Journal of Pediatric Surgery 49 (2014) 1146–1152 Contents lists available at ScienceDirect Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg Development of the human male urethra: A histochemical study on human embryos Ahmed T. Hadidi a,⁎, Jasmin Roessler b, Wiltrud Coerdt b a Hypospadias Clinic, Pediatric Surgery Dept., Emma and Offenbach Hospitals, Germany b Department of Pediatric Pathology, Institute of Pathology, University Medical Center, Mainz, Germany article info abstract Article history: Purpose: Controversy persists regarding the formation of human penile urethra. The classic fusion theory for Received 21 February 2013 the development of the spongy urethra and ectodermal ingrowth or endodermal transformation theories for Received in revised form 31 December 2013 the development of the glanular urethra do not explain the wide spectrum of anomalies seen in patients with Accepted 4 January 2014 hypospadias. This histological study was made to clarify the mechanism of urethral development. Materials & Methods: 15 human male embryos ranging from 6 to 14 weeks were studied. The phalluses were Key words: examined microscopically and photographed. Tissues were prepared as serial histological sections and Urethra stained with haematoxylin and eosin and with special immuno-histochemical stains. Penis Development Results: 1) The penile urethra: At 6 weeks of gestation, the urethral plate which is solid distally and partially Embryology grooved proximally becomes grooved distally and has fused proximally by 8 weeks. At 14 weeks of gestation; Hypospadias the urethral opening migrates only to the middle of the shaft. 2) The glanular urethra: At the 6th week of gestation, a solid epithelial plate reached the tip of the genital tubercle, and a glans cannot be identified. At the 7th week, a central vacuolation appears and the penile urethral groove does not reach the tip of the phallus. At the 8th week; coronal sulcus starts to appear, and a well defined blind central canal was evident in the 13th week. During the 14th week, the floor of the glanular canal degenerated to form a glanular groove. Conclusions: Our observations suggest that the spongy urethra passes through 3 stages of development: a solid epithelial plate, deep urethral groove, and fused urethra. The glanular urethra passes through 4 developmental stages: a solid epithelial plate, a blind central canal, a deep glanular groove, and the floor from the preputial lamella. There was no evidence of ectodermal ingrowth. These observations raise serious questions to the current theories for human urethra development. Further studies on fresh human embryos are needed. © 2014 Elsevier Inc. All rights reserved. The development of the human urethra has been a controversial anus? And why don’t we see skipped segment of fusion (i.e. fused subject in embryology for years [1]. Glenister stated that the penile urethra proximally and distally with a skipped segment in-between) urethra arises by fusion of the primitive urethral groove and the in hypospadias as we see in the case of fusion of the spinal cord (e.g. in secondary urethral groove that develops from the urethral plate [2].In meningocoele)? contrast, Van der Putte and Neeteson concluded that the male penile Similarly, controversy continues to exist regarding the formation urethra is formed by a movement in ventral direction of the urogenital of the glanular urethra [1]. Glenister [2] suggested a dual embryolog- opening relative to the growing perineum and not by fusion of the ical origin with the proximal part of the glanular urethra forming in a genital folds [3]. Kluth et al. could not find evidence of fusion [4,5]. similar way to the penile urethra from the endodermal urethral plate, If the urethra develops through simple fusion (according to the while the terminal part arises from an ectodermal invagination from current classic theory), why do we see chordee? How can torsion the terminal epithelial tag at the tip of the glans — a description that occur? Why do we see variable width and depth of the urethral plate? remains in contemporary embryology textbooks today [6,7].An Why there is a wide range of corpus spongiosum hypoplasia and alternative model proposes that the entire urethra forms from dysplasia in hypospadias? Why there is a variable degree of glans endoderm which undergoes differentiation in glanular portion to clefting (i.e. grooving)? How can simple fusion explain the presence of form squamous epithelium [8,9,2]. Neither model explains the wide ano-cutaneuos tract on the ventral penis in patients with imperforate spectrum of glanular hypospadias that is encountered in clinical practice with different degrees of glans clefting, blind glanular canal and glanular ridge. A clear understanding of the normal development ⁎ Corresponding author at: Max-Planck- Str 2, D-63500 Seligentadt, Germany. of the urethra is important in the elucidation of its malformations [10]. Tel.: +49 174 205 6905; fax: +49 618 28 430 293. E-mail addresses: [email protected], [email protected] The aim of the study is to try to clarify the mechanism of human (A.T. Hadidi). urethra development, based on the principle that the urethra http://dx.doi.org/10.1016/j.jpedsurg.2014.01.009 0022-3468/© 2014 Elsevier Inc. All rights reserved. A.T. Hadidi et al. / Journal of Pediatric Surgery 49 (2014) 1146–1152 1147 with embryos of tolerably exact developmental ages as described by Streeter [11]. All the embryos and foetuses were examined and photographed under magnification using stereomicroscope MZ 16A manufactured by Leica. They were treated in 80% filtered isopropyl alcohol after being fixed in 4% formaldehyde according to the method described by Fahr et al. [12]. Specimens that included the external genitalia, anus and perineum were processed through the standard sequence of dehydration and paraffin embedding. Serial sagittal histological sections were obtained at 3-μm intervals and stained with haematoxylin and eosin and van Gieson stains (Fig. 1). On average, 200 sections were obtained from each specimen. Sections were retained at variable intervals for specific immuno-histochemical stains. The interval depended on the size of the specimens and the importance of the region. The following antibodies were used according to standard immuno-histochemical methods with the VECTASTAIN Elite ABC PEROXYDASE Kit Standard (VC-PK-6100-KI01 AXXORA): 1) Vimentin (Monoclonal Mouse Anti-Vimentin,. Clone V9, 1 ml, M072501, DAKO): diluted 1:200 PBS–BSA, used to label cells of mesenchymal origin Fig. 1. Design of the Study: Sagittal sections were made through the developing penis 2) MIB-1 (monoclonal antibody that reacts with the Ki-67 nuclear during the active period of urethral development. antigen, 1 ml, 7240, DAKO): diluted 1:150 PBS–BSA, used to The study is based on the principle that the urethra develops from proximal to distal. The aim is to record step by step the dynamic process during the critical active period of label proliferating cells, urethral development. Examination of the serial individual sections from distal to 3) Cytokeratin: a combination of AE1/3 (Monoclonal mouse anti- proximal can serve as single films of a moving urethral development. Development of human Cytokeratin, clones AE1/AE3 (500 μg MAB 3412, the glanular urethra goes parallel to that of the spongy urethra: CHEMICON) (diluted 1:100) and CAM 5,2 (BECTON DICKINSON 1) The spongy urethra: The study documented the actual fusion point of the anticytokeratin)(1:25): 100 μl PBS–BSA + 1 μl AE 1/3 + 4 μl spongy urethra (point e). The sections distal to it would show the gradual steps CAM 5,2 that is used to label epithelial cells. of fusion (point d) and sections proximal to it would show an immediately fused urethra (point f). In this way, histological sections of a single embryo Positive and negative controls were done by routine procedures of during active development can show several developmental stages of the patients’ material. Controls on embryos genital area were avoided in spongy urethra (embryos 2, 7, 14). order not to miss serious section slides. 2) The glanular urethra: During the critical active period of development, The dye that labels specifically the apoptotic nuclei (terminal histological sections at the middle of the glans (point b) would document a transferase nuclear end labelling, TUNEL method) was used in one developmental stage that is later than that of the distal glans (point a) and earlier than that of the proximal glans (point c). The aim is to record step by step the embryo but was not conclusive probably due to the fact that the dynamic changes in the glans during active development (embryos 3, 11, 13). specimen was not fresh. For descriptive purposes, the floor of the urethra means the ventral develops from proximal to distal. The present study was designed to urethral surface and roof of the urethra means dorsal surface of the record step by step the dynamic process of urethral development. urethra in an erect penis (Fig. 1). Examination of the serial individual sections from distal to proximal during the critical active period of development can serve as single 2. Results & Observations films of a moving urethral development (Fig. 1). With the help of specific immunohistochemical stains, we attempt Examination of the 15 specimens showed the active sequence of to observe and document the actual fusion point of the urethra (Fig. 1, developmental changes of the human urethra which was persistent in point e). The sections distal to it would show the unfused urethral groove (Fig. 1, point d) and the sections proximal to it would show the gradual steps of fusion (Fig. 1, f). Similarly, histological sections at the Table 1 middle of the glans (Fig. 1b) would document a developmental stage The 15 embryos included in the present study. Their ages ranged from 6 to 14 weeks that is later than that of the distal glans(Fig.