Sry, more than testis determination?
Monte E. Turner, Daniel Ely, Jeremy Prokop and Amy Milsted
Am J Physiol Regul Integr Comp Physiol 301:R561-R571, 2011. First published 15 June 2011;
doi:10.1152/ajpregu.00645.2010
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Am J Physiol Regul Integr Comp Physiol 301: R561–R571, 2011.
First published June 15, 2011; doi:10.1152/ajpregu.00645.2010.
SPECIAL TOPIC
Perspectives
Water and Electrolyte Homeostasis Section Invited Reviews
Sry, more than testis determination?
Monte E. Turner, Daniel Ely, Jeremy Prokop, and Amy Milsted
Department of Biology, The University of Akron, Akron, Ohio
Submitted 24 September 2010; accepted in final form 1 June 2011
SRY; sexual dimorphism; SOX; nervous system
many genes and gene products have
pleiotropic effects, yet for some loci, a known essential function hides this reality. A newly derived function may distract
attention from ancestral functions. In physiology, hormones
have many more functions than that of their original functional
discovery. The Sry locus on the Y chromosome is responsible
for testis determination in placental mammals. The essential
biological importances of sex determination and difficulties in
describing the mechanism have overshadowed the potential of
Sry functions not related to testis determination. In addition,
the prevailing ideas of mammalian sex determination rely on
the ovaries/testes producing estrogens/androgens for the female/male phenotypes. Mammalian sex is determined at the
organismal level instead of the cellular level as in many
invertebrates. The mammalian Sry locus is expressed in many
adult male tissues. Is it possible that this expression acts as a
type of cellular sex determination that then impacts some
male/female differences in mammals? We will examine data
and theory supporting the idea that Sry has other functional
phenotypic effects not involved in the process of testis determination, and these may cause differentiation of male and
female cells or phenotypes.
IT IS WELL UNDERSTOOD THAT
Address for reprint requests and other correspondence: M. E. Turner,
Dept. of Biology, The Univ. of Akron, Akron, OH 44325-3908 (e-mail:
meturner@uakron.edu).
http://www.ajpregu.org
We will not address Sry and testis determination, which has
been summarized elsewhere (100). We will examine the constraints on Sry as a Y chromosome and testis-determining
locus, a SOXB locus, and data on Sry expression in tissues and
times not related to testis determination. What is the evidence
that Sry may have functions in addition to testis determination?
To illustrate the potential of Sry expression in males, we will
use examples of Sry effects on 1) the peripheral nervous system
and neuroendocrine regulation and 2) the central nervous
system.
Although there are different genetic designations for any
particular locus dependent on the organism in which it is
found, throughout this review, we will use Sry to refer to the
locus and SRY to refer to the protein of the Sry locus.
Sry Background
The Sry locus (sex-determining region on the Y chromosome) was the first indentified member of the SOX transcription factor family (86). All of these proteins contain a highmobility group (HMG) box with amino acid sequence conserved at least 46% and a highly conserved hinge region (5).
The HMG box of SRY and SOX proteins recognize a consensus DNA sequence AACAAT, binding in the minor groove of
the DNA (36, 43). Through intercalation of the protein with the
base pairs at the consensus binding site, the major groove is
condensed and minor groove opened, thus inducing a bend in
the DNA. For cases in which a bend already exists, binding of
SRY is enhanced in nonconsensus-binding sequences (26). The
hinge domain is also highly conserved in all SOX members and
is important for nuclear localization (37). Outside the HMG
box and hinge region, Sry shares little homology with other
SOX family members (5).
Sry is a member of the SOXB family of SOX loci, which
includes Sox1, Sox2, Sox3, and Sry. Sox1 and Sox2 are autosomal in most mammals, and Sox3 is X-linked. It is believed
that Sry originated from a mutation of Sox3 on the then
autosomal primordial mammalian X chromosome, leading to
the advent of the mammalian Y chromosome (81). The amount
of HMG amino acid sequence homology between SRY and
other SOX family members supports this theory, with Sox3
having the highest amino acid sequence conservation. Some
regions outside of the HMG box and hinge regions have been
suggested to play a role in SRY-specific function (79). There is
a slight amount of amino acid sequence conservation across
SRY in multiple species in the bridge domain. This bridge
domain has been shown to interact with KRAB-O (64), and
there is no indication that this conserved KRAB-O domain is
involved in testis determination. The conservation in the SRYspecific bridge domain could be involved in long-term transcriptional regulation. Only members of the SOXB group have
0363-6119/11 Copyright © 2011 the American Physiological Society
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Turner ME, Ely D, Prokop J, Milsted A. Sry, more than testis determination? Am J Physiol Regul Integr Comp Physiol 301: R561–R571, 2011.
First published June 15, 2011; doi:10.1152/ajpregu.00645.2010.—The Sry
locus on the mammalian Y chromosome is the developmental switch
responsible for testis determination. Inconsistent with this important
function, the Sry locus is transcribed in adult males at times and in
tissues not involved with testis determination. Sry is expressed in
multiple tissues of the peripheral and central nervous system. Sry is
derived from Sox3 and is similar to other SOXB family loci. The
SOXB loci are responsible for nervous system development. Sry has
been demonstrated to modulate the catecholamine pathway, so it
should have functional consequences in the central and peripheral
nervous system. The nervous system expression and potential function
are consistent with Sry as a SOXB family member. In mammals, Sox3
is X-linked and undergoes dosage compensation in females. The
expression of Sry in adult males allows for a type of sexual differentiation independent of circulating gonadal hormones. A quantitative
difference in Sox3 plus Sry expression in males vs. females could
drive changes in the transcriptome of these cells, differentiating male
and female cells. Sry expression and its transcriptional effects should
be considered when investigating sexual dimorphic phenotypes.
Perspectives
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Sry, MORE THAN TESTIS DETERMINATION?
several amino acids conserved in this domain and may explain
similar functions of SRY and SOXB members. A recent study
by Sato et al. (81) reanalyzed the sequence of the human Sry
gene by searching for homologous DNA sequences. They
concluded that the gene evolved as a hybrid resulting from
insertions of a part of the DGCR8 gene, which encodes DiGeorge syndrome critical region 8 and is involved in biogenesis of microRNA (34), into a region 5= of the HMG box of an
ancestral Sox3 gene.
Sry as a Y Chromosome Locus
AJP-Regul Integr Comp Physiol • VOL
Multiple Copies of Sry on a Single Y Chromosome
In some rodent species, Sry has multiple copies on a single
Y chromosome (60). Multiple copies have been demonstrated
in family Muridae, including African rodents (53, 60), voles
(6), and rats (90). These are true duplicate loci with independent control and coding regions. The exact spatial relationship
between any of these loci is unknown, as none of these Y
chromosomes have been sequenced. In the rat, indirect evidence indicates the loci are not close to each other, as sequenced BACs only contain a single copy. The finding of
copies in many related species indicates these copies are either
very old or their location on the Y chromosome causes duplications to occur at an increased rate. The presence of many
copies in these rodents does not seem to have any obvious
deleterious effects. In voles, the multiple copies are obviously
not functional due to nonsense/frameshift mutations in the
coding region (7), but for these mutations to have occurred and
accumulated, the duplicate Sry loci must be very old.
In other examples, the coding regions do not contain mutations that would lead to nonfunctional proteins or evidence of
Sry pseudogenes (53, 90, 91). Expression patterns have only
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The constraints on mutations and evolution of Sry are
different from an autosomal locus. Sry is located on the
mammalian Y chromosome and is thus constrained by this
location. The Y acts as homologous chromosome with the X
chromosome in meiosis but does not recombine except at
specific regions (pseudoautosomal region). This results in the
majority of the Y chromosome being inherited as a single
locus. The classic view of the Y chromosome is that of a
chromosome that accumulates mutations with selection eliminating Y chromosomes with mutations that have lost essential
functions. A modern version of the Y chromosome includes the
observation that certain regions of the Y chromosome contain
almost identical repeats that have high frequencies of gene
conversion that can “fix” mutations that occur (77).
The human Y chromosome is composed of three types of
sequences: ampliconic, X-transposed, and X-degenerate (87).
The ampliconic regions contain large repeat units in which
gene conversion and homologous recombination occur within
the repeat regions (74). The X-degenerate and X-transposed
regions have origins from ancestral autosomes (X-degenerate)
and more recent transpositions from the X chromosome (Xtransposed) (48). Both the X-degenerate and X-transposed
regions contain primarily single-copy genes, which should
evolve like the classic view of the Y chromosome. The Sry
locus is within the X-degenerate region of the human Y
chromosome. Structural mutations in individual Y chromosomes may have moved the Sry locus into different regions of
the chromosomes in other mammalian species. The location of
Sry in different regions of the Y chromosome may affect the
evolution of the Sry locus in these species.
Because Sry is located in the X-degenerate region of the
human Y chromosome, any mutation not affecting function of
the locus should accumulate among human Y chromosomes.
We might expect the accumulation of synonymous mutations
in the coding region, nonsynonymous mutations outside of the
HMG-box region not affecting DNA binding, and mutations in
the flanking regions not involved in transcriptional control or
stability. The available data demonstrate that the X-degenerate
and X-transposed regions have reduced levels of genetic diversity compared with autosomal regions (78). Consistent with
this observation, the effective population size of a Y chromosome is only ¼ that of an autosomal chromosome, thus reducing the opportunity for mutations.
In a study of the coding regions of genes from 105 human Y
chromosomes, selected to examine the range of human Y
chromosomes, nucleotide diversity was significantly lower for
nonsynonymous sites than synonymous sites, pseudogenes,
or introns, and this is consistent with natural selection
maintaining these gene sequences. It is possible that Sry is
the force driving this purifying selection as any mutation
that eliminates function in an essential developmental
switch will be immediately lost from the population. The
sequence variation in Sry is among the lowest on the Y
chromosome, with only one substitution (synonymous)
found in the coding region of Sry (78).
To examine the variation outside of the coding region, we
can compare the Sry locus in the male personal genomes that
have been published and available. The genomes of KB1 (83),
NB1 (83), ABT (83), George Church (16), Henry Louis Gates
Sr. (16), J. Craig Venter (50), James Watson (97), YRI (17),
Han Chinese Individual (94), Seong-Jin Kim (1), Anonymous
Korean individual (42), Stephen Quake (70), and the 4,000
year old Saqqaq (72) were viewed using the PSU Genome
Browser (http://main.genome-browser.bx.psu.edu/). Only SeongJin Kim genome has an single nucleotide polymorphism (SNP;
synonymous) within the coding region of Sry (chrY:
2,714,896 –2,715,792, numbering from NCBI36/hg18). Looking at 4 kB on each side flanking the Sry-coding sequence
(chrY: 2,710,859 –2,719,828) reveals only four additional
SNPs, but none shared among multiple sequences. Of interest
is that the Saqqaq sequence reveals no SNPs over this stretch
of the Y chromosome, suggesting that the sequence in this
region has remained stable over the past 4,000 years (85). The
Sry flanking region shows the same reduction in variation
consistent with results using only the coding region (78).
Other than the comparison using these genomes, there is no
survey of normal variation in the Sry locus. A study of gonadal
dysgenesis can only identify mutations that disrupt testis determination, but these are lost from the population because the
phenotype is sterile (31). These results from a comparison of
different human Sry sequences, coding region (78) and flanking regions, and coding region of both modern and ancient
genomes (Saqqaq), indicate that the gene does not have the
necessary polymorphism levels to answer either questions of
additional function or to determine sequences necessary for
testis determination using conserved regions because the whole
region seems to be highly conserved.
Perspectives
Sry, MORE THAN TESTIS DETERMINATION?
Sry as a Member of the SOXB Family
In the human and mouse genomes there are 20 members of
the SOX gene family (82), many of which have known roles in
disease etiology. Focusing on the SOXB family (Sox1, Sox2,
and Sox3), which is most similar to Sry, all members are
important for stem-cell maintenance in the central nervous
system (95), and Sox2 and Sox3 are important in pituitary
development (2). All three are coexpressed in both avian and
murine neural progenitor cells and the adult brain (8, 102). Null
mutations of Sox1, Sox2, and Sox3 have different neuronal
phenotypes, indicating differentiation of function (18, 28, 76).
Lost in most discussions of Sry and testis determination is the
idea that Sry was derived from Sox3, and testis determination
is a new derived function rather than the ancestral central
nervous system function (33).
Sox3 is expressed in neural progenitor cells, and both Sox3
and Sry are expressed in adult testis (96). Consistent with Sox3
derivation, Sry expression in the adult testis is an ancestral
condition, not derived. Sox3 deletions in mice have abnormal
development of diencephalon and anterior pituitary gland and
lack differentiated spermatogonia (73). Sox3 has been speculated to activate Sox9 and induce sex reversal in XX transgenic
mice, providing a similar outcome as Sry expression (4, 51,
92). If Sry and Sox3 can behave similarly in testis development,
can they also behave similarly in the brain?
Sry is expressed in the substantia nigra of tyrosine hydroxylase (Th)-expressing cells (15) and has been demonstrated to
increase transcription of the Th promoter (56). Tyrosine hydroxylase is the rate-limiting enzyme for norepinephrine and
dopamine synthesis, suggesting that Sry has a role in malespecific brain development. Sry transcripts have been found in
an ever expanding number of tissues (Table 1), so that understanding the mechanisms by which Sry works becomes increasingly important. The expression pattern of Sry in adult males is
better explained if Sry is examined as a SOXB-derived locus
rather than primarily as a testis determination locus. Does Sry
function similarly to other members of the SOXB family or has
it specialized through alterations in preferred DNA binding
sequences or protein binding partners?
Since Sox3 is an X-linked locus, it is subject to X inactivation. Therefore, mammalian females have SOX3 from one X
AJP-Regul Integr Comp Physiol • VOL
chromosome expression, while males have SOX3 plus SRY in
tissues, where Sry is expressed. Any quantitative relationships
would be enhanced in males over females. This difference is
independent of steroid hormones. Expression of Sry in males
adds a level of cellular sex determination and differentiation
independent of gonads and hormones. The interaction of SRY
with the androgen receptor could further potentiate these affects in male cells vs. female cells (104). This mechanism is
potentially present in the brain and nervous system, both
during development and in adult males due to the expression of
Sry in these tissues and cells.
Sry Expression
Table 1 lists published results and unpublished results from
our laboratory of Sry expression at times and in tissues not
related to testis determination. The number of tissues where
expression has been identified and the conservation of expression in many species are not the expectation or prediction for
a gene involved only in testis determination. Any mutation
eliminating unnecessary expression would be advantageous,
and the expectation would be random loss of expression in
a single species and decreased expression phylogenetically
over evolutionary time in a Muller’s ratchet type of expectation (59).
In SHR and WKY rat strains, we have examined the expression in numerous tissues of the six or seven duplicated copies
of the Sry locus (Table 1) (90). Multiple Sry loci are expressed
in kidney (23, 90), adrenal gland, brain, and other tissues in
young and adult male rats (Table 1) (56, 90, 91). We have used
Sry-specific primers to amplify cDNA from many rat tissues
(Table 1) and find the presence of Sry in tissues tested and no
amplification in no-RT controls or females. Many of these rat
Sry loci are expressed, in patterns that are tissue- and locusdependent. Again, the pattern is one expected with function,
rather than random loss, or the same expression pattern, for all
duplicated loci.
Sry expression is found in many different tissues and times
in addition to the genital ridge at the time of testis determination. For instance, Dewing et al. (15) identified Sry expression
in situ in the rat brain using an antisense Sry probe and SRY
protein with a anti-SRY antibody. Modi et al. (58) identified
Sry mRNA in situ in human adult male testis with an antisense
probe. These results are significant because Sry expression is
not seen in expressed sequence tag (EST) libraries of these
tissues. In fact, Sry expression is missing from all EST library
sequences. If there is enough expression for in situ identification, one would expect multiple EST sequences for Sry from
these and other tissues. This lack of ESTs for Sry is not species
specific but is deficient in all libraries, even from species and
tissues where Sry expression has been identified from methods
that require a much larger number of transcripts. The only
conserved sequence among these species is the HMG box,
hinge region, and bridge domain of the gene; thus, these
regions must have a sequence or secondary structure that
inhibits cloning under standard conditions. Because we are
able to amplify the entire rat Sry sequences in cDNA made
from mRNA, the lack of Sry in EST libraries is probably a
result of inefficient cloning rather than in the process of
creating cDNA.
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been examined in Rattus norvegicus strains, and the multiple
copies on the rat Y chromosome show divergent expression
patterns (90), consistent with either gain or loss of transcriptional components. In addition to transcriptional divergence
these Rattus norvegicus Sry loci have slightly different protein
phenotypes and have potential diverse effects in the adult male
rat.
The occurrence of multiple Sry copies is probably a result of
the repeat organization of the mammalian Y chromosome
leading to many species-specific duplications and deletions.
Multiple Sry copies have been identified in human males
exposed to high background radiation but not in normal control
males (68, 69). The question of how common are multiple
copies of Sry on a single Y chromosome remains largely
unanswered because experiments to identify whether multiple
copies exist have either not been tested or not enough human
Y chromosomes have been sequenced to characterize a population.
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Perspectives
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Sry, MORE THAN TESTIS DETERMINATION?
Table 1. Sry expression in mammalian tissues and method of detection
Tissue
Adrenal gland
Liver
Pancreas
Skeletal muscle
Small intestine
Spleen
Superior cervical ganglia
Thymus
Testis
Gonadal ridge
Preimplantation embryo
Male embryo
Preimplantation embryo
Detection Method
Reference
rat (A)
human (F)
real-time RT-PCR, gel-based
gel-based RT-PCR
56, 90, 91, unpublished data
11
rat (A)
human (F)
rat (A)
rat (A)
rat (A)
mouse (A)
mouse (A)
rat (A)
rat (A)
rat (A)
mouse (A)
rat (A)
rat (A)
rat (A)
rat (A)
rat (A)
real-time RT-PCR
gel-based RT-PCR
real-time RT-PCR
gel-based and real-time RT-PCR
in situ hybridization
gel-based RT-PCR
gel-based RT-PCR
real-time RT-PCR
gel-based PCR
in situ hybridization
gel-based RT-PCR
gel-based PCR
in situ hybridization (ISH)
immunohistochemisty (protein)
gel-based PCR
real-time RT-PCR
unpublished
11
unpublished
unpublished
15
55
55.1
unpublished
56
15
55
56
15
15
56
unpublished
rat
human
real-time RT-PCR
gel-based RT-PCR
gel-based PCR
unpublished data
11
11
rat (A)
rat (A)
human (A)
human (F)
rat (A)
rat (A)
human (A)
rat (A)
human (A)
human (F)
human (F)
rat (A)
human (F)
human (F)
rat (A)
human (F)
rat (A)
bovine (A)
mouse (A)
mouse (A)
human (A)
human (A)
mouse (F)
mouse (F)
porcine (F)
human (F)
real-time RT-PCR
real-time RT-PCR
gel-based RT-PCR
gel-based RT-PCR
real-time RT-PCR
real-time RT-PCR
gel-based RT-PCR
real-time RT-PCR
gel-based RT-PCR
gel-based RT-PCR
gel-based RT-PCR
real-time RT-PCR
gel-based RT-PCR
gel-based RT-PCR
real-time RT-PCR
gel-based RT-PCR
real-time RT-PCR
Northern blot
Northern blot, gel-based RT-PCR
Northern; nuclease protection; ISH
Northern blot
Northern; gel-based PCR
Northern; ISH; gel-based RT-PCR
Gel-based RT-PCR
Gel-based RT-PCR
Gel-based RT-PCR
unpublished data
unpublished data
11
11
unpublished data
91, unpublished data
11
unpublished data
11
11
11
unpublished data
11
11
unpub
11
90, unpublished
13
46
108
86
11
46
107
14
29
data
data
data
data
data
Unpublished data are from the spontaenously hypertensive rat and Wistar-Kyoto strains from our laboratory. Source: A, adult; F, fetal or embryonic.
From our experience with rat Sry sequences, it is obvious
that the Sry sequence does not clone with the same efficiency
as other sequences. In fact, not all of the Sry loci from a single
rat Y chromosome clone equally well. Using primers spanning
the rat Sry coding sequences, the Sry2 amplicon is 39 bp
shorter than the other sequences and can be seen as a separate
band with electrophoresis (91). Using PCR TOPO TA cloning
kit to clone the amplicons, we sequenced more than 100 clones
and found that no clones contained the Sry2 sequence, even
though it would be expected that either 1/6 or 1/7 of the clones
would be Sry2. The Sry2 sequence was eventually determined
from Sry2-specific primers and sequencing of that PCR product (91).
AJP-Regul Integr Comp Physiol • VOL
Sox Loci in the Nervous System
Since the initial discovery of Sry 20 years ago and subsequently the SOX loci, numerous studies have shown a strong
role for SOX transcription factors in the nervous system of
vertebrates and invertebrates. Invertebrates, including Drosophila (61), Amphioxus (38), and the Ptychodera flave (89)
have Sox expression in the nervous system. In all three of these
species, homology of the Sox proteins involved most closely
matches that of the SOXB1 group. All SOXB1 genes have
been shown to be expressed in the central nervous system
(CNS) of vertebrates (12). The most studied of the SOXB1
members is Sox2, which is important for nerve cell formation
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Brain
Amygdala
Brain
Brainstem
Cerebral cortex
Cortex
Cortex
Diencephalon
Hypothalamus
Locus coeruleous
Medial mammilary bodies
Midbrain
Substantia nigraSubstantia nigra
Substantia nigra
Ventral tegmental area
Celiac ganglion
Cultured cells
Neonatal astrocytes
Hep G2
NTERA-2 cl.D1
Heart
Aorta
Atrium
Heart
Heart
Ventricle
Kidney
Source (A, F)
Perspectives
Sry, MORE THAN TESTIS DETERMINATION?
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in the inner ear (63), parenchymal astroglia cell formation (45),
maintenance of neural stem cells (9), and neurogenesis in the
brain (28). Although Sry and Sox2 have similar binding affinities to the consensus binding site (12), some of the processes
in which Sox2 is involved, such as eye formation, require the
transcriptional activation domain found only on the SoxB1
genes (35). SoxB genes are not the only members of the SOX
family to have a role in the nervous system. The SOXE (Sox8,
Sox9, and Sox10) group is known to function in Schwann cell
development (47), oligodendrocyte development (88), and regulation of nicotinic ACh receptors (52). SOXC genes (Sox 4
and Sox11) are expressed in the immature neurons of neural
stem cells in mice (66). Because SRY shares a HMG box, and
thus similar DNA binding sites, with all other SOX proteins,
SRY, when expressed could participate in similar roles in
nervous system development and function in the adult male
nervous system.
Our research demonstrated that Sry has a role in the peripheral nervous system, specifically the sympathetic nervous system. TH is the rate-limiting enzyme for catecholamine biosynthesis both in the brain and periphery. Transient cotransfection
assays in PC12 cells with Sry1 and pTh/Luc(⫺272/⫹27) demonstrated that the product of the Sry1 gene increases expression
of the luciferase gene under control of the Th promoter (56).
When an AP-1 mutated site in the Th promoter was used,
luciferase activity decreased by ⬃75% compared with the
control Th promoter. This suggested that the actions of Sry in
regulating TH transcription are due to interactions at the AP-1
site (75%) and the direct effects of Sry (25%). The impact of
SRY on AP-1 transcription factors is not unexpected, given the
previously identified targets of SRY (30). The impact of Sry on
the sympathetic nervous system has a diverse potential on
multiple phenotypes in humans and other mammals, such as
the fight/flight response, blood pressure, and kidney function.
The sympathetic nervous system is not the only potential
target of Sry in the peripheral nervous system. The reninangiotensin system (RAS) is also a target of Sry regulation
(57). Recently, we have shown that the human SRY protein
also regulates rat angiotensin-converting enzyme (Ace), Ace2,
renin (Ren), and angiotensinogen (Agt) promoter activity
(Fig. 1), suggesting that Sry regulation of target genes is
similar and conserved across species. Our modeling studies
comparing human and rat SRY proteins indicate close structural conservation across these species (Fig. 2). The effects of
rat Sry on the rat RAS were investigated by cloning the coding
sequences of Sry1, Sry2, and Sry3 genes into expression
constructs and cotransfecting each with the promoters of the rat
RAS genes Agt, Ren, Ace, and Ace2 in luciferase reporter
plasmids. All Sry effectors (proteins) increased activity of the
promoters of Agt, Ren, and Ace and decreased activity of Ace2.
The largest effects were seen with Sry3, the locus that activated
the RAS and raised blood pressure after delivery to a rat kidney
(23). Because regulatory control regions in promoters of these
genes are generally conserved across species, we would predict
that the human RAS gene promoters will also respond to
human SRY effectors.
AJP-Regul Integr Comp Physiol • VOL
Fig. 1. The human SRY effector plasmid increases activity of the rat Agt, Ren,
and Ace promoters and tends to decrease Ace2 promoter activity. Student’s
t-tests showed significant effects of human Sry on rat RAS promoters. For Agt,
**P ⫽ 0.0052, for ***Ren, P ⫽ 0.0008, for *Ace, P ⫽0.0141, and for *Ace2,
P ⫽ 0.02 (n ⫽ 3 each).
The RAS consists of both the classical form in the circulatory system and local tissue RASs. In addition to the main
participants in the classical pathway (Agt, Ren, and Ace),
alternate pathways have been described, and enzymes such as
angiotensin I-converting enzyme (ACE2) are known to play
important roles in RAS function (25, 27, 32, 40, 65). To have
a significant effect on the RAS, an effective regulator of
expression of RAS genes would modulate more than one gene.
The more genes in the cascade that are coordinately modulated
by the transcriptional regulator, the larger the overall effect can
be. This is the case with SRY; SRY increases activity of the
promoters of Agt, Ren, and Ace, and it decreases Ace2 promoter activity. The effect of SRY on the RAS is essentially
amplified by up-regulating several genes, including the substrate
angiotensinogen, through the processing enzymes renin and ACE,
while simultaneously down-regulating ACE2. Taken together,
these would favor the increased generation of ANG II.
Hypertension: Effect of Sry on the Peripheral
Nervous System
In the spontaneously hypertensive rat (SHR) model of human hypertension, the Y chromosome and Sry account for a
significant portion of the blood pressure increase in males over
normotensive rat strains (21). We recently published a summary of our Y chromosome, Sry, and hypertension results in
Steroids (24) and only a brief summary is reported here.
Our initial blood pressure studies showed that Wistar-Kyoto
(WKY) males with the SHR Y chromosome had elevated
blood pressure compared with WKY males with a WKY Y
chromosome (19). This was the first evidence that a locus on
the SHR Y chromosome influenced blood pressure. Further
studies examining the mechanism of this blood pressure increase demonstrated that many indices of sympathetic nerve
activity (SNS) activity, such as adrenal gland chromogranin A
and TH, were elevated (20). Pharmacological reduction of plasma
NE reduced the Y chromosome blood pressure effect (99). Next,
our focus switched to identifying the Y chromosome locus that
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Sry, the Peripheral Nervous System
and Neuroendocrine Regulation
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Sry, MORE THAN TESTIS DETERMINATION?
influenced blood pressure. Sry was a candidate locus, and we
found multiple copies of Sry in WKY and SHR; however, SHR
had at least one copy of Sry that was different (90).
When Sry1 was given exogenously to the kidney or adrenal
gland of normotensive WKY males, BP and SNS markers were
elevated (22, 23). When another copy, Sry2 was delivered to
the WKY kidney, BP was not affected (23). However, when a
third copy, Sry3, was delivered to the kidney, the RAS was
activated along with sodium retention and BP elevation, but
SNS markers were not affected (24). The Sry3 locus in SHR
may synergistically act with Sry1 to elevate BP pressure
through an interaction of the SNS and RAS. We speculate that
the differential function of Sry1 vs. Sry3 may be due to the
amino acid substitution in Sry3. These results are consistent
with an increased SNS activity mechanism due to Sry1, and
increased tissue renin angiotensin system activity due to Sry3.
The functions of the other copies of the Sry gene complex on
the Y chromosome remain to be determined. Although the
phenotype originally described and examined was hypertension, the underlying mechanism is the effect of Sry on the
sympathetic nervous system and RAS.
Consistent with a more global response of Sry on Th is the
report that a mutation in a SRY binding site in human Th has
a significant correlation with human blood pressure (106). Sry
also binds and modifies transcription related to a known SNP
in the human chromagranin B locus, consistent with a human
SRY effect on the sympathetic nervous system (105). Because
the human RAS gene promoters also contain conserved SRY
and AP-1 binding sites, they should also respond to SRY. We
suggest that the effects we describe with Sry on RAS gene
regulation are reflected in the effects of human Sry on human
AJP-Regul Integr Comp Physiol • VOL
BP, as well as on numerous other systems and proteins not yet
identified. These results suggest that Sry regulation of target
genes is very similar across species.
Note that these are male effects in neuroendocrine regulation
and the peripheral nervous system that are not related to
androgens or other circulating factors, but to the effects of Sry
expression in these cells. Female cells would not be expected
to have these responses.
Sry in the Central Nervous System
Sry also is present and expressed in the central nervous
system of both humans and rodents. Monoamine oxidase
(MAO) is a degradative enzyme for a number of biogenic
amines, which are involved both in peripheral, as well as
central nervous system function, such as serotonin, norepinephrine, and dopamine. Wu et al. (103) have shown the
presence of a functional binding site for SRY in the MAO A
core promoter both in vitro and in vivo. SRY activates both
MAO A-promoter and catalytic activities in a human male
neuroblastoma BE(2)C cell line. This finding has important
biological and medical implications because the effect of SRY
on MAO A expression could be part of a sexually dimorphic
program in neural structure and physiology. In addition, SRY
effects on MAO A may explain sex differences in cognition,
behavior, and psychiatric disorders that involve the biogenic
amines. Specifically, Sry is known to be expressed in the
midbrain of the rat (15), the hypothalamus and midbrain of the
mouse (54), and the hypothalamus and frontal and temporal
cortex of the human brain (51). Expression in the rat substantia
nigra is associated with agonistic behavior (15). We found Sry
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Fig. 2. Modeled human SRY and rat Sry
bound to DNA. Red denotes high mobility
group (HMG) box; blue denotes hinge domain; green denotes bridge domain; yellow
denotes NH2 terminus, pink denotes COOH
terminus; and gray denotes DNA. Both the
NH2 and COOH terminus are cleaved off for
simplicity but are labeled for total length. A
helix in the bridge domain (green) was predicted in rat SRY (right) but not human SRY
(left).
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Sry, MORE THAN TESTIS DETERMINATION?
Fig. 3. Transfection of Sry1 to WKY female hippocampus increased tyrosine
hydroxylase activity more than 100% compared with control vector (means ⫾
SE, *P ⬍ 0.05).
newly acquired expression or function when, in fact, they are
remnants of the original function of Sry, as a derived Sox3
locus.
Sry is a transcription factor with a consensus binding site the
same as other SOX loci. In any tissue where Sry is expressed,
the transcriptome of that cell would be modified. Is this
modification considered function? This modification does
mean that male cells and female cells do not have the same
transcriptome and this is not only the result of different sex
hormones but includes the effects of Sry expression itself on
gene regulation. Consistent with a nontestis determination
function for Sry is the fact that Fra-1 and Fra-2 are reported
Sry targets (30), which are not involved in testis determination
but are components of the AP-1 transcription factor. Sry
expression would increase AP-1 and hence any pathway controlled by AP-1. This model through AP-1 is consistent with
the results of Sry on the Th promoter in rats (56). In a recent
study examining the effect of human Sry using proteomics and
transcriptome approaches, NT2/D1 cells from human testicular
embryonal cell carcinoma were stably transfected with Sry
(80). Two different lines were studied, SRY1 and SRY2,
both of which overexpressed SRY. Using two-dimensional
gels followed by mass spectrometery analysis to compare
protein profiles in SRY-overexpressing cell lines to controls
showed that SRY downregulated many chaperone proteins
and upregulated laminin, which plays a role in Sertoli cell
differentiation. Comparative analysis of the transcriptomes
of the cell lines by microarray analysis using Affymetrix
GeneChips showed that SRY upregulated many zinc finger
proteins and downregulated cellular growth factors. Cell
proliferation and cell cycle analysis indicated that SRY
overproduction arrested cell cycle progression and inhibited
cell proliferation (80).
Perspectives and Significance: Potential Impacts of Sry
Expression and Function and Unanswered Questions
Whether these effects summarized here are restricted to the
organisms in which they have been identified or are more
widespread is not known, primarily because the experiments
either have not been attempted or are not informative because
of reduced genetic variation or the difficulty of separating sex
determination and sex hormones from Sry effects. In a mouse
Overview of Sry Expression in Adult Males
The acknowledged primary function of Sry is testis determination but evolutionarily, this is a derived function. The Sry
locus was derived from Sox3, which has functions in both the
brain and nervous system. The divergence of Sry into testis
determination did not mean all ancestral Sox3 function was
lost. In fact, it may have required no loss of function. We see
Sry expressed in many brain and nervous system tissues, and
with the focus on testis determination, we examine these as
AJP-Regul Integr Comp Physiol • VOL
Fig. 4. Average serotonin content of the medial amygdala for Sry1 and control
vector transfected WKY males (means ⫾ SE, *P ⬍ 0.05).
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expression in most rat brain areas examined (Table 1).The
following data support the concept that Sry has CNS effects
that may be important for understanding neurotransmitter regulation and nervous system phenotypes.
In the brain of a normotensive WKY rat male, we isolated
eight different brain regions, and for each region, mRNA was
isolated, and primers specific for different Sry loci were used to
determine the expression of the different rat Sry loci in the
mRNA pool (90). There were expression differences in the
different brain regions. The proportion of Sry1 varies according to brain region, varying from about 10% to more than 50%
of total Sry transcripts, while the proportion of the Sry2 varies
from 45% to 90% of total Sry transcripts. Sry1 was transfected
into the hippocampus of a SHR female, and after 5 days, TH
was increased more than 100% compared with vector controls
(Fig. 3).
Sry had not been thoroughly investigated with regard to its
role in the serotonergic system, but it would not be unusual for
transcription factors like Sry to play a role in development of
5-HT neurons. Tryptophan hydroxylase (Tph) is the ratelimiting enzyme for serotonin biosynthesis. When we analyzed
the promoters of the mouse and human Tph genes with
TRANSFAC (101), we found multiple potential SRY consensus binding sites. This suggested that SRY has the potential to
regulate the rat Tph gene by a mechanism similar to that we
described for the tyrosine hydroxylase (Th) gene (56). We then
demonstrated in WKY males that if we added exogenous Sry1
to the amygdala, brain serotonin content decreased. Figure 4
shows that exogenous Sry delivered to the amygdala caused a
significant reduction in serotonin content in the amygdala
compared with controls (23% reduction, P ⬍ 0.05, n ⫽
6/group).
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Sry, MORE THAN TESTIS DETERMINATION?
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AJP-Regul Integr Comp Physiol • VOL
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model, transgenic Sry may allow some testing, but confounding effects, in addition to the effects of other sex determination
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We raise further questions related to nontestis-determining
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• Sry is expressed in the adult testis in only certain cell
types (11). How does this effect the adult testis?
• There are high levels of Sry expression in early mammalian blastula (107). Are XX and XY cell lineages beginning to differ at this early stage or is it a male compensation for the fact that X-inactivation has not yet occurred?
• Is there increased expression of a related Sox locus in
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differences? No, but the transcriptional effects of Sry
expression in adult males should at least be considered as
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