Association of antimullerian hormone with the size of the appendix testis, the androgen and estrogen receptors and their expression in the appendix testis, in congenital cryptorchidism.

OBJECTIVES: Antimullerian hormone (AMH) causes regression of the mullerian ducts in the male fetus. The appendix testis (AT) is a vestigial remnant of mullerian duct origin, containing both androgen (AR) and estrogen (ER) receptors. The role of both AMH and AT in testicular descent is yet to be studied. We investigated the possible association of AMH with AT size, the AR and ER, and their expression in the AT, in congenital cryptorchidism. METHODS: A total of 26 patients with congenital unilateral cryptorchidism and 26 controls with orthotopic testes were investigated, and 21 ATs were identified in each group. AMH and insulin-like three hormone (INSL3) concentrations were measured with spectrophotometry. AR and ER receptor expression was assessed with immunohistochemistry using monoclonal antibodies R441 for AR and MAB463 for ER. For the estimation of receptor expression, the Allred Score method was used. RESULTS: AMH concentrations did not present significant differences between patients with congenital cryptorchidism and the controls. Also, no correlation was found between AMH, INSL3, and AT length. Allred scores did not present significant differences. However, expression percentiles and intensity for both receptors presented significant differences. Three children with cryptorchidism and the highest AMH levels also had the highest estrogen receptor scores in the AT. CONCLUSIONS: No association was found between AMH and the studied major parameters. However, higher AMH concentrations, in combination with higher estrogen receptor scores in the AT, may play a role in cryptorchidism in some children. Larger population samples are needed to verify this observation.

Morphologic alterations of the genital mesentery implicated in testis non-descent in rats prenatally exposed to flutamide.

BACKGROUND: There is an endless debate on whether androgens mediate testis descent through developmental changes in the gubernacular or the cranial suspensory ligament. OBJECTIVE: To investigate the relation of any possible morphologic changes in the genital mesentery, that is, the system of genital peritoneal folds including the gubernacular and cranial suspensory ligaments, with the event of testis non-descent in rats prenatally exposed to the antiandrogen flutamide. MATERIALS AND METHODS: Time-pregnant Sprague Dawley rats received flutamide (100 mg/kg/d) or vehicle subcutaneously on gestational days 16-17. Flutamide-treated male offspring (n = 67), and vehicle-treated male (n = 34) and female (n = 28) offspring were surgically explored under microscope on postnatal day 50. Testicular position was examined bilaterally. Dimensions of genital mesentery parts were also assessed bilaterally. Association of flutamide-induced morphologic changes with descended (n = 61) and undescended (n = 50; 33 cryptorchid and 17 ectopic) testes was investigated with logistic regression analysis. RESULTS: The male genital mesentery comprised a cranial and a caudal fold converging on the vas deferens. Flutamide resulted in enlarged cranial and reduced caudal folds. Of all flutamide-induced alterations, the increased length of the posterior fixation of the cranial fold and the decreased length of the gubernacular ligament of the caudal fold were found to independently increase the odds of testis non-descent. Testicular ectopy, unlike cryptorchidism, was associated with a short gubernacular ligament only. The female genital mesentery consisted of a cranial fold only. CONCLUSION: Our findings showed a combined contribution of both cranial and caudal folds of the genital mesentery to testis non-descent, through an abnormally long mesentery root and an abnormally short gubernacular ligament, respectively. Inhibition of male-specific development of the genital mesentery with flutamide did not result in a feminized architecture.

Spotted hyaenas and the sexual spectrum: reproductive endocrinology and development.

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

Infections of the Male and Female Reproductive System: Spectrum of Imaging Findings.

Infections of the male and female reproductive system can lead to significant morbidity and mortality. This article will review the relevant embryology and anatomy of the male and female reproductive systems and will discuss the imaging findings of different infections. An understanding of the clinical presentation and imaging findings of infections of the reproductive system is critical in order to allow for prompt and accurate diagnosis. A delay in diagnosis for these infections can have significant morbidity, and occasional mortality.

A Conflicted Tale of Two Novel AR Antagonists In Vitro and In Vivo: Pyrifluquinazon Versus Bisphenol C.

Chemicals that disrupt androgen receptor (AR) function in utero induce a cascade of adverse effects in male rats including reduced anogenital distance, retained nipples, and reproductive tract malformations. The objective of this study was to compare the in vitro and in utero activities of two novel AR antagonists, bisphenol C (BPC) and pyrifluquinazon (PFQ). In vitro, BPC was as potent an AR antagonist as hydroxyflutamide. Furthermore, BPC inhibited fetal testis testosterone production and testis gene expression ex vivo. However, when BPC was administered at 100 and 200 mg/kg/d in utero, the reproductive tract of the male offspring was minimally affected. None of the males displayed reproductive malformations. For comparison, in utero administration of flutamide has been shown to induce malformations in 100% of males at 6 mg/kg/d. In vitro, PFQ was several orders of magnitude less potent than BPC, vinclozolin, or procymidone. However, in utero administration of 12.5, 25, 50, and 100 mg PFQ/kg/d on GD 14-18 induced antiandrogenic effects at all dosage levels and 91% of the males displayed reproductive malformation in the high dose group. Overall, BPC was ∼380-fold more potent than PFQ in vitro, whereas PFQ was far more potent than BPC in utero. Incorporating toxicokinetic and toxicodynamic data into in vitro to in vivo extrapolations would reduce the discordance between the in vitro and in utero effects of PFQ and BPC and combining in vitro results with a short-term Hershberger assay would reduce the uncertainty in predicting the in utero effects of antiandrogenic chemicals.

The effect of preconceptional exposure of F0 male mice to di(2-ethylhexyl)phthalate on the induction of reproductive toxicity in F2 generation.

The aim of the study was the estimation of the effects of 8 weeks exposure mature and pubescent male mice to DEHP on the prenatal development of the offspring F2 generation. The F1 offspring, of males exposed for whole cycle of spermatogenesis to DEHP (2000 mg/kg bw or 8000 mg/kg bw) and unexposed females, at 8-9 weeks of age were caged males with females from the same group, but from different litter. Eight weeks preconceptional exposure of mature F0 males to 2000 mg/kg bw DEHP induced the significantly higher number of dead fetuses in the F2 offspring; however, the effect on the sperm count and quality of F1 males was not seen. Contrary, after such exposure of pubescent males not significantly decrease in the number of live implants was noted. Results showed that the subchronical, preconceptional exposure of F0 males to DEHP did not influence strongly on the F2 generation of the offspring. Our study did not confirm higher sensitivity germ cells of pubescent males to harmful effects induced by DEHP. The developmental effect was present as the enhanced number of dead implants of F2 generation after exposure of mature F0 males and slight reduction in the number of live fetuses following the exposure of immature males. It may confirm ability to male mediated developmental toxicity.

Regulation of masculinization: androgen signalling for external genitalia development.

The biology of masculinization is fundamentally important for understanding the embryonic developmental processes that are involved in the development of the male reproductive tract, external genitalia, and also the tumorigenesis of prostate cancer. The molecular mechanisms of masculinization are of interest to many researchers and clinicians involved in varied fields, including molecular developmental biology, cancer research, endocrinology, and urology. Androgen signalling is mediated by the nuclear androgen receptor, which has fundamental roles in masculinization during development. Various modes of androgen signalling, including 5α-dihydrotestosterone-induced regulation of mesenchymal cell proliferation, have been observed in masculinization. Such regulation is essential for regulating urogenital tissue development, including external genitalia development. Androgen-induced genes, such as MAFB, which belongs to the activator protein 1 (AP-1) superfamily of genes, have essential roles in male urethral formation, and disruption of its signalling can interfere with urethral formation, which often results in hypospadias. Another AP-1 superfamily gene, ATF3, could be responsible for some instances of hypospadias in humans. These androgen-dependent signals and downstream events are crucial for not only developmental processes but also processes of diseases such as hypospadias and prostate cancer.

Reproductive system development in male and female horse embryos and fetuses: Gonadal hyperplasia revisited.

In horses, pregnancy is characterized by high levels of maternal estrogens that are produced largely by the interstitial tissue inside the gonads of the offspring, associated with a physiological gonadal hyperplasia, that is uncommon in other species. However, a detailed structural-functional understanding of the early stages of gonadal development and hyperplasia has remained elusive in horse pregnancy because of the lack of substantial data. The goal of this study was to describe the genital organs' development in 19 early horse embryos and fetuses (days 20-140 of gestation) of both sexes by means of anatomy, histology, stereology, and immunohistochemistry, with a specific focus on gonadal hyperplasia and interstitial tissue development. Gonadal hyperplasia with similar amounts of interstitial cells was observed in both sexes, but only during the early stage of development (days 40-90). Surprisingly, a higher degree of hyperplasia, characterized by larger amounts of interstitial cell-rich areas, was seen in fetal ovaries from 90 days of gestation onwards. Another novel aspect was that parallel to the hyperplasia of the interstitial cells, a much more precocious and pronounced differentiation of germinal cells was seen in the ovary, characterized by an earlier peak and decrease of DAZL and OCT protein immune markers. In conclusion, a reduced degree of hyperplasia and interstitial tissue in the fetal testis after 90 days of gestation suggests the existence of a more efficient mechanism regarding the synthesis of estrogen precursors as a structural or physiological difference between both fetal sexes, which warrants further investigation.

Elimination of the male reproductive tract in the female embryo is promoted by COUP-TFII in mice.

The sexual differentiation paradigm contends that the female pattern of the reproductive system is established by default because the male reproductive tracts (Wolffian ducts) in the female degenerate owing to a lack of androgen. Here, we discovered that female mouse embryos lacking Coup-tfII (chicken ovalbumin upstream promoter transcription factor II) in the Wolffian duct mesenchyme became intersex-possessing both female and male reproductive tracts. Retention of Wolffian ducts was not caused by ectopic androgen production or action. Instead, enhanced phosphorylated extracellular signal-regulated kinase signaling in Wolffian duct epithelium was responsible for the retention of male structures in an androgen-independent manner. We thus suggest that elimination of Wolffian ducts in female embryos is actively promoted by COUP-TFII, which suppresses a mesenchyme-epithelium cross-talk responsible for Wolffian duct maintenance.

Expression patterns of Fgf8 and Shh in the developing external genitalia of Suncus murinus.

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.

The evolution of asymmetric genitalia in Coleoptera.

The evolution of asymmetry in male genitalia is a pervasive and recurrent phenomenon across almost the entire animal kingdom. Although in some taxa the asymmetry may be a response to the evolution of one-sided, male-above copulation from a more ancestral female-above condition, in other taxa, such as Mammalia and Coleoptera, this explanation appears insufficient. We carried out an informal assessment of genital asymmetry across the Coleoptera and found that male genital asymmetry is present in 43% of all beetle families, and at all within-family taxonomic levels. In the most diverse group, Cucujiformia, however, genital asymmetry is comparatively rare. We also reconstructed the phylogeny of the leiodid tribe Cholevini, and mapped aspects of genital asymmetry on the tree, revealing that endophallus sclerites, endophallus, median lobe and parameres are, in a nested fashion, increasingly unlikely to have evolved asymmetry. We interpret these results in the light of cryptic female choice versus sexually antagonistic coevolution and advocate further ways in which the phenomenon may be better understood.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

First-trimester determination of fetal gender by ultrasound: measurement of the ano-genital distance.

INTRODUCTION: Early ultrasound fetal sex determination is of obvious interest, particularly in the context of X-linked diseases. In the human, the anogenital distance, i.e., the distance between the caudal end and the base of the genital tubercule is sexually dimorphic. This difference is apparent from 11 weeks of gestation. The aim of this prospective study was to evaluate the accuracy of anogenital distance measurement during the first trimester ultrasound in the early determination of fetal gender. MATERIALS AND METHODS: Fetal gender was assessed by ultrasound in 310 singleton pregnancies at 11-14 weeks of gestation. The optimal cut-off was determined by the minimal p-value technic and validated using bootstrap simulation. RESULTS: 310 women were included. A cut-off of 4.8mm was determined to predict male (≥4.8mm) or female (<4.8mm) fetuses. Sex was correctly determined for 87% of the males and 89% of the females. The inter-observer variability was excellent. CONCLUSION: This study presents a new sonographic sign for early fetal sex determination that has not been previously explored. It appears to be an accurate tool but it requires further validation in larger series.

Hypospadias.

Hypospadias is a hypoplasia of the tissues forming the ventral side of the penis responsible of an ectopic meatus of the urethra. This congenital anomaly results in a fusion defect of the two epithelial surfaces of the urethral groove between the 11th and the 18th weeks of development. The earlier this process arrests, the more the form is proximal and severe. This is the second genital malformation in boys with 1 case per 250 male births. Its origin is often multifactorial (genetic, endocrine, placental and environmental). Three anatomical forms exist: proximal, middle and distal (the most common). Additional exams (endocrine, genetic and morphological) are realized early, before surgery, in case of severe hypospadias, familial, associated with cryptorchidism, bifid scrotum, micropenis and/or skeletal, kidney, and/or heart abnormalities. It clarifies pubertal prognosis. The surgical management is made between 6months and 12months: it limits the functional and aesthetic impact of this malformation. Many surgical techniques are described. They all have in common the three operating time: penile straightening, urethroplasty, reconstruction of the ventral side of penis. They are based on direct sutures, local flaps pedicled, and grafts (skin or mucosa). The rate of postoperative complications is between 6 and 30 %. The two main complications are fistulae and stenoses. The psychological follow-up of these children is necessary to adulthood. Surgery of hypospadias remains a delicate surgery and must be performed by experienced surgeons.

The Reproductive System.

Correct sexual development is arguably the most important trait in an organism's life history since it is directly related to its genetic fitness. The developing gonad houses the germ cells, the only legacy we pass on to subsequent generations. Given the pivotal importance of correct reproductive function, it is confounding that disorders of sex development (DSDs) are among the most common congenital abnormalities in humans (Lee et al. J Pediatr Urol 8(6):611-615, 2012). Urogenital development is a highly complex process involving coordinated interactions between molecular and hormonal pathways in a tightly regulated order. The controls that regulate some of the key events in this process are beginning to be unraveled. This chapter provides an overview of our understanding of urogenital development from the gonads to the urogenital ducts and external genitalia.

Resurrecting embryos of the tuatara, Sphenodon punctatus, to resolve vertebrate phallus evolution.

The breadth of anatomical and functional diversity among amniote external genitalia has led to uncertainty about the evolutionary origins of the phallus. In several lineages, including the tuatara, Sphenodon punctatus, adults lack an intromittent phallus, raising the possibility that the amniote ancestor lacked external genitalia and reproduced using cloacal apposition. Accordingly, a phallus may have evolved multiple times in amniotes. However, similarities in development across amniote external genitalia suggest that the phallus may have a single evolutionary origin. To resolve the evolutionary history of amniote genitalia, we performed three-dimensional reconstruction of Victorian era tuatara embryos to look for embryological evidence of external genital initiation. Despite the absence of an intromittent phallus in adult tuataras, our observations show that tuatara embryos develop genital anlagen. This illustrates that there is a conserved developmental stage of external genital development among all amniotes and suggests a single evolutionary origin of amniote external genitalia.

Dose Addition Models Based on Biologically Relevant Reductions in Fetal Testosterone Accurately Predict Postnatal Reproductive Tract Alterations by a Phthalate Mixture in Rats.

Challenges in cumulative risk assessment of anti-androgenic phthalate mixtures include a lack of data on all the individual phthalates and difficulty determining the biological relevance of reduction in fetal testosterone (T) on postnatal development. The objectives of the current study were 2-fold: (1) to test whether a mixture model of dose addition based on the fetal T production data of individual phthalates would predict the effects of a 5 phthalate mixture on androgen-sensitive postnatal male reproductive tract development, and (2) to determine the biological relevance of the reductions in fetal T to induce abnormal postnatal reproductive tract development using data from the mixture study. We administered a dose range of the mixture (60, 40, 20, 10, and 5% of the top dose used in the previous fetal T production study consisting of 300 mg/kg per chemical of benzyl butyl (BBP), di(n)butyl (DBP), diethyl hexyl phthalate (DEHP), di-isobutyl phthalate (DiBP), and 100 mg dipentyl (DPP) phthalate/kg; the individual phthalates were present in equipotent doses based on their ability to reduce fetal T production) via gavage to Sprague Dawley rat dams on GD8-postnatal day 3. We compared observed mixture responses to predictions of dose addition based on the previously published potencies of the individual phthalates to reduce fetal T production relative to a reference chemical and published postnatal data for the reference chemical (called DAref). In addition, we predicted DA (called DAall) and response addition (RA) based on logistic regression analysis of all 5 individual phthalates when complete data were available. DA ref and DA all accurately predicted the observed mixture effect for 11 of 14 endpoints. Furthermore, reproductive tract malformations were seen in 17-100% of F1 males when fetal T production was reduced by about 25-72%, respectively.

Expression Analysis of DGKK during External Genitalia Formation.

PURPOSE: Genetic variants in diacylglycerol kinase κ (DGKK) have been strongly associated with risk of hypospadias. We investigated the expression pattern of Dgkk during development of mouse external genitalia to better understand its function and mechanism in the etiology of hypospadias. MATERIALS AND METHODS: We performed Dgkk expression analysis via indirect immunofluorescence in histological sections of CD-1 mouse embryonic and postnatal male, female and diethylsilbestrol treated external genitalia. Histological findings were supplemented with DGKK expression analysis using quantitative real-time polymerase chain reaction assays. RESULTS: In mouse external genitalia Dgkk was expressed in the membrane and cytoplasm of differentiating squamous epithelial cells of urethral plate/groove and epidermis but not in the undifferentiated epithelial cells of preputial lamina or basal layer of urethral groove epithelium. CD-1 gestation day 18 male mouse genital tubercle treated with oil or diethylstilbestrol showed similar patterns of Dgkk expression despite many morphological differences, including formation of preputial cleft observed in diethylsilbestrol treated mice. CONCLUSIONS: Dgkk appears to be a marker or mediator of squamous cell differentiation during development of mouse external genitalia. However, no association exists between Dgkk expression and formation of preputial cleft in the genital tubercle of diethylsilbestrol treated mice, suggesting that these 2 events may follow independent pathways in mice. Further studies are needed to elucidate the role of DGKK in hypospadias.

Tissue-specific roles of FGF signaling in external genitalia development.

BACKGROUND: The developmental processes of the genital tubercle (GT), the anlage of the external genitalia, possess several developmental aspects, including GT outgrowth, urethral tube formation, and epithelial differentiation of the urethra. The GT comprises the mesenchyme derived from the lateral mesoderm, ectodermal epithelium, and endodermal epithelium (embryonic urethral epithelium). The three tissue layers develop the GT coordinately. RESULTS: Around the initial stage of GT outgrowth (E11.5), FGF signaling was detected in the mesenchyme of the GT. FGF signaling was detected in the three tissue layers of the GT around the early stage of urethral formation (E13.5). Subsequently, FGF signaling was predominantly detected in the urethral epithelium (E14.5). Tissue-specific roles of FGF signaling in GT development were revealed by conditional Fgfr gene knockout approaches. Mesenchymal FGF signaling in the early-stage GT is required for its outgrowth. Ectodermal FGF signaling in the GT is required for the differentiation of the ectoderm and urethral epithelium at their junction to form the proper urethral tube. Endodermal FGF signaling in the GT is required for the stratification and cell adhesive characteristics of the urethral epithelium. CONCLUSIONS: The current study suggests that spatiotemporally regulated FGF signaling plays tissue-specific roles in multiple processes of external genitalia development.

Class I myosins have overlapping and specialized functions in left-right asymmetric development in Drosophila.

The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left-right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes.