Multiple mono- and polymorphic Y-linked copies of the SRY HMG-box in microtidae.

Sex determination in mammals is controlled by SRY (sex-determining region of the Y chromosome), a single-copy gene located on the Y-specific region. Several exceptions to this rule have been described: some rodent species present Y-specific multiple copies (either mono- or polymorphic) of this gene, and two Ellobius species and one Tokudaia species determine sex without a Y chromosome or the SRY gene. Recently, we have described multiple polymorphic copies of the SRY gene in both males and females of the vole species Microtus cabrerae. The female location and the presence of stop codons in some copies from males and females also suggest that they are nonfunctional copies of this gene (pseudogenes). We have investigated the SRY HMG-box in nine species of the family Microtidae; we report here the presence, in eight of these species, of multiple mono- or polymorphic copies of the SRY gene located on the Y chromosome.     

Origin and spread of the SRY gene on the X and Y chromosomes of the rodent Microtus cabrerae: role of L1 elements

In the rodent species Microtus cabrerae, males as well as females present several copies of the SRY gene, a single-copy gene located on the Y chromosome in most mammals. Using different PCR approaches, we have characterized the sequence, structure, and organization of the SRY copies and their flanking regions distributed on the X and Y chromosomes of this species. All copies of SRY analyzed, including those from the Y chromosome, proved to be nonfunctional pseudogenes, as they have internal stop codons. In addition, we demonstrated the association of SRY pseudogenes with different fragments of L1 and LTR retroelements in both sex chromosomes of M. cabrerae. Examining the possible origin of SRY pseudogene and retroposons association, we propose that retroposons could have been involved in the mechanism of SRY gene amplification on the Y chromosome and in the transference of the Y-linked SRY copies to the X-chromosome heterochromatin.     

棕色田鼠性别决定研究进展

哺乳动物性别决定方式属于雄性异配型性别决定, 依赖于Y染色体, SRY基因是性别决定中最重要的基因。文章报道了棕色田鼠指名亚种有Y染色体, 但是Y染色体上没有SRY基因, 性别决定不依赖于SRY基因, 排除了R-spondin 1基因是性别决定基因, 同时讨论了棕色田鼠指名亚种SRY基因缺失后可能的性别决定 机制。     

Mapping the SRY gene in Microtus cabrerae: a vole species with multiple SRY copies in males and females.

The SRY gene is a single-copy, male-specific gene, located on the Y chromosome in most mammals. However, recently we have described the presence of multiple polymorphic copies of this gene in both males and females of the vole species Microtus cabrerae. Here, we present the chromosomal localization of SRY gene copies in this species by fluorescent in situ hybridization (FISH). This technique localized these gene copies in the short arm, and hence in the euchromatic region, of the Y chromosome. Furthermore, several copies of the SRY gene are located on the X chromosome. These copies are spread along the entire heterochromatic region of the X chromosome, occupying the whole short arm, the centromeric region, and the pericentromeric region of the long arm.     

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。     

Sexual development in marsupials: genetic characterization of bandicoot siblings with scrotal and testicular maldevelopment

In marsupials testis determination requires the presence of a Y chromosome. The sex determining region on the Y gene (SRY) is necessary for testicular development in eutherians and it is assumed to play a similar role in marsupials. Relatively few studies have investigated the genetic basis of sexual development, and as yet there is no direct evidence that SRY is required for testis development in marsupials. Studies on intersexual marsupials have revealed a fundamental difference between marsupial and eutherian sex determination. The scrotum of marsupials is analogous, not homologous, to the eutherian scrotum and is under the control of X-linked genes not androgens. The current study describes two bandicoot (Isoodon macrourus) siblings. Both siblings had underdeveloped male reproductive tracts and testicular dysgenesis, one was ascrotal and the other had a diminutive scrotum. Their karyotypes were normal for this species which eliminates the Y chromosome from some somatic tissues. SRY was detected by Southern blotting. SRY, ubiquitin activating enzyme-1 on the Y (UBE1Y) and glucose 6-phosphate dehydrogenase (G6PD) gene expression were examined. UBE1Y was widely expressed in many tissues. SRY gene expression was much lower than normal in the abnormal siblings and may be responsible for their failure of testicular and epididymal development. The cause of their scrotal abnormalities is unknown. It is possible that the separate defects of scrotal and testis development in the two siblings, which had normal relatives, were due to a mutation in a gene common to both developmental pathways.     

The sex-determining region Y (SRY) gene is mapped to p12-p13 of the Y chromosome in pig (Sus scrofa domestica) by in situ hybridization

The sex-determining region Y is a gene located in the distal portion of the short arm of human (SRY) and mouse (Sry) Y chromosomes and considered to be the best candidate for the testis determining factor (TDF/Tdy). The gene is believed to be the key factor in sex differentiation in mammals and is conserved across mammalian species. We report herein that the SRY/Sry gene has been assigned to pi 2-p13 on the short arm of the Y chromosome in pig by in situ hybridization. The result confirms interspecies conservation of this chromosomal segment in the evolution of mammalian chromosomes, and suggests further use of this gene probe in genomic studies in other mammals. The assignment of the Sry gene is the second physical gene mapping data available for the Y chromosome in pigs. Such data can be used in the effort of constructing the pig gene map and for further establishment of a comparison of sex chromosome morphology in different mammalian species concerning sex-specific and pseudoautosomal regions.     

A sporadic case of the sex-reversed mare (64,XY; SRY-negative): molecular and cytogenetic studies of the Y chromosome

A sex-reversal syndrome appears frequently in the horse. The mare carriers of this syndrome lack of SRY gene. It is suggested that sex-reversal syndrome is probably caused by transfer of the SRY gene from Y to the X chromosome, due to abnormal meiotic exchange. The aim of the study was molecular analysis of the Y-linked genes in a case of the sex-reversed infertile mare with 64,XY karyotype. The karyotype was established on the basis of analysis of 350 metaphase spreads stained by CBG banding. Molecular analysis of the loci assigned to the Y chromosome revealed absence of the SRY gene and presence of the other studied loci (ZFY, AMEL-Y and STS-Y). In this animal all fragments representing X chromosome (ZFX, AMEL-X and STS-X) were detected. External genitalia in the mare were normal, uterus was small and ovaries (examined by ultrasonography) extremely small. The mechanism of sex-reversal syndrome formation was discussed. It is postulated that during spermatogenesis in the sire two crossing-over events between the X and Y chromosomes occurred. One of them took place between the ZFY and SRY loci and another one between the SRY locus and the centromere.     

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。     

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.     

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.     

High levels of nucleotide diversity in the European rabbit (Oryctolagus cuniculus) SRY gene

We have sequenced 2,388 bp of the European rabbit sex determining region Y (SRY) gene. These data provide a 10-fold increase in the coverage of the Y chromosome in this species, including the entire open reading frame of the SRY, the polyadenylation signal, and two repetitive sequences in the 5' -region. A survey of 2021 bp of this gene in eight domestic breeds and four wild individuals revealed a total of nine single nucleotide polymorphisms and one indel, defining two deeply divergent lineages. The resulting estimation of nucleotide diversity (pi=1.34 x10(-3)) is very high when compared with other species, but no variability was detected among the domestic breeds. This study represents a first step in the characterization of the European rabbit Y chromosome and its variability. These sequences can be used in additional phylogeographical analyses of the European rabbit and other Leporid species, as well as in evolutionary studies of sex determination and the Y chromosome in wild species.     

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.     

HUMAN CHROMOSOME Y AND SRY

The precise location of the SRY gene on the human Y chromosome has been revealed through studies of sex reversal cases involving deletion, cross-linking and mutations of the SRY gene. Its DNA sequence and mechanism of action are being understood. Similarity of SRY with Sry of mice and its interaction with other genes in male sex determination are discussed.     

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.     

The role of the Y-chromosome in sex determination.

The basic plan of gonadal development in both sexes is female unless testes are induced by factor(s) of the Y chromosome, known as testis determining factor(s) (TDF). It is not clearly established whether the Y chromosome control is autonomous or under the control of a gene on the X chromosome or autosomes. A gene for the H-Y antigen (Histocompatibility-Y antigen) has been postulated to be the factor determining testicular differentiation. Recent studies have demonstrated that the gene for testis determination and the H-Y determinant are two separate entities. Although earlier cytogenetic observations localized TDF on the pericentric region of the short arm of the Y chromosome, subsequent findings by high-resolution chromosome banding and molecular analysis localise TDF to the distal part of the short arm of the Y chromosome, adjacent to the pseudoautosomal region. A candidate for TDF, the ZFY, was localised within the 140 kb interval where the position of TDF was defined, and considered as the TDF gene. However, a smaller gene sequence of 35 kb, the SRY, situated outside the 140 kb ZFY region, has recently been isolated and proved to be the only and the smallest part of the Y chromosome necessary for male sex determination.     

Mammalian sex--Origin and evolution of the Y chromosome and SRY

Sex determination in vertebrates is accomplished through a highly conserved genetic pathway. But surprisingly, the downstream events may be activated by a variety of triggers, including sex determining genes and environmental cues. Amongst species with genetic sex determination, the sex determining gene is anything but conserved, and the chromosomes that bear this master switch subscribe to special rules of evolution and function. In mammals, with a few notable exceptions, female are homogametic (XX) and males have a single X and a small, heterochromatic and gene poor Y that bears a male dominant sex determining gene SRY. The bird sex chromosome system is the converse in that females are the heterogametic sex (ZW) and males the homogametic sex (ZZ). There is no SRY in birds, and the dosage-sensitive Z-borne DMRT1 gene is a credible candidate sex determining gene. Different sex determining switches seem therefore to have evolved independently in different lineages, although the complex sex chromosomes of the platypus offer us tantalizing clues that the mammal XY system may have evolved directly from an ancient reptile ZW system. In this review we will discuss the organization and evolution of the sex chromosomes across a broad range of mammals, and speculate on how the Y chromosome, and SRY, evolved.     

Evolution of the male-determining gene SRY within the cat family Felidae

In most placental mammals, SRY is a single-copy gene located on the Y chromosome and is the trigger for male sex determination during embryonic development. Here, we present comparative genomic analyses of SRY (705 bp) along with the adjacent noncoding 5' flank (997 bp) and 3' flank (948 bp) in 36 species of the cat family Felidae. Phylogenetic analyses indicate that the noncoding genomic flanks and SRY closely track species divergence. However, several inconsistencies are observed in SRY. Overall, the gene exhibits purifying selection to maintain function (omega = 0.815) yet SRY is under positive selection in two of the eight felid lineages. SRY has low numbers of nucleotide substitutions, yet most encode amino acid changes between species, and four different species have significantly altered SRY due to insertion/deletions. Moreover, fixation of nonsynonymous substitutions between sister taxa is not consistent and may occur rapidly, as in the case of domestic cat, or not at all over long periods of time, as observed within the Panthera lineage. The former resembles positive selection during speciation, and the latter purifying selection to maintain function. Thus, SRY evolution in cats likely reflects the different phylogeographic histories, selection pressures, and patterns of speciation in modern felids.     

Sexing free-ranging brown bears Ursus arctos using hairs found in the field

As an aid to the management of the Pyrenean population of the brown bear Ursus arctos , a sexing method based on the amplification of a Y chromosome specific sequence has been developed, and tested using hairs found in the field as a source of DNA. This method involves a two-step polymerase chain reaction (PCR) which allows the detection of a very small amount of DNA, probably a single SRY gene molecule. The sex can reliably be identified using about 50pg of DNA extract as template. It is possible that this approach could, with adjustments, be used to identify the sex of other species of eutherian mammals.