The normal human diploid cell contains 22 autosomal pairs of chromosomes and two sex chromosomes (two Xs, or one X and one Y). When arranged serially and numbered according to size and centromeric position, they are known as a karyotype. Advances in the techniques of staining chromosomes permit positive identification of each chromosome by its unique banding pattern. A technique called fluorescence in situ hybridization (FISH) is particularly useful in identifying quickly both sex chromosomes, mosaicism and structural abnormalities involving the sex chromosomes, the presence or absence of SRY (the testes determining gene on the Y chromosome), and other deleted genes (Figure 14–1). High-resolution chromosome banding and painting techniques provide precise identification of each chromosome.
FISH (fluorescent in situ hybridization) for SRY in metaphase and interphase cells. Image illustrates localization of the SRY probe on the distal short arm of the Y chromosome (Yp11.3) in spectrum orange. The probe for the centromere region of the X chromosome is shown in spectrum green. Note the presence of both probes in the interphase cell below.
(Reproduced, with permission, from Grumbach MM, Hughes IA, Conte FA. Disorders of sex differentiation. In: Larsen PR, et al, eds. Williams Textbook of Endocrinology. 10th ed. WB Saunders; 2003.)
Studies in animals as well as humans with abnormalities of sexual differentiation indicate that the sex chromosomes (the X and Y chromosomes) and the autosomes harbor genes that influence sex determination and differentiation by causing the bipotential gonad to develop either as a testis or as an ovary. Two intact and normally functioning X chromosomes, in the absence of a Y chromosome or its SRY gene, lead to the formation of an ovary under normal circumstances, whereas a Y chromosome (whose SRY gene is intact) or the translocation of SRY, the testis-determining gene on the short arm of the Y chromosome, to an X chromosome or autosome leads to testicular organogenesis.
In humans, there is a marked discrepancy in size between the X and Y chromosomes. Gene dosage compensation is achieved in all persons with two or more X chromosomes in their genetic constitution by partial inactivation of all X chromosomes except one. This phenomenon is thought to be a random process (except when a structurally abnormal X chromosome is present) that occurs in each cell in the late blastocyst stage of embryonic development, during which either the maternally or the paternally derived X chromosome undergoes heterochromatinization. A result of this epigenetic process is formation of an X chromatin body (Barr body) in the interphase cells of persons having two or more X chromosomes. In patients with two or more X chromosomes, the maximum number of Barr bodies (partially inactivated X chromosomes) seen in interphase cells will be one less than the number of X chromosomes in the karyotype. A gene termed XIST (X-inactive specific transcripts) is located in the X inactivation center at Xq13.2 on the paracentromeric region of the long arm of the X chromosome. XIST is expressed only by the inactive X chromosome. The XIST gene encodes a large RNA that appears to coat the X chromosome and facilitates inactivation of selective genes on the X chromosome.
The distal portion of the short arm of the X chromosome escapes inactivation and has a short (2.5-megabase [mb]) segment homologous to a segment on the distal portion of the short arm of the Y chromosome (Figures 14–2 and 14–3). This segment is called the pseudoautosomal region (PAR); it is these two limited regions of the X and Y that pair during meiosis, undergo obligatory chiasm formation, and allow for exchange of DNA between these specific regions of the X and Y chromosomes. At least 10 genes have been localized to the pseudoautosomal region on the short arm of the X and Y chromosomes. Among these are a gene whose deletion results in the neurocognitive defects observed in Turner syndrome and a gene for short stature, SHOX (short stature homeobox gene), which is expressed in bone. A mutation or deletion of SHOX on either the X or Y chromosome is associated with "idiopathic" short stature as well as dyschondrosteosis (Leri-Weill syndrome). Homozygous mutations of this gene are associated with a more severe form of short stature, Langer mesomelic dwarfism. A pseudoautosomal region has also been described for the distal ends of the long arms of the X and Y chromosomes (see Figures 14–2 and 14–3). The pseudoautosomal region of the long arms of the X and Y chromosomes contains genes that are mostly growth factors and signaling molecules. The Y chromosome (see Figure 14–3) represents only 2% of the human genomic DNA and is about 60 mb in length. It is unique in that it contains few active genes compared to the X and autosomal chromosomes and has a large apparently noncoding heterochromatic region. It contains at least two genes affecting growth, the SHOX gene in the PAR and the growth control gene on the Y chromosome (GCY). The euchromatic region of the short arm of the Y chromosome contains the SRY gene (the male determining factor) distal to the PAR region at Yp11.3. The short arm of the Y chromosome contains genes that when deleted produce the phenotype stigma of Turner syndrome. The pericentromeric region of the Y is the locus for the TSPY (testes-specific protein Y encoded) genes which predispose to gonadoblastoma formation in the presence of dysgenetic testicular development. The PRKY gene is homologous to a gene PRKX (protein kinase X linked) on the X chromosome and is the locus for Y to X translocation observed in 46,XX SRY-positive males. The euchromatic region of the long arm of the Y chromosome has genes that when deleted result in azoospermia.
Diagrammatic representation of G-banded X chromosome. Selected X-linked genes are shown (ATRX, α-thalassemia, X-linked mental retardation; DAX1, DSS-AHC-critical region on the X chromosome gene 1; DIAPH2, human homolog of the Drosophila diaphanous gene; DMD, duchenne muscular dystrophy; GK, glycerol kinase; GPD, glucose-6-phosphate dehydrogenase; MAMLD1, mastermind-like domain-containing 1; MIC2, a cell surface antigen recognized by monoclonal antibody 12E7; PRKX, a member of the cAMP-dependent serine-threonine protein kinase gene family [illegitimate X-Y interchange occurs most frequently between PRKX and PRKY]; RPS4X, ribosomal protein S4; SHOX, short-stature homeobox gene; SOX3, SRY-like HMG box-3; USP9X, human x-linked homolog of the drosophila fat facets-related gene [DFFRX]; XIC, X-inactivation center; XIST, Xi-specific transcripts).
(Reproduced, with permission, from Grumbach MM, Hughes IA, Conte FA. Disorders of sex differentiation. In: Larsen PR, et al, eds. Williams Textbook of Endocrinology. 10th ed. WB Saunders; 2003.)
Diagrammatic representation of a G-banded Y chromosome (AZF, azoospermic factor; DAZ, deleted in azoospermia; MIC2, gene for a cell surface antigen recognized by monoclonal antibody 12E7; PRKY, a member of the cAMP-dependent serine-threonine protein kinase gene family; RPS4Y, ribosomal protein S4; SHOX, short stature homeobox gene; SRY, sex-determining region Y; TSPYA,B, members of the testes-specific factor gene family; ZFY, zinc finger Y).
(Reproduced with permission from Grumbach MM, Hughes IA, Conte FA. Disorders of sex differentiation. In: Larsen PR, et al, eds. Williams Textbook of Endocrinology. 10th ed. WB Saunders; 2003.)
Genes Involved in Organogenesis of the Bipotential gonad
Heterozygous mutations and deletions of the Wilms tumor gene (WT1) located on 11p13 result in urogenital malformations as well as Wilms tumor. Knockout of the WT1 gene in mice results in apoptosis of the metanephric blastema and as a consequence, absence of the kidneys and gonads. Thus, WT1, a transcriptional regulator, appears to act on metanephric blastema early in urogenital development (Figure 14–4A). Dominant-negative point mutations of WT1 in human beings results in the Denys-Drash and Frasier syndromes, whereas a contiguous deletion of the gene and surrounding DNA results in Wilms tumor, aniridia, ambiguous genitalia, and mental retardation—the WAGR syndrome.
Major genes involved in sex determination from the intermediate mesoderm to the bipotential gonad. (DMRT1, doublesex and mab-3-related transcription factor 1; PGC, primordial germ cell [arise from an extragonadal site]; SF-1, steroidogenic factor 1; SOX9, SRY-like HMG-box 9; WT1, Wilms tumor suppressor gene-1) The bipotential gonad contains the precursor cells which differentiate into the supporting cells, steroid hormone-producing cells, and germ cells as shown.
Steroidogenic factor-1 (SF-1) is a nuclear receptor involved in transcriptional regulation of many genes, including those that are involved in gonadal development, adrenal development, steroid synthesis, and reproduction (see Figure 14–4A). SF-1 is located on 9q33 and is expressed in the urogenital ridge as well as in steroidogenic organs. SF-1 is required for the synthesis of testosterone in Leydig cells; in Sertoli cells it regulates the anti-Müllerian hormone (AMH) gene. Homozygous knockout of the gene encoding Sf-1 (Sf-1 is the mouse homologue of mammalian SF-1) in mice results in apoptosis of the cells of the genital ridge that give rise to the adrenals and gonads and thus absence of gonadal and adrenal gland morphogenesis in both males and females. This gene has a critical role in the formation of all steroid-secreting glands (ie, adrenals, testes, and ovaries). Initial studies in humans with SF-1 mutations identified individuals with adrenal insufficiency, a 46,XY karyotype, complete gonadal dysgenesis, and the presence of müllerian derivatives, similar to the mouse phenotype. Subsequently, the spectrum of affected patients has included a range of 46,XY disorders of sexual development without adrenal insufficiency including hypospadias, anorchia, micropenis, complete gonadal dysgenesis, infertility in otherwise normal males, and ovarian failure in 46,XX females.
Haploinsufficiency of DMRT1 (double sex Mab3-related transcription factor gene 1)—a gene related to double sex in drosophila and Mab3 in Caenorhabditis elegans is a candidate for the abnormality in testis organogenesis in patients with 9p deletions (see Figure 14–4A). 46,XY patients invariably have the stigmata of the 9p syndrome (mental retardation, trigonocephaly, upslanting palpebral fissures, etc), as well as female or ambiguous external genitalia and Müllerian structures associated with streak gonads or dysgenetic testes. Recent data suggest that ovarian function may either be compromised or normal in 46,XX females with the 9p syndrome.
Ambiguous genitalia has also been reported in 46,XY patients with deletions of 10q. However, as yet a specific mutant gene(s) causing this DSD has not been identified on the 10q region.
Genes Involved in Testicular Determination
Opposing gene signals (differential activation) determines fate of primordial gonad as a testis or ovary. RSPO1, respondin 1; WNT4, human homolog of Drosophila wingless gene; FOXL-2, forkhead box L2; β-catenin, key-regulated effector of the WNT-signaling pathway. DAX-1 (DSS-AHC-critical region on the X chromosome gene 1) RSPO1 activates the WNT4/β-catenin canonical-signaling pathway which inhibits SOX9 expression and promotes ovarian differentiation. SRY upregulates SOX9 by binding to testicular enhancing elements in the SOX9 gene (TESCO), and both SRY and SOX9 (in mice) inhibit the β-catenin canonical-signaling pathway and promote Sertoli cell and consequent testicular development. A feed-forward loop involving SF-1 and FGF-9 has been demonstrated in mice which maintains SOX9 expression. FOXL2 "knockout" in mice causes gonadal sex reversal, i.e., ovary to testes suggesting that FOXL2 inhibits SOX9 expression. No human homozygous FOXL2 null mutations have been reported as yet. Duplication of DAX1 or WNT4 results in inhibition of testicular development and atypical genitalia—dysgenetic 46,XY DSD. MAP3K4, mitogen-activated protein kinase 4; CBX2, chromobox homologue 2. It has been suggested that CBX2 is upstream of SRY. More complete understanding of the exact function of MAP3K4 remains to be elucidated.
In studies of 46,XX males with very small Y-to-X translocations, a gene was localized to the region just proximal to the pseudoautosomal boundary of the Y chromosome (Yp11.3) (see Figure 14–3) which has been named SRY. Deletions or mutations of the human SRY gene occur in about 15% to 20% of 46,XY females with complete XY gonadal dysgenesis and 90% of 46,XX males have a Y to X translocation which includes the SRY gene. Compelling evidence that SRY is the testis-determining factor is the observation that transfection of the Sry gene into 46,XX mouse embryos results in transgenic 46,XX mice with testes and male sex differentiation.
The SRY gene encodes a DNA-binding protein that has an 80 amino acid domain similar to that found in high-mobility group (HMG) proteins. This domain binds to DNA in a sequence-specific manner (A/TAACAAT). It bends the DNA and thus is thought to facilitate interaction between DNA-bound proteins to affect the transcription of downstream genes. The human SRY gene has no intron; two nuclear localization sites, calmodulin and importin B are critical for the function of the gene. In mice, Sry facilitates the upregulation of Sox9 by binding to multiple enhancing elements in the Sox9 gene and along with Sf-1 establishes a feed-forward pathway resulting in upregulation of Sox9 and its continued expression in Sertoli cells. A feed–forward loop involving Sox9 and Fgf9 has also been demonstrated in the mouse but not yet in the human gonad. Both Sry and Sox9 in mice appear to inhibit the Rspo1 (R-spondin 1)-Wnt4-β-catenin- FOXL2 signaling pathway, facilitating Sertoli cell induction and testicular organogenesis and inhibiting ovarian development (see Figure 14–4B). In the absence of Sry, the Wnt4-β-catenin canonical pathway is stabilized and Sox9 is suppressed which results in ovarian development. Hence, it appears that Sry and Sox9 are repressors of a major ovarian determining pathway—Rspo1-Wnt4-β-catenin—which induces ovarian development along with the downstream gene—Fox L2 and other genes yet to be defined, as well as germ cells which are essential for ovarian development (but not testicular differentiation). Duplications of DAX1 or WNT4 in humans repress testicular development presumably by preventing the repression of β-catenin on its downstream gene FOXL2 by SRY and SOX9. Ovarian determination is a genetically controlled pathway and not, as previously thought, a default pathway.
Most of the mutations thus far described in 46,XY females with gonadal dysgenesis have occurred in the nucleotides of the SRY gene encoding the DNA-binding region (the HMG box) of the SRY protein. Mutations that affect DNA bending as well as nuclear transportation of the SRY protein have also been implicated in defective testicular development.
SOX9 has an HMG box that is more than 60% homologous to that of SRY. It is localized to 17q24.3-q25.1 and is expressed in the developing sex cords and, thereafter, in the Sertoli cells (see Figure 14–4B). It is also expressed in cartilage. Mutations in one allele of the SOX9 gene can result in a bone abnormality called campomelic dysplasia (CMPD) and XY gonadal dysgenesis with ambiguous genitalia in affected 46,XY males. Duplication of the SOX9 gene both in humans and mice results in sex reversal in XX individuals who are SRY negative. It appears that SOX9 is the critical gene acting downstream of SRY for Sertoli cell differentiation and consequent testicular development.
Desert hedgehog (DHH), a gene that codes for a signaling molecule located on chromosome 12q13.1 in humans, is an important gene in mammalian testes organogenesis and function. Mutations in DHH have been reported in individuals with 46,XY gonadal dysgenesis.
GATA4 GATA4 is a transcription factor which interacts with other proteins including SF-1 and FOG2 to regulate the expression of SRY. Recently, a heterozygous missense mutation has been described affecting three male siblings and resulting in 46,XY DSD and congenital heart disease.
Genes Involved in Ovarian Determination
A mutation or deletion of DAX1, which encodes a transcription factor, results in X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism in 46,XY males (see Figures 14–2, 14–4B). Deletion or mutation of the DAX1 gene in 46,XY individuals has not resulted in an abnormality of testicular differentiation in humans. However, XY gonadal dysgenesis has been reported in individuals with duplications of Xp21, a locus that contains the DAX1 gene on the X chromosome. Duplication of the DAX1 gene appears not to affect ovarian morphogenesis and function in 46,XX females. Thus, DAX1 dosage plays a critical role in testicular development and function (see Figure 14–4B).
WNT4 is a signaling molecule encoded by a gene located on the short arm of chromosome region 1 (1p35). Duplication of WNT4 has been associated with dysgenetic testicular development and ambiguous genitalia in 46,XY, SRY, positive individuals. Testis development is inhibited likely as a result of upregulation of DAX1 and stabilization of β-catenin-FOXL2 which would inhibit SOX9 expression in the developing gonad. 46,XY patients who have duplications of WNT4 exhibit a heterogenous phenotype varying from cryptorchidism alone to female external genitalia. WNT4 is expressed in the primordial ovary and is an essential signal for ovarian determination (see Figure 14–4B). In mice, Wnt4 expression in the ovary prevents the migration of steroid (androgen)-producing cells into the ovary, restrains the development of a testis-specific blood vessel (the coelomic vessel), and is critical for the development of the Müllerian ducts and the maintenance of germ cells.
A 46,XX female with absent Müllerian structures (atypical Mayer-Rokitansky-Küster-Hauser syndrome) has been reported who had a loss of function mutation in the WNT4 gene. Her phenotype was similar to that described in Wnt4-mutated mice. She had unilateral renal agenesis as well as clinical signs of androgen excess as manifested by severe acne requiring antiandrogen therapy. Wolffian duct development was not ascertained. Two other similar patients have been reported.
RSPO1 (R-spondin 1), a gene located in 1p34.3, appears to stabilize the expression of WNT4-β-catenin-FOXL2 and thus promotes ovarian determination (see Figure 14–4B). Mutations in RSPO1 cause 46,XX females to develop testes and male sexual development, as well as palmoplantar hyperkeratosis and a predisposition to squamous cell carcinoma of the skin. The fact that a mutation in RSPO1 can redirect gonadal development in a 46,XX individual from an ovary to a testes suggests that it is a critical determinant in ovarian determination in humans.
FOXL2 is a putative forkhead transcription factor, which does not cause a DSD in human. Heterozygous FOXL2 mutations in affected 46,XX human females causes premature ovarian failure with or without blepharophimosis. However, induced FoxL2 deletions in the ovary of the mouse lead to transdifferentiation of the ovary into testes.
A 46,XY phenotypic female with normal ovaries and a mutation in the CBX2 (chromobox homolog 2) gene, the human homolog of mouse m33, an ortholog of the Drosophila Polycomb gene, has recently been reported. It was postulated that this gene is upstream of SRY and that mutations in this gene prevent the repression of the ovarian gene pathway presumably by decreasing SRY/SOX9 expression enabling the expression of RSPO1-WNT4-β-catenin-FOXL2 and other ovarian determining genes.
Our lack of complete understanding of ovarian and testicular determination is illustrated in the study by Dumic et al who reported a familial cohort of 46,XY females. A 46,XY female was reported to have undergone spontaneous menarche and given birth to a 46,XY daughter with gonadal dysgenesis. Studies in this cohort have uncovered a mutation in a gene not previously known to be involved in the sex determination cascade—mitogen-activated protein kinase kinase kinase 4 (MAP3K4). Mutations in this gene involved in the mitogen-activated protein kinase signaling pathway cause a striking decrease in the expression of Sry and Sox9 in the developing mouse gonad resulting in sex reversal. Recently, mutations in MAP3K1 have been reported to 46,XY DSD leading to either partial or complete gonadal dysgenesis. Further studies in humans will be necessary to elucidate the exact mechanism which allows for repression of testicular development and the genesis of an apparently normal functioning ovary in SRY positive 46,XY individuals.