Testis determining factor
|Sex determining region Y|
PDB rendering based on 1hry.
|RNA expression pattern|
Testis-determining factor (TDF), also known as Sex-determining region Y (SRY) protein, is a DNA-binding protein encoded by the SRY gene that is responsible for the initiation of male sex determination in humans. SRY is an intronless sex-determining gene on the Y chromosome in the therians (placental mammals and marsupials)  and mutations in this gene lead to a range of sex-related disorders with varying effects on an individual's phenotype and genotype. TDF is a member of the SOX (SRY-like box) gene family of DNA-binding proteins. When complexed with the SF1 protein, TDF acts as a transcription factor that can upregulate other transcription factors, most importantly SOX9. Its expression causes the development of primary sex cords, which later develop into seminiferous tubules. These cords form in the central part of the yet-undifferentiated gonad, turning it into a testis. The now induced Leydig cells of the testis then start secreting testosterone while the Sertoli cells produce anti-Mullerian hormone.
Gene evolution and regulation
SRY may have arisen from a gene duplication of the X chromosome bound gene SOX3, a member of the Sox family. This duplication occurred after the split between monotremes and therians. Monotremes lack SRY and have a ZW-like sex determination system, likely involving DMRT1, whereas therians (marsupials and placental mammals) use the XY sex determination system. SRY is a rapidly evolving gene and its regulation has been difficult to study because sex determination is not a highly conserved phenomenon within the animal kingdom. 
SRY gene has little in common with sex determination genes of other model organisms, and mice are the main model research organisms that can be utilized for its study . Understanding its regulation is further complicated because even between mammlian species, there is little protein sequence conservation. The only conserved group between mice and mammals is the High-mobility Group (HMG) box region that is responsible for DNA binding. Mutations in this region result in sex reversal, where the opposite sex is produced. Because there is little conservation, the SRY promoter, regulatory elements and regulation are not well understood. Within related mammalian groups there are homologies within the first 400-600 base pairs upstream from the translational start site. In vitro studies of human SRY promoter has shown that a region of at least 310 bp upstream to translational start site are required for SRY promoter function. It's been shown that binding of three transcription factors, Steroidogenic factor 1 (SF1), Specificity Protein 1(Sp1 transcription factor) and Wilms tumor protein 1 (WT1), to the human promoter sequence, influence expression of SRY.
The promoter region has two Sp1 binding sites, at -150 and -13 that function as regulatory sites. Sp1 is a transcription factor that binds GC-rich consensus sequences, and mutation of the SRY binding sites leads to a 90% reduction in gene transcription. Studies of SF1 have resulted in less definite results . Mutations of SF1 can lead to sex reversal and deletion lead to incomplete gonad development. However, it's not clear how SF1 interacts with the SR1 promoter directly. The promoter region also has two WT1 binding sites at -78 and -87 bp from the ATG codon. WT1 is transcription factor that has four C-terminal Zinc fingers and an N-terminal Pro/Glu-rich region and primarily functions as an activator. Mutation of the Zinc fingers or inactivation of WT1 results in reduced male gonad size. Deletion of the gene resulted in complete sex reversal. It is not clear how WT1 functions to up-regulate SRY, but some research suggests that it helps stabilize message processing. However, there are complications to this hypothesis, because WT1 also is responsible for expression of an antagonist of male development, DAX1, which stands for Dosage-sensitive sex reversal, Adrenal hypoplasia critical region, on chromosome X, gene 1 . An additional copy of DAX1 in mice leads to sex reversal. It is not clear how DAX1 functions, and many different pathways have been suggested, including SRY transcriptional destabilization and RNA binding. There is evidence from work on suppression of male development that DAX1 can interfere with function of SF1, and in turn transcription of SRY by recruiting corepressors.
There is also evidence that GATA binding protein 4 (GATA4)and FOG2 contribute to activation of SRY by associating with its promoter. How these proteins regulate SRY transcription is not clear, but FOG2 and GATA4 mutants have significantly lower levels of SRY transcription. FOGs have zinc finger motifs that can bind DNA, but there is no evidence of FOG2 interaction with SRY. Studies suggest that FOG2 and GATA4 associate with nucleosome remodeling proteins that could lead to its activation.
During gestation, the cells of the primordial gonad that lie along the urogenital ridge are in a bipotential state, meaning they possess the ability to become either male cells (Sertoli and Leydig cells) or female cells (follicle cells and Theca cells). TDF initiates testis differentiation by activating male-specific transcription factors that allow these bipotential cells to differentiate and proliferate. TDF accomplishes this by upregulating SOX9, a transcription factor with a DNA-binding site very similar to TDF's. SOX9 leads to the upregulation of fibroblast growth factor 9 (Fgf9), which in turn leads to further upregulation of SOX9 . Once proper SOX9 levels are reached, the bipotential cells of the gonad begin to differentiate into Sertoli cells. Additionally, cells expressing TDF will continue to proliferate to form the primordial testis. While this constitutes the basic series of events, this brief review should be taken with caution since there are many more factors that influence sex differentiation.
Action in the nucleus
The TDF protein consists of three main regions. The central region encompasses the HMG (high-motiliy group) domain, which contains nuclear localization sequences and acts as the DNA-binding domain. The C-terminal domain has no conserved structure, and the N-terminal domain can be phosphorylated to enhance DNA-binding. The process begins with nuclear localization of TDF by acetylation of the nuclear localization signal regions, which allows for the binding of importin β and calmodulin to TDF, facilitating its import into the nucleus. Once in the nucleus, TDF and SF1 (steroidogenic factor 1, another transcriptional regulator) complex and bind to TESCO (testis-specific enhancer of Sox9 core), the testes-specific enhancer element of the Sox9 gene in Sertoli cell precursors, located upstream of the Sox9 gene transcription start site. Specifically, it is the HMG region of TDF that binds to the minor groove of the DNA target sequence, causing the DNA to bend and unwind. The establishment of this particular DNA “architecture” facilitates the transcription of the Sox9 gene. SOX9 protein then initiates a positive feedback loop, involving SOX9 acting as its own transcription factor and resulting in the synthesis of large amounts of SOX9.
SOX9 and testes differentiation
The SF1 protein, on its own, leads to minimal transcription of the SOX9 gene in both the XX and XY bipotential gonadal cells along the urogenital ridge. However, binding of the TDF-SF1 complex to the testis-specific enhancer (TESCO) on SOX9 leads to significant up-regulation of the gene in only the XY gonad, while transcription in the XX gonad remains negligible. Part of this up-regulation is accomplished by SOX9 itself through a positive feedback loop; like TDF, SOX9 complexes with SF1 and binds to the TESCO enhancer, leading to further expression of SOX9 in the XY gonad. Two other proteins, FGF9 (fibroblast growth factor 9) and PDG2 (prostaglandin D2), also maintain this up-regulation. Although their exact pathways are not fully understood, they have been proven to be essential for the continued expression of SOX9 at the levels necessary for testes development.
SOX9 and TDF are believed to be responsible for the cell-autonomous differentiation of supporting cell precursors in the gonads into Sertoli cells, the beginning of testes development. These initial Sertoli cells, in the center of the gonad, are hypothesized to be the starting point for a wave of FGF9 that spreads throughout the developing XY gonad, leading to further differentiation of Sertoli cells via the up-regulation of SOX9. SOX9 and TDF are also believed to be responsible for many of the later processes of testis development (such as Leydig cell differentiation, sex cord formation, and formation of testis-specific vasculature), although exact mechanisms remain unclear. It has been shown, however, that SOX9, in the presence of PDG2, acts directly on Amh (encoding anti-Müllerian hormone) and is capable of inducing testis formation in XX mice gonads, indicating its vital to testes development.
Influence on sex
Embryos are gonadally identical, regardless of genetic sex, until a certain point in development when the testis-determining factor causes male sex organs to develop. Therefore, SRY plays an important role in sex determination. A typical male karyotype is XY. Individuals who inherit a normal Y chromosome and multiple X chromosomes are generally male (such as in Klinefelter Syndrome, which has an XXY karyotype). A genetic recombination event known as crossing over can result in karyotypes that do not match their phenotypic expression.
Crossing over during paternal meiosis prior to conception can cause SRY to be transferred from the Y chromosome to the X chromosome. The Y chromosome that results from this crossover is now lacking an SRY gene, and can no longer initiate testis development. When this chromosome is inherited from the father, the resulting offspring will have Swyer syndrome, characterized by a male karyotype (XY) and a female phenotype. The X chromosome that results from this crossover event now has a SRY gene, and therefore the ability to initiate testis development. Offspring who inherit this chromosome will have a condition called XX male syndrome, characterized by an XX karyotype, and a male phenotype. While most XX males develop phenotypically as males, is it possible for them to experience incomplete differentiation of the testis, resulting in the formation of both testicular and ovarian in the same individual. This true sterile hermaphroditism (not to be confused with the hermaphroditism of hermaphroditic species and taxa such as earthworms, as they can reproduce) is most likely caused by the inactivation (either random or non-random) of the X chromosome containing the SRY in some cells.
While the presence or absence of SRY has generally determined whether or not testis development occurs, it has been suggested that there are other factors that affect the functionality of SRY. Therefore, there are individuals who have the SRY gene, but still develop as females, either because the gene itself is defective or mutated, or because one of the contributing factors is defective. This can happen in individuals exhibiting a XY, XXY, or XX SRY-positive karyotype.
Role in other diseases
SRY has been shown to interact with the androgen receptor and individuals with XY karyotype and a functional SRY gene can have an outwardly female phenotype due to an underlying androgen insensitivity syndrome (AIS). Individuals with AIS are unable to respond to androgens properly due to a defect in their androgen receptor gene, and affected individuals can have complete or partial AIS. SRY has also been linked to the fact that males are more likely than females to develop dopamine-related diseases such as schizophrenia and Parkinson's disease. SRY encodes a protein that controls the concentration of dopamine, the neurotransmitter that carries signals from the brain that control movement and coordination.
Use in Olympic screening
One of the most controversial uses of this discovery was as a means for gender verification at the Olympic Games, under a system implemented by the International Olympic Committee in 1992. Athletes with an SRY gene were not permitted to participate as females, although all athletes in whom this was "detected" at the 1996 Summer Olympics were ruled false positives and were not disqualified. Specifically, eight female participants (out of a total of 3387) at these games were found to have the SRY gene. However, after further investigation of their genetic conditions, all these athletes were verified as female and allowed to compete. These athletes were found to have either partial or full androgen insensitivity, despite having an SRY gene, making them phenotypically female and giving them no advantage over other female competitors. In the late 1990s, a number of relevant professional societies in United States called for elimination of gender verification, including the American Medical Association, stating that the method used was uncertain and ineffective. The screening was eliminated as of the 2000 Summer Olympics. Since this method of testing was eliminated there has been no form of Olympic gender screening that all athletes must undergo. Rather, questions regarding an athlete's gender are now addressed on an individual basis by medical experts, only when there is significant reason to suspect a male athlete may be posing as a female. However, there have been few, if any, instances of such suspicion and evaluation since the system of screening was eliminated, due to the difficulties and impracticality of a male successfully disguising himself as a female athlete (such as the nature of women's event uniforms, which make concealment of a male's body near impossible).
Despite the progress made during the past several decades in the study of sex determination, the Sry gene, and the TDF protein, work is still being done to further our understanding in these areas. There remain factors that need to be identified in the sex-determining molecular network, and the chromosomal changes involved in many other human sex-reversal cases are still unknown. Scientists continue to search for additional sex-determining genes, using techniques such as microarray screening of the genital ridge genes at varying developmental stages, mutagenesis screens in mice for sex-reversal phenotypes, and identifying the genes that transcription factors act on using chromatin immunoprecipitation.
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- GeneReviews/NCBI/NIH/UW entry on 46,XX Testicular Disorder of Sex Development
- OMIM entries on 46,XX Testicular Disorder of Sex Development
- Genes, sry at the US National Library of Medicine Medical Subject Headings (MeSH)
- Sex-Determining Region Y Protein at the US National Library of Medicine Medical Subject Headings (MeSH)