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Background: Measurement of rat anogenital distance (AGD) dates to at least 1912. Increased interest in endocrine disrupting chemicals and the use of AGD as a biomarker for fetal androgen effects have increased the number of studies with this endpoint in recent decades. A literature review revealed different landmarks, methods of measurement, and methods to adjust for body weight differences. AGD is often reported to hundredths of millimeters and as such, deserves precision in all these aspects. This paper presents recommendations for the measurement and analysis of rodent AGD., Methods: Literature and regulatory guidance documents that mentioned or measured rodent AGD were reviewed. Four adjustment methods were evaluated using available online data from three rat studies each with two generations of offspring., Results: Tabulation of studies reveals that species/stocks and time of data collection, but more importantly anatomical landmarks and methods of measurement have produced a variety of results which are difficult to compare. Not all studies have adjusted for test article effects on body weight (and thus size). The four adjustment methods were fairly comparable., Conclusion: Recommendations are as follows. A microscopic method should be used to measure AGD of late rodent fetuses and early postnatal pups. The caudal edge of the genital tubercle and the cranial edge of the anus are clear and identifiable landmarks. The simplest adjustment is to divide individual AGDs by the cube root of animals' body weight. These recommendations will help ensure data consistency and accuracy, and facilitate meaningful comparisons across laboratories and chemical classes., (© 2024 Wiley Periodicals LLC.)

External genital organs are among the most recognizable sexually dimorphic characters. The penis and clitoris develop from the embryonic genital tubercle, an outgrowth at the anterior margin of the cloaca that undergoes an extensive period of development in male and female embryos prior to the onset of sexual differentiation. In mice, differentiation into the penis and clitoris begins around embryonic day (E)15.5. Current knowledge of cell types that comprise the genital tubercle is limited to a few studies that have fate mapped derivatives of endoderm, mesoderm, and ectoderm. Here we use single cell transcriptomics to characterize the cell populations in the genital tubercles of male and female mouse embryos at E14.5, approximately 24 ​h before the onset of sexual differentiation, and we present the first comprehensive atlas of single-cell gene expression during external genital development. Clustering analyses and annotation using marker genes shows 19 distinct cell populations in E14.5 genital tubercles. Mapping of cell clusters to anatomical locations using in situ gene expression patterns revealed granularity of cellular specializations and positional identities. Although E14.5 precedes sexually dimorphic morphogenesis of the genital tubercle, comparative analysis of males and females identified sexual dimorphisms at the single cell level, including male-specific cell clusters with transcriptional signatures of smooth muscle and bone progenitors, both of which are known to be sexually dimorphic in adult genitalia, as well as immune cells. These results provide a new resource for classification of external genital cell types based on gene expression profiles and reveal sex-specific cellular specializations in the early genital tubercle., (Copyright © 2021. Published by Elsevier Inc.)

Birth defects of the external genitalia are among the most common in the world. Proper formation of the external genitalia requires a highly orchestrated process that involves special cell populations and sexually dimorphic hormone signaling. It is clear what the end result of the sexually dimorphic development is (a penis in the male versus clitoris in the female); however, the cell populations involved in the process remain poorly defined. Here, we used single-cell messenger RNA sequencing in mouse embryos to uncover the dynamic changes in cell populations in the external genitalia during the critical morphogenetic window. We found that overall, male and female external genitalia are largely composed of the same core cellular components. At the bipotential stage of development (embryonic day or E14.5), few differences in cell populational composition exist between male and female. Although similar in cell population composition, genetic differences in key sexual differentiation developmental pathways arise between males and females by the early (E16.5) and late (E18.5) differentiation stages. These differences include discrete cell populations with distinct responsiveness to androgen and estrogen. By late sexual differentiation (E18.5), unique cell populations in both male and female genitalia become apparent and are enriched with androgen- and estrogen-responsive genes, respectively. These data provide insights into the morphogenesis of the external genitalia that could be used to understand diseases associated with defects in the external genitalia., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Monozygotic twins with 45,X/46,XY mosaicism, discordant for phenotypic sex, are extremely rare., Methods: This report describes the clinical findings of a rare case of 45,X/46,XY mosaicism in monozygotic twins with different external genitalia. Single nucleotide polymorphism (SNP) array analysis, performed by collecting DNA from each umbilical cord, showed identical SNPs in the autosomal chromosomes of both fetuses., Results: Chorionic villus sampling of a 37-year-old primigravida carrying monozygotic twins revealed a 45,X/46,XY karyotype. Autopsy of the aborted fetuses revealed a penis and testes on one fetus and a vagina, uterus, and ovaries in the other fetus--which also had severe cystic hygroma. Cell counting using fluorescence in situ hybridization with XY probes (XY-FISH) showed 20% and 80% abundance of 45,X cells in the internal genitalia, liver, heart, lung, adrenal gland, bone marrow, and spine of the male and female fetuses, respectively., Conclusion: These results indicated that the fetuses were genetically monozygotic twins and their different degrees of mosaicism may have resulted in different genital phenotypes., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

An adverse outcome pathway (AOP) is a simplified description of the sequence of mechanistic events that lead to a particular toxicological effect, from initial trigger to adverse outcome. Although designed to inform regulatory risk assessors, the AOP framework also provides a platform for innovative collaborations between experts from relevant research fields and the regulatory community. The underpinning for any AOP is basic knowledge about molecular and developmental processes; such knowledge can only be attained by solid bioscientific research. Starting with this fundamental knowledge, the objective is to devise novel testing strategies that focus on key events in a causative pathway. It is anticipated that such a knowledge-based approach will ultimately alleviate many of the burdens associated with classical chemical testing strategies that typically involve large-scale animal toxicity regimens. This hails from the notion that a solid understanding of the underlying mechanisms will allow the development and use of alternative test methods, including both in vitro and in silico approaches. This review is specifically targeted at professionals working in bioscientific fields, such as developmental and reproductive biology, and aims to (i) inform on the existence of the AOP framework and (ii) encourage new cross-disciplinary collaborations. It is hoped that fundamental biological knowledge can thus be better exploited for applied purposes: firstly, an improved understanding of how our perpetual exposure to environmental chemicals is causing human reproductive disease and, secondly, new approaches to screen for harmful chemicals more efficiently. This is not an instructional manual on how to create AOPs; rather, we discuss how to harness fundamental knowledge from the biosciences to assist regulatory toxicologists in their efforts to protect humans against chemicals that harm human reproductive development and function., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Introduction: For decades, ultrasound has enabled determination of foetal sex. Foetal sex is medically indicated in sex-linked diseases, syndromes and multiple pregnancy, but genital malformations are rare. However, guidelines on standard views seem sparse and foetal sex determination is not considered mandatory. The aim of this study was to provide an initial overview on the practice of foetal sex determination in Denmark and the accuracy of the examinations at the second trimester anomaly scan at a regional and a university hospital., Methods: Phone interviews from all 23 Danish foetal medicine departments concerning information, examination, registration, quality criteria and assessment of accuracy. The accuracy of foetal sex determination was assessed in 5,786 singleton pregnancies with live births at two departments by comparison of data from the second trimester anomaly scan (week 18 + 0 - 21 + 6) with the national Danish birth register., Results: Second trimester foetal sex determination was not mandatory but conducted without guideline. Various approaches to information and documentation were used. Foetal sex was assessed in 86.7% of cases and the accuracy was 99.2% with no significant difference between the regional and the university hospital of the study. Sex determination was more inaccurate in females (18/2,118) than in males (18/2,271), p < 0.0001., Conclusions: Foetal sex determination by ultrasound is common practice in Denmark. No guideline existed and incorrect sex was determined in 8/1,000 foetuses, most often females. Foetal sex determination should be standardised extensively to avoid false diagnoses., Funding: none., Trial Registration: This study was approved by the Danish Data Protection Agency (record number: 2012-41-0050)., (Articles published in the DMJ are “open access”. This means that the articles are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits any non-commercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.)

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenals, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography-tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography-mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Link protein is encoded by the Hapln1 gene and is a prototypical protein found in the cartilage matrix. It acts as an important component of the endochondral skeleton during early development. To study its transcriptional regulation, promoter fragments derived from the link protein gene were coupled to the β-galactosidase reporter and used to study in vivo transgene expression in mice. In day 15.5 mouse embryos, a link promoter fragment spanning -1020 to +40 nucleotides demonstrated highly specific β-galactosidase staining of skeletal structures, including the appendicular and axial cartilaginous tissues. Two shorter promoter fragments, spanning -690 to +40 and -315 to +40 nucleotides, demonstrated limb- and genitalia-specific expression resembling that of homeodomain-regulated tissues. Bioinformatic analysis revealed a highly conserved, Hox-like binding site (HLBS) at approximately -220 bp of the promoter, shared by both constructs, which contained the Hox-core consensus sequence TAATTA. Electromobility shift assays demonstrated binding of Hox-B4 recombinant protein to the HLBS, which was eliminated with nucleotide substitutions within the core-binding element. Co-transfection analysis of the HLBS demonstrated a 22-fold transcriptional activation by HoxA9 expression, which was ablated with a substitution within the core HLBS element. Together these findings establish promoter regions within the link protein gene that are important for in vivo expression and identify the potential role of homeodomain-containing proteins in controlling cartilage and limb gene expression., (Published by Elsevier Inc.)

Alfred Jost's work in the 1940s laid the foundation of the current paradigm of sexual differentiation of reproductive tracts, which contends that testicular hormones drive the male patterning of reproductive tract system whereas the female phenotype arises by default. Once established, the sex-specific reproductive tracts undergo morphogenesis, giving rise to anatomically and functionally distinct tubular organs along the rostral-caudal axis. Impairment of sexual differentiation of reproductive tracts by genetic alteration and environmental exposure are the main causes of disorders of sex development, and infertility at adulthood. This review covers past and present work on sexual differentiation and morphogenesis of reproductive tracts, associated human disorders, and emerging technologies that have made impacts or could radically expand our knowledge in this field., (Published by Oxford University Press on behalf of Society for the Study of Reproduction 2019.)

The ability of a single genome to produce distinct and often dramatically different male and female forms is one of the wonders of animal development. In Drosophila melanogaster , most sexually dimorphic traits are controlled by sex-specific isoforms of the doublesex ( dsx ) transcription factor, and dsx expression is mostly limited to cells that give rise to sexually dimorphic traits. However, it is unknown how this mosaic of sexually dimorphic and monomorphic organs arises. Here, we characterize the cis -regulatory sequences that control dsx expression in the foreleg, which contains multiple types of sex-specific sensory organs. We find that separate modular enhancers are responsible for dsx expression in each sexually dimorphic organ. Expression of dsx in the sex comb is co-regulated by two enhancers with distinct spatial and temporal specificities that are separated by a genitalia-specific enhancer. The sex comb-specific enhancer from D. willistoni , a species that primitively lacks sex combs, is not active in the foreleg. Thus, the mosaic of sexually dimorphic and monomorphic organs depends on modular regulation of dsx transcription by dedicated cell type-specific enhancers., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Objective: This study measured anogenital distance (AGD) during late second/early third trimester of pregnancy to confirm previous findings that AGD can be measured noninvasively in the fetus using ultrasound and further showed differences in reference ranges between populations., Method: Two hundred ten singleton pregnancies were recruited at the Rosie Hospital, Cambridge, UK. A 2D ultrasound was performed between 26 and 30 weeks of pregnancy. AGD was measured from the centre of the anus to the base of the scrotum in males and to the posterior convergence of the fourchette in females., Results: A significant difference in AGD between males and females (P < .0001) was found, replicating previous results with a significant correlation between estimated fetal weight (EFW) and AGD in males only (P = .006). A comparison of AGD using reference data from an Israeli sample (n = 118) and our UK sample (n = 208) showed a significant difference (P < .0001) in both males and females, after controlling for gestational age (GA)., Conclusion: Our results confirm that AGD measurement in utero using ultrasound is feasible. In addition, there are strong sex differences, consistent with previous suggestions that AGD is influenced by prenatal androgen exposure. AGD lengths differ between the UK and Israel; therefore, population-specific normative values may be required for accurate clinical assessments., (© 2019 The Authors. Prenatal Diagnosis Published by John Wiley & Sons Ltd.)

A short anogenital distance (AGD) in males is a marker for incomplete masculinization and a predictor of adverse effects on male reproductive health. For this reason, AGD is used to assess the endocrine disrupting potential of chemicals for risk assessment purposes. The molecular mechanisms underpinning this chemically induced shortening of the AGD, however, remains unclear. Although it is clear that androgen receptor-mediated signaling is essential, evidence also suggest the involvement of other signaling pathways. This study presents the first global transcriptional profile of the anogenital tissue in male rat fetuses with chemically induced short AGD, also including comparison to normal male and female control animals. The antiandrogenic drug finasteride (10 mg/kg bw/day) was used to induce short AGD by exposing time-mated Sprague Dawley rats at gestation days 7-21. The AGD was 37% shorter in exposed male fetuses compared with control males at gestation day 21. Transcriptomics analysis on anogenital tissues revealed a sexually dimorphic transcriptional profile. More than 350 genes were found to be differentially expressed between the 3 groups. The expression pattern of 4 genes of particular interest (Esr1, Padi2, Wnt2, and Sfrp4) was also tested by RT-qPCR analyses, indicating that estrogen and Wnt2 signaling play a role in the sexually dimorphic development of the anogenital region. Our transcriptomics profiles provide a stepping-stone for future studies aimed at characterizing the molecular events governing development of the anogenital tissues, as well as describing the detailed Adverse Outcome Pathways for short AGD; an accepted biomarker of endocrine effects for chemical risk assessment., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Development of external genitalia and perineum is the subject of developmental biology as well as toxicology and teratology researches. Cloaca forms in the lower (caudal) end of endoderm. Such endodermal epithelia and surrounding mesenchyme interact with various signals to form the external genitalia. External genitalia (the anlage termed as genital tubercle: GT) formation shows prominent sexually dimorphic morphogenesis in late embryonic stages, which is an unexplored developmental research field because of many reasons. External genitalia develop adjacent to the cloaca which develops urethra and corporal bodies. Developmental regulators including growth factor signals are necessary for epithelia-mesenchyme interaction (EMI) in posterior embryos including the cloaca and urethra in the genitalia. In the case of male type urethra, formation of tubular urethra proceeds from the lower (ventral) side of external genitalia as a masculinization process in contrast to the case of female urethra. Mechanisms for its development are not elucidated yet due to the lack of suitable mutant mouse models. Because of the recent progresses of Cre (recombinase)-mediated conditional target gene modification analyses, many developmental regulatory genes become increasingly analyzed. Conditional gene knockout mouse approaches and tissue lineage approaches are expected to offer vital information for such sexually dimorphic developmental processes. This review aims to offer recent updates on the progresses of these emerging developmental processes for the research field of congenital anomalies., (© 2018 Japanese Teratology Society.)

Exposure to estrogenic endocrine disrupting chemicals (EDCs) during in utero development has been linked to the increasing incidence of disorders of sexual development. Hypospadias, the ectopic placement of the urethra on the ventral aspect of the penis, is one of the most common DSDs affecting men, and can also affect women by resulting in the misplacement of the urethra. This study aimed to comprehensively assess the resulting hypospadias phenotypes in male and female mice exposed in utero from embryonic day 9.5 to 19.5 to the potent estrogenic endocrine disruptor, diethylstilbestrol, at a high, clinically relevant dose, and a low, previously untested dose, administered via water. The anogenital distance of male pups was significantly reduced and hypospadias was observed in males at a high frequency. Females exhibited hypospadias and urethral-vaginal fistula. These results demonstrate the ability of an estrogen receptor agonist to disrupt sexual development in both male and female mice, even at a low dose, administered via drinking water.

Research Question: What is the association between prenatal exposure to persistent organic pollutants, separately and combined, and anogenital distance (in-utero endocrine disruption marker)., Design: A cohort study conducted in Sonora, Mexico. Blood concentrations of polychlorobiphenyls (PCB) 28, 74, 118, 138/158, 153, 170, 180 and the isomers of dichlorodiphenyltrichloroethane (DDT) and its metabolites were determined in women in the third trimester of pregnancy; three variants of anogenital distance were measured on five occasions during the first year of life of their infants: 82 girls (402 observations) and 74 boys (356 observations)., Results: Boys had negative and significant associations between anogenital distance/height and the concentrations of PCB 28 (beta = - 0.005;P = 0.006), PCB 74 (beta = - 0.003;P = 0.013), and PCB 170 (beta = - 0.005;P = 0.001) when analysed individually. Negative and significant associations were also found using statistical models applied to mixtures of compounds. The latter associations were sometimes larger in magnitude and significance, suggesting a possible potentiation of the compounds. No associations were observed between anogenital distance and DDT in either sex or with PCB in girls., Conclusions: The decreased anogenital distance associated with prenatal exposure to the persistent organic pollutants, observed consistently in different analyses, suggests an under-masculinizing effect of these environmental pollutants in boys., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. 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.)

Stevia rebaudiana belongs to the Asteraceae family with high economic and medicinal potential. This article describes and illustrates morphological and histological aspects of leaves and reproductive organs, and the germination process, to provide detailed information on this species and to contribute to taxonomic, phylogenetic and pharmacobotanical projects. The fruit is a cypsela, small, simple, dry, indehiscent, monospermic, light or dark colored, with aristate pappus, and the seed presents a spatulate axile embryo. Germination is phaneroepigeal with a pivotal root system and many absorbing root hairs. The leaves are simple, elliptical to obovate, with two types of trichomes (glandular and tector), with a short petiole, exhibiting an opposite decussate phyllotaxy. Our results showed 37.5% germination after 12 days, only in the dark cypsela, the light colored being considered unviable. The inflorescence is paniculate and the florets are grouped in capitula with isomorphic ends, monoclinous (bisexual), dichlamydeous, heterochlamydeous, pentamerous calyx and corolla, gamossepalous and gamopetalous. The androecium is gamostemone comprised of five stamens with free filaments, isodynamous and epipetalous stamens, synandrous and rimose anthers. The flower presents an inferior ovary, bicarpelar, unilocular and ovules with a basal placentation. The pollen grains are small, isopolar, radial symmetry, tricolporate, with echinate ornamentation.

Genital malformations are among the most common human birth defects, and both genetic and environmental factors can contribute to these malformations. Development of the external genitalia in mammals relies on complex signaling networks, and disruption of these signaling pathways can lead to genital defects. Islet-1 (ISL1), a member of the LIM/Homeobox family of transcription factors, has been identified as a major susceptibility gene for classic bladder exstrophy in humans, a common form of the bladder exstrophy-epispadias complex (BEEC), and is implicated in a role in urinary tract development. We report that deletion of Isl1 from the genital mesenchyme in mice led to hypoplasia of the genital tubercle and prepuce, with an ectopic urethral opening and epispadias-like phenotype. These mice also developed hydroureter and hydronephrosis. Identification of ISL1 transcriptional targets via ChIP-Seq and expression analyses revealed that Isl1 regulates several important signaling pathways during embryonic genital development, including the BMP, WNT, and FGF cascades. An essential function of Isl1 during development of the external genitalia is to induce Bmp4-mediated apoptosis in the genital mesenchyme. Together, these studies demonstrate that Isl1 plays a critical role during development of the external genitalia and forms the basis for a greater understanding of the molecular mechanisms underlying the pathogenesis of BEEC and urinary tract defects in humans., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In multicellular organisms, the specification, maintenance, and transmission of the germ cell lineage to subsequent generations are critical processes that ensure species survival. A number of studies suggest that the Bone Morphogenetic Protein (BMP) pathway plays multiple roles in this cell lineage. We wished to use a comparative framework to examine the role of BMP signaling in regulating these processes, to determine if patterns would emerge that might shed light on the evolution of molecular mechanisms that may play germ cell-specific or other reproductive roles across species. To this end, here we review evidence to date from the literature supporting a role for BMP signaling in reproductive processes across Metazoa. We focus on germ line-specific processes, and separately consider somatic reproductive processes. We find that from primordial germ cell (PGC) induction to maintenance of PGC identity and gametogenesis, BMP signaling regulates these processes throughout embryonic development and adult life in multiple deuterostome and protostome clades. In well-studied model organisms, functional genetic evidence suggests that BMP signaling is required in the germ line across all life stages, with the exception of PGC specification in species that do not use inductive signaling to induce germ cell formation. The current evidence is consistent with the hypothesis that BMP signaling is ancestral in bilaterian inductive PGC specification. While BMP4 appears to be the most broadly employed ligand for the reproductive processes considered herein, we also noted evidence for sex-specific usage of different BMP ligands. In gametogenesis, BMP6 and BMP15 seem to have roles restricted to oogenesis, while BMP8 is restricted to spermatogenesis. We hypothesize that a BMP-based mechanism may have been recruited early in metazoan evolution to specify the germ line, and was subsequently co-opted for use in other germ line-specific and somatic reproductive processes. We suggest that if future studies assessing the function of the BMP pathway across extant species were to include a reproductive focus, that we would be likely to find continued evidence in favor of an ancient association between BMP signaling and the reproductive cell lineage in animals., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The spectrum of congenital anomalies affecting either the upper tract (kidneys and ureters) or lower tract (reproductive organs) of the genitourinary (GU) system are fundamentally linked by the developmental origin of multiple GU tissues, including the kidneys, gonads, and reproductive ductal systems: the intermediate mesoderm. Although ∼31% of DiGeorge/del22q11.2 syndrome patients exhibit GU defects, little focus has been placed on the molecular etiology of GU defects in this syndrome. Among del22q11.2 patients exhibiting GU anomalies, we have mapped the smallest relevant region to only five genes, including CRKL CRKL encodes a src-homology adaptor protein implicated in mediating tyrosine kinase signaling, and is expressed in the developing GU-tract in mice and humans. Here we show that Crkl mutant embryos exhibit gene dosage-dependent growth restriction, and homozygous mutants exhibit upper GU defects at a microdissection-detectable rate of 23%. RNA-sequencing revealed that 52 genes are differentially regulated in response to uncoupling Crkl from its signaling pathways in the developing kidney, including a fivefold up-regulation of Foxd1 , a known regulator of nephron progenitor differentiation. Additionally, Crkl heterozygous adult males exhibit cryptorchidism, lower testis weight, lower sperm count, and subfertility. Together, these data indicate that CRKL is intimately involved in normal development of both the upper and lower GU tracts, and disruption of CRKL contributes to the high incidence of GU defects associated with deletion at 22q11.2., Competing Interests: The authors declare no conflict of interest.

Reciprocal epithelial-mesenchymal interactions and several signalling pathways regulate the development of the genital tubercle (GT), an embryonic primordium of external genitalia. The morphology of the adult male external genitalia of the Asian house musk shrew Suncus murinus (hereafter, laboratory name: suncus) belonging to the order Eulipotyphla (the former order Insectivora or Soricomorpha) differs from those of mice and humans. However, the developmental process of the suncus GT and its regulatory genes are unknown. In the present study, we explored the morphological changes and gene expression patterns during the development of the suncus GT. Morphological observations suggested the presence of common (during the initial outgrowth) and species-specific (during the sexual differentiation of GT) developmental processes of the suncus GT. In gene expression analysis, fibroblast growth factor 8 (Fgf8) and sonic hedgehog (Shh), an indicator and regulator of GT development in mice respectively, were found to be expressed in the cloacal epithelium and the developing urethral epithelium of the suncus GT. This pattern of expression specifically in GT epithelium is similar to that observed in the developing mouse GT. Our results indicate that the mechanism of GT formation regulated by the FGF and SHH signalling pathways is widely conserved in mammals., (© 2017 Society for Reproduction and Fertility.)

Impaired androgen activity induces defective sexual differentiation of the male reproductive tract, including hypospadias, an abnormal formation of the penile urethra. Androgen signaling in the urethral mesenchyme cells (UMCs) plays essential roles in driving dimorphic urethral development. However, cellular events for sexual differentiation remain virtually unknown. In this study, histological analyses, fluorescent staining, and transmission electron microscopy (TEM) were performed to reveal the cellular dimorphisms of UMCs. F-actin dynamics and migratory behaviors of UMCs were further analyzed by time-lapse imaging. We observed a prominent accumulation of F-actin with poorly assembled extracellular matrix (ECM) in female UMCs. In contrast, thin fibrils of F-actin co-aligning with the ECM through membrane receptors were identified in male UMCs. Processes for dimorphic F-actin assemblies were temporally identified during an androgen-regulated masculinization programming window and spatially distributed in several embryonic reproductive tissues. Stage-dependent modulation of the F-actin sexual patterns by androgen in UMCs was also demonstrated by time-lapse analysis. Moreover, androgen regulates coordinated migration of UMCs. These results suggest that androgen signaling regulates the assembly of F-actin from cytoplasmic accumulation to membranous fibrils. Such alteration appears to promote the ECM assembly and the mobility of UMCs, contributing to male type genital organogenesis., (© 2017 S. Karger AG, Basel.)

TYRO3, AXL and MERTK comprise the TAM family of receptor protein tyrosine kinases. Activated by their ligands, protein S (PROS1) and growth-arrest-specific 6 (GAS6), they mediate numerous cellular functions throughout development and adulthood. Expressed by a myriad of cell types and tissues, they have been implicated in homeostatic regulation of the immune, nervous, vascular, bone and reproductive systems. The loss-of-function of TAM signaling in adult tissues culminates in the destruction of tissue homeostasis and diseased states, while TAM gain-of-function in various tumors promotes cancer phenotypes. Combinatorial ligand-receptor interactions may elicit different molecular and cellular responses. Many of the TAM regulatory functions are essentially developmental, taking place both during embryogenesis and postnatally. This review highlights current knowledge on the role of TAM receptors and their ligands during these developmental processes in the immune, nervous, vascular and reproductive systems.

Accurate mouse sexing is vital when conducting research examining sexual dimorphisms. Late fetal and newborn mouse pups are more immature than many previously described sexing methods allow. This study compares the sexing accuracy of a newly described internal gonad sexing method to a recently described peritoneal pigmentation sexing method in embryonic day 20 C57BL/6J mouse pups, using Sry genotyping to confirm the sex. The internal gonad sexing method was found to be highly accurate, while the peritoneal pigmentation method was slightly less accurate. Therefore, while Sry genotyping remains the gold standard, immediate and less expensive sexing methods can be performed accurately as early as the late fetal period in C57BL/6J mice.

Sexual dimorphism in mouse reproductive tissues is observable in adult, post-natal, and embryonic stages. The development of sexually dimorphic tissues starts with an ambisexual structure. It is followed by sex-specific organogenesis as guided by different signaling pathways that occur from late embryonic stages. The measurement of the anogenital distance (AGD), and the observation of the external genitalia are practical ways to distinguish male and female pups at birth and thereafter. Careful observation of the morphological or histological features and the molecular signatures of the external genitalia and perineum enable identification of sex or feminization/masculinization of embryos. Aberrations in hormone signaling via castration or treatment with hormones or hormone disruptors result in dysmorphogenesis of reproductive tissues. Several hormone disruptors have been used to modulate different aspects of hormone action through competitive inhibition and exogenous hormone treatment. Concomitantly, the vast advancement of conditional mutant mouse analysis leads to the frequent utilization of Cre recombination technology in the study of reproductive/urogenital tissue development. Mouse Cre-lines that are tissue-specific and cell-specific are also effective tools in identifying the molecular mechanisms during sexually dimorphic development. Cre-lines applicable to different cell populations in the prostate, seminal vesicles, testis and ovaries, and mammary glands are currently being utilized. In the external genitalia and perineum, Cre lines that examine the signaling pathways of cells of endodermal, ectodermal, and mesenchymal origin reveal the roles of these tissues in the development of the external genitalia. The interaction of hormones and growth factors can be examined further through a variety of techniques available for researchers. Such cumulative information about various technologies is summarized., (Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The primary role of Anti-Müllerian hormone (AMH) during mammalian development is the regression of Müllerian ducts in males. This highly conserved function is retained in birds and is supported by the high levels of AMH expression in developing testes. Mammalian AMH expression is regulated by a combination of transcription factors, the most important being Sry-type high-mobility-group box transcription factor-9 (SOX9). In the chicken embryo, however, AMH mRNA expression precedes that of SOX9, leading to the view that AMH may play a more central role in avian testicular development. To define its role in chicken gonadal development, AMH was overexpressed using the RCASBP viral vector. AMH caused the gonads of both sexes to develop as small and undeveloped structures at both embryonic and adult stages. Molecular analysis revealed that although female gonads developed testis-like cords, gonads lacked Sertoli cells and were incapable of steroidogenesis. A similar gonadal phenotype was also observed in males, with a complete loss of both Sertoli cells, disrupted SOX9 expression and gonadal steroidogenesis. At sexual maturity both sexes showed a female external phenotype but retained sexually dimorphic body weights that matched their genetic sexes. These data suggest that AMH does not operate as an early testis activator in the chicken but can affect downstream events, such as sex steroid hormone production. In addition, this study provides a unique opportunity to assess chicken sexual development in an environment of sex hormone deficiency, demonstrating the importance of both hormonal signaling and direct cell autonomous factors for somatic sex identity in birds.

Genistein (GEN), an isoflavonoid phytoestrogen, is one of the potent estrogenic compounds derived from plants that can cause disrupting effects on sex organ development in non-mammalian and mammalian species. The present study revealed effect of genistein on germ cell number in the genital ridges during gonadogenesis. Genistein (16 and 24 μg/g egg) was injected into the egg yolk prior to incubation. Effect of genistein on quail-primordial germ cells (PGCs) number was examined by counting the number of Wisteria floribunda (WFA)-positive cells localized in both left and right genital ridges compared with the control group. Both concentrations of genistein resulted in significant decrease of PGC number compared with the control group. Percentages of the sterility rate of the embryo treated with 16 and 24 μg of genistein/g egg were 19% and 23%, respectively. These results provide evidence that genistein may be a germ cell toxicant causing sterility later in life of adult birds. This is the first report on the effect of genistein on PGC number in the genital ridges of the avian embryo.

Congenital penile anomalies (CPAs) are among the most common human birth defects. Reports of CPAs, which include hypospadias, chordee, micropenis, and ambiguous genitalia, have risen sharply in recent decades, but the causes of these malformations are rarely identified. Both genetic anomalies and environmental factors, such as antiandrogenic and estrogenic endocrine disrupting chemicals (EDCs), are suspected to cause CPAs; however, little is known about the temporal window(s) of sensitivity to EDCs, or the tissue-specific roles and downstream targets of the androgen receptor (AR) in external genitalia. Here, we show that the full spectrum of CPAs can be produced by disrupting AR at different developmental stages and in specific cell types in the mouse genital tubercle. Inactivation of AR during a narrow window of prenatal development results in hypospadias and chordee, whereas earlier disruptions cause ambiguous genitalia and later disruptions cause micropenis. The neonatal phase of penile development is controlled by the balance of AR to estrogen receptor α (ERα) activity; either inhibition of androgen or augmentation of estrogen signaling can induce micropenis. AR and ERα have opposite effects on cell division, apoptosis, and regulation of Hedgehog, fibroblast growth factor, bone morphogenetic protein, and Wnt signaling in the genital tubercle. We identify Indian hedgehog (Ihh) as a novel downstream target of AR in external genitalia and show that conditional deletion of Ihh inhibits penile masculinization. These studies reveal previously unidentified cellular and molecular mechanisms by which antiandrogenic and estrogenic signals induce penile malformations and demonstrate that the timing of endocrine disruption can determine the type of CPA.

Rapid evolution of genitalia shape, a widespread phenomenon in animals with internal fertilization, offers the opportunity to dissect the genetic architecture of morphological evolution linked to sexual selection and speciation. Most quantitative trait loci (QTL) mapping studies of genitalia divergence have focused on Drosophila melanogaster and its three most closely related species, D. simulans, D. mauritiana, and D. sechellia, and have suggested that the genetic basis of genitalia evolution involves many loci. We report the first genetic study of male genitalia evolution between D. yakuba and D. santomea, two species of the D. melanogaster species subgroup. We focus on male ventral branches, which harm females during interspecific copulation. Using landmark-based geometric morphometrics, we characterized shape variation in parental species, F1 hybrids, and backcross progeny and show that the main axis of shape variation within the backcross population matches the interspecific variation between parental species. For genotyping, we developed a new molecular method to perform multiplexed shotgun genotyping (MSG), which allowed us to prepare genomic DNA libraries from 365 backcross individuals in a few days using little DNA. We detected only three QTL, one of which spans 2.7 Mb and exhibits a highly significant effect on shape variation that can be linked to the harmfulness of the ventral branches. We conclude that the genetic architecture of genitalia morphology divergence may not always be as complex as suggested by previous studies., (Copyright © 2015 Peluffo et al.)

In this issue of Developmental Cell, Infante et al. (2015) compare regulatory DNA sequences in mice, lizards, and limbless snakes to reveal widespread sharing of enhancer activity in developing limbs and genitalia. Genetic deletion of a limb-genital enhancer demonstrates that common regulatory elements affect development of both appendages., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The amniote phallus and limbs differ dramatically in their morphologies but share patterns of signaling and gene expression in early development. Thus far, the extent to which genital and limb transcriptional networks also share cis-regulatory elements has remained unexplored. We show that many limb enhancers are retained in snake genomes, suggesting that these elements may function in non-limb tissues. Consistent with this, our analysis of cis-regulatory activity in mice and Anolis lizards reveals that patterns of enhancer activity in embryonic limbs and genitalia overlap heavily. In mice, deletion of HLEB, an enhancer of Tbx4, produces defects in hindlimbs and genitalia, establishing the importance of this limb-genital enhancer for development of these different appendages. Further analyses demonstrate that the HLEB of snakes has lost hindlimb enhancer function while retaining genital activity. Our findings identify roles for Tbx4 in genital development and highlight deep similarities in cis-regulatory activity between limbs and genitalia., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The sexual development of saurians follows a similar pattern to that described for other amniotes. Changes in the timing or sequence of development events are known as heterochrony. We describe the pattern of sexual development in the viviparous Mexican lizard Barisia imbricata and report heterochrony in the regression of hemipenes in this lizard. We collected gestating females; some lizards were subjected to partial hysterectomy and the embryos were processed using routine histological technique to assess gonadal development; the remaining embryos were used to assess the development of hemipenes. Other pregnant females were kept in captivity in individual terraria until the time of delivery. All neonates were sexed by eversion of hemipenes and some of their body characteristics were recorded. Several neonates were sacrificed and processed to establish gonadal histology and the young of the remaining litters were maintained in captivity to observe the fate of the hemipenes in both sexes. Gonadal development began at embryonic stage 33 and the hemipenes were visible at the same stage. In the neonates, the ovary contained oogonias and ovarian follicles, whereas the testicles showed testicular cords. All neonates had hemipenes and sex could only be established through direct observation of the reproductive ducts and gonadal histology. The hemipenes regression in the females begins after approximately 7 months of postnatal development and concludes at about 15 months of age. We think that the delayed regression of the hemipenes reflects evolutionary differences among reptiles and may be an indication of a stage in the evolutionary process of this species., (© 2015 Wiley Periodicals, Inc.)

Within amniotes, external copulatory organs have undergone extensive morphological diversification. One of the most extreme examples is squamate (lizards and snakes) hemipenes, which are paired copulatory organs that extend from the lateral margins of the cloaca. Here, we describe the development of hemipenes in a basal snake, the ball python (Python regius). Snake hemipenes arise as a pair of lateral swellings on either side of the caudal part of the cloaca, and these paired outgrowths persist to form the left and right hemipenes. In non-squamate amniotes, external genitalia form from paired swellings that arise on the anterior side of the cloaca, which then fuse medially to form a single genital tubercle, the anlagen of the penis or clitoris. Whereas in non-squamate amniotes, Sonic hedgehog (Shh)-expressing cells of the cloacal endoderm form the urethral or sulcus epithelium and are required for phallus outgrowth, the hemipenes of squamates lack an endodermal contribution, and the sulcus does not express Shh. Thus, snake hemipenes differ from the genital tubercles of non-squamate amniotes both in their embryonic origins and in at least part of patterning mechanisms, which raises the possibility that hemipenes may not be direct homologs of the unpaired amniote penis. Nonetheless, we find that some developmental genes show similar expression patterns in snake hemipenes buds and non-squamate genital tubercles, suggesting that homologous developmental mechanisms are involved in aspects of external genital development across amniotes, even when these structures may have different developmental origins and may have arisen independently during evolution.

Epigenetics is the study of changes in gene function that cannot be explained by changes in DNA sequence. A mammalian body contains more than two-hundred different types of cells, all derived from a single fertilized egg. Epigenetic gene regulation mechanisms essentially contribute to various processes of mammalian development. The essence of epigenetic regulation is the modulation of gene activity through changes in chromatin structure. DNA methylation and histone modifications are the major epigenetic mechanisms. Sex determination is the process of establishing a gender. Sry, the sex-determining gene in therian mammals, initiates testis differentiation. Recent studies have provided evidence that epigenetic mechanisms contribute to Sry regulation.

Avian genitalia, particularly in waterfowl, are extremely diverse. Penis morphology varies among species, and penis length and elaboration are associated with the frequency of forced extra-pair copulations, yet the developmental mechanisms responsible for this variation are unknown. In addition, females have a small phallic structure that is homologous to the male phallus, but little is known about when or how sexual differentiation takes place. To determine whether species-specific genital morphologies and sexual differentiation occur during duck embryonic development, we characterized development from the onset of genital tubercle initiation through stages of sexual differentiation in 3 species. Pekin and Laysan ducks have long, thick penises, whereas those of Mandarin ducks are shorter and thinner. Development of the genital tubercle is similar throughout the pre-hatching period across the 3 species, suggesting that differences in penis morphology arise post-hatching. We observed that male and female phallus development is similar at early stages, but the female phallus later regresses. Then, we compared male and female genital development between ducks and chickens, which develop a non-intromittent penis. We found that external genital development in male and female chickens resembles that of female ducks, which raises the possibility that male phallus development became feminized during galliform evolution.

In most animals, reproduction by internal fertilization is facilitated by an intromittent organ, such as the penis in amniote vertebrates. Recent progress has begun to uncover the mechanisms of mammalian external genital development; however, comparatively little is known about the development of the reptilian penis and clitoris. Here, we describe the development of the phallus and cloaca in the American alligator, Alligator mississippiensis. The embryonic precursor of the penis and clitoris is the genital tubercle, which forms by the budding of genital mesenchyme beneath the ventral body wall ectoderm, adjacent to the cloacal membrane. The cloacal lips develop from another pair of outgrowths, the lateral swellings. Early development of the alligator phallus, cloaca, and urogenital ducts generally resembles that of other reptiles, suggesting that differences in adult reptilian phallus and cloacal anatomy arise at later stages. The phallic sulcus is derived from the cloacal endoderm, indicating that the crocodilian sulcus is functionally and developmentally homologous to the mammalian urethra. Initial external genital outgrowth and patterning occur prior to temperature-dependent sex determination. Our analysis of alligator phallus and cloaca development suggests that modifications of an ancestral program of urogenital development could have generated the morphological diversity found in the external genitalia of modern amniotes.

In most amniotes, the intromittent organ is a single phallus; however, squamates (lizards, snakes, and amphisbaenians) have paired hemiphalluses. All amniotes studied to date initiate external genital development with the formation of paired genital swellings. In mammals, archosaurs, and turtles, these swellings merge to form a single genital tubercle, the precursor of the penis and clitoris; however, in squamates, the paired genital buds remain separate, giving rise to the hemiphalluses (hemipenes in males and hemiclitores in females). Although the molecular genetics and sexual differentiation of the genital tubercle have been investigated in mammals and birds, little is known about hemiphallus development. Here we describe development of the cloaca and hemiphallus in the green anole, Anolis carolinensis. Each hemiphallus originates as a protuberance that emerges at the ventral base of the hindlimb bud. Development of the hemipenes resembles penis development; however, differences exist in their tissue composition, morphogenesis, and gene expression patterns. These findings reveal aspects of phallus development that appear to be evolutionarily labile, both within squamates and more broadly among reptiles, and identify features that are conserved across amniotes. Our results, together with parallel studies in other reptilian taxa, suggest potential mechanisms for the diversification of external genital form.

The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention, little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the limb and genitals; however, no underlying developmental mechanism has been identified. We re-examined this question using micro-computed tomography, lineage tracing in three amniote clades, and RNA-sequencing-based transcriptional profiling. Here we show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing centre. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, they suggest that a change in the relative position of the cloacal signalling centre during evolution has led to an altered developmental route for external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry owing to a common evolutionary origin.

The birth of a baby with malformations of the genitalia urges medical action. Even in cases where the condition is not life-threatening, the identification of the external genitalia as male or female is emotionally essential for the family, and genital malformations represent one of the most stressful situations around a newborn. The female or male configuration of the genitalia normally evolves during fetal life according to the genetic, gonadal, and hormonal sex. Disorders of sex development occur when male hormone (androgens and anti-Müllerian hormone) secretion or action is insufficient in the 46,XY fetus or when there is an androgen excess in the 46,XX fetus. However, sex hormone defects during fetal development cannot explain all congenital malformations of the reproductive tract. This review is focused on those congenital conditions in which gonadal function and sex hormone target organ sensitivity are normal and, therefore, not responsible for the genital malformation. Furthermore, because the reproductive and urinary systems share many common pathways in embryo-fetal development, conditions associating urogenital malformations are discussed., (© 2014 Wiley Periodicals, Inc.)

Genital malformations occur at a high frequency in humans, affecting ~1:250 live births. The molecular mechanisms of external genital development are beginning to be identified; however, the origin of cells that give rise to external genitalia is unknown. Here we use cell lineage analysis to show that the genital tubercle, the precursor of the penis and clitoris, arises from two populations of progenitor cells that originate at the lateral edges of the embryo, at the level of the posterior hindlimb buds and anterior tail. During body wall closure, the left and right external genital progenitor pools are brought together at the ventral midline, where they form the paired genital swellings that give rise to the genital tubercle. Unexpectedly, the left and right external genital progenitor pools form two lineage-restricted compartments in the phallus. Together with previous lineage studies of limb buds, our results indicate that, at the pelvic level, the early lateral mesoderm is regionalized from medial to lateral into dorsal limb, ventral limb, and external genital progenitor fields. These findings have implications for the evolutionary diversification of external genitalia and for the association between external genital defects and disruption of body wall closure, as seen in the epispadias-extrophy complex.

Bladder exstrophy-epispadias complex (BEEC), the severe end of the urorectal malformation spectrum, has a profound impact on continence as well as sexual and renal functions. It is widely accepted that for the majority of cases the genetic basis appears to be multifactorial. Here, we report the first study which utilizes genome-wide association methods to analyze a cohort comprising patients presenting the most common BEEC form, classic bladder exstrophy (CBE), to identify common variation associated with risk for isolated CBE. We employed discovery and follow-up samples comprising 218 cases/865 controls and 78 trios in total, all of European descent. Our discovery sample identified a marker near SALL1, showing genome-wide significant association with CBE. However, analyses performed on follow-up samples did not add further support to these findings. We were also able to identify an association with CBE across our study samples (discovery: P = 8.88 × 10(-5); follow-up: P = 0.0025; combined: 1.09 × 10(-6)) in a highly conserved 32 kb intergenic region containing regulatory elements between WNT3 and WNT9B. Subsequent analyses in mice revealed expression for both genes in the genital region during stages relevant to the development of CBE in humans. Unfortunately, we were not able to replicate the suggestive signal for WNT3 and WNT9B in a sample that was enriched for non-CBE BEEC cases (P = 0.51). Our suggestive findings support the hypothesis that larger samples are warranted to identify association of common variation with CBE., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This study examined late-life effects of perinatal exposure of rats to a mixture of endocrine-disrupting contaminants. Four groups of 14 time-mated Wistar rats were exposed by gavage from gestation day 7 to pup day 22 to a mixture of 13 anti-androgenic and estrogenic chemicals including phthalates, pesticides, u.v.-filters, bisphenol A, parabens, and the drug paracetamol. The groups received vehicle (control), a mixture of all 13 chemicals at 150-times (TotalMix150) or 450-times (TotalMix450) high-end human exposure, or 450-times a mixture of nine predominantly anti-androgenic chemicals (AAMix450). Onset of puberty and estrous cyclicity at 9 and 12 months of age were assessed. Few female offspring showed significantly regular estrus cyclicity at 12 months of age in the TotalMix450 and AAMix450 groups compared with controls. In 19-month-old male offspring, epididymal sperm counts were lower than controls, and in ventral prostate an overrepresentation of findings related to hyperplasia was observed in exposed groups compared with controls, particularly in the group dosed with anti-androgens. A higher incidence of pituitary adenoma at 19 months of age was found in males and females in the AAMix450 group. Developmental exposure of rats to the highest dose of a human-relevant mixture of endocrine disrupters induced adverse effects late in life, manifested as earlier female reproductive senescence, reduced sperm counts, higher score for prostate atypical hyperplasia, and higher incidence of pituitary tumors. These delayed effects highlight the need for further studies on the role of endocrine disrupters in hormone-related disorders in aging humans.

Although several syndromes include abnormalities of both the ventral body wall and external genitalia, the developmental bases of this correlation are largely unknown. Naturally occurring mutations in Aristaless-like 4 (Alx4, Strong's luxoid: Alx4Lst) have ventral body wall and pelvic girdle abnormalities. We sought to determine whether the development of the genital tubercle (GT) and its derivatives, the external genitalia, is affected by this mutation. We thus performed genetic and tissue labeling analyses in mutant mice. Alx4Lst/Lst mutants displayed hypoplasia of the dorsal GT and reduced expression of Fibronectin. We analyzed cell migration during GT formation by tissue labeling experiments and discovered that the cells located in the proximal segment of the umbilical cord (infra-umbilical mesenchyme) migrate toward the dorsal part of the GT. The Alx4Lst/Lst mutants also displayed augmented expression of Hh signal-related genes. Hence, we analyzed a series of combinatorial mutants for Alx4, Sonic hedgehog (Shh) and GLI-Kruppel family member 3 (Gli3). These phenotype-genotype analyses suggested a genetic interaction between Alx4 and Hh signaling during GT formation. Moreover, Hh gain-of-function mutants phenocopied some of these phenotypes. These observations reveal novel information regarding the pathogenic mechanisms of syndromic lower ventral body malformations, which are largely unknown.

Development of the mammalian external genitalia is controlled by a network of signaling molecules and transcription factors. Because FGF signaling plays a central role in this complicated morphogenetic process, we investigated the role of Sprouty genes, which are important intracellular modulators of FGF signaling, during embryonic development of the external genitalia in mice. We found that Sprouty genes are expressed by the urethral epithelium during embryogenesis, and that they have a critical function during urethral canalization and fusion. Development of the genital tubercle (GT), the anlage of the prepuce and glans penis in males and glans clitoris in females, was severely affected in male embryos carrying null alleles of both Spry1 and Spry2. In Spry1(-/-);Spry2(-/-) embryos, the internal tubular urethra was absent, and urothelial morphology and organization was abnormal. These effects were due, in part, to elevated levels of epithelial cell proliferation in Spry1(-/-);Spry2(-/-) embryos. Despite changes in overall organization, terminal differentiation of the urothelium was not significantly affected. Characterization of the molecular pathways that regulate normal GT development confirmed that deletion of Sprouty genes leads to elevated FGF signaling, whereas levels of signaling in other cascades were largely preserved. Together, these results show that levels of FGF signaling must be tightly regulated during embryonic development of the external genitalia in mice, and that this regulation is mediated in part through the activity of Sprouty gene products., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The study of the external genitalia is divided into 2 developmental stages: the formation and growth of a bipotential genital tubercle (GT) and the sexual differentiation of the male and female GT. The sexually dimorphic processes, which occur during the second part of GT differentiation, are suggested to be governed by androgen signaling and more recently crosstalk with other signaling factors. The process of elucidating the regulatory mechanisms of hormone signaling towards other signaling networks in the GT is still in its early stages. Nevertheless, it is becoming a productive area of research. This review summarizes various studies on the development of the murine GT and the defining characteristics of a masculinized GT and presents the different signaling pathways possibly involved during masculinization.

For almost 2000 years, human beings have been discussing about gender. New scientific evidences give interesting new points of view, partially subverting the normal dichotomy described by the "two-gender" theory. In this article, we are going to critically review the history of the approach towards people born with a Sexual-Differentiation-Disorder, passing through the analysis of the Italian National Ethics Committee's opinion, describing the modern scientific evidences on the gender-identity development, furthermore ruling out the new approach borned from the femminist philosophies, and the new biogiuridical experiments borned in Australia and Germany. Would it be possible a world where a person could be more then a male or a female?

External genitalia are found in each of the major clades of amniotes. The phallus is an intromittent organ that functions to deliver sperm into the female reproductive tract for internal fertilization. The cellular and molecular genetic mechanisms of external genital development have begun to be elucidated from studies of the mouse genital tubercle, an embryonic appendage adjacent to the cloaca that is the precursor of the penis and clitoris. Progress in this area has improved our understanding of genitourinary malformations, which are among the most common birth defects in humans, and created new opportunities for comparative studies of other taxa. External genitalia evolve rapidly, which has led to a striking diversity of anatomical forms. Within the past year, studies of external genital development in non-mammalian amniotes, including birds, lizards, snakes, alligators, and turtles, have begun to shed light on the molecular and morphogenetic mechanisms underlying the diversification of phallus morphology. Here, we review recent progress in the comparative developmental biology of external genitalia and discuss the implications of this work for understanding external genital evolution. We address the question of the deep homology (shared common ancestry) of genital structures and of developmental mechanisms, and identify new areas of investigation that can be pursued by taking a comparative approach to studying development of the external genitalia. We propose an evolutionary interpretation of hypospadias, a congenital malformation of the urethra, and discuss how investigations of non-mammalian species can provide novel perspectives on human pathologies.