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In human, the SRY gene is located on short (p) arm of the
Y chromosome at position 11.2
Testis-determining factor (
TDF), also known as
sex-determining region Y (
SRY)
protein, is a
DNA-binding
protein (also known as gene-regulatory protein/transcription factor) encoded by the
SRY gene that is responsible for the initiation of male sex determination in humans.
[3] SRY is an
intronless sex-determining
gene on the
Y chromosome in
therians (placental mammals and marsupials);
[4] 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.
[5] 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-Müllerian hormone.
[6]
SRY gene effects normally take place 6–8 weeks after foetus formation
and inhibits the female anatomical structural growth in males. It also
works towards developing the dominant male characteristics.
Gene evolution and regulation
Evolution
SRY may have arisen from a
gene duplication of the X chromosome bound gene
SOX3, a member of the
Sox family.
[7] This duplication occurred after the split between
monotremes and
therians. Monotremes lack SRY and some of their sex chromosomes share homology with bird sex chromosomes.
[8] SRY
is a quickly evolving gene and its regulation has been difficult to
study because sex determination is not a highly conserved phenomenon
within the animal kingdom.
[9]
Regulation
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 mammalian species, there is little
protein sequence conservation. The only conserved group between mice and
other 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.
[10] 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 have 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.
[10]
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.
[11] 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.
[11] 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.
[10]
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.
[12] 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.
[13]
Function
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-mobility 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.
[11] 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.
[5]
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.
[11] 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.
[11]
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.
[5]
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.
[14]
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.
[15]
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.
[14]
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). Atypical
genetic recombination during
crossover when a sperm cell is developing can result in karyotypes that do not match their phenotypic expression.
Most of the time, when a developing sperm cell undergoes crossover
during its meiosis, the SRY gene stays on the Y chromosome. If it is
transferred to the X chromosome, however, the resulting Y chromosome
will not have an SRY gene and can no longer initiate testis development.
Offspring which inherit this Y chromosome will have
Swyer syndrome,
characterized by an XY karyotype 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 X chromosome will have a condition called
XX male syndrome,
characterized by an XX karyotype, and a male phenotype. While most XX
males develop testis, it is possible for them to experience incomplete
differentiation resulting in the formation of both testicular and
ovarian tissues in the same individual. XX male syndrome results in
infertility, most likely caused by the inactivation (either random or
non-random) of the X chromosome containing the SRY in some cells.
[16]
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.
[17] 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.
[18] 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).
[19]
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.
[20] 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.
[21]
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.
[22]
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.
[23] Chromosomal screening was eliminated as of the
2000 Summer Olympics,
[23][24][25] but this was later followed by other forms of testing based on hormone levels.
Ongoing research
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.
[11]
