Testis-determining Gene Research Articles

The role of Sry in mammalian sex determination.

The testis-determining gene is the Y-linked gene responsible for initiating the developmental pathway leading to testis formation in males. A strategy based on determining the precise chromosomal location of this locus has been used to clone a new gene which has been called SRY in humans (Sry in mice). A variety of studies now show that this is indeed the testis-determining gene. Sry has a spatial and temporal pattern of expression which correlates with the initiation of testis differentiation. The amino acid sequence encoded by the gene suggests that the protein may function as a transcription factor, which fits well with models of sex determination. Some cases of XY sex reversal in humans and mouse have been attributed to mutations in SRY/Sry, indicating that it is normally necessary for testis determination. The finding that a genomic fragment carrying Sry can cause male development in XX mice has proved that Sry is the only gene from the Y chromosome necessary for testis determination.

Characterisation of Urogenital Ridge Gene Expression in the Human Embryonal Carcinoma Cell Line NT2/D1

The study of the mammalian sex-determining pathway has been hampered by the lack of cell culture systems to investigate the underlying molecular relationships between sex-determining genes. Recent approaches using high-throughput genome-wide studies have revealed a number of sexually dimorphic genes expressed in the developing mouse gonad. Here, we investigated a human testicular cell line in terms of its expression of known sex-determining genes and newly identified candidates. The human embryonal carcinoma cell line NT2/D1 was screened for the expression of 46 genes with known or potential roles in the sex-determining and differentiation pathway. Forty genes tested were expressed in NT2/D1 cells including the testis-determining genes SRY, SOX9, SF-1, DHH and FGF9. Genes not expressed included WT1, DAX1 and the ovary-specific genes FOXL2 and WNT4. Cell-specific markers demonstrate that NT2/D1 cells reflect a number of cell types in the gonad including Sertoli, Leydig and germ cells. Our results suggest that male pathways initiated by SRY, SOX9 and SF-1 remain intact in these cells. Lack of expression of ovary-specific genes is consistent with a commitment of these cells to the male lineage. Manipulation of gene expression in this cell line could be an important new in vitro tool for the discovery of new human sex-determining genes.

Cloning of the full-length coding sequence and expression analysis of Sox9b in guppy (Poecilia reticulata)

Sox9 is an SRY-related testis-determining gene. It has been identified in different species, thereby testifying to its conserved role in evolution. In this study, we isolated and characterized the guppy sox9b coding sequence and then analyzed its expression pattern in various adult guppy tissues by reverse transcription (RT)-PCR. The guppy sox9b encoded a 480-amino acid (aa) protein containing a SOX-TCF_HMG-box from aa 103 to 174 in the N-terminus region. The predicted molecular weight and the isoelectric point of this protein were 52.7 kDa and 6.61, respectively. The deduced amino acid sequence of guppy Sox9b showed 91.0% similarity to that of Oryzias latipes and 88.3% similarity to that Gasterosteus aculeatus. The detection of Sox9b mRNA in both the testis and ovary suggests that this gene might be involved in the gonad development of guppy.

R-spondin1 is essential in sex determination, skin differentiation and malignancy

R-spondins are a recently characterized small family of growth factors. Here we show that human R-spondin1 (RSPO1) is the gene disrupted in a recessive syndrome characterized by XX sex reversal, palmoplantar hyperkeratosis and predisposition to squamous cell carcinoma of the skin. Our data show, for the first time, that disruption of a single gene can lead to complete female-to-male sex reversal in the absence of the testis-determining gene, SRY.

Goat SRY induces testis development in XX transgenic mice

The testis-determining gene SRY is not well-conserved among mammals, particularly between mouse and other mammals, both in terms of protein structure and of expression regulation. To evaluate SRY phylogenic conservation in regards to its function, we expressed the goat gene ( gSRY) in XX transgenic mouse gonads. Here, we show that gSRY induces testis formation, despite a goat expression profile. Our results demonstrate that sex-reversal can be induced in XX-mice by a non-mouse SRY thus suggesting a conserved molecular mechanism of action of this testis-determining gene across mammalian species.

Isolation and Sequence Analysis of the Sox-1, -2, -3 Homologs in Trionyx sinensis and Alligator sinensis Having Temperature-Dependent Sex Determination

Members of the Sox gene family are characterized by an HMG-box that shows sequence similarity with that of the mouse testis-determining gene Sry. Using degenerate primers PCR, seven and eight HMG-box motifs of Sry-related genes were cloned and sequenced from genomic DNA of Trionyx sinensis (termed TS41-47) and Alligator sinensis (AS41-48) with TSD (temperature-dependent sex determination). Among 15 Sry-related genes, TS41, TS42, AS41, and AS42 shared 80, 72, 81, and 79% amino acid identity, respectively, with each HMG-box domain of the mouse Sox-1, -2, and -3 genes by Blast analysis. Molecular phylogenetic analysis showed that the clustering of TS41-42 and AS41-42 was distant to the clustering of the nonreptilian vertebrate Sox-1, -2, -3 homologs, including fish, amphibian, bird, and mammals. The amino acid identity among TS41-42, AS41-42, and the nonreptilian vertebrate Sox-1, -2, -3 homologs is lower than identities among the Sox-1, -2, -3 homologs, suggesting that the sequence changes in TS41-42 of Trionyx sinensis and AS41-42 of Alligator sinensis might have occurred after the diversification of amniotes.

Sexually Dimorphic Regulation of Inhibin Beta B in Establishing Gonadal Vasculature in Mice1

Sexually dimorphic differentiation of gonads is accomplished through balanced interactions between positive and negative regulators. One of the earliest features of gonadal differentiation is the divergent patterning of the vasculature. A male-specific coelomic vessel develops on the anterior to posterior of the XY gonad, whereas this vessel is absent in XX gonads. It is postulated that the testis-determining gene Sry controls formation of the coelomic vessel, but the exact molecular mechanism remains unknown. Here we reveal a novel role for inhibin beta B in establishing sex-specific gonad vasculature. In the testis, inhibin beta B contributes to proper formation of the coelomic vessel, a male-specific artery critical for testis development and, later in development, hormone transportation. On the other hand, in the ovary, inhibin beta B is repressed by WNT4 and its downstream target follistatin, leading to the absence of the coelomic vessel. When either Wnt4 or follistatin was inactivated, the coelomic vessel appeared ectopically in the XX ovary. However, when inhibin beta B was also removed in either the Wnt4-null or follistatin-null background, normal ovarian development was restored and no coelomic vessel was found. Our results indicate that the sex-specific formation of the coelomic vessel is established by positive components in the testis as well as an antagonizing pathway from the ovary. Inhibin beta B is strategically positioned at the intersection of these opposing pathways.

Is the coronary risk different, in our men and women?

In mammals, sex determination defines the differentiation of the bipotential genital ridge into either testes or ovaries. Sry, the mammalian Y-chromosomal testis-determining gene, is a master regulator of male sex determination. It acts to switch the undifferentiated genital ridge towards testis development, triggering the adoption of a male fate. Sry initiates a cascade of gene networks through the direct regulation of Sox9 expression and promotes supporting cell differentiation, Leydig cell specification, vasculature formation and testis cord development. In the absence of Sry, alternative genetic cascades, including female sex-determining genes RSPO1, Wnt4/β-catenin and Foxl2, are involved in the formation of female genitalia and the maintenance of female ovarian development. The mutual antagonisms between male and female sex-determining pathways are crucial in not just the initiation but also the maintenance of the somatic sex of the gonad throughout the organism’s lifetime. Any imbalances in above sex-determining genes can cause disorders of sex development in humans and mice. In this review, we provide a detailed summary of the expression profiles, biochemical properties and developmental functions of Sry and SoxE genes in embryonic testis development and adult gonadal development. We also briefly summarize the dedicate balances between male and female sex-determining genes in mammalian sex development, with particular highlights on the molecular actions of Sry and Sox9 transcription factors.

Sexual development of a model marsupial male

In eutherian mammals sexual differentiation occurs during fetal development, making experimental manipulation difficult, unlike in marsupials. We are investigating the roles of several key genes and hormones whose exact role in gonadal differentiation is still unclear using the tammar wallaby (Macropus eugenii) as a model. As in humans, unlike in mice, the testis-determining gene SRY is expressed in male tammar fetuses in many tissues over an extended period. Not all sexual differentiation depends on testicular hormones. Scrotum and mammary glands are under the control of X-linked gene(s). Our demonstration of DMRT1 expression in tammar and mouse ovaries suggests it has a wider role than previously thought. The Y-borne copy of ATRX (ATRY) is coexpressed with DMRT1 in developing testis. Gonadal sex reversal can be induced in males by neonatal oestrogen treatment and in females by grafting developing ovaries to males or culturing them in minimal medium. Treatments of developing young with various androgens, and studies of steroid metabolism have shown that the steroid androstenediol may have a previously unrecognised role in virilisation. Our studies using a marsupial model have given some surprising insights into the evolution and control of sexual development in all mammals.

Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes

Despite the identification of SRY as the testis-determining gene in mammals, the genetic interactions controlling the earliest steps of male sex determination remain poorly understood. In particular, the molecular lesions underlying a high proportion of human XY gonadal dysgenesis, XX maleness and XX true hermaphroditism remain undiscovered. A number of screens have identified candidate genes whose expression is modulated during testis or ovary differentiation in mice, but these screens have used whole gonads, consisting of multiple cell types, or stages of gonadal development well beyond the time of sex determination. We describe here a novel reporter mouse line that expresses enhanced green fluorescent protein under the control of an Sf1 promoter fragment, marking Sertoli and granulosa cell precursors during the critical period of sex determination. These cells were purified from gonads of male and female transgenic embryos at 10.5 dpc (shortly after Sry transcription is activated) and 11.5 dpc (when Sox9 transcription begins), and their transcriptomes analysed using Affymetrix genome arrays. We identified 266 genes, including Dhh, Fgf9 and Ptgds, that were upregulated and 50 genes that were downregulated in 11.5 dpc male somatic gonad cells only, and 242 genes, including Fst, that were upregulated in 11.5 dpc female somatic gonad cells only. The majority of these genes are novel genes that lack identifiable homology, and several human orthologues were found to map to chromosomal loci implicated in disorders of sexual development. These genes represent an important resource with which to piece together the earliest steps of sex determination and gonad development, and provide new candidates for mutation searching in human sexual dysgenesis syndromes.

Three New 46,XX Male Patients: A Clinical, Cytogenetic and Molecular Analysis

XX males range phenotypically from completely masculinised individuals to true hermaphrodites and include a subset of SRY negative patients. The correlation between genotype (SRY+/-) and phenotype is still unclear. To report three new patients with this rare condition, one of whom was diagnosed prenatally and another was SRY negative, and to verify in our patients whether the presence of SRY results in a more masculinised phenotype. We present two phenotypically normal XX male patients (10 and 13.5 years) and one 3.1 years old XX male with ambiguous external male genitalia Prader IV. The patients were diagnosed by clinical, hormonal, sonographic, genetic and histological criteria. Basal hormonal status was normal for phenotype but an excessive response to GnRH testing was noticed in the second patient together with insufficient hCG stimulation in all three patients. Pelvic ultrasound displayed male structures without Müllerian ducts; testicular biopsy, performed only in the intersex patient, showed Sertoli and Leydig cell hypoplasia. Chromosome analysis confirmed 46,XX karyotype. FISH analysis and molecular analysis by PCR were positive for Yp fragments/SRY gene on Xp in two patients and negative in the patient with ambiguous external genitalia. In our observation Y chromosome-specific material containing the SRY gene translocated to the X chromosome results in a completely masculinised phenotype. In the intersex patient, incomplete masculinisation without SRY suggests a mutation of one or more downstream non-Y testis-determining genes.

An Analysis of the Genetic Factors Involved in Testicular Descent in a Cohort of 14 Male Patients with Anorchia

Anorchia, or the "vanishing testis syndrome," is characterized by the absence of testis in a 46,XY individual with a male phenotype. The etiology is unknown; however, the familial occurrence of the disease and the association of this phenotype with 46,XY gonadal dysgenesis has led to the suggestion that genetic factors, which play a role in testicular determination, may be involved. Alternatively, exploratory laparoscopy has suggested that anorchia may be caused by a prenatal testicular vascular accident associated with torsion during testicular descent. We screened a cohort of 14 boys with bilateral anorchia for mutations in the Y chromosome-linked testis-determining gene SRY (sex-determining region, Y chromosome); in the gene necessary for correct testicular descent, INSL3; and in the gene of its receptor (LGR8). Mutations in the INSL3 gene and the LGR8 T222P mutation are known to cause cryptorchidism. We confirmed previous reports that mutations in the SRY gene are not associated with anorchia. Although a common polymorphism was identified in the INSL3 gene, no mutations were observed. The recurrent T222P mutation in the LGR8 gene was not found in any of the patients. These data show for the first time a lack of association between genetic factors necessary for correct testicular descent and anorchia.

Wnt4 is required for proper male as well as female sexual development

Genes previously implicated in mammalian sexual development have either a male- or female-specific role. The signaling molecule WNT4 has been shown to be important in female sexual development. Lack of Wnt4 gives rise to masculinization of the XX gonad and we showed previously that the role of WNT4 was to inhibit endothelial and steroidogenic cell migration into the developing ovary. Here we show that Wnt4 also has a function in the male gonad. We find that Sertoli cell differentiation is compromised in Wnt4 mutant testes and that this defect occurs downstream of the testis-determining gene Sry but upstream of Sox9 and Dhh, two early Sertoli cell markers. Genetic analysis shows that this phenotype is primarily due to the action of WNT4 within the early genital ridge. Analysis of different markers identifies the most striking difference in the genital ridge at early stages of its development between wild-type and Wnt4 mutant embryos to be a significant increase of steroidogenic cells in the Wnt4 −/− gonad. These results identify WNT4 as a new factor involved in the mammalian testis determination pathway and show that genes can have a specific but distinct role in both male and female gonad development.

Testis-like development of gonads in female moles. New insights on mammalian gonad organogenesis

Moles are unique among mammals because all females of several species of genus Talpa have bilateral ovotestes (gonads with both ovarian and testicular tissue). Based on the analysis of a large sample of embryos, foetuses and infants over a 13-year period, we have studied the development of the gonads in male and female moles of the species Talpa occidentalis. Several new field and laboratory procedures were developed specifically to obtain and manage this singular material. Our results reveal that gonads of female moles develop according to a testis-like pattern, which includes cord formation and mesonephric cell migration, and begins at the same time as testis differentiation in males. The first signs of sex differentiation do not appear in males but in females. Female (but not male) gonads are regionalised with a cortex (precursor of the ovarian tissue) and a medulla (precursor of the testicular tissue). Germ cells concentrate only in the cortex, so that the medulla soon becomes sterile. Testicular tissue development is transiently retarded in females for about a week before birth, and resumes afterwards. Development of the ovarian tissue in females is considerably delayed with respect to that of testicular tissue in both males and females. The molecular characterisation of peritubular myoid cells, which are exclusive of testes, evidences the presence of testicular tissue in the gonads of female moles, which also contain Leydig cells. However, the absence of fully differentiated Sertoli cells indicates that these cells are not responsible for triggering the differentiation of such a testicular tissue. Our results are also discussed regarding the definition of Sertoli cell morphology and function, and the possible role of germ cells in the sex-reversal process. Differences observed between XX and XY gonad development in moles suggest that the mammalian testis-determining gene, SRY, has an “anti-regionalisation” role during gonadal development, at least in those mammalian species in which regionalisation of the female gonad occurs.

Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation.

SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis, overexpression of genes coding for structural myelin proteins such as P0 due to mutant SOX10 may explain the dysmyelination phenotype observed in the patients with an additional neurological disorder.

The molecular action and regulation of the testis-determining factors, SRY (sex-determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9

Despite 12 yr since the discovery of SRY, little is known at the molecular level about how SRY and the SRY-related protein, SOX9 [SRY-related high-mobility group (HMG) box 9], initiate the program of gene expression required to commit the bipotential embryonic gonad to develop into a testis rather than an ovary. Analysis of SRY and SOX9 clinical mutant proteins and XX mice transgenic for testis-determining genes have provided some insight into their normal functions. SRY and SOX9 contain an HMG domain, a DNA-binding motif. The HMG domain plays a central role, being highly conserved between species and the site of nearly all missense mutations causing XY gonadal dysgenesis. SRY and SOX9 are architectural transcription factors; their HMG domain is capable of directing nuclear import and DNA bending. Whether SRY and SOX9 activate testis-forming genes, repress ovary-forming genes, or both remains speculative until downstream DNA target genes are identified. However, factors that control SRY and SOX9 gene expression have been identified, as have a dozen sex-determining genes, allowing some of the pieces in this molecular genetic puzzle to be connected. Many genes, however, remain unidentified, because in the majority of cases of XY females and in all cases of XX males lacking SRY, the mutated gene is unknown.

Mapping a gene for 46,XY gonadal dysgenesis by linkage analysis.

46,XY gonadal dysgenesis was transmitted as an autosomal-dominant trait in a large family with multiple affected members. Expressivity of the trait was highly variable, ranging from pure to partial gonadal dysgenesis associated with normal female genitalia or sexual ambiguity, to mild hypospadias in otherwise normal males. The phenotypic features of this trait appeared to be confined to the genitourinary system. Multipoint parametric analysis using markers D5S664, D5S633, and D5D2102 yielded an LOD score of 4.47, assuming sex-limited, autosomal-dominant inheritance with a penetrance of 0.6. Because mutation in testis-determining genes leads to gonadal dysgenesis in 46,XY individuals, we postulate that the gene mapped by this study normally plays a role in gonadal differentiation.

The role of androgens in development of the scrotum of the grey short-tailed Brazilian opossum ( Monodelphis domestica).

In eutherian mammals, sex differentiation is initiated by expression of the testis-determining gene on the Y chromosome. Subsequent phenotypic development of the reproductive tract and genitalia depends on the production of hormones by the differentiated testis. In marsupials the mechanisms of phenotypic development may vary from this pattern, as differentiation of the scrotal primordia has been shown to occur before that of the gonad. Thus, the development of the scrotum in the marsupial has been regarded as an androgen-independent process. We have sought to clarify the ontogeny of scrotal development and the appearance of androgen receptor immunoreactivity by examining Monodelphis domesticaembryos/pups from 1 day prior to birth until 2 days after birth. We have also used immunocytochemistry to determine the expression of the key steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase as an indicator of when the developing gonad may be capable of synthesizing androgens. Expression of this enzyme was first detected in the gonads and adrenals of both sexes 1 day prior to birth and before the appearance of scrotal bulges. Androgen receptor immunoreactivity was detected in the scrotal anlagen of male opossum pups as early as 1 day following birth. This finding is significantly earlier than previous reports and coincides with the appearance 1 day after birth of distinct scrotal bulges. Androgen receptor immunoreactivity was also observed in the genital tubercles of male pups, but not female pups, 2 days after birth. These results suggest that androgens may play an important role in the development of the male genitalia at a much earlier stage than that indicated by previously published work and that scrotal development in this species may not be androgen-independent.

DMRT1 is upregulated in the gonads during female-to-male sex reversal in ZW chicken embryos.

Sex in birds is chomosomally based (ZZ male, ZW female), but the mechanism underlying sex determination remains unknown. An unresolved question is whether Z gene dosage plays a role in avian sex determination. DMRT1 is an avian Z-linked gene that shows higher expression in male gonads during embryogenesis and has been proposed as a putative testis-determining gene in birds. The Z-linkage of this gene makes it an ideal candidate for testing the question of gene dosage in avian testis determination. A higher level of DMRT1 expression in male (ZZ) versus female (ZW) embryonic gonads may reflect the presence of two Z-linked copies in the male, or it may be due to specific and active upregulation of DMRT1 during testis formation. A functional interventionist strategy was used to distinguish between these two possibilities. DMRT1 expression was analyzed in chicken embryos during experimentally induced female-to-male sex reversal, using the aromatase enzyme inhibitor fadrozole. DMRT1 expression was analyzed by whole mount in situ hybridization and reverse transcription polymerase chain reaction (for mRNA) and indirect immunofluorescence (for protein). Female-to-male sex-reversed embryos (genetically ZW) showed elevated levels of DMRT1 expression similar to those of normal males (with two copies of the Z chromosome). Elevated levels of DMRT1 are therefore associated with testis development, both in normal males (ZZ) and in sex-reversed females (ZW). SOX9 expression was also activated during female-to-male sex reversal but appeared delayed relative to DMRT1 upregulation. These results show that testis development does not require two Z-linked copies of DMRT1, but it does involve active upregulation of the gene. Higher levels of DMRT1 expression during testis differentiation therefore do not simply reflect a gene dosage difference between the two sexes but imply active involvement in male development.

46,XY agonadism associated with adrenal adenoma/myelolipoma: report of a case analyzing Y-chromosome-specific sequences

A unique patient with 46,XY agonadism associated with adrenal adenoma/myelolipoma is described. The patient was an 18-year-old female with primary amenorrhea, lack of secondary sexual development and an aldosterone-producing adrenocortical adenoma associated with foci of myelolipoma. Molecular analyses of Y-chromosome-specific regions, including automated sequencing of the entire coding region of SRY, the Y-linked testis-determining gene, were performed. Our results excluded the possibility that a mutation in SRY was responsible for this unusual clinical combination.