A 46,XY female DSD patient with bilateral gonadoblastoma, a novel SRY missense mutation combined with a WT1 KTS splice-site mutation

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY gonadal dysgenesis cases (i.e., Swyer syndrome), SRY mutations, residing in the HMG (High Mobility Group) domain, are found to affect nuclear transport or binding to and bending of DNA. Frasier syndrome (FS) is characterized by gonadal dysgenesis with a high risk for development of GB as well as chronic renal failure in early adulthood, and is known to arise from a splice site mutation in intron 9 of the Wilms' tumor 1 gene (WT1). Mutations in SRY as well as WT1 can lead to diminished expression and function of SRY, resulting in sub-optimal SOX9 expression, Sertoli cell formation and subsequent lack of proper testicular development. Embryonic germ cells residing in this unfavourable micro-environment have an increased risk for malignant transformation. Here a unique case of a phenotypically normal female (age 22 years) is reported, presenting with primary amenorrhoea, later diagnosed as hypergonadotropic hypogonadism on the basis of 46,XY gonadal dygenesis with a novel missense mutation in SRY. Functional in vitro studies showed no convincing protein malfunctioning. Laparoscopic examination revealed streak ovaries and a normal, but small, uterus. Pathological examination demonstrated bilateral GB and dysgerminoma, confirmed by immunohistochemistry. Occurrence of a delayed progressive kidney failure (focal segmental glomerular sclerosis) triggered analysis of WT1, revealing a pathogenic splice-site mutation in intron 9. Analysis of the SRY gene in an additional five FS cases did not reveal any mutations. The case presented shows the importance of multi-gene based diagnosis of DSD patients, allowing early diagnosis and treatment, thus preventing putative development of an invasive cancer.     

Global Genome Analysis of the Downstream Binding Targets of Testis Determining Factor SRY and SOX9

A major event in mammalian male sex determination is the induction of the testis determining factor Sry and its downstream gene Sox9. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. A modified ChIP-Chip analysis using a comparative hybridization was used to identify 71 direct downstream binding targets for SRY and 109 binding targets for SOX9. Interestingly, only 5 gene targets overlapped between SRY and SOX9. In addition to the direct response element binding gene targets, a large number of atypical binding gene targets were identified for both SRY and SOX9. Bioinformatic analysis of the downstream binding targets identified gene networks and cellular pathways potentially involved in the induction of Sertoli cell differentiation and testis development. The specific DNA sequence binding site motifs for both SRY and SOX9 were identified. Observations provide insights into the molecular control of male gonadal sex determination.     

性腺母细胞瘤的分子遗传机制研究进展

性腺母细胞瘤(Gonadoblastoma, GB)是一种由性索和生殖细胞演化而来的罕见原位性腺肿瘤,与性腺遗传物质异常有密切联系。80%的GB患者表现为46,XY女性表型,其余为45,XY 和46,XX性别发育异常患者等。35%的GB会进一步演化为无性细胞瘤和精原细胞瘤等恶性肿瘤。由于表型与遗传的异质性,GB的分子遗传机制还未完全揭示。越来越多的研究显示GB的发生与性别分化和决定调控基因(如SRY、WT1、SOX9、Foxl2和TSPY等)之间存在密切关联,且表现出遗传与表观遗传调控相互作用。本文综述了GB的临床表现、病理特征、诊断与治疗措施,总结了性腺遗传异常导致GB的分子遗传与表观遗传调控机制,分析并归纳参与GB形成相关基因的共同表达调控网络,指出了当前研究中的障碍与不足,为进一步研究GB致病分子机制提供新思路。     

SRY protein is expressed in ovotestis and streak gonads from human sex-reversal

In mammals, a master gene located on the Y chromosome, the testis-determining gene SRY, controls sex determination. SRY protein is expressed in the genital ridge before testis determination, and in the testis it is expressed in Sertoli and germ cells. Completely sex-reversed patients are classified as either 46,XX males or 46,XY females. SRY mutations have been described in only 15% of patients with 46,XY complete or partial gonadal dysgenesis. However, although incomplete or partial sex-reversal affects 46,XX true hermaphrodites, 46,XY gonadal dysgenesis, and 46,XX/46,XY mosaicism, only 15% of the 46,XX true hermaphrodites analyzed have the SRY gene. Here, we demonstrate that the SRY protein is expressed in the tubules of streak gonads and rete testis, indicating that the SRY protein is normally expressed early during testis determination. Based on these results, we propose that some factors downstream from SRY may be mutated in these 46,XY sex-reversal patients. We have also analyzed SRY protein expression in the ovotestis from 46,XX true hermaphrodites and 46,XX/46,XY mosaicism, demonstrating SRY protein expression in both testicular and ovarian portions in these patients. This suggests that the SRY protein does not inhibit ovary development. These results confirm that other factors are needed for complete testis development, in particular, those downstream of the SRY protein.     

Mutation analysis of subjects with 46, XX sex reversal and 46, XY gonadal dysgenesis does not support the involvement of SOX3 in testis determination

Despite the identification of an increasing number of genes involved in sex determination and differentiation, no cause can be attributed to most cases of 46, XY gonadal dysgenesis, approximately 20% of 46, XX males and the majority of subjects with 46, XX true hermaphroditism. Perhaps the most interesting candidate for involvement in sexual development is SOX3, which belongs to the same family of proteins (SOX) as SRY and SOX9, both of which are involved in testis differentiation. As SOX3 is the most likely evolutionary precursor to SRY, it has been proposed that it has retained a role in testis differentiation. Therefore, we screened the coding region and the 5 and 3 flanking region of the SOX3 gene for mutations by means of single-stranded conformation polymorphism and heteroduplex analysis in eight subjects with 46, XX sex reversal (SRY negative) and 25 subjects with 46, XY gonadal dysgenesis. Although no mutations were identified, a nucleotide polymorphism (1056C/T) and a unique synonymous nucleotide change (1182A/C) were detected in a subject with 46, XY gonadal dysgenesis. The single nucleotide polymorphism had a heterozygosity rate of 5.1% (in a control population) and may prove useful for future X-inactivation studies. The absence of SOX3 mutations in these patients suggests that SOX3 is not a cause of abnormal male sexual development and might not be involved in testis differentiation.An erratum to this article can be found at     

St?rungen der m?nnlichen Gonadendifferenzierung

XY gonadal dysgenesis is characterized by a failure of testis differentiation and can be caused either by disturbed development of the urogenital ridge to the bipotential gonad or by impaired differentiation of the bipotential gonad to testis. Genes responsible for early gonadal development like WT1 and SF1 can be distinguished from genes involved in testis differentiation such as SRY, SOX9, DMRT, DAX1, WNT4, DHH, CBX2, TSPYL1, ATRX and ARX. In complete XY gonadal dysgenesis, M??llerian but no Wolffian structures are present. In partial XY gonadal dysgenesis, remnants of M??llerian and Wolffian ducts can be present and virilization of the external genitalia can take place. In about a third of cases, XY gonadal dysgenesis occurs in a syndromic form. In these syndromic forms, the extragenital phenotypes can indicate the causative genes, but these genes can also cause non-syndromic forms of XY gonadal dysgenesis.     

SOX9 regulates prostaglandin D synthase gene transcription in vivo to ensure testis development

In mammals, male sex is determined by the Y-chromosomal gene Sry (sex-determining region of Y chromosome). The expression of Sry and subsequently Sox9 (SRY box containing gene 9) in precursors of the supporting cell lineage results in the differentiation of these cells into Sertoli cells. Sertoli cells in turn orchestrate the development of all other male-specific cell types. To ensure that Sertoli cells differentiate in sufficient numbers to induce normal testis development, the early testis produces prostaglandin D(2) (PGD(2)), which recruits cells of the supporting cell lineage to a Sertoli cell fate. Here we show that the gene encoding prostaglandin D synthase (Pgds), the enzyme that produces PGD(2), is expressed in Sertoli cells immediately after the onset of Sox9 expression. Promoter analysis in silico and in vitro identified a paired SOX/SRY binding site. Interestingly, only SOX9, and not SRY, was able to bind as a dimer to this site and transactivate the Pgds promoter. In line with this, a transgenic mouse model showed that Pgds expression is not affected by ectopic Sry expression. Finally, chromatin immunoprecipitation proved that SOX9 but not SRY binds to the Pgds promoter in vivo.     

Dax1 regulates testis cord organization during gonadal differentiation

Mutations of the DAX1 nuclear receptor gene cause adrenal hypoplasia congenita, an X-linked disorder characterized by adrenal insufficiency and hypogonadotropic hypogonadism. Targeted deletion of Dax1 in mice also reveals primary testicular dysgenesis, which is manifest by obstruction of the rete testis by Sertoli cells and hyperplastic Leydig cells, leading to seminiferous tubule dilation and degeneration of germ cells. Because Dax1 is expressed early in gonadal development, and because Sertoli and Leydig cells are located ectopically in the adult, we hypothesized that these testis abnormalities are the result of an early defect in testis development. In Dax1(-/Y) males, the gonad develops normally until 12.5 dpc. However, by 13.5 dpc, the testis cords are disorganized and incompletely formed in Dax1-deficient mice. The number of germ and Sertoli cells is unchanged, and the expression of Sertoli-specific markers appears to be normal. However, the number of peritubular myoid cells, which normally surround the testis cords, is reduced. BrdU labeling of peritubular myoid cells is low, consistent with decreased proliferation. The basal lamina produced by peritubular myoid and Sertoli cells is disrupted, leading to open and incompletely formed testis cords. Leydig cells, which normally reside in the peritubular space and extend from the coelomic surface to the dorsal surface of the gonad, are restricted to the coelomic surface of Dax1-deficient testis. We conclude that Dax1 plays a crucial role in testis differentiation by regulating the development of peritubular myoid cells and the formation of intact testis cords. The developmental abnormalities in the Dax1-deficient testis lay the foundation for gonadal dysgenesis and infertility in adult mice and, potentially in humans with DAX1 mutations.     

Adenomatous polyposis coli (APC) is essential for maintaining the integrity of the seminiferous epithelium

Sertoli cells provide the microenvironment necessary for germ cell development and spermatogenesis; disruption of Sertoli cell morphology or function can lead to germ cell aplasia, which is observed in testicular dysgenesis syndrome. Mutation of the adenomatous polyposis coli (APC) gene has been associated with various human cancers, including testicular cancer, but its involvement in nonmalignant testicular pathologies has not been reported. We have developed a mouse model (APC(cko)) that expresses a truncated form of APC in Sertoli cells. Despite normal embryonic and early postnatal testicular development in APC(cko) mice, premature germ cell loss and Sertoli cell-only seminiferous tubules were observed in mutant testes without affecting Sertoli cell quiescence, apoptosis, or differentiation, which were confirmed by the absence of both proliferating cell nuclear antigen, DNA strand breaks, and anti-Müllerian hormone, respectively. We show that mutant Sertoli cells lose their apical extensions, which would normally enclose germ cells during various stages of spermatogenesis, and were unable to maintain the blood-testis barrier because of disrupted expression of junctional proteins. We also observed an up-regulation of Snail and Slug, markers suggestive of epithelial-mesenchymal transition in the Sertoli cells, but tumorigenesis was not observed. No comparable phenotype was observed with Sertoli cell-specific loss-of-function mutations in β-catenin, leading us to speculate that truncation of APC in Sertoli cells results in progressive degeneration of the seminiferous tubules by a mechanism that disrupts the integrity of Sertoli cell junctions independently of APC-regulated β-catenin activities and leads to development of a Sertoli cell-only phenotype.     

Mutations in MAP3K1 cause 46,XY disorders of sex development and implicate a common signal transduction pathway in human testis determination

Investigations of humans with disorders of sex development (DSDs) resulted in the discovery of many of the now-known mammalian sex-determining genes, including SRY, RSPO1, SOX9, NR5A1, WT1, NR0B1, and WNT4. Here, the locus for an autosomal sex-determining gene was mapped via linkage analysis in two families with 46,XY DSD to the long arm of chromosome 5 with a combined, multipoint parametric LOD score of 6.21. A splice-acceptor mutation (c.634-8T>A) in MAP3K1 segregated with the phenotype in the first family and disrupted RNA splicing. Mutations were demonstrated in the second family (p.Gly616Arg) and in two of 11 sporadic cases (p.Leu189Pro, p.Leu189Arg)-18% prevalence in this cohort of sporadic cases. In cultured primary lymphoblastoid cells from family 1 and the two sporadic cases, these mutations altered the phosphorylation of the downstream targets, p38 and ERK1/2, and enhanced binding of RHOA to the MAP3K1 complex. Map3k1 within the syntenic region was expressed in the embryonic mouse gonad prior to, and after, sex determination. Thus, mutations in MAP3K1 that result in 46,XY DSD with partial or complete gonadal dysgenesis implicate this pathway in normal human sex determination.     

Expression of the human SRY protein during development in normal male gonadal and sex-reversed tissues.

Sex determination in mammals is controlled by the SRY gene located on the Y chromosome. It encodes a protein containing a DNA-binding and DNA-bending domain. In spite of recent advances in the identification of the mechanisms that regulate male sex determination in mammals, the expression profile of the SRY protein in normal and sex-reversed human tissues is not well established. In order to localize the SRY protein and determine its cellular distribution and expression at different stages of development, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One of these antibodies, LSRY1.1, recognizes a protein of 27 kDa in total lysates of HeLa SRYB3, a human cell line transfected with the SRY gene under the control of the SV40 promoter. Immunocytochemical analysis in the cell lines shows nuclear localization of the SRY protein. We have studied SRY protein expression in human tissues at different stage of fetal development until adult life and have demonstrated that the SRY protein is located in the nuclei of somatic cells and germ cells in the genital ridge during testis development. After testis determination, it can be detected until the adult stage in both germ cells and Sertoli cells. The presence of the SRY protein was also analyzed in biopsies of gonadal tissues of sex-reversal patients such as SRY-positive 46,XX males or SRY-positive 46,XX true hermaphrodites. SRY protein is detected in the nuclei of Sertoli cells of the testis and in the nuclei of granulosa cells in the ovotestis in these patients and in the nuclei of germ cells of both tissue types. These results suggest a common cellular origin for both Sertoli cells and granulosa cells.     

Abnormal gonadal differentiation in two subjects with ambiguous genitalia,Mullerian structures,and normally developed testes: Evidence for a defect in gonadal ridge development

Among a group of patients with abnormal sexual differentiation, we have identified two subjects who had a 46,XY karyotype, ambiguous genitalia, and well-developed Müllerian structures, but normal appearing testes. The presence of ambiguous genitalia and persistent Müllerian structures implied both Leydig cell and Sertoli cell dysfunction, hence, gonadal dysgenesis. However, the normal testicular histology suggested that the underlying abnormality was not a defect in testis determination itself but an abnormality in timing of gonadal ridge and testis development. In one of the two subjects genomic DNA was available. The sequence of the SRY gene was normal. Because rare patients with partial androgen insensitivity may have a similar phenotype, the AR gene was evaluated by denaturing gradient gel electrophoresis (DGGE) and was normal. Some subjects with mutation of the WT1 gene or with deletion of the distal short arm of chromosome 9 may have similar phenotypes. The WT1 gene was studied by single-strand conformation polymorphism (SSCP) analysis and was normal. In addition, there was no loss of heterozygosity of polymorphic markers in distal 9p. The gene for Müllerian inhibiting substance (MIS) was also studied by SSCP and was normal. Although the exact mechanism for the defect in the two subjects is unknown, it may be due to an abnormality in a gene or genes involved in the timing of gonadal ridge development. Received: 5 August 1994 / Revised: 25 January 1995, 3 April 1995     

Monozygotic twins of opposite sex

Although discordant karyotypes are known in identical twins, cases involving differences in sex phenotype are rare. We studied identical twins with the 46,XY karyotype - a male with mixed gonadal dysgenesis and a female with "pure" gonadal dysgenesis. The testis-determining SRY gene was present in DNA from both twins but no mutations were detected in the SRY conserved motif. Monozygosity was indicated by short tandem repeat polymorphism analysis. These observations could be attributed to (i) mutation and mosaicism involving "downstream" sex-determining loci, (ii) variable penetrance of genes such as DSS/NR0B1, duplication of which can disrupt the male-determining pathway, or (iii) occurrence of cryptic 45,X gonadal cell lines.     

Cloning of cDNA for newt WT1 and the differential expression during spermatogenesis of the Japanese newt, Cynops pyrrhogaster

To investigate the function of Wilms' tumor 1 (WT1) during spermatogenesis, cDNA for newt WT1 homolog was cloned and the expression of WT1 in newt testes was examined. The cDNA is 2089 bp in length and encodes 426 amino acid (aa) residues. The deduced aa sequence shares 76 and 79% homology with human and Xenopus WT1, respectively. Northern blot analysis shows that WT1 mRNA, 3.2 and 4.5kb in length, are expressed in the testis and kidney. Both WT1 mRNA species are detected in various stages of spermatogenesis, but the 3.2kb mRNA is highly expressed in spermatogonia and mature sperm stages, while the amount of 4.5kb mRNA is almost constant throughout spermatogenesis. In situ hybridization reveals that WT1 mRNA is localized in Sertoli cells. Moreover, immunohistochemical analysis shows that WT1 protein is highly expressed in the nuclei of Sertoli cells in early spermatogonia and mature sperm stages, but not in pericystic cells or germ cells. These results suggest that WT1 is involved in the regulation of gene expression in Sertoli cells, depending on the spermatogenic stage.