Accidental X-Y recombination and the aetiology of XX males and true hermaphrodites

Accidental recombination between the differential segments of the X and Y chromosomes in man occasionally allows transfer of Y-linked sequences to the X chromosome leading to testis differentiation in so-called XX males. Loss of the same sequences by X-Y interchange allows female differentiation in a small proportion of individuals with XY gonadal dysgenesis. A candidate gene responsible for primary sex determination has recently been cloned from within this part of the Y chromosome by Page and his colleagues. The observation that a homologue of this gene is present on the short arm of the X chromosome and is subject to X-inactivation, raises the intriguing possibility that sex determination in man is a quantitative trait. Males have two active doses of the gonad determining gene, and females have one dose. This hypothesis has been tested in a series of XX males, XY females and XX true hermaphrodites by using a genomic probe, CMPXY1, obtained by probing a Y-specific DNA library with synthetic oligonucleotides based on the predicted amino-acid sequence of the sex-determining protein. The findings in most cases are consistent with the hypothesis of homologous gonad-determining genes, GDX and GDY, carried by the X and Y chromosomes respectively. It is postulated that in sporadic or familial XX true hermaphrodites one of the GDX loci escapes X-inactivation because of mutation or chromosomal rearrangement, resulting in mosaicism for testis and ovary-determining cell lines in somatic cells. Y-negative XX males belong to the same clinical spectrum as XX true hermaphrodites, and gonadal dysgenesis in some XY females may be due to sporadic or familial mutations of GDX.     

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.     

Normal structure and expression of Zfy genes in XY female mice mutant in Tdy.

Zfy-1 and Zfy-2 are candidate genes for Tdy, the testis-determining gene in mice. We have analysed these genes in a line of XY female mice that have been shown to be mutated in Tdy. We have used Southern blot analysis to show that the Zfy genes have not undergone any major structural alterations, and have also demonstrated that both genes are transcribed normally from the mutant Y chromosome (Y) in both adult XYY testis and XY female embryonic gonads. The fact that these genes show a normal structure and expression pattern in mice with a Y chromosome known to carry a mutation in Tdy and that mutant embryos develop into females despite Zfy-1 expression, strongly supports other recent evidence that Zfy genes are not directly involved in primary testis determination.     

Wild-derived XY sex-reversal mutants in the Medaka, Oryzias latipes

The medaka, Oryzias latipes, has an XX/XY sex-determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a sex-determining gene in this species. Previously, we found 23 XY sex-reversed females from 11 localities by examining the genotypic sex of wild-caught medaka. Genetic analyses revealed that all these females had Y-linked gene mutations. Here, we aimed to clarify the cause of this sex reversal. To achieve this, we screened for mutations in the amino acid coding sequence of DMY and examined DMY expression at 0 days after hatching (dah) using densitometric semiquantitative RT-PCR. We found that the mutants could be classified into two groups. One contained mutations in the amino acid coding sequence of DMY, while the other had reduced DMY expression at 0 dah although the DMY coding sequence was normal. For the latter, histological analyses indicated that YwOurYwOur (YwOur, Y chromosome derived from an Oura XY female) individuals with the lowest DMY expression among the tested mutants were expected to develop into females at 0 dah. These results suggest that early testis development requires DMY expression above a threshold level. Mutants with reduced DMY expression may prove valuable for identifying DMY regulatory elements.     

C57BL/6J-T-associated sex reversal in mice is caused by reduced expression of a Mus domesticus Sry allele

C57BL/6J-T-associated sex reversal (B6-TAS) in XY mice results in ovarian development and involves (1) hemizygosity for Tas, a gene located in the region of Chromosome 17 deleted in T(hp) and T(Orl), (2) homozygosity for one or more B6-derived autosomal genes, and (3) the presence of the AKR Y chromosome. Here we report results from experiments designed to investigate the Y chromosome component of this sex reversal. Testis development was restored in B6 T(Orl)/+ XY(AKR) mice carrying a Mus musculus Sry transgene. In addition, two functionally different classes of M. domesticus Sry alleles were identified among eight standard and two wild-derived inbred strains. One class, which includes AKR, did not initiate normal testis development in B6 T(Orl)/+ XY mice, whereas the other did. DNA sequence analysis of the Sry ORF and a 5' 800-bp segment divided these inbred strains into the same groups. Finally, we found that Sry is transcribed in B6 T(Orl)/+ XY(AKR) fetal gonads but at a reduced level. These results pinpoint Sry as the Y-linked component of B6-TAS. We hypothesize that the inability of specific M. domesticus Sry alleles to initiate normal testis development in B6 T(Orl)/+ XY(AKR) mice results from a biologically insufficient level of Sry expression, allowing the ovarian development pathway to proceed.     

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.     

Sex reversal in C57BL/6J XY mice caused by increased expression of ovarian genes and insufficient activation of the testis determining pathway

Sex reversal can occur in XY humans with only a single functional WT1 or SF1 allele or a duplication of the chromosome region containing WNT4. In contrast, XY mice with only a single functional Wt1, Sf1, or Wnt4 allele, or mice that over-express Wnt4 from a transgene, reportedly are not sex-reversed. Because genetic background plays a critical role in testis differentiation, particularly in C57BL/6J (B6) mice, we tested the hypothesis that Wt1, Sf1, and Wnt4 are dosage sensitive in B6 XY mice. We found that reduced Wt1 or Sf1 dosage in B6 XY(B6) mice impaired testis differentiation, but no ovarian tissue developed. If, however, a Y(AKR) chromosome replaced the Y(B6) chromosome, these otherwise genetically identical B6 XY mice developed ovarian tissue. In contrast, reduced Wnt4 dosage increased the amount of testicular tissue present in Sf1+/- B6 XY(AKR), Wt1+/- B6 XY(AKR), B6 XY(POS), and B6 XY(AKR) fetuses. We propose that Wt1(B6) and Sf1(B6) are hypomorphic alleles of testis-determining pathway genes and that Wnt4(B6) is a hypermorphic allele of an ovary-determining pathway gene. The latter hypothesis is supported by the finding that expression of Wnt4 and four other genes in the ovary-determining pathway are elevated in normal B6 XX E12.5 ovaries. We propose that B6 mice are sensitive to XY sex reversal, at least in part, because they carry Wt1(B6) and/or Sf1(B6) alleles that compromise testis differentiation and a Wnt4(B6) allele that promotes ovary differentiation and thereby antagonizes testis differentiation. Addition of a "weak" Sry allele, such as the one on the Y(POS) chromosome, to the sensitized B6 background results in inappropriate development of ovarian tissue. We conclude that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, this dosage-sensitivity is genetic background-dependant, and the mouse strains described here are good models for the investigation of human dosage-sensitive XY sex reversal.     

Quantitative analysis of sex-chromosome mosaicism with X-Y DNA probes.

Sex-chromosome mosaicism was quantitatively analyzed in two patients using DNA probes specific for human X and Y chromosomes. Both patients were female with stigmata of the Turner syndrome, and both had a 45,X cell line and a 46,XY cell line. One of the patients had a morphologically abnormal, nonfluorescent Y chromosome, dic(Y)(q11). Hybridization of DNA from this patient with two repetitive DNA sequences specific for the heterochromatic region of the Y chromosome indicated that most of the Y-heterochromatic sequences were deleted. DNA from both patients was hybridized with a probe for the DXYS1 locus and found to have the X- and Y-linked loci. Densitometric measurements of the relative intensities of the X- and Y-linked bands were used to calculate the degree of mosaicism in each case. The percentages of 45,X cells obtained by DNA analysis agreed with those obtained by chromosome analysis. DNA analysis provides a way to quantitate mosaicism at the DNA level and in nondividing tissue.     

Molecular dissection of the human Y-chromosome

Human Y chromosome, earlier thought to be gene deficient, has attracted a great deal of attention owing to its supremacy in male sex determination and unique haplotype status in the genome. Studies on Y chromosome have shown the presence of different types of satellite DNA and several genes implicated with a variety of physical and physiological functions. The interaction of these repetitive DNA with genes in normal individuals and in patients with Y-chromosome-related genetic anomalies is still an unresolved issue and is actively being pursued. The fast changing scenario of the human genome project is likely to effect our overall understanding of the Y chromosome and Y-linked genetic anomalies in a big way. We provide a brief overview of the organization of Y chromosome with respect to several important loci encompassing both the arms and their likely involvement/modulation in genetic anomalies. The experimental approaches discussed here are envisaged to be of clinical relevance for the molecular diagnosis of the Y-linked disorders.     

Molecular heterogeneity of XY sex reversal in horses

Male-to-female 64,XY sex reversal is a frequently reported chromosome abnormality in horses. Despite this, the molecular causes of the condition are as yet poorly understood. This is partially because only limited molecular information is available for the horse Y chromosome (ECAY). Here, we used the recently developed ECAY map and carried out the first comprehensive study of the Y chromosome in XY mares (n=18). The integrity of the ECAY in XY females was studied by FISH and PCR using markers evenly distributed along the euchromatic region. The results showed that the XY sex reversal condition in horses has two molecularly distinct forms: (i) a Y-linked form that is characterized by Y chromosome deletions and (ii) a non-Y-linked form where the Y chromosome of affected females is molecularly the same as in normal males. Further analysis of the Y-linked form (13 cases) showed that the condition is molecularly heterogeneous: the smallest deletions spanned about 21 kb, while the largest involved the entire euchromatic region. Regardless of the size, all deletions included the SRY gene. We show that the deletions were likely caused by inter-chromatid recombination events between repeated sequences in ECAY. Further, we hypothesize that the occurrence of SRY-negative XY females in some species (horse, human) but not in others (pig, dog) is because of differences in the organization of the Y chromosome. Finally, in contrast to the Y-linked SRY-negative form of equine XY sex reversal, the molecular causes of SRY-positive XY mares (5 cases) remain as yet undefined.     

XY female mice resulting from a heritable mutation in the primary testis-determining gene, Tdy

Chimeric mice constructed with XY embryonic stem (ES) cells that had been multiply infected with a retroviral vector were used in a genetic screen to look for mutations affecting the sex determination pathway in mice. From a small number of chimeras screened one was identified that gave rise to a low proportion of XY females amongst his offspring. Analysis of the segregating patterns of retroviral insertions demonstrated that the mutation was found in a subset of the offspring derived from one originally infected ES cell. However, the mutation appeared to have occurred subsequent to the infection. Some of the XY females proved to be fertile, and the mutant phenotype was found to segregate exclusively with the Y chromosome. Analysis of the offspring also confirmed the absence of any retroviral insertion that could be correlated with the mutation. Further characterisation of the Y chromosome carrying the mutation by karyotypic analysis, and by Southern blotting with a range of Y-specific DNA probes suggested that there has been no gross deletion or rearrangement of the Y carrying the mutation. There also appeared to be no loss of Y-specific gene functions apart from that of testis determination. Moreover, the mutation is complemented by Sxr', the minimum portion of the mouse Y known to carry Tdy. From the phenotype and deduced location of the mutation, we conclude that it is within the Tdy locus. This is the first such mutation to be described in mice.     

Minor abnormalities of testis development in mice lacking the gene encoding the MAPK signalling component, MAP3K1

In mammals, the Y chromosome is a dominant male determinant, causing the bipotential gonad to develop as a testis. Recently, cases of familial and spontaneous 46,XY disorders of sex development (DSD) have been attributed to mutations in the human gene encoding mitogen-activated protein kinase kinase kinase 1, MAP3K1, a component of the mitogen-activated protein kinase (MAPK) signal transduction pathway. In individuals harbouring heterozygous mutations in MAP3K1, dysregulation of MAPK signalling was observed in lymphoblastoid cell lines, suggesting a causal role for these mutations in disrupting XY sexual development. Mice lacking the cognate gene, Map3k1, are viable and exhibit the eyes open at birth (EOB) phenotype on a mixed genetic background, but on the C57BL/6J genetic background most mice die at around 14.5 dpc due to a failure of erythropoiesis in the fetal liver. However, no systematic examination of sexual development in Map3k1-deficient mice has been described, an omission that is especially relevant in the case of C57BL/6J, a genetic background that is sensitized to disruptions to testis determination. Here, we report that on a mixed genetic background mice lacking Map3k1 are fertile and exhibit no overt abnormalities of testis development. On C57BL/6J, significant non-viability is observed with very few animals surviving to adulthood. However, an examination of development in Map3k1-deficient XY embryos on this genetic background revealed no significant defects in testis determination, although minor abnormalities were observed, including an increase in gonadal length. Based on these observations, we conclude that MAP3K1 is not required for mouse testis determination. We discuss the significance of these data for the functional interpretation of sex-reversing MAP3K1 mutations in humans.     

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.     

Substitution rates in a new Silene latifolia sex-linked gene, SlssX/Y

Dioecious white campion Silene latifolia has sex chromosomal sex determination, with homogametic (XX) females and heterogametic (XY) males. This species has become popular in studies of sex chromosome evolution. However, the lack of genes isolated from the X and Y chromosomes of this species is a major obstacle for such studies. Here, I report the isolation of a new sex-linked gene, Slss, with strong homology to spermidine synthase genes of other species. The new gene has homologous intact copies on the X and Y chromosomes (SlssX and SlssY, respectively). Synonymous divergence between the SlssX and SlssY genes is 4.7%, and nonsynonymous divergence is 1.4%. Isolation of a homologous gene from nondioecious S. vulgaris provided a root to the gene tree and allowed the estimation of the silent and replacement substitution rates along the SlssX and SlssY lineages. Interestingly, the Y-linked gene has higher synonymous and nonsynonymous substitution rates. The elevated synonymous rate in the SlssY gene, compared with SlssX, confirms our previous suggestion that the S. latifolia Y chromosome has a higher mutation rate, compared with the X chromosome. When differences in silent substitution rate are taken into account, the Y-linked gene still demonstrates significantly faster accumulation of nonsynonymous substitutions, which is consistent with the theoretical prediction of relaxed purifying selection in Y-linked genes, leading to the accumulation of nonsynonymous substitutions and genetic degeneration of the Y-linked genes.     

Three loci on mouse chromosome 5 and 10 modulate sex determination in XX Ods/+ mice

In mouse, XY embryos are committed to the male sex determination pathway after the transient expression of the Y-linked Sry gene in the Sertoli cell lineage between 10.5 and 12.5 dpc. In the C57BL/6J strain, male sex determination program can be modulated by some autosomal genes. The C57BL/6J alleles at these autosomal loci can antagonize male sex determination in combination with specific Sry alleles. In this report, the authors have identified an effect of these C57BL/6J specific alleles in combination with a mutated Sox9 allele, Sox9(Ods). Authors report the mapping of three of these genetic loci on mouse chromosome 5 and 10 in a backcross of the Ods mutation to the C57BL/6J background. Our study confirms the importance of the strain C57BL/6J for the investigation of the genetic mechanisms that control sex determination.     

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.     

The inter-specific hybrid Silene latifolia x S. viscosa reveals early events of sex chromosome evolution

The dioecious plant species Silene latifolia has a sex determination mechanism based on an active Y chromosome. Here, we used inter-specific hybrids in the genus Silene to study the effects of gene complexes on the Y chromosome. If the function of Y-linked genes has been maintained in the same state as in the hermaphrodite progenitor species, it should be possible to substitute such genes by genes coming from a related hermaphrodite species. In the inter-specific hybrid, S. latifolia x S. viscosa, anthers indeed develop far beyond the early bilobal stage characteristic of XX S. latifolia female plants. The S. viscosa genome can thus replace the key sex determination gene whose absence abolishes early stamen development in females (loss of the stamen-promoting function, SPF), so that hybrid plants are morphologically hermaphrodite. However, the hybrids have two anther development defects, loss of adhesion of the tapetum to the endothecium, and precocious endothecium maturation. Both these defects were also found in independent Y-chromosome deletion mutants of S. latifolia. The data support the hypothesis that the evolution of complete gender dimorphism from hermaphroditism involved a major largely recessive male-sterility factor that created females, and the appearance of new, dominant genes on the Y chromosome, including both the well-documented gynoecium-suppressing factor, and two other Y specific genes promoting anther development.     

Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations

The medaka, Oryzias latipes, has an XX/XY sex determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a prime candidate for the sex-determining gene. Furthermore, the crucial role of DMY during male development was established by studying two wild-derived XY female mutants. In this study, to find new DMY and sex-determination related gene mutations, we conducted a broad survey of the genotypic sex (DMY-negative or DMY-positive) of wild fish. We examined 2274 wild-caught fish from 40 localities throughout Japan, and 730 fish from 69 wild stocks from Japan, Korea, China, and Taiwan. The phenotypic sex type agreed with the genotypic sex of most fish, while 26 DMY-positive (XY) females and 15 DMY-negative (XX) males were found from 13 and 8 localities, respectively. Sixteen XY sex-reversals from 11 localities were mated with XY males of inbred strains, and the genotypic and phenotypic sexes of the F(1) progeny were analyzed. All these XY sex-reversals produced XY females in the F(1) generation, and all F(1) XY females had the maternal Y chromosome. These results show that DMY is a common sex-determining gene in wild populations of O. latipes and that all XY sex-reversals investigated had a DMY or DMY-linked gene mutation.