Development Of Genitalia Research Articles

A Preterm Newborn with Abnormal Development of the Lower Abdomen and Genitalia

A preterm newborn is noted after delivery to have abnormal development of the lower abdomen and genitalia. ### Prenatal and Birth Histories ### Presentation (Newborn Day) Immediately after birth, the neonatology team that was present at delivery examined the infant and noted a lower abdominal mass and ambiguous genitalia (Fig 1). The infant was transferred to the NICU for further evaluation. On admission, the newborn was noted to have central cyanosis and given nasal prong continuous positive airway pressure (CPAP). Figure 1. Exstrophy and epispadiasis. ### Vital Signs ### Physical Examination

High-throughput tool to discriminate effects of NMs (Cu-NPs, Cu-nanowires, CuNO3, and Cu salt aged): transcriptomics in Enchytraeus crypticus

The current testing of nanomaterials (NMs) via standard toxicity tests does not cover many of the NMs specificities. One of the recommendations lays on understanding the mechanisms of action, as these can help predicting long-term effects and safe-by-design production. In the present study, we used the high-throughput gene expression tool, developed for Enchytraeus crypticus (4 × 44k Agilent microarray), to study the effects of exposure to several copper (Cu) forms. The Cu treatments included two NMs (spherical and wires) and two copper-salt treatments (CuNO3 spiked and Cu salt field historical contamination). To relate gene expression with higher effect level, testing was done with reproduction effect concentrations (EC20, EC50), using 3 and 7 days as exposure periods. Results showed that time plays a major role in the transcriptomic response, most of it occurring after 3 days. Analysis of gene expression profiles showed that Cu-salt-aged and Cu-nanowires (Nwires) differed from CuNO3 and Cu-nanoparticles (NPs). Functional analysis revealed specific mechanisms: Cu-NPs uniquely affected senescence and cuticle pattern formation, which can result from the contact of the NPs with the worms’ tegument. Cu-Nwires affected reproduction via male gamete generation and hermaphrodite genitalia development. CuNO3 affected neurotransmission and locomotory behavior, both of which can be related with avoidance response. Cu salt-aged uniquely affected phagocytosis and reproductive system development (via different mechanisms than Cu-Nwires). For the first time for Cu (nano)materials, the adverse outcome pathways (AOPs) drafted here provide an overview for common and unique effects per material and linkage with apical effects.

Wilms’ Tumor 1 Overexpression in Granulosa Cells Is Associated with Polycystic Ovaries in Polycystic Ovary Syndrome Patients

Background: Polycystic ovary syndrome (PCOS) is a heterogeneous disorder characterized by chronic ovulatory dysfunction, hyperandrogenism, and polycystic ovaries. Wilms’ tumor 1 (WT1) encoding a transcription factor involved in the differentiation of granulosa cells (GCs) regulates androgen receptor in the development of male genitalia. However, the expression pattern and possible role of WT1 in ovaries of PCOS patients are still unknown. Methods: GCs from 95 PCOS patients (PCOS group) and 62 healthy controls (control group) were isolated. The expression of WT1 in GCs was quantified using the reverse transcription-polymerase chain reaction. The correlation between WT1 expression and clinical characteristics was evaluated in PCOS patients. Results: WT1 expression was increased in PCOS patients compared with the normal controls. The expression of WT1 was moderately correlated with testosterone (r = 0.334, p = 0.001) and luteinizing hormone (r = 0.357, p = 0.001) levels and the antral follicle counts (r = 0.337, p = 0.001). Conclusions: Our study provided novel insights into the relationship between hyperandrogenism and polycystic ovaries of PCOS and WT1.

Description of mutation spectrum and polymorphism of Wilms' tumor 1 (WT1) gene in hypospadias patients in the Indonesian population

Hypospadias is one of the most common congenital anomalies of the penis. Previous studies reported mutation of the Wilms' tumor 1 (WT1) gene as a cause of hypospadias. The aim of this study is to describe the WT1 mutation spectrum and polymorphism in hypospadias patients in Indonesia. DNA was isolated from 74 hypospadias patients at the Division of Pediatric Surgery, Department of Surgery Hasan Sadikin Hospital. All exons in the WT1 gene were amplified by a PCR method, followed by Sanger sequencing. Mutation analysis was performed using BioEdit software and in silico analysis using Mutation Taster, Polymorphism Phenotyping-2 (PolyPhen-2), and Sorting Intolerant from Tolerant (SIFT). DNA analysis results showed two types of heterozygous mutations in five subjects (Table), hence the frequency of WT1 mutations was 6.7% (10/148 allele). The first mutation was a missense mutation identified in twin boys. The second was a novel heterozygous alteration in the non-coding region nine bp upstream of exon 6 (c.366-9T>C), which was identified in three patients. One heterozygous polymorphism in the coding region of exon 7 (c.471A>G/rs16754) was identified in 10 subjects. This variant did not cause any change in amino acid products (silence polymorphism). Allele frequency for the G allele (mutant allele) and A allele (wild type) was 13.5% and 86.5%, respectively. WT1 is one of the best known hypospadias genes. The WT1 gene is involved in male genital development in the early and late periods of sex determination, and hence is known as a long-term expression gene in genitalia development. Mutation analysis of WT1 in a Chinese population identified that the WT1 mutation frequency was 4.4%. The WT1 mutation frequency identified in the present study was higher, at 6.7%. Coincidentally, research subjects with p.R158H variants were monozygotic twin siblings with midshaft hypospadias accompanied by undescended testis in one and penoscrotal hypospadia with micropenis in the other. The incidence of familial hypospadias in male siblings suffering from hypospadias was reported to be 9.6% in a study conducted by Sorensen etal. Moreover, in the present study polymorphism c.471A>G(rs16754) at exon 7 was identified heterozygously in 10 research subjects (minor allele frequency 13.5%). WT1 mutations were identified in only a few cases of hypospadias and most of these were syndromic. This result implies that mutation of WT1 is not a common cause of hypospadias in the Indonesian population.

Expression of androgen, estrogen, and progesterone hormone receptors in the penile tissues of children with different types of hypospadias.

OBJECTIVE:Androgen (AR), Estrogen (ER) and Progesterone (PR) hormones play an important role in the prenatal and postnatal development of urogenital tract and especially the penis. The expressions of AR, ER and PR receptors in penile tissues in children with hypospadiases had also been shown previously. In this leading study, to demonstrate of the sex hormone receptor expression in cases with different types of hypospadias were aimed.METHODS:This study was designed in children operated due to hypospadiases without DSD. Biopsy samples of 3 mm’s were obtained from three different sytes as the lateral parameatal tissue and the anterior corner of the prepuce, and inner layer of posterior prepuce. The presence of AR, ER and PR receptors was investigated immunehistochemically.RESULTS:Mean age was 5.4 years in 18 children with hypospadiases; in totally 33 specimens were taken in 5 subcoronal as 5 specimens, and 7 penile as 15 specimens, and 6 penoscrotal as 13 specimens. According to sytes of samples; 13 samples were from lateral para-meatal tissues, and 13 were from anterior corners of prepuces, and 7 were from inner layers of posterior prepuces. In regard to receptor expression; ER and AR receptors were positive in 29 (87.8%) and 12 (36.4%) respectively; PR receptors were negative.CONCLUSION:This study emphasized the dominant expression of estrogen receptors in penile tissues of children with hypospadias. Although there was not a manifest correlation of androgen receptors absence in regard to the severity of hypospadias patients, there was a marked estrogen receptors presence in penile tissues. These findings suggest that the disrupted androgen and estrogen receptor interaction and/or balance could play a role during the development of external genitalia in hypospadias patients. Progesterone receptor was not present and therefore the active role in the postnatal development of hypospadias is still debatable.

Evolution of the cloacal and genital musculature, and the genitalia morphology in liolemid lizards (Iguania: Liolaemidae) with remarks on their phylogenetic bearing

In this study, we describe the intra- and interspecific anatomical variations of cloacal and related muscles of male and female genitalia in species of five iguanian genera (three liolemid: Ctenoblepharys, Liolaemus, and Phymaturus plus Diplolaemus leopardinus and Tropidurus melanopleurus as outgroups). We found variations (seventeen characters) in topology, origin and insertion areas, tendon morphology and size of the musculature of this region. We also describe the variations of hemipeneal morphology, which is especially notable for the hemipenis of C. adspersa, D. leopardinus, and T. melanopleurus, as this is first time they are described in the literature. Among the most significant findings are the identification of three new muscles, two of them inserted on the roof of the cloacal chamber (anterior and posterior cloacal retractor) and the third inserted superficially in the floor of the cloaca, just before anterior to the precloacal glands row (superficialis cloacalis retractor). We report sexual dimorphism in seven muscle characters. Musculature related to hemiclitoris is reduced in proportion to its size in comparison to the degree of development of male genitalia and associated musculature. The evolution of characters was traced on the known phylogenetic hypotheses of relationships among families. Characters taken from the cloacal/genital myology bring similar support to the liolaemid tree even rooting the analysis using different outgroups. In addition, a phylogenetic study using only myological characters was performed. In this case, C. adspersa was found to be more related to Liolaemus species instead of being basal to Liolaemus plus Phymaturus.

Sexual Maturation, Attitudes towards Sexual Maturity, and Body Esteem in Elementary-School Children.

The purpose of this study is to evaluate sexual maturation, attitudes toward sexual maturity, and body esteem in the sexual development of Korean elementary-school boys and girls. A descriptive cross-sectional study was conducted with 399 fifth and sixth graders (192 boys and 207 girls). The data were analysed with a χ2 test, t-test, and Pearson correlation coefficients. Among the 207 girls, 70.5% had pubic hair growth, 68.1% had breast development, and 56.0% had a menstrual period. In boys, 59.4% of the 192 subjects experienced the development of external genitalia and 52.6% had pubic hair growth. Sexual maturation was related to grade (boys, t=7.07, p=.008; girls, t=12.76, p < .001), age (t=-2.20, p=.030; t=-4.11, p < .001), height (t=-5.16, p < .001; t=-7.52, p < .001), and weight (t=-2.89, p=.004; t=-5.19, p < .001) in both boys and girls. Girls were more likely to have sexual maturity than boys (χ2=22.29, p < .001). Boys showed more positive attitudes toward sexual maturity (t=2.10, p=.036) and higher body esteem (t=2.12, p=.035) than girls. This study shows that sexual maturation, attitude toward sexual maturity, and body esteem in sexual development differ between boys and girls. The findings indicate that it is necessary to develop a tailored sex-education program according to the sex of elementary-school children.

Nuclear Androgen Receptor Regulates Testes Organization and Oocyte Maturation in Zebrafish

Androgens act through the nuclear androgen receptor (AR) to regulate gonad differentiation and development. In mice, AR is necessary for spermatogenesis, testis development, and formation of external genitalia in males and oocyte maturation in females. However, the extent to which these phenotypes are conserved in nonmammalian vertebrates is not well understood. Here, we generate zebrafish with a mutation in the ar gene (aruab105/105) and examine the role of AR in sexual determination and gonad development. We found that zebrafish AR regulates male sexual determination, because the majority of aruab105/105 mutant embryos developed ovaries and displayed female secondary sexual characteristics. The small percentage of mutants that developed testes displayed female secondary sexual characteristics, exhibited structurally disorganized testes, and were unable to release or produce normal levels of sperm, demonstrating that AR is necessary for zebrafish testis development and fertility. In females, we found that AR regulates oocyte maturation and fecundity. The aruab105/105 mutant females developed ovaries filled primarily with immature stage I oocytes and few mature stage III oocytes. Two genes whose expression is enriched in wild-type ovaries compared with testes (cyp19a1a, foxl2a) were upregulated in ar mutant testes, and two genes enriched in testes (amh, dmrt1) were upregulated in ar mutant ovaries. These findings demonstrate that AR regulates sexual determination, testis development, and oocyte maturation and suggest that AR regulates sexually dimorphic gene expression. The ar mutant we developed will be useful for modeling human endocrine function in zebrafish.

A study of splicing mutations in disorders of sex development

The presence of splicing sequence variants in genes responsible for sex development in humans may compromise correct biosynthesis of proteins involved in the normal development of gonads and external genitalia. In a cohort of Brazilian patients, we identified mutations in HSD17B3 and SRD5A2 which are both required for human sexual differentiation. A number of these mutations occurred within regions potentially critical for splicing regulation. Minigenes were used to validate the functional effect of mutations in both genes. We evaluated the c.277 + 2 T > G mutation in HSD17B3, and the c.544 G > A, c.548-44 T > G and c.278delG mutations in SRD5A2. We demonstrated that these mutations altered the splicing pattern of these genes. In a genomic era these results illustrate, and remind us, that sequence variants within exon-intron boundaries, which are primarily identified for diagnostic purposes and have unknown pathogenicity, need to be assessed with regards to their impact not only on protein expression, but also on mRNA splicing.

Clinical Approach for Evaluation and Manage¬ment of Disorders of Sex Development

"It's a boy" and "It's a girl" are words that are heard every second of every day all around the world. However, it is very distressing when the birth attendants are unable to make such a pronouncement because of disorders of sex development (DSD). DSD are congenital conditions associated with an atypical development of chromosomal, gonadal or anatomical sex. Normal sex development progresses in steps from conception to the complete development of the fetal external genitalia; any disturbance in any of these steps can lead to DSD. Ambiguous genitalia are the most common type of DSD and it is a challenging clinical diagnosis for the pediatric endocrinologist. A newborn baby with ambiguous genitalia is often a surprise for both the medical team and the parents, frequently described as an emergency. The condition needs a special approach in terms of counseling the parents appropriately, evaluation and management. The Chicago Conference (2006) recommended new nomenclature and a classification for DSD, as the old nomenclature was confusing for doctors and parents, and sometimes pejorative. The new classification is based on karyotyping and gonadal structure, improving understanding of the underlying pathogenic mechanisms. The rapid progression of genetic diagnosis of DSD using advanced techniques such as next-generation sequencing (NGS) allows more appropriate diagnosis and genetic counseling for families.&#x0D; The focus of the article is a review of normal sex development, DSD classification, clinical approach, genetic assessment, sex assignment, surgical management and risk of germ cell tumor development.

De la Chapelle syndrome: clinical and laboratory characteristics of 4 patients

We describe 4 cases of 46,XX testicular disorder of sex development. Depending on the presence of the SRY gene, two variants of this disease are distinguished: SRY-positive and SRY-negative ones. The SRY (sex-determining region Y) gene is involved in differentiation of Sertoli cells and development of the testes. The pathogenesis of the disease in SRY-positive patients is related to translocation of the SRY gene from the Y chromosome to the X chromosome. The pathogenesis of the disease in SRY-negative patients is associated with overexpression of genes (SOX9, SOX3, etc.) responsible for the development of male genitalia or inadequate gene expression (WNT4, RSPO1, etc.) responsible for the development of female genitalia. The patients had a hermaphroditic genital structure with a masculinization score (EMS) of 6 and 11 and were SRY-negative in 50% of cases; 50% of the patients were SRY-positive with full masculinization, microorchidism, and gynecomastia. All children underwent a hormonal examination in the neutral and pubertal periods. Patients who reached pubertal age developed partial and total hypergonadotropic hypogonadism. The patients have had no need in substitution therapy with testosterone. One of the patients underwent a semiological analysis that revealed necrozoospermia and oligoasthenoteratozoospermia. A SRY-negative patient with cryptorchidism underwent biopsy of the cryptorchid gonad; no Leydig cells were found.

Validity of Self-Assessed Sexual Maturation Against Physician Assessments and Hormone Levels

To compare self-report and physician assessments of sexual maturation against serum hormone markers to evaluate the hypothesis that the validity of self-assessed sexual maturation is underestimated in traditional validation studies. We adapted a self-assessment instrument that 248 Mexican children and adolescents, aged 8-13 years, completed. The participants were examined by a trained pediatrician and provided fasting blood samples for measurement of reproductive hormones (eg, testosterone, estradiol, sex hormone-binding globulin, inhibin B) and other hormones (eg, C-peptide, insulin-like growth factor 1, leptin, dehydroepiandrosterone sulfate) known to change during adolescence. Spearman correlations (r) were calculated among the average rank of all hormones and self-assessed and physician-assessed Tanner stage. The method of triads was used to assess the validity of self-reports by estimating correlations between self-assessments and true but unobservable sexual maturation based on all available data. Bootstrap sampling was used to construct 95% CIs. The validity of self-reported genitalia staging for boys was modest (r = 0.50; 95% CI, 0.31-0.65) and inferior to physician assessment (r = 0.75; 95% CI, 0.56-0.93). Breast stage was well reported (r = 0.89; 95% CI, 0.79-0.97) and superior to physician assessment (r = 0.80; 95% CI, 0.70-0.89). Pubic hair stage reported by boys (r = 0.91; 95% CI, 0.79-0.99) and girls (r = 0.99; 95% CI, 0.96-1.00) was superior to physician assessment (r = 0.79; 95% CI, 0.57-0.97 and r = 0.91; 95% CI, 0.83-0.97, respectively). Self-assessment can be validly used in epidemiologic studies for evaluating sexual maturation in children; however, physician assessment may be necessary for accurate assessment of genitalia development in boys.

Function of Nanos1 gene in the development of reproductive organs of Schistosoma japonicum.

Nanos is a necessary factor in the differentiation and migration of primordial germ cells. It is closely associated with the development of genitalia in a wide range of species. We questioned whether Nanos was involved in the reproductive organ development of Schistosoma japonicum. Firstly, by in situ hybridization, S. japonicum Nanos1 (SjNanos1) gene was expressed mainly in reproductive organs of S. japonicum. Then, the paired schistosome of 28days post-infection (dpi) was transfected with SjNanos1 small interfering RNA three times and cultured in vitro for 10days. SjNanos1 expression suppression in the mRNA and protein levels were confirmed compared to that of the controls. The morphological changes in reproductive organs and egg production were observed after SjNanos1 gene knockdown. The results observed by confocal laser scanning microscopy showed significant changes in the morphology of reproductive organs of parasites, especially the female ovaries, vitellarium, and the male testes, after RNAi. In addition, SjNanos1 silencing also induced the reduction of eggs, and affected the changes of reproduction-related genes, like Pumilio, CNOT6L, and Fs800. Therefore, our findings demonstrate that the SjNanos1 gene is essential in the development of reproductive organs and the egg production of S. japonicum.

Perineal lipoma mimicking an accessory penis with scrotum

A case of accessory penis with scrotum in a 4 months old boy is reported because of its rarity. The infant presented with a tumour mimicking an accessory penis with scrotum between the normal sited scrotum and anus. Both testes had descended into the scrotum. After complete evaluation, there was no other urological anomaly. The tumour was excised and the histo-pathological findings of the tumor indicated a perineal lipoma. An overview of normal development of male external genitalia has been provided and the deranged mechanism resulting in this anomaly has been reviewed with hypothesis regarding etiology of accessory scrotum.

Effect of Number of Players and Maturity on Ball-Drills Training Load in Youth Basketball.

This study aimed to assess the basketball ball-drills workload analyzing: (1) the effect of varying the number of players involved on physiological and technical demands; (2) the temporal changes in players’ responses across bouts; and (3) the relationship of players’ workload with their maturation status and training age. Twelve young male basketball players (mean ± SD; age 13.9 ± 0.7 years; height 1.76 ± 0.06 m; body mass 65.7 ± 12.5 kg; HRmax 202 ± 8 beat·min−1) completed three bouts of 4 min interspersed by 2 min of passive recovery of two vs. two and four vs. four ball-drills. The mean percentage of HRmax (%HRmax) and ratings of perceived exertion (RPE) were collected. Technical actions (TAs) (dribbles, passes, shots, interceptions, steals, rebounds, and turnovers) were calculated through notational analysis. Players’ genitalia development (GD) and pubic hair (PH) growth were assessed using Tanner scale. Results showed a higher %HRmax (p = 0.018), RPE (p = 0.042), dribbles (p = 0.007), shots (p = 0.003), and rebounds (p = 0.006) in two vs. two compared to four vs. four condition. Furthermore, a statistical difference was found for %HRmax (p = 0.005) and number of passes (p = 0.020) between bouts. In addition, no correlation between GD, PH, and training age with %HRmax, RPE, and TAs was found. These findings suggest that variations of the number of players involved affect ball-drills workload and that ball-drills training intensity varies across bouts. Finally, ball-drills elicit an adequate training stimulus, regardless of players’ maturation status and training age.

Is the technical performance of young soccer players influenced by hormonal status, sexual maturity, anthropometric profile, and physical performance?

The aim of this study was to examine the influence of hormonal status, anthropometric profile, sexual maturity level, and physical performance on the technical abilities of 40 young male soccer players during small-sided games (SSGs). Anthropometric profiling, saliva sampling, sexual maturity assessment (Tanner scale), and physical performance tests (Yo-Yo and vertical jumps) were conducted two weeks prior to the SSGs. Salivary testosterone was determined by the enzyme-linked immunosorbent assay method. Technical performance was determined by the frequency of actions during SSGs. Principal component analyses identified four technical actions of importance: total number of passes, effectiveness, goal attempts, and total tackles. A multivariate canonical correlation analysis was then employed to verify the prediction of a multiple dependent variables set (composed of four technical actions) from an independent set of variables, composed of testosterone concentration, stage of pubic hair and genitalia development, vertical jumps and Yo-Yo performance. A moderate-to-large relationship between the technical performance set and the independent set was observed. The canonical correlation was 0.75 with a canonical R2 of 0.45. The highest structure coefficient in the technical performance set was observed for tackles (0.77), while testosterone presented the highest structure coefficient (0.75) for the variables of the independent set. The current data suggest that the selected independent set of variables might be useful in predicting SSG performance in young soccer players. Coaches should be aware that physical development plays a key role in technical performance to avoid decision-making mistakes during the selection of young players.

Flutamide-induced hypospadias in rats: A critical assessment

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.

Expression of steroidogenic enzymes and steroid receptors in foetal gonads of domestic cat-Sex similarities and differences.

Foetal gonads already produce steroid hormones and by this influence the further development of external and internal genitalia as well as of the brain. Beside this, foetal gonads themselves can be influenced by foetal or maternal hormones. The time course of foetal gonadal development can differ between species. As knowledge on processes in domestic cats is very limited, the steroidogenic enzyme expressions as well as these of steroid receptors were analysed in foetal gonads of domestic cats. We investigated a period from beginning of the second half of pregnancy to the beginning of the third trimester; a phase, where also gonadal development proceeds. The mRNA expression of most of the steroidogenic enzymes was remarkably higher in male gonads compared to female ones on all analysed days. The enzyme mRNA expression in female gonads shows a tendency for an increase towards the beginning of the third trimester, except that of aromatase gene CYP19A1-it shows the opposite trend. CYP19A1 was detectable just in female gonads, indicating that only female foetal gonads are capable of producing oestrogens. Gene expressions of genomically and non-genomically acting steroid receptors for progesterone, androgen and oestrogen reception were observed in gonads of both genders. Slightly higher expressions of some receptors were detected in female compared to male gonads; only for the non-genomically oestrogen receptor GPER, we observed the opposite. The protein staining for progesterone receptor membrane component 1 (PGRMC1) exposed a potential function of it on steroid-producing cell and/or cells that suppose early oogenesis.

Ancestral androgenic differentiation pathways are repurposed during the evolution of adult sexual plasticity.

Although early exposure to androgens is necessary to permanently organize male phenotype in many vertebrates, animals that exhibit adult sexual plasticity require mechanisms that prevent early fixation of genital morphology and allow for genital morphogenesis during adult transformation. In Lythrypnus dalli, a teleost fish that exhibits bi-directional sex change, adults display dimorphic genitalia morphology despite the absence of sex differences in the potent fish androgen 11-ketotestosterone. Based on conserved patterns of vertebrate development, two steroid-based mechanisms may regulate the early development and adult maintenance of dimorphic genitalia; local androgen receptor (AR) and steroidogenic enzyme expression. Consistent with the ancestral pattern of AR expression during the multipotential phase of differentiation, juvenile differentiation into either sex involved high mesenchymal AR expression. In adults, AR expression was high throughout the male genitalia, but low or absent in females. Consistent with the hypothesis that adult sexual plasticity repurposes pathways from primary differentiation, we show that adults with transitioning genitalia also exhibited higher AR expression relative to females. Local androgen biosynthesis may also participate in genitalia transformation, as transitioning adults had greater 11β-HSD-like immunoreactivity in the epithelial layer of the dorsal lumen compared to both sexes. By administering an AR antagonist to adult males, we show AR is necessary to maintain male-typical morphology. In a species that is resistant to early sexual canalization, early androgenic differentiation mechanisms are consistent with other vertebrates and the tissue-specific regulation of AR expression appears to be repurposed in adulthood to allow for transitions between sexual phenotypes.

Altered SOX9 genital tubercle enhancer region in hypospadias

Human mutations in the SOX9 gene or its regulatory region can disrupt testicular development, leading to disorders of sex development (DSDs). Our previous work involving the genomic analysis of isolated DSD patients revealed a 78kb minimal sex determining region (RevSex) far upstream of SOX9 that was duplicated in 46,XX and deleted in 46,XY DSDs. It was postulated that RevSex contains a gonadal enhancer. However, the most highly conserved sub-region within RevSex, called SR4, was neither responsive to sex determining factors in vitro nor active in the gonads of transgenic mice, suggesting that SR4 may not be functioning as a testicular enhancer. Interestingly, SR4 transgenic mice showed reporter activity in the genital tubercle, the primordium of the penis and clitoris, a previously unreported domain of Sox9 expression. SOX9 protein was detected in the genital tubercle, notably in the urethral plate epithelium, preputial glands, ventral surface ectoderm and corpus cavernosa. SR4 may therefore function as a Sox9 genital tubercle enhancer, mutations of which could possibly lead to hypospadias, a birth defect seen in the DSD patients in the RevSex study. SR4 activity and the observed SOX9 expression pattern suggest that SR4 may function as a Sox9 genital tubercle enhancer. However, conditional ablation of Sox9 in the genital tubercle using Shh-Cre/+;Sox9flox/flox mice revealed no genital tubercle abnormalities, possibly due to compensation by similar Sox factors. To conclude, we have identified a novel regulatory enhancer driving Sox9 expression during external genitalia development.