Evidence for increased prevalence of SRY mutations in XY females with complete rather than partial gonadal dysgenesis.

The Y chromosome gene SRY (sex-determining region, Y gene) has been equated with the mammalian testis-determining factor. The SRY gene of five subjects with 46,XY complete gonadal dysgenesis (46,XY karyotype, completely female external genitalia, normal Müllerian ducts, and streak gonads) was evaluated for possible mutations in the coding region by using both single-strand conformation polymorphism (SSCP) assay and DNA sequencing. Mutations were identified in three subjects, of which two gave altered SSCP patterns. Two of them were point mutations causing amino acid substitutions, and the third was a single-base deletion causing a frameshift. All three mutations caused alterations in the putative DNA-binding region of the SRY protein. Genomic DNA was obtained from the fathers of two of the three mutant patients: one mutation was demonstrated to be de novo, and the other was inherited. The presence of SRY mutations in three of five patients suggests that the frequency of SRY mutations in XY females is higher than current estimates.     

Testis-determining factor and Y-linked sex reversal.

A gene named SRY, isolated last year from the sex-determining region of the human Y chromosome, satisfies many of the criteria expected of the testis-determining factor gene. Mutations in SRY have been found in XY females, strongly implicating SRY as the testis-determining gene.     

Molecular analysis of the sex-determining region from the Y chromosome in two patients with Frasier syndrome.

In the Frasier syndrome there is an association between XY gonadal dysgenesis and chronic renal failure. Owing to an observed sex reversal, the Y chromosomes of two girls with this syndrome have been analyzed. Using molecular-biology techniques, no major alterations of the known sex-determining area of the Y chromosome were found. Furthermore, the sequence did not reveal impairment of the recently described testis-determining factor SRY. These data suggest that in the Frasier syndrome, XY sex reversal and renal failure could be the result of either faulty gene(s) located downstream in the sex differentiation pathway during embryogenesis, or impaired SRY regulation. Preliminary results on the Wilms' tumor suppressor gene WT1, a candidate for acting downstream to SRY, are also provided.     

中国人SRY基因的分离、克隆和核苷酸序列分析

睾丸决定因子基因(Testis-determining factor,TDF)位于Y染色体短臂上,它的表达产物诱导睾丸组织的发生。SRY基因(Sex-determining Region of the Y)位于Y染色体的性别决定区内,许多特征显示SRY就是TDF。我们选用与SRY基因相应的引物,用PCR技术对正常人男女各10例的基因组DNA进行扩增。将特异扩增的男性SRY基因片段重组到质粒PUC12中,得到含有中国人SRY基因片段的克隆,命名为PSY-1、PSY-2。用[~(32)p]标记重组质粒中的SRY基因片段作探针,与PCR结果进行Southern杂交,男性样品均显示特异杂交带,女性样品为阴性。用末端终止法测定克隆的SRY基因片段的全部核苷酸序列为299bp,含有SRY基因中高度保守及功能特异性区域的240bp,我们对此进行了讨论。     

Is ZFY the sex-determining gene on the human Y chromosome?

The sex-determining region of the human Y chromosome contains a gene, ZFY, that encodes a zinc-finger protein. ZFY may prove to be the testis-determining factor. There is a closely related gene, ZFX, on the human X chromosome. In most species of placental mammals, we detect two ZFY-related loci: one on the Y chromosome and one on the X chromosome. However, there are four ZFY-homologous loci in mouse: Zfy-1 and Zfy-2 map to the sex-determining region of the mouse Y chromosome, Zfx is on the mouse X chromosome, and a fourth locus is autosomal.     

Molecular analysis in true hermaphroditism: demonstration of low-level hidden mosaicism for Y-derived sequences in 46,XX cases

True hermaphroditism (TH) is an unusual form of sex reversal, characterized by the development of testicular and ovarian tissue in the same subject. Approximately 60% of the patients have a 46,XX karyotype, 33% are mosaics with a second cell line containing a Y chromosome, while the remaining 7% are 46,XY. Molecular analyses have demonstrated that SRY is present in only 10% of TH with a 46,XX karyotype; therefore, in the remaining 90%, mutations at unknown X-linked or autosomal sex determining loci have been proposed as factors responsible for testicular development. True hermaphroditism presents considerable genetic heterogeneity with several molecular anomalies leading to the dual gonadal development as SRY point mutations or SRY hidden gonadal mosaicism. In order to identify genetic defects associated with subjects with the disease, we performed molecular analyses of the SRY gene in DNA from blood leukocytes and gonadal tissue in 12 true hermaphrodites with different karyotypes. Our results using PCR and FISH analyses reveal the presence of hidden mosaicism for SRY or other Y sequences in some patients with XX true hermaphroditism and confirms that mosaicism for SRY limited to the gonads is an alternative mechanism for testicular development in 46,XX true hermaphrodites.     

A novel missense mutation (S18N) in the 5′ non-HMG box region of the SRY gene in a patient with partial gonadal dysgenesis and his normal male relatives

Mutations in the sex-determining region of the Y chromosome (the SRY gene) have been reported in low frequency in patients with 46,XY gonadal dysgenesis. We investigated 21 Brazilian 46,XY sex-reversed patients, who presented either complete or partial gonadal dysgenesis or embryonic testicular regression syndrome. Using Southern blotting, polymerase chain reaction, denaturing gradient gel electrophoresis and direct sequencing, we analyzed deletions and point mutations in the SRY gene. We found a missense mutation at codon 18 upstream of the 5′ border of the HMG box of the SRY gene in one patient with partial gonadal dysgenesis. This variant sequence was also found in DNA obtained from blood and sperm cells of his father and in blood cells of his normal brother. The S18N mutation was not found in 50 normal males, ruling out the possibility of a common polymorphism. We identified a novel familial missense mutation (S18N) in the 5’ non-HMG box of the SRY gene in 1 of 21 patients with 46,XY sex reversal. Received: 6 May 1997 / Accepted: 2 October 1997     

XY sex reversal associated with a nonsense mutation in SRY.

Sex determination in humans is mediated through the expression of a testis-determining gene on the Y chromosome. In humans, a candidate gene for the testis-determining factor (TDF) that encodes a protein with a putative DNA-binding motif and has been isolated is termed SRY. Here we describe an XY sex-reversed female with pure gonadal dysgenesis who harbors a de novo nonsense mutation in the SRY open reading frame (SRY-orf). This single-basepair substitution results directly in the formation of a termination codon in the putative SRY DNA-binding motif, presumably leading to a nonfunctional gene product. This brings the number of reported XY sex-reversed females with de novo mutations in the known SRY-orf to three, each occurring in the putative DNA-binding domain. This provides further evidence to support SRY being TDF in humans and also indicates the functional importance of the putative DNA-binding domain of the SRY protein.     

XY gonadal dysgenesis and gonadoblastoma: a study in two sisters with a cryptic deletion of the Y chromosome involving the SRY gene

This paper reports a case of XY gonadal dysgenesis in two sisters. Both patients presented an eunochoid female phenotype with normal external genitalia. At laparotomy, the elder sister was found to have bilateral gonadoblastoma. Cytogenetic studies, which included G and C banding and in situ hybridization, showed that the patients had an apparently normal 46, XY karyotype. PCR analyses revealed absence of the conserved portion (HMG box) of the SRY gene and of the Y chromosome pseudoautosomal boundary region sequence in both patients. The presence of the ZFY sequence was detected by Southern hybridization in the two affected sisters. The patients' father (46, XY, no mosaicism detected in peripheral blood lymphocytes) was positive for SRY and ZFY sequences. The occurrence of gonadoblastoma is discussed in terms of the genetic factors that may lead to tumor development.     

The rise and fall of SRY

Comparisons between species reveal when and how SRY, the testis-determining gene, evolved. SRY is younger than the Y chromosome, and so was probably not the original mammal sex-determining gene that defined the Y. SRY is typical of genes on the Y chromosome. It arose from a gene on the proto-sex chromosome pair with a function (possibly brain-determination) in both sexes. It has been buffeted in evolution, and shows variation in copy number, structure and expression. And it is dispensable, having been lost at least twice independently in different rodent lineages. At the observed rate of attrition, the human Y chromosome will be gone in 5-10 million years. This could lead to the extinction of our species or to a burst of hominid speciation.     

No evidence of mutations in four candidate genes for male sex determination/differentiation in sex-reversed XY females with campomelic dysplasia.

Campomelic dysplasia (Cd) occurs combined with sex reversal resulting in XY females. The recent identification of candidate genes for sex determination/differentiation and of a sex determining region on the human Y chromosome prompted the authors to study these genes for mutations in patients with Cd and sex reversal. In a total of five cases, no evidence for a mutation in the genes SRY, ZFY, ZFX, MEA and some anonymous Y-linked sequences was found. In addition to Southern analysis, gene expression of ZFY, ZFX and MEA was found to be normal as well. It is concluded that sex reversal in this condition is due to mutation in a so far unidentified gene which may act secondary to the testis-determining factor (TDF).     

The sex-determining zinc finger sequences in XY females of Akodon azarae (Rodentia, Cricetidae)

Wild populations of Akodon azarae comprise females with a karyotype indistinguishable from that of males. These individuals were formerly assumed to be Xx, the x being an X chromosome with a deletion of most of its long arm. By using a DNA probe derived from the testis-determining region of the human Y chromosome (comprising a candidate gene for the testis-determining factor, Y-linked zinc finger [ZFY]), we demonstrate that A. azarae gonosomally variant females are XY and not Xx. The ZFY sequences in A. azarae are amplified and located in two different families of EcoRI fragments derived from Y-chromosome DNA. No rearrangement or change in the state of methylation of ZFY or ZFX (X-linked zinc finger) sequences were found in XY females. We propose that sex reversal in A. azarae may be mediated by a gene or genes other than ZFX or ZFY.     

一例性反转畸形SRY基因片段的扩增、克隆和核苷酸序列分析

在哺乳动物中,位于Ｙ染色体上的指导雄性性别分化的基因被命名为睾丸决定因子（Ｔｅｓｔｉｓ－ｄｅｔｅｒｍｉｎｉｎｇｆａｃｔｏｒ,ＴＤＦ）１９９０年６月分离获得的ＳＲＹ基因（Ｓｅｘ－ｄｅｔｅｒｍｉｎｉｎｇｒｅｇｉｏｎｏｆｔｈｅＹ）被认为是ＴＤＦ基因最好的候选者［１－４］。ＳＲＹ基因为单拷贝,位于Ｙ染色体短臂末端１Ａ１Ａ区,靠近假常染色体配对区（ＰＡＰＲ）的交界处,其部分顺序编码８０个保守性氨基酸组成的多肽。本实验使用与ＳＲＹ基因相应的引物,利用ＰＣＲ技术以一例性反转畸形病人的基因组ＤＮＡ为模板分离ＳＲＹ基因保守区顺序,并将特异扩增出的此ＳＲＹ基因片段重组到质粒ｐＵＣ１２中,得到含有ＳＲＹ基因片段的克隆。经测序表明其ＳＲＹ基因保守顺序上有Ｔ→Ｃ（Ｓｅｒ→Ｐｒｏ）突变。ＳＲＹ基因的存在及其突变可能是导致性反转畸形发病的原因。     

Monozygotic twins of opposite sex

Although discordant karyotypes are known in identical twins, cases involving differences in sex phenotype are rare. We studied identical twins with the 46,XY karyotype - a male with mixed gonadal dysgenesis and a female with "pure" gonadal dysgenesis. The testis-determining SRY gene was present in DNA from both twins but no mutations were detected in the SRY conserved motif. Monozygosity was indicated by short tandem repeat polymorphism analysis. These observations could be attributed to (i) mutation and mosaicism involving "downstream" sex-determining loci, (ii) variable penetrance of genes such as DSS/NR0B1, duplication of which can disrupt the male-determining pathway, or (iii) occurrence of cryptic 45,X gonadal cell lines.     

SRY-negative XX sex reversal in a pony: a case report

A three year old pony with sexually ambiguous external genitalia was found to have a normal female karyotype (64, XX) and bilateral inguinal testes. The PCR analysis of blood samples revealed the absence of the Y chromosome sequences SRY, eTSPY and ZFY. No Y chromosome sequences were identified in DNA extracted from the gonads. The mechanism whereby XX sex reversal occurs in the absence of SRY is unknown.     

Tracing the emergence of a novel sex-determining gene in medaka, Oryzias luzonensis

Three sex-determining (SD) genes, SRY (mammals), Dmy (medaka), and DM-W (Xenopus laevis), have been identified to date in vertebrates. However, how and why a new sex-determining gene appears remains unknown, as do the switching mechanisms of the master sex-determining gene. Here, we used positional cloning to search for the sex-determining gene in Oryzias luzonensis and found that GsdfY (gonadal soma derived growth factor on the Y chromosome) has replaced Dmy as the master sex-determining gene in this species. We found that GsdfY showed high expression specifically in males during sex differentiation. Furthermore, the presence of a genomic fragment that included GsdfY converts XX individuals into fertile XX males. Luciferase assays demonstrated that the upstream sequence of GsdfY contributes to the male-specific high expression. Gsdf is downstream of Dmy in the sex-determining cascade of O. latipes, suggesting that emergence of the Dmy-independent Gsdf allele led to the appearance of this novel sex-determining gene in O. luzonensis.     

Sex chromosomes and sex determination in weird mammals

Weird mammals are of two types. Highly divergent mammals, such as the marsupials and monotremes, have informed us of the evolutionary history of the Y chromosome and sex-determining gene, and the recently specialized rodents can help us predict its future. The Y chromosome has had a short but eventful history, and is already heading briskly for oblivion. It originated as a homologous partner of the X when it acquired a sex-determining gene (not necessarily SRY). Most of the genes on the Y, even those with a male-specific function, evolved from genes now on the X. At the mercy of a high rate of variability and the forces of drift and selection, the Y has lost genes at a rate of 3-6 genes/million years, sparing those that acquired critical male-specific functions. Even these genes have disappeared from one mammalian lineage or another as their functions were usurped by genes elsewhere in the genome. The mammalian testis-determining gene, SRY, is a typical Y-borne gene. It arose by truncation of a gene (SOX3) on the X that is expressed in brain development, and it may work by interacting with (inhibiting?) related genes, including SOX9. Variant sex-determining systems in rodents show that the action of SRY can change, as it evidently has in the mouse, and SRY can be inactivated, as in akodont rodents, or even completely superseded, as in mole voles.     

Mutations in the Testis-Specific Enhancer of SOX9 in the SRY Independent Sex-Determining Mechanism in the Genus Tokudaia

SRY (sex-determining region Y) is widely conserved in eutherian mammals as a sex-determining gene located on the Y chromosome. SRY proteins bind to the testis-specific enhancer of SOX9 (TES) with SF1 to upregulate SOX9 expression in undifferentiated gonads of XY embryos of humans and mice. The core region within TES, named TESCO, is an important enhancer for mammalian sex determination. We show that TESCO of the genus Tokudaia lost enhancer activity caused by mutations in its SRY and SF1 binding sites. Two species of Tokudaia do not have the Y chromosome or SRY, and one species has multiple SRYs located on the neo-Y chromosome consisting of the Y fused with an autosome. The sequence of Tokudaia TESCO exhibited more than 83% identity with mouse TESCO, however, nucleotide substitution(s) were found in two out of three SRY binding sites and in five out of six SF1 binding sites. TESCO of all species showed low enhancer activity in cells co-transfected with SRY and SF1, and SOX9 and SF1 in reporter gene assays. Mutated TESCO, in which nucleotide substitutions found in SRY and SF1 binding sites were replaced with mouse sequence, recovered the activity. Furthermore, SRYs of the SRY-positive species could not activate the mutated TESCO or mouse TESCO, suggesting that SRYs lost function as a sex-determining gene any more. Our results indicate that the SRY dependent sex-determining mechanism was lost in a common ancestor of the genus Tokudaia caused by nucleotide substitutions in SRY and SF1 binding sites after emergence of a new sex-determining gene. We present the first evidence for an intermediate stage of the switchover from SRY to a new sex-determining gene in the evolution of mammalian sex-determining mechanism.     

Z and W chromosomes of chickens: studies on their gene functions in sex determination and sex differentiation

Since the discovery of SRY/SRY as a testis-determining gene on the mammalian Y chromosome in 1990, extensive studies have been carried out on the immediate target of SRY/SRY and genes functioning in the course of testis development. Comparative studies in non-mammalian vertebrates including birds have failed to find a gene equivalent to SRY/SRY, whereas they have suggested that most of the downstream factors found in mammals including SOX9 are also involved in the process of gonadal differentiation. Although a gene whose function is to trigger the cascade of gene expression toward gonadal differentiation has not been identified yet on either W or Z chromosomes of birds, a few interesting genes have been found recently on the sex chromosomes of chickens and their possible roles in sex determination or sex differentiation are being investigated. It is the purpose of this review to summarize the present knowledge of these sex chromosome-linked genes in chickens and to give perspectives and point out questions concerning the mechanisms of avian sex determination.