Regional assignments of the zinc finger Y-linked gene (ZFY) and related sequences on human and mouse chromosomes

Recent chromosome walking experiments have identified a candidate gene (ZFY) for the testis-determining factor on the human Y chromosome (Page et al., 1987). We report here the regional assignments of the ZFY gene and related sequences in the human and the mouse. By in situ hybridization, we assigned ZFX and ZFY to human chromosome bands Xp21 and Yp11.3, respectively. Although the mouse harbors two Zfy genes, only one site at band A1 of its Y chromosome was significantly labeled. The mouse Zfx gene and the Zfa gene on chromosome 10 were assigned to bands XD and 10B5, respectively. These assignments of the ZFX gene in human and mouse add another marker to the conserved syntenic group for evaluating the evolutionary relationship of the human and mouse X chromosomes.     

Patterns of Y and X chromosome DNA sequence divergence during the Felidae radiation.

The 37 species of modern cats have evolved from approximately eight phylogenetic lineages within the past 10 to 15 million years. The Felidae family has been described with multiple measures of morphologic and molecular evolutionary methods that serve as a framework for tracking gene divergence during brief evolutionary periods. In this report, we compare the mode and tempo of evolution of noncoding sequences of a large intron within Zfy (783 bp) and Zfx (854 bp), homologous genes located on the felid Y and X chromosomes, respectively. Zfy sequence variation evolves at about twice the rate of Zfx, and both gene intron sequences track feline hierarchical topologies accurately. As homoplasies are infrequent in patterns of nucleotide substitution, the Y chromosome sequence displays a remarkable degree of phylogenetic consistency among cat species and provides a highly informative glimpse of divergence of sex chromosome sequences in Felidae.     

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.     

Characterization of the Xp21-23 region in the wood lemming, a region involved in XY sex reversal.

The wood lemming (Myopus schisticolor) harbors two types of X chromosome, a normal X and a variant X, designated X*. The X* chromosome contains a mutation that causes XY sex reversal. We have previously demonstrated that the Xp21-23 region is deleted from X* and is associated with XY sex reversal. To further analyze the deleted region, we have constructed and characterized seven X chromosome- and region-specific recombinant DNA libraries. Further, we have screened mouse fetal gonad cDNA libraries with the microdissected Xp21-23 DNA as a probe in an attempt to identify homologous and expressed sequences from the deletion. Fourteen positive clones were isolated, and sequence analyses showed that ten of these contained identical sequences homologous to mouse gamma-satellite sequences. One of the remaining four was perfectly homologous to the mouse gene Ccth (chaperonin containing t-complex polypeptide 1, eta subunit). Southern blot indicated that the Ccth cDNA was located on the X chromosome, not deleted from the X* but closely linked to the deletion region. Although the role of the Ccth containing region in sex determination of the wood lemming requires additional studies, the isolation of the mouse Ccth gene by the deletion Xp21-23 probe could be important since this gene is mainly expressed in testis.     

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.     

Zfa is an expressed retroposon derived from an alternative transcript of the Zfx gene.

ZFY, a gene on the Y chromosome encoding a zinc finger protein, has been proposed as a candidate for the human testis determining gene. Sequences related to ZFY, called ZFX, are present on the X chromosome of a wide range of placental mammals. Unlike most mammals the mouse has four genes homologous to ZFY; two on the Y chromosome, Zfy-1 and Zfy-2, an X-linked gene, Zfx, and an autosomal gene, Zfa. We show here that Zfa has arisen recently by retroposition of one of at least three alternatively spliced mRNAs transcribed from the Zfx gene. Zfa is an unusual retroposon in that it has retained an open reading frame and is expressed, although its function may be limited or altered by the presence of a potentially inactivating mutation in the third of its zinc fingers. This mutation must have occurred at the same time or soon after the retroposition event as it is also present in the Zfa gene of Mus spretus. Interestingly the third finger of the M. musculus musculus Zfy-2 gene has also sustained a mutation suggesting that this gene family may be rapidly evolving in mice.     

Retroposon compensatory mechanism hypothesis not supported: Zfa knockout mice are fertile

It is hypothesized that autosomal retroposons compensate for the loss of their inactivated essential X-chromosome progenitors during spermatogenesis. Here we test this Retroposon Compensatory Mechanism (RCM) hypothesis using the Zfy gene family. The mouse autosomal retroposon Zfa is expressed in testes at the same developmental time points at which Zfx levels decline, which correspond to the time of male sex chromosome inactivation, suggesting that Zfa may compensate for the loss of Zfx during spermatogenesis. We examined the effect of Zfa-targeted mutagenesis on spermatogenesis in three genetically distinct mouse strains. Surprisingly, Zfa knockout mice showed no detectable fertility, sperm count, or testes morphology defects. We therefore conclude that Zfa is not an essential gene for spermatogenesis and fertility. This surprising finding now challenges the RCM hypothesis at least for the Zfy gene family. It also forces us to reevaluate the original data underpinning the RCM hypothesis for this family and to propose alternative hypotheses.     

On the evolutionary stability of the female-biased sex ratio in the wood lemming (Myopus schisticolor): the effect of inbreeding.

The evolutionary stability of the female-biased sex ratio observed in the wood lemming (Myopus schisticolor) is discussed. The hypothesis analysed is that the skewed sex ratio is maintained as a result of partial and/or recurrent inbreeding. Fredga et al. (1976, 1977) have suggested that an X-linked mutant gene, X, affects the male-determining action of the Y chromosome, thus converting some XY individuals into females. By a mechanism of selective non-disjunction in the foetal ovary only X-carrying eggs are produced. In particular the stability of that genetic mechanism (or the X chromosome) is analysed by considering the introduction of a "suppressing" sex-linked mutant gene Y. Several deterministic simulation models assuming father-daughter and/or brother-sister matings have been developed and analysed. It is concluded that in the case of extremely strong inbreeding, the hypothesised genetic mechanism may, as a result, be evolutionarily stable. Interpreting field observations on microtine rodents in general it is concluded that only a few species are likely to experience such extreme cases of inbreeding. The wood lemming and the related collared lemming (Dicrostonyx troquatus), another case which seems to have XY-females, are likely to exhibit sufficiently strong inbreeding.     

Aberrant chromosomal sex-determining mechanisms in mammals, with special reference to species with XY females

Both mouse and man have the common XX/XY sex chromosome mechanism. The X chromosome is of original size (5-6% of female haploid set) and the Y is one of the smallest chromosomes of the complement. But there are species, belonging to a variety of orders, with composite sex chromosomes and multiple sex chromosome systems: XX/XY1Y2 and X1X1X2X2/X1X2Y. The original X or the Y, respectively, have been translocated on to an autosome. The sex chromosomes of these species segregate regularly at meiosis; two kinds of sperm and one kind of egg are produced and the sex ratio is the normal 1:1. Individuals with deviating sex chromosome constitutions (XXY, XYY, XO or XXX) have been found in at least 16 mammalian species other than man. The phenotypic manifestations of these deviating constitutions are briefly discussed. In the dog, pig, goat and mouse exceptional XX males and in the horse XY females attract attention. Certain rodents have complicated mechanisms for sex determination: Ellobius lutescens and Tokudaia osimensis have XO males and females. Both sexes of Microtus oregoni are gonosomic mosaics (male OY/XY, female XX/XO). The wood lemming, Myopus schisticolor, the collared lemming, Dirostonyx torquatus, and perhaps also one or two species of the genus Akodon have XX and XY females and XY males. The XX, X*X and X*Y females of Myopus and Dicrostonyx are discussed in some detail. The wood lemming has proved to be a favourable natural model for studies in sex determination, because a large variety of sex chromosome aneuploids are born relatively frequently. The dosage model for sex determination is not supported by the wood lemming data. For male development, genes on both the X and the Y chromosomes are necessary.     

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.     

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.     

Human and mouse amelogenin gene loci are on the sex chromosomes

Enamel is the outermost covering of teeth and is the hardest tissue in the vertebrate body. The enamel matrix is composed of enamelin and amelogenin classes of protein. We have determined the chromosomal locations for the human and mouse amelogenin (AMEL) loci using Southern blot analyses of DNA from human, mouse, or somatic cell hybrids by hybridization to a characterized mouse amelogenin cDNA. We have determined that human AMEL sequences are located on the distal short arm of the X chromosome in the p22.1----p22.3 region and near the centromere on the Y chromosome, possibly at the proximal long arm (Yq11) region. These chromosomal assignments are consistent with the hypothesis that perturbation of the amelogenin gene is involved in X-linked types of amelogenesis imperfecta, as well as with the Y-chromosomal locations for genes that participate in regulating tooth size and shape. Unlike the locus in humans, the mouse AMEL locus appears to be assigned solely to the X chromosome. Finally, together with the data on other X and Y chromosome sequences, these data for AMEL mapping support the notion of a pericentric inversion occurring in the human Y chromosome during primate evolution.     

Comparative molecular phylogeny and evolution of sex chromosome DNA sequences in the family Canidae (Mammalia: Carnivora)

To investigate the molecular phylogeny and evolution of the family Canidae, nucleotide sequences of the zinc-finger-protein gene on the Y chromosome (ZFY, 924-1146 bp) and its homologous gene on the X chromosome (ZFX, 834-839 bp) for twelve canid species were determined. The phylogenetic relationships among species reconstructed by the paternal ZFY sequences closely agreed with those by mtDNA and autosomal DNA trees in previous reports, and strongly supported the phylogenetic affinity between the wolf-like canids clade and the South American canids clade. However, the branching order of some species differed between phylogenies of ZFY and ZFX genes: Cuon alpinus and Canis mesomelas were included in the wolf-like canid clades in the ZFY tree, whereas both species were clustered in a group of Chrysocyon brachyurus and Speothos venaticus in the ZFX tree. The topology difference between ZFY and ZFX trees may have resulted from the two-times higher substitution rate of the former than the latter, which was clarified in the present study. In addition, two types of transposable element sequence (SINE-I and SINE-II) were found to occur in the ZFY final intron of the twelve canid species examined. Because the SINE-I sequences were shared by all the species, they may have been inserted into the ZFY of the common ancestor before species radiation in Canidae. By contract, SINE-II found in only Canis aureus could have been inserted into ZFY independently after the speciation. The molecular diversity of SINE sequences of Canidae reflects evolutionary history of the species radiation.     

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.     

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.     

Akodon sex reversed females: the never ending story

The existence of fertile A. azarae females with a chromosome sex pair indistinguishable from that of males was reported more than 35 years ago. These heterogametic females were initially thought to occur due to an extreme process of dosage compensation in which X inactivation was restricted to Xp and complemented by a deletion of Xq (Xx females). Later on, a C-banding analysis of A. mollis variant females showed that these specimens were in fact XY* sex reversed and not Xx females. The finding of positive testing for Zfy and Sry multiple-copy genes in Akodon males and heterogametic females confirmed the XY* assumption. At the present time, XY* sex reversed females have been found to exist in nine Akodon species. Akodon heterogametic females produce X and Y* oocytes, which upon sperm fertilization give rise to viable XX (female), XY* (female), and XY (male) embryos, and to non-viable Y*Y zygotes. Heterozygous females exhibit a better reproductive performance than XX females in order to compensate the Y*Y zygote wastage. XY* sex reversed females are assumed to occur due to a deficient Sry expression resulting in the development of ovaries instead of testes. Moreover, the appearance of Y* elements is a highly recurrent event. It is proposed that homozygosity for an autosomal or pseudoautosomal recessive mutation (s-) inhibits Sry expression giving rise to XY* embryos with ovary development. Location of the Y* chromosome in the female germ cell lineage produces an ovary-specific imprinting of the Sry* gene maintaining its defective expression through generations independently from the presence or absence of s- homozygosity. By escaping the ovary-specific methylation some Y* chromosomes turn back to normal Ys producing Y oocytes capable of generating normal male embryos when fertilized by an X sperm. Fluctuations in the rate of variant females in field populations and in laboratory colonies of Akodon depend on the balance between the appearance of new variant females (s-/s-, XY* specimens) and the extinction of sex reversed specimens due to imprinting escape.