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XY C57BL/6J (B6) mice harboring a Mus musculus domesticus -type Y chromosome (Y POS ), known as B6.Y POS mice, commonly undergo gonadal sex reversal and develop as phenotypic females. In a minority of cases, B6.Y POS males are identified and a proportion of these are fertile. This phenotypic variability on a congenic B6 background has puzzled geneticists for decades. Recently, a B6.Y POS colony was shown to carry a non-B6-derived region of chromosome 11 that protected against B6.Y POS sex reversal. Here. we show that a B6.Y POS colony bred and archived at the MRC Harwell Institute lacks the chromosome 11 modifier but instead harbors an ∼37 Mb region containing non-B6-derived segments on chromosome 13. This region, which we call Mod13 , protects against B6.Y POS sex reversal in a proportion of heterozygous animals through its positive and negative effects on gene expression during primary sex determination. We discuss Mod13 's influence on the testis determination process and its possible origin in light of sequence similarities to that region in other mouse genomes. Our data reveal that the B6.Y POS sex reversal phenomenon is genetically complex and the explanation of observed phenotypic variability is likely dependent on the breeding history of any local colony., (Copyright © 2020 Livermore et al.)

Disorders of sex development (DSDs) are congenital conditions in which chromosomal, gonadal and sex is atypical. It is difficult to diagnose and manage patients with DSD in clinical practice, and the molecular etiology of DSD is still not completely understood. Here, we identified two novel pathogenic mutations from three unrelated Chinese patients with 46,XY complete gonadal dysgenesis (CGD) that is a clinical subgroup of DSD by whole exome sequencing. A novel mutation in the SRY gene (c.161delG) was identified in the first patient, and the second patient carried a novel missense mutation in the MAP3K1 gene (c.2117T>G). Bioinformatics analysis found that the deletion of SRY (c.161delG) led to a premature stop codon at amino acid 59 in the SRY protein, which resulted in lacking the DNA binding domain of SRY protein. Functional studies found that the missense mutation in the MAP3K1 gene (c.2117T>G) could interfere with the gene function through increasing the phosphorylation of the downstream targets of MAP3K1, ERK1/2 and p38, which resulted in reducing testis-determining factor SOX9 expression and increasing ovary-promoting factor β-catenin activity. According to the American college of medical genetics and genomics (ACMG) standards and guidelines, these mutations were categorized as "pathogenic" mutations. Thus, our findings provide two novel pathogenic mutations associated with 46,XY CGD that can improve the etiological diagnosis for 46,XY CGD. ABBREVIATIONS., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: To identify the genetic cause of a pedigree with four patients with 46,XY pure gonadal dysgenesis (PGD)., Design: Genetic mutation study., Setting: Academic medical center., Patient(s): Four first cousins, from three households of a Chinese pedigree, affected by 46,XY PGD., Intervention(s): None., Main Outcome Measure(s): The patients were studied from clinical and genetic perspectives. Whole-genome sequencing was conducted in family members., Result(s): Four first cousins in the third generation were affected by 46,XY PGD. A specific familial characteristic was the prevalence of as high as 100% of gonadal tumors in patients. Whole-genome sequencing identified a new ferritin heavy chain-like 17 (FTHL17) mutation, c.GA442_443TT (p.E148L), which has the potential to interfere with protein function and cause 46,XY PGD. Moreover, the location (Xp21.2) of the FTHL17 gene proves that the family is X-linked recessive. In vitro functional study revealed that the perturbation of FTHL17 caused the decrease of protein expression and cell proliferation., Conclusion(s): We describe the first 46,XY PGD pedigree that may be attributed to mutations of the FTHL17 gene. We speculated that the FTHL17 gene is involved in the testis-determining pathway and tumorigenesis., (Copyright © 2019 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In humans, mutations of Desert Hedgehog gene (DHH) have been described in patients with 46,XY gonadal dysgenesis (GD), associated or not with polyneuropathy. In this study, we describe two patients diagnosed with GD, both harboring novel DHH compound heterozygous mutations p.[Tyr176*];[Asn337Lysfs*24] and p.[Tyr176*];[Glu212Lys]. To investigate the functional consequences of p.(Asn337Lysfs*24) and p.(Glu212Lys) mutations, located within the C-terminal part of DHh on auto-processing, we performed in vitro cleavage assays of these proteins in comparison with Drosophila melanogaster Hedgehog (Hh). We found that p.(Glu212Lys) mutation retained 50% of its activity and led to a partially abolished DHh auto-processing. In contrast, p.(Asn337Lysfs*24) mutation resulted in a complete absence of auto-proteolysis. Furthermore, we found a different auto-processing profile between Drosophila Hh and human DHh, which suggests differences in the processing mechanism between the two species. Review of the literature shows that proven polyneuropathy and GD is associated with complete disruption of DHh-N, whereas disruption of the DHh auto-processing is only described with GD. We propose a model that may explain the differences between Schwann and Leydig cell development by autocrine versus paracrine DHh signaling. To our knowledge, this is the first study investigating the effect of DHH mutations on DHh in vitro auto-processing., (© 2018 Wiley Periodicals, Inc.)

The male hypogonadism-related bone mass loss is often under diagnosed. Peak bone mass is severely affected if the hypogonadism occurs during puberty and is left untreated. We present an interesting; almost bizarre case of a male with non-functional testes early during childhood and undiagnosed and untreated hypogonadism until his fifth decade of life. Forty six year male is referred for goitre, complaining of back pain. Phenotype suggested intersexuality: gynoid proportions, micropenis, no palpable testes into the scrotum, no facial or truncal hair. His medical history had been unremarkable until the previous year when primary hypothyroidism was diagnosed and levothyroxine replacement was initiated. Later, he was diagnosed with ischemic heart disease, with inaugural unstable angina. On admission, the testosterone was 0.2 ng/mL (normal: 1.7-7.8 ng/mL), FSH markedly increased (56 mUI/mL), with normal adrenal axis, and TSH (under thyroxine replacement). High bone turnover markers, and blood cholesterol, and impaired glucose tolerance were diagnosed. The testes were not present in the scrotum. Abdominal computed tomography suggested bilateral masses of 1.6 cm diameter within the abdominal fat that were removed but no gonadal tissue was confirmed histopathologically. Vanishing testes syndrome was confirmed. The central DXA showed lumbar bone mineral density of 0.905 g/cm2, Z-score of -2.9SD. The spine profile X-Ray revealed multiple thoracic vertebral fractures. Alendronate therapy together with vitamin D and calcium supplements and trans-dermal testosterone were started. Four decades of hypogonadism associate increased cardiac risk, as well as decreased bone mass and high fracture risk.

Mutations of Desert hedgehog (DHH) have been associated to 46,XY pure gonadal dysgenesis (PGD) and to mixed gonadal dysgenesis (MGD); however, there have been no functional studies of mutations described in DHH. To determine if mutations p.L162P and Δ1086delG yield functional impairment, we performed in vitro and in silico analysis of both DHH mutants. In complementary DNA of DHH, we performed site-directed mutagenesis, which was confirmed by DNA sequencing. Protein extracts were obtained from HEK293cells transfected with different constructs and analyzed by Western blot; besides, densitometric analysis of chemiluminescent signals was performed. In addition, the structure of the wt-DHH and its two mutant proteins was inferred using in silico protein molecular modeling. In the Western blot analysis, we observed the absence of signal for p.L162P in DHH-N and a diminished signal for Δ1086delG in DHH-C, when compared to wt-DHH. Protein modeling showed notable conformational changes for the side chains of p.L162P, while the secondary structure was drastically modified in Δ1086delG, when compared to wt-DHH. To our knowledge, this is the first study focused to determine by in vitro studies, the effect of two specific mutations in DHH associated with 46,XY PGD and MGD. Our results suggest that both mutations have a deleterious effect on the expression of the DHH mutant proteins.

Background: The steroidogenic acute regulatory protein (StAR) is essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause lipoid congenital adrenal hyperplasia., Objective and Methods: To identify causative mutations in a patient presenting with adrenal failure during early infancy. The objective was to study the functional and structural consequences of the novel StAR mutation p.Trp147Arg in a Turkish patient detected in compound heterozygosity with the p.Glu169Lys mutation., Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.Trp147Arg mutant. The previously described p.Glu169Lys mutant led to significantly lower cholesterol conversion than wild-type StAR protein. As derived from three-dimensional protein modeling, the residue W147 is stabilizing the C-terminal helix in a closed conformation hereby acting as gatekeeper of the ligand cavity of StAR., Conclusions: The novel mutation p.Trp147Arg causes primary adrenal insufficiency and complete sex reversal in the 46,XY patient. Clinical disease, in vitro studies and three-dimensional protein modeling of the mutation p.Trp147Arg underscore the relevance of this highly conserved residue for StAR protein function., (Copyright © 2013 S. Karger AG, Basel.)

Loss of the kinase MAP3K4 causes mouse embryonic gonadal sex reversal due to reduced expression of the testis-determining gene, Sry. However, because of widespread expression of MAP3K4, the cellular basis of this misregulation was unclear. Here, we show that mice lacking Gadd45γ also exhibit XY gonadal sex reversal caused by disruption to Sry expression. Gadd45γ is expressed in a dynamic fashion in somatic cells of the developing gonads from 10.5 days postcoitum (dpc) to 12.5 dpc. Gadd45γ and Map3k4 genetically interact during sex determination, and transgenic overexpression of Map3k4 rescues gonadal defects in Gadd45γ-deficient embryos. Sex reversal in both mutants is associated with reduced phosphorylation of p38 MAPK and GATA4. In addition, embryos lacking both p38α and p38β also exhibit XY gonadal sex reversal. Taken together, our data suggest a requirement for GADD45γ in promoting MAP3K4-mediated activation of p38 MAPK signaling in embryonic gonadal somatic cells for testis determination in the mouse., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Male sex determination in mammals is induced by Sry, a gene whose regulation is poorly understood. Here we show that mice mutant for the stress-response gene Gadd45g display complete male-to-female sex reversal. Gadd45g and Sry have a strikingly similar expression pattern in the genital ridge, and they are coexpressed in gonadal somatic cells. In Gadd45g mutants, Sry expression is delayed and reduced, and yet Sry seemed to remain poised for expression, because its promoter is demethylated on schedule and is occupied by active histone marks. Instead, p38 MAPK signaling is impaired in Gadd45g mutants. Moreover, the transcription factor GATA4, which is required for Sry expression, binds to the Sry promoter in vivo in a MAPK-dependent manner. The results suggest that a signaling cascade, involving GADD45G → p38 MAPK → GATA4 → SRY, regulates male sex determination., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: SRY, located on the Y chromosome, is one of the key genes involved in human sex determination. SRY mutations are responsible for 10-15% of all cases of 46,XY gonadal dysgenesis (GD) but are rarely implicated in the pathogenesis of mixed GD., Methods: SRY was analyzed by sequence analysis of DNA extracted from blood leukocytes. SRY activity was evaluated by SOX9 immunostaining, one of the targets of SRY., Results: We report a case of mixed GD due to a novel SRY point mutation in a patient with a 46,XY karyotype, without mosaicism or submicroscopic genomic imbalances. Hormonal studies showed low anti-müllerian hormone and histological examination of the gonads showed a streak gonad on the right side and a left dysgenetic testis, thus permitting the diagnosis of mixed GD. Immunostaining for SOX9, a target of SRY, was positive in nuclei of Sertoli and epididymal cells in the left gonad and negative on the right, thus indicating asymmetric activation of SRY., Conclusion: Mixed GD can result from SRY mutations without mosaicism, neither in peripheral blood, nor within the gonads. The asymmetric effect of the point mutation implies the presence of local factors modulating SRY expression or action., (Copyright © 2012 S. Karger AG, Basel.)

Background: The early gonad is bipotential and can differentiate into either a testis or an ovary. In XY embryos, the SRY gene triggers testicular differentiation and subsequent male development via its action on a single gene, SOX9. The supporting cell lineage of the bipotential gonad will differentiate as testicular Sertoli cells if SOX9 is expressed and conversely will differentiate as ovarian granulosa cells when SOX9 expression is switched off., Results: Through copy number variation mapping this study identified duplications upstream of the SOX9 gene in three families with an isolated 46,XX disorder of sex development (DSD) and an overlapping deletion in one family with two probands with an isolated 46,XY DSD. The region of overlap between these genomic alterations, and previously reported deletions and duplications at the SOX9 locus associated with syndromic and isolated cases of 46,XX and 46,XY DSD, reveal a minimal non-coding 78 kb sex determining region located in a gene desert 517-595 kb upstream of the SOX9 promoter., Conclusions: These data indicate that a non-coding regulatory region critical for gonadal SOX9 expression and subsequent normal sex development is located far upstream of the SOX9 promoter. Its copy number variations are the genetic basis of isolated 46,XX and 46,XY DSDs of variable severity (ranging from mild to complete sex reversal). It is proposed that this region contains a gonad specific SOX9 transcriptional enhancer(s), the gain or loss of which results in genomic imbalance sufficient to activate or inactivate SOX9 gonadal expression in a tissue specific manner, switch sex determination, and result in isolated DSD.

Patients with XY gonadal dysgenesis are at increased risk of developing gonadal tumors. The etiology of several cases of XY gonadal dysgenesis remains unknown, but X/XY gonadal mosaicism has been hypothesized to play a role. At the histologic level, the presence of persistent primitive sex cords containing immature germ cells in dysgenetic gonads (an entity called undifferentiated gonadal tissue, or UGT) was recently described, and these immature germ cells are thought to be at risk of neoplastic transformation. To further investigate both these aspects, we retrospectively studied the gonads from 30 patients with pure (22) and mixed (8) gonadal dysgenesis. Cytogenetic analyses performed on 35 gonads revealed that structurally abnormal Y chromosomes were lost in a majority of cells from the gonads, explaining the gonadal dysgenesis of patients bearing a rearranged Y chromosome. On the other hand, normal Y chromosomes were less often lost in gonads of patients with gonadal dysgenesis. At the histologic level, 43 of the 51 gonads presented areas characteristic of a streak; 13 of these streak gonads also presented areas of UGT. Structures resembling sex cords but without germ cells were found in many of the streaks not containing UGT, suggesting that UGT was initially present. Of the 13 gonads containing both UGT and a streak, 9 developed a tumor. The proximity of UGT with the tumors as well as the immunostaining patterns (PLAP+, OCT3/4+, and CD117/KIT+) suggests that germ cells found in UGT are a risk factor for gonadal tumors.

Objective: To investigate the familial segregation, role, and function of a novel SRY missense mutation c.347T>C in two half-sisters affected by 46,XY complete gonadal dysgenesis (CDG) compatible with a successful pregnancy outcome., Design: Phenotypic, mutational, and functional study., Setting: Academic research unit., Patient(s): Two half-sisters, their common father, and 100 healthy control individuals., Intervention(s): Chromosome, molecular cytogenetic analysis, and Sanger sequencing of the SRY gene in blood lymphocytes of the proband, her affected half-sister, and in inflammatory tissue of the father postmortem. Cloning and expression of high mobility group box carboxy-terminal domains of Sry and electrophoretic mobility shift assay were performed., Main Outcome Measure(s): Not applicable., Result(s): A novel SRY missense mutation c.347T>C (p.Leu116Ser) was identified in two half-sisters and segregates with the CGD phenotype. It is present in the common healthy father in a mosaic state. Functional analyses demonstrate the pathogenic effect of the mutation by a strong reduction of DNA affinity for the mutant p.Leu116Ser SRY protein., Conclusion(s): The missense mutation c.347T>C in the high mobility group domain of SRY causes 46,XY CGD. Paternal gonadal mosaicism is likely to explain the familial occurrence of 46,XY CGD suggesting a de novo mutational event during the early stages of embryonic development. This novel mutation is compatible with a successful pregnancy outcome., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

SRY on the Y-chromosome acts as a transcription factor to initiate testicular differentiation in mammals. Sox9 is a SRY target gene, upregulated immediately after Sry expression, and plays a key role in testicular differentiation. In the present study, we examined the expression of SRY and SOX9 proteins in the B6.Y(TIR) gonad, which undergoes partial or complete sex reversal. The results show that the ontogeny of SRY expression in the B6.Y(TIR) gonad was comparable with that in the B6.XY gonad. On the other hand, while SOX9 expression immediately followed SRY expression in the B6.XY gonad, it was considerably delayed compared to SRY expression in the B6.Y(TIR) gonad or SOX9 expression in the B6.XY gonad. Although SOX9 expression reached the entire gonad at a time point, it was downregulated and became restricted to the central area in which testis cords were organized. MIS, a marker of Sertoli cells, appeared only in well-organized testis cords. We speculate that the SRY protein from the Y(TIR)-chromosome is inefficient in upregulating the Sox9 gene on the B6 background, allowing the initiation of ovarian differentiation., (Copyright © 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Context: Undervirilization in males, i.e. 46,XY disordered sex development (46,XY DSD), is commonly caused by either lack of androgen action due to mutant androgen receptor (AR) or deficient androgen synthesis, e.g. due to mutations in 17alpha-hydroxylase (CYP17A1). Like all other microsomal cytochrome P450 (CYP) enzymes, CYP17A1 requires electron transfer from P450 oxidoreductase (POR)., Objective: The objective of the study was to analyze the clinical and biochemical phenotype in a 46,XY individual carrying concomitant POR and AR mutations and to dissect their impact on phenotypic expression., Methods: We characterized the clinical and biochemical phenotype, genetic identification, and functional analysis of POR missense mutation by yeast micrososomal coexpression assays for CYP17A1, CYP21A2 and CYP19A1 activities., Results: The patient presented neonatally with 46,XY DSD and was diagnosed as partial androgen insensitivity syndrome carrying a disease causing AR mutation (p.Q798E). She was raised as a girl and gonadectomized at the age of 4 yr. At 9 yr progressive clitoral enlargement prompted reassessment. Urinary steroid analysis was indicative of POR deficiency, but surprisingly androgen production was normal. Genetic analysis identified compound heterozygous POR mutations (p.601fsX12/p.Y607C). In vitro analysis confirmed p.Y607C as a pathogenic mutation with differential inhibition of steroidogenic CYP enzymes., Conclusion: Both mutant AR and POR are likely to contribute to the neonatal presentation with 46,XY DSD. Virilization at the time of adrenarche appears to suggest an age-dependent, diminishing disruptive effect of both mutant proteins. This case further highlights the importance to assess both gonadal and adrenal function in patients with 46,XY DSD.

XY females are rare individuals who carry a Y chromosome but are phenotypically female. In approximately 80-90% of these cases, there are no mutations in the SRY gene, a testis-determining gene on the short arm of the Y chromosome, and the pathophysiology of XY females without SRY mutation remains unclear. In the present study, we used a molecular data mining technique to analyze the pathophysiology of an XY female with functional SRY and pericentric inversion of the Y chromosome, and compared the results with those of a normal male. Interestingly, upregulations of numerous genes included in the development category of the Biological Process ontology, including genes associated with sex determination and organ morphogenesis, were seen in the patient. Additionally, the transforming growth factor-beta (TGF-beta) signaling pathway and Wnt signaling pathway, in which most cell-cell interactions during embryonic development are involved, were altered. Alterations in the expression of numerous genes at the developmental stage, including alterations at both the gene and pathway levels, may persist as a vestige of anomalies of sex differentiation that presumably began in the fetal period. The present study indicates that a data mining technique using bioinformatics contributes to identification of not only genes responsible for birth defects, but also disorders of sex development (DSD)-specific pathways, and that this kind of analysis is an important tool for clarifying the pathophysiology of human idiopathic XY gonadal dysgenesis. Our findings could serve as one of the basic datasets which will be used for future follow-up investigations.

Mutations of SRY are the cause of 46,XY complete pure gonadal dysgenesis (PGD) in 10-15% of patients. In this study, DNA was isolated and sequenced from blood leukocytes and from paraffin-embedded gonadal tissue in five patients with 46,XY complete PGD. DNA binding capability was analyzed by three different methods. The structure of the full length SRY and its mutant proteins was carried out using a protein molecular model. DNA analysis revealed two mutations and one synonymous polymorphism: in patient #4 a Y96C mutation, and a E156 polymorphism; in patient #5 a S143G mosaic mutation limited to gonadal tissue. We demonstrated, by all methods used, that both mutant proteins reduced SRY DNA binding activity. The three-dimensional structure of SRY suggested that besides the HMG box, the carboxy-terminal region of SRY interacts with DNA. In conclusion, we identified two SRY mutations and a polymorphism in two patients with 46,XY complete PGD, demonstrating the importance of the carboxy-terminal region of SRY in DNA binding activity.

Objective: Steroidogenic factor 1 (SF1), officially designated NR5A1, is a nuclear receptor that plays key roles in endocrine development and function. Previous reports of human SF1 mutations revealed a spectrum of phenotypes affecting adrenal function and/or gonadal development and sex differentiation. We present the clinical phenotype and functional effects of a novel SF1 mutation., Patient: The patient is a 22-year-old 46, XY Japanese patient who presented with dysgenetic testes, atrophic vasa deferentia and epididymides, lack of Müllerian structures, and clitoromegaly. Endocrine studies revealed normal adrenal function., Results: Analysis of the SF1 gene revealed compound heterozygosity for a previously described p.G146A polymorphism and a novel missense mutation (p.R84C) in the accessory DNA-binding domain. The father carried the p.G146A polymorphism and the mother had the p.R84C mutation; both were clinically and reproductively normal. Functional studies demonstrated that the p.R84C SF1 had normal nuclear localization but decreased DNA-binding affinity and transcriptional activity compared with wild-type SF1; it did not exhibit any dominant negative activity., Conclusions: These results describe the human phenotype that results from compound heterozygosity of the p.G146A polymorphism and a novel p.R84C mutation of SF1, thereby extending the spectrum of human SF1 mutations that impair testis development and sex differentiation in a sex-limited manner while preserving normal adrenal 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., (2007 S. Karger AG, Basel)

The majority of genetic variations in the androgen receptor (AR) gene are point mutations leading to impairment of the DNA- or hormone-binding domains. The N-terminus encoded by the first exon of the AR-gene usually harbors disruptive mutations associated with complete androgen insensitivity syndrome (CAIS) while missense mutations related with partial androgen insensitivity syndrome (PAIS) are seemingly rare. We present a 46,XY male with scrotal hypospadias in whom we detected a S432 F point mutation within the N-terminus. Transient transfections of an AR expression plasmid carrying the S432 F mutation using Chinese Hamster Ovary (CHO) cells revealed a significant partial reduction in transactivation of the co-transfected androgen responsive (ARE) (2)TATA luciferase reporter gene thus confirming PAIS. In two further 46, XY patients with slight to moderate virilization defects, we detected an S411 N mutation, and a 9 base pair deletion leading to the loss of amino acids 409 to 411 (L-A-S), respectively. These mutations did not compromise AR-function under the chosen experimental settings. The S432 F-patient supports particular significance of the AR-N-terminus for mild forms of AIS while the functional role of the two further mutations remains unclear. The N-terminus is a species-specific AR-domain possibly also involved in contributing to target tissue selectivity of AR-actions via mediating co-regulator interactions. Therefore, mild molecular defects of the AR-N-terminus may not necessarily inhibit general transactivation properties using currently established reporter gene models.

Context: Maturation delay of germ cells and their progression into carcinoma in situ (CIS) frequently occurs in intersex patients. A developmentally delayed germ cell resembles a CIS cell and displays prolonged expression of immunohistochemical markers used for the diagnosis of CIS. This questions their applicability in young children., Objective: The objective of the study was the elaboration of tools to distinguish germ cells with maturation delay and CIS., Design: The design was a qualitative and quantitative analysis of the expression of diagnostic markers for CIS in gonads of young patients with undervirilization syndromes., Setting: The study was conducted in the pathology department of a university center, specializing in germ cell tumor pathogenesis., Patients: Fifty-eight formalin-fixed, paraffin-embedded testicular tissue samples of 30 undervirilized patients (1 month to 23 yr of age) were analyzed., Interventions: INTERVENTIONS included hematoxylin-eosin staining, immunohistochemistry for octamer binding transcription factor (OCT)3/4, gene encoding the stem cell factor receptor that has tyrosine kinase activity c-KIT, placental/germ alkaline phosphatase (PLAP), testis-specific protein Y encoded (TSPY), and VASA, double staining for OCT3/4 and VASA, with ploidy determination by fluorescent in situ hybridization., Main Outcome Measure: Maturation delay and CIS are characterized by the staining patterns of the immunohistochemical markers., Results: CIS was diagnosed in three of 30 patients (10%) and four of 58 gonads (6.9%). Patient age, distribution of OCT3/4-positive cells throughout the gonad, and their position within the seminiferous tubule differ between maturation delay and CIS. Abnormal OCT3/4 and testis-specific protein Y encoded expression appear to be of pathogenetic relevance in the development of these lesions., Conclusion: The dimorphic expression of OCT3/4 allows distinction between maturation delay and CIS. Studies in larger patient series are essential before a biopsy to evaluate the neoplastic risk can eventually be proposed as an alternative for gonadectomy.

The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding/bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein in vitro and in vivo, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMPbeta. The SRY-R133W mutant shows reduced IMPbeta binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMPbeta binding, whereas SRY-R75N and SRY-R76P show normal IMPbeta binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.

Sex determining Region of the Y chromosome (SRY) is the Y-borne gene required for male sex determination. Many XY females with complete gonadal dysgenesis carry SRY mutations. We describe here the effects of eight clinically isolated point mutations on the DNA-binding and -bending functions of SRY. We found that the seven mutations in the HMG domain affected the protein's DNA-binding and -bending activities to varying degrees, although all cause complete gonadal dysgenesis. DNA binding was abolished by the R75N and L94P mutations, severely disrupted by the F67V mutation and reduced by the M64R (6-fold), R76P (4-fold), A113T (3-fold), and M78T (1.7-fold) mutations. Of these, variant M64R showed no DNA-bending activity, while M78T caused a mild reduction in DNA bending. The S18N mutation, a familial mutation that lies outside the HMG domain and caused partial gonadal dysgenesis in one patient, had minimal effect on DNA binding and bending. Analysis of the NMR solution structure of the SRY HMG domain bound to DNA suggests that mutations disrupt the protein's conformation (helicity, packing), or interactions at the DNA interface. The degree to which mutations causing complete gonadal dysgenesis affect the DNA-binding activity varies. We propose that there is a threshold level of SRY activity or expression required for testis determination, as we observe that familial mutations have the least effect on SRY activity.

Objective: To investigate the responses of two patients previously diagnosed as Reifenstein's syndrome to graded high-dose testosterone in terms of hormone levels, nitrogen balance and sebum secretion and to attempt to correlate these parameters with the properties of their androgen receptors and mutations in the androgen receptor gene., Design: Nitrogen balance was determined by comparing controlled nitrogen intake to the amount excreted. Sebum excretion was measured on the forehead. Patients were studied during control periods (no treatment) and during administration of testosterone propionate. Blood samples were used as a source of genomic DNA and to measure peripheral hormone levels; androgen receptor binding was determined using genital skin fibroblasts., Patients: Two patients of XY karyotype, with ambiguous external genitalia and problems of testicular descent who had required mastectomy as teenagers. Normal male controls of proven fertility., Measurements: Nitrogen balance, sebum excretion rate and peripheral hormone levels (testosterone, dihydrotestosterone, LH and FSH) were studied before and after testosterone therapy (1 or 5 mg/kg/day). Genomic DNA was extracted from peripheral blood leucocytes and regions of the androgen receptor gene amplified by polymerase chain reaction using pairs of specific primers. Mobility of amplified DNA from patients was analysed on denaturing gradient acrylamide gels and fragments differing in mobility from those of normal controls were sequenced. Fibroblasts were cultured from scrotal skin biopsies and androgen receptor binding parameters, subcellular localization and up-regulation were determined., Results: Testosterone therapy resulted in raised plasma testosterone, dihydrotestosterone and oestradiol in both patients. In patient 1 (lesser genital abnormality), LH was suppressed by 5 mg/kg/day testosterone to the upper limit of the normal range but FSH remained low normal. Both LH and FSH were suppressed by testosterone treatment in patient 2 (greater genital abnormality). Nitrogen retention was increased in both patients (4.2 and 3.0 g/24 h respectively); sebum excretion rate increased to normal in patient 1 but showed no change in patient 2. Mutations in the androgen receptor gene were identified in both patients. In patient 1 a single nucleotide change from adenosine to guanosine resulted in the substitution of glycine for glutamic acid at position 772 within the hormone binding domain of the receptor. In patient 2 a single nucleotide mutation from guanosine to adenosine resulted in the substitution of lysine for arginine at position 608 (exon 3) situated in the second zinc finger of the DNA binding domain. Both patients had a normal number of androgen binding sites in genital skin fibroblasts but those in patient 1 showed reduced binding affinity and rapid dissociation of receptor/ligand complexes while those in patient 2 showed defective nuclear localization., Conclusion: In patients with partial androgen insensitivity syndrome the type of androgen receptor mutation and responses to short-term androgen treatment can be correlated with the individual's potential to virilize. If there is a mutation in the androgen receptor DNA binding domain the patient may show little ability to virilize either spontaneously at puberty or after androgen treatment. Sebum excretion appears to be more discriminating than nitrogen balance or gonadotrophin suppression as an index of tissue response to androgens.

We have characterized the androgen receptor in a Japanese infant with complete androgen insensitivity syndrome (or androgen resistance), and have investigated the molecular basis. Androgen binding was undetectable in cultured genital skin fibroblasts from this patient by whole-cell androgen receptor binding assay. Sequence analysis of the entire coding region of the androgen receptor gene from this patient revealed a single nucleotide substitution (G-->T) at nucleotide position 2676 in exon E (or 5), resulting in conversion of glutamine codon (GAG) to amber stop codon (TAG) at amino acid position 772 within the hormone-binding domain of the androgen receptor. This premature termination mutation (or nonsense mutation), introducing a truncated androgen receptor that lacks most of its androgen binding capacity, is though to cause the receptor-negative form of complete androgen insensitivity syndrome in this patient.

The biochemical and histological features of two related patients with the complete form of the androgen insensitivity syndrome (AIS) coexisting with incomplete regression of the Müllerian ducts are described. Both patients presented unilateral Müllerian derivatives (fallopian tube) identified by microscopic examination of surgically excised internal genital tissue. Biochemical studies performed in genital skin-derived fibroblasts from one of the affected subjects showed the existence of a specific and saturable 8.2 to 8.4 S cytosolic and 3.4 S nuclear androgen receptor exhibiting a Kd of 1.32 nmol/L. These mutant cells, however, clearly presented a significantly low maximal nuclear [3H]-5 alpha-dihydrotestosterone uptake (71.0 fmol/mg of deoxyribonucleic acid [DNA]; control strain, 284 fmol/mg DNA). Thus, an impaired uptake of the androgen receptor complex at the nuclear level was probably the cause of the complete absence of phenotypic expression of androgen action in this family. The overall findings are on line with the well-demonstrated genetic and molecular heterogeneity of the AIS.

Among women with 46,XY gonadal dysgenesis, there is a high incidence of gonadal tumors. Because of evidence of a connection between occurrence of those tumors, H-Y phenotype, and breast development, we surveyed 55 cases of 46,XY gonadal dysgenesis and 12 related cases involving chromosomal and/or skeletal abnormalities. Our survey, including three new cases presented here, indicates that H-Y phenotype but not breast development may be related to the development of the gonadoblastoma-dysgerminoma. Thus among women with 46,XY gonadal dysgenesis, there are H-Y- and H-Y+ classes, but gonadal tumors are found almost exclusively in the H-Y+ class. Yet one of our patients may represent an exception to the association of H-Y+ phenotype and gonadal tumors in this syndrome.

A 19-year-old phenotypic female with gonadal agenesis and XY karyotype underwent an endocrinologic study. She lacked secondary sexual characteristics and there was posterior labial fusion, absence of vagina, and no gonadal or gonadal duct tissues present at laparotomy. Elevated levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responded well to intravenous injection of LH-releasing hormone (LH-RH) but did not respond to clomiphene citrate. Both OH and FSH levels were suppressed by administration of testosterone or estrogen. Low serum concentration of testosterone did not respond to human chorionic gonadotropin (hCG) injection. Dihydrotestosterone binding by cultured skin fibroblast revealed the existence of a binding component with low capacity and high affinity. This is the first report of a patient with XY gonadal agenesis in whom androgen receptors in the target cells are demonstrated.

A 17 year old patient with female phenotype and 46,XY karyotype presented because of primary amenorrhoea. Enlarged clitoris and pubic hair were observed and no breast development. Gonadotrophin values were greatly raised into the castrate range, while testosterone was at the upper limit of the normal range for females. No uterus, Fallopian tubes or remnants of Mullerian and Wolffian products were found at laparotomy. Bilateral inguinal masses were removed and proved to be testicular tissue. This case report shows that gonads can be present in patients with the clinical and endocrinological characteristics of the syndrome of 'true agonadism'.

Diagnosis of XY pure gonadal dysgenesis was established in a patient of female phenotype, with female internal genitalia, but with a chromosomal constitution of 46 XY. Streak gonads had undergone neoplastic transformation--gonadoblastoma and dysgerminoma. Before operation the concentrations of gonadotrophins in plasma were high and of oestradiol was low. Administration of oestradiol benzoate initially suppressed and then stimulated an increase in the plasma concentration of LH. These changes were not accompanied by changes in blood levels of endogenous sex steroids. A single injection of hCG failed to stimulate steroid secretion. The activities in vitro of steroid-metabolizing enzymes in the dysgenetic gonadal tissue more closely resembled those of ovarian tissue from a premenopausal and from a postmenopausal women than those in testes from two androgen-insensitive patients. However, aromatase activity was higher in the dysgenetic gonads than in the pre or post-menopausal ovaries. Examination of enzymes in genital skin fibroblasts demonstrated normal activities of 3 alpha/beta-beta-hydroxysteroid dehydrogenase and 17 beta-hydroxysteroid dehydrogenase (oxidative and reductive directions). However, 5 alpha-reductase activity was low in minces and fibroblasts of genital skin from the patient. Androgen binding was within the range for male controls.

A patient with clinical manifestations of the incomplete androgen insensitivity syndrome was studied with respect to peripheral blood levels of steroids and steroid sulphates before, during and after gonadectomy. Steroid and steroid sulphate concentrations were also analyzed in spermatic venous blood and gonadal tissue collected during surgery. The metabolic capacity of gonadal tissue was also studied in vitro using progesterone, dehydroepiandrosterone sulphate and oestrone sulphate as substrates. Profound differences between the two gonads were noted with respect to both steroid content and release into pampiniform veins and to in vitro conversion of progesterone and oestrone sulphate. Histological examination revealed the presence of seminiferous tubules with carcinoma in situ in both gonads. It is suggested that the differences between the gonads may be due to an autonomous steroid production in the right gonad in spite of adequate or even elevated gonadotrophic stimulation resulting in a steroidogenic situation resembling the complete androgen insensitivity syndrome, while the conditions found in the left gonad more resembles the incomplete form of the disease.

The common epithelial tumors of the human ovary have rarely been found in the gonads of intersex patients with gonadal dysgenesis or true hermaphroditism. This report describes a patient with ovarian serous cystadenocarcinoma and mixed gonadal dysgenesis (45,X/46,XY) and reviews other reported cases. Intersex patients require early evaluation with treatment based on the karyotypic risk of malignant gonadal transformation. Epithelial ovarian tumors arising in dysgenetic gonads, which lack ova and are incapable of ovulating, provide a unique model for understanding the role of pituitary gonadotropins in ovarian epithelial tumorigenesis.

Two cases of XY dysgenesis are reported. In one case, that of a 13 year-old girl presenting with impuberism, a gonadoblastoma was detected by histologic examination of the streak. In the second case, that of a 6 month-old girl, short stature and dysplasia of the nails suggested the diagnosis.

The endocrine and biochemical characteristics of four related 46,XY pseudohermaphrodite patients with the Reifenstein Syndrome are presented. All of them (6 and 9 years old, first generation, and 9 and 12 months old, second generation) exhibited ambiguity of external genitalia and a family pedigree characteristic of an X-linked pattern of inheritance. Serum basal levels of LH, FSH, testosterone (T), androstenedione and 5 alpha-dihydrotestosterone (DHT) were within normal limits. Administration of hCG induced a normal response in terms of serum T in three of the patients, with a concomitant increase in serum DHT. However, an abnormally elevated T: DHT ratio was found in two of these subjects on the day of maximal T response (T: DHT ratio, 24 and 27; normal range, 4-21). Genital skin-derived fibroblasts from all patients were studied for [3H]DHT uptake in a whole-cell monolayer assay. Three of the mutant strains exhibited values of [3H]DHT uptake at 37 degrees C within the lower limits of normality (39.4-47.05 fmol/mg protein/h; normal strains, 36-101 fmol/mg protein/h), whereas fibroblasts from the remaining patient presented a slightly decreased uptake (31.66 fmol/mg protein); when studied at 42 degrees C, all mutant strains behaved as the normal controls. The existence of a specific 4.6 S cytosol androgen receptor was clearly seen in the two mutant strains when analysed by sucrose gradient centrifugation. Nevertheless, in one of the mutant strains, a significantly low maximal nuclear [3H]DHT uptake was detected (173.6 fmol/mg DNA; control strain, 301.6 fmol/mg DNA). The overall data were interpreted as demonstrating the existence of an impaired uptake of the androgen-receptor complex at the nuclear levels as the cause of the incomplete phenotypic expression of androgen action in this family. In this setting, the presence of low peripheral 5 alpha-reductase activity may be considered as a secondary manifestation of the androgen insensitivity.

A phenotypic female patients with 46,XY true hermaphroditism and clitoromegaly was found at laparotomy to have normal müllerian structures with a right streak gonad and a left dysgenetic ovotestis that contained a gonadoblastoma. Karyotypes of peripheral lymphocytes and cells from labial skin and both gonads were all 46,XY, so an unrecognized mosaic or chimera was unlikely. Tissue from both gonads was studied in vitro and estrogen and testosterone production measured. The streak produced 186.6 pg/mg/24 hr of estrogen and 78 pg/mg/24 hr of testosterone, whereas the dysgenetic ovotestis produced 85.8 pg/mg/24 hr of estrogen and 184.6 pg/mg/24 hr of testosterone (more than double the testosterone produced by the streak). Comparisons between these data and those previously published suggest that dysgenetic streak gonadal tissue is more active steroidogenically than postmenopausal ovarian stroma.