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.     

Molecular aspects of sex determination in mice: an alternative model for the origin of the Sxr region

Using a combination of in situ mapping and DNA analysis with recombinant DNA probes specific for the Sxr region of the mouse Y chromosome, we show that both the gene(s) controlling primary sex determination and the expression of the male-specific antigen H-Y (Tdy and Hya respectively) are located on the minute short arm of the mouse Y chromosome. We demonstrate that the H-Y- variant of Sxr (Sxr') arose by a partial deletion within the Sxr region and propose an alternative model for the generation of the original Sxr region.     

Localization of Murine X and Autosomal Sequences Homologous to the Human Y Located Testis-Determining Region

Recently a candidate gene for the primary testis-determining factor (TDF) encoding a zinc finger protein (ZFY) has been cloned from the human Y chromosome. A highly homologous X-linked copy has also been identified. Using this human sequence it is possible to identify two Y loci, an X and an autosomal locus in the mouse (Zfy-1, Zfy-2, Zfx and Zfa, respectively). Suprisingly ZFY is more homologous to the mouse X and autosomal sequences than it is to either of the Y-linked loci. Both Zfy-1 and Zfy-2 are present in the Sxr region of the Y but Zfy-2 is absent in the Sxr deletion variant Sxrb (or Sxr") suggesting it is not necessary for male determination. Extensive backcross analyses map Zfa to mouse chromosome 10 and Zfx to a 5-cM interval between anonymous X probe MDXS120 and the tabby locus (Ta). We also show that the mouse androgen receptor locus (m-AR) believed to underlie the testicular feminization mutation (Tfm) shows complete linkage to Zfx. Comparative mapping indicates that in man these genes lie in separate conserved DNA segments.     

A ZFY-negative 46,XX true hermaphrodite is positive for the Y pseudoautosomal boundary

Summary Two loci on the short arm of the human Y chromosome have recently been described as candidates for the testis determining factor (TDF); namely, ZFY, and a locus distal to ZFY, near the pseudoautosomal boundary. We have previously reported on seven 46,XX true hermaphrodites and one 45,X mixed gonadal dysgenesis case all presenting with testicular tissue in their gonads in the apparent absence of Y-specific DNA sequences. A reanalysis of these cases shows them all to lack ZFY, but one 46,XX true hermaphrodite carries sequences next to the Y pseudoautosomal boundary. This case provides further evidence for assigning the TDF locus very close to the pseudoautosomal region on Yp.     

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 ZFY gene family in humans and mice

For several years, ZFY (zinc finger gene on the Y chromosome) was considered the best candidate for the human testis-determining gene TDF. This gene and its close relatives have been intensely studied in the hope of understanding the molecular biology of sex determination, particularly in humans and mice. Now that there is overwhelming evidence that ZFY and TDF are distinct loci, we are left with a large body of data, and a question: what do these genes really do?     

Normal Testis Determination in the Mouse Depends on Genetic Interaction of a Locus on Chromosome 17 and the Y Chromosome

We previously described a locus on chromosome (Chr) 17 of the mouse that is critical for normal testis development. This locus was designated "T-associated sex reversal" (Tas) because it segregated with the dominant brachyury allele hairpin tail (Thp) and caused gonads of C57BL/6J XY, Thp/+ individuals to develop as ovaries or ovotestes rather than as testes. To clarify the inheritance of Tas, we investigated the effects of T-Orleans (TOrl), another brachyury mutation, on gonad development. We found that gonads of C57BL/6J XY, Thp/+ and TOrl/+ mice develop ovarian tissue if the Y chromosome is derived from the AKR/J inbred strain, whereas normal testicular development occurs in the presence of a Y chromosome derived from the C57BL/6J inbred strain. From these observations we conclude that: (1) Tas is located in a region on Chr 17 common to the deletions associated with Thp, and TOrl, and (2) the Y-linked testis determining gene, Tdy, carried by the AKR/J inbred strain differs from that of the C57BL/6J inbred strain. We suggest that in mammals Tdy is not the sole testis determinant because autosomal loci must be genetically compatible with Tdy for normal testicular development.     

Sex inversion as a model for the study of sex determination in vertebrates

As a consequence of genetic sex determination, the indifferent gonadal blastema normally becomes either a testis or an ovary. This applies to mammals and to the majority of non-mammalian vertebrates. With the exception of placental mammals, however, partial or complete sex inversion can be induced in one sex by sexual steroid hormones of the opposite sex during a sensitive period of gonadogenesis. There is evidence that also during normal gonadogenesis in these species, in the XY/XX mechanism of sex determination testicular differentiation is induced by androgens, and in the ZZ/ZW mechanism, ovarian differentiation by oestrogens. In either case, the hormones may act via serological H-Y antigen as a morphogenetic factor. In contrast, in placental mammals including man, primary gonadal differentiation is independent of sexual steroid hormones, and factors directing differential gonadal development have not yet been conclusively identified. However, various mutations at the chromosome or gene level, resulting respectively in sex inversion or intersexuality, have provided clues as to some genes involved and their possible nature. In this context also, serological H-Y antigen is discussed as a possible factor acting on primordial gonadal cells and inducing differential growth or morphogenesis or both. The data available at present allow a tentative outline of the genetics of sex determination in placental mammals.     

Mammalian genome evolution: new clues from comparisons of eutherians, marsupials and monotremes.

1. Comparisons of chromosomes and gene maps of different mammals are yielding a big picture of the evolution of mammalian genome form and function. It has been particularly instructive to compare gene arrangements on the sex chromosomes between the three major groups of mammals. Eutheria (so-called placental mammals). Metatheria (marsupials) and Prototheria (monotremes), which diverged 150 and 170 Myr BP respectively. 2. A region amounting to 3% of the haploid genome is located on the X chromosome in all three groups, implying that this region must have been part of the original X in a common ancestor. This region comprises the long arm of the human X. 3. A region represented by the short arm of the human X is common to the X in all eutherians, but is autosomal in marsupials and monotremes; thus it was not a part of the original X, and must have been acquired by the X early in the eutherian radiation. 4. This recently acquired region was probably translocated to a pseudoautosomal region shared by the eutherian X and Y. Thus it was originally paired and exempt from X chromosome inactivation; stepwise deletion of this region from the Y and recruitment of the newly unpaired region of the X into the inactivation system could account for some of the peculiarities of this region of the human X. 5. The sex-determining gene TDF must lie on the Y in all mammals in which the Y is male determining. The autosomal location of the candidate gene ZFY in marsupials and monotremes eliminates it from consideration. The recently described candidate gene SRY has yet to pass the "marsupial test".     

A bird zinc-finger protein closely related to ZFY

The ZFY gene is thought to reside in the "sex-determining" region of the mammalian Y chromosome and encodes a zinc-finger protein that may function in determining the sex of embryos. Although birds have a ZZ(male)/ZW(female) sex-determination system, they possess a gene, Zfb, that is highly homologous to ZFY. We used ZFY as a hybridization probe to clone the zinc-finger domain of the chicken Zfb gene. Chicken Zfb is widely transcribed in male and female tissues and encodes a protein with a zinc-finger domain that is 93% identical in amino acid sequence to the zinc-finger domain of ZFY. Thus, the putative DNA-binding domains of the Zfb and ZFY proteins diverged little from a common ancestral protein that existed prior to birds and mammals, suggesting that the DNA binding site has been similarly conserved. The absence of sex differences in the hybridization patterns of Zfb raises the question of whether this gene is present on the Z/W sex chromosomes in birds.     

The role of the sex-determining region of the Y chromosome (SRY) in the etiology of 46,XX true hermaphroditism

Summary The syndrome of 46,XX true hermaphroditism is a clinical condition in which both ovarian and testicular tissue are found in one individual. Both Mullerian and Wolffian structures are usually present, and external genitalia are often ambiguous. Two alternative mechanisms have been proposed to explain the development of testicular tissue in these subjects: (1) translocation of chromosomal material encoding the testicular determination factor (TDF) from the Y to the X chromosome or to an autosome, or (2) an autosomal dominant mutation that permits testicular determination in the absence of TDF. We have investigated five subjects with 46,XX true hermaphroditism. Four individuals had a normal 46,XX karyotype; one subject (307) had an apparent terminal deletion of the short arm of one X chromosome. Genomic DNA was isolated from these individuals and subjected to Southern blot analysis. Only subject 307 had Y chromosomal sequences that included the pseudoautosomal boundary, SRY (sex-determining region of Y), ZFY (Y gene encoding a zinc finger protein), and DXYS5 (an anonymous locus on the distal short arm of Y) but lacked sequences for DYZ5 (proximal short arm of Y) and for the long arm probes DYZ1 and DYZ2. The genomic DNA of the other four subjects lacked detectable Y chromosomal sequences when assayed either by Southern blotting or after polymerase chain reaction amplification. Our data demonstrate that 46,XX true hermaphroditism is a genetically heterogeneous condition, some subjects having TDF sequences but most not. The 46,XX subjects without SRY may have a mutation of an autosomal gene that permits testicular determination in the absence of TDF.     

Molecular Method for Determining Sex of Walruses

Abstract: We evaluated the ability of a set of published trans-species molecular sexing primers and a set of walrus-specific primers, which we developed, to accurately identify sex of 235 Pacific walruses (Odobenus rosmarus divergens). The trans-species primers were developed for mammals and targeted the X- and Y-gametologs of the zinc finger protein genes (ZFX, ZFY). We extended this method by using these primers to obtain sequence from Pacific and Atlantic walrus (O. r. rosmarus) ZFX and ZFY genes to develop new walrus-specific primers, which yield polymerase chain reaction products of distinct lengths (327 and 288 base pairs from the X- and Y-chromosome, respectively), allowing them to be used for sex determination. Both methods yielded a determination of sex in all but 1–2% of samples with an accuracy of 99.6–100%. Our walrus-specific primers offer the advantage of small fragment size and facile application to automated electrophoresis and visualization.     

Sry promoters from domesticus (Tirano) and C57BL/6 mice function similarly in embryos and adult animals.

Introduction of the Y chromosome from a Mus musculus domesticus (Tirano) subspecies into the Mus musculus musculus C57BL/6 (B6) inbred strain background results in sex reversal in XY offspring. It has been hypothesized that the domesticus testis-determining Y (Tdy) locus is misregulated in B6 genome, thereby impairing sex determination in B6.Y(Dom) animals. The identification of a gene in the sex-determining region on the Y chromosome (Sry) as the Tdy has provided a means to experimentally examine this hypothesis. We have generated several lines of B6 transgenic mice harboring a green fluorescent protein gene directed by a Sry promoter from the domesticus (Tirano) Y chromosome. Detailed analysis of the transgene expression was conducted in both fetal and adult tissues of the transgenic mice. The domesticus Sry promoter was capable of directing the expression of the green fluorescent protein gene in a pattern similar, if not identical, to that of the endogenous B6 Sry gene. These observations suggest that the domesticus Sry promoter is not involved in the postulated misregulation of the domesticus (Tirano) Sry gene in the B6 genomic background. These results are discussed with reference to a second hypothesis invoking incompatible protein interaction(s) as a mechanism of aberrant sex determination in B6.Y(Dom) animals.     

X-linked genes of the H-Y antigen system in the wood lemming (Myopus schisticolor)

Summary H-Y antigen was investigated in 18 specimens representing six different sex chromosome constitutions of the wood lemming (Myopus schisticolor). The control range of H-Y antigen was defined by the sex difference between normal XX females (H-Y negativeper definitionem) and normal XY males (H-Y positive, full titer). H-Y antigen titers of the X^*Y and X^*0 females were in the male control range, while in the X^*X and X0 females the titers were intermediary. Data were obtained with two different H-Y antigen assays: the Raji cell cytotoxicity test and the peroxidase-antiperoxidase (PAP) method. Fibroblasts, gonadal cells, and spleen cells were checked. Presence of full titers of H-Y antigen in the absence of testis differentiation is readily explained by the assumption of a deficiency of the gonadspecific receptor of H-Y antigen. Since sex reversal is inherited as an X-linked trait, genes for this receptor are most likely X-linked. The implications of our findings are discussed in connection with earlier findings concerning H-Y antigen in XY gonadal dysgenesis in man and the X0 situation in man and mouse.     

True hermaphroditism in a 46,XY individual, caused by a postzygotic somatic point mutation in the male gonadal sex-determining locus (SRY): molecular genetics and histological findings in a sporadic case.

Recently, the gene for the determination of maleness has been identified in the sex-determining region on the short arm of the Y chromosome (SRY) between the Y-chromosomal pseudoautosomal boundary (PABY) and the ZFY gene locus. Experiments with transgenic mice confirmed that SRY is a part of the testis-determining factor (TDF). We describe a sporadic case of a patient with intersexual genitalia and the histological finding of ovotestes in the gonad, which resembles the mixed type of gonadal tissue without primordial follicle structures. The karyotype of the patient was 46,XY. By PCR amplification, we tested for the presence of PABY, SRY, and ZFY by using DNA isolated from peripheral blood leukocytes and for the presence of SRY by using DNA obtained from histological gonadal slices. The SRY products of both DNA preparations were further analyzed by direct sequencing. All three parts of the sex-determining region of the Y chromosome could be amplified from leukocytic DNA. The patient's and the father's SRY sequences were identical with the published sequence. In the SRY PCR product of gonadal DNA, the wild-type and two point mutations were present in the patient's sequence, simulating a heterozygous state of a Y-chromosomal gene: one of the mutations was silent, while the other encoded for a nonconservative amino acid substitution from leucine to histidine. Subcloning procedures showed that the two point mutations always occurred together. The origin of the patient's intersexuality is a postzygotic mutation of the SRY occurring in part of the gonadal tissue. This event caused the loss of the testis-determining function in affected cells.     

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.     

Gene SRY et anomalies de la determination genetique du sexe chez l’homme

Normal sexual development in man is the consequence of a complex process. This review focuses on the translation of genedal sex (XX or XY karyotype) into gonadal sex (testis or ovary). During the last three years attempts to identify and clone the testis determining factor (TDF) have exploited detailed maps of the Y chromosome established by geneticists over the last decade. A candidate gene, named SRY (sex determining region, Y) located at the tip of the short arm of the Y chromosome, shows many characteristics in common with TDF in that it is the sole element of the Y chromosome required for male development. The discovery of TDF led us to analyse sex-reversed individuals, i.e. XX males and XY females, with the aim of constructing a model for the processes regulating the development of an organ as complex as the testis. This SRY gene is now the subject of intense molecular biological effort by various groups, effort which we hope will elucidate the mechanism(s) of sex determination.     

Interfamilial characterization of a region of the ZFX and ZFY genes facilitates sex determination in cetaceans and other mammals

Sequence polymorphism of homologues ZFX and ZFY, in a 604-base pair exon region, was examined in 10 known males and 10 known females across seven cetacean families and used to design a simple, highly sensitive and widely applicable fluorescent 5' exonuclease assay for gender determination in cetaceans. Multiplex amplification, cloning, and sequencing of these previously uncharacterized regions revealed (i) eight fixed differences between ZFX and ZFY, (ii) 29 variable sites between ZFX and ZFY and (iii) very low interspecific nucleotide diversity for both ZFX and ZFY across all families examined. We developed a 5' exonuclease assay that produces a small (105 bp) polymerase chain reaction (PCR) product from both the X and the Y chromosome orthologs, and used double-labelled fluorescent probes to distinguish between the two genes in a real-time PCR assay that is highly reproducible and sensitive. We demonstrated sex specificity for 33 cetacean species in nine families. Given the availability of conserved primers and sequence information for many mammalian species, this approach to designing sexing assays for a wide range of species is both practical and efficient.