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Introduction: The presence of an ovotestis is a rare difference of sex development. The diagnosis can be difficult with the gold standard being the presence of both testicular cords and ovarian follicles within the same gonad., Objective: Herein we describe two new markers of ovotesticular syndrome: ovotesticular cords and ovotesticular follicles., Study Design: Twenty human gonads with a previous diagnosis of ovotestis were re-stained with markers for testicular cords (SOX9, TSPY, SALL4, DDX4, cP450, AR, α-actin) and ovarian tissue (FOXL2, SALL4, DDX4). Ovotesticular cords were defined as structures expressing both testicular Sertoli cell marker (SOX9) and an ovarian follicular cell marker (FOXL2), and in Y chromosome positive specimens, TSPY-positive testicular germ cells. Ovotesticular follicles were defined as a hybrid ovarian follicle containing FOXL2-positive granulosa cells and a central oocyte, but also containing cells expressing the testicular Sertoli cell marker, SOX9, intermingled within FOXL2-positive granulosa cells and male and female germ cells., Results: Six of twenty ovotestis did not meet our criterion for the diagnosis of ovotestis lacking the histologic evidence of both testicular and ovarian tissue. The remaining 13 patients in which 14 separate specimens were evaluated, contained ovotestis defined by the presence of testicular cords and ovarian follicles. Eleven of the 14 ovotestis specimens (79 %) contained ovotesticular cords. Four of 11 ovotestis specimens (36 %) contained ovotesticular follicles., Discussion: We recommend using eight immunohistochemical markers to diagnose an ovotestis: 1) SOX9, TSPY, SALL4, DDX4, cytochrome P450, AR, smooth muscle α-actin for the testicular component and FOXL2 and SALL4, DDX4 for the ovarian component. SOX9 and TSPY (useful only in the presence of a Y karyotype) are specific testicular markers and FOXL2 the only specific ovarian marker. We found ovotesticular cords and ovotesticular follicles in both human bipolar and mixed ovotestis specimens both with and without the presence of the Y chromosome. The clinical significance of ovotesticular cords and follicles remains unknown. We did not observe any obvious abnormalities in cellular architecture with the juxtaposition of testicular cells and ovarian cells., Conclusion: We have identified two new structures in humans with ovotestis, ovotesticular cords and ovotesticular follicles (Figure), which appears to be additional markers to facilitate the diagnosis of ovotesticular gonads., Competing Interests: Conflicts of interest None., (Copyright © 2024 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Benign prostatic hyperplasia (BPH) is the nodular proliferation of the prostate transition zone in older men, leading to urinary storage and voiding problems that can be recalcitrant to therapy. Decades ago, John McNeal proposed that BPH originates with the "reawakening" of embryonic inductive activity by adult prostate stroma, which spurs new ductal proliferation and branching morphogenesis. Here, by laser microdissection and transcriptional profiling of the BPH stroma adjacent to hyperplastic branching ducts, we identified secreted factors likely mediating stromal induction of prostate glandular epithelium and coinciding processes. The top stromal factors were insulin-like growth factor 1 (IGF1) and CXC chemokine ligand 13 (CXCL13), which we verified by RNA in situ hybridization to be coexpressed in BPH fibroblasts, along with their cognate receptors (IGF1R and CXCR5) on adjacent epithelium. In contrast, IGF1 but not CXCL13 was expressed in human embryonic prostate stroma. Finally, we demonstrated that IGF1 is necessary for the generation of BPH-1 cell spheroids and patient-derived BPH cell organoids in 3D culture. Our findings partially support historic speculations on the etiology of BPH and provide what we believe to be new molecular targets for rational therapies directed against the underlying processes driving BPH.

Female mice were treated for 35 days from birth to 60 days postnatal (P0, [birth], P5, P10, P20 and adult [∼P60]) with dihydrotestosterone (DHT). Such treatment elicited profound masculinization the female external genitalia and development of penile features (penile spines, male urogenital mating protuberance (MUMP) cartilage, corpus cavernosum glandis, corporal body, MUMP-corpora cavernosa, a large preputial space, internal preputial space, os penis). Time course studies demonstrated that DHT elicited canalization of the U-shaped clitoral lamina to create a U-shaped preputial space, preputial lining epithelium and penile epithelium adorned with spines. The effect of DHT was likely due to signaling through androgen receptors normally present postnatally in the clitoral lamina and associated mesenchyme. This study highlights a remarkable male/female difference in specification and determination of urogenital organ identity. Urogenital organ identity in male mice is irreversibly specified and determined prenatally (prostate, penis, and seminal vesicle), whereas many aspects of the female urogenital organogenesis are not irreversibly determined at birth and in the case of external genitalia are not irreversibly determined even into adulthood, the exception being positioning of the female urethra, which is determined prenatally., (Copyright © 2023 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

We present a comprehensive description of the differentiating somatic cell types (Sertoli, Leydig, and peritubular myoid cells) of the mouse testis from embryonic day 10.5 (E10.5) to adulthood, postnatal day 60 (P60). Immunohistochemistry was used to analyze expression of: Sox9 (a Sertoli cell marker), 3βHSD-1 (a fetal Leydig cell marker), 3βHSD-6 (an adult Leydig cell marker), α-actin (a peritubular myoid cell marker), and androgen receptor (a marker of all three somatic cell types). The temporal-spatial expression of these markers was used to interrogate findings of earlier experimental studies on the origin of Sertoli, Leydig and peritubular myoid cells, as well as extend previous descriptive studies across a broader developmental period (E10.5-P60). Such comparisons demonstrate inconsistencies that require further examination and raise questions regarding conservation of developmental mechanisms across higher vertebrate species., (Copyright © 2022 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

A definition of normal human fetal and early postnatal ovarian development is critical to the ability to accurately diagnose the presence or absence of functional ovarian tissue in clinical specimens. Through assembling an extensive histologic and immunohistochemical developmental ontogeny of human ovarian specimens from 8 weeks of gestation through 16 years of postnatal, we present a comprehensive immunohistochemical mapping of normal protein expression patterns in the early fetal through post-pubertal human ovary and detail a specific expression-based definition of the early stages of follicular development. Normal fetal and postnatal ovarian tissue is defined by the presence of follicular structures and characteristic immunohistochemical staining patterns, including granulosa cells expressing Forkhead Box Protein L2 (FOXL2). However, the current standard array of immunohistochemical markers poorly defines ovarian stromal tissue, and additional work is needed to identify new markers to advance our ability to accurately identify ovarian stromal components in gonadal specimens from patients with disorders of sexual differentiation., (Copyright © 2022 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Human gonadal development culminating in testicular differentiation is described through analysis of histologic sections derived from 33-day to 20-week human embryos/fetuses, focusing on early development (4-8 weeks of gestation). Our study updates the comprehensive studies of Felix (1912), van Wagenen and Simpson (1965), and Juric-Lekic et al. (2013), which were published in books and thus are unsearchable via PubMed. Human gonads develop from the germinal ridge, a thickening of coelomic epithelium on the medial side of the urogenital ridge. The bilateral urogenital ridges contain elements of the mesonephric kidney, namely the mesonephric duct, mesonephric tubules, and mesonephric glomeruli. The germinal ridge, into which primordial germ cells migrate, is initially recognized as a thickening of coelomic epithelium on the urogenital ridge late in the 4th week of gestation. Subsequently, in the 5th week of gestation, a dense mesenchyme develops sub-adjacent to the epithelium of the germinal ridge, and together these elements bulge into the coelomic cavity forming bilateral longitudinal ridges attached to the urogenital ridges. During development, primordial cells migrate into the germinal ridge and subsequently into testicular cords that form within the featureless dense mesenchyme of the germinal ridge at 6-8 weeks of gestation. The initial low density of testicular cords seen at 8 weeks remodels into a dense array of testicular cords surrounded by α-actin-positive myoid cells during the second trimester. Human testicular development shares many features with that of mice being derived from 4 elements: coelomic epithelium, sub-adjacent mesenchyme, primordial germ cells, and the mesonephros., (Copyright © 2022 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The mouse has been used as a model of human organogenesis with the tacit assumption that morphogenetic and molecular mechanisms in mice are translatable to human organogenesis. While many morphogenetic and molecular mechanisms are shared in mice and humans, many anatomic, morphogenetic, and molecular differences have been noted. Two critical gaps in our knowledge prevent meaningful comparisons of mouse versus human testicular development: (a) human testicular development is profoundly under-represented in the literature, and (b) an absence of a detailed day-by-day ontogeny of mouse testicular development from E11.5 to E16.5 encompassing the ambisexual stage to seminiferous cord formation. To address these deficiencies, histologic and immunohistochemical studies were pursued in comparable stages of mouse and human testicular development with a particular emphasis on Leydig, Sertoli and myoid cells through review of the literature and new observations. For example, an androgen-receptor-positive testicular medulla is present in the developing human testis but not in the developing mouse testis. The human testicular medulla and associated mesonephros were historically described as the source of Sertoli cells in seminiferous cords. Consistent with this idea, the profoundly androgen receptor (AR)-positive human testicular medulla was shown to be a zone of mesenchymal to epithelial transition and a zone from which AR-positive cells appear to migrate into the human testicular cortex. While mouse Sertoli and Leydig cells have been proposed to arise from coelomic epithelium, Sertoli (SOX9) or Leydig (HSD3B1) cell markers are absent from the immediate coelomic zone of the developing human testis, perhaps because Leydig and Sertoli cell precursors are undifferentiated when they egress from the coelomic epithelium. The origin of mouse and human myoid cells remains unclear. This study provides a detailed comparison of the early stages of testicular development in human and mouse emphasizing differences in developmental processes., (Copyright © 2022 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The role of the mesonephros in testicular development was re-evaluated by growing embryonic day 11.5 (E11.5) mouse testes devoid of mesonephros for 8-21 days in vivo under the renal capsule of castrated male athymic nude mice. This method provides improved growth conditions relative to previous studies based upon short-term (4-7 days) organ culture. Meticulous controls involved wholemount examination of dissected E11.5 mouse testes as well as serial sections of dissected E11.5 mouse testes which were indeed shown to be devoid of mesonephros. As expected, grafts of E11.5 mouse testes with mesonephros attached formed seminiferous tubules and also contained mesonephric derivatives. Grafts of E11.5 mouse testes without associated mesonephros also formed seminiferous tubules and never contained mesonephric derivatives. The consistent absence of mesonephric derivatives in grafts of E11.5 mouse testes grafted alone is further proof of the complete removal of the mesonephros from the E11.5 mouse testes. The testicular tissues that developed in grafts of E11.5 mouse testes alone contained canalized seminiferous tubules composed of Sox9-positive Sertoli cells as well as GENA-positive germ cells. The seminiferous tubules were surrounded by α-actin-positive myoid cells, and the interstitial space contained 3βHSD-1-positive Leydig cells. Grafts of E11.5 GFP mouse testes into wild-type hosts developed GFP-positive vasculature indicating that E11.5 mouse testes contain vascular precursors. These results indicate that the E11.5 mouse testis contains precursor cells for Sertoli cells, Leydig cells, myoid cells and vasculature whose development and differentiation are independent of cells migrating from the E11.5 mesonephros., (Copyright © 2021 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

A comprehensive immunohistochemical ontogeny of the developing human fetal testis has remained incomplete in the literature to date. We collected human fetal testes from 8 to 21 weeks of fetal age, as well as postnatal human testes at minipuberty, pre-pubertal, and pubertal stages. Immunohistochemistry was performed with a comprehensive panel of antigens targeting gonadocytes, Sertoli cells, fetal Leydig cells, peritubular myoid cells, and other hormonal and developmental targets. Testicular cords, precursor structures to seminiferous tubules, developed from 8 to 14 weeks of fetal age, separating the testis into the interstitial and intracordal compartments. Fetal gonadocytes were localized within the testicular cords and evaluated for Testis-Specific Protein Y, Octamer-binding transcription factor 4, Sal-like protein 4, and placental alkaline phosphatase expression. Fetal Sertoli cells were also localized in the testicular cords and evaluated for SRY-box Transcription Factor 9, inhibin, and anti-Mullerian hormone expression. Fetal Leydig cells were present in the interstitium and stained for cytochrome p450c17 and calretinin, while interstitial peritubular myoid cells were examined using smooth muscle α-actin staining. Androgen receptor expression was localized close to the testicular medulla at 8 weeks and then around the testicular cords in the interstitium as they matured in structure. Postnatal staining showed that Testis-Specific Protein Y remained positive of male gonadocytes throughout adulthood. Anti-Mullerian hormone, SRY-box Transcription Factor 9, and Steroidogenic factor 1 are expressed by the postnatal Sertoli cells at all ages examined. Leydig cell markers cytochrome p450c17 and calretinin are expressed during mini-puberty and puberty, but not expressed during the pre-pubertal period. Smooth muscle α-actin and androgen receptor were not expressed during mini-puberty or pre-puberty, but again expressed during the pubertal period. The ontogenic map of the human fetal and postnatal testicular structure and expression patterns described here will serve as a reference for future investigations into normal and abnormal testicular development., (Copyright © 2022 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Stromal androgen-receptor (AR) action is essential for prostate development, morphogenesis and regeneration. However, mechanisms underlying how stromal AR maintains the cell niche in support of pubertal prostatic epithelial growth are unknown. Here, using advanced mouse genetic tools, we demonstrate that selective deletion of stromal AR expression in prepubescent Shh-responsive Gli1-expressing cells significantly impedes pubertal prostate epithelial growth and development. Single-cell transcriptomic analyses showed that AR loss in these prepubescent Gli1-expressing cells dysregulates androgen signaling-initiated stromal-epithelial paracrine interactions, leading to growth retardation of pubertal prostate epithelia and significant development defects. Specifically, AR loss elevates Shh-signaling activation in both prostatic stromal and adjacent epithelial cells, directly inhibiting prostatic epithelial growth. Single-cell trajectory analyses further identified aberrant differentiation fates of prostatic epithelial cells directly altered by stromal AR deletion. In vivo recombination of AR-deficient stromal Gli1-lineage cells with wild-type prostatic epithelial cells failed to develop normal prostatic epithelia. These data demonstrate previously unidentified mechanisms underlying how stromal AR-signaling facilitates Shh-mediated cell niches in pubertal prostatic epithelial growth and development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Mouse penile development is androgen-dependent. During development of male and female external genitalia, an internal ectodermal epithelial structure forms called the preputial lamina. At puberty the male preputial lamina canalizes to create the preputial space, effectively splitting into two layers: (a) the epithelial lining of the prepuce and (b) the surface epithelium of the penis. The female preputial lamina does not canalize, and instead remodels into the inverted U-shaped clitoral lamina of the adult female mouse. Androgen-dependent penile development was studied in transgenic mice with pathway-selective AR mutant transgenes through which AR signaling was activated either via the classical (AR-C) or the nonclassical pathway (AR-NC). Penile development and canalization of the preputial lamina was observed in AR-C and wild-type male mice naturally having both AR-C and AR-NC pathways. Conversely, clitoral development occurred in AR null (lacking both AR-C and AR-NC pathways) and AR-NC mice. The process of canalization of the preputial lamina seen in wild-type, AR-C and AR-C/AR-NC male mice involved cornification of the preputial lamina which involved up-regulation of keratin 10 and loricrin. Such up-regulation of these epidermal proteins was absent in the developing and adult clitoral lamina seen in wild-type female mice and AR-NC and AR null male (XY) mice. Thus, signaling through AR-C is sufficient to initiate and promote penile development and canalization of the preputial lamina, a process involving epithelial cornification., (Copyright © 2021 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Objective: To determine whether human fetal urogenital sinus mesenchyme (UGM) can induce prostatic development in a responsive mouse epithelium., Method: Male and female human fetal UGM was combined with mouse urinary bladder epithelium (BLE), and the resultant human UGM + mouse BLE tissue recombinants were grown under renal capsules of male athymic mice. Human male and female UGM was derived from reproductive tracts 9 and 14 weeks of gestation obtained following elective termination of pregnancy. At these ages prostatic ducts had already emerged from the urogenital sinus epithelium, and the human UGM remained contaminated with human prostatic epithelium. This unavoidable problem was tolerated because the induced mouse prostatic epithelium could be distinguished from contaminating human prostatic epithelium., Results: The simple columnar epithelium induced from mouse bladder epithelium by human male and female UGM resembled mouse prostatic epithelium by: (a) histology, (b) the pattern of basal cell distribution, (c) Hoechst dye nuclear staining, (d) expression of NKX3.1, (e) the pattern of androgen receptor expression and (f) the expression of probasin, a mouse prostatic secretory protein. Summary/Interpretation: These findings provide validation for mouse as a model of human prostatic development as the molecular dialogue involved in mesenchymal-epithelial interactions are sufficiently conserved that human UGM can induce mouse bladder epithelium to undergo prostatic development., Competing Interests: None., (AJCEU Copyright © 2021.)

The Jost hypothesis states that androgens are necessary for normal development of the male external genitalia. In this review, we explore the complementary hypothesis that estrogens can elicit abnormal development of male external genitalia. Herein, we review available data in both humans and mice on the deleterious effects of estrogen on external genitalia development, especially during the "window of susceptibility" to exogenous estrogens. The male and female developing external genitalia in both the human and mouse express ESR1 and ESR2, along with the androgen receptor (AR). Human clinical data suggests that exogenous estrogens can adversely affect normal penile and urethral development, resulting in hypospadias. Experimental mouse data also strongly supports the idea that exogenous estrogens cause penile and urethral defects. Despite key differences, estrogen-induced hypospadias in the mouse displays certain morphogenetic homologies to human hypospadias, including disruption of urethral fusion and preputial abnormalities. Timing of estrogenic exposure, or the "window of susceptibility," is an important consideration when examining malformations of the external genitalia in both humans and mice. In addition to a review of normal human and mouse external genital development, this article aims to review the present data on the role of estrogens in normal and abnormal development of the mouse and human internal and external genitalia. Based on the current literature for both species, we conclude that estrogen-dependent processes may play a role in abnormal genital development., (Copyright © 2020 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

This paper reviews and provides new observations on the ontogeny of estrogen receptor alpha (ESR1) and estrogen receptor beta (ESR2) in developing human male and female internal and external genitalia. Included in this study are observations on the human fetal uterine tube, the uterotubal junction, uterus, cervix, vagina, penis and clitoris. We also summarize and report on the ontogeny of estrogen receptors in the human fetal prostate, prostatic urethra and epididymis. The ontogeny of ESR1 and ESR2, which spans from 8 to 21 weeks correlates well with the known "window of susceptibility" (7-15 weeks) for diethylstilbestrol (DES)-induced malformations of the human female reproductive tract as determined through examination of DES daughters exposed in utero to this potent estrogen. Our fairly complete mapping of the ontogeny of ESR1 and ESR2 in developing human male and female internal and external genitalia provides a mechanistic framework for further investigation of the role of estrogen in normal development and of abnormalities elicited by exogenous estrogens., (Copyright © 2020 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

This paper addresses a confusing issue of preputial anatomy of the mouse. The term "internal prepuce" was used in 2013 to describe a preputial structure integral to the mouse glans penis. Subsequently in 2015 the same term was applied by another group to describe entirely different morphology, generating confusion in the literature. Because it is inappropriate to use the same term to describe entirely different structures, we take this opportunity to provide further descriptive information on the internal prepuce of the mouse employing gross dissection, analysis of serial histologic section sets, three-dimensional reconstruction, scanning electron microscopy and immunohistochemistry. For this purpose, we review and illustrate the relevant literature and provide some additional new data using standard morphological techniques including immunohistochemistry. The mouse internal prepuce is integral to the glans penis and clearly is involved in sexual function in so far as it contains a major erectile body innervated by penile nerves. The development of the mouse internal prepuce is described for the first time and related to the development of the corpus cavernosum glandis., (Copyright © 2020 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The spotted hyaena (Crocuta crocuta) is a unique species, even amongst the Hyaenidae. Extreme clitoral development in female spotted hyaenas challenges aspects of the accepted framework of sexual differentiation and reproductive function. They lack a vulva and instead urinate, copulate and give birth through a single, long urogenital canal that traverses a clitoris superficially resembling a penis. Recent and historical evidence is reviewed to describe our changing understanding of the biology of this species. Expanding upon observations from hyaenas in nature, much has been learned from studies utilising the captive colony at the University of California, Berkeley. The steroid environment of pregnancy is shaped by placental androgen and oestrogen secretion and a late gestational increase in sex hormone binding globulin, the regulated expression and steroid-binding characteristics of which are unique within the Hyaenidae. While initial external genital development is largely free of androgenic influence, the increase in testosterone concentrations in late gestation influences foetal development. Specifically, anti-androgen (AA) treatment of pregnant females reduced the developmental influence of androgens on their foetuses, resulting in reduced androstenedione concentrations in young females and easier birth through a 'feminised' clitoris, but precluded intromission and mating by 'feminised' male offspring, and altered social interactions. Insight into the costs and benefits of androgen exposure on spotted hyaena reproductive development, endocrinology and behaviour emphasises the delicate balance that sustains reproductive success, forces a re-evaluation of how we define masculine vs feminine sexual characteristics, and motivates reflection about the representative value of model species.

The goal of this paper is to explore the ability of the human female urogenital sinus immediately below the bladder (proximal urethra) to undergo prostatic development in response to dihydrotestosterone (DHT). To establish this idea, xenografts of human fetal female proximal urethra were grown in castrated nude mouse hosts receiving a subcutaneous DHT pellet. To verify the prostatic nature of the resultant glands, DHT-treated human fetal female urethral xenografts were compared with human fetal prostatic xenografts (derived from male specimens) grown in untreated and DHT-treated castrated mouse hosts and human fetal female proximal urethral xenografts grown in untreated castrated hosts. The resultant glands observed in DHT-treated human fetal female proximal urethral xenografts expressed 3 prostate-specific markers, NKX3.1, prostate specific antigen and prostatic acid phosphatase as well as the androgen receptor. Glands induced by DHT exhibited a protein expression profile of additional immunohistochemical markers (seven keratins, RUNX1, ESR2, TP63 and FOXA1) consistent with the unique spatial pattern of these proteins in prostatic ducts. Xenografts of human fetal female proximal urethra grown in DHT-treated hosts also expressed one of the salient features of prostatic development, namely androgen responsiveness. The experimental induction of prostatic differentiation from human fetal female proximal urethra makes possible future in-depth analysis of the molecular pathways directly involved in initiation of human prostatic development and subsequent epithelial differentiation, and more important whether the molecular pathways involved in human prostatic development are similar/identical versus different from that in murine prostatic development., (Copyright © 2020 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

To better understand how the human fetal penis and clitoris grows and remodels, we undertook an investigation to define active areas of cellular proliferation and programmed cell death spatially and temporally during development of human fetal external genitalia from the indifferent stage (8 weeks) to 18 weeks of gestation. Fifty normal human fetal penile and clitoral specimens were examined using macroscopic imaging, scanning electron microscopy and immunohistochemical localization for the cellular proliferation and apoptotic markers, Ki67 and Caspase-3, respectively. A number of hot spots of cellular proliferation characterized by Ki67 localization are present in the penis and clitoris especially early in development, most notably in the corporal body, glans, remodeling glanular urethra, the urethral plate, the roof of the urethral groove and the fully formed penile urethra. The 12-fold increase in penile length over 10 weeks of growth from 8 to 18 weeks of gestation based on Ki67 labelling appears to be driven by cellular proliferation in the corporal body and glans. Throughout all ages in both the developing penis and clitoris Ki67 labeling was consistently elevated in the ventral epidermis and ventral mesenchyme relative to the dorsal counterparts. This finding is consistent with the intense morphogenetic activity/remodeling in the ventral half of the genital tubercle in both sexes involving formation of the urethral/vestibular plates, canalization of the urethral/vestibular plates and fusion of the urethral folds to form the penile urethra. Areas of reduced or absent Ki67 staining include the urethral fold epithelium that fuses to form the penile tubular urethra. In contrast, the urethral fold mesenchyme is positive for Ki67. Apoptosis was rarely noted in the developing penis and clitoris; the only area of minimal Caspase-3 localization was in the epithelium of the ventral epithelial glanular channel remodeling., (Copyright © 2019. Published by Elsevier B.V.)

Prostate embryonic development, pubertal and adult growth, maintenance, and regeneration are regulated through androgen signaling-mediated mesenchymal-epithelial interactions. Specifically, the essential role of mesenchymal androgen signaling in the development of prostate epithelium has been observed for over 30 years. However, the identity of the mesenchymal cells responsible for this paracrine regulation and related mechanisms are still unknown. Here, we provide the first demonstration of an indispensable role of the androgen receptor (AR) in sonic hedgehog (SHH) responsive Gli1-expressing cells, in regulating prostate development, growth, and regeneration. Selective deletion of AR expression in Gli1-expressing cells during embryogenesis disrupts prostatic budding and impairs prostate development and formation. Tissue recombination assays showed that urogenital mesenchyme (UGM) containing AR-deficient mesenchymal Gli1-expressing cells combined with wildtype urogenital epithelium (UGE) failed to develop normal prostate tissue in the presence of androgens, revealing the decisive role of AR in mesenchymal SHH responsive cells in prostate development. Prepubescent deletion of AR expression in Gli1-expressing cells resulted in severe impairment of androgen-induced prostate growth and regeneration. RNA-sequencing analysis showed significant alterations in signaling pathways related to prostate development, stem cells, and organ morphogenesis in AR-deficient Gli1-expressing cells. Among these altered pathways, the transforming growth factor β1 (TGFβ1) pathway was up-regulated in AR-deficient Gli1-expressing cells. We further demonstrated the activation of TGFβ1 signaling in AR-deleted prostatic Gli1-expressing cells, which inhibits prostate epithelium growth through paracrine regulation. These data demonstrate a novel role of the AR in the Gli1-expressing cellular niche for regulating prostatic cell fate, morphogenesis, and renewal, and elucidate the mechanism by which mesenchymal androgen-signaling through SHH-responsive cells elicits the growth and regeneration of prostate epithelium., Competing Interests: The authors have declared that no competing interest exist.

The common view on penile development is that it is androgen-dependent, based first and foremost on the fact that the genital tubercle forms a penis in males and a clitoris in females. However, critical examination of the complex processes involved in human penile development reveals that many individual steps in development of the genital tubercle are common to both males and females, and thus can be interpreted as androgen-independent. For certain developmental events this conclusion is bolstered by observations in androgen-insensitive patients and androgen receptor mutant mice. Events in genital tubercle development that are common to human males and females include: formation of (a) the genital tubercle, (b) the urethral plate, (c) the urethral groove, (d) the glans, (e) the prepuce and (f) the corporal body. For humans 6 of 13 individual developmental steps in penile development were interpreted as androgen-independent. For mice 5 of 11 individual developmental steps were found to be androgen-independent, which were verified through analysis of androgen-insensitive mutants. Observations from development of external genitalia of other species (moles and spotted hyena) provide further examples of androgen-independent events in penile development. These observations support the counter-intuitive idea that penile development involves both androgen-independent and androgen-dependent processes., (Copyright © 2019 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

To better understand how the human fetal penis and clitoris grows and remodels, we undertook an investigation to define active areas of cellular proliferation and programmed cell death spatially and temporally during development of human fetal external genitalia from the indifferent stage (8 weeks) to 18 weeks of gestation. Fifty normal human fetal penile and clitoral specimens were examined using macroscopic imaging, scanning electron microscopy and immunohistochemical localization for the cellular proliferation and apoptotic markers, Ki67 and Caspase-3. A number of hot spots of cellular proliferation characterized by Ki67 localization are present in the penis and clitoris especially early in development, most notably in the corporal body, glans, remodeling glanular urethra, the urethral plate, the roof of the urethral groove and the fully formed penile urethra. The 12-fold increase in penile length over 10 weeks of growth from 8 to 18 weeks of gestation based on Ki67 labelling appears to be driven by cellular proliferation in the corporal body and glans. Throughout all ages in both the developing penis and clitoris Ki67 labeling was consistently elevated in the ventral epidermis and ventral mesenchyme relative to the dorsal counterparts. This finding is consistent with the intense morphogenetic activity/remodeling in the ventral half of the genital tubercle in both sexes involving formation of the urethral/vestibular plates, canalization of the urethral/vestibular plates and fusion of the urethral folds to form the penile urethra. Areas of reduced or absent Ki67 staining include the urethral fold epithelium that fuses to form the penile tubular urethra. In contrast, the urethral fold mesenchyme is positive for Ki67. Apoptosis was rarely noted in the developing penis and clitoris; the only area of minimal Caspase-3 localization was in the epithelium of the ventral epithelial glanular channel remodeling., (Copyright © 2019 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Development of the human prepuce was studied over the course of 9-17 weeks of gestation in 30 specimens. Scanning electron microscopy revealed subtle surface features that were associated with preputial development, namely the appearance of epidermal aggregates that appeared to be associated with formation of the preputial fold. Transverse and sagittal sections revealed that the epidermis of the glans is considerably thicker than that of the penile shaft. We described a novel morphogenetic mechanism of formation of the preputial lamina, namely the splitting of the thick epidermis of the glans into the preputial lamina and the epidermis via the intrusion of mesenchyme containing red blood cells and CD31-positive blood vessels. This process begins at 10-11 weeks of gestation in the proximal aspect of the glans and extends distally. The process is likely to be androgen-dependent and mediated via androgen receptors strategically localized to the morphogenetic process, but signaling through estrogen receptor may play a role. Estrogen receptor alpha (ESR1) has a very limited expression in the developing human glans and prepuce, while estrogen receptor beta (ESR2) is expressed more broadly in the developing preputial lamina, epidermis and urethra. Examination of the ontogeny of innervation of the glans penis and prepuce reveals the presence of the dorsal nerve of the penis as early as 9 weeks of gestation. Nerve fibers enter the glans penis proximally and extend distally over several weeks to eventually reach the distal aspect of the glans and prepuce by 14-16 weeks of gestation., (Copyright © 2019 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The goal of this report is (a) to provide the first detailed description of mouse clitoral development, and (b) to compare mouse and human clitoral development. For this purpose, external genitalia of female mice were examined by wholemount microscopy, histology and immunohistochemistry from 14 days of gestation to 10 days postnatal. Human clitoral development was examined by these techniques as well as by scanning electron microscopy and optical projection tomography from 8 to 19 weeks of gestation. The adult mouse clitoris is an internal organ defined by a U-shaped clitoral lamina whose development is associated with the prenatal medial and distal growth of the female preputial swellings along the sides of the genital tubercle to form the circumferential preputial lamina. Regression of the ventral aspect of the preputial lamina leads to formation of the U-shaped clitoral lamina recognized as early as 17 days of gestation. While the adult U-shaped mouse clitoral lamina is closely associated with the vagina, and it appears to be completely non-responsive to estrogen as opposed to the highly estrogen-responsive vaginal epithelium. The prominent perineal appendage in adult females is prepuce, formed via fusion of the embryonic preputial swellings and is not the clitoris. The human clitoris is in many respects a smaller anatomic version of the human penis having all of the external and internal elements except the urethra. The human clitoris (like the human penis) is derived from the genital tubercle with the clitoral glans projecting into the vaginal vestibule. Adult morphology and developmental processes are virtually non-comparable in the mouse and human clitoris., (Copyright © 2019 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The study of male and female reproductive tract development requires expertise in two separate disciplines, developmental biology and endocrinology. For ease of experimentation and economy, the mouse has been used extensively as a model for human development and pathogenesis, and for the most part similarities in developmental processes and hormone action provide ample justification for the relevance of mouse models for human reproductive tract development. Indeed, there are many examples describing the phenotype of human genetic disorders that have a reasonably comparable phenotype in mice, attesting to the congruence between mouse and human development. However, anatomic, developmental and endocrinologic differences exist between mice and humans that (1) must be appreciated and (2) considered with caution when extrapolating information between all animal models and humans. It is critical that the investigator be aware of both the similarities and differences in organogenesis and hormone action within male and female reproductive tracts so as to focus on those features of mouse models with clear relevance to human development/pathology. This review, written by a team with extensive expertise in the anatomy, developmental biology and endocrinology of both mouse and human urogenital tracts, focusses upon the significant human/mouse differences, and when appropriate voices a cautionary note regarding extrapolation of mouse models for understanding development of human male and female reproductive tracts., (Copyright © 2019 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Androgen signaling is essential for prostate development, morphogenesis, and regeneration. Emerging evidence also indicates a regulatory role of Notch signaling in prostate development, differentiation, and growth. However, the collaborative regulatory mechanisms of androgen and Notch signaling during prostate development, growth, and regeneration are largely unknown. Hairy and Enhancer of Split 1 (Hes1) is a transcriptional regulator of Notch signaling pathways, and its expression is responsive to Notch signaling. Hes1-expressing cells have been shown to possess the regenerative capability to repopulate a variety of adult tissues. In this study, we developed new mouse models to directly assess the role of the androgen receptor in prostatic Hes1-expressing cells. Selective deletion of AR expression in embryonic Hes1-expressing cells impeded early prostate development both in vivo and in tissue xenograft experiments. Prepubescent deletion of AR expression in Hes1-expressing cells resulted in prostate glands containing abnormalities in cell morphology and gland architecture. A population of castration-resistant Hes1-expressing cells was revealed in the adult prostate, with the ability to repopulate prostate epithelium following androgen supplementation. Deletion of AR in Hes1-expressing cells diminishes their regenerative ability. These lines of evidence demonstrate a critical role for the AR in Notch-responsive cells during the course of prostate development, morphogenesis, and regeneration, and implicate a mechanism underlying interaction between the androgen and Notch signaling pathways in the mouse prostate., (Copyright © 2019 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Emerging evidence has shown that both prostatic basal and luminal cells are able to initiate oncogenic transformation. However, despite the diversity of tumor-initiating cells, most prostate cancer cells express the androgen receptor (AR) and depend on androgens for their growth and expansion, implicating an essential role of androgen signaling in prostate tumorigenesis. Prostatic basal cells express p63 and are able to differentiate into luminal, neuroendocrine, and basal cells. Here, we directly assessed the essential role of androgen signaling in prostatic p63-expressing cell initiated oncogenic transformation and tumor formation. Using novel and relevant mouse models, we demonstrated that, with stabilized β-catenin expression, prostatic p63-expressing cells possess the ability to initiate oncogenic transformation and, in the presence of androgens, they further transdifferentiate into luminal-like tumor cells and develop adenocarcinomas. Castration prior to activating stabilized β-catenin sensitizes p63-expressing cells and increases their sensitivity to androgens, resulting in aggressive and fast growing tumor phenotypes. These findings are consistent with what have been observed in human prostate cancers, demonstrating an essential role for androgen signaling in prostate cancer initiation and progression. This study also provides fresh insight into developing new therapeutic strategies for better treating prostate cancer patients.

The urinary bladder collects urine from the kidneys and stores it until the appropriate moment for voiding. The trigone and ureterovesical junctions are key to bladder function, by allowing one-way passage of urine into the bladder without obstruction. Embryological development of these structures has been studied in multiple animal models as well as humans. In this report we review the existing literature on bladder development and cellular signalling with particular focus on bladder development in humans. The bladder and ureterovesical junction form primarily during the fourth to eighth weeks of gestation, and arise from the primitive urogenital sinus following subdivision of the cloaca. The bladder develops through mesenchymal-epithelial interactions between the endoderm of the urogenital sinus and mesodermal mesenchyme. Key signalling factors in bladder development include shh, TGF-β, Bmp4, and Fgfr2. A concentration gradient of shh is particularly important in development of bladder musculature, which is vital to bladder function. The ureterovesical junction forms from the interaction between the Wolffian duct and the bladder. The ureteric bud arises from the Wolffian duct and is incorporated into the developing bladder at the trigone. It was previously thought that the trigonal musculature developed primarily from the Wolffian duct, but it has been shown to develop primarily from bladder mesenchyme. Following emergence of the ureters from the Wolffian ducts, extensive epithelial remodelling brings the ureters to their final trigonal positions via vitamin A-induced apoptosis. Perturbation of this process is implicated in clinical obstruction or urine reflux. Congenital malformations include ureteric duplication and bladder exstrophy., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

We have studied the ontogeny of the developing human male and female urogenital tracts from 9 weeks (indifferent stage) to 16 weeks (advanced sex differentiation) of gestation by immunohistochemistry on mid-sagittal sections. Sixteen human fetal pelvises were serial sectioned in the sagittal plane and stained with antibodies to epithelial, muscle, nerve, proliferation and hormone receptor markers. Key findings are: (1) The corpus cavernosum in males and females extends into the glans penis and clitoris, respectively, during the ambisexual stage (9 weeks) and thus appears to be an androgen-independent event. (2) The entire human male (and female) urethra is endodermal in origin based on the presence of FOXA1, KRT 7, uroplakin, and the absence of KRT10 staining. The endoderm of the urethra interfaces with ectodermal epidermis at the site of the urethral meatus. (3) The surface epithelium of the verumontanum is endodermal in origin (FOXA1-positive) with a possible contribution of Pax2-positive epithelial cells implying additional input from the Wolffian duct epithelium. (4) Prostatic ducts arise from the endodermal (FOXA1-positive) urogenital sinus epithelium near the verumontanum. (5) Immunohistochemical staining of mid-sagittal and para-sagittal sections revealed the external anal sphincter, levator ani, bulbospongiosus muscle and the anatomic relationships between these developing skeletal muscles and organs of the male and female reproductive tracts. Future studies of normal human developmental anatomy will lay the foundation for understanding congenital anomalies of the lower urogenital tract., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

This paper provides a detailed compilation of human prostatic development that includes human fetal prostatic gross anatomy, histology, and ontogeny of selected epithelial and mesenchymal differentiation markers and signaling molecules throughout the stages of human prostatic development: (a) pre-bud urogenital sinus (UGS), (b) emergence of solid prostatic epithelial buds from urogenital sinus epithelium (UGE), (c) bud elongation and branching, (d) canalization of the solid epithelial cords, (e) differentiation of luminal and basal epithelial cells, and (f) secretory cytodifferentiation. Additionally, we describe the use of xenografts to assess the actions of androgens and estrogens on human fetal prostatic development. In this regard, we report a new model of de novo DHT-induction of prostatic development from xenografts of human fetal female urethras, which emphasizes the utility of the xenograft approach for investigation of initiation of human prostatic development. These studies raise the possibility of molecular mechanistic studies on human prostatic development through the use of tissue recombinants composed of mutant mouse UGM combined with human fetal prostatic epithelium. Our compilation of human prostatic developmental processes is likely to advance our understanding of the pathogenesis of benign prostatic hyperplasia and prostate cancer as the neoformation of ductal-acinar architecture during normal development is shared during the pathogenesis of benign prostatic hyperplasia and prostate cancer., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Recent studies in our lab have utilized three imaging techniques to visualize the developing human fetal urogenital tract in three dimensions: optical projection tomography, scanning electron microscopy and lightsheet fluorescence microscopy. We have applied these technologies to examine changes in morphology and differential gene expression in developing human external genital specimens from the ambisexual stage (<9 weeks fetal age) to well-differentiated male and female organs (>13 weeks fetal age). This work outlines the history and function of each of these three imaging modalities, our methods to prepare specimens for each and the novel findings we have produced thus far. We believe the images in this paper of human fetal urogenital organs produced using lightsheet fluorescence microscopy are the first published to date., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

We present a detailed review of fetal development of the male and female human urogenital tract from 8 to 22 weeks gestation at the macroscopic and morphometric levels. Human fetal specimens were sexed based on macroscopic identification of fetal testes or ovaries, Wolffian or Müllerian structures and the presence of the SRY gene in the specimens at or near the indifferent stage (8-9 weeks). Specimens were photographed using a dissecting microscope with transmitted and reflected light. Morphometric measurements were taken of each urogenital organ. During this time period, development of the male and female urogenital tracts proceeded from the indifferent stage to differentiated organs. The kidneys, ureters, and bladder developed identically, irrespective of sex with the same physical dimensions and morphologic appearance. The penis, prostate and testis developed in males and the clitoris, uterus and ovary in females. Androgen-dependent growth certainly influenced size and morphology of the penile urethra and prostate, however, androgen-independent growth also accounted for substantial growth in the fetal urogenital tract including the clitoris., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Androgen signaling is essential for prostate development, morphogenesis, and regeneration. Emerging evidence indicates that Wnt/β-catenin signaling also contributes to prostate development specifically through regulation of cell fate determination. Prostatic Axin2-expressing cells are able to respond to Wnt signals and possess the progenitor properties to regenerate prostatic epithelium. Despite critical roles of both signaling pathways, the biological significance of androgen receptor (AR) in Axin2-expressing/Wnt-responsive cells remains largely unexplored. In this study, we investigated this important question using a series of newly generated mouse models. Deletion of Ar in embryonic Axin2-expressing cells impaired early prostate development in both ex vivo and tissue implantation experiments. When Ar expression was deleted in prostatic Axin2-expressing cells at pre-puberty stages, it results in smaller and underdeveloped prostates. A subpopulation of Axin2 expressing cells in prostate epithelium is resistant to castration and, following androgen supplementation, is capable to expand to prostatic luminal cells. Deletion of Ar in these Axin2-expressing cells reduces their regenerative ability. These lines of evidence demonstrate an indispensable role for the Ar in Wnt-responsive cells during the course of prostate development, morphogenesis, and regeneration, which also imply an underlying interaction between the androgen and Wnt signaling pathways in the mouse prostate. Stem Cells 2018;36:891-902., (© 2018 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

This paper addresses the developmental mechanisms of formation of the mouse and human penile urethra and the possibility that two disparate mechanisms are at play. It has been suggested that the entire penile urethra of the mouse forms via direct canalization of the endodermal urethral plate. While this mechanism surely accounts for development of the proximal portion of the mouse penile urethra, we suggest that the distal portion of the mouse penile urethra forms via a series of epithelial fusion events. Through review of the recent literature in combination with new data, it is unlikely that the entire mouse urethra is formed from the endodermal urethral plate due in part to the fact that from E14 onward the urethral plate is not present in the distal aspect of the genital tubercle. Formation of the distal portion of the mouse urethra receives substantial contribution from the preputial swellings that form the preputial-urethral groove and subsequently the preputial-urethral canal, the later of which is subdivided by a fusion event to form the distal portion of the mouse penile urethra. Examination of human penile development also reveals comparable dual morphogenetic mechanisms. However, in the case of human, direct canalization of the urethral plate occurs in the glans, while fusion events are involved in formation of the urethra within the penile shaft, a pattern exactly opposite to that of the mouse. The highest incidence of hypospadias in humans occurs at the junction of these two different developmental mechanisms. The relevance of the mouse as a model of human hypospadias is discussed., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The role of tissue interactions was explored to determine whether epithelial differentiation within the developing human reproductive tract is induced and specified by mesenchyme in tissue recombinants composed of mouse vaginal mesenchyme + human uterine tubal epithelium (mVgM+hTubE). The tissue recombinants were grown in DES-treated ovariectomized athymic mice. After 2-4 weeks of in vivo growth, several vaginal specific features were expressed in the human tubal epithelium. The mesenchyme-induced effects included morphological change as well as expression of several immunohistochemical markers. Although the mesenchyme-induced shift in vaginal differentiation in the human tubal epithelium was not complete, the partial induction of vaginal markers in human tubal epithelium verifies the importance of mesenchymal-epithelial interactions in development of the human female reproductive tract. In a separate experiment, DES-induction of uterine epithelial progesterone receptor (PGR) and estrogen receptor 1 (ESR1) was explored in tissue recombinants composed of wild-type or Esr1KO mouse uterine mesenchyme + human fetal uterine epithelium (wt UtM+hUtE and Esr1KO UtM+hUtE). The rationale of this experiment was to determine whether DES-induction of PGR and ESR1 is mediated directly via epithelial ESR1 or indirectly (paracrine mechanism) via mesenchymal ESR1. DES-induction of uterine epithelial ESR1 and PGR in Esr1KO UtM+hUtE tissue recombinants (devoid of mesenchymal ESR1) formally eliminates the paracrine mechanism and demonstrates that DES induction of human uterine epithelial ESR1 and PGR is directly mediated via epithelial ESR1., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Human female fetal reproductive tracts 9.5-22 weeks of gestation were grown for 1 month in ovariectomized athymic adult female mouse hosts that were either untreated or treated continuously with diethylstilbestrol (DES) via subcutaneous pellet. Normal morphogenesis and normal patterns of differentiation marker expression (KRT6, KRT7, KRT8, KRT10, KRT14, KRT19, ESR1, PGR, TP63, RUNX1, ISL1, HOXA11 and α-ACT2) were observed in xenografts grown in untreated hosts and mimicked observations of previously reported (Cunha et al., 2017) non-grafted specimens of comparable age. DES elicited several notable morphological affects: (a) induction of endometrial/cervical glands, (b) increased plication (folding) of tubal epithelium, (c) stratified squamous maturation of vaginal epithelium and (d) vaginal adenosis. DES also induced ESR1 in epithelia of the uterine corpus, cervix and globally induced PGR in most cells of the developing human female reproductive tract. Keratin expression (KRT6, KRT7, KRT8, KRT14 and KRT19) was minimally affected by DES. Simple columnar adenotic epithelium was devoid of TP63 and RUNX1, while DES-induced mature vaginal epithelium was positive for both transcription factors. Another striking effect of DES was observed in grafts of human uterine tube, in which DES perturbed smooth muscle patterning. These results define for the first time IHC protein markers of DES action on the developing human reproductive tract, which provide bio-endpoints of estrogen-induced teratogenesis in the developing human female reproductive tract for future testing of estrogenic endocrine disruptors., (Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

We present a detailed review of the embryonic and fetal development of the human female reproductive tract utilizing specimens from the 5th through the 22nd gestational week. Hematoxylin and eosin (H&E) as well as immunohistochemical stains were used to study the development of the human uterine tube, endometrium, myometrium, uterine cervix and vagina. Our study revisits and updates the classical reports of Koff (1933) and Bulmer (1957) and presents new data on development of human vaginal epithelium. Koff proposed that the upper 4/5ths of the vagina is derived from Müllerian epithelium and the lower 1/5th derived from urogenital sinus epithelium, while Bulmer proposed that vaginal epithelium derives solely from urogenital sinus epithelium. These conclusions were based entirely upon H&E stained sections. A central player in human vaginal epithelial development is the solid vaginal plate, which arises from the uterovaginal canal (fused Müllerian ducts) cranially and squamous epithelium of urogenital sinus caudally. Since Müllerian and urogenital sinus epithelium cannot be unequivocally identified in H&E stained sections, we used immunostaining for PAX2 (reactive with Müllerian epithelium) and FOXA1 (reactive with urogenital sinus epithelium). By this technique, the PAX2/FOXA1 boundary was located at the extreme caudal aspect of the vaginal plate at 12 weeks. During the ensuing weeks, the PAX2/FOXA1 boundary progressively extended cranially such that by 21 weeks the entire vaginal epithelium was FOXA1-reactive and PAX2-negative. This observation supports Bulmer's proposal that human vaginal epithelium derives solely from urogenital sinus epithelium. Clearly, the development of the human vagina is far more complex than previously envisioned and appears to be distinctly different in many respects from mouse vaginal development., (Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The penile and clitoral anatomy of four species of Talpid moles (broad-footed, star-nosed, hairy-tailed, and Japanese shrew moles) were investigated to define penile and clitoral anatomy and to examine the relationship of the clitoral anatomy with the presence or absence of ovotestes. The ovotestis contains ovarian tissue and glandular tissue resembling fetal testicular tissue and can produce androgens. The ovotestis is present in star-nosed and hairy-tailed moles, but not in broad-footed and Japanese shrew moles. Using histology, three-dimensional reconstruction, and morphometric analysis, sexual dimorphism was examined with regard to a nine feature masculine trait score that included perineal appendage length (prepuce), anogenital distance, and presence/absence of bone. The presence/absence of ovotestes was discordant in all four mole species for sex differentiation features. For many sex differentiation features, discordance with ovotestes was observed in at least one mole species. The degree of concordance with ovotestes was highest for hairy-tailed moles and lowest for broad-footed moles. In relationship to phylogenetic clade, sex differentiation features also did not correlate with the similarity/divergence of the features and presence/absence of ovotestes. Hairy-tailed and Japanese shrew moles reside in separated clades, but they exhibit a high degree of congruence. Broad-footed and hairy-tailed moles reside within the same clade but had one of the lowest correlations in features and presence/absence of ovotestes. Thus, phylogenetic affinity and the presence/absence of ovotestes are poor predictors for most sex differentiation features within mole external genitalia., (© 2017 Wiley Periodicals, Inc.)

This paper provides the first detailed description of flutamide-induced hypospadias in the rat based upon wholemount, histologic, three-dimensional reconstruction, scanning electron microscopic, and immunocytochemical analysis. The penile malformations elicited by this potent anti-androgen include a substantial proximal shift in the urethral meatus that clearly conforms to the definition of hypospadias based upon specific morphological criteria for this malformation. Through examination of the normal penile development and flutamide-induced abnormal penile development observed in prenatally oil- and flutamide-treated rats, our analysis provides insights into the morphogenetic mechanism of development of hypospadias. In this regard, a common theme in normal penile development is midline fusion of epithelia followed by removal of the epithelial seam and establishment of midline mesenchymal confluence during development of the penile urethra and prepuce, processes which are impaired as a result of prenatal flutamide treatment. The developmental processes occurring in normal penile development, through comparison with development of female external genitalia and those impaired due to prenatal flutamide treatment, are consistent with critical role of androgen receptors in normal penile development in the rat, and the specific penile abnormalities embodied in flutamide-induced rat hypospadias., (Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Hypospadias is a common malformation whose etiology is based upon perturbation of normal penile development. The mouse has been previously used as a model of hypospadias, despite an unacceptably wide range of definitions for this malformation. The current paper presents objective criteria and a definition of mouse hypospadias. Accordingly, diethylstilbestrol (DES) induced penile malformations were examined at 60 days postnatal (P60) in mice treated with DES over the age range of 12 days embryonic to 20 days postnatal (E12-P20). DES-induced hypospadias involves malformation of the urethral meatus, which is most severe in DES E12-P10, DES P0-P10 and DES P5-P15 groups, and less so or absent in the other treatment groups. A frenulum-like ventral tether between the penis and the prepuce was seen in the most severely affected DES-treated mice. Internal penile morphology was also altered in the DES E12-P10, DES P0-P10 and DES P5-P15 groups (with little effect in the other DES treatment groups). Thus, adverse effects of DES are a function of the period of DES treatment and most severe in the P0-P10 period. In "estrogen mutant mice" (NERKI, βERKO, αERKO and AROM+) hypospadias was only seen in AROM+ male mice having genetically-engineered elevation is serum estrogen. Significantly, mouse hypospadias was only seen distally at and near the urethral meatus where epithelial fusion events are known to take place and never in the penile midshaft, where urethral formation occurs via an entirely different morphogenetic process., (Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)