Purpose We describe the “double zipper” mechanism of human male urethral formation, where the distal zipper opens the urethral groove through canalization of the urethral plate, and a second closing zipper follows behind and closes the urethral groove to form the tubular urethra. Materials and Methods Anonymous human fetal genital specimens were acquired and gender was determined by polymerase chain reaction of the Y chromosome. Specimens were processed for optical projection tomography, stained with E-cadherin, Ki67 and caspase 3, and imaged. Results Eight developing male fetal specimens from 6.5 to 16.5 weeks of gestation were analyzed by optical projection tomography, and an additional 5 specimens by serial sections. Phallus length ranged from 1.3 to 3.7 mm. The urethral plate canalized into a groove with 2 epithelial edges that subsequently fused. Ki67 staining was localized to the dorsal aspect of the urethral plate. In contrast, caspase 3 staining was not observed. The entire process was completed during a 10-week period. Conclusions The human male urethra appears to form by 2 mechanisms, an initial “opening zipper” that facilitates distal canalization of the solid urethral plate to form the urethral groove, which involves a high rate of epithelial proliferation (apoptosis not observed), and a “closing zipper” facilitating fusion of the 2 epithelial surfaces of the urethral groove, and thus extending the penile urethra distally. Improved knowledge of the molecular mechanisms of these processes is critical to understanding mechanisms of abnormal urethral development, such as hypospadias.

Human female reproductive tract development rests mostly upon hematoxilyn and eosin stained sections despite recent advances on molecular mechanisms in mouse studies. We report application of immunohistochemical methods to explore the ontogeny of epithelial and mesenchymal differentiation markers (keratins, homobox proteins, steroid receptors), transcription factors and signaling molecules (TP63 and RUNX1) during human female reproductive tract development. Keratins 6, 7, 8, 10, 14 and 19 (KRT6, KRT7, KRT8, KRT10, KRT14, KRT19) were expressed in a temporally and spatially dynamic fashion. The undifferentiated Müllerian duct and uterovaginal canal, lined by simple columnar epithelia, expressed KRT7, KRT8 and KRT19. Glandular derivatives of the Müllerian duct (uterine tube, uterine corpus and endocervix) maintained expression of these keratins, while tissues that undergo stratified squamous differentiation (exocervix and vagina) expressed KRT6, KRT14 and KRT10 during development in an age-dependent fashion. TP63 and RUNX1 were expressed prior to KRT14, as these two transcription factors are known to be upstream from KRT14 in developing Müllerian epithelium. In the vagina, KRT10, a marker of terminal differentiation, appeared after endogenous estrogens transformed the epithelium to a thick glycogenated squamous epithelium. Uroplakin, a protein unique to urothelium, was expressed only in the bladder, urethra and vaginal introitus, but not in the female reproductive tract itself. Mesenchymal differentiation was examined through immunostaining for HOXA11 (expressed in uterine mesenchyme) and ISL1 (expressed in vaginal mesenchyme). A detailed ontogeny of estrogen receptor alpha (ESR1), progesterone receptor (PGR) and the androgen receptor (AR) provides the mechanistic underpinning for the teratogenicity of estrogens, progestins and androgens on female reproductive tract development. Immunohistochemical analysis of differentiation markers and signaling molecules advance our understanding of normal development of the human female reproductive tract. These observations demonstrate remarkable similarities in mouse and human female reproductive tract development, but also highlight some key differences.

The human penis and clitoris develop from the ambisexual genital tubercle. To compare and contrast the development of human penis and clitoris, we used macroscopic photography, optical projection tomography, light sheet microscopy, scanning electron microscopy, histology and immunohistochemistry. The human genital tubercle differentiates into a penis under the influence of androgens forming a tubular urethra that develops by canalization of the urethral plate to form a wide diamond-shaped urethral groove (opening zipper) whose edges (urethral folds) fuse in the midline (closing zipper). In contrast, in females, without the influence of androgens, the vestibular plate (homologue of the urethral plate) undergoes canalization to form a wide vestibular groove whose edges (vestibular folds) remain unfused, ultimately forming the labia minora defining the vaginal vestibule. The neurovascular anatomy is similar in both the developing human penis and clitoris and is the key to successful surgical reconstructions.

Development of the human female reproductive tract is reviewed from the ambisexual stage to advanced development of the uterine tube, uterine corpus, uterine cervix and vagina at 22 weeks. Historically this topic has been under-represented in the literature, and for the most part is based upon hematoxylin and eosin stained sections. Recent immunohistochemical studies for PAX2 (reactive with Müllerian epithelium) and FOXA1 (reactive with urogenital sinus epithelium and its known pelvic derivatives) shed light on an age-old debate on the derivation of vaginal epithelium supporting the idea that human vaginal epithelium derives solely from urogenital sinus epithelium. Aside for the vagina, most of the female reproductive tract is derived from the Müllerian ducts, which fuse in the midline to form the uterovaginal canal, the precursor of uterine corpus and uterine cervix an important player in vaginal development as well. Epithelial and mesenchymal differentiation markers are described during human female reproductive tract development (keratins, homeobox proteins (HOXA11 and ISL1), steroid receptors (estrogen receptor alpha and progesterone receptor), transcription factors and signaling molecules (TP63 and RUNX1), which are expressed in a temporally and spatially dynamic fashion. The utility of xenografts and epithelial-mesenchymal tissue recombination studies are reviewed.

The urethra within the human penile shaft develops via (1) an "Opening Zipper" that facilitates distal canalization of the solid urethral plate to form a wide urethral groove and (2) a "Closing Zipper" that facilitates fusion of the epithelial surfaces of the urethral folds. Herein, we extend our knowledge by describing formation of the human urethra within the glans penis as well as development of the prepuce. Forty-eight normal human fetal penile specimens were examined using scanning electron microscopy and optical projection tomography. Serial histologic sections were evaluated for morphology and immunohistochemical localization for epithelial differentiation markers: Cytokeratins 6, 7, 10, FoxA1, uroplakin and the androgen receptor. As the closing zipper completes fusion of the urethral folds within the penile shaft to form a tubular urethra (~ 13 weeks), canalization of the urethral plate continues in proximal to distal fashion into the glans penis to directly form the urethra within the glans without forming an open urethral groove. Initially, the urethral plate is attached ventrally to the epidermis via an epithelial seam, which is remodeled and eliminated, thus establishing mesenchymal confluence ventral to the glanular urethra. The morphogenetic remodeling involves the strategic expression of cytokeratin 7, FoxA1 and uroplakin in endodermal epithelial cells as the tubular glanular urethra forms. The most ventral epithelial cells of the urethral plate are pinched off from the glanular urethra and are reabsorbed into the epidermis ultimately losing expression of their markers, a process undoubtedly regulated by androgens. The prepuce initially forms on the dorsal aspect of the glans at approximately 12 weeks of gestation. After sequential proximal to distal remodeling of the ventral urethral plate along the ventral aspect of glans, the prepuce of epidermal origin fuses in the ventral midline.