49 3 Embryology for the Urologist Allan Johnston1, Maike F. Eylert2, Tarik Amer1, and Omar M. Aboumarzouk1, 3 1 Glasgow Urological Research Unit, Department of Urology, Queen Elizabeth University Hospital, Glasgow, UK Department of Urology, University Hospital of Wales, Cardiff, UK 3 University of Glasgow, School of Medicine, Dentistry & Nursing, Glasgow, UK 2 Abstract The urogenital tract is largely derived from the mesoderm. Initial kidney structures appear in the fourth week of embryogenesis, but definitive kidney development occurs from the 5th-10th week. The bladder and urethra start to form at the same time as the definitive kidney. Sexual development is indifferent until the sixth week of gestation, with the sex-determining region Y (SRY) gene being the primary driver for male foetal development. This is largely through further action of anti-müllerian hormone (AMH) and testosterone. The mesonephric ducts become the male genital duct system, and the paramesonephric ducts become the female genital duct system. Testicular descent is initially dependent on AMH and insulin-like hormone 3, and later testosterone. Dihydrotestosterone drives male external genital development. The overall sequence of urogenital development is well established and can explain the majority of congenital malformations that may present to the urologist. Additional details will only be of interest to the subspecialist and the researcher Keywords anti-müllerian hormone (AMH); development; dihydrotestosterone; embryology; genital tubercle; gonadal ridges; gubernaculum; insulin-like hormone 3; labioscrotal folds; mesonephric duct; mesonephros; metanephros; paramesonephric duct; pronephros; SRY gene; testicular descent; testosterone; trigone; ureteric bud; urethral plate; urogenital sinus; Key Points ●● ●● ●● ●● ●● Three kidney systems: pronephros, mesonephros, and metanephros. The ureteric bud arises from the mesonephric duct and forms the entire upper tract drainage system. The mesonephric ducts become the male genital duct system. The paramesonephric ducts become the female genital duct system. The urogenital sinus gives rise to the bladder and the urethra with the exception of the fossa navicularis and male urethral meatus. Blandy’s Urology, Third Edition. Edited by Omar M. Aboumarzouk. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd. ●● ●● ●● The sex‐determining region Y (SRY) gene, anti‐müllerian hormone (AMH), insulin‐like hormone 3, testosterone, and dihydrotestosterone are essential in the normal development of the male foetus. Testicular descent happens in two phases; initially dependent on AMH and insulin‐like hormone 3 and then dependent on testosterone. Dihydrotestosterone drives male external genital development. 50 3 Embryology for the Urologist 3.1 ­Historical Consideration Embryology stems from the Greek words of ‘embry’, meaning the ‘unborn’, and –‘ology’, which is the subject of study or a branch of knowledge. [1, 2] The Greeks were the first to describe their thoughts on the origins of man in the womb. More notably, Aristotle postulated that the foetus was formed in the uterus from a coagulum of blood (menstrual blood), and the foetus itself was fully developed in miniaturised form in the sperm. This chain of thought was carried on for centuries through Europe, even as late as the seventeenth century, when Marcello Malphigi (1672) described poultry eggs as containing a miniature chick and extrapolated that humans were fully formed in the sperm. In 1694, 21 years after the invention of the microscope by Antonie van Leeuwenhoek, did Nicolaas Harsoeker postulate that humans were formed from the joining of the spermatozoon and ovum, further described by Lazzaro Spallanzani in 1775. Interestingly, a revelation to the Prophet Mohamed (peace be upon him) in the seventh century detailed descriptions for embryological development, including the formation of the embryo from both the male and female ‘drops’ (i.e. sperm and ovum). Multiple Quranic verses were revealed detailing the formation of the embryo to the fully developed foetus. However, it was not until the nineteenth and twentieth centuries when these revelations were confirmed as fact and held as a true representation of embryo‐foetal development. From the experimental works of Karl Ernst von Baer, Charles Darwin, and Ernest Haeckel, to the chemical and mechanical discoveries of embryology by Otto Warburg, paving the way for Ross Harrison, Frank R. Lillie, and Glomeruli Cortex Medulla Ducts of bellini Papilla Calix Figure 3.1 The basic unit of the kidney is the pyramid, with a flower bunched arrangement in a vase. Hans Spemann to describe the more detailed mechanisms of embryonic development throughout the early twentieth century. It was late in the twentieth century, with the advent of more sophisticated instruments such as the electron microscope and spectrophotometer, that embryology in its modern form took place. More notably the work of Keith Moore described the embryological and foetal development to the finest detail, giving rise to the established current knowledge of embryology. 3.2 ­Introduction A description of each urological organ will be described individually; hence, overlap and repetition will inevitably exist to allow a detailed understanding of the development of each organ. Three basic cell layers comprise the embryonic disc which becomes the embryo: the ectoderm (originates from the amniotic surface), the mesoderm (originates form inpouring cells from the ectoderm), and the endoderm (yolk sac). The majority of the urogenital tract is derived from the mesoderm. Organ development in general occurs between the 3rd and 10th week of gestation (Table 3.1). 3.3 ­Embryology of the Kidneys and Ureters The basic unit of the kidney is the renal pyramid, which is arranged like a bunch of flowers in a vase (Figure 3.1). The flowers are the glomeruli; the stalks are the collecting tubules; and the vase is the calix. The design of the normal pyramid is important in preventing reflux of urine up into the renal parenchyma. There are three paired kidney systems during foetal development (Figure 3.2), with only the third system being of functional importance. First, the pronephros forms and rapidly regresses in the cervical region of the intermediate mesoderm during the fourth week. The pronephros in humans is both rudimentary and segmented. Later in the fourth week, the unsegmented mesonephros forms from the intermediate mesoderm in the upper thoracic to upper lumbar segments. These appear as a pair of sausage‐shaped swellings on the posterior abdominal wall on either side of the mesentery – the genitourinary ridges. A faint groove demarcates each ridge into a medial gonadal and lateral nephrogenic part (Figures 3.3 and 3.4). These swellings lengthen and acquire primitive nephron‐like structures, which is a collection of capillaries that form a glomerulus at the medial extremity, and Bowman’s capsule, which forms around 3.3 Embryology of the Kidneys and Ureters Table 3.1 Table of developmental timings. Structure Development starts Disappears Comments, ultimate structures Pronephros 4th week 4th week No functional relevance Mesonephros 4th week 8th week No permanent function Mesonephric duct (male) 4th week — Central zone of the prostate, ejaculatory ducts, vas deferens, seminal vesicles, epididymis, and efferent ductules Mesonephric duct (female) 4th week 8th week Remnants: epoophoron, paroophoron, Gartner cyst Metanephros 5th week — Permanent kidney including glomeruli, convoluted tubules, loop of Henle. Urine production from 10th week. Ureteric bud 5th week — Collecting ducts, minor calyces, major calyces, renal pelvis, ureter Urogenital sinus 5th week — Bladder, posterior urethra (male) or entire urethra (female), anterior urethra (male) up to the edge of the glans penis Gonadal ridges Germ cells migrate in 6th week — Testes or ovaries Paramesonephric duct (male) 6th week 8th week Remnants: appendix testes, utricle Paramesonephric duct (female) 6th week — Fallopian tubes, uterus, and the upper two‐thirds of the vagina Genital tubercle enlargement (male) 6th week — Penis. Urethral plate closes to form tubular urethra 12th week Urethral endoderm and surrounding 13th week mesoderm (male) — Prostate Ingrowth of ectoderm from the tip of the glans penis (male) 15th week — Fossa navicularis and urethral meatus Testicular descent (abdominal) 8–15th week — AMH and insulin‐like hormone 3 dependent Testicular descent (inguinal) 24–28th week — Testosterone dependent Testicular descent (scrotal) 28–33rd week — Testosterone dependent AMH, anti‐müllerian hormone. the glomerulus, forming the renal corpuscle. These simple excretory units may function briefly before regressing in the eighth week. The mesonephros functions for a short time during early foetal life by producing urine from the sixth through to the eighth weeks of development. On the lateral aspect and adjacent to the mesonephros, the mesonephric ducts advance distally to drain into the cloaca (this is the primitive hindgut which goes on to form the bladder and rectum) at the caudal end of the embryo. Whilst the caudal aspects of the tubules are differentiating, the cranial tubules and the glomeruli degenerate, with the majority of the mesonephros absent by the end of the second month of gestation. The mesonephric system disappears completely in the female around the eighth week. In the male, the ­mesonephric ducts (also known as the wolffian ducts) persist, giving rise to the efferent ductules of the testes, the epididymis, vasa, seminal vesicles, and appendix epididymis. Pronephros Mesonephros Metanephros Figure 3.2 Locations of pronephros (fourth week), mesonephros (fourth to eighth weeks), and metanephros (from fifth week) in the embryo. 51 52 3 Embryology for the Urologist Gonadal ridge Mesonephros Urogenital ridge Gut Figure 3.3 The mesonephros runs along the length of the foetus on the lateral side of the genitourinary ridge. Heart Lung Mesonephros Gonadal ridge Figure 3.4 The mesonephros can be identified in the four‐week‐ old human embryo. Third, the metanephros (the permanent kidneys) develops from the fifth week from metanephric mesoderm in the most caudal region of the nephrogenic ridge, the lateral aspect of the genitourinary ridge. As the tail end of the foetus curls up, the hindgut is curled with it and so are the nephrogenic ridges with their wolffian ducts, which twist upwards and inwards. Branches from the most caudal part of the wolffian (mesonephric) ducts enter the metanephros. These branches, or outgrowths, are called the ‘ureteric buds’ (Figure 3.5). In contrast to the first two systems, excretory units only form by a process called ‘reciprocal induction’ between the ureteric bud and metanephric tissue caps. The collecting ducts develop from the ureteric bud (fifth week, Figure 3.6). The ureteric bud subdivides and induces formation of the glomeruli in the mesenchyme of the metanephros. The branches of the bud then grow peripherally into the cortex, dilating and splitting repeatedly until about 15 generations of ducts have formed (Figure 3.7). The first four or five generations of the dividing ureteric branches become dilated and incorporated in the eventual renal pelvis (Figure 3.8). The next four or five generations form the major calices and ­collecting tubules (Figure 3.8) [3]. The successive generations elongate and converge on the minor calyx (seventh week), thereby forming the renal pyramid in the flower– vase configuration described previously. Subsequent generations elongate and converge to form renal ­pyramids, and ultimately, they form around one million collecting ducts per kidney (until the fifth month). 3.3 Embryology of the Kidneys and Ureters (a) (b) Genital ridge Mesonephros Mesonephros Wolffian duct Cloaca Wolffian duct Metanephros Metanephros Ureteric bud Figure 3.5 (a) The caudal part of the mesonephros becomes the metanephros. It receives its own branch from the mesonephric (wolffian) duct, the ureteric bud. (b) As the foetus curls up, the wolffian duct and the ureteric bud are bent around. Figure 3.6 The ureteric bud develops as an outgrowth of the mesonephric duct close to the urogenital sinus (fifth week). Mesonephros Allantois Foetal hindgut Mesonephric duct Urogenital sinus Metanephric tissue cap Ureteric bud The metanephric tissue caps covering each collecting tubule form renal vesicles which develop into nephrons. Capillaries grow into the opposite end of the nephron giving rise to glomeruli (Figure 3.9). From about the 10th week, urine is produced by the metanephros; however, nephrons continue to form until birth. After birth, no further nephrons will form (approximately 700 000 per kidney), but existing ones will continue to grow. This growth is responsible for the change from lobulated kidneys at birth to kidneys with a smooth outline. The metanephros ascends during weeks 6–10 as a result of the elongation of the sacral and lumbar regions of the embryo as well as loss of the initial curvature of the embryo. The arterial supply originates from the aorta, 53 54 3 Embryology for the Urologist Generations 6–15 Fifth generation First generation Second generation Forth generation Third generation Figure 3.7 The ureteric bud splits repeatedly until about 15 generations of ducts have formed. The first generation becomes the renal pelvis, the second the major calyces, the third to fifth minor calyces, and the remainder become renal pyramids and collecting ducts. Collecting tubules Metanephros and serial arteries are formed and regress during renal ascent. Some vessels may remain as accessory renal arteries. During the fourth to sixth weeks of gestation, while the caudal end of the foetus curls up and bends the hindgut into a U configuration, the mesoderm grows down into the gap between the future rectum and bladder, thus forming the urorectal septum (Figure 3.10). This septum separates the cloaca into a primary urogenital sinus (ventrally) and rectum (dorsally). The wolffian ducts lie in this wedge‐shaped septum, and grow down with it. They also become bent into a loop and take the ureteric buds with them (Figure 3.10) [4]. Part of this septum is incorporated into the bladder to form the trigone, and because of the incorporated loop of the wolffian duct, the ureteric duct comes to open into the bladder cephalad to the duct. In males, the wolffian duct becomes the vas deferens and seminal vesicles, whilst in the females these ducts regress in the absence of testosterone [4]. At this stage, the tail end of the foetus is roughly 1‐cm long and the space between the tail and the umbilical cord is filled by the cloacal membrane. On either side of this membrane are the two small genital tubercles (Figure 3.11). This membrane is formed by tightly packed ectoderm and endoderm cells with no intervening mesoderm. As this Collecting tubule Metanephric tissue cap Calices Pelvis Capillary Renal vesicle Nephron Figure 3.8 As the ureteric bud approaches the metanephros, it branches repeatedly. The branches induce the formation of glomeruli. The first four or five generations of branches become incorporated into the renal pelvis. Glomerulus Figure 3.9 The metanephric tissue caps covering each collecting tubule form renal vesicles which develop into nephrons. Capillaries grow into the opposite end of the nephron, giving rise to glomeruli. 3.3 Embryology of the Kidneys and Ureters Figure 3.10 The urorectal septum grows down between the future bladder and rectum. Mesonephros Wolffian duct Bladder Ureter Cloacal membrane Urorectal septum Rectum Figure 3.11 The cloacal membrane disappears and the phallic tubercles meet in the midline. Umbilical cord Genital tubercle Cloacal membrane Urorectal septum Tail Bladder Phallus Tail Rectum Urorectal septum Rectum Tail 55 56 3 Embryology for the Urologist membrane dissolves, the two genital tubercles fuse to form the united phallic tubercle (Figure 3.11). While all this is taking place, the mesonephros is withering away, but as it regresses, the wolffian duct generates a second duct, parallel and lateral to it, the müllerian duct (Figure 3.12). As the urogenital ridge twists round, so the müllerian ducts approach each other, meet in the midline in front of the wolffian ducts, and burrow down in the urorectal septum (Figure 3.12). The urorectal septum is partially absorbed into the trigone, and the müllerian ducts open into the urethra medial to, and in front of, the wolffian ducts. The subsequent fate of the wolffian and müllerian ducts is determined by the X and Y chromosomes. Until the fourth week, the urogenital ridge is neuter. At four weeks it is invaded by gonadal cells, which migrate by amoeboid movements from the yolk sac across the coelom and burrow into the gonadal ridge (Figure 3.13). By the sixth week, it is estimated that there are about 60 000 gonadal cells in each gonadal ridge. The male ones are active at once; the female gonadocytes stay dormant for another two weeks. 3.3.1 Relevant Congenital Malformations Partial or complete ureteric duplication results from early splitting of the ureteric bud. In a complete duplex system, the Weigert‐Meyer rule states that the ureter (a) Gonadal ridge Wolffian duct Mesonephros Müllerian duct Ureter Metanephros (b) Ovary Müllerian ducts Metanephros Figure 3.12 (a) A second (müllerian) duct forms on the lateral side of the mesonephros. (b) The müllerian duct roll towards each other, meet in the midline in front of the wolffian ducts, and burrow down into the urorectal septum to form the uterus and fallopian tubes in the female. 3.4 Embryology of the Bladder draining the lower moiety tends to reflux as a result of a shorter submucosal tunnel positioned laterally and superior; the upper pole tends to obstruct, be ectopic, or form ureteroceles and is positioned medially and inferiorly (Figure 3.14). An ectopic ureter may drain into the bladder, bladder neck, or prostatic urethra in males, or into the vagina, uterus, or ovary in females (Figure 3.15). The pathophysiology of duplex kidney is explained by the insertion of the ureter into the bladder, whilst the lower pole tends to reflux due to a shorter submucosal tunnel, and the upper pole tends to obstruct, be ectopic, or form ureteroceles. Genital ridge Failure of renal ascent gives rise to a pelvic kidney (Figure 3.16). Midline fusion of both kidneys during their ascent gives rise to a horseshoe kidney, with further ascent limited by the root of the inferior mesenteric artery. The midpoint joining both kidneys is known as the isthmus (Figure 3.17). Crossed fused renal ectopia results from both kidneys ascending on the same side of the body and fusing in the process (Figure 3.18). Renal agenesis results from failed reciprocal induction, intrinsic defects within the mesenchyme, or involution of a multicystic dysplastic kidney. Multicystic dysplastic kidney may be the result of faulty ureteric bud development. Renal dysplasia results from defects in reciprocal induction or from obstruction during the foetal period. 3.4 ­Embryology of the Bladder Germ cells Coelom Yolk sac Figure 3.13 Migration of the germ cells from the yolk sac to the genital ridge. The foetal hindgut is curled, following the outline of the tail end of the embryo, in the shape of a hook (Figure 3.19). The caudal most part of the hindgut remains in communication with the allantois and will form the bladder. As stated, the urorectal septum descends at four to six weeks to separate the cloaca into the urogenital sinus anteriorly and the anal canal posteriorly. The cloacal membrane dissolves to open both canals (Figures 3.20 and 3.21). As the allantois shrinks it becomes a solid cord – the urachus – linking the apex of the bladder to the umbilicus. In clinical terms, the urachus becomes the median umbilical ligament upon closing. If it remains patent, the patient will have a congenital umbilical fistula. The urogenital Figure 3.14 Complete duplex system on the left. The Wiegert‐Meyer rule states that the ureter from the upper moiety will enter inferomedial to the lower moiety ureter. Upper moiety Lower moiety Normal kidney Normal ureter of lower moiety Normal ureter Normal ureteric orifices Bladder neck Prostate Ectopic ureter of lower moiety (showing two possible terminations) 57 58 3 Embryology for the Urologist Figure 3.15 The ureter from the upper ­ half‐kidney may open into the vagina below the sphincter and cause incontinence. Ureter from upper half-kidney draining into ectopic orifice External sphincter Ectopic ureter Superior mesenteric artery Pelvic kidney Horseshoe kidney Figure 3.17 If the lower end of the metanephros fuse together, they remain ‘rotated’, and their course up the abdomen is held by the superior or, rarely, the inferior mesenteric arteries. Figure 3.16 If the kidney remains in the pelvis, it stays ‘rotated’. sinus itself will give rise to the bladder, the posterior urethra (male) or entire urethra (female), and the anterior urethra (male) up to the edge of the glans penis. These are all endodermal (originates from yolk sac) in origin. The male anterior urethra is not initially tubular, but rather a flattened urethral plate, which is pulled forwards with the growing phallus (Figure 3.22). It then folds in sideways and closes along the midline at around 12 weeks (Figure 3.23 and 3.24). The terminal part of the male 3.4 Embryology of the Bladder ­ rethra (fossa navicularis and external urethral meatus) u is formed by ingrowth of ectoderm (derived from amniotic sac) from the tip of the glans penis (15 weeks). The lower ends of the mesonephric ducts (due to become the vasa efferentia) and the lower ends of the ureters become incorporated into the posterior wall of the bladder, and thus form the trigone (Figures 3.25 and 3.26). Descent of the testes then causes the vas deferens on either side to swing anteriorly over the ureter (‘water under the bridge’). 3.4.1 Relevant Congenital Malformations Failure of fusion of the urethral folds results in hypospadias. Epispadias results if the genital tubercle (see discussion in this chapter) forms in the urorectal septum with part Urogenital septum Cloaca Figure 3.19 The foetal hindgut bends round and the urogenital septum descends to separate the bladder from the rectum; the patent urachus keeps the future cladder in continuity with the allantois. Figure 3.18 Crossed renal ectopia. Allantois Urogenital sinus Cloacal membrane (dissolving) Urorectal septum Anal canal Figure 3.20 The urorectal septum descends at four to six weeks to separate the cloaca into the urogenital sinus anteriorly and the anal canal posteriorly. The cloacal membrane dissolves to open both canals. 59 60 3 Embryology for the Urologist Figure 3.21 As the urogenital septum reaches the perineum, the cloacal membrane dissolves to reveal two openings, the urethra and the rectum. Wolffian mesonephric duct Urogenital sinus Metanephros Bladder Allantois Urachus Ureter Genital tubercle Cloacal membrane Ureteric bud Rectum Urorectal septum Urethral plate Phallus Phallus Urethral plate Urogenital sinus Hindgut Figure 3.23 The male anterior urethra folds in sideways and closes along the midline at around 12 weeks. The genital and labioscrotal folds move caudally and fuse to form the scrotum with the midline scrotal septum. Figure 3.22 The male anterior urethra is not initially tubular, but rather a flattened urethral plate, which is pulled forwards with the growing phallus. of the membrane cranial to the genital tubercle. Exstrophy of the bladder (which always includes epispadias) is caused by failure of the abdominal wall to form. 3.5 ­Embryology of the Indifferent Genital System The gonads begin their development from the urogenital ridge, located behind the coelom. They divide longitudinally, in the third week, to form the gonadal or genital Inrolling folds forming urethra Figure 3.24 The skin rolls in on either side to form the urethra. 3.6 Embryology of the Male Genital System Figure 3.25 The lower ends of the mesonephric ducts (due to become the vasa efferentia) and the lower ends of the ureters become incorporated into the posterior wall of the bladder, and thus form the trigone. Descent of the testes then causes the vas deferens on either side to swing anteriorly over the ureter. Ureter Bladder Allantois Vas deferens Kidney Trigone Testis Phallus Wolffian duct Bladder Rectum Figure 3.26 The urogenital septum brings down the wolffian ducts, which will become the ureters. ridge on the medial side and the embryonic mesonephros on the lateral side, which later becomes the urinary tract. Paramesonephric ducts (müllerian ducts) develop from these genital ridges. Between weeks five and six of embryonic development, the germ cells arising from the yolk sac integrate into the gonadal ridge after travelling by amoeboid movement through the umbilical cord and the coelom (Figures 3.27–3.28). Upon the arrival of germ cells, primitive sex cords form in the still indifferent gonad. Failure of gonadal development occurs if the germ cells do not reach the gonadal ridges because they trigger the development of the gonad into ovary or testis [5]. In the human embryo, the gonads remain undifferentiated until about week seven of development. Depending on the XY genetic constitution they then differentiate into the testes or the ovaries [6]. Labioscrotal fold Figure 3.27 Cloacal folds form in the third week and will become genital folds. The genital tubercle forms at the same time. Indifferent labioscrotal folds form lateral to the genital folds. 3.6 ­Embryology of the Male Genital System Once in the gonadal ridge, the presence of the s­ex‐ determining region Y (SRY) gene located on the short arm of the Y chromosome leads to testicular development and male phenotyping. Down streaming from SRY 61 62 3 Embryology for the Urologist differentiate to become the seminiferous tubules. The tubuli recti are formed from the narrowing of the deeper portion of the semiferous tubules, and it converges into the tubuli recti. The tubules’ connections with the germinal epithelium are discontinued with the formation of a dense network of fibrous connective tissue known as the ‘tunica albuginea’ [5]. Male genital development depends on the SRY gene, anti‐müllerian hormone (AMH; also called müllerian inhibitory substance [MIS]), insulin‐like hormone 3, ­testosterone, and dihydrotestosterone (DHT). The gene does two things consecutively: Yolk sac Coelom Urogenital ridge Figure 3.28 The germ cells pass from the yolk sac across the coelom to the gonadal ridge. ­ roduces steroidogenesis factor (SF1) and SOX9 that p ­stimulate testicular cellular differentiation. This induces the first step of the organogenesis of the testes, the formation of gonadocytes, and proliferation of Leydig cells and sustentacular cells of Sertoli (Figures 3.29 and 3.30) [7]. This development not only relies on the presence of the SRY gene, but also the lack of the DAX1 gene (which can down regulate the SRY gene action) and WNT4 gene, which is responsible for female gonadal development. These cells then proliferate with the aid of SRY gene proteins and form the primary sex cords. These further proliferate and extend into the medulla of the gonad to form the testis or medullary cords. Once here, the cords branch with their deep ends anastomosing to form the tubules of the rete testis. The gonadal cords further develop to give rise to the testicular cords which 1) It makes the gonadocytes differentiate into Sertoli cells which secrete another simple polypeptide, the müllerian duct‐inhibiting factor. This has a dramatic effect: the entire müllerian duct system disappears within a single day, leaving behind only the tiny vestige of the utriculus masculinis in the verumontanum (Figure 3.31). MIS also inhibits the formation of the uterus and fallopian tubes (the müllerian structures) [7–9]. 2) Approximately one week later (approximately week eight), the SRY gene enables the differentiation of the germ cells located between the testicular cords into Leydig cells derived from the mesenchyme of the gonadal ridge. These contain 17‐ketosteroid reductase which is involved in the synthesis of testosterone, which is activated by the enzyme 5‐alpha reductase to 5‐alpha DHT. This active substance reacts with a cytosol receptor in the phallic tubercles and wolffian ducts to secrete growth factor. This results in the two changes necessary to convert the neuter foetus into the male. The cytosol receptor factor is a product of one of the genes in the X chromosome. In the presence of testis determining factor (SRY ­protein), medullary cords of the testis, and the rete testis Figure 3.29 Sertoli cells secrete müllerian duct inhibiting factor. About a week later, Leydig cells secrete testosterone, which is activated to dihydrotestosterone, and causes descent of the testicles and formation of the penis and urethra. Gonadocytes Sertoli cells Leydig cells 17 – ketoreductase Testosterone Müllerian duct inhibitory factor 3.6 Embryology of the Male Genital System SRY gene: Foetal pituitary LH Leydig cells Testicular development FSH Foetal testicles Sertoli cells Regression of the müllerian ducts Anti-müllerian hormone Testosterone Dihydrotestoste Differentiation of genital tubercle and urogenital sinus: External Genitalia Prostate Differentiation of the wolffian ducts: Testicular descent Seminal Vesicles Vas Deferens Epididymis Figure 3.30 Male hormone dependence. Wolffian duct Utriculus masculinus (Müllerian) Müllerian duct Ureteric bud Wolffian duct Testis Figure 3.31 The müllerian duct‐inhibiting factor from Sertoli cells cause the müllerian ducts to disappear except for the pit in the verumontanum. form (Figure 3.32). Formation of the tunica albuginea follows. Sertoli cells (from epithelium) and Leydig cells (from mesenchyme) form dependent on the SRY protein. SRY protein stimulates AMH production by Sertoli cells (seventh week), which in turn causes Leydig cells to produce testosterone and insulin‐like hormone 3 (ninth week). The testis cord continues to remain solid until the onset of puberty, when it acquires a lumen to form the 63 64 3 Embryology for the Urologist persist (i.e. appendix testes, utricle). Testosterone influences development of the mesonephric ducts and male external genitalia. Mesonephric ducts differentiate into the central zone of the prostate, ejaculatory ducts, vas deferens, seminal vesicles, epididymis, and efferent ductules (Figures 3.33 and 3.34) Mesonephros Vitelline duct Gonadal ridge Germ cells Hindgut seminiferous tubules, which connects with the rete testis and enters the ductuli efferentes, which are remnants of the mesonephric system. The ductus deferens is formed when rete testis is joined to the wolffian duct with the aid of the ductuli efferentes [5]. AMH causes involution of the paramesonephric ducts. Only small remnants from the paramesonephric ducts (b) Testicular descent happens in two phases, guided by the action on the gubernaculum (Figure 3.35). 1) Passive phase: Dependent on AMH/MIS and insulin‐ like hormone 3 guiding abdominal descent to the inguinal ring (8–15th week) 2) Active phase: Dependent on testosterone through the inguinal canal (24–28th week), and to the base of the scrotum (28th‐33rd week). Figure 3.32 Upon arrival of germ cells, primitive sex cords form in the then‐indifferent gonad. In the presence of testis determining factor (SRY protein), medullary cords of the testis and the rete testis form. (a) 3.6.1 The Descent of the Testis (c) During their descent, the testes acquire a layer of ­peritoneum, which becomes the tunica vaginalis. During development the testis begins its journey in the lumbar area of the retroperitoneum. It transfers from here to the scrotum near the end of the third month of pregnancy. This is mediated by testosterone and the gubernaculum (Figure 3.36), a lump of jelly, which forms an expanding track, which leads to and inserts into the genital swelling, the future scrotum [10]. Ectopic testes are formed when the gubernaculum leads them in the wrong direction, such as towards the penis or the thigh, and incomplete descent occurs if the gubernaculum fails to form a path to the scrotum (Figure 3.37) [11–13]. (d) Phallic tubercles Figure 3.33 (a–d) Testosterone from the Leydig cells cause the phallus to grow and the urethra to roll in from either side. 3.6 Embryology of the Male Genital System Figure 3.34 In males, the wolffian duct becomes the vas deferens, epididymis, and seminal vesicles. Vas deferens Seminal vesicle Appendix epididymis [Müllerian] [Müllerian] appendix testis Utriculus masculinus Peritoneum Testicle Gubernaculum Tunica vaginalis Figure 3.35 Testicular descent happens in two phases: (1) dependent on AMH and insulin‐like hormone 3 during abdominal descent to the inguinal ring (8–15th week) and (2) dependent on testosterone through the inguinal canal (24–28th week),and to the base of the scrotum (28–33rd week). Both phases are guided by the gubernaculum. During their descent, the testes acquire a layer of peritoneum, which becomes the tunica vaginalis. Figure 3.36 Normal migration of the testicle. Peritoneum Processus vaginalis Gubernaculum 65 66 3 Embryology for the Urologist Ecotopic (‘off course’) Incompletely descended (‘on course’) Abdominal Abdominal Entrant inguinal Penile High retractile Perineal Low retractile Crural Figure 3.37 The maldescended testis may be off its normal course of descent (ectopic) or on the normal course (incomplete) descent. Encysted hydrocele of the cord Congenital hydrocele Hernia magna Intra-abdominal hydrocele Figure 3.38 Varieties of hydrocele and hernia. 3.6.2 Relevant Congenital Malformations As the testis descends, it is accompanied by the processus vaginalis. The lumen of the processus vaginalis is normally obliterated within a few weeks of birth; ­however, if it persists, it gives rise to defects such as a congenital hernia, hydrocele, an encysted hydrocele of the cord, or an abdominoscrotal hydrocele (Figures 3.38 and 3.39) [14]. Maldescent of a testis results in an undescended testis. Malposition of the gubernaculum or an abnormally long 3.7 Embryology of the Prostate (a) (b) (c) (d) (e) (f) Figure 3.39 Various types of hernia and hydrocele. (a) Common hydrocele, (b) encysted hydrocele, (c) ‘double’ hydrocele, (d) hernia and hydrocele, (e) ‘hernia magna’, and (f ) abdominoscrotal hydrocele. gubernaculum result in an ectopic testis. Failure of, incomplete, or inappropriate development of the genital system leads to disorders of sexual differentiation. In males, the müllerian duct lingers as two tiny vestiges, the utriculus masculinis in the verumontanum and the appendix testis, neighbouring the appendix epididymis which is a vestige of the wolffian duct (Figures 3.31 and 3.34) [5]. If there is a congenital deficiency of MIS, phenotypical males are born with fallopian tubes and a uterus, usually found by chance at laparoscopic orchidopexy or hernia repair, and occasionally, associated with testicular tumours [7]. 3.7 ­Embryology of the Prostate As described previously, the male foetus develops in the presence of a Y chromosome, which encodes the SRY protein, thus enabling testicular differentiation and the production of androgens such as testosterone. In addition to the actions of SRY protein and androgens, a third factor is required for male development, AMH, also known as MIS [15]. This causes the müllerian (paramesonephric) duct to degenerate, forming the prostatic utricle (or utriculus masculinis). Blandy originally described this as, ‘a volcanic crater on the summit of the verumontanum’. 67 68 3 Embryology for the Urologist Testis Figure 3.40 The prostate begins to form in the mesenchyme of the urogenital septum in the vicinity of the opening of the wolffian ducts. Wolffian duct Bladder Metanephros Ureter Urorectal septum Rectum Tail The prostate forms in the mesenchyme of the urogenital septum. The cloacal membrane, a thin film which covers the convexity of the hindgut, regresses to leave gaps in front of and behind the urogenital septum, and in so doing, forming the urethra and anus. Running down in this septum are the müllerian and wolffian ducts and the ureteric buds (Figure 3.40). At approximately week eight, the Leydig cells of the foetal testis secrete testosterone [16, 17], and as a consequence, the human prostate begins its development at about the 10th week of gestation. The initial outgrowths of the epithelial ‘prostatic’ buds from the urethra into the urogenital sinus (UGS) occur in response to the binding of 5α‐dihydrotestosterone to androgen receptors localised in the surrounding ­mesenchymal tissue [18–21]. There are five pairs of buds. The top pairs are derived from mesoderm (i.e. wolffian structures) and form the transitional, periurethral, and central zones of the prostate, whereas, the bottom pairs are derived from endoderm and form the peripheral zone. These prostatic buds begin as solid cords of epithelial cells that elongate and undergo extensive branching morphogenesis during the latter stages of foetal growth to develop primitive lumens [22]. During weeks 13–15, serum testosterone elevates and remains high until week 25, which in turn induces epithelial differentiation. At this point, the three important epithelial populations, other than the stem cells, are distinct: luminal, basal, and neuroendocrine cells [22]. The stromal component compromises of fibroblasts, smooth muscles, and myofibroblasts. At week 25, the testosterone level diminishes, and the gland remains in a quiescent state until puberty. The central zone is primarily sensitive to testosterone, whereas the peripheral and transitional/periurethral are sensitive to DHT. Summary: DHT prompts development of the prostate from urethral endoderm and surrounding mesoderm (13–16th weeks), giving rise to the transitional zone and peripheral zone. The central zone is formed from the mesonephric duct. 3.8 ­Embryology of the Penis and Urethra Two crucial events occur in the male embryo, between the fifth and seventh week: ●● ●● the disappearance of the müllerian ducts the transformation of the phallic tubercles Both events are orchestrated by the two sex chromosomes. On the Y chromosome, the SRY gene controls germ cell differentiation into Sertoli cells (whose müllerian inhibiting factor causes the müllerian ducts to ­disappear). By the eighth week of gestation, the mesenchymal cells of the genital ridge differentiate into Leydig cells which secrete testosterone, which enters the wolffian ducts and the phallic and genital tubercles. These tissues contain 5‐ alpha reductase, an enzyme which activates testosterone to a more potent form, DHT. DHT binds to a cytosol receptor protein and sets off the changes in growth, which allow the wolffian ducts to develop into the vasa efferentia, seminal vesicles, and epididymis. The phallic and genital tubercles become the penis, scrotum, and urethra (Figure 3.41). A gene on the X chromosome codes the cytosol receptor 3.8 Embryology of the Penis and Urethra Figure 3.41 The Y chromosome produces the HY gene, which turns germ cells either into Sertoli cells, which secrete the müllerian duct‐inhibiting factor, or Leydig cells. The Leydig cells secrete testosterone, which is hydrogenated to dihydrotestosterone, and binds to receptors in the wolffian ducts and phallic tubercles. Y chromosome HY gene Germ cells Sertoli cell Leydig cells Testosterone Müllerian ductinhibiting factor Müllerian Dihydrotestosterone Wolffian Urogenital sinus Penis and urethra Umbilical cord Phallic tubercle Urogenital sinus Genital tubercles Urogenital septum Rectum Figure 3.42 When the cloacal membrane dissolves, the bladder opens behind the genital tubercle. protein. Each step on this ladder of events is carried out by a certain enzyme coded by a single gene. In the absence of this organised testicular development, the differentiated gonads will develop into ovaries by the 13th and 14th weeks of gestation [23]. Each step may go wrong and result in one of the variations of intersex. By week seven, the cloacal membrane dissolves, and the primitive bladder opens on the ventral aspect of the Dihydrotestosterone Figure 3.43 Under the influence of dihydrotestosterone, the genital tubercle elongates, and a groove folds in on either side to form the urethra, up to the groove behind the solid glans penis. genital tubercle (Figure 3.42). This elongates to form the penis under which a groove is folded in from either side to form the urethra (Figure 3.43). Rods of mesenchyme in each fold differentiate into the corpora cavernosa and corpus spongiosum. At the tip of the penis, a groove demarcates the glans through which a solid cord extends and then becomes canalised as, the terminal urethra (Figure 3.44). Skin grows forwards from the coronal sulcus to enclose the glans in the prepuce and then becomes adherent. 69 70 3 Embryology for the Urologist Figure 3.44 A cord extends through the solid glans and then becomes canalised to form the terminal urethra. Skin grows forwards from the coronal sulcus to enclose and adhere to the glans. Figure 3.45 The scrotum is formed by the ­ in‐rolling of the two genital tubercles over the urethra. The scrotum is formed by the meeting together in the midline of the two genital tubercles over the urethra (Figure 3.45). All these processes must be complete within a critical window of time. If the genital folds and penis are not completed by the 12th week they never will be. However much androgen is given later on, all it can do is slightly enlarge the penis [24, 25]. Summary: Under the influence of DHT (formed from testosterone by the action of 5‐α‐reductase), the penis, scrotum, and prostate form. DHT causes elongation of the genital tubercle after six weeks to form the phallus with the urethral plate. The genital and labioscrotal folds on either side move caudally and fuse along the midline, which carries the urethra to the tip of the formed penis and forms the scrotum with the midline scrotal septum. DHT sets off the train of events leading to the growth and fusion of the phallus, in‐rolling of the urethral tube, formation of the scrotal sac, and the downward migration of the testes [7, 8]. The wolffian ducts are dependent on t­estosterone to develop and form the epididymis, vas deferens, and seminal vesicles. 3.9 ­Neuter State Without a Y chromosome or its SRY genes, the foetus stays neuter. The neuter state seems at first glance to be female: There is no phallus; the müllerian ducts persist; and the wolffian ducts fail to turn into the vas deferens. 3.10 ­Embryology of the Female Genital System The paramesonephric ducts form the basis of the female genital system. In the absence of the SRY gene, default development is down the female pathway. The mesonephric ducts regress and only leave a few remnants, including Gartner’s cysts, paroophoron, and epoophoron. The paramesonephric ducts develop into fallopian tubes, uterus, and the upper two‐thirds of the vagina (Figure 3.46). With regards to external genitalia, the genital tubercle develops into the clitoris, the urogenital sinus forms the introitus and vestibule of the vagina (distal one‐third), the genital folds become the labia minora, and the labioscrotal folds become the labia majora (Figure 3.47). 3.11 Embryology of the Adrenal Gland Fallopian tube Genital tubercle/clitoris Genital folds/labia minora Uterus Ovary Labioscrotal folds/labia majora Vagina Sinovaginal bulb Figure 3.47 Female external genital development: the genital tubercle develops into the clitoris, the genital folds become the labia minora, and the labioscrotal folds become the labia majora. Figure 3.46 Derivatives of the paramesonephric duct in females: fallopian tubes, uterus, and the upper two‐thirds of the vagina. Neural tube Migration of neuroblasts (Week 7) Adrenal cortex Adrenal medulla Fetal cortex arising from genitourinary ridge (Week 5) Genitourinary ridge Figure 3.48 Migration of neuroectodermal cells from neural crest. 3.11 ­Embryology of the Adrenal Gland There are two distinct components of the adrenal gland: cortex and medulla. The cortex is derived from the mesoderm. At about the fifth to sixth weeks of life, the foetal cortex develops arising from the genitourinary ridge, subsequently surrounded by a second wave of mesothelial cells, which will eventually form the definitive cortex near the developing gonads and kidneys; tiny rests of adrenal cortical tissue are common in the renal cortex, retroperitoneum, and testis as well as the broad ligament near the ovary (Figures 3.48 and 3.49). After birth, the foetal cortex regresses except for its outermost layer, which differentiates into the reticular zone. The adrenal cortex shares many of the enzymes of gonads – notably those for the synthesis of steroids – so that some inborn errors of metabolism affect them both. 71 72 3 Embryology for the Urologist Figure 3.49 The cortex arises in the mesoderm of the ‘intermediate cell mass’, which later forms the genitourinary ridge. Neuroectodermal cells migrate into it from the neural crest to form the medulla. Ectoderm Neural crest Intermediate cell mass Gut Coelom Ingrowth of neuroectoderm Genitourinary ridge Cortex Medulla In the seventh week of foetal life, neuroblasts from the sympathetic system of the neural crest (ectodermal cells) invade the medial aspect of the developing adrenal ­cortex to form the medulla. After a week, they differentiate into sympathicoblasts and pheochromocytes ­containing the intracellular catecholamines, adrenaline, and noradrenaline. Cells derived from the neural crest migrate on each side of the aorta to form the sympathetic chains. It is from these paraganglia cells that extra‐adrenal neuro‐ ectodermal tumours (paragangliomas) arise. Expert Opinion The overall sequence of urogenital development is well established and can explain the majority of congenital malformations that may present to the urologist. Additional details will only be of interest to the subspecialist and the researcher. In foetal life, the adrenals are larger than the kidneys and are still about one‐third of their size at birth. ­References 1 Saaddat, S. (2009). Human embryology and the holy 2 3 4 5 Quran: an overview. International Journal of Health Sciences 3 (1): 103–109. MG Mott The Developing Human, Clinically Oriented Embrology, 5e. Elsevier. Osathanondh, V. and Potter, E.L. (1964). Pathogenesis of polycystic kidney: survey of results of microdissection. Archives of Pathology 77: 502. Stephens FD (1982). Embryopathy of malformations. The Journal of Urology 127: 13. Tilmann, C. and Capel, B. (1999). Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad. Development 126 (13): 2883–2890. 6 Mittwoch, U. (1992). Sex determination and sex reversal: genotype, phenotype, dogma and semantics. Human Genetics 89 (5): 467–479. 7 Behringer, R.R., Finegold, M.J., and Cate, R.L. (1994). Mullerian‐inhibiting substance function during mammalian sexual development. Cell 79 (3): 415–425. 8 Haqq, C.M., King, C.Y., Ukiyama, E. et al. (1994). Molecular basis of mammalian sexual determination: activation of Mullerian inhibiting substance gene expression by SRY. Science 266 (5190): 1494–1500. 9 McElreavey, K., Vilain, E., Abbas, N. et al. (1993). A regulatory cascade hypothesis for mammalian sex determination: SRY represses a negative regulator of male ­ References 10 11 12 13 14 15 16 17 development. Proceedings of the National Academy of Sciences of the United States of America 90 (8): 3368–3372. Heyns, C.F. and Hutson, J.M. (1995). Historical review of theories on testicular descent. The Journal of Urology 153 (3 Pt 1): 754–767. McLoughlin, P.V. and Chisholm, G.D. (1980). Pubic ectopic testicle. British Journal of Urology 52 (2): 164. Wensing, C.J. (1986). Testicular descent in the rat and a comparison of this process in the rat with that in the pig. The Anatomical Record 214 (2): 154–160. Whitaker, R.H. (1992). Undescended testis – the need for a standard classification. British Journal of Urology 70 (1): 1–6. Klin, B., Efrati, Y., Mor, A., and Vinograd, I. (1992). Unilateral hydroureteronephrosis caused by abdomin­ oscrotal hydrocele. The Journal of Urology 148 (2 Pt 1): 384–386. Munsterberg, A. and Lovell‐Badge, R. (1991). Expression of the mouse anti‐mullerian hormone gene suggests a role in both male and female sexual differentiation. Development 113 (2): 613–624. Siitery, P.K. and Wilsond, J.D. (1974). Testosterone fromation and metabolism during male sexual differentiation in the human embryo. The Journal of Clinical Endocrinology and Metabolism 38: 113–125. Cunha, G.R., Fujii, H., Neubauer, B.L. et al. (1983). Epithelial‐mesenchymal interactions in prostatic development, I. Morphological observations on prostatic induction by urogenital sinus mesenchyme in epithelium of adult rodent urinary bladder. The Journal of Cell Biology 96: 1662–1670. 18 Shannon, J.M. and Cunha, G.R. (1983). 19 20 21 22 23 24 25 Autoradiographic localization of androgen binding in the developing mouse prostate. The Prostate 4 (4): 367–373. Lasnitzki, I., Takeda, H., and Mizuno, T. (1989). Autoradiographic studies of androgen‐binding sites in human benign prostatic hyperplasia in vitro. The Journal of Endocrinology 120 (1): 167–170. Takeda, H., Mizuno, T., and Lasnitzki, I. (1985). Autoradiographic studies of androgen‐binding sites in the rat urogenital sinus and postnatal prostate. The Journal of Endocrinology 104 (1): 87–92. Takeda, H. and Chang, C. (1991). Immunohistochemical and in‐situ hybridization analysis of androgen receptor expression during the development of the mouse prostate gland. The Journal of Endocrinology 129 (1): 83–89. Timms, B.G. (2008). Prostate development: a historical perspective. Differentiation; Research in Biological Diversity 76 (6): 565–577. Mittemeyer, B.T. and Haynes, A.L. (2011). Embryology for Urologists. In: Practical Urology: Essential Principles and Practice (ed. C. Chapple and W. Steers), 3–28. London: Springer‐Verlag. Marshall, F.F. (1978). Embryology of the lower genitourinary tract. The Urologic Clinics of North America 5 (1): 3–15. Vaughan, E.D. Jr. and Middleton, G.W. (1975). Pertinent genitourinary embryology: review for practicing urologist. Urology 6 (2): 139–149. 73
