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Two specialized functions of cholesterol during fetal development include serving as a precursor to androgen synthesis and supporting hedgehog (HH) signaling activity. Androgens are produced by the testes to facilitate masculinization of the fetus. Recent evidence shows that intricate interactions between the HH and androgen signaling pathways are required for optimal male sex differentiation and defects of either can cause birth anomalies indicative of 46,XY male variations of sex development (VSD). Further, perturbations in cholesterol synthesis can cause developmental defects, including VSD, that phenocopy those caused by disrupted androgen or HH signaling, highlighting the functional role of cholesterol in promoting male sex differentiation. In this review, we focus on the role of cholesterol in systemic androgen and local HH signaling events during fetal masculinization and their collective contributions to pediatric VSD.

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3XtJ mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3XtJ testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3XtJ mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3XtJ testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract., Author summary Disorders in male sex differentiation (DSD) are among the most common defects in all live births, yet in many cases, pediatric patient families are reluctant to address the issue and endure lifelong consequences. Urogenital tract development, as in many organ systems, depends on exquisite timing among layers of a number of signaling pathways. Here, we show that interactions between the hedgehog and androgen signaling pathways are required for the development of internal and external male sex characteristics, but results for each tissue is distinct. This new knowledge will aid in discovering the means by which congenital malformations might occur, identify potential developmental targets that might be vulnerable to environmental exposures, and promote new ideas for how they might be prevented.

Aromatase (encoded by the cyp19a1a and cyp19a1b genes) plays a central role in sex differentiation in fish, but its precise roles during sex differentiation are still largely unknown. Here, we systematically generated cyp19a1a and cyp19a1b mutant lines as well as a cyp19a1a;cyp19a1b double mutant line in zebrafish using transcription activatorlike effector nucleases. Our results showed that cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants, but not cyp19a1b mutants, had impaired sex differentiation, and all cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants were males. During sex differentiation, the ovary-like gonads were not observed and the male sex differentiation program was delayed in the cyp19a1a-null fish, and these phenotypes could be partially rescued by 17β-estradiol treatment. Gene expression analysis indicated that male and female sex differentiation-related genes were significantly decreased in the cyp19a1a mutant. Collectively, our results revealed dual functions of the cyp19a1a gene during sex differentiation: cyp19a1a is not only indispensable for female sex differentiation but also required for male sex differentiation.

International audience; A 5,6 Mb de novo 19q12-q13.12 interstitial deletion was diagnosed prenatally by array-comparative genomic hybridization in a 26 weeks male fetus presenting with intra-uterine growth retardation, left clubfoot, atypical genitalia and dysmorphic features. Autopsic examination following termination of pregnancy identified a severe disorder of sex development (DSD) including hypospadias, micropenis, bifid scrotum and right cryptorchidism associated with signs of ectodermal dysplasia: scalp hypopigmentation, thick and frizzy hair, absence of eyelashes, poorly developed nails and a thin skin with prominent superficial veins. Other findings were abnormal lung lobation and facial dysmorphism.This new case of DSD with a 19q12q13 deletion expands the phenotypic spectrum associated with this chromosomal rearrangment and suggests that WTIP is a strong candidate gene involved in male sex differentiation.

Anti-müllerian hormone (AMH) is produced by Sertoli cells and signals through 2 transmembrane receptors (AMHR), specific types I and II, leading to regression of müllerian ducts during fetal male sex differentiation. Mutations in AMH and AMHR2 lead to the persistence of müllerian ducts in males which is transmitted in a recessive pattern. Here, we report 2 Egyptian DSD (disorder of sex development) patients reared as males who presented with bilateral cryptorchidism and otherwise normal male external genitalia and who both had a 46,XY karyotype. The first patient presented at the age of 2 years. Laparoscopic surgery revealed a uterus and fallopian tubes with the presence of 2 gonads, and biopsy and pathology revealed prepubertal testicular tissue showing small-sized tubules with mostly Sertoli cells and very few spermatogonia, edematous stroma, and no detectable ovarian tissue. The second patient presented at the age of 3 years. Laparoscopic surgery revealed a uterus and fallopian tubes, and serum AMH was very low (0.1 ng/mL). Molecular studies revealed a novel missense mutation in the AMHR2 gene in the first patient (c.767A>C; p.H256P) and a novel frameshift mutation in the AMH gene in the second patient (c.203delC; p.L70Cfs*7). We conclude that persistent müllerian ducts should be included in the differential diagnosis of cryptorchidism.

Male sex differentiation is driven by 2 hormones, testosterone and anti-müllerian hormone (AMH), responsible for the regression of müllerian ducts in male fetuses. Mutations inactivating AMH or its receptor AMHRII lead to the persistent müllerian duct syndrome (PMDS) in otherwise normally virilized 46,XY males. Our objective was to review the clinical, anatomical, and molecular features of PMDS based upon a review of the literature and upon 157 personal cases. Three clinical presentations exist: bilateral cryptorchidism, unilateral cryptorchidism with contralateral hernia, and transverse testicular ectopia. Abnormalities of male excretory ducts are frequent. Testicular malignant degeneration occurs in 33% of adults with the disorder, while cancer of müllerian derivatives is less frequent. Fertility is rare but possible if at least one testis is scrotal and its excretory ducts are intact. Eighty families with 64 different mutations of the AMH gene have been identified, mostly in exons 1, 2, and 5. AMHRII gene mutations representing 58 different alleles have been discovered in 75 families. The most common mutation, a 27-bp deletion in the kinase domain, was found in 30 patients of mostly Northern European origin. In 12% of cases, no mutation of AMH or AMHRII has been detected, suggesting a disruption of other pathways involved in müllerian regression.

Fetal sex differentiation of the male involves complex processes and is dependent on the androgen production from fetal Leydig cells triggered by placental human chorionic gonadotropin (hCG) [1, 2]. It is later replaced by luteinizing hormone (LH) that is secreted by the fetal pituitary gland during gestation [3]. The hormones, such as antimullerian hormone (AMH) synthesized by Sertoli cells of the testes, testosterone produced by the Leydig cells and dihydrotestosterone, are also essential for the formation of male genitalia [4–6]. Because the action of both hCG and LH is through the luteinizing hormone/chorionic gonadotropin receptor (LHCGR), its presence and smooth function is crucial for male sex differentiation and characteristics in fetal and adult life. Any changes in the DNA or gene expression levels of LHCGR are associated with a variety of phenotypic as well as clinical symptoms and pathologies. Human LHCGR gene is located on chromosome 2 and encodes 11 exons (NCBI GeneID 3,973; http://www.ncbi.nlm.nih.gov/). Exons 1–10 and a part of exon 11 encode the extracellular domain which is responsible for ligand binding, and the remaining of exon 11 encodes a transmembrane domain and intracellular C-terminal domain which is responsible for signal transduction [7]. Leydig cell hypoplasia (LCH) or Leydig cell agenesis is a very rare disease with an approximate incidence of 1:1,000,000 occurring with inactivating mutations in the LHCGR gene (6). These mutations mainly affect the amino acid sequence, leading to the alterations of receptor protein structure and function. Depending on the severity of the functional incompetence, a spectrum of phenotypes has been described [8]. The phenotype of males with LCH varies from micropenis, ambiguous genitalia, hypospadias to pseudohermaphroditism. In females, LH insensitivity is also presented with irregular menstrual cycles, infertility, recurrent miscarriage loss, polycystic ovarian syndrome (PCOS), and genital malformations [9–13]. The most severe form of LCH, type I, occurs due to luteinizing hormone/human chorionic gonadotropin receptor (LHCGR) mutations that completely prevent hCG/LH signal transduction [10, 14, 15]. On the other hand, patients with LCH type II have milder symptoms caused by LHCGR mutations that partially affect the responsiveness of hCG [9, 10, 16]. In the last decade, several mutations including homozygous and compound heterozygous mutations in the LHCGR gene have been reported in both familial and sporadic cases of LCH. A number of these studies showed an impaired or absent ligand binding and cAMP production in response to hCG stimulation, suggesting that some of these mutations have inactivating roles [11–13, 16]. In such cases, the basal hormone status, usually with normal FSH, elevated LH, and low testosterone, provides an initial diagnostic hint of the underlying defect. Diagnosis confirmed by testicular biopsy showed LCH and by techniques of molecular biology revealed mutations of the LH receptor gene [17]. To date, no standard treatment approaches have been suggested or developed in LCH. Here, we report our experience with a man with LCH that resulted in successful sperm recovery and live birth with IVF treatment.

International audience; Müllerian inhibiting substance (MIS, also known as anti-Müllerian hormone), is a key factor of male sex differentiation in vertebrates. In amniotes, it is responsible for Müllerian duct regression in male embryos. In fish, despite the absence of Müllerian ducts, MIS is produced and controls germ cell proliferation during gonad differentiation. Here we show for the first time the presence of MIS in an amphibian species, Pleurodeles waltl. This is very astonishing because in caudate amphibians, Müllerian ducts do not regress in males. Phylogenetic analysis of MIS P. waltl ortholog revealed that the deduced protein segregates with MIS from other vertebrates and is clearly separated from other TGF-β family members. In larvae, MIS mRNA was expressed at higher levels in the developing testes than in the ovaries. In the testis, MIS mRNA expression was located within the lobules that contain Sertoli cells. Besides, expression of MIS was modified in the case of sex reversal: it increased after masculinizing heat treatment and decreased after estradiol feminizing exposure. In addition to the data obtained recently in the fish medaka, our results suggest that the role of MIS on Müllerian ducts occurred secondarily during the course of evolution.

Male sex development is a complex process of events involving genetic, epigenetic, and hormonal factors with androgens playing a major role. The formation of testes in 46,XY individuals establishes the male gonadal sex. Hormones produced by the testes, especially testosterone and anti-Mullerian hormone, play essential roles in male sex differentiation; conversely, defects in the production and action of these hormones result in disorders of sex development (DSD). In this chapter, the genetic, epigenetic, and hormonal factors controlling male sex determination and differentiation are reviewed. By discussing subjects with defects in androgen production and action, particularly those with 17beta-hydroxysteroid dehydrogenase-3 deficiency, 5alpha-reductase-2 deficiency, and androgen insensitivity syndrome, the roles of androgens in male sex differentiation, gender identity, and cognitive function are emphasized. The issue of the complex interaction of nature versus nurture is addressed.

Androgens are required for normal male sex differentiation and development of male secondary sexual characteristics. Mutations in AR gene are known to cause defects in male sexual differentiation. In current study, we enrolled a 46,XY phenotypically female patient bearing testes in inguinal canal. DNA sequencing of the AR gene detected a missense mutation C.1715A > G (p. Y572C) in exon 2 which is already known to cause complete androgen insensitivity syndrome (CAIS). We focused on the effects of this mutation on the testicular histopathology of the patient. Surface spreading of testicular tissues showed an absence of spermatocytes while H&E staining showed that seminiferous tubules predominantly have only Sertoli cells. This meiotic failure is likely due to the effect of the AR mutation which ultimately leads to Sertoli cell only syndrome. Tubules were stained with SOX9 and AMH which revealed Sertoli cells maturation arrest. Western blot and realtime PCR data showed that patient had higher levels of AMH, SOX9 and inhibin-B in the testis. Therefore, we suggest that the dysfunctioning of AR by mutation enhances AMH expression which ultimately leads to the failure in maturation of Sertoli cells.

Intersex, or the presence of oocytes in the testes, has been documented in fish following exposure to wastewater effluent and estrogenic compounds. However, the molecular networks underlying the intersex condition are not completely known. To address this, we exposed male fathead minnows to a low, environmentally-relevant concentration of 17alpha-ethinylestradiol (EE2) (15ng/L) and measured the transcriptome response in the testis after 96h to identify early molecular initiating events that may proceed the intersex condition. The short-term exposure to EE2 did not affect gonadosomatic index and proportion of gametes within the testes. However, the production of 11-ketotestosterone and testosterone from the testis in vitro was decreased relative to controls. Expression profiling using a 8×60K fathead minnow microarray identified 10 transcripts that were differentially expressed in the testes, the most dramatic change being that of coagulation factor XIII A chain (20-fold increase). Transcripts that included guanine nucleotide binding protein (Beta Polypeptide 2), peroxisome proliferator-activated receptor delta, and WNK lysine deficient protein kinase 1a, were down-regulated by EE2. Subnetwork enrichment analysis revealed that EE2 suppressed transcriptional networks associated with steroid metabolism, hormone biosynthesis, and sperm mobility. Most interesting was that gene networks associated with doublesex and mab-3 related transcription factor 1 (dmrt1) were suppressed in the adult testis, despite the fact that dmrt1 itself was not different in expression from control males. Transcriptional networks involving forkhead box L2 (foxl2) (transcript involved in ovarian follicle development) were increased in expression in the testis. Noteworthy was that a gene network associated to granulosa cell development was increased over 100%, suggesting that this transcriptome network may be important for monitoring estrogenic exposures. Other cell processes rapidly downregulated by EE2 at the transcript level included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway. Conversely, lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased. These data provide new insight into the transcriptional responses that are perturbed prior to gonadal remodeling and intersex following exposure to estrogens. These data demonstrate that low concentrations of EE2 (1) rapidly suppresses male hormone production, (2) down-regulate molecular networks related to male sex differentiation, and (3) induce transcriptional networks related to granulosa cell development in the adult testis. These responses are hypothesized to be key molecular initiating events that occur prior to the development of the intersex phenotype following estrogenic exposures.

The marmoset monkey is a valuable model in reproductive medicine. While previous studies have evaluated germ cell dynamics in the postnatal marmoset, the features of testicular somatic cells remain largely unknown. Therefore, the aim of this study was to establish marmoset-specific markers for Sertoli and peritubular cells (PTCs) and to compare protocols for the enrichment and culture of testicular cell types. Immunohistochemistry of Sertoli and PTC-specific markers – anti-müllerian hormone (AMH), vimentin (VIM), α-smooth muscle actin (SMA) – was performed and corresponding RNA expression profiles were established by quantitative PCR analysis (SOX9,AMH, FSHR,VIM, and SMA). For these analyses, testicular tissue from newborn (n = 4), 8-week-old (n = 4) and adult (n = 3) marmoset monkeys was used. Protocols for the enrichment and culture of testicular cell fractions from the 8-week-old marmoset monkeys (n = 3) were evaluated and cells were analyzed using germ cell- and somatic cell-specific markers. The expression of AMH, VIM and SMA reflects the proportion and differentiation status of Sertoli and PTCs at the RNA and the protein levels. While applied protocols did not support the propagation of germ cells in vitro, our analyses revealed that PTCs maintain their proliferative potential and constitute the dominant cell type after short- and long-term culture. Expression of functionally meaningful testicular somatic markers is similar in the human and the marmoset monkey, indicating that this primate can indeed be used as model for human testicular development. The PTC culture system established in this study will facilitate the identification of factors influencing male sex differentiation and spermatogenesis.

Background Over the past decades, an increasing trend in male external genital malformations such as cryptorchidism and hypospadias has led to the suspicion that environmental chemicals are detrimental to male fetal sexual development. Several environmental pollutants, including organochlorine pesticides, polychlorinated biphenyls, bisphenol A, phthalates, dioxins and furans have estrogenic and anti-androgenic activity and are thus considered as endocrine-disrupting chemicals (EDCs). Since male sex differentiation is critically dependent on the normal production and action of androgens during fetal life, EDCs may be able to alter normal male sex differentiation. Objective The objective of this study was to determine the incidence of external genital malformations in a population of full-term newborn males in southern France. We also performed a case-control study to identify the risk factors for male external genital malformations, with a focus on parental occupational exposure to EDCs. Methods Over a 16-month period, 1615 full-term newborn males with a birth weight above 2500 g were registered on a level-1 maternity ward, and the same pediatrician systematically examined 1442 of them (89%) for cryptorchidism, hypospadias and micropenis. For every male newborn with genital malformation, we enrolled nearly two males matched for age, parity and term. All parents of the case and control newborns were interviewed about pregnancy aspects, personal characteristics, lifestyle and their occupational exposure to EDCs using a detailed questionnaire. RESULTS We report 39 cases of genital malformation (2.70%), with 18 cases of cryptorchidism (1.25%), 14 of hypospadias (0.97%), 5 of micropenis (0.35%) and 2 of 46,XY disorders of sexual differentiation (DSD; 0.14%). We observed a significant relationship between newborn cryptorchidism, hypospadias or micropenis and parental occupational exposure to pesticides [odds ratio (OR) = 4.41; 95% confidence interval (95% CI), 1.21-16.00]. Familial clustering for male external genital malformations (OR = 7.25; 95% CI, 0.70-74.30) and medications taken by mothers during pregnancy (OR = 5.87; 95% CI, 0.93-37.00) were associated with the risk of cryptorchidism, hypospadias and micropenis, although the association was not statistically significant. Conclusions Although the causes of male genital malformation are multifactorial, our data support the hypothesis that prenatal contamination by pesticides may be a potential risk factor for newborn male external genital malformation and it should thus be routinely investigated in all undervirilized newborn males.

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5-7 μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13-15 μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease.

In normal ontogenetic development, the expression of the sex-determining region of the Y chromosome (SRY) gene, involved in the first step of male sex differentiation, is spatiotemporally regulated in an elaborate fashion. SRY is expressed in germ cells and Sertoli cells in adult testes. However, only few reports have focused on the expressions of SRY and the other sex-determining genes in both the classical organ developing through these genes (gonad) and the peripheral tissue (skin) of adult XY females. In this study, we examined the gonadal tissue and fibroblasts of a 17-year-old woman suspected of having disorders of sexual differentiation by cytogenetic, histological, and molecular analyses. The patient was found to have the 46,X,inv(Y)(p11.2q11.2) karyotype and streak gonads with abnormally prolonged SRY expression. The sex-determining gene expressions in the patient-derived fibroblasts were significantly changed relative to those from a normal male. Further, the acetylated histone H3 levels in the SRY region were significantly high relative to those of the normal male. As SRY is epistatic in the sex-determination pathway, the prolonged SRY expression possibly induced a destabilizing effect on the expressions of the downstream sex-determining genes. Collectively, alterations in the sex-determining gene expressions persisted in association with disorders of sexual differentiation not only in the streak gonads but also in the skin of the patient. The findings suggest that correct regulation of SRY expression is crucial for normal male sex differentiation, even if SRY is translated normally.

Insulin-like peptide 3 (INSL3) is a hormone produced by fetal and adult Leydig cells of the mammalian testis. During embryonic life INSL3 is required for testicular descent, whereas in adults it is involved in bone metabolism and male germ cell survival. Despite these important roles, the molecular mechanisms regulating INSL3 expression remain poorly understood. So far, two transcription factors have been implicated in INSL3 transcription: the nuclear receptors SF1 and NUR77. Circumstantial evidence also points to a role for androgens. Using transient transfections in MA-10 Leydig cells, we found that testosterone regulates in a time- and dose-dependent manner the human INSL3 promoter. The INSL3 promoter, however, does not contain a classical androgen-responsive element. Testosterone responsiveness was found to be mediated through an element located in the proximal INSL3 promoter, which also contains a NUR77-SF1-binding site. Furthermore, we found that posttranslational modifications, such as phosphorylation and acetylation, modulate transcription factor activity and therefore also contribute to INSL3 promoter activity in Leydig cells. All together, these data provide new insights into the molecular mechanisms regulating INSL3 expression in the mammalian testis.

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle, with male sex differentiation at the juvenile stage, a bisexual gonad during first 2 yr of life, and a male-to-female sex change (with vitellogenic oocytes) at 3 yr of age. The present study investigated the role of aromatase (cyp19a1a/Cyp19a1a) in gonadal development in this species, especially in relation to sexual differentiation and sex change. Fish of various ages were treated with estradiol (E2) or aromatase inhibitor (AI) to determine whether manipulation of the hormonal environment has an impact on these processes. We report an integrative immunohistochemical, cellular, and molecular data set describing these interesting phenomena. During male sex differentiation, high levels of cyp19a1a/Cyp19a1a expression were observed in the undifferentiated gonad (4 mo of age), in marked contrast to the low cyp19a1a/Cyp19a1a levels detected in the differentiated testis at the age of 5–6 mo. A low dose of E2 (0.25 mg/kg ...

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle, with a mono-male sex differentiation at the juvenile stage and male-to-female sex change at 3 yr of age. We report for the first time integrative molecular data on these interesting phenomena. Sex differentiation occurred between 4 and 5 mo of age. Testicular nr5a4 transcripts increased to high levels during sex differentiation (5 mo old), whereas nr0b1 (Dax1) did not increase until the age of 8 mo. High nr5a4 and nr0b1 expression in testicular tissue, in contrast to low nr5a4 and high nr0b1 expression in ovarian tissue, were found in the male phase of 0 + - to 2-yr-old fish (before sex change). Increased nr5a4, decreased nr0b1, and increased cyp19a1a were found in the ovarian tissues undergoing development from primary oocytes to vitellogenic oocytes during the natural sex change in 2 + -yrold fish. Removal of testicular tissue in 1 + -yr-old fish resulted in both increased ovarian nr5a4 and genes in the steroidogenic pathway and decreased nr0b1 together with the appearance of vitellogenic oocytes. Ovary developed into the active stage with the increased expression of star and steroidogenic enzymes, including aromatase, in concordance with the decreased expression of nr0b1 in the testis-excised fish. Long-term estradiol (E2) administration resulted in early sex change, but the ovaries were mainly with primary oocytes. Low nr5a4, high nr0b1, and low steroidogenic enzymes, including cyp19a1a expression, were also observed in these E2-fed ovarian tissues. Thus, nr5a4 but not nr0b1 was associated with male sex differentiation. Testicular development required cooperative functions of both nr5a4 and nr0b1. The present study suggests that nr5a4 and nr0b1 have an antagonistic interaction for the oocyte development. Testicular tissue exerted inhibitory effects on ovarian development. It is probable that nr0b1 regulates the timing of vitellogenic development and sex change in black porgy. early development, gene regulation, oocyte development, ovary, testis

Mutations in the X-linked AR gene cause androgen insensitivity syndrome (AIS) by impairing androgen-dependent male sex differentiation to various degree. Here we describe a partial AIS patient with confliction with the assigned female sex. Although the patient was noticed to have ambiguous genitalia at birth, the patient was reared as a female with no medical intervention. At the age of 31 years, the patient visited us because the patient was dissatisfied with the assigned female sex. The patient was treated with systemic testosterone and topical dihydrotestosterone, but the external genitalia responded only minimally to the treatment. The genetic analysis revealed a novel missense K580R mutation in the P-box of the DNA-binding domain of androgen receptor, which was the first missense mutation shared by AIS and prostate cancer. Although the best predictor of the adult gender identity is documented to be the initial gender assignment in patients with partial AIS as well as those with complete AIS, deciding gender assignment for infants with partial AIS is still challenging.

The nuclear receptor steroidogenic factor 1 (SF-1 or NR5A1) and the zinc finger protein GATA4 mediate key events in the early steps of gonadal development and sex differentiation, presumably by activating the expression of essential target genes. An important SF-1 target in male sex differentiation is the gene encoding the anti-Müllerian hormone (AMH), which induces regression of the Müllerian ducts in the developing male embryo. In cell transfection studies, there is apparent cooperation between GATA4 and SF-1 in the regulation of both human and mouse Amh promoters. We hypothesized that compound haploinsufficiency of both SF-1 and GATA4, by reducing their synergism, might cause a more severe phenotype than that seen in mice that were heterozygous for either SF-1 or Gata4 alone. Surprisingly, in adult and embryonic mice, compound haploinsufficiency of SF-1 and GATA4 caused no gonadal or reproductive abnormalities beyond those seen in SF-1+/– mice. Thus, although cooperation between SF-1 and GATA4 very likely is important for regulation of their target genes, such synergy was not revealed in our in vivo studies of gonadal development and function.

Normal synthesis and action of androgens is essential for normal male sex differentiation. 17Beta-hydroxysteroid dehydrogenase (17beta-HSD) and 5alpha-reductase isoenzymes play essential roles in normal androgen biosynthesis. We hypothesized that differences in expression of these enzymes in genital skin could contribute to the pathogenesis of 46,XY disorders of sex development (DSD). We investigated the mRNA transcription patterns of 17beta-hydroxysteroid dehydrogenase-isoenzymes type 1, 2, 3, 4, 5, 7, and 10, 5alpha-reductase type 1 and 2 and the androgen receptor in genital skin fibroblasts from foreskin and scrotal skin obtained from healthy males and patients with unclassified 46,XY DSD. mRNA expression was semi-quantified by real-time PCR. Although no systematic differences of gene expression of any enzyme between normal controls and hypospadias patients could be detected, we found in nearly half of all investigated patients' samples noticeable differences in the transcription profiles of 17beta-hydroxysteroid dehydrogenase type 5. In scrotal skin samples of patients a significantly higher transcription of the androgen receptor was detected. A role for an altered expression pattern of different enzymes of steroidogenesis in the etiology of genital malformations in some patients may be postulated.

Insulin-like 3 (INSL3) is a hormone produced by fetal and adult Leydig cells of the testis and by theca and luteal cells of the adult ovary. In males, INSL3 regulates testicular descent during fetal life, whereas in adults, it acts as a germ cell survival factor. In the ovary, INSL3 regulates oocyte maturation. Despite its importance for male sex differentiation and reproductive function in both sexes, very little is known regarding the molecular mechanisms that regulate Insl3 expression. So far, the nuclear receptor NR5A1 is the only transcription factor known to regulate the mouse Insl3 promoter in Leydig cells. NR5A1 by itself, however, cannot explain the spatiotemporal expression pattern of the Insl3 gene. In the present study, we have identified the orphan nuclear receptor NR4A1 as a novel regulator of INSL3 transcription in Leydig cells. Using RT-PCR, we found that Nr4a1 is coexpressed with Insl3 in purified Leydig cells and in several Leydig cell lines. Through detailed analyses of the mouse and human INSL3 promoter in Leydig cells, we have mapped a novel regulatory element located at -100 bp that is essential and sufficient to confer NR4A1 responsiveness. Consistent with a role for NR4A1 in Insl3 transcription, chromatin immunoprecipitation assays revealed that endogenous NR4A1 binds to the proximal Insl3 promoter in vivo. Finally, we found that NR4A1 is also implicated in cAMP-induced Insl3 transcription in Leydig cells. Taken together, our identification of NR4A1 as an important regulator of mouse and human INSL3 promoter activity helps us to better define the tissue-specific regulation of the INSL3 gene in gonadal cells.

In mammals, the anti-Müllerian hormone (Amh) is responsible for the regression of the Müllerian ducts; therefore, Amh is an important factor of male sex differentiation. The amh gene has been cloned in various vertebrates, as well as in several teleost species. To date, all described species show a sexually dimorphic expression of amh during sex differentiation or at least in differentiated juvenile gonads. We have identified the medaka amh ortholog and examined its expression pattern. Medaka amh shows no sexually dimorphic expression pattern. It is expressed in both developing XY male and XX female gonads. In adult testes, amh is expressed in the Sertoli cells and in adult ovaries in granulosa cells surrounding the oocytes, like in mammals. To better understand the function of amh, we cloned the anti-Müllerian hormone receptor type II (amhrII) ortholog and compared its expression pattern with amh, aromatase (cyp19a1), and scp3. During gonad development, amhrII is coexpressed with medaka amh in somatic cells of the gonads and shows no sexually dimorphic expression. Only the expression level of the Amh type II receptor gene was decreased noticeably in adult female gonads. These results suggest that medaka Amh and AmhrII are involved in gonad formation and maintenance in both sexes.

Denys-Drash syndrome (DDS) and Frasier syndrome (FS) are caused by mutations of the WT1 gene. These disorders are characterized by renal disease, abnormality of male sex differentiation, and Wilms’ tumor and gonadoblastoma. There have been few reports on gonadal function in a large series of patients with mutations of the WT1 gene. Here, we evaluated the relation between gonadal function and the phenotype of external genitalia in 15 Japanese patients with WT1 mutations. We confirmed three sets of information. First, if a diagnosis of DDS and FS is arrived at by genetic analysis, there are some overlaps in the phenotypes of external genitalia and renal complications. Second, the responses of serum T for the human CG (HCG) loading test coincided with the phenotype of external genitalia in both DDS and FS, except two patients. One DDS patient had male type external genitalia with a low level of serum T response, and one FS patient had complete female external genitalia despite a definite serum T response to HCG stimulation. Third, four FS patients had incomplete development of pubic hair, together with low DHEA-S levels.

SYNOPSIS. Over the past thirty years, a global occurrence of sexual aberration has occurred whereby females among populations of prosobranch snails exhibit male sex characteristics. This condition, called imposex, has been causally associated with exposure to the biocide tributyltin. Tributyltin-exposed, imposex snails typically have elevated levels of testosterone which have led to the postulate that this endocrine dysfunction is responsible for imposex. This overview describes recent evidence that supports this postulate. Gastropods maintain circulating testosterone levels and administration of testosterone to females or castrates stimulates male sex differentiation in several snail species. Studies in the mud snail ( Ilyanassa obsoleta) have shown that gastropods utilize a unique strategy for regulating free testosterone levels. Excess testosterone is converted to fatty acid esters by the action of a testosterone-inducible, high capacity/low affinity enzyme, acyl-CoA:testosterone acyl transferase, and stored within the organisms. Free testosterone levels are regulated during the reproductive cycle apparently due to changes in esterification/desterification suggesting that testosterone functions in the reproductive cycle of the organisms. Testosterone esterification provides a unique target in the testosterone regulatory machinery of snails that is altered by tributyltin. Indeed, imposex and free testosterone levels were elevated in field collected snails containing high tin levels, while testosteronefatty acid ester pools were reduced in these organisms. These observations indicate that tributyltin elevates free testosterone by reducing the retention of testosterone as fatty acid-esters. This endocrine effect of tributyltin may be responsible for imposex.

The first morphological event after initiation of male sex determination is seminiferous cord formation in the embryonic testis. Cord formation requires migration of pre-peritubular myoid cells from the adjacent mesonephros. The embryonic Sertoli cells are the first testicular cells to differentiate and have been shown to express neurotropin-3 (NT3), which can act on high-affinity trkC receptors expressed on migrating mesonephros cells. NT3 expression is elevated in the embryonic testis during the time of seminiferous cord formation. A trkC receptor tyrophostin inhibitor, AG879, was found to inhibit seminiferous cord formation and mesonephros cell migration. Beads containing NT3 were found to directly promote mesonephros cell migration into the gonad. Beads containing other growth factors such as epidermal growth factor (EGF) did not influence cell migration. At male sex determination the SRY gene promotes testis development and the expression of downstream sex differentiation genes such as SOX-9. Inhibition of NT3 actions caused a reduction in the expression of SOX-9. Combined observations suggest that when male sex determination is initiated, the developing Sertoli cells express NT3 as a chemotactic agent for migrating mesonephros cells, which are essential to promote embryonic testis cord formation and influence downstream male sex differentiation.

From early embryonic life, anti-Mullerian hormone (AMH) is produced by Sertoli cells and is essential for male sex differentiation. In females, AMH is produced by immature granulosa cells (GCs) but a definitive function in females is uncertain. We have assessed the cellular localization and specificity of a panel of five novel high-affinity AMH monoclonal antibodies. Two recognize the mature C-terminal form of AMH, whereas three recognize the active pro-mature form of AMH in human tissue. The antibodies were tested on fetal male testicular and mesonephric tissue aged 8- 19 weeks post conception (pc), fetal male serum aged 16 -26 weeks pc and human immature GCs by immunofluorescence, immu- nohistochemistry, ELISA and western blotting. The active pro-mature forms of AMH were expressed in both Sertoli cells from human fetal testis and human immature GCs. In contrast, the mature C-terminal form of AMH was hardly detected in Sertoli cells, but was readily detected in GCs. This particular form was also located to the nucleus in GCs, whereas the other investigated AMH forms remained in the cytoplasm. Interestingly, the distribution of the AMH forms in the fetal serum of boys showed that the fraction of inactive precursor AMH only accounted for 4.5%+ 0.6 (mean+ SD) of the total AMH measured, and the remaining AMH was the active pro-mature form. Furthermore, western blot analysis demon- strated a number of previously unrecognized molecular forms of AMH. The present findings suggest that processing of AMH is a tightly regulated process, which is likely to be important for the function of AMH and which differs between the two sexes.

Androgens are essential for normal male sex differentiation and are responsible for the normal development of male secondary sexual characteristics at puberty. The physiological effects of androgens are mediated by the androgen receptor (AR). Mutations in the AR gene are the most common cause of androgen insensitivity syndrome. The present study undertook a genetic analysis of the AR gene in two unrelated families affected by complete androgen insensitivity syndrome (CAIS) in China. In family 1, a previously reported nonsense mutation (G-to-A; p.W751X) was identified in exon 5 of the AR gene. In addition, a novel missense mutation was detected in exon 6 of the AR gene from family 2; this mutation resulted in a predicted amino acid change from phenylalanine to serine at codon 804 (T-to-C; p.F804S) in the ligand-binding domain (LBD) of AR. Computer simulation of the structural changes generated by the p.F804S substitution revealed marked conformational alterations in the hydrophobic core responsible for the stability and function of the AR-LBD. In conclusion, the present study identified two mutations from two unrelated Chinese families affected by CAIS. The novel mutation (p.F804S) may provide insights into the molecular mechanism underlying CAIS. Furthermore, it expands on the number of mutational hot spots in the international AR mutation database, which may be useful in the future for prenatal diagnosis and genetic counseling.

Although gonads are not required for development or survival, defects in gonadal development undoubtedly have a profound influence on affected individuals. Recent complementary studies in the fields of cytology, biochemistry and molecular genetics have revealed that normal gonad development involves an exquisitely regulated network of gene expression and protein-protein interactions. The initial event of gonadogenesis, in both males and females, involves the formation of a bipotential primordium. A Y chromosome then activates the male-specific pathway. The demonstration that mutations in the SOX proteins, SRY and SOX9, are responsible for disorders associated with male-to-female sex reversal showed dramatically that SRY and SOX9 have an essential role in male sex differentiation. This was emphasized when it was shown that female mice carrying transgenes that encode these proteins developed as males. SRY and SOX9 proteins have been characterized extensively and aspects of their function and regulation are now known.

In early fetal development, the testis secretes – independent of pituitary gonadotropins – androgens and anti-Müllerian hormone (AMH) that are essential for male sex differentiation. In the second half of fetal life, the hypothalamic–pituitary axis gains control of testicular hormone secretion. Follicle-stimulating hormone (FSH) controls Sertoli cell proliferation, responsible for testis volume increase and AMH and inhibin B secretion, whereas luteinizing hormone (LH) regulates Leydig cell androgen and INSL3 secretion, involved in the growth and trophism of male external genitalia and in testis descent.This differential regulation of testicular function between early and late fetal periods underlies the distinct clinical presentations of fetal-onset hypogonadism in the newborn male: primary hypogonadism results in ambiguous or female genitalia when early fetal-onset, whereas it becomes clinically undistinguishable from central hypogonadism when established later in fetal life. The assessment of the hypothalamic–pituitary–gonadal axis in male has classically relied on the measurement of gonadotropin and testosterone levels in serum. These hormone levels normally decline 3–6 months after birth, thus constraining the clinical evaluation window for diagnosing male hypogonadism. The advent of new markers of gonadal function has spread this clinical window beyond the first 6 months of life. In this review, we discuss the advantages and limitations of old and new markers used for the functional assessment of the hypothalamic–pituitary–testicular axis in boys suspected of fetal-onset hypogonadism. Fil: Grinspon, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Loreti, Nazareth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Braslavsky, Débora. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Valeri, Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Schteingart, Helena Fedora. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Ballerini, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Bedecarras, Patricia Gladys. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Ambao, Veronica Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Gottlieb, Silvia Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Ropelato, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Bergadá, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Campo, Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina Fil: Rey, Rodolfo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina

Leydig cell steroidogenesis is controlled by the pituitary gonadotropin LH that activates several signaling pathways, including the Ca2+/calmodulin kinase I (CAMKI) pathway. In other tissues, CAMKI regulates the activity of the myocyte enhancer factor 2 (MEF2) transcription factors. MEF2 factors are essential regulators of cell differentiation and organogenesis in numerous tissues but their expression and role in the mammalian gonad had not been explored. Here we show that MEF2 factors are expressed in a sexually dimorphic pattern in the mouse gonad. MEF2 factors are present in the testis throughout development and into adulthood but absent from the ovary. In the testis, MEF2 was localized mainly in the nucleus of both somatic lineages, the supporting Sertoli cells and the steroidogenic Leydig cells. In Leydig cells, MEF2 was found to activate the expression of Nr4a1, a nuclear receptor important for hormone-induced steroidogenesis. In these cells MEF2 also cooperates with forskolin and CAMKI to enhance Nr4a1 promoter activity via two MEF2 elements (−318 and −284 bp). EMSA confirmed direct binding of MEF2 to these elements whereas chromatin immunoprecipitation revealed that MEF2 recruitment to the proximal Nr4a1 promoter was increased following hormonal stimulation. Modulation of endogenous MEF2 protein level (small interfering RNA-mediated knockdown) or MEF2 activity (MEF2-Engrailed active dominant negative) led to a significant decrease in Nr4a1 mRNA levels in Leydig cells. All together, our results identify MEF2 as a novel testis-specific transcription factor, supporting a role for this factor in male sex differentiation and function. MEF2 was also positioned upstream of NR4A1 in a regulatory cascade controlling Leydig cell gene expression.

The formation of a testis from the indifferent gonad is the prelude to sequential steps in male sex differentiation orchestrated by time-dependent androgen biosynthesis and action. Information about the cellular and molecular mechanisms of androgen action can be obtained by the study of disorders of sex differentiation in males. The pivotal role of the androgen receptor as a ligand-induced transcription factor is emphasised and preliminary studies are described which attempt to identify developmentally regulated androgen-responsive genes. That androgen action can be modulated by gene polymorphisms is illustrated by the influence of an androgen receptor polyglutamine repeat in the multi-factorial causation of less severe forms of male under-masculinization.

Anti-Mullerian hormone (AMH) is a member of the TGF-β family which elicits its main action during male sex differentiation. This hormone is probably the most convenient marker of Sertoli cell differentiation and maturation throughout testicular development. Studying AMH gene regulation may thus be one way of identifying effectors of Sertoli cell differentiation. To this end we first tried to locate and then to characterise DNA elements responsible for in vivo transcriptional control of AMH expression. We obtained transgenic mice expressing a reporter gene (LacZ), under control of various putative AMH regulatory sequences. Analysis of transgenic animals revealed that activation of the AMH gene probably requires a two-step regulatory process. The first step corresponds to the initial activation of the AMH gene occurring at around 12.0-dpc. It requires the presence of regulatory DNA encompassed within a maximum of 370-bp upstream of the translation start site of the gene, delimited by the presence of an upstream housekeeping gene (SAP-62). Following this initial transient phase, a second phase seems to account for the persistence of AMH gene expression until the onset of puberty. As the 370-bp regulatory region is not sufficient on its own to allow the triggering of this second phase, it seems possible that additional control elements are required for normal AMH expression throughout testicular development. The complete array of regulatory elements remains to be located. Mol. Reprod. Dev. 59:256–264, 2001. © 2001 Wiley-Liss, Inc.

The present study was designed to evaluate the altered gene expression in neonatal rat testis after antenatal exposure of cyclophosphamide (one time single dose of either 2, 10, or 20 mg/kg body weight) to the developing fetuses, especially at the time of male sex differentiation. The rationale behind these experiments is to know about the involvement of Y-chromosome gene-dependent product(s) associated with gonadal dysfunction. Using SDS-polyacrylamide gel electrophoresis and photosensitive silver staining technique, the study shows that the variety of proteins of different molecular weight ranges 40,000 to 127,000 Da are modulated by cyclophosphamide exposure to the developing testis. Interestingly, the overexpression of one protein of 74,500 Da was observed both in supernatant as well as in pallet fractions. The qualitative and quantitative regulation of newly synthesized protein appearance or disappearance is observed in a dose-dependent manner.

The clinical use of diethylstilbestrol (DES) by pregnant women has resulted in an increased incidence of genital carcinoma in the daugh- ters born from these pregnancies. Also, in the so-called DES-sons abnormalities were found, mainly, the presence of Mullerian duct remnants, which indicates that fetal exposure to DES may have an effect on male sex differentiation. Fetal regression of the Mullerian ducts is under testicular control through anti-Mullerian hormone (AMH). In male mice, treated in utero with DES, the Mullerian ducts do not regress completely, although DES-exposed testes do produce AMH. We hypothesized that incomplete regression in DES-exposed males is caused by a diminished sensitivity of the Mullerian ducts to AMH. Therefore, the effect of DES on temporal aspects of Mullerian duct regression and AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in male mouse fetuses was studied. It was observed that Mullerian duct regression was incomplete at E19 (19 days post coitum), upon DES administration during preg- nancy from E9 through E16. Furthermore, analysis of earlier time points of fetal development revealed that the DES treatment had clearly delayed the onset of Mullerian duct formation by approxi- mately 2 days; in untreated fetuses, Mullerian duct formation was complete by E13, whereas fully formed Mullerian ducts were not observed in DES-treated male fetuses until E15. Using in situ hybridization, no change in the localization of AMH and AMHRII mRNA expression was observed in DES-exposed male fetuses. The mRNA expression was quantified using ribonuclease protection assay, showing an increased expression level of AMH and AMHRII mRNAs at E13 in DES-exposed male fetuses. Furthermore, the mRNA expression levels of Hoxa 11 and steroidogenic factor-1 (SF-1) were determined as a marker for fetal development. Prenatal DES exposure had no effect on Hoxa 11 mRNA expression, indicating that DES did not exert an overall effect on the rate of fetal develop- ment. In DES-exposed male fetuses, SF-1 showed a similar increase in mRNA expression as AMH, in agreement with the observations that the AMH gene promoter requires an intact SF-1 DNA binding site for time- and cell-specific expression, although an effect of DES on SF-1 expression in other tissues, such as the adrenal and pituitary gland, cannot be excluded. However, the increased expression levels of AMH and AMHRII mRNAs do not directly explain the decreased sensitivity of the Mullerian ducts to AMH. Therefore, it is concluded that prenatal DES exposure of male mice delays the onset of Mulle- rian duct development, which may result in an asynchrony in the timing of Mullerian duct formation, with respect to the critical period of Mullerian duct regression, leading to persistence of Mullerian duct remnants in male mice. (Endocrinology 139: 4244 - 4251, 1998)

The cranial suspensory ligament is located on the border of the cranial (mesonephric) mesentery in adult female mammals, which runs between the cranial pole of the internal genitalia and the dorsal abdominal wall. Absence of the cranial suspensory ligament in male mammals depends upon exposure of its primordium to fetal testicular androgens and is a prerequisite for testis descent. Female rats were exposed to 5alpha-dihydrotestosterone propionate at different stages of genital development, and cranial suspensory ligament development was studied in neonatal and in adult animals. Androgens suppressed cranial suspensory ligament development when exposure started during the early stages of genital development, until day 19 postconception (pc). Androgen receptor expression was immunohistochemically detected in the cranial mesentery of both sexes from day 16 pc onwards. A decrease of androgen receptor expression in female fetuses from day 18 pc onwards coincided with the appearance of a differentiated cranial suspensory ligament, as evidenced by the expression of two cell differentiation markers: alpha-smooth muscle (alpha-SM) actin and desmin. alpha-SM actin was located on the outer border of the cranial mesentery of both sexes at day 17 pc, and expression increased only in female fetuses. On day 19 pc, desmin expression was also detectable in the a-SM actin-positive cells. Proliferation and apoptosis indices of cells in the cranial mesentery, as analysed by 5'-bromodeoxyuridine incorporation and by detection of DNA strand breaks (TUNEL method) respectively, did not show any difference between the sexes, neither on day 17 nor on day 18 pc. Since primordial cells of the cranial suspensory ligament highly express the androgen receptor during the period of gestation when androgens can suppress cranial suspensory development, altered morphogenesis of these cells may be a direct consequence of androgen action.

Industrial chemicals and environmental pollutants can disrupt reproductive development in wildlife and humans by mimicking or inhibiting the action of the gonadal steroid hormones, estradiol and testosterone. The toxicity of these so-called environmental endocrine disruptors is especially insidious during sex differentiation and development due to the crucial role of gonadal steroid hormones in regulating these processes. This review describes the mechanism of toxicity and clinical implications of a new class of environmental chemicals that inhibit androgen-mediated sex development. For several of these chemicals, including the agricultural fungicide vinclozolin and the ubiquitous and persistent 1,1,1-trichloro-2,2-bis (p-chlorophenyl)ethane metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene, the molecular mechanism of action and the adverse developmental effects on male sex differentiation have been elucidated and are used as examples. Environmental chemicals with antiandrogenic activity offer profound implications with regard to recent clinical observations that suggest an increasing incidence of human male genital tract malformations, male infertility, and female breast cancer. Finally, in light of increasing concern over the potential endocrine disrupting effects of environmental pollutants, an in vitro/in vivo investigational strategy is presented which has proved useful in identifying chemicals with antiandrogen activity and their mechanism of action.

A de novo apparently balanced translocation involving chromosomes 8 and 20 was found in a 14-year-old boy with minor anomalies, mild skeletal abnormalities and ambiguous external genitalia including perineoscrotal hypospadias, rudimentary fused labioscrotal folds, bilateral cryptorchidism, and small penis. The karyotype was 46,XY, t(8;20)(q22.3-23;p13). No signs of other conditions known to be associated with structural anomalies of either chromosome 8 or 20 were present and incomplete masculinisation of the external genitalia appears to be the main component of the phenotype. Clinical and biological studies showed apparently normal testicular function in utero and after birth. Examinations excluded 5{alpha}-reductase deficiency or a block in any enzymatic steps of testosterone, glucocorticoid and mineralocorticoid biosynthesis. Coding sequences of the sex-determining gene (SRY) and androgen receptor gene (AR) were found to be identical to those of a normal male excluding their role in the cause of the present condition. Since several other reports describe the association of hypospadias and hypertelorism with deletions or translocations involving 8q, we suggest that a locus necessary for male sex differentiation is located at distal 8q. 24 refs., 3 figs.

The synthetic estrogen 17α-ethinyl estradiol (EE2) disturbs reproduction and causes gonadal malformation in fish. Effects on the transcription of genes involved in gonad development and function that could serve as sensitive biomarkers of reproductive effects in the field is, however, not well known. We have studied mRNA expression in testes and liver of adult zebrafish (Danio rerio) males treated with 0, 5 or 25ng/L EE2for 14days. qPCR analysis showed that the mRNA expression of four genes linked to zebrafish male sex determination and differentiation, Anti-Mullerian Hormone, Double sex and mab-related protein, Sry-related HMG box-9a and Nuclear receptor subfamily 5 group number 1b were significantly decreased by 25ng/L, but not 5ng/L EE2 compared with the levels in untreated fish. The decreased transcription was correlated with a previously shown spawning failure in these males (Reyhanian et al., 2011. Aquat Toxicol 105, 41–48), suggesting that decreased mRNA expression of genes regulating male sexual function could be involved in the functional sterility. The mRNA level of Cytochrome P-45019a, involved in female reproductive development, was unaffected by hormone treatment. The transcription of the female-specific Vitellogenin was significantly induced in testes. While testicular Androgen Receptor and the Estrogen Receptor-alpha mRNA levels were unchanged, Estrogen receptor-beta was significantly decreased by 25ng/L EE2. Hepatic Estrogen Receptor-alpha mRNA was significantly increased by both exposure concentrations, while Estrogen Receptor-beta transcription was unaltered. The decreased transcription of male-predominant genes supports a demasculinization of testes by EE2 and might reflect reproductive disturbances in the environment.

Heterozygous mutations in SOX9 lead to a human dwarfism syndrome, Campomelic dysplasia. Consistent with a role in sex determination, we find that Sox9 expression closely follows differentiation of Sertoli cells in the mouse testis, in experimental sex reversal when fetal ovaries are grafted to adult kidneys and in the chick where there is no evidence for a Sry gene. Our results imply that Sox9 plays an essential role in sex determination, possibly immediately downstream of Sry in mammals, and that it functions as a critical Sertoli cell differentiation factor, perhaps in all vertebrates.

Genetic control of male sex differentiation involves sex-determining region Y (SRY) (Y chromosome) derepressing an autosomal region to allow SOX9 (17q24.1) expression. In the absence of SRY and SOX9, ovaries develop under influence of FOXL2 (3q21). Many perturbations resulting in disorders of sex development (DSD), including sex-reversed males (46,XX) with bilateral testes and sex-reversed females (46,XY) with streak gonads or, rarely, ovaries. 46,XY males with undervirilization result from 45,X/46,XY mosaicism, deficiencies of adrenal or gonadal enzymes or impeded cellular response to androgenesis. In ovotesticular DSD (true hermaphroditism), both ovarian and testicular tissues exist. In XX ovarian dysgenesis, many different conditions and genes are recognized.

The antimullerian hormone (AMH) e located in the short arm of chromosome 19 with 2750 nucleotide bases e is a homodimeric glycoprotein that weighs 140 kDa and is a member of the transforming growth factor-s superfamily, which is involved in male sex differentiation and is responsible for the regression of Mullerian ducts. 1 AMH is exclusively produced by granulosa cells and is the only marker that is thought to be stable throughout the menstrual cycle. AMH has been suggested as a marker for the quantity of oocytes remaining within the ovaries as it is a better marker of ovarian reserve than age, basal FSH, estradiol and inhibin. 2 AMH also enables: (1) prediction of both over and poor response in the controlled ovarian stimulation environment; (2) the most appropriate stimulation regimen to be determined; and (3) pretreatment counseling helping couples make an appropriate and informed choices. 3 Recent reports further suggest that AMH may be useful in the following situations, including: (1) prediction of long-term fertility; (2) prediction of the age of menopause; (3) prediction of ovarian ageing in women prior to or following chemotherapy; (4) prediction of long-term fertility following ovarian surgery; and (5) screening for polycystic ovaries (PCO). 3 In this issue, Chao and colleagues aimed to respond to two questions simulta- neously, that is, assessing the age-related changes of AMH levels in Taiwanese women with and without PCO. 4 At first, Chao and colleagues found that AMH levels revealed an age-related decline in the regular menstrual cycle of healthy women. 4 Furthermore, the AMH level rapidly dropped between 30 and 40 years of age, followed by a slow decrease after 40 years of age. This substantiates previous findings that the AMH concentration declines significantly with increasing age in premenopausal women. 5 Polycystic Ovaries Syndrome (PCOS), a common endo- crinopathy characterized by oligo- or anovulation, clinical or biochemical hyperandrogenemia, and polycystic ovaries on ultrasonography, affects 5e10% of women of reproductive age. 6 Recent studies have shown that 50% of women with PCOS fulfill the criteria of metabolic syndrome and that PCOS is frequently associated with insulin resistance accompanied by compensatory hyperinsulinemia, resulting in an increased risk for the development of type 2 diabetes mellitus and cardiovascular disease. 7 PCOS was reported to have a higher level of AMH compared with healthy controls. 8 Pigny et al indicated that serum AMH levels were three-fold higher in PCOS patients than in controls, and the elevated levels of AMH were significantly related to the follicle number in women with PCOS. 9 Chao's report was in agreement with the above finding e AMH levels were significantly higher in women with PCOS than in healthy fertile controls. The sensitivity and specificity of AMH in detecting PCOS was calculated using an AMH cut-off value of 3.5 ng/mL. 4 AMH levels were found to be positively correlated with testosterone, androstendione and the free androgen index. 9 The data in Chao's study were similar; however, the Chao and colleagues failed to investigate the relationship between AMH and insulin sensitivity in Taiwanese women with PCOS. If additional data addressing the above question were avail- able, this report from Chao and colleagues would be more appealing. Based on other reports, we believed this correlation may be present, since the La Marca group found that AMH levels were positively correlated with the homeostasis model's assessment (HOMA) index, 10 and the use of metformin e an insulin-sensitizer e can significantly reduce both androgen and AMH levels in obese women with PCOS. 11

Introduction Androgen is a generic term usually applied to describe a group of sex steroid hormones. Androgens are responsible for male sex differentiation during embryogenesis at the sixth or seventh week of gestation, triggering the development of the testes and penis in male fetuses, and are directed by the testicular determining factor: the gene SRY (sex determining region on Y chromosome) located on the short arm of chromosome Y. The differentiation of male external genitalia (penis, scrotum and penile urethra) occurs between the 9th and 13th weeks of pregnancy and requires adequate concentration of testosterone and the conversion of this to another more potent androgen, dihydrotestosterone, through the action of 5α-reductase in target tissues. Case presentation This report describes the case of a teenage girl presenting with a male karyotype, and aims to determine the extension of the mutation that affected the AR gene. A Caucasian girl aged 15 was referred to our laboratory for genetic testing due to primary amenorrhea. Physical examination, karyotype testing and molecular analysis of the androgen receptor were critical in making the correct diagnosis of complete androgen insensitivity syndrome. Conclusions Sex determination and differentiation depend on a cascade of events that begins with the establishment of chromosomal sex at fertilization and ends with sexual maturation at puberty, subsequently leading to fertility. Mutations affecting the AR gene may cause either complete or partial androgen insensitivity syndrome. The case reported here is consistent with complete androgen insensitivity syndrome, misdiagnosed at birth, and consequently our patient was raised both socially and educationally as a female. It is critical that health care providers understand the importance of properly diagnosing a newborn manifesting ambiguous genitalia. Furthermore, a child with a pseudohermaphrodite phenotype should always undergo adequate endocrine and genetic testing to reach a conclusive diagnosis before gender is assigned and surgical interventions are carried out. Our results show that extreme care must be taken in selecting the genetic tools that are utilized for the diagnosis for androgen insensitivity syndrome.

Anti-Mullerian hormone (AMH) plays an important role in male sex differentiation in vertebrates. AMH produced by Sertoli cells of the fetal testis induces regression of the Mullerian duct in mammalian species. In alligators, sexual differentiation is controlled by the temperature during egg incubation, termed temperature-dependent sex determination (TSD). The TSD mechanism inducing sex differentiation is thought to be unique and different from that of genetic sex determination as no gene such as the SRY of mammals has been identified. However, many of the genes associated with gonadal differentiation in mammals also are expressed in the developing gonads of species exhibiting TSD. To clarify the molecular mechanisms associated with gonad formation during the temperature-sensitive period (TSP), we have cloned the full length AMH gene in the alligator, and quantitatively compared mRNA expression patterns in the gonad–adrenal–mesonephros (GAM) complex isolated from alligator embryos incubated at male and female producing temperatures. The deduced amino acid sequence of the alligator AMH cDNA showed high identity (59–53%) to avian AMH genes. AMH mRNA expression was high in the GAM of male alligator embryos at stage 24 (immediately after sex determination) and hatchlings, but suppressed in the GAM of estrogen-exposed hatchlings incubated at the male-producing temperature. In the alligator AMH proximal promoter, a number of transcriptional factors (for SF-1. GATA, WT-1 and SOX9) binding elements were also identified and they exhibit a conserved pattern seen in other species. SOX9 up-regulates transcriptional activity through the amAMH promoter region. These results suggested that AMH and SOX9 play important roles in TSD of the American alligator.

The luteinizing hormone receptor (LHR), one of the three glycoprotein hormone receptors, is necessary for critical reproductive processes, including gonadal steroidogenesis, oocyte maturation and ovulation, and male sex differentiation. Moreover, it has been postulated to contribute to certain neoplasms, particularly ovarian cancer. A member of the G protein-coupled receptor family, LHR contains a relatively large extracellular domain responsible for high affinity hormone binding; transmembrane activation then leads to G protein coupling and subsequent second messenger production. This review deals with recent advances in our understanding of LHR structure and structure-function relationships, as well as hormone-mediated changes in gene expression in ovarian cancer cells expressing LHR. Suggestions are also made for critical gaps that need to be filled as the field advances, including determination of the three-dimensional structure of inactive and active receptor, elucidation of the mechanism by which hormone binding to the extracellular domain triggers the activation of Gs, clarification of the putative roles of LHR in non-gonadal tissues, and the role, if any, of activated receptor in the development or progression of ovarian cancer.

It has been suggested that sex differentiation in vertebrates is steroid hormone dependent, that estrogens play a critical role in ovarian differentiation, and that male sex differentiation will occur in the absence of estrogens. Using the model of the alligator in which sex can be manipulated by incubation conditions (eggs incubated at a constant temperature of 30 degrees produce 100% females, and at 33 degrees produce 100% males), a series of experiments using antiestrogens, antiandrogen, estradiol-17 beta, dihydrotestosterone (DHT), and aromatase inhibitors were performed. Test substances were injected into alligator eggs prior to gonadal sex differentiation and the eggs were incubated at male or female temperatures until just before expected date of hatching. Gonads were excised and the sex was verified histologically. Control embryos injected with vehicle produced the expected sex: females at 30 degrees and males at 33 degrees. Estradiol in eggs at 33 degrees (male temperature) produced 100% females and did not alter female development in eggs at 30 degrees. Antiandrogen, DHT, and a steroid antiestrogen had no discernible effect in either the 30 degrees or the 33 degrees eggs at the doses tested. The aromatase inhibitors aminoglutethimide and 4-hydroxyandrostenedione caused a moderate disruption of ovarian development and had no apparent effect on testicular development. The nonsteroidal aromatase inhibitor, Ciba Geigy 16949A, caused inhibition of ovarian development in all treated embryos. The Mullerian ducts did not appear to be affected by this treatment, or by any of the other treatments, and the gonads did not appear masculinized. We conclude that estrogen appears to be necessary for normal ovarian development, but that inhibition of estrogen synthesis alone is insufficient to cause masculinization. Likewise, exogenous androgens appear unable to masculinize embryonic gonads. The evidence suggests that testicular differentiation in amniote vertebrates is dependent on factors other than androgens or level of estrogens.

Recent discoveries in molecular biology have much clarified the regulation and function of steroid-converting enzymes. Most progress has been made in the area of cytochromes, which regulate the side chain cleavage of cholesterol (P-450 SCC) and the 17α-hydroxylase and 17,20-desmolase (or 17,20-lyase) activities (P-450 17α), as well as in 3β-hydroxysteroid dehydrogenase. Nevertheless, there are some discrepancies between fundamental knowledge and clinical experience, which are difficult to understand: why is it for example possible that cases with ‘pure’ 17α-hydroxylase or 17,20-desmolase deficiency exist, when there is only one cytochrome regulating both steps? After a brief review of clinical and biochemical findings in the various defects of testosterone biosynthesis, a case is discussed, which is of interest in this respect. This XY patient with female external genitalia, who has been shown to have compound heterozygous mutations, had ‘pure’ 17,20-desmolase deficiency up to adolescence, but additional 17α-hydroxylase deficiency with hypertension developed thereafter. From this observation, it has to be concluded that as yet unknown, possibly age-dependent modulating factors exist, which influence the activity of the cytochrome. Also the estrogen replacement given to the patient might have played a role in this change.

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle with a male sex differentiation at the juvenile stage and male-to-female sex change at 3 years of age. We had characterized the sex differentiation and sex change in this species by the integrative approaches of histology, endocrine and molecular genetics. The fish differentiated in gonad at the age around 4-months and the gonad further developed with a bisexual gonad for almost for 3 years and sex change at 3 year of age. An antagonistic relationship in the testicular and ovarian tissues was found during the development of the gonadal tissue. Male- (such as sf-1, dmrt1, dax-1 and amh) and female- (such as wnt4, foxl2 and cyp19a1a) promoting genes were associated with testicular and ovarian development, respectively. During gonadal sex differentiation, steroidogenic pathway and estrogen signaling were also highly expressed in the brain. The increased expression of sf-1 and wnt4, cyp19a1a in ovarian tissue and decreased expression of dax-1 in the ovarian tissue may play important roles in sex change from a male-to-female. Endocrine factors such as estradiol and luteinizing hormone may also involve in the natural sex change. Estradiol induced the expression of female-promoting genes and resulted in the precocious sex change in black porgy. Our series of studies shed light on the sex differentiation and sex change in protandrous black porgy and other animals.

Objective To present a case of complete androgen insensitivity syndrome that initially presented as incarcerated inguinal hernia and to investigate the expression of antimullerian hormone (AMH), SOX-9 (SRY-box–containing gene 9), prostaglandin D synthase (PGDS), and androgen receptors (AR). Design Case report. Setting District hospital. Patient(s) A 12-year-old girl with negative past medical and family history, who presented with a 6-hour history of progressive left groin pain. Intervention(s) Open gonadectomy. Main Outcome Measure(s) Immediate surgery, diagnosis, and referral to a multidisciplinary team for further management. Result(s) Pathologic analysis revealed a left twisted and infarcted testicle, and a normal right one containing immature seminiferous tubules with a decreased number of spermatogonia. Immunohistochemical analysis revealed strong cytoplasmic and nuclear expression of AMH and SOX-9, respectively, by the Sertoli cells as well as focal weak cytoplasmic PGDS expression. The spermatogonia showed focal weak nuclear PGDS expression. The Leydig cells showed no immunoreactivity at all. No AR immunoreactivity was observed. Conclusion(s) Negative AR immunostaining could either reflect Sertoli-cell immaturity or a mutation resulting in no protein production. The AMH immunodetection, consistent with its high serum levels, could potentially reflect Sertoli-cell immaturity. The SOX-9 nuclear detection in the Sertoli cells was consistent with its role, inducing male sex differentiation, including AMH expression. The nuclear localization of PGDS in the spermatogonia needs further investigation.

We describe here the isolation of cDNA and genomic clones corresponding to the mouse gene encoding anti-Müllerian hormone, and the use of these clones as molecular probes to study AMH gene expression. We constructed a 14.5 days post coitum (dpc) mouse fetal testes library and isolated a cDNA clone using bovine, human and rat partial cDNAs as probes. This clone contained a 1 kb insert, which was confirmed by sequencing to be the mouse homologue of AMH. Probes derived from the mouse cDNA clone were used to screen genomic libraries and a 12 kb DNA fragment containing the complete coding region of mouse AMH was isolated. In situ hybridisation was used to determine the precise timing and localisation of AMH expression in male and female embryos and postnatal testes and ovaries. AMH transcripts were first detected in fetal testes at 12.5 Ape when differences between testes and ovaries first become visible. The signal was specific for the Sertoli cells of the testes. Other fetal tissues or female embryos were negative for AMH transcripts. During male development, AMH expression is shut off postnatally. In the female, the expression of AMH was first detected at day 6 after birth and is restricted to granulosa cells. We have correlated the pattern of AMH expression in both sexes with cellular events occurring in gonadal development and discuss some implications that this may have for its function and regulation.