doi:10.1152/ajpregu.00645.2010 301:R561-R571, 2011. First published 15 June 2011;Am J Physiol Regul Integr Comp Physiol

Monte E. Turner, Daniel Ely, Jeremy Prokop and Amy Milsted, more than testis determination?Sry

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SPECIAL TOPIC Water and Electrolyte Homeostasis Section Invited Reviews

Sry, more than testis determination?

Monte E. Turner, Daniel Ely, Jeremy Prokop, and Amy MilstedDepartment of Biology, The University of Akron, Akron, Ohio

Submitted 24 September 2010; accepted in final form 1 June 2011

Turner ME, Ely D, Prokop J, Milsted A. Sry, more than testis deter-mination? Am J Physiol Regul Integr Comp Physiol 301: R561–R571, 2011.First published June 15, 2011; doi:10.1152/ajpregu.00645.2010.—The Srylocus on the mammalian Y chromosome is the developmental switchresponsible for testis determination. Inconsistent with this importantfunction, the Sry locus is transcribed in adult males at times and intissues not involved with testis determination. Sry is expressed inmultiple tissues of the peripheral and central nervous system. Sry isderived from Sox3 and is similar to other SOXB family loci. TheSOXB loci are responsible for nervous system development. Sry hasbeen demonstrated to modulate the catecholamine pathway, so itshould have functional consequences in the central and peripheralnervous system. The nervous system expression and potential functionare consistent with Sry as a SOXB family member. In mammals, Sox3is X-linked and undergoes dosage compensation in females. Theexpression of Sry in adult males allows for a type of sexual differen-tiation independent of circulating gonadal hormones. A quantitativedifference in Sox3 plus Sry expression in males vs. females coulddrive changes in the transcriptome of these cells, differentiating maleand female cells. Sry expression and its transcriptional effects shouldbe considered when investigating sexual dimorphic phenotypes.

SRY; sexual dimorphism; SOX; nervous system

IT IS WELL UNDERSTOOD THAT many genes and gene products havepleiotropic effects, yet for some loci, a known essential func-tion hides this reality. A newly derived function may distractattention from ancestral functions. In physiology, hormoneshave many more functions than that of their original functionaldiscovery. The Sry locus on the Y chromosome is responsiblefor testis determination in placental mammals. The essentialbiological importances of sex determination and difficulties indescribing the mechanism have overshadowed the potential ofSry functions not related to testis determination. In addition,the prevailing ideas of mammalian sex determination rely onthe ovaries/testes producing estrogens/androgens for the fe-male/male phenotypes. Mammalian sex is determined at theorganismal level instead of the cellular level as in manyinvertebrates. The mammalian Sry locus is expressed in manyadult male tissues. Is it possible that this expression acts as atype of cellular sex determination that then impacts somemale/female differences in mammals? We will examine dataand theory supporting the idea that Sry has other functionalphenotypic effects not involved in the process of testis deter-mination, and these may cause differentiation of male andfemale cells or phenotypes.

We will not address Sry and testis determination, which hasbeen summarized elsewhere (100). We will examine the con-straints on Sry as a Y chromosome and testis-determininglocus, a SOXB locus, and data on Sry expression in tissues andtimes not related to testis determination. What is the evidencethat Sry may have functions in addition to testis determination?To illustrate the potential of Sry expression in males, we willuse examples of Sry effects on 1) the peripheral nervous systemand neuroendocrine regulation and 2) the central nervoussystem.

Although there are different genetic designations for anyparticular locus dependent on the organism in which it isfound, throughout this review, we will use Sry to refer to thelocus and SRY to refer to the protein of the Sry locus.

Sry Background

The Sry locus (sex-determining region on the Y chromo-some) was the first indentified member of the SOX transcrip-tion factor family (86). All of these proteins contain a high-mobility group (HMG) box with amino acid sequence con-served at least 46% and a highly conserved hinge region (5).The HMG box of SRY and SOX proteins recognize a consen-sus DNA sequence AACAAT, binding in the minor groove ofthe DNA (36, 43). Through intercalation of the protein with thebase pairs at the consensus binding site, the major groove iscondensed and minor groove opened, thus inducing a bend inthe DNA. For cases in which a bend already exists, binding ofSRY is enhanced in nonconsensus-binding sequences (26). Thehinge domain is also highly conserved in all SOX members andis important for nuclear localization (37). Outside the HMGbox and hinge region, Sry shares little homology with otherSOX family members (5).

Sry is a member of the SOXB family of SOX loci, whichincludes Sox1, Sox2, Sox3, and Sry. Sox1 and Sox2 are auto-somal in most mammals, and Sox3 is X-linked. It is believedthat Sry originated from a mutation of Sox3 on the thenautosomal primordial mammalian X chromosome, leading tothe advent of the mammalian Y chromosome (81). The amountof HMG amino acid sequence homology between SRY andother SOX family members supports this theory, with Sox3having the highest amino acid sequence conservation. Someregions outside of the HMG box and hinge regions have beensuggested to play a role in SRY-specific function (79). There isa slight amount of amino acid sequence conservation acrossSRY in multiple species in the bridge domain. This bridgedomain has been shown to interact with KRAB-O (64), andthere is no indication that this conserved KRAB-O domain isinvolved in testis determination. The conservation in the SRY-specific bridge domain could be involved in long-term transcrip-tional regulation. Only members of the SOXB group have

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).

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several amino acids conserved in this domain and may explainsimilar functions of SRY and SOXB members. A recent studyby Sato et al. (81) reanalyzed the sequence of the human Srygene by searching for homologous DNA sequences. Theyconcluded that the gene evolved as a hybrid resulting frominsertions of a part of the DGCR8 gene, which encodes Di-George syndrome critical region 8 and is involved in biogen-esis of microRNA (34), into a region 5= of the HMG box of anancestral Sox3 gene.

Sry as a Y Chromosome Locus

The constraints on mutations and evolution of Sry aredifferent from an autosomal locus. Sry is located on themammalian Y chromosome and is thus constrained by thislocation. The Y acts as homologous chromosome with the Xchromosome in meiosis but does not recombine except atspecific regions (pseudoautosomal region). This results in themajority of the Y chromosome being inherited as a singlelocus. The classic view of the Y chromosome is that of achromosome that accumulates mutations with selection elimi-nating Y chromosomes with mutations that have lost essentialfunctions. A modern version of the Y chromosome includes theobservation that certain regions of the Y chromosome containalmost identical repeats that have high frequencies of geneconversion that can “fix” mutations that occur (77).

The human Y chromosome is composed of three types ofsequences: ampliconic, X-transposed, and X-degenerate (87).The ampliconic regions contain large repeat units in whichgene conversion and homologous recombination occur withinthe repeat regions (74). The X-degenerate and X-transposedregions have origins from ancestral autosomes (X-degenerate)and more recent transpositions from the X chromosome (X-transposed) (48). Both the X-degenerate and X-transposedregions contain primarily single-copy genes, which shouldevolve like the classic view of the Y chromosome. The Srylocus is within the X-degenerate region of the human Ychromosome. Structural mutations in individual Y chromo-somes may have moved the Sry locus into different regions ofthe chromosomes in other mammalian species. The location ofSry in different regions of the Y chromosome may affect theevolution of the Sry locus in these species.

Because Sry is located in the X-degenerate region of thehuman Y chromosome, any mutation not affecting function ofthe locus should accumulate among human Y chromosomes.We might expect the accumulation of synonymous mutationsin the coding region, nonsynonymous mutations outside of theHMG-box region not affecting DNA binding, and mutations inthe flanking regions not involved in transcriptional control orstability. The available data demonstrate that the X-degenerateand X-transposed regions have reduced levels of genetic di-versity compared with autosomal regions (78). Consistent withthis observation, the effective population size of a Y chromo-some is only ¼ that of an autosomal chromosome, thus reduc-ing the opportunity for mutations.

In a study of the coding regions of genes from 105 human Ychromosomes, selected to examine the range of human Ychromosomes, nucleotide diversity was significantly lower fornonsynonymous sites than synonymous sites, pseudogenes,or introns, and this is consistent with natural selectionmaintaining these gene sequences. It is possible that Sry is

the force driving this purifying selection as any mutationthat eliminates function in an essential developmentalswitch will be immediately lost from the population. Thesequence variation in Sry is among the lowest on the Ychromosome, with only one substitution (synonymous)found in the coding region of Sry (78).

To examine the variation outside of the coding region, wecan compare the Sry locus in the male personal genomes thathave been published and available. The genomes of KB1 (83),NB1 (83), ABT (83), George Church (16), Henry Louis GatesSr. (16), J. Craig Venter (50), James Watson (97), YRI (17),Han Chinese Individual (94), Seong-Jin Kim (1), AnonymousKorean individual (42), Stephen Quake (70), and the 4,000year old Saqqaq (72) were viewed using the PSU GenomeBrowser (http://main.genome-browser.bx.psu.edu/). Only Seong-Jin 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). Look-ing at 4 kB on each side flanking the Sry-coding sequence(chrY: 2,710,859–2,719,828) reveals only four additionalSNPs, but none shared among multiple sequences. Of interestis that the Saqqaq sequence reveals no SNPs over this stretchof the Y chromosome, suggesting that the sequence in thisregion has remained stable over the past 4,000 years (85). TheSry flanking region shows the same reduction in variationconsistent with results using only the coding region (78).

Other than the comparison using these genomes, there is nosurvey of normal variation in the Sry locus. A study of gonadaldysgenesis can only identify mutations that disrupt testis de-termination, but these are lost from the population because thephenotype is sterile (31). These results from a comparison ofdifferent human Sry sequences, coding region (78) and flank-ing regions, and coding region of both modern and ancientgenomes (Saqqaq), indicate that the gene does not have thenecessary polymorphism levels to answer either questions ofadditional function or to determine sequences necessary fortestis determination using conserved regions because the wholeregion seems to be highly conserved.

Multiple Copies of Sry on a Single Y Chromosome

In some rodent species, Sry has multiple copies on a singleY chromosome (60). Multiple copies have been demonstratedin family Muridae, including African rodents (53, 60), voles(6), and rats (90). These are true duplicate loci with indepen-dent control and coding regions. The exact spatial relationshipbetween any of these loci is unknown, as none of these Ychromosomes have been sequenced. In the rat, indirect evi-dence indicates the loci are not close to each other, as se-quenced BACs only contain a single copy. The finding ofcopies in many related species indicates these copies are eithervery old or their location on the Y chromosome causes dupli-cations to occur at an increased rate. The presence of manycopies in these rodents does not seem to have any obviousdeleterious effects. In voles, the multiple copies are obviouslynot functional due to nonsense/frameshift mutations in thecoding region (7), but for these mutations to have occurred andaccumulated, the duplicate Sry loci must be very old.

In other examples, the coding regions do not contain muta-tions that would lead to nonfunctional proteins or evidence ofSry pseudogenes (53, 90, 91). Expression patterns have only

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been examined in Rattus norvegicus strains, and the multiplecopies on the rat Y chromosome show divergent expressionpatterns (90), consistent with either gain or loss of transcrip-tional components. In addition to transcriptional divergencethese Rattus norvegicus Sry loci have slightly different proteinphenotypes and have potential diverse effects in the adult malerat.

The occurrence of multiple Sry copies is probably a result ofthe repeat organization of the mammalian Y chromosomeleading to many species-specific duplications and deletions.Multiple Sry copies have been identified in human malesexposed to high background radiation but not in normal controlmales (68, 69). The question of how common are multiplecopies of Sry on a single Y chromosome remains largelyunanswered because experiments to identify whether multiplecopies exist have either not been tested or not enough humanY chromosomes have been sequenced to characterize a popu-lation.

Sry as a Member of the SOXB Family

In the human and mouse genomes there are 20 members ofthe SOX gene family (82), many of which have known roles indisease etiology. Focusing on the SOXB family (Sox1, Sox2,and Sox3), which is most similar to Sry, all members areimportant for stem-cell maintenance in the central nervoussystem (95), and Sox2 and Sox3 are important in pituitarydevelopment (2). All three are coexpressed in both avian andmurine neural progenitor cells and the adult brain (8, 102). Nullmutations of Sox1, Sox2, and Sox3 have different neuronalphenotypes, indicating differentiation of function (18, 28, 76).Lost in most discussions of Sry and testis determination is theidea that Sry was derived from Sox3, and testis determinationis a new derived function rather than the ancestral centralnervous system function (33).

Sox3 is expressed in neural progenitor cells, and both Sox3and Sry are expressed in adult testis (96). Consistent with Sox3derivation, Sry expression in the adult testis is an ancestralcondition, not derived. Sox3 deletions in mice have abnormaldevelopment of diencephalon and anterior pituitary gland andlack differentiated spermatogonia (73). Sox3 has been specu-lated to activate Sox9 and induce sex reversal in XX transgenicmice, 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 hydrox-ylase (Th)-expressing cells (15) and has been demonstrated toincrease transcription of the Th promoter (56). Tyrosine hy-droxylase is the rate-limiting enzyme for norepinephrine anddopamine synthesis, suggesting that Sry has a role in male-specific brain development. Sry transcripts have been found inan ever expanding number of tissues (Table 1), so that under-standing the mechanisms by which Sry works becomes increas-ingly important. The expression pattern of Sry in adult males isbetter explained if Sry is examined as a SOXB-derived locusrather than primarily as a testis determination locus. Does Sryfunction similarly to other members of the SOXB family or hasit specialized through alterations in preferred DNA bindingsequences or protein binding partners?

Since Sox3 is an X-linked locus, it is subject to X inactiva-tion. Therefore, mammalian females have SOX3 from one X

chromosome expression, while males have SOX3 plus SRY intissues, where Sry is expressed. Any quantitative relationshipswould be enhanced in males over females. This difference isindependent of steroid hormones. Expression of Sry in malesadds a level of cellular sex determination and differentiationindependent of gonads and hormones. The interaction of SRYwith the androgen receptor could further potentiate these af-fects in male cells vs. female cells (104). This mechanism ispotentially present in the brain and nervous system, bothduring development and in adult males due to the expression ofSry in these tissues and cells.

Sry Expression

Table 1 lists published results and unpublished results fromour laboratory of Sry expression at times and in tissues notrelated to testis determination. The number of tissues whereexpression has been identified and the conservation of expres-sion in many species are not the expectation or prediction fora gene involved only in testis determination. Any mutationeliminating unnecessary expression would be advantageous,and the expectation would be random loss of expression ina single species and decreased expression phylogeneticallyover evolutionary time in a Muller’s ratchet type of expec-tation (59).

In SHR and WKY rat strains, we have examined the expres-sion in numerous tissues of the six or seven duplicated copiesof the Sry locus (Table 1) (90). Multiple Sry loci are expressedin kidney (23, 90), adrenal gland, brain, and other tissues inyoung and adult male rats (Table 1) (56, 90, 91). We have usedSry-specific primers to amplify cDNA from many rat tissues(Table 1) and find the presence of Sry in tissues tested and noamplification in no-RT controls or females. Many of these ratSry loci are expressed, in patterns that are tissue- and locus-dependent. Again, the pattern is one expected with function,rather than random loss, or the same expression pattern, for allduplicated loci.

Sry expression is found in many different tissues and timesin addition to the genital ridge at the time of testis determina-tion. For instance, Dewing et al. (15) identified Sry expressionin situ in the rat brain using an antisense Sry probe and SRYprotein with a anti-SRY antibody. Modi et al. (58) identifiedSry mRNA in situ in human adult male testis with an antisenseprobe. These results are significant because Sry expression isnot seen in expressed sequence tag (EST) libraries of thesetissues. In fact, Sry expression is missing from all EST librarysequences. If there is enough expression for in situ identifica-tion, one would expect multiple EST sequences for Sry fromthese and other tissues. This lack of ESTs for Sry is not speciesspecific but is deficient in all libraries, even from species andtissues where Sry expression has been identified from methodsthat require a much larger number of transcripts. The onlyconserved sequence among these species is the HMG box,hinge region, and bridge domain of the gene; thus, theseregions must have a sequence or secondary structure thatinhibits cloning under standard conditions. Because we areable to amplify the entire rat Sry sequences in cDNA madefrom mRNA, the lack of Sry in EST libraries is probably aresult of inefficient cloning rather than in the process ofcreating cDNA.

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From our experience with rat Sry sequences, it is obviousthat the Sry sequence does not clone with the same efficiencyas other sequences. In fact, not all of the Sry loci from a singlerat Y chromosome clone equally well. Using primers spanningthe rat Sry coding sequences, the Sry2 amplicon is 39 bpshorter than the other sequences and can be seen as a separateband with electrophoresis (91). Using PCR TOPO TA cloningkit to clone the amplicons, we sequenced more than 100 clonesand found that no clones contained the Sry2 sequence, eventhough it would be expected that either 1/6 or 1/7 of the cloneswould be Sry2. The Sry2 sequence was eventually determinedfrom Sry2-specific primers and sequencing of that PCR prod-uct (91).

Sox Loci in the Nervous System

Since the initial discovery of Sry 20 years ago and subse-quently the SOX loci, numerous studies have shown a strongrole for SOX transcription factors in the nervous system ofvertebrates and invertebrates. Invertebrates, including Dro-sophila (61), Amphioxus (38), and the Ptychodera flave (89)have Sox expression in the nervous system. In all three of thesespecies, homology of the Sox proteins involved most closelymatches that of the SOXB1 group. All SOXB1 genes havebeen shown to be expressed in the central nervous system(CNS) of vertebrates (12). The most studied of the SOXB1members is Sox2, which is important for nerve cell formation

Table 1. Sry expression in mammalian tissues and method of detection

Tissue Source (A, F) Detection Method Reference

Adrenal gland rat (A) real-time RT-PCR, gel-based 56, 90, 91, unpublished datahuman (F) gel-based RT-PCR 11

BrainAmygdala rat (A) real-time RT-PCR unpublished dataBrain human (F) gel-based RT-PCR 11Brainstem rat (A) real-time RT-PCR unpublished dataCerebral cortex rat (A) gel-based and real-time RT-PCR unpublished dataCortex rat (A) in situ hybridization 15Cortex mouse (A) gel-based RT-PCR 55Diencephalon mouse (A) gel-based RT-PCR 55.1Hypothalamus rat (A) real-time RT-PCR unpublished dataLocus coeruleous rat (A) gel-based PCR 56Medial mammilary bodies rat (A) in situ hybridization 15Midbrain mouse (A) gel-based RT-PCR 55Substantia nigra- rat (A) gel-based PCR 56Substantia nigra rat (A) in situ hybridization (ISH) 15Substantia nigra rat (A) immunohistochemisty (protein) 15Ventral tegmental area rat (A) gel-based PCR 56

Celiac ganglion rat (A) real-time RT-PCR unpublished dataCultured cells

Neonatal astrocytes rat real-time RT-PCR unpublished dataHep G2 human gel-based RT-PCR 11NTERA-2 cl.D1 gel-based PCR 11

HeartAorta rat (A) real-time RT-PCR unpublished dataAtrium rat (A) real-time RT-PCR unpublished dataHeart human (A) gel-based RT-PCR 11Heart human (F) gel-based RT-PCR 11Ventricle rat (A) real-time RT-PCR unpublished data

Kidney rat (A) real-time RT-PCR 91, unpublished datahuman (A) gel-based RT-PCR 11

Liver rat (A) real-time RT-PCR unpublished datahuman (A) gel-based RT-PCR 11human (F) gel-based RT-PCR 11

Pancreas human (F) gel-based RT-PCR 11Skeletal muscle rat (A) real-time RT-PCR unpublished dataSmall intestine human (F) gel-based RT-PCR 11Spleen human (F) gel-based RT-PCR 11Superior cervical ganglia rat (A) real-time RT-PCR unpubThymus human (F) gel-based RT-PCR 11Testis rat (A) real-time RT-PCR 90, unpublished

bovine (A) Northern blot 13mouse (A) Northern blot, gel-based RT-PCR 46mouse (A) Northern; nuclease protection; ISH 108human (A) Northern blot 86human (A) Northern; gel-based PCR 11

Gonadal ridge mouse (F) Northern; ISH; gel-based RT-PCR 46Preimplantation embryo mouse (F) Gel-based RT-PCR 107Male embryo porcine (F) Gel-based RT-PCR 14Preimplantation embryo human (F) Gel-based RT-PCR 29

Unpublished data are from the spontaenously hypertensive rat and Wistar-Kyoto strains from our laboratory. Source: A, adult; F, fetal or embryonic.

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in the inner ear (63), parenchymal astroglia cell formation (45),maintenance of neural stem cells (9), and neurogenesis in thebrain (28). Although Sry and Sox2 have similar binding affin-ities to the consensus binding site (12), some of the processesin which Sox2 is involved, such as eye formation, require thetranscriptional activation domain found only on the SoxB1genes (35). SoxB genes are not the only members of the SOXfamily to have a role in the nervous system. The SOXE (Sox8,Sox9, and Sox10) group is known to function in Schwann celldevelopment (47), oligodendrocyte development (88), and reg-ulation of nicotinic ACh receptors (52). SOXC genes (Sox 4and Sox11) are expressed in the immature neurons of neuralstem cells in mice (66). Because SRY shares a HMG box, andthus similar DNA binding sites, with all other SOX proteins,SRY, when expressed could participate in similar roles innervous system development and function in the adult malenervous system.

Sry, the Peripheral Nervous Systemand Neuroendocrine Regulation

Our research demonstrated that Sry has a role in the periph-eral nervous system, specifically the sympathetic nervous sys-tem. TH is the rate-limiting enzyme for catecholamine biosyn-thesis both in the brain and periphery. Transient cotransfectionassays in PC12 cells with Sry1 and pTh/Luc(�272/�27) dem-onstrated that the product of the Sry1 gene increases expressionof 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 thecontrol Th promoter. This suggested that the actions of Sry inregulating TH transcription are due to interactions at the AP-1site (75%) and the direct effects of Sry (25%). The impact ofSRY on AP-1 transcription factors is not unexpected, given thepreviously identified targets of SRY (30). The impact of Sry onthe sympathetic nervous system has a diverse potential onmultiple phenotypes in humans and other mammals, such asthe fight/flight response, blood pressure, and kidney function.

The sympathetic nervous system is not the only potentialtarget of Sry in the peripheral nervous system. The renin-angiotensin system (RAS) is also a target of Sry regulation(57). Recently, we have shown that the human SRY proteinalso regulates rat angiotensin-converting enzyme (Ace), Ace2,renin (Ren), and angiotensinogen (Agt) promoter activity(Fig. 1), suggesting that Sry regulation of target genes issimilar and conserved across species. Our modeling studiescomparing human and rat SRY proteins indicate close struc-tural conservation across these species (Fig. 2). The effects ofrat Sry on the rat RAS were investigated by cloning the codingsequences of Sry1, Sry2, and Sry3 genes into expressionconstructs and cotransfecting each with the promoters of the ratRAS genes Agt, Ren, Ace, and Ace2 in luciferase reporterplasmids. All Sry effectors (proteins) increased activity of thepromoters of Agt, Ren, and Ace and decreased activity of Ace2.The largest effects were seen with Sry3, the locus that activatedthe RAS and raised blood pressure after delivery to a rat kidney(23). Because regulatory control regions in promoters of thesegenes are generally conserved across species, we would predictthat the human RAS gene promoters will also respond tohuman SRY effectors.

The RAS consists of both the classical form in the circula-tory system and local tissue RASs. In addition to the mainparticipants in the classical pathway (Agt, Ren, and Ace),alternate pathways have been described, and enzymes such asangiotensin I-converting enzyme (ACE2) are known to playimportant roles in RAS function (25, 27, 32, 40, 65). To havea significant effect on the RAS, an effective regulator ofexpression of RAS genes would modulate more than one gene.The more genes in the cascade that are coordinately modulatedby the transcriptional regulator, the larger the overall effect canbe. This is the case with SRY; SRY increases activity of thepromoters of Agt, Ren, and Ace, and it decreases Ace2 pro-moter activity. The effect of SRY on the RAS is essentiallyamplified by up-regulating several genes, including the substrateangiotensinogen, 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 PeripheralNervous System

In the spontaneously hypertensive rat (SHR) model of hu-man hypertension, the Y chromosome and Sry account for asignificant portion of the blood pressure increase in males overnormotensive rat strains (21). We recently published a sum-mary of our Y chromosome, Sry, and hypertension results inSteroids (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 elevatedblood pressure compared with WKY males with a WKY Ychromosome (19). This was the first evidence that a locus onthe SHR Y chromosome influenced blood pressure. Furtherstudies examining the mechanism of this blood pressure in-crease demonstrated that many indices of sympathetic nerveactivity (SNS) activity, such as adrenal gland chromogranin Aand TH, were elevated (20). Pharmacological reduction of plasmaNE reduced the Y chromosome blood pressure effect (99). Next,our focus switched to identifying the Y chromosome locus that

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’st-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).

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influenced blood pressure. Sry was a candidate locus, and wefound multiple copies of Sry in WKY and SHR; however, SHRhad at least one copy of Sry that was different (90).

When Sry1 was given exogenously to the kidney or adrenalgland of normotensive WKY males, BP and SNS markers wereelevated (22, 23). When another copy, Sry2 was delivered tothe WKY kidney, BP was not affected (23). However, when athird copy, Sry3, was delivered to the kidney, the RAS wasactivated along with sodium retention and BP elevation, butSNS markers were not affected (24). The Sry3 locus in SHRmay synergistically act with Sry1 to elevate BP pressurethrough an interaction of the SNS and RAS. We speculate thatthe differential function of Sry1 vs. Sry3 may be due to theamino acid substitution in Sry3. These results are consistentwith an increased SNS activity mechanism due to Sry1, andincreased tissue renin angiotensin system activity due to Sry3.The functions of the other copies of the Sry gene complex onthe Y chromosome remain to be determined. Although thephenotype originally described and examined was hyperten-sion, the underlying mechanism is the effect of Sry on thesympathetic nervous system and RAS.

Consistent with a more global response of Sry on Th is thereport that a mutation in a SRY binding site in human Th hasa significant correlation with human blood pressure (106). Sryalso binds and modifies transcription related to a known SNPin the human chromagranin B locus, consistent with a humanSRY effect on the sympathetic nervous system (105). Becausethe human RAS gene promoters also contain conserved SRYand AP-1 binding sites, they should also respond to SRY. Wesuggest that the effects we describe with Sry on RAS generegulation are reflected in the effects of human Sry on human

BP, as well as on numerous other systems and proteins not yetidentified. These results suggest that Sry regulation of targetgenes is very similar across species.

Note that these are male effects in neuroendocrine regulationand the peripheral nervous system that are not related toandrogens or other circulating factors, but to the effects of Sryexpression in these cells. Female cells would not be expectedto have these responses.

Sry in the Central Nervous System

Sry also is present and expressed in the central nervoussystem of both humans and rodents. Monoamine oxidase(MAO) is a degradative enzyme for a number of biogenicamines, which are involved both in peripheral, as well ascentral nervous system function, such as serotonin, norepi-nephrine, and dopamine. Wu et al. (103) have shown thepresence of a functional binding site for SRY in the MAO Acore promoter both in vitro and in vivo. SRY activates bothMAO A-promoter and catalytic activities in a human maleneuroblastoma BE(2)C cell line. This finding has importantbiological and medical implications because the effect of SRYon MAO A expression could be part of a sexually dimorphicprogram in neural structure and physiology. In addition, SRYeffects on MAO A may explain sex differences in cognition,behavior, and psychiatric disorders that involve the biogenicamines. Specifically, Sry is known to be expressed in themidbrain of the rat (15), the hypothalamus and midbrain of themouse (54), and the hypothalamus and frontal and temporalcortex of the human brain (51). Expression in the rat substantianigra is associated with agonistic behavior (15). We found Sry

Fig. 2. Modeled human SRY and rat Srybound to DNA. Red denotes high mobilitygroup (HMG) box; blue denotes hinge do-main; green denotes bridge domain; yellowdenotes NH2 terminus, pink denotes COOHterminus; and gray denotes DNA. Both theNH2 and COOH terminus are cleaved off forsimplicity but are labeled for total length. Ahelix in the bridge domain (green) was pre-dicted in rat SRY (right) but not human SRY(left).

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expression in most rat brain areas examined (Table 1).Thefollowing data support the concept that Sry has CNS effectsthat may be important for understanding neurotransmitter reg-ulation and nervous system phenotypes.

In the brain of a normotensive WKY rat male, we isolatedeight different brain regions, and for each region, mRNA wasisolated, and primers specific for different Sry loci were used todetermine the expression of the different rat Sry loci in themRNA pool (90). There were expression differences in thedifferent brain regions. The proportion of Sry1 varies accord-ing to brain region, varying from about 10% to more than 50%of total Sry transcripts, while the proportion of the Sry2 variesfrom 45% to 90% of total Sry transcripts. Sry1 was transfectedinto the hippocampus of a SHR female, and after 5 days, THwas increased more than 100% compared with vector controls(Fig. 3).

Sry had not been thoroughly investigated with regard to itsrole in the serotonergic system, but it would not be unusual fortranscription factors like Sry to play a role in development of5-HT neurons. Tryptophan hydroxylase (Tph) is the rate-limiting enzyme for serotonin biosynthesis. When we analyzedthe promoters of the mouse and human Tph genes withTRANSFAC (101), we found multiple potential SRY consen-sus binding sites. This suggested that SRY has the potential toregulate the rat Tph gene by a mechanism similar to that wedescribed for the tyrosine hydroxylase (Th) gene (56). We thendemonstrated in WKY males that if we added exogenous Sry1to the amygdala, brain serotonin content decreased. Figure 4shows that exogenous Sry delivered to the amygdala caused asignificant reduction in serotonin content in the amygdalacompared with controls (23% reduction, P � 0.05, n �6/group).

Overview of Sry Expression in Adult Males

The acknowledged primary function of Sry is testis deter-mination but evolutionarily, this is a derived function. The Srylocus was derived from Sox3, which has functions in both thebrain and nervous system. The divergence of Sry into testisdetermination did not mean all ancestral Sox3 function waslost. In fact, it may have required no loss of function. We seeSry expressed in many brain and nervous system tissues, andwith the focus on testis determination, we examine these as

newly acquired expression or function when, in fact, they areremnants of the original function of Sry, as a derived Sox3locus.

Sry is a transcription factor with a consensus binding site thesame as other SOX loci. In any tissue where Sry is expressed,the transcriptome of that cell would be modified. Is thismodification considered function? This modification doesmean that male cells and female cells do not have the sametranscriptome and this is not only the result of different sexhormones but includes the effects of Sry expression itself ongene regulation. Consistent with a nontestis determinationfunction for Sry is the fact that Fra-1 and Fra-2 are reportedSry targets (30), which are not involved in testis determinationbut are components of the AP-1 transcription factor. Sryexpression would increase AP-1 and hence any pathway con-trolled by AP-1. This model through AP-1 is consistent withthe results of Sry on the Th promoter in rats (56). In a recentstudy examining the effect of human Sry using proteomics andtranscriptome approaches, NT2/D1 cells from human testicularembryonal cell carcinoma were stably transfected with Sry(80). Two different lines were studied, SRY1 and SRY2,both of which overexpressed SRY. Using two-dimensionalgels followed by mass spectrometery analysis to compareprotein profiles in SRY-overexpressing cell lines to controlsshowed that SRY downregulated many chaperone proteinsand upregulated laminin, which plays a role in Sertoli celldifferentiation. Comparative analysis of the transcriptomesof the cell lines by microarray analysis using AffymetrixGeneChips showed that SRY upregulated many zinc fingerproteins and downregulated cellular growth factors. Cellproliferation and cell cycle analysis indicated that SRYoverproduction arrested cell cycle progression and inhibitedcell proliferation (80).

Perspectives and Significance: Potential Impacts of SryExpression and Function and Unanswered Questions

Whether these effects summarized here are restricted to theorganisms in which they have been identified or are morewidespread is not known, primarily because the experimentseither have not been attempted or are not informative becauseof reduced genetic variation or the difficulty of separating sexdetermination and sex hormones from Sry effects. In a mouse

Fig. 3. Transfection of Sry1 to WKY female hippocampus increased tyrosinehydroxylase activity more than 100% compared with control vector (means �SE, *P � 0.05).

Fig. 4. Average serotonin content of the medial amygdala for Sry1 and controlvector transfected WKY males (means � SE, *P � 0.05).

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model, transgenic Sry may allow some testing, but confound-ing effects, in addition to the effects of other sex determinationloci, include the amount of control region included in thetransgene and expression and epigenetic effects of moving Sryfrom heterochromatin to euchromatin. Sry expression is con-served in many adult male mammalian tissues; the transcrip-tional effect of this expression on the cellular transcriptomeneeds to be established using current techniques like those ofSato et al. (80).

We raise further questions related to nontestis-determiningactions and potential clinical relevance of Sry expression.

• Sry is expressed in the adult testis in only certain celltypes (11). How does this effect the adult testis?

• There are high levels of Sry expression in early mamma-lian blastula (107). Are XX and XY cell lineages begin-ning to differ at this early stage or is it a male compen-sation for the fact that X-inactivation has not yet oc-curred?

• Is there increased expression of a related Sox locus infemales that compensates for Sry expression in males?

• Does Sry, derived from Sox3, still need to maintain a Sox3expression level until X-inactivation?

• Do Sry and Sox3 differences in the nervous system act ina sex determination mechanism during development ofthe nervous system?

• Do effects of Sry on the sympathetic nervous systempredispose males to an increased response level?

• What is the developmental expression of Sry and how arethese expression profiles conserved across mammalianevolution?

• Is Sry responsible for sex differences in human disease?Sex differences have been described for many diseases inaddition to hypertension, including myocardial infarction(93), stroke (3), and many immune disorders (98).

• Is Sry involved in autism risks since there are a maleexcess and Y chromosome haplotype associations (84).Could Sry expression in the nervous system be responsi-ble?

• Could Sry expression affect chronic kidney disease?Males have a higher incidence and progress to end-stagerenal disease faster than women (49, 62). Diabetic malesare more likely than females to show renal damage (75).

• Is Sry involved in other conditions that have a major sexdifferences? For example, males have higher mortalityfollowing hip fracture (67), shorter time to secondaryprogressive multiple sclerosis (44), faster disease progres-sion following HIV seroconversion (39), higher incidencerate of small intestinal cancer (71), worse prognosis inidiopathic pulmonary fibrosis (10), and increased risk ofabdominal aortic aneurysm (41).

• Will Sry expression be shown to be responsible for all sexdifferences? No, but the transcriptional effects of Sryexpression in adult males should at least be considered asa potential option. While some of these differences maybe a reflection of hormonal status or environment, otherslikely represent genetic or epigenetic differences.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

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Perspectives

R571Sry, MORE THAN TESTIS DETERMINATION?

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