highly suggestive pictures in median sections through 12–17-mm embryos, appears to be related to the underlying idea that the cloaca is in essence a chamber-like structure being divided into two parts. However, if the information from the developing epithelial and mesenchymal components of the cloaca is combined, it emerges that the cloaca is basically a tubular structure which becomes increasingly more bent toward the surface. Frontal sections clearly demonstrate the tubular character of urogenital and anal structures surrounded by their mantles and illustrate that the just-mentioned pictures of median sections are obtained only in a very narrow median zone (van der Putte 1986). The present study demonstrates the cloaca to be a bending tube undergoing a rapid and disproportionately strong growth of the mantle around the ventral urogenital part as compared with the mantle around the dorsal part; an alternative mechanism for division of the cloaca is thus offered. It is possible that changes in the shape of the caudal region in a process of caudal “unfolding” as suggested by Nievelstein et al. (1998), or a more general process of “transformation” as suggested by Paidas et. al. (2000), may contribute but it appears that the marked disproportionate growth of the mantle of the cloaca is the major and most direct factor in its division. According to this concept there is no “septum,” but rather a region consisting of the dorsal part of the mantle around the urogenital compartment of the cloaca, and the ventral part of the mantle around the gut and the anal compartment. Separated by a caudal extension of the coelomic cavity at a deep level, these layers are in close contact with each other more superficially where they constitute a midperineal region which has no clear demarcation other than the lumina of the urogenital and anal parts of the cloaca. This region is therefore more a topographical feature than a well defined morphological structure. It is the interlocking of both mantles which will greatly contribute to the strength of the female and male perineum. The idea that the “urorectal septum” would finally fuse with the cloacal membrane thereby completing the division of the cloaca and establishing separate urogenital and anal membranes is expressed in textbooks of embryology (Hamilton and Mossman 1972; Sadler 1995; Larsen 1997; Moore and Persaud 1998) and features prominently in theories about the pathogenesis of anorectal malformations such as “imperforate anus” (Bill and Johnson 1958; Duhamel et al. 1966; Stephens et al. 1988), notwithstanding regular dismissal (Politzer 1931; Ludwig 1965; van der Putte 1986; Nievelstein 1998). The present analysis demonstrates that the most superficial part of the bending cloaca remains an open communication between the urogenital and anal compartments until it becomes part of a single cloacal orifice first and

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Development of the Sexually Indifferent Perineum

forms a short groove on the surface later. This groove usually disappears without leaving a trace, but may persist as has been observed in the “female perineum” of premature babies (Stephens 1968). This concept on the division of the cloaca fits well into the new explanation of the pathogenesis of “imperforate anus,” with or without communications with the surface of the perineum or the urogenital system, as has been established on the basis of observations in pig embryos with congenital hereditary imperforate anus (van der Putte and Neeteson 1984; van der Putte 1986). 3.3.4 Urogenital Sinus The term “urogenital sinus” has been applied to different structures. Originally it was used to indicate that portion of the ventral cloaca situated between the orifices of the mesonephric ducts and the cloacal membrane, later (after disintegration of this membrane) urogenital orifice. The short cranial segment of the cloaca between the mesonephric orifices and the bladder was named “urethra” (Mijsberg 1924), “primary urethra” (Chwalla 1927), or “primitive urethra” (Hamilton and Mossman 1972), thus illustrating the opinion of the investigators that this part would transform into the female urethra (and the most cranial part of the male urethra). The urogenital sinus itself is subdivided by them into a deeper “pelvic” part cranial to the bulbourethral gland primordia, and a superficial “phallic” part caudally on the basis of marked differences in shape. Nomenclature in more recent literature is even more confusing. The ventral part of the cloaca before the partition of the cloaca has been completed has been designated as “urogenital sinus” (Wartenburg 1993; Moore and Persaud 1998) or “primitive urogenital sinus” (Sadler 1995; Larsen 1997). After the partition, that (primitive) urogenital sinus is considered to become the urinary bladder and pelvic and phallic parts of the urogenital sinus (Wartenburg 1993; Moore and Persaud 1998), or bladder and definitive urogenital sinus (Sadler 1995), or pelvic urethra and definitive urogenital sinus (Larsen 1997). It is evident that the view on the development of the ventral part of the cloaca, and allantois in particular as presented here, cannot be brought in line with any of these terms. The present study does not offer arguments in favor of a specific segment between the bladder and the (definitive) urogenital sinus, and positioned superficial to the orifices of the mesonephric ducts, that has been thought to transform into the upper part of the male urethra and the whole of the female urethra (Chwalla 1927; Hamilton and Mossman 1972; Larsen 1997). The present investigation stresses the nature of this segment as a borderland with elements of the bladder prevailing ventrally, elements of the compound trigone and cranial part of the tuberculum Mlleri dorsally, and elements of

Discussion

37

the urogenital sinus caudally. During male differentiation this complex construction remains visible in the relatively short segment of the male urethra cranial to the orifices of the ejaculatory ducts. In the female it is found that this cranial segment is not simply the precursor of the female urethra, as is general belief. As will be demonstrated, the female urethra develops as a composite structure in fetuses between 60 and 110 mm CRL (see Sect. 4.2.2). The present investigation supports the subdivision of the urogenital sinus into two parts, with distinct differences in the histology of the surrounding stroma added to the already reported differences in shape. However, the traditional nomenclature of “pelvic” and “phallic” parts has been abandoned now. Investigations into the later development of this area demonstrate that both the female urethra and the male prostatic urethra and prostate, including their mantles of smooth and striated musculature, derive from the deep part of the ventral cloaca (later urogenital sinus) and can therefore not be separated from the more superficial “perineal” structures. This relationship to the perineum is also illustrated by their common vascular supply and innervation. It makes the designation “pelvic part” for the deeper portion of the urogenital sinus inappropriate and even misleading. For this reason the non-committal adjectives “deep” and “superficial” are preferred. Information about the development of the urogenital sinus has mainly been based on the detailed work of Chwalla (1927) and on some preambulatory data in original work on the development of the male urethra (Herzog 1904; Johnson 1920) and female vestibulum (Mijsberg 1924). These data yield hardly any information on the differentiation and organization of the surrounding tissue, which is generally described as “mesenchyme” without further specification. From the present study emerges a much more complicated picture. In the development of the at-first-sight simple looking deep urogenital sinus two aspects deserve special attention. The first is the complexity of the developing coat, a complexity which at this stage is mainly manifested by differences in pattern rather than by cellular differentiation. However, if brought into the perspective of histological development after this sexually indifferent period, it can be established that even at this early stage the anlage for all main elements distinguished in older fetuses, i.e., the trigonumrelated vesical sphincter, predominantly ventral internal end external urethral sphincter, prostate-related musculature, ventral and dorsal extensions of the muscle layers of the bladder, minor contributions of the mantles of mesonephric and paramesonephric ducts, and stromal tissue of the tuberculum, are already present. No information on this composition at this stage could be retrieved from the literature. The second aspect is the invasion of the paramesonephric system into the tuberculum Mlleri. The present observations confirm the earliest events in the development of the paramesonephric ducts, which have been reported to originate from coelomic epithelium covering the most cranial parts of the

38

Development of the Sexually Indifferent Perineum

genital ridges, grow caudalward guided by the epithelium of the mesonephric ducts, and finally reach the tuberculum while fusing with each other in the median plane just before contacting the dorsal wall of the urogenital sinus (Gruenwald 1941; Frutiger 1969). The present study stresses, however, that the intimate and direct contact between the solid ends of the paramesonephric ducts and the epithelium of the most distal parts of the mesonephric ducts on both lateral sides persists, and that only a minor contact, if any, is established between the paramesonephric epithelium and the sinus epithelium, as is also illustrated by the temporary column of mesenchyme separating both. This configuration has apparently not been appreciated yet and will form an essential step in the development of the vagina or the prostatic utricle later (see Sects. 4.2.1, 5.2.1). In the development of the superficial part of the urogenital sinus there are also two aspects which deserve special attention. The first relates to the fate of the ventral part of the cloacal membrane, which is shown to transform into the structurally differing urogenital and glans plates. This plate-like ventral part of the cloacal membrane has featured prominently in texts on the development of the urogenital system as the “urethral plate,” which has been presumed to play a major role in the formation of the male spongy urethra (Glenister 1954, 1958; Arey 1965; Hamilton and Mossman 1972; Wartenberg 1993; Moore and Persaud 1998; Sadler 1995; Larsen 1997). The present study shows its formation to be associated with the strong growth of the ventral part of the cloacal eminence, which transforms the ventral part of the cloacal membrane into a large superficial plate separating the cloacal and amniotic cavities, and also forming an elongating slender extension along the ventral wall of the cloaca, later urogenital sinus. Later dehiscence in the central layer of the larger superficial plate enlarges the cavity of the cloaca and also widens the already dorsally existing urogenital orifice. This is the final situation in both the female and the male, the plate area to become part of the superficial vestibulum in the female and part of the navicular fossa in the male. A similar dehiscence in the extension of the urogenital plate along the ventral wall of the urogenital sinus will later form a median ventral longitudinal groove in the vestibulum and in the male urethra, from which will originate the primordia of the lacunae Morgagni. It will be shown that there is no indication of urogenital plate transformation into the male spongy urethra, as has been constructed by Glenister (1954, 1956). Its role in the formation of the vestibulum and its rather limited role in the development of the male urethra make the generally used term “urethral plate” less appropriate than its replacement, “urogenital plate.” The study identifies a hitherto overlooked, relatively small, most ventral part of the cloacal membrane as different from the urogenital plate. It is named “glans plate” because of its close relationship to the developing glans. During the sexually indifferent developmental stage of the perineum, histological differences between the glans plate and the urogenital plate are subtle

Discussion

39

and the structure as a whole rather inconspicuous, which is most likely the reason for its escape from earlier detection. However, the identification of this specific part of the cloacal plate is important because it solves an ongoing dispute about the formation of the terminal urethra in the male. Central in that discussion is the question as to whether the terminal segment develops by canalization of the solid distal part of the entodermal urogenital plate, which then is thought to extend to the tip of the glans from the beginning and by canalization either provides the urethra with its definitive external orifice (Nagel 1892; Van de Broek 1910; Williams 1952) or enlarges the original urethral orifice (Herzog 1904; Kurzrock et al. 1999; Baskin 2000; Penington 2001), or derives from an isolated ectodermal ingrowth from the surface of the tip of the glans, which canalizes and makes contact with the more proximal urethra derived from the entodermal “urethral plate” (Hart 1908; Wood Jones 1910; Glenister 1954, 1958; Altemus and Hutchins 1991; Stephens et al. 1996). It is the last theory which has found general acceptance in the textbooks of embryology (Hamilton and Mossman 1972; Wartenberg 1993; Sadler 1995; Larsen 1997; Moore and Persaud 1998). The glans plate identified in the present investigation combines the major elements of both main theories: it is part of the original cloacal plate but differs from the undoubtedly entoderm-derived more proximal urogenital plate by features that suggest an ectodermal derivation such as a distinctly different type of epithelium that reveals differentiation into str.sq. epithelium in fetuses of 45 mm and remains a distinct str.sq. epithelium thereafter. While decreasing in the female, the glans plate becomes an important structure in the male. Matching the considerable growth of the glans penis, it becomes a relatively large comb-like extension of the roof of the navicular fossa and, after splitting, greatly widens the preexisting fossa and external urethral orifice. These latter remain lined by original entoderm-derived ps.str.col. epithelium at the side of the venter (see also Sect. 5.2.1). The second major aspect in the development of the superficial part of the urogenital sinus relates to the differentiation of its broad mantle. No information about this layer is available other than that it consists of “mesenchymal proliferations.” It is now demonstrated that even at this early stage of development, differences in the structure of the mantle can be identified which make it possible to distinguish a ventral and a dorsal component. The ventral mantle forms a fibrovascular zone that embraces the urogenital sinus on the ventral side and consists of the erectile structures in sensu strictu, i.e., the glans and corpora cavernosa ventrally, the bulbi spongiosi/vestibulares and urogenital labia laterally. The dorsal mantle has a strikingly parallel and predominantly ventralward orientation in tissue that embraces the superficial urogenital sinus and the vascular zone from dorsally. Its various elements will differentiate into an intricate system of septa and fasciae in female and male fetuses. This construction of two embracing sets of stromal tissues necessarily creates a basically cylindrical shape, i.e., the phallic struc-

40

Development of the Sexually Indifferent Perineum

ture which will elongate by strong growth into the male penis and will become relatively flattened into the female vulva. 3.3.5 Anal Canal The present observations on the early development of the anal canal confirm the detailed descriptions by Johnson (1914) and the complementary work by Chwalla (1927). These authors have given special attention to the relationship between the shape of the canal and its epithelial lining in order to establish exactly the disputed extent of entoderm-derived and ectoderm-derived parts. Johnson considered the str.sq. epithelium inside the canal to be derived from the original cloaca, thereby positioning the original localization of the “anal membrane” at the level of the anocutaneous line. In contrast, Chwalla was of the opinion that only the deeper cuboidal to columnar epithelium derives from the entodermal cloaca and that str.sq. epithelium is proof of a derivation from the ectoderm-derived “proctodeum,” thereby positioning the anal membrane at the level of the pectinate line. That proctodeum (synonyms: anal or ectodermal pit, ectodermal depression, external cloaca) has been thought to have formed as a depression by the proliferation of surrounding mesenchyme related to the postanal swellings (Stephens et al. 1996) and external anal sphincter (Otis 1905). Although the concept of a “proctodeum” has been criticized by Ludwig (1965), who stated that as a counterpart of the stomatodeum such a structure should include the larger urogenital part of the external groove between the cloacal labia as well, and by De Vries and Friedland (1974) who dismissed the idea of a proctodeum as a misinterpretation of developmental events in the chick, the idea still finds general favor. The present study clearly demonstrates that the anal canal is a complicated and even ambiguous structure as the cloaca-derived anal epithelium and lamina propria become surrounded by the muscularis propria of the rectum for most of its length. The originally cloaca-derived structure that ends where the cloacal membrane disintegrates is lengthened considerably by the addition of an external labia-related segment, which creates a new orifice more superficially. In agreement with Ludwig (1964) and De Vries and Friedland (1974), this external segment is not here considered to be a “proctodeum” in the sense of a caudal counterpart of the stomatodeum. As has also been noted by Ludwig (1964), the developmental stage is not correct and such a “proctodeum” should comprise the whole external groove outside the cloacal membrane, and cannot represent a specific anal portion of this groove. The neutral term “anal pit” is a better choice in that respect. However, the present investigation demonstrates that as a “macroscopic” structure, this pit comprises also a portion of the cloaca-derived anal canal in its deepest part and cannot be considered to represent exclusively the ec-

Discussion

41

todermal component of the cloaca as is most commonly stated in the literature. On histologic and ontogenetic grounds the non-committal terms “deep and superficial anal segments” are preferred to indicate the histologically different cloaca-derived and labia-related parts, respectively. This study shows that the transition from ps.str.col. epithelium typical of the cloaca into the broad str.sq. epithelium characteristic of the inner sides of the cloacal labia is at the original position of the cloacal membrane and therefore forms the border between the entoderm- and ectoderm-derived mucosal segments of the anal canal. This would suggest that in the definitive anal canal that border is situated at the dentate line where str.sq. epithelium meets ps.str.col., as is indeed the general view. However, as will be demonstrated in the next section on the further development of the anal canal after the sexually indifferent period, the appearance of a sort of intermediate zone, which for some time combines str.sq. epithelium and remnants of ps.str.col. epithelium, still leaves some cause for uncertainty (see Sect. 4.2.8). A noteworthy phenomenon observed during this investigation is the pseudo-occlusion of the anal orifice by the broad str.sq. epithelium near its border with the cloaca-derived ps.str.col. epithelium. That pseudo-occlusion has been interpreted either as a primary anal membrane persisting in embryos as large as 22–32 mm (8–9 weeks; Johnson 1914; Chwalla 1927; Tench 1936), or as a secondary true occlusion in embryos up to 27 mm (Ludwig 1964) or between 18 and 32 mm (Nievelstein et al. 1998). However, as has been shown in the present study a separate “anal membrane” does not form; the related dorsal part of the cloacal membrane disappears in embryos of 15 mm and when the pseudo-occlusion is scrutinized a tiny lumen is observed in each of the embryos. This temporary situation is apparently the result of a rapidly and markedly broadening epithelium within a narrowing passage. At most, a small focal adherence between the opposing surfaces may occur. It is evident from these observations that an “imperforate anal membrane” cannot be considered a cause for most types of anorectal malformations as is generally assumed. It should be stressed that the present observations clearly demonstrate that the anal canal has a character of its own which is quite different from the adjacent rectum as the most distal part of the intestine, and should not be designated as “rectum” as is almost general practice. 3.3.6 Perineal Striated Musculature Information on the development of the perineal striated muscles during the sexually indifferent period is scarce and provides only a few data about the appearance of the external anal sphincter muscle only (Otis 1905; Bourdelat et al. 1990).

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Development of the Sexually Indifferent Perineum

Central to the development of the perineal musculature as a whole is the idea of Popowsky (1899) of a basic structure derived from a cloacal sphincter. His theory was confirmed by Power (1948) who paid special attention to phylogenetic aspects. The present observation of primordia developing bilaterally from sacral somites and forming two series lateral to the cloacal structure, which become linked to each other both dorsal and ventral to the cloaca, makes his concept attractive indeed. However, it has to be realized that Popowskys diagrams demonstrating a circular band of (striated) muscle tissue around the cloaca, which had been based on the findings in dissection of 3-month-old fetuses, is an oversimplification at the cloacal stage and not realistic in man. During the period that the cloaca exists, the primordial muscle tissue is still part of the right and left dorsolateral perineal complexes and positioned as small, poorly defined masses in the dorsolateral base of the cloacal eminence. Only at the end of that period do these masses extend ventralward, and then are still difficult to distinguish as they form only small concentrations of dense tissue identifiable by position and nerve supply only. Identification by the appearance of myoblasts and by a better delimitation is not possible before the 22-mm stage, after the cloaca has ceased to exist as such. There is no support for Otiss idea that the external anal sphincter muscle develops from the postanal swellings, which in his view would extend ventralward while encircling the anal orifice. It is now found that the primordial tissue of the sphincter is situated deep to the postanal swellings. From the earliest stage of development from sacral dermatomes the tissue of these swellings is easily identified by their special structure and position immediately beneath the epidermis, finally to disappear completely in embryos of about 22 mm. No separate anlage for the transverse perineal muscles can be identified. 3.3.7 Vascular System Information on the development of the vascular system of the perineum appears to be restricted to the results of an investigation into the arterial supply of the anorectum in relation to the pathogenesis of anorectal malformations by Bourdelat et al. (1988). The present analysis confirms their data, and notably the participation of the median sacral artery in the vascularization of the anorectum, its subsequent reduction and the simultaneous progressive appearance of the medial and inferior rectal arteries. However, our analysis also reveals that from an embryological point of view, the involvement of the median sacral artery is greater and more fundamental than suggested by their work. The development of a pair of large ventral branches at a stage in which the median sacral artery is actually the caudal part of the dorsal aorta strongly suggests that the two ventral branches supplying the dorsal cloaca, as a derivative of the most caudal part of the hind gut, may

Introduction

43

well represent the most caudal set of arteries that supply the early gut and allantois and is therefore homologue to the other ventral branches of the aorta, namely celiac, superior and inferior mesenteric, and umbilical arteries. In accord with this concept, the median sacral system supplies the whole of the dorsal half of the cloaca and its derivatives. It is interesting to note that the transverse artery of the perineum is a remnant of this early system that has been incorporated into the now-dominant internal pudendal arterial system. This drastic change appears closely linked to the decrease of the sacral vessels and to the strong development of the ventral cloaca, perineal musculature, and labioscrotal swellings, all of which from an early stage onward are supplied by the internal pudendal arteries. 3.3.8 Labioscrotal Swellings The development of the labioscrotal swellings has apparently drawn attention only for their conspicuous external shape. Their origin has not been specified yet. Making use of their remarkably extensive nerve supply they are now traced back to an early stage when their mesenchyme was still part of both dorsolateral perineal complexes, as is the anlage for the perineal striated musculature. Following this dense subepidermal mesenchyme backward in its development makes a derivation from the dermatomes of sacral somites most likely. The close association to the muscles persists throughout life and is expressed in their common vascularization and innervation by branches of the internal pudendal arteries and perineal nerves. Their mesenchyme is specific and differs from the usual subepidermal type in that it differentiates into the dartos stroma first and smooth muscular dartos fascia later.

4 Development of the Female Perineum 4.1 Introduction Descriptions of developmental events in the female perineum demonstrate a high degree of unanimity. A compilation of information from textbooks of embryology sketch as major events: (a) the transformation of the urogenital sinus into the urethra cranial to the Mllerian tubercle and a wide but shallow vestibulum vaginae caudally; (b) the outgrowth of greater (Bartholins) and smaller (Littrs) glands from the vestibulum; (c) the formation of the vestibular bulbs from the bulbar portions of the corpus spongiosum, leaving the rest of the corpus as a vestigial structure; (d) the transformation of the sexually indifferent phallus into the clitoris by lagging in development and

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Development of the Female Perineum

by bending caudalward; (e) the growth of the urogenital folds (syn. genital folds, urethral folds) which develop into the labia minora, form the prepuce by fusing near the glans clitoridis ventrally, and form the frenulum by fusing dorsally; (f) the expansion of the genital swellings (syn. labial swellings, labioscrotal swellings) into labia majora, which form the posterior commissure by fusing in front of the anus. The original literature about these events offers few extra data. This information is also fragmented as it focuses on certain aspects or selected structures only. It comprises the appearance of the external genitalia, the shape of the urogenital sinus and early vestibulum in relation to the developing vagina, the vestibular epithelium, the periurethral and vestibular glands, the smooth musculature of the urethra, and the prepuce. Differences of opinion concern minor aspects such as the derivation of the epithelial lining of the vestibulum and the formation of the prepuce. Even about the major controversial issue concerning the development of the vagina, consensus seems to have been achieved. But although the predominant theory as voiced by textbooks of embryology and relevant clinical literature still sketches its derivation from fused paramesonephric ducts in which the original paramesonephric epithelium is replaced by intruding epithelium from the urogenital sinus, differing opinions regularly emerge. Data about the differentiation of non-epithelial components are practically limited to the development of the urethral and anal sphincters. In that respect the recognition of the basic construction of these tissues during the sexually indifferent period as described in the preceding section, offers a firm base for their analysis in the female, and the more so because in the female, but not the male, the sexually indifferent configuration is to a large extent preserved. 4.2 Observations Changes indicating the transition of the sexually indifferent perineum into the female perineum occur late and gradually. During that early period (f.32 mm–60 mm) the urogenital ventral part of the perineum still actually shows a basically sexually indifferent structure (Fig. 18a). The transformation of this sexually indifferent configuration into the female architecture is most strongly influenced by drastic changes in the mesonephric-paramesonephric complex. The growth of the paramesonephric element into the vagina (f.60–110 mm), which is associated with an absolute shift of that caudal part of the developing vagina and its orifice towards the superficial urogenital sinus, has striking consequences for the fate of the urogenital sinus as a whole, as it is this process which exerts the most profound influence on its transformation into the urethra and vestibulum.

Observations

45

Fig. 18a–d Schematic median sections through the perineum of female fetuses of 40 mm to 190 mm to demonstrate major median structures during the transformation of the urogenital sinus into the urethra and vestibulum, in association with the transformation of the mesonephric-paramesonephric complex into the vagina. In a major structures indicated are: the urogenital sinus (1), anal canal (2), mesonephric-paramesonephric complex (3), urinary bladder (4), muscular coat of the deep urogenital sinus (5), symphysis pubis (6), corpora cavernosa (7), glans of the clitoris (8), rectum (9), muscularis of the rectum (also surrounding part of the deep anal canal; 10), puborectalis (11), and external anal sphincter (12). Special attention is given to the complexity of the epithelial zones in the cloaca-derived structures: a shows the zones of characteristic cloaca-derived ps.str.col. epithelium of the deep (a) and superficial (a) urogenital sinus, with the urogenital plate (arrowhead) and primordium of the greater vestibular gland (small circle), and of the deep anal canal (a"). Also depicted are zones of str.sq. epithelium originally related to the cloacal labia (b) and the glans plate (arrow). In b a new transitional zone of str.sq. epithelium, covered by a remnant of ps.str.col. epithelium at the luminal side (c), has appeared, and lacunae of Morgagni (arrowheads) have developed from the ventral urogenital plate extension that has split into a groove. In c squamous metaplasia has begun in the high ps.str.col. epithelium of the deep vestibulum and adjacent urethra (d) and (d) this changing epithelium lines the deep part of the vestibulum. Original magnifications, 17 (a), 12 (b), 7 (c), 4 (d)

46

Development of the Female Perineum

The development of the female perineum will be demonstrated by describing the main underlying processes, i.e., the formation of the vagina, urethra, vestibulum, labia minora and hood, labia majora, anal canal, and external perineum. 4.2.1 Vagina The following major developmental aspects are distinguished in the formation of the vagina: (a) regression of the mesonephric ducts, (b) incorporation of the mesonephric orifices into the orifice of the developing vagina, (c)

Fig. 19 Schematic transverse sections of female fetuses of 40 mm to 180 mm, and median sections of fetuses of 40 mm to 150 mm, to illustrate the development of the vagina. They show the relationship between the epithelia of the paramesonephric structure, the mesonephric ducts, and the urogenital sinus. In a the original (sexually indifferent) complex demonstrates the fused paramesonephric ducts (1), with their solid distal ends in direct contact with the mesonephric epithelium (2), but still separated from the epithelium of the urogenital sinus (3) by a column (4) of dense stroma of the tuberculum M
lleri. An arrow indicates a regressing nerve that becomes entrapped by the fusing paramesonephric ducts. In b the distal paramesonephric epithelium has thickened into lateral “wings” of solid str. epithelium, while sinus epithelium extends over a short distance into the orifices of the regressing mesonephric ducts as they pass the now decreasing stromal column. In c the lateral wings of the paramesonephric epithelium have greatly broadened, the central lumen narrowed, and the epithelium transformed into str.sq.; the stromal column has further regressed. In d the volume of the vagina has greatly increased while its glycogen-rich epithelium disintegrates centrally to form the definitive lumen of the vagina. The same events are illustrated in median reconstructions from e to i. Arrow, regressing entrapped nerve. Original magnifications, 80 (a), 55 (b–d, h and i), 65 (e–g)

Observations

47

transformation of paramesonephric duct epithelium, (d) development of the lamina propria and muscularis, (e) descent of the developing vagina, (f) formation of the hymen (Fig. 19). Regression of the Mesonephric Ducts. This is a slow process which begins early during the transformation of the mesonephric-paramesonephric complex (Fig. 19a) into the vagina in the form of individual cell necrosis in the epithelium (f.60 mm). It is followed by the complete regression of increasingly larger cranial segments of the ducts, leaving no more than some caudal remnants adhering to the developing vagina at a later stage in some fetuses (f.150 mm; Fig. 19b, c). Incorporation of the Mesonephric Orifices into the Orifice of the Developing Vagina. Early during the regression of the mesonephric ducts, the epithelium inside the orifices is undermined and replaced by urogenital-sinus-derived epithelium from the surface of the Mllerian tubercle inward (Figs. 19b, g, h, 20a). This replacement is well illustrated by an alteration in the immunohistochemical profile of the area, which demonstrates small remnants of mesonephric epithelium reactive with anti-cytokeratin 07 and anti-vimentin on top of urogenital sinus epithelium reactive with anti-cytokeratin 07 but not with anti-vimentin. These remnants often surround original mesonephric lumina filled with some eosinophilic debris. As a consequence, direct contacts are now established between the paramesonephric epithelium (which is in direct contact with the regressing mesonephric epithelium) and the replacing urogenital sinus epithelium, such contacts extending over a short distance through the two orifices. As a reminder of the original situation, a median column of mesenchyme originally surrounded by the urogenital sinus, mesonephric orifices, and the paramesonephric “bridge” remains. Simultaneously with the formation of a small diverticulum-like extension of the developing urethra here, the urogenital-sinus-derived epithelium extends somewhat further into the regressing mesonephric ducts. It remains, however, well-distinguishable from the adjacent paramesonephric epithelium by its more strictly oriented columnar cells with more basal crowding of nuclei, less eosinophilia, and negative reactions with vimentin (Fig. 20b). In addition, the basal layer of the sinus epithelium is negative in the anti-cytokeratin 07 immuno reaction, in contrast with the positivity of this layer in the paramesonephric epithelium. An extra indication of the boundary between the two epithelia is given by the remnant of a nerve locked inside the epithelium of the fused solid ends of the paramesonephric ducts. This nerve originally formed a loop in the mesenchyme between the still separate caudal ends of the paramesonephric ducts but became entrapped inside the fusing epithelium (Figs. 19a–c, e–g, 20b). This nerve is a constant finding in fetuses up to 180 mm. It remains visible long after the basic structure of the vaginal anlage has formed and the difference between

48

Development of the Female Perineum

Fig. 20a–d Development of the vagina in female fetuses of 70 mm sagittal section (s; a), 85 mm s (b), 125 mm s (c) and 160 mm s (d). In a, a paramedian section through the anlage for the distal vagina demonstrates the short extension of epithelium of the urogenital sinus (1) into the orifice of a regressing mesonephric duct (remnant indicated by arrowhead) meeting a distinctly differently structured paramesonephric epithelium (2; border indicated by arrows). In b an entrapped shrunken nerve (asterisk) also indicates the border (arrows) between sinus epithelium (1) and paramesonephric epithelium (2). In c gradual transformation of the originally columnar type of paramesonephric epitheli-

Observations

49

urogenital-sinus-derived and paramesonephric epithelia has become increasingly more vague due to squamous metaplasia and glycogen accumulation in both. The mesenchymal column between the two extensions of urogenital-sinus-derived epithelium inside the original mesonephric orifices becomes smaller and eventually disappears (f.140 mm; Fig. 19d, h). The original lumina of the mesonephric openings persist in the urogenital-sinus-derived extensions and will be incorporated into to the definitive lumen of the vagina later. Transformation of the Paramesonephric Duct Epithelium into Vaginal Epithelium. The paramesonephric epithelium of the anlage for the vagina is altered in four steps. The earliest change (f.60 mm) is a thickening of the original two- to three-layered ps.str.col. epithelium of the fused ducts into an irregular stratified epithelium very similar to that of the partially fused solid ends. The transformation gradually proceeds cranialward (Fig. 20c). The anlage thereby expands greatly, especially in a lateral direction where solid “wings” are formed with a later characteristic configuration of buds growing into irregular ridges (f.90 mm; Fig. 19c). In a second step this epithelium transforms into a str.sq. epithelium that narrows the preexistent lumen to near obliteration (f.125 mm). During this differentiation the original positivity for vimentin disappears. This transformation too proceeds from the caudal part near the urethra cranialward. In a third development an even more striking expansion of the vagina follows when the squamous cells begin to accumulate glycogen and greatly increase their volume (f.150 mm; Figs. 19d, i, 20d). Because, simultaneously, adjacent urogenital-sinus-derived epithelium inside the urethra and vestibulum also transforms into glycogen-storing str.sq. epithelium, and vimentin reactivity in the paramesonephric epithelium decreases and finally disappears, differences between the two epithelia become increasingly less distinct. The marked increase in volume of the epithelial ridges by this process creates the typical vaginal outline characterized by “bulging” epithelium between thin stromal papillae. A simultaneous loss of cohesion between the squamous cells in the central area, which results in the formation of a wide and definitive lumen partially filled by desquamated cells (f.180 mm), marks the final step toward the formation of the definitive vagina.

t um (a) through intermediate stages (b and c) into a str. type (d) is illustrated. In d a greatly expanded vagina shows the beginning of central luminization; intracellular glycogen accumulation obscures the border between the str.sq. sinus epithelium (1) and the paramesonephric epithelium (2). 3, Hymen; 4, dorsal groove of the vestibulum

50

Development of the Female Perineum

Development of the Lamina Propria and Muscularis of the Vagina. A mantle of mesenchyme originally surrounds both mesonephric and paramesonephric ducts with the disappearing mesonephric elements in a peripheral lateral position. The peripheral layer of this mesenchyme first develops in a more or less longitudinal orientation, then becomes spirally configured (f.70 mm), followed by differentiation into smooth muscle tissue (f.120 mm). An inner layer rich in blood vessels and nerves transforms into a thin sub-epithelial lamina propria and a broader middle layer consisting of abundant vessels and nerves mixed with smooth muscle tissue (f.175 mm). The pattern becomes more complicated in the most caudal part, where stromal components related to the dorsal wall of the original urogenital sinus, namely, a cuff of lamina propria and an external circumferential sling of internal and external urethral sphincter, become incorporated into the wall of the vagina as a consequence of the descent of the vagina. Descent of the Developing Vagina. During the just-described processes, the position of the caudal part of the developing vagina and its orifice changes considerably in relation to surrounding structures, namely, from a level just cranial to the pubic symphysis (f.60 mm) to a level just caudal to the crura of the corpora cavernosa (f.110 mm; Fig. 18). In absolute measurements the distance to the surface of the midperineal region remains the same while the area as a whole doubles its size. This first shift is followed by a second one when the expanding vagina bulges into the vestibulum and may even reach the vestibular orifice (f.175 mm). Because of this protrusion, the caudal part of the vagina becomes flanked by the vestibular bulbs and bulbospongiosus muscles, which were originally directly related to the wall of the developing vestibulum. Formation of the Hymen. The development of the hymen begins on the dorsal side of the orifice of the vagina and is the result of its descent in combination with the deepening of the dorsal groove of the vestibulum in statu nascendi (f.90 mm; Fig. 20d). This mechanism creates a semilunar structure consisting of the small-cell stromal tissue of the Mllerian tubercle. The process then extends lateralward involving the cranial ends of the original lateral grooves of the urogenital sinus. The ventral part of the hymen develops from the stromal tissue of the tuberculum in the increasingly more acute angle between the urethra and vagina. As a consequence of this derivation from the stroma of the tubercle, the hymen is built of a finely fibrillar connective tissue without the smooth muscle element predominant in all of its surrounding tissues. 4.2.2 Transformation of the Urogenital Sinus into the Urethra and Vestibulum The descent of the caudal part of the vagina and its orifice (f.60 –110 mm) has as a major consequence that parts of the wall of the original urogenital

Observations

51

Fig. 21a–c Schematic median sections through female fetuses of 40 mm to 125 mm to demonstrate the rearrangement of segments of the wall of the urogenital sinus during its transformation into the urethra and vestibulum in association with the descent of the vagina. These demonstrate how the urethra is composed by the combination of the whole of the ventral wall of the deep urogenital sinus (fine dots) with the supravaginal part of the dorsal wall related to the trigone of the bladder (oblique stripes), while the vestibulum forms by the combination of the remaining infravaginal part of the dorsal wall of the deep urogenital sinus with the whole of the superficial urogenital sinus (coarse dots). Broken lines, bladder; small circles, orificia of greater vestibular glands. Original magnifications, 25 (a), 16 (b), 15 (c)

sinus become rearranged (Figs. 18, 21). The shift of the vaginal orifice caudalward necessarily causes a corresponding lengthening of the dorsal wall of the deep urogenital sinus cranial to that orifice because the position of the adjacent part of the bladder and the ventral wall of the deep urogenital sinus do not alter, as can be deduced from their relationship to the “static” pubic symphysis. As a result the supravaginal part of the dorsal wall becomes increasingly more positioned opposite the whole of the ventral wall including the ventral part originally caudal to the level of the vaginal orifice. The combination of the supravaginal part of the dorsal wall of the deep urogenital sinus and the whole of its ventral wall forms the female urethra. The remaining part of the dorsal wall caudal to the vaginal orifice combines with the superficial urogenital sinus to form the vestibulum. After the transformation of the superficial urogenital sinus into the vestibulum vaginae, the urogenital labia are renamed labia minora and, correspondingly, the labial swellings are now named labia majora.

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Development of the Female Perineum

Fig. 22a, b Trigone of the urinary bladder and urethra in female fetuses of 150 mm t (a) and 140 mm t (b). In a the contrast is shown between the more transitional type of epithelium of the trigone (upper) and the ps.str. col. epithelium with intraepithelial acini (arrow) of the urethra (lower). In b the urethra (1) demonstrates primordia of prostatehomologue urethral glands (2), and trigone-related dense primordial vesical sphincter (3) that merges with bundles of the internal urethral sphincter ventrally (4); 5, lamina propria

4.2.3 Urethra The length of the urethra increases considerably during the process of rearrangement and more longitudinal grooves and ridges appear. The high ps.str.col. epithelium shows a distinct increase in lightly stained clear cytoplasm at the level of the Mllerian tubercle. Elsewhere it differentiates into a typical ps.str.col. urethral epithelium2 characterized by crowding of nuclei at the base and an eosinophilic cytoplasmic zone with the formation later of intraepithelial acini at the luminal side (f.120 mm) (Fig. 22a). Thereafter, squamous metaplasia supervenes in the caudal part in continuity with the same alteration in the adjacent vagina and vestibulum 2

This epithelium is, indeed, “pseudostratified” as is confirmed by a test with antibodies against low-molecular-weight cytokeratins (Ck 07 and Cam 5.2) in a 140-mm fetus. The reaction which outlines the individual cells, demonstrates after three-dimensional reconstruction by a confocal laser scan microscope that all cells including the luminal cells have their base on the basement membrane. This is in contrast with the situation in the transitional epithelium of the adjacent bladder and defines the epithelium as pseudostratified.

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Fig. 23 Schematic paramedian sections through the wall of the developing urethra of female fetuses of 40 mm to 125 mm. They illustrate the complicated structure of the muscular coat of the urethra as the result of rearrangement (see also Fig. 21). Indicated are: caudal bundles of the detrusor vesicae (1), dense and fine trigonum-related primordial smooth muscle tissue, with a ventralcaudalward orientation of the vesical sphincter (2), which forms most of the dorsal urethral wall, bundles of smooth muscle tissue with a transverse to dorsalcaudalward orientation of the internal urethral sphincter (3), which predominates in the ventral wall and is peripherally mixed with striated muscle tissue of the external urethral sphincter in the same pattern (4 in b) and, caudally, surround at first the deep future vestibulum (a and b) and later the descending vagina (c). The outlines of the vestibulum and vagina are indicated by thin broken lines for orientation; compare with Fig. 21. Also shown in a are the small-cell stromal tissue of the M
llerian tubercle (5) and the temporary “prostatic” stromal tissue (6). Original magnifications, 18 (a and b), 15 (c)

(f.90 mm). At the side of the bladder and in the trigone in particular, the ps.str.col. epithelium gradually passes into the strikingly different transitional epithelium of the bladder (Fig. 22a). Primordial periurethral (paraurethral) glands sprout from the dorsolateral and, in particular, the lateral urethral grooves (f.60 mm; Fig. 22b). They are at first solid, vary greatly in number and in extension and often show a conspicuous bladderward direction. The largest develop in the caudal part of the urethra and become the ducts of Skene. Their nature as glands becomes later increasingly more obscured by squamous metaplasia of their ps.str.col. epithelium in combination with little tendency to form acini (f.300 mm). The rearrangement of parts of the wall of the deep urogenital sinus has a more profound effect on the structure of the mantle mesenchyme, at the start of the process consisting of a thin longitudinally structured lamina propria and a composite primordial muscle layer (Fig. 23). The density and longitudinal orientation of the lamina propria becomes more marked thereafter. This longitudinal pattern is only interrupted by a consistently present

54

Development of the Female Perineum

remarkable band of transversely oriented stromal tissue embracing the future urethra from ventrally and spreading into the primordial muscular layer dorsally. This band too illustrates the descent of the vagina as it seems to move cranialward from its original position at the level of the vaginal orifice during the descent of the vaginal orifice, and later becomes a semicircular smooth muscle band in the caudal definitive urethra (f.200 mm). Further differentiation accentuates preexistent, rather vague differences between the original five major stromal constituents of the coat of the deep urogenital sinus. It shows that the trigone-related primordial vesical sphincter, with its ventralcaudalward orientation embracing the urethra dorsally, will lengthen to form the major component of the dorsal wall of the urethra. This element preserves its dense and fine structure while differentiating into smooth muscle tissue at a relatively late stage (f.100 mm). Bending ventralward it meets another main element positioned caudally and ventrally and shows a contrasting predominantly dorsalcaudalward orientation in its tissue. This primarily ventral internal urethral sphincter differentiates into more distinct bundles of smooth muscle cells, which process had already started at a much earlier stage (f.25 mm) than that just described concerning the dorsal musculature. The internal sphincter is so intimately linked to the external urethral sphincter, which develops somewhat later (f.60 mm), that their fibers become intertwined at the interface and have the same orientation and extension. Both become circumferential in the caudal urethral segment only and will finally surround the most distal part of the descending vagina as a sling. At the side of the bladder both the vesical and internal urethral sphincters pass gradually into the bundles of the detrusor vesicae which had already differentiated before the beginning of fetal development. At the level of the transition of bladder and urethra some conspicuous ventral and dorsal longitudinal bundles of this detrussor vesicae form the anlage for the pubovesical and vesicovaginal muscles, respectively. At the side of the future vestibulum some elements of the ventral internal urethral sphincter extend along the ventral wall of the vestibulum to end in its broad mantle of fibrovascular tissue more superficially. The ventral internal sphincter also spreads in a more irregular pattern around the ducts of the greater vestibular glands and the vestibular bulbs laterally. After the descent of the vagina (f.110 mm) the internal and external urethral sphincters thicken considerably. The internal sphincter forms increasingly more longitudinal bundles at its inner side, which makes its structure more complicated, and its distinction from the longitudinally oriented fibromuscular lamina propria more difficult (f.200 mm). The stromal tissue, which is originally present in the dorsal wall caudal to the Mllerian tubercle, and which is not characterized by a special orientation and an apparently homologous relationship to the primordial prostaterelated stroma in the male, stays behind in growth and cannot be identified

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later on (f.90 mm). The stroma of the tubercle itself becomes part of the hymen. 4.2.4 Vestibulum The descent of the caudal vagina and its orifice not only combines the dorsal wall of the original deep urogenital sinus caudal to the orifice with the whole of the superficial urogenital sinus in the formation of the vestibulum, but also influences an alteration in its shape from an irregular trumpet-like duct into a wider and more shallow chamber (Figs. 18, 21). Additional factors determining its shape are a deepening of the dorsal groove and the adjacent parts of the cranially converging lateral grooves, a broadening of the ventral wall resulting from an increase in the volume of the ventral ridge, a deepening of the flanking ventrolateral ridges, and a marked bending of the deeper part of the future vestibulum ventralward (f.70–110 mm). The splitting of the remnant of the urogenital plate extension along the ventral wall (f.60 mm) does not contribute significantly to the width of the vestibulum because the resulting groove stays behind in growth. The final remodeling takes place when the greatly expanding vagina bulges into the vestibulum, and grooves and ridges disappear in an increasingly more shallow structure (f.175 mm). The epithelium of the future vestibulum varies from a high four-layered ps.str.col. urethral type in the deep part, which like the urethra is derived from the deep segment of the urogenital sinus, to a lower mostly two-layered ps.str.col. epithelium in the remaining part derived from the superficial part. This distinction in origin remains visible in that the urethra-type of epithelium occurring in the deep dorsolateral wall later demonstrates the same squamous metaplasia, leaving small foci of ps.str.col. epithelium, as does the epithelium in the adjacent urethra (f.300 mm; Fig. 24a, b). At the deep ventral side of the vestibulum another epithelium develops that is very similar to the dorsal epithelium at first sight but differs in the absence of foci of ps.str.col. epithelium and in the presence of small acinar glands protruding from the basal layer into the lamina propria (Fig. 24b). The squamous metaplasia extends in the direction of the orifice somewhat beyond the orifices of the greater vestibular glands where it meets another type of squamous transformation that involves the epithelium of the superficial vestibulum. That other type of transformation starts early (f.50 mm) at the vestibular orifice where basal cells differentiate into squamous cells, lifting a layer of the original columnar epithelium, the remnant of which is observed for a considerable length of time (f.50–175 mm; Fig. 24c). Unlike the adjacent str.sq. epithelium on the labia minora, this epithelium does not cornify. It differs from the metaplastic str.sq. epithelium of the deeper vestibulum by not having foci of ps.str.col. epithelium or forming basal glands, by having a less

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Development of the Female Perineum

Fig. 24a–d Differentiation of the epithelium of the vestibulum in female fetuses of 175 mm f (a), 320 mm t (b and d) and 105 mm t (c). In a, a survey of the vestibulum (1) shows the original ps.str.col. epithelium (arrowheads) partially replaced by a urethratype of str.sq. epithelium (2) on the side of the urethra, and by “transitional” str.sq. epithelium (3) on the side of the vestibular orifice; 4, lateral grooves; 5, vagina. In b squamous transformation in the deep vestibulum reveals two different forms, namely (upper) a mixture of ps.str.col. (arrow) and str.sq. (arrowheads) epithelium in the dorsal part, and (lower) a broad non-keratinizing str.sq. epithelium with small basal glands (asterisk)

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clear cytoplasm with less storage of glycogen (Fig. 24d), and by later developing of a parakeratotic layer. Both types of epithelium differ from the noncornifying and more compact str.sq. epithelium of the glans plate, which forms only a narrow median strip beneath the glans clitoridis. The remnant of the ventral urogenital plate extension splits to form a groove and becomes less distinct, but not before it has given rise to three to four solid buds representing primordial lacunae of Morgagni in some fetuses (f.70 mm). Later these buds may acquire lumina and show squamous metaplasia (f.190 mm). The original ps.str.col. epithelium of the vestibulum persists only in the excretory ducts of the greater vestibular glands. These glands grow from the original solid buds lateralward (f.30–45 mm), form branches reaching the caudal extensions of the internal urethral sphincter and the vestibular bulbs (f.45–60 mm), and develop acini with clear mucous cells (f.180 mm). The lamina propria is dense, longitudinally oriented, and spreads into the vascular tissue in the ventral wall of the superficial part of the vestibulum. It reveals some reactivity for anti-smooth muscle actin in older fetuses (f.200 mm). In accordance with its derivation from the deep urogenital sinus, the deep part of the future vestibulum is for some time (f.70–110 mm) surrounded by the most caudal and circumferential part of the internal and external urethral sphincters. Together with the related cuff of lamina propria, this band is passed on its inner side by the descending vagina, the caudal part of which will thereafter become surrounded by it (Fig. 23). The vestibulum does not develop a true circumferential muscularis propria. It preserves its original composition of a broad mantle consisting of fibrovascular tissue embracing ventrally and a composite stromal tissue including smooth muscle tissue embracing dorsally. 4.2.5 Erectile Structures The erectile glans, corpora cavernosa, vestibular bulbs, and labia minora develop their own characteristic microscopic anatomy and histology. The glans of the clitoris alters from a relatively large cap-like structure which forms almost a third of the original phallic structure into a relatively

t and without ps.str.col. epithelium in the ventral part. In c and d the superficial vestibulum reveals in a “transitional” zone near the orifice how at an early stage (c), ps.str.col. epithelium (arrowhead) overlies (at arrow) str.sq. epithelium (3), which differentiates later (d) into a broad non-cornifying str.sq. epithelium (3), contrasting in staining intensity with the metaplastic str.sq. epithelium rich in glycogen (2) of the deep vestibulum

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Development of the Female Perineum

small nodular structure that becomes partially covered by the glandopreputial lamella and prepuce (f.60–85 mm). Its original thin peripheral extension becomes subdivided by the developing lamella into a proximal part that becomes a thin fibrovascular layer on the inner side of the prepuce, and a more distal part that expands into the corona of the glans (see Sect. 4.2.6). Histogenesis is a slow process with an intricate pattern of small, indistinct arteries and veins extending from larger vessels centrally into gradually loosening dense stromal tissue (f.70 mm–140 mm), avoiding only a vague zone of connective tissue under the epithelium. This architecture undergoes few changes during further development. The initially almost straight corpora cavernosa, with their laterally bending crura proximal and unpaired ends distal, become curved in a dorsalcaudalward direction. The dense small-cell stroma differentiates into vascular cores centrally that are surrounded by a thick bi-laminate tunica albuginea of very dense connective tissue (f.60 mm).The vascular core of each cylinder becomes increasingly more distinct in a distalward direction, with complicated vascular structures sprouting from a central artery enveloped by nerves. The more definitive pattern is characterized by small arterial branches in a loose connective tissue between plexiform veins with thick muscular mantles from which small bundles radiate, as a typical phenomenon, into the surrounding connective tissue (f.260 mm). The vestibular bulbs enlarge from the initially rather vague primordia into bulb-like structures that point toward the bending parts of the corpora cavernosa and shift from an originally lateral position into a more ventral position in relation to the vestibulum. They do not develop a sheath but become more or less defined by the differentiation of the lamina propria of the future vestibulum medially, the bulbospongiosus laterally, the internal urethral sphincter cranially, and dorsal urogenital stromal tissue caudally. The relatively coarsely structured dense fibrovascular tissue differentiates into a conspicuously loose myxomatous connective tissue with an increasingly more distinct vascular component consisting of a relatively large number of arteries branching into networks of arterioles and capillaries (f.70–200 mm). Thereafter the stromal tissue becomes rich in collagen, with the vascular components increasing in diameter rather than in numbers and complexity. The labia minora increase their size without altering form and structure substantially. From the dense mesenchyme develops a complicated fibrovascular tissue in which arteries and veins equally abound (f.70 mm). This tissue gradually passes into the lateral walls of the vestibulum where it acquires some longitudinal orientation similar to the deeper lamina propria. The development of these more or less circumscribed erectile structures is associated with the persistence of rich networks of anastomosing vessels from the original highly vascular area embracing the vestibulum ventrally. Into this system materializes a bilateral pair of larger vascular channels par-

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Fig. 25a, b Development of a highly vascular area in the ventral wall of the vestibulum in fetuses of 70 mm s (a) and 190 mm s (b). This area is characterized by the appearance of a strikingly regular pattern of (a) small parallel vessels (arrow) connecting branches of the internal pudendal arteries, and veins in the ventral wall of the vestibulum (1), to minute vessels in the corpora cavernosa (2), which vessels (b) have later differentiated into predominantly venous anastomoses (arrowhead) between the two systems (1 and 2; cf. male Fig. 40d)

allel to the ventral wall of the vestibulum beneath the corpora cavernosa (f.40–60 mm). Differentiation into arteries and veins is followed by a strong predominance of the venous element (f.70–175 mm) (Fig. 25a, b) that results in a well-structured, highly vascular area with close vascular links to the erectile structures in sensu strictu. 4.2.6 Fascial Tissues Composite stromal tissue, including deep and superficial dorsal urogenital stroma, superficial stroma of the shaft, and dartos stroma of the labia majora, embraces the future vestibulum dorsally, then differentiates into smooth muscle and fibrous tissue. The deep dorsal urogenital stromal preserves its original orientation parallel to the superficial urogenital sinus, bending ventralward to end lateral to the corpora cavernosa (Fig. 26). It expands considerably (f.32 mm), and a dense peripheral zone takes shape which is so intimately connected to adjacent structures that, after differentiation (f.125–175 mm), smooth muscle

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Development of the Female Perineum

Fig. 26a–d Schematic illustration of the deep dorsal urogenital stromal tissue shown from the left side of the fetuses (a and c), and in transverse sections through the midperineal region between the urogenital/vestibular and anal orifices (b and d, at levels indicated in a and c) of female fetuses of 40 mm and 130 mm The deep dorsal urogenital stroma (1) embraces the future vestibulum (2) from dorsally and differentiates into a fibrous and smooth muscle tissue with a predominant ventralward orientation. In a and c the stroma demonstrates a broad ventralward sheath that bends superficial to the bulbi vestibulares (3) and ends lateral to the corpora cavernosa (4). In b and d the stroma (1) expands dorsally into a fibromuscular tissue that considerably increases the distance between the two sides, is interlocked with longitudinal bundles (5) of the muscularis propria of the rectum, and extends into the striated muscle tissue of the left and right puborectalis (6), bulbospongiosus (7), and external urethral sphincter (8) muscles. Original magnifications, 20 (a), x35 (b), 9 (c), 23 (d)

cells expand between striated muscle cells of the bilateral puborectal, of adjacent deep portions of the bulbospongiosus, of caudal extremities of the internal and external urethral sphincters, and also into the greater vestibular glands and vestibular bulbs. In the central area, which is less dense, end the ventral longitudinal bundles of the outer layer of the muscularis propria of the rectum in a strikingly abrupt way. This central area expands greatly (f.32 mm), which results in a wide separation between the bilateral parts of the puborectal and bulbospongiosus muscles (along with related superficial transverse perineal muscles, as identifiable in some fetuses larger than 90 mm), and between the greater vestibular glands and vestibular bulbs (Figs. 26b, d, 27a). After differentiation of the central area tissue into smooth muscle, it forms a dorsal muscular layer of the vestibulum with bundles spreading dorsalward and into the septa of the adipose tissue of the midperineal region between vestibulum and anal canal (Fig. 27b). In the ventrallybending component fibrous tissue predominates, but scattered small bundles of smooth muscle cells may occur as far as the area lateral to the corpora cavernosa. The small element of superficial dorsal urogenital stroma, which connects the dorsal extremities of the labia minora, forms a thin band of transversely oriented, finely fibrillar connective tissue. It becomes the frenulum of the labia minora in the dorsal margin of the vestibular orifice (Fig. 28).

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Fig. 27a, b Stromal tissue dorsal to the vestibulum in female fetuses of 130 mm t (a) and 210 mm s (b). In a, a transverse section through the midperineal region demonstrates the predominant central and denser peripheral deep dorsal urogenital stroma (1) that extends (arrows) into the puborectalis (2) and bulbospongiosus (3) muscles, and by its expansion moves muscles and greater vestibular glands (4) lateralward; 5, vagina. In b a median sagittal section immunostained by anti-smooth muscle actin antibody demonstrates deep dorsal urogenital stroma differentiated into a smooth muscle layer (1) in the dorsal wall of the vestibulum (6), and spreading dorsalward as the “perineal body” (aster7, longitudinal muscularis propria of the rectum

The superficial shaft stroma of the original phallic structure develops into a characteristic subepidermal layer of finely fibrillar connective tissue with a prominent parallel ventralward orientation in the sulcus between the labia minora and majora, and in the hood of the clitoris, which includes the prepuce distally (Fig. 29a). The proliferation of this superficial shaft stroma is a major element in the formation of the prepuce (Fig. 30). It is part of a concerted growth which also includes the formation of a solid glandopreputial lamella and the expansion of the corona of the glans. The process starts with an increased proliferation of epithelium in a vague groove well within the thin peripheral extension of the glans. This growth keeps in step with a proliferation of superficial shaft stroma of the developing prepuce proximally and with an increase in fibrovascular tissue of the corona of the glans distally. The result is a lengthening glandopreputial lamella. This growth is outward, as is indicated by the fact that the deepest and most cellular part of the lamella remains in the same position in relation to underlying structures such as the tunica

62

Development of the Female Perineum

Fig. 28a–d Schematic representation of the superficial stromal tissues of the perineum in relation to the developing external form that changes from the original phallic configuration in female fetuses of 40 mm and 70 mm into a female shape in fetuses of 135 mm and 190 mm. The dorsal stromal elements embrace the (primordial) glans of the clitoris (1) and urogenital labia/labia minora (2) in a ventralward orientation, and comprise: 1. a small band of superficial dorsal urogenital stroma becoming the frenulum (3) of the labia minora; 2. superficial stroma of the phallic shaft (4, shown in c and d at the right side of the fetuses only), becoming a special superficial layer of connective tissue in the hood of the clitoris and interlabial sulci; and 3. the deep dartos stroma (5, partially illustrated at the right side of the fetuses), which forms a deep lamina of relatively large bundles of smooth muscle. The last fan out from the midperineal region between the urogenital and anal orifices into the labia majora, where they gradually pass into a superficial lamina with a more dense, irregular, and transverse structure (6, shown at the right side of the fetuses only). Note also the lengthening ventralward extension of labia-derived fibrovascular stroma (7), related to perianal fibrovascular stroma. Original magnifications, 21 (a), 7 (b), 5 (c), 3 (d)

albuginea of the corpora cavernosa and the main nerves and vessels to the glans (Fig. 30). The lamella together with the prepuce extend over most of the glans. Built of glans epithelium, the lamella thus remains on both sides and closely related to the original fibrovascular stroma of the glans. On the sides of the prepuce this results in a thin but permanent subepithelial layer of glans-derived stroma (Fig. 30). From its deepest part two to six tubular glandopreputial glands may develop which may penetrate deep into the dense stromal tissue around the corpora cavernosa. Before they show any differentiation that would allow classification, they regress; but they may persist as small abortive remnants with squamous metaplasia and/or cystic alteration. Thereafter, the prepuce shows a relative reduction in size in correlation with the relative decrease of the glans. Most of the lamella is still solid at birth.

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Fig. 29a, b Superficial shaft stroma in a female fetus of 50 mm f (a) and dartos fascia in a labium majus of a fetus of 330 mm s (b). In a the obliquely transverse oriented superficial shaft stroma (1) is shown in the dorsum of the hood of the clitoris (2) and under the interlabial sulcus (3); 4, ventral extension of the urogenital plate; 5, corpora cavernosa; 6, labial swelling. In b is seen a sagittal section of the bi-laminate dartos fascia with longitudinally oriented large bundles of the deep lamina (7a) and more dense and irregularly transverse tissue of the superficial lamina (7b), between the epidermal appendages (antismooth muscle actin antibody)

The dartos stroma, which initially constitutes most of the labia majora, differentiates into a finely fibrillar tissue in which at a late stage of development (f.200 mm) small bundles of smooth muscle cells appear, with preservation of the original bi-laminate structure (Fig. 29b). This stroma thereby shows the development of a deep lamina of relatively large and more or less individual bundles of smooth muscle cells in a predominantly parallel pattern, with a ventralward orientation spreading from the ventral part of the midperineal region. Situated under the epidermal appendages it splits into finer bundles that form a denser, more irregularly structured superficial lamina between the appendages, with an orientation in a more ventrolateral direction and toward the epidermis. The dartos fascia does not extend into the sulcus between the labia majora and minora nor into the hood of the clitoris.

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Development of the Female Perineum

Fig. 30a, b Development of the prepuce in female fetuses of 65 mm f (a) and 125 mm f (b). The prepuce (1), glandopreputial lamella (2), and corona of the glans (3) demonstrate their outward expansion, as indicated by the fixed position of the deep margin of the lamella in relation to underlying structures such as the dorsal nerves of the clitoris (arrows); note that the dense stroma of the glans forms a thin inner layer in the prepuce (arrowheads)

4.2.7 Labia Majora The labia majora expand greatly. Initially this growth is the result of proliferation of dartos stroma, its almost exclusive component. Later “tongues” of lipoblasts extend into the labia from lateral and deeper areas (f.180 mm). These increase in volume and become the predominant tissue restricting the bundles of the dartos fascia (see above) to the intermediate septa and the skin. 4.2.8 Anal Canal Initially the tube-like anal canal still reveals the histologically different cloaca-derived deep and labia-related superficial segments of the sexually indifferent configuration (Fig. 17a). The deep anal segment is wide at the side of the rectum, narrows about halfway at the level of the distal border of the muscularis propria, which had descended from the rectum, and then widens again before passing into the superficial anal segment. Like the urogenital

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sinus, this deep segment is lined by cloaca-derived ps.str.col. epithelium and has a dense lamina propria with a longitudinal orientation of its stroma and the formation of grooves and ridges, i.e., anal sinuses and columns. This mucosa is distinctly different from the rectal mucosa, with its goblet cells, crypt formation, and loose lamina propria. It is, however, largely surrounded by the muscularis propria of the rectum flanked by the puborectal muscles. The superficial anal segment has a totally different structure as it is lined by a broad str.sq. epithelium on a base of fibrovascular stroma derived from the cloacal labia overlying the external anal sphincter. Where both segments meet, ps.str.col. epithelium extends somewhat over the str.sq. epithelium in a sort of transitional zone similar to the configuration met in the vestibulum. At the anal orifice the str.sq. epithelium passes into the thinner epidermis and the fibrovascular stroma into the less dense early dermis. That anal canal shows a distinct relative decrease in length as the distance between the anorectal border and the orifice remains the same whereas the perineum as a whole becomes slightly more than two times its original size (f.32–125 mm). As a striking phenomenon, the muscularis descends towards the anal orifice with the distance between the muscle and the orifice halved during the same period. It thereby inserts itself between the superficial anal segment and the striated external anal sphincter, which at the same time greatly enlarges and, in contrast to the muscularis, extends into the direction of the deep segment (Fig. 31a). The outer longitudinal bundles of the muscularis propria differentiate later than the inner circular layer, with distal extensions spreading ventrally into the deep dorsal urogenital stroma of the midperineal region and also laterally and dorsally into the striated external anal sphincter. These events are associated with a disproportionate increase in the median diameter of the canal, especially at an early stage, that transforms the pin-point orifice into a split that is forked on the ventral side because of a broad ventral ridge. This process involves the deep anal segment more than the superficial segment, creating an ampulla with anal sinuses and columns developing (Fig. 31a). Thereafter the anal canal shows the following developments: (a) the diminishment of the original ps.str.col. epithelium into a narrow band; (b) the formation of tubular anal glands from this band (f.140 mm), their extension into the muscularis propria (f.175 mm), and differentiation of characteristic acinar cells producing clear mucus (f.230 mm); (c) the transformation of the adjacent zone of epithelium, with its remarkable combination of str.sq. epithelium covered by a layer of cuboidal cells extending from the deeper ps.str.col. epithelium, into a zone of non-keratinizing str.sq. epithelium (Fig. 31b); (d) the keratinization of the broad labia-related str.sq. epithelium of the last zone inside the superficial anal segment, which forms abortive primordial epidermal appendages in some fetuses (f.130 mm), and a strikingly thick horn layer (f.175 mm), but which also remains different from the

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Development of the Female Perineum

Fig. 31a, b Anal canal in female fetuses of 70 mm t (a) and 85 mm t (b). In a the deep segment (1a) has descended toward the surface of the perineum, forming a wide ampulla with anal columns (arrows) between sinuses, and is now completely surrounded by alsodescended muscularis propria of the rectum (internal anal sphincter) (2), positioned well inside the external anal sphincter (3); 4, lamina propria; 5, rectum; arrowhead, border with superficial anal segment (1b). In b is shown how the ps.str.col. epithelium of the deep segment (1a) overlies str.sq. epithelium via a transitional zone (between arrowheads) toward the superficial segment (1b). Note also the vascular subepithelial stroma derived from the cloacal labia (asterisk)

outer and thinner epidermis characterized by more delicate rete ridges, well-formed hair follicle-sebaceous-apocrine units, and eccrine glands; (e) the widening of blood vessels and the appearance of smooth muscle actin in the lamina propria of the deep anal segment (f.230 mm); (f) the enlargement of dense vascular networks of fine blood vessels in the originally labia-related subepithelial stroma of the superficial segment (Fig. 31b), and the transformation of a ventralward extension of the lamina into a cushion-like elevation of the midperineal region as far as the fourchette of the vestibular opening (f.110 mm); (g) the further descent of the muscularis propria of the rectum until the inner circular smooth muscle layer comes almost level with the orifice itself, investing both the deep and superficial segment while passing the external anal sphincter on its inner side (f.110 mm). It thickens and forms the internal anal sphincter, while the outer longitudinal bundles reveal a rapidly increased volume with major extensions into the external anal sphincter and dorsal muscular layer of the vestibulum (f.125 mm).

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4.2.9 Perineal Striated Musculature The original configuration of a left and right series of primordial perineal muscles is only partially preserved. It only persists fully in the ischiocavernosus muscles related to the bilateral crura of the clitoris. The bulbospongiosus muscles related to the bilateral bulbi vestibulares remain for the larger part separate but with a small bridge dorsally where their primordial tissue is combined with that of the deep external anal sphincter. In the external urethral sphincter and external anal sphincter the original bilateral character is lost when fibers encircle the urethra and anal canal, respectively. The superficial part of the external anal sphincter retains its original horseshoe shape to a great extent. Bending inward, the fascicles of striated muscle tissue of this part become intertwined by the longitudinally oriented fibrous tissue of the lamina propria and smooth muscle tissue of the muscularis propria (f.60 mm).The transversus perinei superficialis muscles do not develop from specific primordia. In all primordial muscles with the exception of the external urethral sphincter, differentiation into striated muscle fibers had already started in the sexually indifferent period (f.22–25 mm). In the external urethral sphincter increased sarcoplasm and multinucleation are observed in fetuses of 45 mm and 60 mm, respectively. The bilateral transversus perinei superficialis muscles can be identified in fetuses as large as 90 mm, in which they extend from the meeting points of fibers of the deep external anal sphincter and the lateral sides of the bulbospongiosus lateralward. The muscles acquire their basic form early in the fetal period. 4.2.10 External Perineum The external shape of the female perineum, which is characterized by a relatively short distance between the wide anal and vestibular orifices, prominent labia minora, voluminous labia majora, and a small clitoris, is to a large extent determined by the differential growth of the main stromal components that originally surrounded the superficial urogenital sinus (Figs. 18, 28). Most striking in this process is the flattening of the originally prominent phallic structure, which change is caused by a relative reduction in growth of most erectile structures, accentuated by the caudal bending of the corpora cavernosa. The primordial erectile urogenital labia, later labia minora, in contrast, demonstrate a (slight) increase in size in proportion with the persistently wide urogenital, later vestibular, orifice. The growth of the deep dorsal urogenital stromal tissue transforming into a thick layer of smooth muscle tissue between the vestibulum and anal canal in particular broadens the originally narrow midperineal region between the vestibular

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Development of the Female Perineum

and anal orifices. In this region superficial dorsal urogenital stroma gives rise to the dorsal frenulum of the labia minora ventrally. Dorsally, labia-derived fibrovascular tissue from around the anal orifice extends ventralward and forms a more or less distinct broad elevation occupying most of the region. The great increase in the labial swellings, later labia majora, is due to proliferation of the dartos stroma (f.100 mm) first, and the labia further expand later by the invasion of adipose tissue from the adjacent subcutis and deeper adipose tissues (f.230 mm). The analysis of the histogenesis of the surface of the perineum shows that the usual type of skin develops in a peripheral zone only. The rest of the perineum develops a different epithelial and stromal composition, notably with: (a) labia-derived fibrovascular stroma covered by epithelium without cutaneous appendages in a median zone from around the anal opening to the midperineal region as far as the labia minora; (b) parallel zones with strikingly large sebaceous glands, some apocrine glands and, in the interlabial sulci, deep penetrating non-eccrine, non-apocrine tubular glands, and also a dense subepidermal connective tissue dorsally and parallel oriented, finely fibrillar superficial shaft connective tissue ventrally extending into the sulci between the labia minora and majora; (c) lateral areas with fewer and smaller epidermal appendages, and instead of a dermis, dartos fascia in the labia majora and superficial shaft connective tissue in the hood of the clitoris. Subcutaneous adipose tissue develops under the usual type of skin at the periphery of the perineum (f.175 mm) and extends from there into the labia majora (f.230 mm). 4.3 Discussion This comprehensive histologic study of the developing female perineum, based on the analysis of epithelial as well as mesenchymal elements and also on findings in the sexually indifferent perineum, offers a new insight into the formation of all of its major structures, namely, vagina, urethra, vestibulum, anal canal, and external perineum. This investigation demonstrates that two major processes determine the architecture of the female perineum: (a) the development of a predominating vagina, which greatly influences the subdivision of the urogenital sinus into the urethra and vestibulum in the deep perineum; and (b) the differentiation of the mantle around the superficial urogenital sinus into fibrovascular erectile structures, which embrace the future vestibulum from the ventral side, and fibrous and smooth muscle fascial elements, which embrace it from the dorsal side, in the superficial perineum. Their differential growth also determines the external shape of the area.

Discussion

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4.3.1 Vagina Investigations into the development of the vagina have led to a profusion of different and often controversial theories (ORahilly 1977). These ideas will be addressed in the context of the present observations arranged in the sequence of developmental events. The present observations confirm the earliest events, i.e., the formation of the paramesonephric ducts, as have been described by Gruenwald (1941) and Frutiger (1969). The present study reveals, however, that the distal tips of the paramesonephric system remain intimately linked to the mesonephric ducts and separated from the epithelium of the urogenital sinus by a bar of mesenchyme. This study also confirms earlier data about a following developmental phase during which inside and around the orificia of the mesonephric ducts a broad layer of pale-staining epithelium is formed. This “clear” epithelium forms two dorsal extensions that become broad contacts between the epithelium of the urogenital sinus and the solid paramesonephric tips. As for the controversy about the nature of these extensions considered to be mesonephric (Mijsberg 1924; Forsberg 1965, 1973; Witschi 1970), urogenital sinus-related (Matejka 1959; Minh and Herv de Sigalony 1989), or a combination of both (Vilas 1932; Meyer 1934, 1937, 1938; Kempermann 1935; Bulmer 1957), immunological tests applied in this investigation reveal the profile of sinus epithelium with some remnants of mesonephric epithelium on the luminal side. Confirming some earlier observations (Koff 1933; Bulmer 1957; Witschi 1970), the extensions apparently expand inside the most caudal parts of the regressing mesonephric ducts as is indicated by topographical relations such as their positions in relation to the intermediate column of mesenchyme, by the finding of remnants of mesonephric epithelium centrally, and by the attachment later of occasionally persisting parts of mesonephric ducts to these extensions at some distance from the urogenital sinus. The sinus-related epithelium reaches the inner, vaginal side of the column of mesenchyme to a variable extent. It is the role of these two extensions of “clear” epithelium in the formation of the vagina as a whole that has been a major element in the controversy about the development of the vagina. In the present, investigation it is concluded that these two extensions are involved only in the formation of the entrance to the vagina, and that nearly the whole vagina is built by the paramesonephric system, including its epithelium, which later transforms into a str.sq. epithelium. Analysis of the substantial literature on the subject reveals that the disputes center on the nature and derivation of that str.sq. epithelium. It is noticed that the discussion is more about interpretation than observation, as descriptions hardly differ; the same vaginal epithelium has been interpreted as either mesonephric (Kempermann 1931), meso-

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Development of the Female Perineum

nephric or sinus-related (Forsberg 1973), mesonephric and paramesonephric (Mijsberg 1924; Burkl and Politzer 1952; Witschi 1970), paramesonephric (Hunter 1930; Von Lippman 1939; Waltz 1958), paramesonephric and sinusrelated (Koff 1933; Bulmer 1957), or sinus-related (Vilas 1932, 1934; Meyer 1934, 1937, 1938; Kempermann 1935; Zuckerman 1941; Politzer 1955; Matejka 1959; Fluhman 1960; Uhlfelder and Robboy 1976; Minh and Herv de Sigalony 1989). The supposed derivation from the mesonephric epithelium has been based on the position of the two extensions of broad epithelium inside the mesonephric orifices (Mijsberg 1924; Witschi 1970) and on enzyme histochemical patterns (Forsberg 1973). The theory about the derivation of the vagina from sinus epithelium favored by most investigators comprises in itself greatly differing ideas, such as: sinus epithelium filling up space provided by cranialward-retreating paramesonephric epithelium (Vilas 1932, 1934; Meyer 1934; 1937, 1938; Kempermann 1935; Politzer 1955; Matejka 1959); sinus epithelium constructing the lateral “wings” of the vaginal anlage with the central part derived from the original paramesonephric uterovaginal canal (Bulmer 1957); and sinus epithelium replacing the paramesonephric epithelium after the latter had itself already been transformed into a broad stratified squamous epithelium (Prins et al 1976). The conclusion presented here that the str.sq. epithelium of almost the entire vagina develops directly from the original columnar paramesonephric epithelium of the uterovaginal canal is based on the following observations and considerations: a. There is during the main developmental events a distinct difference between the str.sq. epithelium derived from sinus epithelium and the str.sq. epithelium derived from paramesonephric epithelium, with the latter demonstrating a more irregular structure, distinctly more eosinophilic cytoplasm similar to the original solid epithelium of the (partially) fused tips of the paramesonephric ducts, the presence of intracellular vimentin during the process of vaginal formation, and the formation of characteristic epithelial ridges later (f.65–135 mm). The immuno reactivity for anti-vimentin antibodies is highly indicative of its derivation from the paramesonephric system, since the latter originates from the vimentin-positive mesodermal coelomic lining. This is in striking contrast to the consistent vimentin-negativity of the entoderm-derived sinus epithelium, but in conformity with reactivity for anti-uroplakin antibody in the evaginating urogenital sinus epithelium that forms sinuvaginal bulbs and the lower part of the vagina (Shapiro et al 2000). b. The so-called dividing line between str.sq. epithelium and columnar paramesonephric epithelium does migrate cranialward indeed, but it in fact forms the progressing front of transformation of the originally columnar paramesonephric epithelium into a stratified type. The true “dividing line”

Discussion

c.

d.

e.

f.

g.

71

between sinus epithelium and paramesonephric epithelium remains just inside the orifice where the two extensions of sinus epithelium meet the different paramesonephric stratified epithelium, and it shows no more than a minimal progression inward. Through the whole process of the formation of the vagina, the position of this line is also indicated by a shrunken nerve [interpreted as a vessel by Meyer (1934, 1937, 1938)] that had been entrapped by the fusing distal ends of the paramesonephric ducts. It is difficult to understand how such a tiny regressing nerve would survive a rapid and massive upward migration of sinus epithelium as has been suggested. The observation of (remnants of) lumina in the sinus-derived extensions separated from the narrow remaining lumen in the paramesonephric part of the vagina by only a small barrier of originally solid paramesonephric epithelium is in accordance with the results of silicone injections by Terruhn (1980), who demonstrated the presence of a lumen throughout the vaginal anlage at least as early as the 14th week (f.105 mm). The thin solid barrier and the initially very small diameter of the lumina as observed in histological sections have apparently been overcome by the pressure exerted. These findings are contrary to the idea of an up-growing solid sinus-derived epithelium forming a “vaginal plate,” as proposed in the “sinus theory.” Transformation of columnar paramesonephric epithelium into a stratified epithelium may occur multifocally as is observed in a 130-mm fetus studied presently. This pattern does not fit into the idea of an upward-growing sinus-derived str.sq. epithelium. But it does offer a better explanation for the so-called adenosis developing after exposure to diethylstilbestrol (Uhlfelder and Robboy 1976). Such squamous transformation is found to be a gradual process involving the whole layer of original columnar paramesonephric epithelium, without leaving remnants of columnar epithelium on the luminal side. Such remnants should remain detectable at least for a short time if there was a rapid and massive replacement by intruding sinus epithelium. Such squamous transformation itself can no longer be considered as an argument in favor of a derivation from sinus epithelium, because this phenomenon has also been demonstrated in paramesonephric epithelium (Bulmer 1957; Forsberg 1973; Cunha et al. 1983). It is the intracytoplasmic accumulation of large quantities of glycogen in the stratified epithelium of this area that gives the sinus-related and paramesonephric types of epithelia a similar appearance and obscures the original dividing line. This cranialward-progressing alteration into large pale polygonal cells can, however, not be considered as a late replacement of paramesonephric stratified epithelium by mesonephric or sinus epithelium as has been suggested by Forsberg (1965, 1973). Such a replacement at that time of development in fetuses of about 120 mm would be massive and therefore easily to detect. No trace whatsoever of such a process is found.

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Development of the Female Perineum

h. The vaginal sac anomaly can no longer be considered proof of the “sinus theory,” as has been suggested (Zuckerman 1941; Politzer 1955; Cunha 1975; Uhlfelder and Robboy 1976), because after a mouse model revealed that this blind-ending vagina, which was thought to survive regression of the paramesonephric system because of its derivation from sinus epithelium, is actually a persisting part of an only partially regressing paramesonephric structure (Mauch et al. 1985). i. The hymen imperforatum condition is better explained by the development of the vagina from an intruding paramesonephric system. Its pathogenesis is hard to conceive if the vagina was formed by up-growing sinus epithelium, as the way upward is apparently blocked. j. Analysis of congenital malformations involving the vagina support the concept that the complete vagina is of paramesonephric origin (Stephens et al. 1996). k. The concept that the human vagina derives from the paramesonephric system for almost its entire length is in line with the findings in all mammals studied by Kemperman (1935) and Forsberg (1973), who themselves considered the derivation from sinus epithelium in man an exception.

The view expressed in the present study corresponds best with the ideas of Koff (1933). The contribution of sinus epithelium to the vagina appears, however, still smaller than the one fifth of the total length estimated by him. Based on the differences between sinus-related and paramesonephric epithelia before they are fully glycogenated, and also on the position of the regressing entrapped nerve in the borderland, it is concluded that only a very small area, namely, the inner side of the hymen, becomes lined by sinus-related epithelium. The present analysis demonstrates that the hymen is mainly formed by the extraordinary growth of the vagina itself as compared with its orifice, and by the descent of the vagina which makes it finally bulge into the vestibulum. It confirms that folds in the urogenital sinus contribute to the formation of the lateral folds of the hymen, and that an active deepening of the dorsal vestibulum accentuates the dorsal segment, as has been suggested before (Mijsberg 1924, Meyer 1938). The present data indicating that the epithelium of both sides of the hymen derives from the urogenital sinus confirm the ideas of Koff (1933), Villas (1933a), and Meyer (1938), and refute a mesonephric Mijsberg (1924), Witschi (1970) or paramesonephric (Bloomfield and Frazer 1928; Von Lippmann 1939; Walz 1958) character for that inner side. The data shown here are in line with the absence of the typical thin stromal papillae of the vaginal mucosa and with the presence on the inner side of the hymen of an occasional urethral gland primordium. The development of the latter on either the inner or outer side of the hymen may be the source of hymenal cysts as later described by Merlob et al. (1978). Contrary to the opinion of earlier investigators who thought the hymenal stromal tis-

Discussion

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sue to derive from the adjacent wall of the urogenital sinus (Villas 1933a; Koff 1933), from the urogenital sinus and vagina (Bloomfield and Frazer 1928), or from the urogenital sinus and tuberculum Mlleri (Meyer 1938), the present study points to the stromal tissue of the temporary tuberculum Mlleri as at least its major constituent. This is based on topographical and structural arguments that make a contribution from surrounding walls, in which smooth muscle tissue abounds, less likely (Mahran and Saleh 1964). The variation in position and form of the orifice itself (Mor et al. 1986) may well depend on the size of the stromal column between the paramesonephric structure and the urogenital sinus. The more voluminous the intermediate median column of mesenchyme, the greater the chance that it will not regress completely and will persist as a median band or even as an (almost) completely imperforate hymen. 4.3.2 Transformation of the Urogenital Sinus into the Urethra and Vestibulum According to current opinion, the female urethra derives from the pelvic part of the urogenital sinus and the vestibulum from its phallic part (Wartenburg 1993; Sadler 1993; Larsen 1997). The basis for this idea is not clear, as references to original work are lacking and information from such original studies offers a different picture. These studies distinguish a primary (primitive) urethra as a separate entity between the bladder and the urogenital sinus, with that sinus positioned caudal to the orifices of the mesonephric ducts (Mijsberg 1924; Chwalla 1927). According to this theory, mainly based on the interpretation of three-dimensional reconstructions of the epithelial structures, this primary urethra would become the definitive female urethra while the pelvic and phallic parts of the urogenital sinus together would form the vestibulum. The present analysis of epithelial and stromal components by histological and immunohistological techniques reveals, however, that this subdivision is more complicated than previously assumed. It shows a rearrangement of parts of the wall of the urogenital sinus caused by the descent of the vagina, which descent may well be due to the growth of the caudal part of the vagina, in particular, as has been suggested by Witschi (1970). The descent makes the cranial part of the dorsal wall of the deep urogenital sinus, which is situated between the orifices of the mesonephric ducts, with associated vaginal anlage and the urinary bladder elongate. This elongation caudalward brings it opposite the ventral wall, which is originally positioned for its largest part caudal to the orifices. It is this combination that creates the urethra. The part of the dorsal wall caudal to the orifices and vaginal anlage combines with the whole of the superficial urogenital sinus to form the vestibulum.

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Development of the Female Perineum

4.3.3 Urethra The present study confirms the very detailed observations on the shape of the epithelial part of the urethra including the time of appearance, form and topography of its longitudinal folds and peri- and paraurethral glands, which were considered a homologue to the prostatic glands of the male (Johnson 1922; Chwalla 1927; Huffman 1943). The more comprehensive histological study presented here explains the composite character of the lining of the urethra as observed in adults (Petrowa et al. 1937). The rearrangement of originally different parts of the urogenital sinus, which is hardly reflected in the epithelial lining and lamina propria, has a major effect, however, on the structure of the muscular coat. The marked difference between the ventral and dorsal parts of the urethral musculature has been noticed before (Chwalla 1927; Dros et al. 1974). The present analysis recognizes in addition also the strikingly contrasting orientation of the muscle fibers. As for the trigone-related ventralcaudalward-oriented fine fibers in the dorsal wall, these are possibly related to the separation of the orifices of the ureters and mesonephric ducts. The predominantly dorsalcaudalward oriented bundles in the ventral wall are likewise possibly related to the caudalward migration of the vaginal orifice. The features of the trigonerelated smooth muscle tissue fit well into the description of the “vesical sphincter” described by Dorschner et al. (2001) both in its fine structure and in the changing of the orientation of its fibers from transverse to oblique ventralcaudalward. The origin of this trigonum-related musculature could not be traced precisely because of the absence of differentiation at a sufficiently early stage, i.e., during the partition of mesonephric ducts and ureters in the sexually indifferent period. With respect to the ventral musculature, the present study shows a distinctly different origin and early development of the smooth muscle from the striated muscle components. It is therefore preferred to distinguish them as separate entities notwithstanding some intertwinement of fibers in their borderland and the likelihood that they form a functional unit, and unlike Bourdelat et al. (1992) and Dorschner et al. (2001) to maintain the current terminology of internal and external urethral sphincters. It is also found that the longitudinal bundles of smooth muscle fibers, described by Dorschner et al. (2001) in the adult as part of the complicated structure of the dilator urethrae muscle, develop as part of the internal urethral sphincter and also originate from the fibromuscular lamina propria. No outer component linking these structures to the symphysis as described by them has been found in the present fetuses. The originally bilaterally striated external urethral sphincter develops into a single broadening muscle layer which is intimately linked to the ventral part of the smooth muscular internal urethral sphincter. The observa-

Discussion

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tions are in accordance with those of Dros et al. (1974), Oelrich (1983), and Tichy (1989). The most caudal segment of the muscle, which originally surrounds the deep urogenital sinus, thereafter the cranial part of the future vestibulum, and finally the descending vagina, apparently becomes the urethrovaginal sphincter as identified by Oelrich (1983). In accordance with the observations by Dorschner et al. (2001) in the adult, also during fetal development there is no indication of the existence of a urogenital diaphragm, an entity recognized by clinicians. There is no sign of a developing perinei profundus muscle. As is demonstrated, the external urethral sphincter originates from the dorsolateral perineal complexes, is strictly separated from the puborectal muscles, and does not retrieve myogenic cells from those muscles as suggested by Bourdelat et al. (1992). 4.3.4 Vestibulum Current information in textbooks about the development of the vestibulum is brief. It describes the formation of a simple cavity, which derives from the phallic part of the urogenital sinus (Wartenburg 1993; Sadler 1995; Larsen 1997), keeps the original wide external orifice, is the origin of the bilateral greater vestibular glands, and becomes finally an increasingly more shallow entrance to the predominating vagina. Original work did offer more details, such as precise information about changes in shape with the temporary existence of a complicated pattern of folds, the appearance of greater and smaller vestibular glands (Johnson 1923; Mijsberg 1924), and its epithelial lining (Bulmer 1959). It did, however, lack data about the combined epithelial and mesenchymal differentiation as addressed in the present investigation. This analysis reveals in the first place that the vestibulum derives not only from the superficial part of the urogenital sinus, but also includes an adjacent part of the deep part. This explains why the deep dorsal part of the vestibulum is lined by the same type of mixed str.sq. and ps.str.col. epithelium that lines the caudal adjacent part of the urethra, and also why it is surrounded by the most caudal segment of both the internal and external urethral sphincters before this muscular sling shifts to embrace the descending vagina and become the urethrovaginal sphincter (Oelrich 1983). This “urethral” type of epithelium has been reported as “glandular” epithelium by Bulmer (1959), who described the lining of the vestibulum in detail and rejected previous suggestions of a down-growth of str.sq. epithelium from the vagina (Hunter 1930), or an up-growth of ectodermal epithelium (Walz 1958), or “sex skin” (Zuckerman 1940). His “glandular” epithelium appears to comprise also the slightly different epithelium described in the present report as lining the ventral wall of the vestibulum, which seems to show the same type of str.sq. epithelium as observed dorsally but differs by

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Development of the Female Perineum

the absence of remnants of ps.str.col. epithelium and by the presence of small acinar glands basally. Since this composition can be observed in the spongy urethra of the (adult) male if squamous transformation supervenes, it appears that this type of vestibular epithelium develops by squamous transformation of ps.str.col. epithelium special to the superficial part of the urogenital sinus. His “vaginal” type of epithelium lining the superficial part of the vestibulum does resemble the epithelium of the vagina but contains less glycogen and does not show the characteristic thin stromal papillae typical of the vagina. The precise mechanism of the forming of this epithelium could not be established with certainty in the present investigation. The early configuration characterized by the overlap of original ps.str.col. and str.sq. epithelium is highly suggestive of a process by which labia-related str.sq. epithelium from outside the orifice replaces the preexistent ps.str.col. epithelium of the developing vestibulum. But this idea causes two problems: a progressing border is not found during the relevant period (which may, however, be caused by the lack of sufficiently marked differences in the stillimmature epithelia at that early stage); and the definitive epithelium that derives from this zone is distinctly different from the epithelium outside the orifice by the absence of cornification and by a tendency to form sebaceous glands during adolescence. Transformation of the preexistent ps.str.col. epithelium itself may be an alternative option. Such a process may then be related to the special nature of the epithelium of the preceding urogenital plate, and /or may be due to inductive influences by adjacent tissues such as underlying stroma, as has been demonstrated in experiments elsewhere in the genital tract (Cunha et al. 1985). It should be noted that the same process occurs in the anal canal (see Sect. 4.3.6) and the navicular fossa of the male (see Sect. 5.3.1). Hardly any information is available in the literature about the differentiation of the stromal elements of the wall of the vestibulum. Derived from the broad mantle of dense mesenchyme around the superficial urogenital sinus, the wall shows, apart from the formation of a markedly dense longitudinally oriented lamina propria, the development of a zone of fibrovascular stromal tissue, including the primordial erectile structures embracing the superficial urogenital sinus ventrally, and a complex of stromal components with a marked ventralward orientation of their fibers embracing it dorsally. As for the fibrovascular zone, the present study draws attention to its important role in the structure and, possibly, the physiology of the ventral and lateral walls of the vestibulum, and in the external shape of the area. The study demonstrates that this vascular area provides abundant vascular networks among the enlarging labia minora and the glans of the clitoris, the corpora cavernosa, and the vestibular bulbs, and reveals not only smaller size but also “underdevelopment” of internal structures as compared with homologue structures in the male. This area of high vascularity between the vestibulum and the clitoris, with its vague delimitation from erectile struc-

Discussion

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tures in sensu strictu, is probably identical to the “bulbs of the clitoris” recently described by OConnell et al. (1998) in adults, although the urethral dimensions indicated by them is not identified in the fetuses studied here. Taking into account the developmental process and relationships, it is clear that their proposed renaming of “vestibular bulbs” as “bulbs of the clitoris” is not justified. Analysis of the stromal tissue which embraces the vestibulum dorsally is greatly facilitated by variations in the plane of sectioning and by the application of antibodies that identify smooth muscle cell differentiation. Together, these techniques enable early recognition of a number of different components within the rather inaccessible original dense “mesenchymal” tissue, which plays a special role in the forming of the superficial perineum. In this respect the superficial dorsal urogenital stroma is least distinctive, as it constitutes only the rather inconspicuous fourchette. The deep dorsal urogenital stroma, in contrast, becomes the predominant smooth muscle component of the midperineal region. Smooth muscle tissue in this region is usually considered in the context of the perineal body, which separates the vagina from the anorectum and is considered homologue to the perineal body of the male (Williams et al. 1989). Looking in detail, however, reveals the development of this area to be a remarkably subtle and intricate process comprising three elements: (a) a marked increase in the volume of the stromal tissue, which keeps all bilateral structures in the area (such as the greater vestibular glands, vestibular bulbs, and bulbospongiosus and puborectalis muscles) widely apart; (b) a differentiation into smooth muscle tissue that forms a dorsal smooth muscular coat of the vestibulum, and (c) an intimate relationship between this smooth muscle tissue and adjacent structures, such as some striated muscles into which it extends. This combination of cohesion and separation, with smooth muscle tissue as a cement, appears an important factor to make the female perineum strong and flexible as seems essential for its physiologic functions. It is interesting to note that this development in the female stands in sharp contrast with the situation in the male, where the central component of all stromal tissue regresses resulting in an approximation of the various bilateral structures to the midline where they become strongly fixed to a small perineal body and thin perineal septum (see Sect. 5.3.3). The superficial shaft stroma is special because it forms a subepidermal layer of fine connective tissue with markedly parallel and ventralward-oriented fibers, unlike the usual dermal tissue, and has no underlying subcutaneous adipose tissue, either. Its distribution from the labia minora ventralward, spreading into the interlabial sulci and converging into the hood of the clitoris and its prepuce directly relates to its origin between the labioscrotal swellings, the urogenital labia, and the thin peripheral extension of the glans, which area constitutes the shaft of the sexually indifferent phallic structure. This configuration persists in its essence, notwithstanding the rel-

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Development of the Female Perineum

ative flattening of the female perineum. The same layer developing in the male becomes greatly protracted as the result of the formation of the long shaft of the penis occupying the whole area between the free edge of the prepuce and the scrotum. In both sexes this tissue plays a major role in the formation of the prepuce. Although the prepuce is directly connected to the labia minora there is no indication that these labia produce the prepuce as has been suggested (Hamilton and Mossman 1973; Wartenberg 1993). Its formation will be discussed in relation to the male prepuce (see Sect. 5.3.4). Unlike the situation in the male, the female prepuce remains small, does not become circumferential, and has no dartos fascia. The glandopreputial lamella also remains small, and solid in its deeper part. 4.3.5 Labia Majora No information is available in the literature about the precise development of the labia majora other than that they derive from the labioscrotal (syn. genital) swellings. According to some authors, these labial swellings grow together between the vestibular and anal orifices to form the posterior labial commissure (Spaulding 1921; Szenes 1925; Arey 1965; Hamilton and Mossman 1972; Moore and Persaud 1998) and may encircle the vestibular opening ventrally (Moore and Persaud 1998). Other authors do not mention the formation of a posterior commissure (Sadler 1995; Larsen 1997) or consider the labia to extend dorsal to the anal orifice (Wartenberg 1993). It is suggested by their descriptions that these opinions are based on observations of the external appearances of the fetuses only. The present histological analysis reveals a specific origin, i.e., the dorsolateral perineal complexes derived from the sacral somites, and a further development intimately related to the smooth muscle tissue of the dartos fascia. When this future labioscrotal mesenchyme extends from its original position dorsolateral to the cloacal eminence ventralward, a deep component becomes involved with the more general process of ventralward orientation within the stromal tissues dorsal to the urogenital orifice, while in a superficial part the original, indifferent pattern is preserved. This apparently forms the basis for its bi-laminate structure, similar to the male construction (see Sect. 5.3.3), but differs in: (a) relatively late differentiation into smooth muscle cells with no smooth muscle actin detected before the fetus has reached a length of about 230 mm (28 postovulatory weeks), as compared with 140 mm (18 weeks) in male fetuses; (b) a far smaller volume mainly consisting of a thin superficial layer, as most of the deep lamina is split into elements within the septa between the immigrating adipose tissue, whereas in the scrotum the fascia is built as a thick two-layered structure and no adipose tissue intervenes; (c) its absence from the prepuce and hood (syn. ante-

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rior labial commissure) of the clitoris, whereas in the male it becomes involved in the process which creates the shaft and prepuce of the penis and provides it with a distinct layer throughout the shaft as far as the free margin of the prepuce. The presence of smooth muscle tissue deep in the septa between the increasingly more predominant adipose tissue of the labia majora indicates that the latter is a secondary acquisition. 4.3.6 Anal Canal Knowledge about the development of the anal canal is almost exclusively based on the detailed descriptions by Johnson (1914). Chwalla (1927) confirmed his observations but differed in opinion on his interpretation of the str.sq. epithelium lining the superficial part of the definitive anal canal. According to Johnson, this epithelium is derived from the “bulbus terminalis,” which develops from the cloaca inside the anal membrane, is therefore entodermal, and brings the entodermal-ectodermal border to the anocutaneous line. Chwalla disagreed by considering the “bulbus terminalis” to provide stratified columnar epithelium only, the str.sq. epithelium between the dentate and anocutaneous line deriving from the ectodermal “proctodeum,” which thus positioned the original anal membrane at the dentate line as is general opinion at present. More recently those “static” theories have been challenged by observations by Jit (1975), who found that ectodermal epithelium creeps cranialward from the original position of the anal membrane and reaches the dentate line in fetuses of 180 mm CRL. The observations in the present study match those in these earlier reports to a large extent. With respect to the dispute about the derivation of its nonkeratinizing str.sq. epithelium, and thereby the position of the ento-ectodermal border, the present study offers no definitive answer. As has been shown, that border is clearly at the original position of the cloacal membrane at the beginning of sexual differentiation of the perineum, which is represented by the transition of cloacal ps.str.col. epithelium into labia-related str.sq. epithelium. However, it is also demonstrated that shortly afterwards a new zone of str.sq. epithelium appears between these two epithelia. This zone consists of str.sq. epithelium characteristically covered by a single layer of cuboidal-to-flat cells continuous with the adjacent ps.str.col. epithelium. This pattern is in fact similar to the configuration observed in the female vestibulum and navicular fossa of the male urethra. It starts in the narrow zone which originally holds the attachment of the cloacal membrane. The configuration here suggests that str.sq. epithelium indeed “creeps up cranialward,” as Jit (1975) worded. This author did not, however, mention the special nature of this epithelium and had drawn his conclusion from the cranialward movement of the upper border of this str.sq. epithelium with respect to the lower border of the internal anal sphincter. He apparently did

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not notice that, in fact, the lower border of the sphincter moves caudalward toward the anal orifice, and that the position of the str.sq. epithelium hardly changes in relation to the underlying lamina propria and anal columns during that process (f.32–60 mm). Because the pattern within the composite epithelium does not alter for a long period (f.45–200 mm), and the zone lengthens in proportion to the growth of the region as a whole, it cannot be decided if the process is an extension of ectodermal str.sq. epithelium cranialward or a transformation of ps.str.col. epithelium into str.sq. epithelium. It has its own character, different from the more external str.sq. epithelium as it does not cornify and does not form epidermal appendages. This investigation also demonstrates that morphological elements directly related to their derivation from the cloaca, such as the ps.str.col. epithelium forming tubular glands, a dense lamina propria of longitudinally oriented stromal tissue forming ridges, and fibrovascular tissue surrounding the orifice, persist in the definitive anal canal. These characteristics explain a number of special features of that canal, such as: a. The narrow zone of ps.str.col. epithelium deep to the dentate line (“anal transitional zone” of Fenger, 1988) from which extend anal glands very similar in morphology to the urethral and vestibular glands; b. The so-called musculus canalis recti (syn. Treitz muscle, musculus sustentator tunicae mucosae, musculus submucosae ani), which develops from the dense longitudinally oriented lamina propria with its characteristic extension into the internal anal sphincter and its differentiation of smooth muscle elements, a derivation which contradicts the assumption that this “muscle” spreads from that sphincter into the lamina propria and “corpus cavernosum recti” (Thomson 1975; Hansen 1976), and which explains the particular strong bond between the mucosa and the internal sphincter that prevents mucosal prolapse; c. The “corpus cavernosum recti,” which has been described as a specially structured vascular tissue near the anal orifice that keeps this opening wellclosed between defecations (Stelzner et al. 1962), and is now demonstrated to derive from a true “erectile” structure, i.e., from the dorsal parts of the cloacal labia that form a fibrovascular cuff around the anal opening.

4.3.7 External Perineum The external appearance of the perineum had been studied in detail to enable early sex determination (Spaulding 1921; Szenes 1925; Wilson 1926; Glenister 1954). The present study provides the microscopic base for the sequential changes in shape of the perineal area by demonstrating the pattern of differential growth in specific underlying stromal elements derived from the cloacal eminence.

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The observation that most of the midperineal region between vestibulum and anal canal becomes occupied by a ventralward extension of labia-derived stroma of variable volume from the anal orifice fits well into the continuous spectrum of variations in shape observed by Stephens (1968) in the “female perineum” of 62 unselected premature babies. Interestingly, he also found a concave type of “perineum” varying from a central groove to a wet, red depression, which may be grooved ventrally and raised dorsally. In the light of the early development of this area during the sexually indifferent period it therefore appears that in some female fetuses remnants of the original cloacal groove remain. The special character of the region as a whole is also reflected in the structure of its surface, with the usual type of skin developing at the periphery only. In addition to the variation in thickness of the epidermis, in number and size of the epidermal appendages, and in the presence of the specific subepidermal stromal tissues, there is also a development of strikingly large tubular glands in the sulci between the labia minora and majora which, by their position, size and deep extension, most likely represent the anlage of glands that may assume mammary-like features during adulthood (van der Putte 1994). Other glandopreputial tubular glands developing from the deep margin of the glandopreputial lamella are apparently abortive. As they show a tendency to squamous metaplasia, these glands may be the source of some poorly understood epidermoid cysts which are occasionally found near the clitoris in the adult (Merlob et al. 1978). Such glands have not been described before. They are of interest in relation with the dispute about the presence or absence of so-called Tyssons glands in the male preputial sac (Barieto et al. 1992). However, the tubular glands regularly observed in the female fetuses are a rare occurrence in male fetuses, and Tyssons glands are anyhow supposed to be sebaceous in nature.

5 Development of the Male Perineum 5.1 Introduction The formation of the male perineum is currently described as a complicated process in which: (a) the pelvic part of the urogenital sinus, with its issuing bladder and mesonephric ducts, becomes surrounded by the prostate and urethral sphincter and forms the prostatic and membranous urethrae; (b) the urethral plate of the urogenital sinus lengthens by the elongation of the genital tubercle into the penis, hollows and tubularizes to form the spongy urethra by the fusion of flanking urethral folds, from behind forward, leaving a median raphe on the surface; (c) the external urethral orifice and adja-

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Development of the Male Perineum

cent navicular fossa are constituted from a solid cord or plate that grows inward from the epithelium of the glans, canalizes, and makes contact with the already formed proximal spongy urethra; (d) the prepuce develops from a preputial fold which extends from the dorsum of the penis lateralward to fuse at the side of the venter, where it forms the frenulum on its inner side; (d) scrotal swellings lateral to the developing penis migrate dorsalward and fuse or merge into a single scrotum. It was during our preceding studies into the normal and abnormal development of the anorectum in the pig that it became clear that not only traditional ideas on the division of the cloaca and the early development of the perineum were incorrect, but that likewise a major element in current ideas on the development of the male perineum, i.e., the formation of the male spongy urethra, should be questioned. Such demurral had been suggested before, but that signal has apparently been ignored, most likely because of highly suggestive changes in the external appearance of the developing region and the existence of malformations such as hypospadias. It is striking that ideas on the development of the male perineum were mostly based on changes in the external form, whereas observations challenging their correctness came from histological studies. In the original literature on the development of the male perineum, more detailed information about aspects of the genesis of the urethra, internal and external urethral sphincters, prostate, prostatic utricle, bulbourethral glands, and prepuce were present, with conflicting opinions about the formation of the prostatic utricle, glandular urethra, and prepuce. With the exception of that concerning formation of the urethral sphincters, data on the differentiation of the erectile, fascial, and muscular structures are practically non-existent. 5.2 Observations The first signs of the development of a male type of perineum are identified at the very start of the fetal period. The sexually indifferent perineum at that stage reveals a marked preponderance of urogenital structures derived from the ventral part of the cloaca over anal elements derived from its dorsal part. Where both structures meet, a small midperineal region is formed. It is the transformation of that urogenital part which will give the perineum its conspicuous male composition, major characteristic structures being the urethra, raphe, septal and fascial elements, penis and prepuce, scrotum, and external surface of the perineum (Fig. 32).

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Fig. 32a–c Schematic midsagittal sections through the perineum of male fetuses of 35 mm to 65 mm to show major median structures. They demonstrate: urethra (1), anal canal (2), paramesonephric structure (3); urinary bladder (4), urethral sphincter (5), symphysis pubis (6), corpora cavernosa (7), glans (8), rectum (9), muscularis propria of the rectum (also surrounding the deeper part of the anal canal; 10), puborectalis (11), external anal sphincter (12). Arrowhead, urogenital plate; arrow, glans plate; small circle, orificium of the bulbourethral gland. Note the preservation of the original configuration due to the simple lengthening of the urogenital sinus into the definitive urethra. In this respect, special attention is given to the epithelial zones in cloaca-derived structures. In a is shown the characteristically cloacal high (a) and low (a) ps.str.col. epithelium of the deep and superficial urogenital-sinus-like urethra, respectively; ps.str.col. epithelium (a") of the deep anal canal; and str.sq. epithelium (b) originally related to the cloacal labia. In b the position of the dorsal urethral groove, transforming into a dorsal ridge, is indicated between short arrows. In c all zones have lengthened further, and a new transitional zone of str.sq. epithelium, with a remnant of ps.str.col. epithelium at the luminal side (c), has appeared; the ventral urogenital plate has split into a groove from which develop the lacunae of Morgagni (arrowheads). Original magnification, 25

5.2.1 Urethra During the development of the male urethra, differences between the deep and superficial segments become marked. The deep segment transforms into both the prostatic and the membranous urethra, and the superficial segment into the spongy urethra, including its terminal navicular fossa. 5.2.1.1 Prostatic Urethra The prostatic urethra develops from the deepest part of the early urethra. It characteristically shows the transformation of the mesonephric-paramesonephric complex into the ejaculatory ducts and the prostatic utricle, and also the formation of the glandular complex of the prostate, which is enveloped by an intricate mass of fibrous and smooth muscle tissue.

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Development of the Male Perineum

In more detail, the crescentic lumen of the lengthening deep early urethra becomes more irregular as the Mllerian tubercle becomes much more prominent, (f.45 mm) forming the colliculus seminalis; and a large number of small oblique longitudinal grooves appear (f.90 mm). The epithelium becomes more varied. The typical high ps.str.col. epithelium, which reveals an eosinophilic luminal zone and intraepithelial acini later (f.140 mm), remains the basic element. It is highest near the bladder, where it gradually passes into the thinner epithelium of the urinary bladder, with its more abundant cytoplasm, which later becomes a broader and true transitional type of epithelium (f.100 mm). That bladder type of epithelium extends relatively far into the urethra ventrally and may be mixed with small patches of the urethral type in the trigone of the bladder dorsally. At the colliculus seminalis, and to a lesser degree in the adjacent urethra, the superficial layer of columnar cells of the urethral epithelium develops an abundant, clear cytoplasm rich in glycogen. A similar alteration takes place in small tongue-like extensions of mesonephric epithelium from the mesonephric ducts into the urethral lining cranial to the orifices. This epithelium distinguishes itself from the adjacent clear urethral epithelium by its display of a contrasting nucleus-free basal zone (Fig. 33a). Inside the colliculus the mesonephric ducts differentiate into ejaculatory ducts and the paramesonephric ductal structure into the prostatic utricle (Figs. 33, 34). Initially there is no contact between the urogenital-sinus-derived urethral epithelium on the colliculus and the paramesonephric structure between the mesonephric ducts inside the colliculus (Fig. 12b). That structure remains when the main, cranial, part of the system regresses and disappears (f.35–60 mm). Derived from the two fused paramesonephric ducts, it consists of a short cranial ductal part and a solid split distal end near the surface of the tubercle. It remains at first (f.35–70 mm) in direct contact with the epithelium of the mesonephric ducts on both sides, while separated from the urethral epithelium by a median column of mesenchyme. However, simultaneously with the regression and disappearance of the column of mesenchyme, the original contacts between the paramesonephric structure (named prostatic utricle from now on) and the lateralward-shifting distal mesonephric ducts (named ejaculatory ducts) are lost, and a variably large direct contact is established between the paramesonephric and urethral epithelia. That contact is restricted to the caudal side only, as the small “tongues” of mesonephric epithelium extending upward into the urethra of the orifices prevent such a contact on the cranial side. This step is followed by the replacement of regressing remaining paramesonephric epithelium by a small-cell epithelium derived from the urethral epithelium from the contact zone inward (f.70 mm). The prostatic utricle thus becomes a blind-ending urethral appendage occasionally showing the remnant of the original lumen in some fetuses and for some time still traversed near the orifice by a nerve entrapped during the process of fusion of the distal paramesonephric ducts

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Fig. 33a, b Development of the prostatic utricle in male fetuses of 73 mm t (a) and 200 mm t (b). In a the colliculus seminalis shows the regressing paramesonephric structure (1), now separated from the mesonephric ducts (now renamed ejaculatory ducts; 2), the external epithelium, (2a) of which is “clear” like the adjacent urethral epithelium with its characteristic apical nuclei (3a). The paramesonephric epithelium is partially replaced by urethra-derived epithelium (3), recognizable by small dark nuclei (border indicated by arrows), and surrounding shrunken, entrapped nerves (asterisk). In b the prostatic utricle (1) shows squamous transformation of the preceding str.col. urethral epithelium, which latter is still identifiable by budding (arrows) and by glandular rosettes (arrowheads) inside and outside the utricle

(f.70–140 mm) (Fig. 33a).The urethral type of epithelium thereafter transforms into a str.sq. epithelium, revealing a central area of large pale-staining cells (Fig. 33b) with a tendency to dissociate and to form a cavity later (f.140–200 mm). Remarkably, this transformed epithelium also reveals glandular features in the form of isolated rosettes of columnar cells throughout, and of larger solid sprouts very similar to those forming the prostate gland. At the same time, the structure may grow to be relatively large and “cystic,” with a variably wide orifice into the urethra, and giving rise to a highly variable number of radiating sprouts. The same squamous metaplasia also takes place in the urethral epithelium on the colliculus between the ejaculatory ducts, where intraepithelial glandular elements leave similar rosettes (Fig. 33b), as they occasionally do in the large ducts of the prostate glands. From the urethral epithelium arises a highly variable number of solid buds (f.50 mm) as a first phase in the development of the prostate. From these buds series of four to five large glands develop at each side of the col-

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Development of the Male Perineum

Fig. 34a–e Schematic transverse sections through the colliculus seminalis of male fetuses of 35 mm to 190 mm. They illustrate the relationship between the epithelia of the urethra, mesonephric ducts/ejaculatory ducts, and the paramesonephric structure during the transformation of the last into the prostatic utricle. In a the original sexually indifferent configuration is shown with the median paramesonephric epithelium (1) in direct contact with the mesonephric epithelium (2), and separated from the epithelium of the early urethra (3) by a column of mesenchyme (4); arrow, nerve entrapped by the fusing paramesonephric ducts. In b a direct contact is established between the sinus-derived urethral and paramesonephric epithelia after the column has regressed. In c epithelium of the urethra begins to replace regressing paramesonephric epithelium while mesonephric and paramesonephric structures separate. In d the replacement has been completed and some buds develop. In e the initially solid structure becomes the “prostatic utricle” by luminization of the epithelium (which shows squamous transformation); its derivation from the urethral epithelium is recognizable by (prostatic?) buds and also rosettes of glandular epithelium (arrowheads). Original magnifications, 15 (a–c) and 55 (d and e)

liculus, with one or two buds extending cranialward and the remaining ones growing lateralward and lateralcaudalward. In some fetuses, one or two relatively large glands also sprout from the ventral epithelium and extend into the ventral wall. On the colliculus itself and throughout the prostatic urethra up to the border with the bladder, many more smaller glands and buds appear. In some smaller glands caudal to the colliculus, mucous cells similar to those in the bulbourethral and urethral glands differentiate from a lower columnar epithelium. The glands of the prostate form a lumen, branch (f.75 mm), and penetrate the smooth muscle coat (f.90–140 mm). The lamina propria of the prostatic urethra consist of a markedly dense, longitudinally oriented stromal tissue that later differentiates into a connective tissue with a smooth muscle element (f. 200 mm). It is dorsally interrupted by the initially small-cell mesenchyme of the colliculus that becomes a fibrous tissue (f.150 mm). On the ventral side and caudal to the level of the orifices of the ejaculatory ducts, the longitudinal pattern is interrupted by a relatively thin but conspicuous band, with a more transverse pattern similar to the structure observed in the female (see Sect. 4.2.3). Differentiation in the mantle around the prostatic urethra starts with the appearance of initially vague differences in pattern (Fig. 35). Apart from a

Observations

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Fig. 35a–e Schematic parasagittal sections through the lateral wall of the prostatic urethra of male fetuses of 50 mm and 65 mm, and transverse sections through that urethra at 65 mm at levels indicated in b. They demonstrate the composite structure of the developing muscular mantle, which at these stages consists of: fine primordial muscle tissue of the vesical sphincter (1), with a ventralcaudalward orientation and relationship to the trigone of the bladder; ventral bundles of smooth muscle cells of the internal urethral sphincter (2), with a predominant dorsalcaudalward orientation and peripherally mixed with striated muscle tissue of the external urethral sphincter of the same pattern (3 in b), and both surrounding the urethra almost completely on the caudal side; a dorsolateral mass of prostate-related tissue (4); and minor contributions from the detrusor vesicae (5) and from the coats ( curved arrows in b and d) around the ejaculatory ducts (6). 7, Bladder; 8, urethra; 9, colliculus seminalis. Original magnifications, 23 (a and b) and 33 (c–e)

distinctive longitudinal orientation in the lamina propria, these differences consist of an oblique ventralcaudalward orientation in the dorsal part closely related to the trigonum, a transverse-to-oblique dorsalcaudalward orientation in the ventral area, and a tissue without a special orientation caudal to the small-cell stroma of the colliculus (f. 35–40 mm). This proves to be a basic configuration that develops into smooth muscle components thereafter, as follows (Fig. 35): a. The trigonum-related part forms a layer in the trigone of the bladder on the cranial side and embraces the most cranial part of the urethra on the caudal side as the vesical sphincter. It blends with the ventral smooth muscle component (see b) but differs from that component in its ventralcaudalward orientation, much finer structure, and much later smooth muscle differentiation (f.110 mm versus 23 mm). The tissue is intimately linked to parallelrunning oblique transverse bundles of smooth muscle cells of the detrusor vesica around the internal urethral orifice, which had already differentiated during the postcloacal sexually indifferent period.

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Development of the Male Perineum

b. The largest caudal part of the internal urethral sphincter originating on the ventral side of the prostatic and membranous urethra differentiates early (f.23 mm) into bundles of smooth muscle cells with a transverse orientation cranially and an increasingly more oblique dorsalcaudalward orientation caudally. This pattern becomes more marked during the expansion of the prostate, when caudal bundles assume an almost longitudinal course (f.65 mm). Cranial bundles finally surround the urethra for about three quarters of its circumference, whereas the caudal bundles become almost circumferential as those on both sides converge into the perineal body in congruity with those of the more distalward membranous urethra. On the outside, the smooth muscle cells intermingle with fibers of the striated external urethral sphincter, which appear in fetuses of about 60 mm and demonstrate the same orientation and about the same extension. c. The prostate-related smooth musculature, derived from dense stroma without a special orientation dorsalcaudal to the colliculus seminalis, shows its greatest expansion dorsallateralward, where it becomes the stroma of the two lateral lobes of the prostate. It differentiates into a dense intricate network of smooth muscle tissue, and within its meshes ductuli of the gland penetrate. The latter are directly surrounded by a finely fibrillar connective tissue rich in blood vessels (f.150 mm).

These various smooth muscle elements gradually pass into each other, notably so inside the prostate. The prostate is thus built from smooth muscle tissue related to the vesical sphincter cranially (middle lobe), from internal urethral sphincter elements ventrally and laterally, from prostate-related smooth muscle tissue dorsally and dorsolaterally, from minor contributions from the detrusor vesicae dorsally, and from the ejaculatory ducts centrally. The detrusor vesicae contributes external longitudinal bundles on the ventral and dorsal sides of the cranial part of the urethra. which represent the pubovesical musculus and vesicoprostatic muscle, respectively. The latter may extend far into the prostate. 5.2.1.2 Membranous Urethra The membranous part becomes the narrowest segment of the urethra. From a very short element at first, it lengthens considerably (f.65–90 mm). It is flanked externally by the bilateral puborectal muscles. Its crescentic lumen cranially changes into an H-form caudally as the ventral groove is followed caudalward by the temporary ventral ridge, and as the colliculus seminalis on the dorsal side becomes more prominent and continues into a newly formed dorsal ridge that replaces the original dorsal groove (f.50 mm). Thereafter, that H shape becomes obscured by numerous smaller grooves and ridges.

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Its originally high (two- to three-layered) ps.str.col. epithelium becomes a two-layered cuboidal type. In some fetuses a few tubular mucous glands develop (f.50 mm). The lamina propria is a continuation of the lamina in the prostatic urethra, which here gradually changes into the lamina of the spongy urethra, characterized by many longitudinal vessels. The membranous urethra is surrounded by the internal and external urethral sphincters in the form of a horseshoe, its narrow opening connected to the perineal body. The orientation of the fibers is transverse to oblique transverse with a dorsalcaudalward orientation. Later (f.65 mm), longitudinal bundles appear at the border with the lamina propria. The peripherally developing striated muscle tissue of the external urethral sphincter (f.60 mm) shows the same pattern and extension as the outer layer of the internal sphincter. A great increase in length of this segment (f.65 mm) is associated with a distinct thickening of both components of that sphincter. 5.2.1.3 Spongy Urethra3 The spongy urethra develops from a relatively short, wide, and markedly grooved superficial part of the early urethra that is almost perpendicular to the surface of the perineum, into a much longer, relatively narrow tube that runs almost parallel to the surface (Fig. 32). Its terminal part, or navicular fossa, has a distinctly different structure and will be described separately. The lumen of the spongy urethra reveals initially the configuration of the sexually indifferent period. But this soon changes drastically when this part of the urethra begins to lengthen. It becomes relatively narrow and bends ventralward due to a disproportionately strong growth of the stromal tissue dorsal to the early spongy urethra (f.40–60 mm). Its relief changes simultaneously when: (a) the median dorsal groove is incorporated into a developing dorsal ridge and disappears (Fig. 36a); (b) the two lateral grooves move medialward toward the dorsal ridge, bringing the orifices of the bulbourethral glands and their lengthening excretory ducts close to the midline; (c) the ventral ridge disappears (f. 50 mm); and (d) the remaining ventral extension of the urogenital plate elongates and transforms into a ventral urethral groove distalward. Thereafter, the lumen of the urethra widens and new longitudinal ridges and grooves develop, varying considerably in number and size. The epithelium remains two- to three-layered ps.str.col.. At the end of gestation, focal to widespread squamous metaplasia is observed in some fetus3 In relation to the developing penis the terms “cranial” and “caudal” will be replaced by “proximal” and “distal,” respectively. In line with general use, the ventral side of the penis will be named “dorsum” and, as a consequence, the dorsal side “venter.”

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Development of the Male Perineum

Fig. 36a, b Spongy urethra in male fetuses of 82 mm t (a) and 330 mm t (b). In a the spongy urethra (1) reveals the dorsal ridge (arrow), urogenital plate-derived ventral groove (asterisk), urethral gland primordia (arrowhead), and derivatives of the deep dorsal urogenital stroma: deep perineal septum (2), sheath (3) of the corpus spongiosum (4), deep fascia of the penis (5) embracing the corpora cavernosa (6), and deep stroma of the scrotum (7). In b the spongy urethra (1) demonstrates the dorsal ridge (arrow) and a large urethral gland (arrowhead) derived from the ventral urethral groove. 4, Corpus spongiosum

es. Primordial urethral glands bud in greatly varying numbers from the more solid deepest parts of the grooves. The earliest and largest primordia develop from the distal part of the remnant of the ventral extension of the urogenital plate (f.50 mm). They demonstrate from the beginning a striking tendency to bladderward orientation, and form often long and wide ducts with a contrastingly poor differentiation of acini. They become the lacunae of Morgagni but are basically similar to the other urethral glands. The latter appear later and bud predominantly from the ventral, ventrolateral, and lateral grooves, with their highest concentration in the middle segment of the spongy urethra (f.75 mm). They branch, extend into the adjacent part of the corpus spongiosum (f.150 mm), and develop acini lined by mucous epithelium (f.200 mm; Fig. 36b). The orifices of the right and left bulbourethral glands, which had already budded from the lateral grooves during the sexually indifferent period (25 mm), shift toward the developing dorsal ridge together with the lateral grooves (f.40–50 mm). This process brings the excretory ducts into a para-

Observations

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median position parallel to this greatly lengthening segment of the urethra. At the same time, the ducts elongate as the distance increases between their orifices and their newly formed distal branches, which hold their original dorsolateral position to the urethra in the borderland between the cranial corpus spongiosum and the caudal extensions of the internal urethral sphincter. The ducts keep their lining of ps.str.col epithelium after their branches have developed a lining of mucous cells (f.125 mm). Some variation is noticed, with hypoplasia of one gland seen in one fetus and some adjacent accessory glands in another. In the dense longitudinal stroma of the lamina propria appear numerous wide longitudinal veins. These veins become so closely linked to the vascular plexuses of the corpus spongiosum that this (outer) part of the lamina propria becomes part of that erectile structure, leaving only a thin subepithelial layer of a more usual type of lamina beneath the urethral epithelium (Fig. 36b). In the median line where the dorsal ridge is formed, the stroma forms a narrow though conspicuous band of increased density that forms the deepest element of the perineal septum. The spongy urethra does not develop a true muscular coat. It receives, however, extensions from the internal urethral sphincter at the ventral side. This muscular element retains to some extent the basic pattern of the sphincter, with most outer bundles revealing a transverse orientation while inner bundles take a longitudinal course. It thins down toward the navicular fossa with only very few bundles developing distally. On the dorsal side extensions from the internal urethral sphincter surround the branches of the bulbourethral glands, with a minor component spreading outward around the cranial end of the corpus spongiosum and a major component converging into the thinning septum of the deep dorsal urogenital stroma between the bulbar parts of the corpus spongiosum, which greatly expand dorsalward. 5.2.1.4 Navicular Fossa The development of the navicular fossa differs in many respects from the rest of the spongy urethra. Initially, it has a relatively wide lumen with little relief as the grooves and ridges of the rest of the spongy urethra hardly extend to this terminal part. The same holds for the dense longitudinally oriented stroma of the lamina propria, which passes here into fibrovascular tissue related to the developing erectile tissue embracing the fossa on the dorsum of the penis, and into dense tissue with a transverse orientation embracing it on the venter (f.32–40 mm). The specific structure of the fossa becomes more marked when male development accelerates (f.40–70 mm). Its lumen becomes much wider in a transverse direction, as is clearly illustrated by the urethral orifice altering

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Development of the Male Perineum

Fig. 37a, b Early distal urethra in male fetuses of 42 mm t and 49 mm s. In a the lumen of the urethra (1) has broadened dorsally, where the epithelium forms micropapillary protrusions (arrowhead), while ventrally the ventral extension of the urogenital plate (2) persists. Note the transverse dorsal stroma of the navicular fossa (3) directly beneath the epithelium. Arrow, remnant of ps.str.col. epithelium on the surface just proximal to the urethral orifice; 4, bulbus spongiosus; 5, deep dorsal urogenital stroma. In b a median sagittal section shows urethra-derived ps.str.col. epithelium (arrow) trailing behind in the midline on the distalward progressing stroma; 3, transverse dorsal stroma of the navicular fossa; 6, raphe; 7, lamina propria; 8, orificium of the urethra

from longitudinal slit-like into an increasingly more diamond shape, with the widest part on the venter side. The fossa is lined by a high ps.str.col. epithelium with micropapillary protrusions on a band of transversely oriented stroma (Fig. 37a). The ps.str.col. epithelium often forms a short median extension outside the orifice in a small groove in the early perineal raphe, which itself is covered by a broad str.sq. epithelium (Fig. 37b). In one fetus a small isolated remnant was observed at a short distance from the edge. In the “roof ” of the fossa the ventral remnant of the urogenital plate ends as it passes into the glans plate. The latter is like a comb embedded in the dense mesenchyme of the glans and extends to the tip of the glans. It differentiates into a str.sq. epithelium at an early stage (f.45 mm). This configuration becomes reversed during a following developmental phase (f.70–90 mm) when the lumen on the venter side shows a relative decrease in size in association with the regressing urethral labia, whereas the glans plate and adjacent parts of the fossa on the dorsum side greatly increase their size in congruity with the rapidly growing glans (Fig. 38a). Like

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Fig. 38a, b Navicular fossa in male fetuses of 90 mm t (a) and 140 mm f (b). In a the navicular fossa (1) shows micropapillary ps.str.col. epithelium (arrow) on the “venter” of the penis, and str.sq. epithelium of the glans plate (2) on the “dorsum.” Also shown is the transverse dorsal stroma of the navicular fossa (3), the glandopreputial lamella (4), with the corona of the glans (5) on one side and the originally peripheral extension of glans stroma (6) on the other side. In b the original urethral channel (1), lined by ps.str.col. epithelium (arrow) on the venter side, contrasts with a greatly broadened glans plate (2) of str.sq. epithelium that will open later; arrowhead: str.sq. epithelium covered by ps.str.col. epithelium

in the anal canal and vestibulum vagina of the female (see Sects. 4.2.4, 4.2.8), a narrow “transitional” zone of str.sq. epithelium covered by a single layer of cuboidal cells continuous with the adjacent urethral ps.str.col. epithelium appears just inside the urethral orifice and bordering the glans plate (Fig. 38b). The str.sq. epithelium thickens to such an extent that only a tiny lumen lined by the original ps.str.col. is left on the venter side. The cuboidal cells later disappear and the epithelium differentiates into a non-cornifying str.sq. epithelium. It is only shortly before term that the greatly enlarged glans plate breaks down centrally and the definitive wide navicular fossa with its orifice extending to the tip of the glans is formed. Where the rapidly growing glans plate borders the more proximal urogenital plate a rather abrupt transition may develop that will become the “valvula Gurin.” Outside the spongy urethra and its lamina propria, the original mantle of mesenchyme of the urogenital sinus has differentiated during the sexually indifferent period into a series of erectile structures that embrace the urethra on the dorsum side, and also into a combination of stromal tissues be-

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Development of the Male Perineum

coming septa and fasciae that embrace the urethra on the venter side. In this process participate superficial shaft stroma and dartos stroma, which originally do not form part of the mantle in sensu strictu. 5.2.2 Erectile Structures The development of the erectile structures in the male demonstrates a strong contrast between the great expansion of the glans, corpora cavernosa, and corpus spongiosum and the decrease to near disappearance of the urogenital/urethral labia (Fig. 39). The glans of penis of the sexually indifferent phallic structure persists as a relatively large part of the (non-erected) penis. Its initial cap-like form, ending in a large, thin peripheral extension proximalward, is markedly altered by the formation of the glandopreputial lamella (f.50–90 mm), which forms the dividing line between a distal expanding corona and a thin layer of vascular connective tissue on the inner side of the developing prepuce

Fig. 39 Schematic drawings of the external perineum of male fetuses of 35 mm to 90 mm showing the glans (1) and regressing urethral labia (2) embracing the decreasing urethral orifice (3) ventrally, and also the projection of the patterns of superficial stromal elements that embrace the orifice dorsally. The latter comprise transverse dorsal stroma of the navicular fossa (4), developing scrotal-and-penile raphe (5), superficial stroma of the shaft of the penis (6, shown at the right side of the fetus only), and dartos layers consisting of a deep lamina (7a, shown at the left side of the fetus) passing into a superficial lamina (7b) confined to the scrotum (shown at the right side only). 8, Anal orifice; 9, preanal raphe. Note that the configuration of the stromal elements does not change during the shift of the urethral orifice to the tip of the penis, which argues strongly against the formation of the urethra by fusion of the urogenital labia distalward. Original magnification, 25

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(see Sect. 5.2.6). The glans penis thereby surrounds the tip of the outward growing penis almost completely. Its developing structure reveals the formation of a conspicuous broad zone of connective tissue beneath the glandopreputial lamella and an intricate pattern of arteries and veins. Anastomosing veins widen and develop smooth muscle layers of strikingly varying thickness, culminating in the formation of true cushions in more central veins at the end of pregnancy. This differentiation of glans tissue is distinctly slower in the corona, in which undifferentiated stroma persists until a late developmental stage. The glans does not form a sheath. It is strongly linked to the corpora cavernosa and the corpus spongiosum by extensions of the sheaths of those two structures. In addition, there are also numerous vascular anastomoses between these erectile bodies. The corpora cavernosa become large trunks with a thick double-layered tunica albuginea on the outside and increasingly denser vascular networks centrally. In this tissue veins become the most prominent feature as they form large and complicated anastomosing channels that develop thick smooth muscle coats, with small bundles radiating into the surrounding connective tissue as a characteristic feature (f.270 mm). This vascular system is also supplied by contributions from the dorsal arteries of the penis and becomes a significant contributor to the vascular system of the corpus spongiosum (see below). The corpus spongiosum of the male starts from the pair of ovoid primordia named bulbi spongiosi lateral to the deepest part of the spongy urethra. The further development of the corpus spongiosum is characterized by four major processes: a. Expansion of the two bulbi spongiosi into a single bulbus penis. The primordia expand into a dorsal direction, in particular, where they approach each other in the midline dorsal to the urethra, but remain separated by a thin septum, the deep perineal septum (f. 50 mm). Together they form the bulbus penis which is enveloped by extensions of the internal urethral sphincter proximally, deep dorsal urogenital stroma distally, and by the bulbospongiosus laterally. The cranial part is variably penetrated by branches of the bulbourethral glands. b. Differentiation of the bulbar vascular tissue. This process begins with the appearance of a conspicuous component of loose connective tissue that becomes rich in ground substance and surrounds an abundant vascular network (f. 50 mm). The arterial element becomes especially prominent (f.65– 140 mm) and remains a highly characteristic feature, even after it has become surrounded by venous plexuses forming large, labyrinthine cavities (Fig. 40a, b). c. Elongation of the distal tips of the bulbar parts along a lengthening spongy urethra. These tapering extensions are positioned laterally between the lamina propria and outer sheathes made of deep dorsal urogenital stroma that

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Development of the Male Perineum

Fig. 40a–d Development of the corpus spongiosum in male fetuses of 140 mm t (a), 330 mm t (b, c) and 160 mm (d). In a is illustrated the striking contrast in the corpus between a peripheral bulbar structure (1), characterized by numerous arteries (arrow) and sinusoid veins (arrowhead), and a lamina propria-related component (2), characterized by wide longitudinal veins. Their border is indicated by an asterisk; 3, urethra; 4, sheath of the corpus. In b and c is demonstrated how the proximal corpus consists of typical bulbar tissue with prominent arteries (arrows) and labyrinth-like veins (arrowheads), while the distal corpus (c) shows almost exclusively lamina-propria-related longitudinal venous channels. In d parallel arteries (arrows), developed in the original vascular zone between the corpus spongiosum (1) and corpora cavernosa (5), form arterial anastomoses through the tunica albuginea of the corpora cavernosa (asterisk)

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originates from the deep perineal septum and that embraces the corpus spongiosum distal to the bulbospongiosus. d. Integration of longitudinal venous plexuses of the lamina propria into the distal corpus spongiosum. The bulbar extensions become increasingly thinner distalward and may even disappear at some distance from the urethral orifice. In this part of the corpus, the initial separation between the bulbar tissue and the numerous wide longitudinal veins in the lamina propria becomes less strict, and gradually this lamina-propria-related system becomes predominant, or even completely takes over from the bulbar element (Fig. 40c).

The bulbar parts of the corpus spongiosum remain separated dorsally by the deep septum of the perineum, which forms an obstacle to the development of communicating vessels between left and right. More distally the septum is incomplete and allows many anastomoses. The corpus spongiosum receives an extra arterial supply from longitudinal vascular plexuses that develop between the urethra and corpora cavernosa. Similar to the situation in the female, at first a right and left series of parallel, small, straight vessels (f.65 mm) develop, and these later transform into parallel arteries and veins between the vascular systems of the corpora cavernosa and the bilateral primordia of the corpus spongiosum (f.140 mm). Most of these vessels are lost but, in contrast to the situation in the female, some relatively large arteries remain as branches of the central arteries of the corpora cavernosa. These branches perforate the tunica albuginea and supply large distal parts of the corpus spongiosum (f.160 mm; Fig. 40d). The originally prominent urethral labia flanking the urethral orifice decrease rapidly (f.60–90 mm). The original arteries later become involved in the blood supply to the most distal parts of the corpus spongiosum (f.160 mm). 5.2.3 Fascial Tissues The strong effect of the marked growth of the erectile structures on the construction of the typical male perineum is matched by the strong and fast proliferation of stromal tissues embracing the urethra dorsally. This proliferation in the midperineal region makes the distance between the orifices of the anal canal and the urethra increase rapidly, creates the perineal raphe, septum and major fasciae, and contributes significantly to the formation of the scrotum, shaft of the penis, and prepuce (Figs. 39, 41). These stromal elements are at first difficult to identify in the very dense tissue dorsal to the early urethra. When they become increasingly better defined, it can be established that the superficial dorsal urogenital stroma transforms into the ventral raphe, superficial perineal septum, and trans-

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Development of the Male Perineum

Fig. 41a–g Schematic drawings to demonstrate the formation of the septum and fasciae in male fetuses of 35 mm to 82 mm In a, a lateral projection in an early male perineum shows the ventralcaudalward orientation in the dorsal component (1a), of the deep dorsal urogenital stroma and the change in direction of its ventral part (1b), which embraces the urethra (2) and ends lateral to the corpora cavernosa (3). In b a transverse section demonstrates that ventral component (1b) as it splits into the sheath (1c) of the bulbar parts of the corpus spongiosum (4) and the deep fascia of the penis (1d), which also embraces the corpora cavernosa (3). Also shown is the deep perineal septum (5) and stroma that spreads lateralward, constituting the deep scrotum (6). In c a median section through the midperineal region between the urethral and anal orifices shows the perineal septum to consist of: 1. deep dorsal urogenital stroma (1), with a ventralcaudalward orientation, forming the perineal body and deep perineal septum; 2. more interwoven stroma (7) between adjacent bilateral parts of the external anal sphincter (8) and bulbospongiosus (9); and 3. superficial dorsal urogenital stroma with a ventralcranialward orientation, forming the raphe (10) and superficial perineal septum (11); 4, outline of the corpus spongiosum; 12, transverse superficial dorsal urogenital stroma of the navicular fossa; 13, muscularis propria of the rectum. In d–g the process of approximation is demonstrated in transverse sections through the spongy urethra (d and e) and the midperineal region (f and g). In d and e the spongy urethra (2) shows the transformation of the dorsal urethral groove (asterisk in d) into a ridge (asterisk in e) and the medialward shift of the orificia (arrowheads) of the primordial excretory ducts of the bulbourethral glands. Likewise shifting toward the middle are the bulbi spongiosi (4) and bulbospongiosus (7), thereby transforming the lamina propria and ventral part of the deep urogenital stroma (1) into the thin deep perineal septum (e). In the midperineal region (f and g), the approximation transforms the deep dorsal urogenital stroma into the perineal body (1), to which converge the longitudinal bundles of the muscularis propria of the rectum (13), puborectalis (14), urethral sphincter (15 in g), superficial transverse muscle of the perineum (16 in g), and bulbospongiosus (8), and which stroma passes into the superficial perineal septum (10) and raphe (11). Original magnifications, 13 (b), 15 (a), 25 (g), 32 (c–e) and 38 (f)

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verse dorsal stroma of the navicular fossa, and that the deep dorsal urogenital stroma differentiates into the perineal body, deep perineal septum, and deep fasciae. The superficial stroma of the shaft of the penis forms a special subepidermal layer in the shaft, and the dartos stroma transforms into the dartos fascia. These elements will be described in more detail in the context of the major perineal structures of the male, i.e., perineal septum including perineal body and raphe, penis including prepuce and frenulum, and scrotum. 5.2.4 Perineal Raphe, Septum, Body, and Fasciae The perineal raphe, septum, body, and fasciae are mainly constituted by contributions of the superficial and deep dorsal urogenital stroma. The superficial dorsal urogenital stroma forms a complex which comprises elements forming the ventral perineal raphe, superficial perineal septum, and transverse dorsal stroma of the navicular fossa. The ventral perineal raphe originates from the fibrovascular tissue of the urethral labia just dorsal to the urogenital orifice, where for a short time the cloacal groove existed as a remnant of the original communication between the urogenital and anal compartments of the cloaca. From a short V-shape it lengthens into the narrow median raphe (Fig. 39). The raphe is characterized by dense fibrovascular tissue with a conspicuous parallel and longitudinal orientation (Fig. 42a, b). It may for some time reveal a groove in the midline in which some ps.str.col. epithelium may extend from the dorsal margin of the urethra. It shows the broad str.sq. epithelium that characteristically covers the derivatives of the cloacal labia. This epithelium does not form epidermal appendages later and, as a result, a variably narrow median zone of the raphe is permanently devoid of hair follicles and glands. This long ventral raphe is complemented dorsally by a similar extension, i.e., dorsal perineal raphe, built of stroma derived from the anus-related dorsal parts of the labia. The parallel longitudinal tissue of the raphe gradually bends inward into the underlying superficial part of the perineal septum thus demonstrating a structural unity (Figs. 41c, 42a). The precise origin of this stroma is difficult to trace. From its position and (future) relationship to adjacent stromal components such as the dartos stroma and deep dorsal urogenital stroma, and to blood vessels crossing laterally towards the urogenital labia, it appears that the most likely source is the portion of the superficial dorsal urogenital stroma that develops just dorsal to the transverse dorsal stroma of the navicular fossa, and may derive from stroma related to the labia. The stroma forms an initially broad (f.45–90 mm) but later increasingly thin sheet in the midline of this part of the perineum. The orientation of the fibers within this sheet is parallel and ventralcranialward and appears a continuation of the same in the raphe (Fig. 42a, c). On the side of the urethra

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Development of the Male Perineum

Fig. 42a–d Development of the raphe and perineal septum in male fetuses of 65 mm s (a), 90 mm f (b), 65 mm t (c) and 82 mm (d). In a the raphe (1) and superficial septum (2) demonstrate parallel ventralward- and ventralcranialward-orientated fibers, contrasting with interfering (asterisk) ventralcaudalward-oriented fibers of the deep septum (3). In b the ventralward-oriented fibers of the raphe contrast with transverse fibers of the superficial lamina of the dartos layer (4) in the scrotal halves. In c (left) from the scrotal septum (2), which extends from the dorsal ridge (arrow) of the urethra to the raphe (1), spread the sheath of the corpus spongiosum (5), the deep fascia of the penis (6), the deep connective tissue of the scrotum, (7) and the dartos layer (8). A detail of the septum

Observations

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this septal stroma shows a distinct interference with contrasting ventralcaudalward-oriented deep dorsal urogenital stroma (Figs. 41c, 42a). This original superficial septal element becomes less distinct later, when it develops into a thinning central core flanked by broader zones of homogenous collagen, lateralward-spreading connective tissue, smooth muscle tissue of the dartos fascia, and numerous blood vessels and nerves. These blood vessels and nerves run in a distinct and contrasting ventralcaudalward direction, which had been their original direction toward the urogenital labia and scrotal swellings. All elements together form the definitive (anatomical) septum. This structure of the raphe and superficial septum is far less distinct in the penis than in the scrotum. Near the urethral orifice the septum ends in the transverse dorsal stroma of the navicular fossa. This component is small, reveals a conspicuous transversely oriented structure, and is in direct contact with the dorsal ps.str.col. epithelium of the terminal urethra situated between the labia-derived tissue at the orifice and the markedly longitudinally oriented lamina propria deeper (Figs. 37, 38a, 41c). Although small and temporary, the transverse dorsal stroma is important because it forms the dorsal stromal confinement of the terminal urethra during the whole process of urethral lengthening, moving distalward with the orifice (Fig. 39). The deep dorsal urogenital stroma forms the perineal body, deep perineal septum, and also fascial tissues spreading lateralward and becoming a sheath for the distal part of the corpus spongiosum, deep fascia of the penis, and deep scrotal connective tissue. The perineal body and adjacent deep septum develop from that portion of the deep dorsal urogenital stroma initially positioned dorsal to the urogenital sinus. The original distinction between a dense peripheral zone that extends into the adjacent medial sides of the puborectal and bulbospongiosus and the caudal extremity of the external urethral sphincter muscles, and a less dense central area (Fig. 41f) in which ventral longitudinal bundles of the muscularis propria of the rectum abruptly end, disappears with the regression of the central area (f.45– 80 mm). As a result, all neighboring bilateral structures positioned laterally, i.e., the bulbi spongiosi, bulbourethral glands, and puborectal, internal and external urethral sphincters, bulbospongiosus, transversus perinei superficialis (recognizable in fetuses larger than 90 mm), and deep external anal sphincter muscles, shift to the midline dorsal to the spongy urethra t (right) reveals its central core of ventralcranialward-oriented stroma (arrow) and numerous flanking vessels and nerves (arrowheads). In d a decrease of deep dorsal urogenital stroma (1) has caused approximation of the bilateral puborectalis (9) and bulbospongiosus (10) muscles in the midline, creating the perineal body (asterisk) and septum (between arrowheads). Arrow, raphe

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Development of the Male Perineum

(Fig. 41d–g), coming together with longitudinal bundles of the outer layer of the muscularis propria of the rectum. The dorsal urogenital stroma of this “contracting” area then differentiates into a mixture of connective and smooth muscle tissue (f.160 mm). It becomes the perineal body dorsally, and the deepest part of the perineal septum between the bulbar halves of the corpus spongiosum and of the bulbospongiosus ventrally, with preservation of the original ventralcaudalward-oriented texture. Dorsally it passes into an interwoven stromal tissue differentiating into connective tissue that links the bilateral dorsal parts of the bulbospongiosus to the deep part of the external anal sphincter (Fig. 41c). On the ventral side, the deep septum gives origin to deep dorsal urogenital stroma that embraces the urethra. This septum splits into an inner layer forming a dorsal fibrous sheath for that part of the corpus spongiosum distal to the bulbus, and an outer layer extending lateral to the corpora cavernosa, forming the deep fascia of the penis (Figs. 36a, 41a, b). Both layers consist almost exclusively of connective tissue. A zone of increased density in the midline indicates the position of the deep perineal septum. More superficially the ventral candalward-oriented stroma of the deep septum interferes with the ventralcranialward-oriented tissue of the superficial part of the perineal septum. Lateralward-spreading stroma from this part of the septum forms the finely fibrillar connective tissue of the deep scrotum in which no adipose tissue will develop (Fig. 42c). Briefly summarized, the analysis demonstrates that the septum is basically a thin triangular vertical condensation of stroma between the perineal raphe and the dorsal ridge of the spongy urethra, and between the dorsal edge of the urethral orifice and the muscularis propria of the rectum (Fig. 41c). It has a complicated structure which comprises from the urethra to the surface: a. b. c. d.

A dense zone of lamina propria in the dorsal ridge of the urethra The deep septum including the perineal body The superficial septum and the raphe Interwoven connective tissue between the dorsal parts of the originally bilateral bulbospongiosus, and also between the ventral extensions of the external anal sphincter

5.2.5 Scrotum The scrotum develops by the combination of the two scrotal swellings, originally positioned on both sides of the early penis, with the median stromal elements dorsal to the urethra, which by their strong proliferation make the urethral orifice shift ventralward. This ventralward progression of stromal tissue and orifice is absolute if correlated to a “fixed” structure such as the pubic symphysis (Fig. 43). That strong proliferation on the scrotal side of

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Fig. 43a–d Schematic drawings demonstrating the ventralward shift of the developing penis in male fetuses of 35 mm to 90 mm They demonstrate that, with the strong disproportionate growth of median stromal tissues dorsal to the urethral orifice (dorsal edge indicated by arrow), the orifice shifts ventralward with respect to the more stationary scrotal swellings (1; finely stippled) on both sides, with the corpora cavernosa (2) rotating in relation to the symphysis pubis (3). Original magnification, 15

the developing penis is also associated with a rotation of the penis ventralward, as is illustrated by the changing angle between the axis of the corpora cavernosa and the ventral side of the pubic symphysis from 160 degrees (f.35 mm) to 90 degrees (f.90 mm). As the position of the laterally positioned scrotal swellings does not alter in relation to the symphysis, it is the lengthening penis that actually moves ventralward between these two swellings. As a result, the swellings become almost entirely positioned dorsal to the penis and become united into a single scrotum by the strongly growing stromal elements in the medial parts of the two swellings, i.e., the lengthening and subsequently thinning scrotal raphe and septum with the associated deep stroma of the scrotum and dartos stroma (f.40–50 mm). The dartos stroma, which originally formed the main constituent of both scrotal swellings, keeps its bi-laminate structure. The deep lamina, with its characteristic ventralward orientation, differentiates into relatively large ventralward-oriented and isolated bundles of smooth muscle cells embedded in a finely fibrillar connective tissue rich in blood vessels (f.130 mm). The bundles begin at the ventral extremity of the external anal sphincter, fan out into the scrotal halves, converge toward the shaft of the penis where the pattern becomes predominantly oblique-transverse because the bundles maintain their ventralward direction around a penis which stands almost perpendicular to the scrotum, and end at the free margin of the prepuce (Fig. 39). As found only in the scrotum, this deep lamina passes through complicated acute angles into a denser mass of finer bundles with a predominantly transverse lateralward pattern , more or less directed toward the surface and situated between the epidermal appendages. That mass constitutes the superficial lamina of the dartos fascia.

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Development of the Male Perineum

5.2.6 Penis, Prepuce, Preputial Sac and Frenulum Basic to the formation of the penis is the combined growth of the fibrovascular glans, corpora cavernosa, and corpus spongiosum embracing the early urethra ventrally, and of the stromal tissues that embrace the early urethra dorsally and are situated in the base of the early penis. The growth of the dorsal stromal elements not only makes the urethral orifice shift distalward but also envelops the lengthening urethra and ventral erectile structures in a number of fascial layers. This combined circumferential growth gives the penis its distinct cylindrical shape. The dorsal stromal components extend from the region of the developing scrotum into the shaft of the initially perpendicular penis in an oblique direction. Thus the penis has a deep septum derived from dorsal urogenital stroma. From this septum spread the sheath of the corpus spongiosum and the more outward deep fascia of the penis embracing the corpora cavernosa (Fig. 44a). In the distal part of the penis, the sheath of the corpus spongiosum extends for a short length into the glans before meeting the fascia that extends from the tunica albuginea of the corpora cavernosa and links the three erectile structures to each other. The deep fascia of the penis ends just outside the deepest part of the glandopreputial lamella and related glans stroma. It borders the dartos layer because the shaft lacks the fibrous tissue that develops on both sides of the deep septum of the scrotum. Superficial fascial layers in the shaft also differ from those in the scrotum. The raphe and septum lack the distinct parallel orientation. This different structure in the penis as compared with its scrotal homologue is probably due to the circumstance that they both develop from a less regularly structured original labial stroma near the preceding urogenital orifice. The deeper part of the superficial septum is related to the originally deep dartos lamina, which continues into the prepuce (Figs. 44, 45a). The smooth muscular superficial dartos lamina of the scrotum is absent in the penis where, instead, the superficial shaft stroma develops into a special subepidermal layer of finely fibrillar, transversely oriented connective tissue (Figs. 44a, 45a). The superficial shaft stroma. At the beginning of male differentiation, a specially structured subepidermal stroma surrounds the base of the phallic structure from the urethral labia ventralward, occupying the area between the glans and the labioscrotal swellings, which area will become the shaft of the penis (Fig. 39). Situated directly beneath the epidermis, it differentiates into a broad layer of finely fibrillar connective tissue with a marked transverse circular orientation of its fibers and extending from the penile septum to the dorsum of the penis (Fig. 44a). This stroma lengthens with the growing shaft (f.60–80 mm), envelops those fascial layers that extend from the scrotum into the shaft, and forms the superficial layer of the prepuce.

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Fig. 44a, b Shaft and prepuce of the penis in male fetuses of 82 mm t (a) and 140 mm f (b). In a is demonstrated stromal tissue embracing the urethra (1) on the side of the venter side, namely, superficial shaft stroma (2), sheath of the corpus spongiosum (3), deep fascia of the penis (4), and dartos layer (5), all of which are linked to the raphe (arrow) and penile septum (arrowhead). In b the dartos fascia (5) in the male prepuce (antismooth muscle actin antibody) is situated between a thin inner layer of glans-derived stroma (6) and a broad layer of superficial shaft stroma (2); 4, deep fascia of the penis; 7, glandopreputial lamella; 8, corona of the glans

The early stages of the formation of the prepuce are similar to those described in the female (see Sect. 4.2.6). In the male the prepuce, glandopreputial lamella, and corona of the glans of penis reach the remnants of the urethral labia on the venter side in 90-mm fetuses. They envelop the terminal part of the distalward-growing urethra and its directly related tissues almost completely, but remain separated by the rapidly proliferating stroma proximal to the urethral orifice (Figs. 45a, 46a–c). This stroma, consisting of the original deep penile septum flanked by the thin layers of glans stroma related to these expanding parts of the lamella, becomes a thin median septum when the medial ends of the lamella on both sides of that tissue expand disproportionately (Fig. 45b). Superficial to these expanding medial parts of the lamella remains the associated distalward-progressing prepuce, closely linked to the equally progressing superficial median septum (Fig. 45). It thus appears that those medial parts of the lamella “carry” the overlying parts of the prepuce and the bridging medial tissues distalward beyond the urethral orifice (f.110 mm) (Fig. 46d, e).

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Development of the Male Perineum

Fig. 45a, b Formation of the frenulum in male fetuses of 82 mm t (a) and 155 mm t (b). In a, a penile septum (1), extending between the most distal sheath of the corpus spongiosum (2) and the raphe (3), is locally flanked by glans-derived stroma (arrows) and distal medial parts of the glandopreputial lamella (4), which in b have expanded, thereby demarcating the median stromal elements laterally, and transforming them into a future frenulum; 5, dartos fascia; 6, superficial shaft stroma; 7, urethra; 8, transverse dorsal stroma of the navicular fossa; 9, corona of the glans

The still-solid epithelium of the lamella then covers the glans completely and leaves only a narrow open canal from the urethral orifice toward the opening created by the protruding prepuce. This canal lies on top of a narrow ridge that is in fact the most distal part of the extended raphe on the inner surface of the protruding circumferential prepuce. This raphe lies in line with the thin septum which had formed between the expanding medial parts of the original lamella. Together they persist and, after the preputial sac has formed by luminization of the lamella, constitute the frenulum of the penis. The epithelium of this area then forms a sort of mosaic with a short temporary tongue of ps.str.col. epithelium extending from the orifice on the frenulum, and various types of str.sq. epithelium related to the regressing urethral labia, the glans plate, and glandopreputial lamella. The lamella itself develops an irregular outline by the formation of relatively large whorls (f.80–135 mm). Later, as the result of keratinization within the whorls, small cavities are formed (f.200–230 mm). These cavities become confluent, come into contact with the narrow channel from the urethral orifice, and form the open preputial sac. The lining of the sac, which includes the surface of the

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Fig. 46a–e Schematic drawings to illustrate the development of the glandopreputial lamella and frenulum in frontal views of the area in male fetuses of 75 mm to 110 mm, and in median sections of fetuses of 140 mm and 200 mm. In a–c is seen the expansion (arrows) of the glandopreputial lamella (shaded area) into proximal and medial directions on the venter side in a concerted growth, with the prepuce (1) and corona of the glans (2) (outline of glans indicated by broken lines). This expansion of lamella epithelium proximal to the orifice (3) results in a demarcation of the median stromal components (asterisk), including the penile septum, into a thinning future frenulum, while a further expansion distalward lengthens the frenulum and “carries” the linked prepuce beyond the urethral orifice (directions indicated by arrows in c). This latter phase is also demonstrated in d and e, with the outline of the glandopreputial lamella indicated by a broken line with dots, and the area of demarcation, which will become the lateral surface of the definitive frenulum after luminization of the solid lamella, by double shading; 2, glans; 3, urethral orifice; 4, glans plate; 5, raphe. Original magnification, 15

glans, then consists of a broad cornifying epithelium, with a minor noncornifying component, related to the epithelium of the glans plate. The epithelium of the sac does not form hair follicles or glands as a rule, with but a single tubular glandopreputial gland similar to those regularly occurring in the female (see Sect. 4.2.6) originating from the deep margin of the lamella in a 150-mm fetus as an exception. 5.2.7 Anal Canal The development of the anal canal is similar in the male and female (see Sect. 4.2.8). 5.2.8 Perineal Striated Musculature The development of the ischiocavernosus muscle, transversus perinei superficialis muscle, and external anal sphincter is similar to that of their female counterparts (see Sect. 4.2.9). The construction of the external urethral sphincter differs in that the formation of the prostate interrupts the horseshoe-like pattern of the ventrally embracing muscle, and the marked length-

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Development of the Male Perineum

ening of the membranous urethra is associated with an almost complete encirclement. The bilateral anlage of the bulbospongiosus muscle shifts toward the midline during the general process of approximation. But the two parts remain separated by the deep perineal septum, a structure later to become a median raphe to which the two muscles are intimately linked. 5.2.9 External Perineum The development of the external shape of the male perineum is determined by: (a) the disproportionate and strong growth of stromal tissue embracing the superficial part of the early urethra dorsally, which greatly increases the distance between the anal and urethral orifices (f.35–90 mm), with the formation of a lengthening raphe dorsal to a ventralward migrating and decreasing urethral orifice, and with the penis rotating ventralward between the scrotal swellings; (b) the combination of this dorsally embracing stroma with the likewise disproportionately growing fibrovascular glans, corpora cavernosa, and bulbi spongiosi embracing the urethra ventrally, and transforming the sexually indifferent phallic structure into the cylindrical shaft of the penis (with the prepuce; f.60–80 mm); (c) the approximation of initially separated bilateral perineal structures, manifested externally in the combination of the scrotal swellings into one scrotum and accentuated by a marked increase in ground substance and loosening of central connective tissue (f.50–90 mm). The histogenesis of the surface of the perineum shows that the usual type of skin develops in a peripheral zone only. The central area demonstrates a more specific structure with these major aspects: a. A narrow median zone of cloacal-labia-derived fibrovascular stroma and cornifying str.sq. epithelium, without epidermal appendages, on top of the raphe that reaches from the anal orifice to the free margin of the prepuce b. Narrow flanking zones showing large sebaceous glands often combined with apocrine glands embedded in dense connective tissue c. Lateral areas comprising most of the scrotum and the shaft of the penis, demonstrating few and small appendages surrounded by smooth muscular dartos fascia in the scrotum and by the special, finely fibrillar superficial connective tissue in the shaft and prepuce

Subcutaneous adipose tissue develops only in relation to the usual skin in the periphery of the perineum. Built of fascial tissue, most of the scrotum and the shaft of the penis remain practically without adipose tissue.

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5.3 Discussion This study demonstrates how the male perineum develops by differential growth of epithelial and mesenchymal structures from a postcloacal sexually indifferent basic configuration. It highlights the role of the stromal components, which have until now apparently been underestimated as compared with the epithelial elements. Like the parallel study of the development of the female perineum, this approach alters current ideas on major aspects of sexual differentiation, which in the male perineum are the development: of the urethra; of specialized erectile structures (i.e., glans, corpora cavernosa and corpus spongiosum); of fascial elements i.e., perineal raphe, septum, and fasciae) from which the penis (including the prepuce, preputial sac, and frenulum) is formed; and of the scrotum. The development of the anal canal will not be discussed as it is addressed in the section on the development of the perineum in the female. 5.3.1 Urethra The present observations on the development of the deep urethra, i.e., prostatic and membranous urethra differ from earlier data only in detail. They are in conformity with information on its developing shape (Johnson 1920; Chwalla 1927) and the formation of the prostate (Herzog 1904; Lowsley 1912; Chwalla 1927). Confirming the opinion of Herzog (1904), it is established that its epithelium is pseudostratified columnar and not transitional as is still generally believed. This is a characteristic of cloaca-derived epithelium and is similar to the epithelium that originally lines the female vestibulum, and is also a permanent feature of a narrow zone in the anal canal. The present data on the fate of the mesonephric–paramesonephric complex inside the colliculus are basically in line with ideas of Vilas (1933b), who suggested a complete replacement of regressing paramesonephric epithelium by urogenital-sinus-derived epithelium, and its isolation as the so-called prostatic utricle from the lateralward-shifting ejaculatory ducts. The material available does not endorse the interpretation of Glenister (1962) that the “utricle” is a rudiment of composite paramesonephric and sinus-derived epithelium, but it cannot be excluded either that in a portion of the utriculi, especially those without the formation of prostate glands, metaplastic squamous paramesonephric epithelium is present, possibly as the result of incomplete involution. The present study demonstrates a late stage during which the epithelium has a mixed composition of str.sq. epithelium and glandular rosettes in combination with solid sprouts extending outward. The rosettes are found to be similar to those formed in the urogenital epithelium covering the colliculus when the original ps.str.col. epithelium was

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replaced by str.sq. epithelium, leaving remnants of intra-epithelial acini characteristic of this segment of the urethral lining, and also similar to those occasionally observed in large excretory ducts of prostate glands when squamous metaplasia supervenes. The large sprouts extending outward from the diverticulum appear to be similar to those growing from the urethra itself, forming the glands of the prostate. This fits well into observations in adults in which this “utricle” varies from single diverticulum to an elaborate prostatic gland (Vintemberger 1926; Glenister 1962). The observations support the concept by Dros et al. (1974) and Oelrich (1980) of a urethral muscular coat built of trigonum-related smooth muscle tissue dorsally [named vesical sphincter by Dorschner et al. (2001)], prostate-related smooth muscle tissue laterally, and dorsally, and internal urethral sphincter intimately linked to striated muscle tissue of the external urethral sphincter ventrally (Dros et al. 1974; Oelrich 1980). Other aspects of the muscular coat can be found in the discussion on the development of the female urethra (Sect. 4.3.3). The ejaculatorius muscle, recognized by Dorschner et al. (2001) in the sexually mature male, cannot be identified as an entity during development. The tissue they describe is present, indeed, but is considered here as a lamina propria (which is very similar to the situation with respect to the dilator urethra muscle ventrally; see Sect. 4.3.3). This tissue is at this stage still considered to be lamina propria, notwithstanding some smooth muscle differentiation in fetuses of about 200 mm, because it is a continuation of the lamina propria of the bladder on the cranial side and passes gradually into the more vascular lamina propria of the spongy urethra on the caudal side. These findings, in combination with the derivation of the epithelial elements from the deep urogenital sinus, indicate that the prostate is a perineal structure judged from its developmental history. The early development of the spongy urethra, including the navicular fossa, has dominated the development of the male perineum. It is a general view expressed in textbooks that, in contrast with the prostatic, membranous, and bulbar part of the spongy urethra, most of the spongy urethra is not formed by lengthening of the preexistent urogenital sinus, but by the hollowing of an initially solid urethral plate into an urethral groove, which is then followed by closure of this groove from the amniotic cavity by the fusion of flanking urethral folds from dorsally toward the tip of the penis. It is generally thought that this process is completed by a separate growth of epithelium from the surface of the glans into its substance, where it meets the urethral-plate-derived urethra, and after canalization develops into the navicular fossa and external urethral orifice. This “fusion theory” has a long history dating back into the second half of the nineteenth century. Its prevailing influence appears to have been bolstered by the highly suggestive changes in the developing external appearance of the area (Retterer 1890; Herzog 1904; Felix 1911, Spaulding 1921,;

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Szenes 1925; Wilson 1926). It also offered an embryological base for the explanation of congenital malformations such as hypospadias (Johnson 1930). Doubt about its validity arising from histological embryological research in lower mammals by Fleischmann and coworkers (1907), and later in man by Politzer (1932), were apparently ignored. On the contrary, the theory became thereafter even more firmly established by histological investigations by Glenister (1954, 1956, 1958) who stressed a crucial role for the urethral plate, as had been observed before in the red squirrel by Barnstein and Mossman (1938). Current opinion mirrors the fusion theory and Glenisters ideas in particular. The results of the present comprehensive histological analysis of stromal as well as epithelial perineal components are incompatible with the fusion theory. No indications have been found in favor of the process of fusion itself, such as adhering surfaces of the urethral labia (syn. urethral folds), epithelium in a state of regression, or a pattern in the underlying stromal tissue consistent with fusing labia. It is most unlikely that such phenomena would go unnoticed in the present study taking into account the large number of fetuses from the period that were examined, a consideration that has been indicated to be relevant by previous research. And the more so because the surfaces of the supposedly fusing urethral labia are at the time covered by a very thick str.sq. epithelium which has to disappear completely before stromal tissue from both sides can make contact and cause a permanent closure. If this epithelium is excluded from the closing urethra itself, as it is said to be, this would mean that fusion should also take place where ps.str.col. and str.sq. epithelia meet (Glenister 1954, 1956, 1958), which, in the specimens studied now, is not where the opening is narrowest, but at a deeper level where the walls recede lateralward as the str.sq. epithelium often extends inward over a variable distance. Fusion at this level would leave a considerable mass of labial stromal tissue and related str.sq. epithelium protuberant on the raphe for at least some time. Such features of regression and tissue surplus have not been observed now and have not been reported in previous histologic studies (Van der Broek 1910; Williams 1952; Glenister 1954, 1956 1958; Altemus and Hutchins 1991). The microphotograph which comes nearest to illustrating fusion (Glenister 1954, his Fig. 18) shows in actuality the tangentially cut strip of micropapillary ps.str.col. epithelium that may remain for a considerable length of time at the dorsal rim of the urethral orifice, as is demonstrated in the present study. This epithelium may even extend outward and has no relationship to the exclusion of surface epithelium from the lining of the urethra. This configuration, and the occasional presence of an isolated remnant of ps.str.col. epithelium on the raphe at some distance from the orifice, rather suggest a ventralward “pushing” of the orifice by underlying rapidly proliferating stromal tissue with some epithelium trailing behind. The latter process can also explain the remarkable change in form within a very short pe-

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riod of the originally longitudinal slit-like urethral orifice into a broader diamond-shaped opening. A hint to such a process has been given by Altemus and Hutchins (1991), who stressed the importance of mesenchymal proliferation to roll up the epithelium in causing the closure of the urethral groove—but they too still believed the epithelium to fuse. Such a process of lengthening of the spongy urethra and related ventral part of the midperineal region between the urethral and anal orifices, without fusion, is well in accord with other observations presented here showing that the complicated configuration of epithelial and stromal components of the urethra, and its distal part in particular, remains the same throughout the whole process of the formation of the urethra in fetuses between 35 mm and 140 mm. This concept pertains especially to the terminal part of the urethra, where a subtle succession of epithelial and stromal elements is shown on the venter side of the penis just dorsal to the orifice. This succession comprises, from deep to superficial: the strictly longitudinally oriented tissue of the (deeper) lamina propria, with the original dorsal urogenital groove transforming into a dorsal urethral ridge; a small band of dense, transversely oriented superficial urogenital stroma that embraces a conspicuously widening part of the most distal urethra lined by a distinct micropapillary ps.str.col. epithelium; and, finally, fibrovascular tissue diverging into the urethral labia forming the raphe. This short segment of intricate architecture “follows” the ventralward-“pushed” urethral orifice. Such a process appears incompatible with a lengthening of the urethra through closure of the urethral groove by fusing urethral labia (syn. urethral folds). In that situation the progressing urethral orifice would become separated from the succession described above by a lengthening stretch of uniform fibrovascular, labia-derived tissue flanked by the related characteristic superficial stromal tissue of the shaft of the penis over the full length of the raphe. This is clearly not the case (see Fig. 39). The development of the navicular fossa is still a major subject of discussion. The prevalent theory in textbooks of embryology (Arey 1965; Hamilton and Mossman 1972; Sadler 1995; Larsen 1997; Moore and Persaud 1998) and related clinical literature (Stephens et al. 1996) is that the segment and its orifice derive from an isolated ingrowth of solid ectodermal surface epithelium at the tip of the penis. This solid outgrowth later canalizes and fuses with the proximal urethra. That part of the urethra had already formed by canalization of the entodermal “urethral plate” and subsequent fusion of the flanking “urethral folds” ( Hart 1908; Wood Jones 1910; Glenister 1954; Sommers and Stephens 1980). As an alternative mechanism, it has recently been suggested that the solid ingrowth was originally continuous with the urethral plate, but became temporarily isolated from this plate before contacting the definitive urethra (Van der Werff et al. 2000). However, there are also conflicting theories arguing that the distal urethra derives from the most distal part of the entodermal “urethral plate,” either by canalization of its solid dis-

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tal end. thus providing the urethra with an external orifice at the tip of the penis (Nagel 1892; Van de Broek 1910; Williams 1952), or by the splitting of that part of the plate, which merely enlarges the preexistent terminal urethra and orifice (Herzog 1904; Kurzrock et al. 1999; Baskin 2000; Penington and Hutson 2001). The present investigation demonstrates that the original urogenital orifice remains patent during the whole process of the formation of the urethra and is therefore also the definitive urethral opening, as has been stated before (Herzog 1904; Kurzrock and Baskin 1999; Baskin 2000). This observation confirms the existence of a solid plate of str.sq. epithelium and demonstrates that this glans plate is a comb-like extension in the roof of the fossa and that its luminization only causes a widening, albeit a very considerable one, thereby advancing the original urethral orifice to the tip of the glans. In the section on sexually indifferent perineum, it is shown that this plate appears as part of the cloacal membrane in embryos as small as 9 mm; it has been part of the urogenital sinus as precursor of the urethra from the beginning of its formation. This precludes its development as an isolated solid ingrowth from the surface epithelium of the glans. It was also demonstrated that the epithelium of the glans plate differs from both the surface epithelium of the glans and the epithelium of the urogenital plate. In its differentiation this epithelium comes, however, closest in character to the ectodermal epithelium at the surface of the glans and may well have been derived from the superficial ectodermal component of the cloacal membrane. The recent suggestion by Kurzrock and Baskin (1999) and Baskin (2000) that the plate is the result of differentiation of the entodermal “urethral plate” (“urogenital plate” in the present article) into a str.sq. epithelium on the basis of immunostaining cannot be endorsed. Epithelium inside the navicular fossa does show luminal cells with the immunoprofile of entodermal urothelium on top of a basal layer revealing the immunoprofile of str.sq. epithelium but, as has been shown, it is situated in a part of the fossa adjacent to the glans plate. This combination of epithelia is a phenomenon also occurring in the anal canal and in the vestibulum of the female, and has no direct relevance to the formation of the glans plate. 5.3.2 Erectile Structures Information about the development of the erectile tissues is scarce and inconsistent. It provides mainly data about the time of origin of the erectile tissues in sensu strictu, and of their vascularization (Herzog 1904; Lichtenberg 1906; Felix 1911; Johnson 1920). The present analysis stresses the derivation of the various erectile structures from a fibrovascular zone. This explains the presence later of numerous vascular anastomoses between these structures, which includes promi-

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nent arterial connections between the corpora cavernosa and the corpus spongiosum as a most striking phenomenon. The study also demonstrates that each erectile structure develops its own characteristic histologic picture, the histogenesis of which is more subtle than suggested by the generally employed description, to wit, “differentiation from dense mesenchyme.” The subtlety is most distinctly exemplified in the present investigation by the complicated formation of the corpus spongiosum. 5.3.3 Perineal Raphe, Septum, Body, and Fasciae No information was available from the literature about the differentiation of stromal tissues of the penis complementary to the erectile structures. The present study demonstrates the special orientation of the fibers and their arrangement into the perineal raphe and septum and into the various fasciae. The analysis presented here demonstrates that the perineal raphe, perineal septum and body, and fasciae form a composite histological structure based on the diversity in tissues involved in its development. Information about this development is scarce and pertains to the raphe only. The perineal raphe is generally considered to mark the line of fusion between the so-called urethral folds (Glenister 1954; Hamilton and Mossman 1972), or of a combination of urethral folds and scrotal swellings (Szenes 1925; Wartenberg 1993), or between urethral folds for the penile raphe and between scrotal swellings for the scrotal raphe (Arey 1965; Stephens et al. 1996; Moore and Persaud 1998a). However, Fleischman (1907) and Politzer (1932) rejected the idea of fusion as a developmental mechanism, with Politzer suggesting that the raphe is formed by embryonic tissue filling the temporary cloacal groove that had remained from the regressing middle part of the cloaca between the urogenital and anal compartments. The present investigation indicates that the raphe cannot possibly mark the line of fusion between the urethral labia (syn. urethral folds) because such fusion does not take place, as has been discussed above. Other arguments against its formation by the fusing of “urethral folds” are: (a) the existence of a dorsal part of the raphe just ventral to the anal orifice, which region is never implicated in any fusion; (b) the distinct longitudinal orientation in the texture of most of the raphe, which structure does not fit into a pattern to be expected from supposedly fused urethral labia built of a much more irregularly structured fibrovascular tissue; and (c) the structural unity of the raphe and the superficial part of the septum, which part of the septum shows an intimate relationship to the deep septum and the dartos fascia laterally. The subtle groove often observed in the midline of the ventral part of the raphe, which has also been illustrated before (Spaulding 1921; Szenes 1925;

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Wartenberg 1993), is not the result of fusion but due to elongation of a split originally present between the diverging dorsal parts of the urethral labia. The formation of the dorsal and ventral segments of the raphe is found to be directly related to the great lengthening of the midperineal region between the anal and urethral orifices. The current investigation indicates that both develop from the same type of tissue, namely, the mesenchyme that originally formed the cloacal labia. After the division of the cloaca, this fibrovascular tissue becomes divided into components, related to the urogenital and the anal orifices, that meet where the originally connecting cloacal groove regressed. It is now shown that the component ventral to the anal orifice elongates into the dorsal raphe, while the component dorsal to the urogenital orifice becomes involved in the markedly proliferating superficial dorsal urogenital stroma and forms the ventral, or scrotal and penile, raphe. The derivation of these components from the labia is also illustrated by permanent histological characteristics, such as high vascularity and broad, markedly cornifying str.sq. epithelium, which in the midline of the raphe does not form appendages, in contrast to the strikingly large sebaceous glands in flanking zones. This histological pattern is similar to that developing around the anal orifice (both sexes) and vestibular opening in the female. The absence of a dorsal raphe in the female is due to a marked lateralward enlargement of the midperineal region between the orifices, which may later broaden the structure homologous to the dorsal raphe of the male into a convex, flat, or concave hairless area in the traditional “female perineum” (Stephens 1968). The structure in the female which is homologue to the ventral raphe of the male remains very short since no strong proliferation of superficial dorsal urogenital stroma takes place. It forms no more than the dorsal frenulum (fourchette) of the vestibular opening. It is surprising that the dual origin of the raphe has not been recognized before, although the two parts are well illustrated in works on the development of the external genitalia (Spaulding 1921; Szenes 1925; Glenister 1956; Wartenburg 1993). The perineal septum including the perineal body proves to be a complicated structure derived from a number of primordial stromal tissues. It separates and at the same time unifies the structures of the right and left halves of the perineum. This strong fixation to the median septum is in the first place due to the weak demarcation of the median primordial stromal components, which have extended into all adjacent developing structures, including striated muscles such as the puborectalis and bulbospongiosus, the most caudal parts of the urethral sphincter musculature, external longitudinal bundles of the muscularis propria of the rectum, the bulbourethral glands, and the bulbar parts of the corpus spongiosum. This aspect is accentuated by the regression of the central area of the deep dorsal urogenital stroma and further thinning of the septal tissue, which causes approximation of the initially still widely separated bilateral structures, and results in a particularly strong coherence of the two sides in a rather rigid construction.

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The process forms an interesting contrast with the events in the female perineum, which develops in an opposite direction, with the central area of deep dorsal urogenital stroma expanding into a broad fibromuscular mass widely separating the right and left perineal structures in an equally strong but much more flexible construction. No data are available about the development of the dartos fascia. The present analysis of its development reveals a much more interesting basic structure than is suggested by the traditional description of the tissue as a thin layer of smooth muscle in the scrotum and penis. Attention is here drawn to its likely origin in the bilateral dorsolateral perineal complex, derived from the dermatomes of the same somites that provide the tissue for the striated muscles of the perineum. Focus is also on its double-layered construction. This construction is shown to be the result of an early division into two components, superficial and deep. The latter participates in the strong proliferation of the stroma dorsal to the urethral orifice, thereby forming a continuous layer of parallel bundles of smooth muscle cells extending from the external anal sphincter to the free margin of the prepuce. Meanwhile, the superficial component keeps its distinct structure and remains restricted to the skin of the scrotum. Together, the two components provide the dartos fascia of the scrotum with its remarkably intricate bilaminate histologic structure, as also recognized in the adult (Holstein et al. 1974). 5.3.4 Penis, Prepuce, Preputial Sac, and Frenulum The penis is generally considered to develop by elongation of the so-called genital tubercle (Arey 1965; Hamilton and Mossman 1972; Sadler 1995; Larsen 1997; Moore and Persaud 1998). As has already been demonstrated in the section on the sexually indifferent period, the “genital tubercle” is poorly defined as either the whole area between the umbilical cord and the tail (Arey 1965), or as the most prominent part of this area, including the cloacal labia and superficial urogenital sinus (Wartenberg 1993), or most often, as only the tip of the prominence, which is in part the future glans (Hamilton and Mossman 1972; Sadler 1995; Larsen 1997; Moore and Persaud 1998). The statement that the phallus and, later, the penis is the result of elongation of this tubercle appears a simplification in the light of the present observations, which show the participation of many different tissues in forming a most intricate structure. The present work demonstrates that the developmental process is in essence a combination of regression of the urethral labia and strong growth of ventrally embracing fibrovascular and dorsally embracing fascial tissues. This combined strong growth around the lengthening urethra makes the narrowing urethral orifice move distalward and creates the cylindrical penis. The (obliquely) transverse orientation of the fibers in

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the penis is a reminder of this process in an initially perpendicular organ, and appears to be quite appropriate for the future function of the organ. After long-lasting disputes about the development of the prepuce, preputial sac, and frenulum (Hunter 1935; Glenister 1956), those structures are nowadays generally thought to be formed by a combination of preputial fold formation, i.e., overgrowth of tissue originating from just proximal to the glans, and growth of the glandopreputial lamella as anlage for the preputial sac. According to Glenister (1956) the process ends after the most distal parts of the urethral labia (syn. urethral folds) had fused to form the frenulum. The present observations do not support this theory. There is no indication that the prepuce lengthens by inward growth of the glandopreputial lamella, because throughout its formation, the position of its deepest margin does not alter in relation to underlying permanent structures, such as the large nerves and blood vessels supplying the glans, as had been observed before (Johnson 1920). Successive developmental stages demonstrate instead a combined and simultaneous growth of the prepuce, lamella, and corona of the glans “outward.” The growth of the prepuce is the result of proliferation of stromal tissues proximal to the glans, i.e., dartos stroma, superficial shaft stroma and, as a minor component, peripheral glans stroma directly extending from the corona. The lamella remains solid when the prepuce gradually extends over previously uncovered glans surface, which suggest a process of rearrangement within the epithelium and precludes a flowing over, as has been proposed (Hart 1908; Hunter 1935). The present investigation also shows that there is no role for fusion of the urogenital labia in either the development of the prepuce or the frenulum (Hunter 1935; Williams 1952; Glenister 1956), or for fusion of “preputial skin folds” in the formation of the frenulum (Altemus and Hutchins 1991). This idea of fusion was the logical consequence of the theory that the spongy urethra is formed by the distalward fusion of “urethral folds,” which idea has been rejected, above. The formation of the prepuce and frenulum on the venter side is now shown to be based on synchronous distalward growth of the median raphe and septum, combined with the closely linked medial parts of the prepuce that are “carried” beyond the urethral orifice by the disproportionate growth of the medial ends of the glandopreputial lamella, which process at the same time isolates most of the frenulum. These results indicate that the defective development of prepuce and frenulum, as observed in hypospadias, is not the result of incomplete fusion of “urethral folds” (Hunter 1935; Glenister 1956), but is part of the same underdevelopment of the stromal tissue dorsal to the urethral orifice that probably causes hypospadias itself.

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5.3.5 Scrotum There is a general understanding that the scrotum develops by a merging or fusing of the scrotal swellings at the base of the penis after they have moved dorsalward from their original position on both sides of the phallic structure. The scrotal septum is thought to be formed where they meet (Arey 1965; Hamilton and Mossman 1972; Wartenberg 1993; Sadler 1995; Larsen 1997; Ammini et al. 1997; Moore and Persaud 1998; Van der Werff et al. 2000). It now appears, however, that the alterations in the external appearance of the area on which the idea of dorsalward migrating is based are deceiving. Reference of the position of these swellings to a fixed point, such as the pubic symphysis, demonstrates that they retain their position while the shaft of the penis moves ventralward (and at the same time rotates from perpendicular to more parallel to the surface of the perineum). This process is explained by the marked proliferation of the stromal elements dorsal to the superficial part of the early urethra, with the midline elements constituting the scrotal septum. Further growth of all components involved, in association with a thinning of the septum, explains the formation of a single scrotum. It demonstrates that the single scrotum is formed neither by a process of fusion, nor by a merge of the two scrotal swellings, but by apposition of the growing scrotal tissue to the rapidly proliferating stromal tissues in the midline dorsal to the distal urethra, to which tissues it had been linked from the beginning. That median tissue becomes the scrotal septum, which therefore does not derive from fusing or merging scrotal swellings. The circumstance that the medial two-thirds of the scrotum on both sides of the septum are formed by primordial fascial tissue, i.e., dartos stroma and deep dorsal urogenital stroma, and not from the usual type of subcutis observed in the lateral one-third, is the most likely explanation for the fact that no adipose tissue develops in most of the scrotum. This supposition is supported by the different situation found in the basically homologous labia majora of the female, in which a poor development of the dartos fascia and ventral part of the deep dorsal urogenital stroma is associated with the introduction of an abundance of adipose tissue from the surroundings.

6 Summary Sexually Indifferent Perineum. The earliest indication of the development of the human perineum is the appearance of the cloacal eminence. This structure is formed by the increase of mantle mesenchyme around a cloaca originating from the blind-ending part of the hindgut after the mesonephric ducts have come into contact with it. The mantle mesenchyme protrudes

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along the cloacal membrane as the cloacal labia and, by a markedly disproportionate growth ventrally, combines with a transformation of the related part of the cloacal membrane to form a plate, leaving the dorsal part thin. In its growth, this mesenchyme also forces the cloaca to bend into an increasingly more acute U form, with a relatively large ventral urogenital compartment into which issue the allantois and mesonephric ducts, and a shorter gut-related anal compartment that becomes tubular after the early regression of the blind-ending tailgut. The two compartments are connected by a diminishing communication directly beneath the cloacal membrane. The urogenital compartment widens lateralward into “cornua” into which issue the mesonephric ducts, with newly formed ureteric diverticula growing into ureters. Incorporation of the terminal segments of these ducts into cloacal cornua, resulting from regression of their mesonephric epithelium and replacement by cloacal epithelium from the cornua, causes the mesonephric ducts and ureters to acquire separate openings into the cloaca near the entrance of the allantois. The anal compartment hardly changes. By the end of the cloacal period the ventral cloacal eminence has grown into a phallic structure that accentuates the U form of the essentially tubular cloaca. The enlargement then brings about the appearance of a urogenital plate, a specific glans plate, a further narrowing of the communication between the compartments, and a further thinning of the dorsal part of the cloacal membrane. Subsequent disintegration of that dorsal part creates a single cloacal orifice for the urogenital and anal compartments and their communication. The cloaca is thereby turned into a urogenital sinus and an anal canal separated by a bar-like midperineal region, with the communication remaining as an open and median cloacal groove. Subsequent regression of this groove results in the definitive separation between urogenital and anorectal systems. Simultaneously, vague differences within the mantle mesenchyme indicate early structural differentiation. The mesenchyme is thereby complemented on both sides by a bilateral pair of dorsolateral perineal complexes. These are derived from sacral somites and extend ventralward along the cloaca, medial to the anlage of the puborectalis. They are innervated by sacral spinal nerves and supplied by internal pudendal blood vessels. In addition to allantois-related vessels, this pudendal blood vascular system also comes to supply the ventral cloacal structures, while the dorsal cloaca and mantle are vascularized by the median sacral system. Autonomous nerve plexuses which had appeared dorsolateral to the anal compartment during the early cloacal period extend ventralward to the urogenital compartment. Early during the postcloacal period, the orifices of the mesonephric ducts and ureters part. The orifices of the ducts hold their position inside the deep segment of the urogenital sinus, while those of the ureters shift into the domain of the allantois, which has a histology quite different from that of the sinus, and which becomes the urinary bladder. The parting is the result of

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Summary

growth of the area between the orifices, which simultaneously exchanges some of its cloaca-related features for those of the bladder to form the structurally specific trigone of the bladder. Shortly afterwards the M
llerian tubercle develops in the dense mesenchyme at the dorsal side of the deep urogenital sinus around the orifices of the mesonephric ducts. This tubercles growth includes an increase in stromal tissue and the invasion of the paramesonephric ducts, guided by the epithelium of the mesonephric ducts. Fusing into a single median structure, the paramesonephric ducts remain separated from the epithelium of the urogenital sinus, but retain their intimate contact with the mesonephric epithelium distally, and thus constitute the mesonephric-paramesonephric complex. Cranially appears the primordial vesical sphincter. The mantle mesenchyme on the ventral side of this deep urogenital sinus differentiates into the smooth muscle tissue of the internal urethral sphincter, which is in close contact with the still-undifferentiated, originally bilateral primordia of the external urethral sphincter. The much wider trumpet-like superficial part of the urogenital sinus develops a characteristic pattern of grooves and ridges. The urogenital plate splits, widening the urethral orifice but retaining its solid character in a median extension on the ventral wall of the sinus. The latter ends in the histologically different solid glans plate, which is covered by a conspicuous epithelial tag. From lateral grooves sprout primordia of the bulbourethral/ greater vestibular glands. In the mantle mesenchyme of this superficial part appears a distinctive configuration, with: (a) primordial erectile structures that embrace this part of the sinus ventrally and distinguish themselves as glans, corpora cavernosa, bulbi spongiosi/vestibulares, and urogenital labia; and (b) primordial fascial elements that embrace the sinus dorsally, can be subdivided into deep and superficial dorsal urogenital stroma, and become associated with the specific superficial shaft stroma and dartos stroma. The anal canal is lengthened outward by a deepening anal depression that is formed by flattening dorsal derivatives of the cloacal labia and underlying bilateral primordia of the external anal sphincter laterally and dorsally, and by the expanding midperineal region between the urogenital and anal orifices ventrally. The deep cloaca-derived anal canal becomes for the larger part surrounded by the descending muscularis propria of the rectum. The pelvic autonomic nerve plexuses reach the area of the deep urogenital sinus and adjacent bladder. The originally predominating median sacral blood vascular system is taken over by the internal pudendal system. From the dorsolateral perineal complexes, which extend ventralward to the anlage for the corpora cavernosa, emerge the bilateral series of primordia of the external anal sphincter, bulbospongiosus, ischiocavernosus, and external urethral sphincter from the deeper components, and the increasingly more prominent labioscrotal swellings from the superficial components.

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Female Urogenital System. In female fetuses the sexually indifferent configuration persists for a considerable period of time, until the transformation of the mesonephric-paramesonephric complex into a vaginal anlage, and the descent of that anlage towards the surface of the perineum, transforms the urogenital sinus into a urethra and a vestibulum vaginae. The vaginal anlage develops from the fused paramesonephric ducts, which now contact epithelium of the urogenital sinus, and replace regressing mesonephric epithelium inside the orifices of the mesonephric ducts. When the preexisting column of mesenchyme between these two intrusions from the sinus has disappeared, a single vaginal opening is established. The originally columnar paramesonephric epithelium thickens into a stratified epithelium that expands into lateral “wings,” narrows the lumen to almost obliteration, becomes glycogenated, and dissociates centrally to form the vagina. From stroma of the tuberculum Mlleri at the vaginal orifice develops the hymen. The simultaneous descent of the vaginal orifice and adjacent part of the anlage towards the surface not only lengthens the supravaginal part of the deep segment of the urogenital sinus (its dorsal wall in particular) and widens the infravaginal part, but also causes rearrangement of segments of the original wall of the sinus. During this process the urethra is formed by the recombination of the supravaginal part of the dorsal wall of the deep segment of the urogenital sinus and the whole of its ventral wall. The vestibulum is created in this process from the infravaginal part of the dorsal wall of the deep segment of the urogenital sinus in combination with the latters entire superficial segment. In the urethra the rearrangement has little effect on its lining of ps.str.col. epithelium, which is preserved, with some squamous metaplasia later supervening in a zone adjacent to the vestibule. Glands homologous to the prostate glands in the male become large peri-urethral glands and/or involute. However, as a consequence of this rearrangement, the internal and external urethral sphincters in the ventral wall are combined with a trigone-related vesical sphincter in the dorsal wall over the full length of the urethra, while the circumferential caudal part of the internal and external urethral sphincters at first embrace the deep vestibulum, and later the most superficial part of the descending vagina, as well. In the deep dorsal vestibulum, the rearrangement remains visible in the form of an area with a urethra-type epithelium. This differs from two other types of epithelium that gradually replace the original pseudostratified columnar epithelium of the vestibulum. From lateral grooves greater vestibular glands extend right and left. Erectile and fascial tissues around the superficial segment of the urogenital sinus, which gradually transforms into the vestibulum, retain the basic configuration of the sexually indifferent period to a considerable extent. Differential growth, which is relatively inhibited for the glans clitoridis, the caudalward-bending corpora cavernosa, and the ventralward-extending vestibu-

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Summary

lar bulbs, result in a flattening of the initially still-prominent phallic structure, whereas the proportional or slightly increased growth of the urogenital labia into the labia minora is associated with a permanently large vestibular orifice. These erectile structures develop complicated vascular systems that are characteristic of each of them. Anastomosing plexuses between them form a highly vascular area in the ventrolateral wall of the vestibulum. Within the group of differentiating fascial tissues, the deep dorsal urogenital stroma shows, in its component dorsal to the vestibulum, a growing distinction between a peripheral component, which due to its weak delimitating powers extends into adjacent structures such as striated perineal muscles, and a central component that expands and thereby shifts adjacent bilateral structures widely apart. The dorsal urogenital stroma differentiates into predominantly smooth muscle tissue dorsally where it forms a major component of the midperineal region, including a dorsal muscular layer of the vestibulum, and is pervaded by longitudinal bundles of the muscularis of the rectum, resulting in a strong bond with the anorectum, and ventral connective tissue that extends as an ill-defined fascia on each side of the vestibulum superficial to the vestibular bulbs and related bulbospongiosus muscles, to end in the area lateral to the corpora cavernosa. Superficial dorsal urogenital stroma forms the frenulum of the labia minora. An underlying deep lamina of dartos stroma fans out into the labia majora, where it combines with a slightly differently structured superficial lamina, to form a bi-laminate dartos layer. Although the dartos stroma initially constitutes most of the labial swellings that become labia majora, their contribution becomes relatively smaller when, during the second half of gestation, adipose tissue invades from neighboring regions. The adipose element increase the volume of the labia considerably at the cost of the dartos tissue, which differentiates relatively late in comparison with its male counterpart into a relatively thin smooth muscular dartos fascia. The superficial stroma of the shaft becomes a superficial layer of special connective tissue in the sulcus between the labia minora and majora and forms the major constituent of the hood of the clitoris, including the prepuce. The prepuce develops in conjunction with the glandopreputial lamella and the corona of the glans clitoridis by outward growth. Most of the superficial midperineal region between the vestibular and anal orifices is formed by labia-derived stroma and stratified squamous epithelium extending from the anal opening to the frenulum of the clitoris. This combination of stroma and epithelium forms a median mucosal strip without epidermal appendages, the central part of a zonal composition of the surface of the perineum. The originally bilateral configuration of primordial striated muscles of the perineum persists for the ischiocavernosus, related to the crura of the clitoris, and the bulbospongiosus, related to the bulbi vestibulares, with the bulbospongiosus muscles interconnected by a narrow band also linked to the

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deep part of the external anal sphincter, which becomes truly circumferential surrounding the anal canal. The muscles of the external urethral sphincter become almost circumferential in only its caudal part, i.e., the urethrovaginal sphincter. The transversus perinei superficialis develops from muscle fibers extending lateralward from where bulbospongiosus and external anal sphincter meet. Male Urogenital System. Sexual differentiation of the male perineum manifests itself at the very start of fetal development. It is characterized by a pronounced elongation of the urogenital sinus and its mantle, during which process its preexistent architecture is in essence preserved. For the deep segment of the urogenital sinus this means the preservation of a high ps.str.col. epithelium, forming small intra-epithelial glands and large tubular glands of the prostate later. In the ventral wall the internal urethral sphincter differentiates into bundles of smooth muscle cells that extend dorsalward, together with the later-differentiating striated muscle fibers of the external urethral sphincter. These fibers are complemented cranially by the trigone-related vesical sphincter and by specific prostate-related muscle tissue differentiating later. This part of the urogenital sinus forms the prostatic urethra. In the prostatic urethra the Mllerian tubercle transforms into the prominent colliculus seminalis, with the mesonephric ducts separating from the mesonephric-paramesonephric complex to become the ejaculatory ducts. In the paramesonephric component of the complex, paramesonephric epithelium is replaced by urethra-derived epithelium capable of forming prostate glands; this structure persists as the prostatic utricle. Caudally, the combined internal and external urethral sphincters surround the urethra almost completely, establishing the membranous urethra. The elongation of the male urethra is most marked in the transformation of the short trumpet-like superficial urogenital sinus into the long tubular spongy urethra. This segment shows a considerable and disproportionate growth of the future erectile structures that embrace the early urethra ventrally. An even greater increase occurs in the future fascial tissues that embrace the urethra dorsally. This combined circumferential growth makes the narrowing urethral orifice shift ventralward and positions it at the tip of a lengthening penis. It also causes the penis itself to shift ventralward between the scrotal swellings, with the dorsal stroma providing the median tissues that link the two swellings into a single scrotum dorsal to the penis. During the elongation of the superficial urogenital sinus into the spongy urethra, the basic configuration of its epithelial zones, grooves, and ridges (with lacunae Morgagni, urethral, and bulbourethral glands) though becoming more protracted, remains essentially unaltered. There are two exceptions: (a) a dorsal groove transforms into a dorsal ridge as the result of a more general process of approximation; and (b) the terminal urethra and its orifice decrease in size on the venter side in correlation with the diminish-

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ment of the urethral labia, while this part of the urethra strikingly increases in size on the dorsum side, where the glans plate acquires a comb-like shape in correlation with the marked growth of the glans. It is the luminization of this glans plate at the end of gestation that gives this navicular fossa its considerable volume, thrusting the original external orifice to the tip of the glans. The basic pattern of the related mesenchyme-derived structures is also preserved in essence, although their configuration seems to undergo drastic changes. With respect to the future erectile tissues, the changes are the result of differential growth with the glans, and its corona in particular, greatly increasing in size, the corpora cavernosa lengthening, and the corpus spongiosum enlarging greatly. As to the latter enlargement, the originally bilateral bulbi spongiosi expand dorsalward and meet behind the urethra to form the bulbus penis proximally and combine with an extensive vascular component of the lamina propria to form the rest of the corpus. In contrast, the urethral labia decrease in size and disappear as such. Each of these future erectile structures develops its own characteristic morphology but remains closely linked to the others by an abundance of anastomosing vessels. With respect to the developing fascial tissues, the deep dorsal urogenital stroma greatly lengthens both in its median deepest component dorsal to the urethra, and in the lateral components that embrace ventral elements. The dorsal component (which is intimately linked to the urethral sphincters, the muscularis propria of the anorectum, the bilateral striated perineal muscles, the bulbourethral glands, and the bulbi spongiosi) decreases in width and thereby causes approximation of these structures to the midline, forming the smooth muscular perineal body and deep perineal septum. The septum divides the bulbus of the penis and continues ventralward as the fibrous deep perineal septum of the scrotum and penis, from which spread the embracing fascial sheath of the distal half of the corpus spongiosum, deep fascia of the penis, connective tissue of the deep scrotum, and (toward the surface) into connective tissue linking the originally bilateral halves of the bulbospongiosus and external anal sphincter. From the superficial fascial tissues develop the temporary transverse dorsal stroma of the navicular fossa, which confines the dorsal dimension of the distalward-shifting external urethral orifice. Labia-derived stroma lengthens into the ventral raphe and probably into the related superficial perineal septum. This superficial septum is connected in the scrotum to the deep lamina of the dartos stroma, which fans out into the scrotal halves. The dartos stroma is complemented there by a differently structured superficial lamina in constituting the bi-laminate dartos fascia of the scrotum, and continues into the penis. In the shaft of the penis this septum is also linked to the superficial shaft stroma, which differentiates into a subepidermal layer of special connective tissue and, with the dartos fascia, forms the major constituents of the prepuce.

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The development of the prepuce is part of a concerted outward growth of stroma of the shaft, glandopreputial lamella, and corona of the glans. The process begins on the dorsum of the penis and gradually extends toward the venter side. Disproportionate expansion of the medial ends of the lamella here demarcates the deep penile septum proximal to the urethral orifice as a future frenulum, and “carries” the superficial septum and related tissue, including the medial parts of the prepuce, over the orifice, thereby extending the frenulum and prepuce beyond the tip of the glans. Luminization of the glandopreputial lamella makes it the preputial sac. From the originally bilateral anlage for the striated muscles, remains the ischiocavernosus muscles, the only overtly bilateral pair of muscles of the perineum. As for the bulbospongiosus, the originally bilateral character becomes obscured by the process of approximation dorsal to the urethra, during which the muscles and related bulbi spongiosi shift to the midline but remain separated by the perineal septum. The external urethral sphincter differentiates relatively late, is intimately linked to the internal urethral sphincter at their interphase, acquires the shape of a horseshoe in the urethra prostatica, and becomes almost circumferential in the urethra membranacea. The transversus perinei superficialis develops from muscle fibers extending lateralward from where bulbospongiosus and external anal sphincter meet. Anal Canal During Female and Male Differentiation. In the anal canal the difference between the cloaca-derived deep segment and labia-related superficial segment becomes a permanent feature. In the deep segment the ps.str.col. epithelium remains, gives rise to anal glands, and forms the anal sinuses and columns in association with the dense longitudinal stroma of the lamina propria. The superficial segment keeps its broad stratified squamous epithelium and dense subepithelial connective tissue, with abundant blood vessels derived from the dorsal parts of the cloacal labia. This segment remains relatively narrow when the anal canal shortens, and its deeper segment widens, forming the ampulla in combination with the adjacent part of the rectum. The precise border between the two anal segments remains, however, elusive because of the appearance of an intermediate composite epithelium in their borderland. The anal canal becomes, over its full length, surrounded by the descending muscularis propria of the rectum, which inserts itself between the mucosa and the upward-growing external anal sphincter and expands distally into the internal anal sphincter. The original two parts of the external anal sphincter fuse early into a complete sphincter. The original relationship to the urogenital derivatives of the cloaca remains recognizable by similarity in the zonal configuration of its epithelium and underlying stromal tissue, in the extension of labia-derived tissue components ventralward forming a strip of mucosa in the midperineal region of

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the female and a dorsal raphe in the male, and in bundles of the external longitudinal layer of the muscularis propria of the rectum intertwining with the deep dorsal urogenital stroma of the perineal body.

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Subject Index

allantois 5, 17, 29, 33 – vessels 13 anal – canal 16, 25, 40, 79, 107 – – deep segment 25, 41, 64 – – orifice 11, 26 – – superficial segment 26, 41, 65 – column 28 – compartment 7 – gland 65 artery – inferior rectal 29 – internal pudendal 29 – median sacral 28, 42 – transverse artery of the perineum 29, 43

– labium 12 – membrane 5, 9 – orifice 11 – sphincter 42 corpus – cavernosum 23, 58, 76, 95 – spongiosum 94, 101 – – sheath 104 dartos – fascia 78, 103, 116 – lamina 104 – stroma 117, 118 dorsolateral perineal complex ejaculatory duct

13, 78

84

bulb(us) – penis 95 – spongiosum 95 – spongiosi/vestibulares 24 – vestibular 58, 76 bulbourethral vestibular gland 21

fascia – dartos 78, 103, 116 – deep fascia of the penis 101, 104 fourchette 77 frenulum 60, 106, 117

clitoris – deep dorsal vein 29 – glans 57 cloaca 5, 32 – anal compartment 7 – communication between compartments 7, 12 – cornu 7 – nervous system 14 – subdivision 34 – urogenital compartment 7 – vascular system 13 cloacal – eminence 5, 11, 15, 32 – groove 11, 12

gland – anal 65 – bulbourethral 90 – bulbourethral/greater vestibular 21 – glandopreputial 64, 81, 107 – greater vestibular 57 – periurethral (paraurethral) 53, 74 – prostatic 85 – urethral 90 glandopreputial lamella 61, 81, 105 glans – of clitoris 57, 76 – of penis 94, 105 – of the phallic structure 11, 22 – plate 11, 20, 37, 38, 57, 92, 113

134 hind gut 5 hymen 50, 72 – imperforatum 72, 73 hypospadias 117 imperforate anus 36 labium – cloacal 12 – majus 64, 78, – minus 51, 58, 60, 76 – urethral 97 – urogenital 16, 23, 51 lacunae Morgagni 37, 57, 90 lymph vessel 29 mesonephric duct 5, 7, 19, 69, 84 – partition from ureter 17, 33 – regression in female 47 – transformation into ejaculatory duct 84 mesonephric-paramesonephric complex 19, 46, 109 Mllerian tubercle 19, 47, 50, 84 muscle – bulbospongiosus 24, 28, 67, 108 – detrusor vesicae 17, 54, 87 – dilator urethrae 7, 110 – ejaculatorius 110 – external anal sphincter 28, 67, 107 – external urethral sphincter 19, 28, 37, 54, 67, 74, 89, 107, 110 – internal anal sphincter 66, 79 – internal urethral sphincter 19, 37, 54, 74, 88, 89, 110 – ischiocavernosus 28, 67, 107 – perinei profundus 75 – prostate-related 37, 88, 110 – puborectalis 60, 77, 101, 106 – pubovesical 54, 88 – transversus perinei superficialis 67, 107 – urethrovaginal 75 – vesical sphincter 19, 37, 74, 87, 110 – vesicoprostatic 88 – vesicovaginal 54 navicular fossa 91, 110, 112 – transverse dorsal stroma 101

Subject Index nerve – autonomic pelvic plexus – perineal 30 – pudendal 30 – spinal 30 nervous system 14, 30

30

orifice 92 – of anal canal 11, 26 – of cloaca 11 – of urogenital sinus 11, 20, 113 – urethral 91, 92, 113 paramesonephric duct 19, 37, 69, 84 penis 104, 116 – bulb 95 – deep dorsal vein 29 – deep fascia 101, 104 perineal – body 101, 115 – musculature 28, 41, 67, 107 – septum 115 perineum – definition 3, 32 – external shape 15, 30, 67, 80, 108 – midperineal region 3, 30, 32, 66, 81, 97 – nervous system 30 – surface 15, 31, 68, 81, 108 – transverse artery 29, 43 – vascular system 13, 28 phallic structure 16, 30, 67 prepuce – of clitoris 61, 78 – of penis 105, 117 preputial sac 106, 117 proctodeum 40, 79 prostate 85, 110 – smooth musculature 88 prostatic utricle 84, 109 pudendal system 14 raphe perinei 99, 104, 114 rectum 25, 40 scrotal – raphe 103 – septum 103, 118 – swelling 102, 118 scrotum 102, 118 – deep connective tissue

101

Subject Index septum – penile 104 – perineal 99, 115 – scrotal 118 – urorectal 35 sphincter – anal – – external 28, 67, 107 – – internal 66, 79 – cloacal 42 – urethral – – external 19, 28, 37, 54, 67, 74, 89, 107, 110 – – internal 19, 37, 54, 74, 88, 89, 110 – urethrovaginal 75 – vesical 19, 37, 74, 87, 110 spinal nerve 14 stroma – dartos 25, 63, 103, 118 – dorsal urogenital – – deep 25, 59, 77, 101, 118 – – superficial 24, 60, 77, 97, 115 – superficial stroma of the shaft 25, 61, 77, 104, 117 swelling – labial 68, 78 – labioscrotal 15, 29, 31, 43 – postanal 12, 42 – scrotal 102, 118 tail gut 5, 7 trigone 18, 34 urachus 17 ureter 8 ureteric diverticulum 8 urethra – female 51, 74

135 – male 83, 109 – membranous 88, 109 – prostatic 83, 109 – spongy 89, 110 urethral – gland 90 – labium 97 – orifice 91, 92, 113 urinary bladder 17, 33 – trigone 18, 33, 34 urogenital – labium 16, 23, 51 – plate 9, 37 – – ventral extension 20, 37, 55, 57, 89 – sinus 16, 17, 19, 35 – – deep segment 19, 37 – – orifice 11, 20, 113 – – superficial segment 20, 37, 38 urorectal septum 35 vagina 46, 69 – derivation 69 – descent 50, 73 valvula Gurin 93 vascular – area 58, 76 – plexus – – internal pudendal 13 – – median sacral 13 – system 13, 28 vein – deep dorsal vein of the clitoris/penis 29 – median sacral 29 – of allantois 29 vestibulum vaginae 55, 75 – dorsal smooth muscular coat 77 – (ventral) vascular area 58, 76