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Regular Article |
a Department of Genetics, La Trobe University, Melbourne, Victoria 3083, Australia
ABSTRACT
In this review I want to argue that, far from being a macho entity with an all-powerful role in male development, the human Y chromosome is a "wimp." It is merely a relic of the X chromosome, and most or all of the genes it bears—including the genes that determine sex and control spermatogenesis—are relics of genes on the X chromosome that have other functions altogether.
Sertoli cells, spermatogenesis
HUMAN SEX, GENES, AND CHROMOSOMES
In humans, as in other mammals, sex determination depends on the testis, and testis differentiation depends on the Y chromosome.
Sex Determination
The event that marks human male determination is the differentiation of the testis at about 5 wk of gestation. The embryonic testis then churns out testosterone and Müllerian-inhibiting substance, powerful hormones that divert developmental pathways along male lines. The pivotal genetic event is the switching of the indifferent gonad (the genital ridge) to testis differentiation by the action of a gene called the testis-determining factor (TDF). After this event, male development unfolds automatically, unless something goes wrong. A number of genetic accidents—anything from absence or mutation of TDF to a block of the action of testosterone—can interrupt the male-determining pathway, and when this happens, the phenotype of the baby is female. Female development has therefore been said to be the "default pathways," and females have even been called "mutant males."
The production of a male is likely to require many more genes than just the TDF gene. Some of these are becoming known through studies of patients with a variety of sex reversal syndromes. There are also likely to be many genes—hundreds or thousands—required for germ-cell differentiation and male fertility. Of course, there are likely to be just as many genes required for ovarian differentiation and egg development, and so far we know rather little about these genes or how they are switched on in the absence of testis development.
Sex Chromosomes
In mammals, the male-specific Y chromosome plays a pivotal role in sex determination, and also bears genes that are required for spermatogenesis. However, not all the genes that are needed to make a testis or to make germ cells need to be on the Y chromosome, and many are known to be located on the X chromosome or on the autosomes (chromosomes other than the X and Y).
Like other mammals, human females have two X chromosomes (XX) and males have a single X and a single Y chromosome (XY). The X is large (5% of the total length of a single set of chromosomes) and bears a proportional number of genes (3000 or 4000), which have a variety of functions much like those of genes located on other chromosomes. To ensure fair play between the sexes, only one X chromosome is genetically active in female cells. The set of genes on the X chromosome is almost completely conserved between different species of eutherian ("placental") mammals, probably because breaking it up would disrupt this chromosome-wide X-inactivation mechanism.
The Y chromosome is much smaller than the X chromosome and contains only a few genes. It is largely composed of repetitive sequences that preferentially bind fluorescent dyes, so that it literally glows in the dark. These sequences have no known coding function and are good candidates for hard-core "junk DNA." The X and Y chromosomes are homologous over a tiny region at the end of the short arms, and they regularly pair and undergo crossing over within this 2600-kilobase-pair (kbp) pseudoautosomal region) (PAR). The human X and Y chromosomes also have a 500-kbp PAR at the ends of their long arms.
A grand total of 33 genes have been characterized on the Y chromosome, nine of these within PAR1 and four within PAR2. Many are inactive pseudogenes. This may be a reasonably exhaustive list, because the human Y chromosome was thoroughly searched by screening a testis cDNA library with large fragments of the human Y chromosome cloned into yeast artificial chromosomes (Y-specific YACs) [1]. The 19 genes on the nonrecombining, Y-specific (differentiated) region of the Y chromosome are a peculiar lot. Several are expressed only in the testis, suggesting a male-specific function in spermatogenesis. The Y chromosome is unique in its "functional coherence."
Lahn and Page [1] divided up the 19 genes on the nonrecombining region of the Y chromosome into two classes. Class I genes are single-copy genes on the Y chromosome that are ubiquitously expressed and have homologues on the X chromosome. Class II genes (the interesting ones) are multicopy and testis specific, and have no homologues on the X chromosome. However, as I will point out, this classification breaks down when we consider the origin of two of the best-characterized male-specific genes, one involved in spermatogenesis and the other, TDF itself.
The Y chromosome is therefore peculiar in that it contains few active genes and a lot of junk, and it is unique in that it is male specific and bears several genes important for male functions. Let us consider alternative models of the organization, function, and evolution of the human Y chromosome.
MODELS OF THE Y CHROMOSOME
There are many models put forth to account for the peculiarities of the Y chromosome, and I have distilled them into three categories (Fig. 1). The first represents a concept that we have all grown up with—the concept of a dominant entity, acting to determine a male, regardless of which other chromosomes are present. An alternative model is that the Y is a selfish entity, which somehow accumulates genes that are handy in a male and/or bad in a female. The third model is the one for which I shall argue. It holds that the Y chromosome is a "wimp," a pale shadow of its former self, having degraded to almost nothing. The genes that it contains are just relics of genes that were originally on an autosome and have been retained intact on the X chromosome.
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The Dominant Y Chromosome
The reason for the traditional view of the human Y chromosome as an all-powerful entity is the dominant role it plays in sex determination. This is evident from the sex of patients with abnormal numbers of sex chromosomes. Males with Klinefelter syndrome (XXY) have two X chromosomes, like a female, plus a Y chromosome, like a male [2], whereas females with Turner syndrome (XO) have a single X chromosome and no Y chromosome [3]. Even patients with multiple X chromosomes (up to five) are male, if they possess a Y chromosome, and female, if they lack a Y chromosome. The phenotype (outward appearance) of patients is remarkably normal because all but one X chromosome is largely inactive anyway; the major effect of abnormal numbers of X chromosomes is infertility.
Variations in the number of X chromosomes have also been observed in other eutherian mammals. Monkeys, cats, horses, sheep, cattle, pigs, rats, and mice with an XO karyotype are female, and cats and mice with an XXY karyotype are male. As in humans, the abnormal number of X chromosomes has little effect, except on fertility. Thus, the rule for eutherian mammals is that the presence of a Y chromosome makes the mammal a male, no matter how many copies of the X chromosome are present. This has been thought to imply that there is a gene on the Y chromosome (TDF) that has a positive action in determining the testis, and that there is no dosage effect of the X chromosome. We therefore think of the Y chromosome as dominant, because it bears TDF, which determines the testis even in the presence of several X chromosomes.
How dominant is the Y chromosome really? Given that only one of the X chromosomes is active anyway, the phenotypes of individuals with XO and XXY karyotypes say little about a potential balance. There is at least one report of a human fetus with an XXXY karyotype that was female; perhaps there are non-inactivated genes on the X chromosome that balance the effect of the testis-determining factor. We know of at least one gene, DAX1 on the short arm of the X chromosome, that causes XY female sex reversal when duplicated [4]. At other Y loci that control spermatogenesis, the Y chromosome is certainly not dominant over extra X chromosomes, for any organism—man or mouse—with two X chromosomes is sterile [5]. In this case, the presence of two X chromosomes is a killer of fertility, if not of virility!
In marsupial mammals, a dosage effect of the X chromosome is yet more obvious, because mammals with XXY and XO karyotypes are intersexes, neither completely male, nor completely female. Marsupial mammals with XXY karyotypes have intra-abdominal testes, but their scrotum is replaced by a pouch with mammary glands. Mammals with an XO karyotype have no testes, but their pouch and mammary glands are replaced with an empty scrotum [6]. Thus, the testis does not control all of the downstream events in marsupials, and at least some sexual differentiation (scrotal or mammary development) seems to be a function of the numbers (or parental origin) of the X chromosomes [7]. Even so, the Y chromosome controls whether animals have a testis or not, implying that marsupials also have a dominant TDF on the Y chromosome. Thus, eutherian and marsupial TDF has what looks like a male-dominant action, although the evidence for this action is less convincing than has been appreciated. This dominant action has traditionally been interpreted to mean that TDF codes for some kind of activator that turns on transcription of other genes in the male-determining pathway. However, I shall argue that this male-dominant action results from completely the opposite action—the testis-determining gene on the Y chromosome acts as a spoiler that turns off genes that turn off testis determination.
The Selfish Y Chromosome
The Y chromosome is unique in that it is found only in males. This chromosome would be the perfect place for evolution to relocate genes that have a function only in males, as well as genes that would have an adverse effect in females. Has the Y chromosome become a repository for such genes?
Hurst [8] argued that the Y chromosome acts as an "attractor for selfish growth factors." Such selfish, Y-borne genes favor implantation and embryonic growth, even at the expense of the future reproduction of the female (perhaps with another male), as has been suggested for the action of imprinted genes expressed only from the paternal genome. An embryo carrying a Y chromosome containing such a gene will be selected, and this selfish Y chromosome will spread through the population.
As predicted by this hypothesis, there are some genes on the Y chromosome that affect growth. However, three of these, the osteogenesis gene (SHOX) and three growth factor receptor genes (IL3RA, CSF2RA in PAR1, and Il9R in PAR2), lie within the PAR, and are therefore shared with and recombine with genes on the X chromosome. Another factor has been identified only as a region that, when deleted, causes overgrowth of the undifferentiated gonad (gonadoblastoma), so this factor is also more likely to represent a growth inhibitor (or gonad differentiation promoter) than a growth factor.
The selfish Y hypothesis also requires that selfish genes on the Y chromosome are truly Y specific and would therefore correspond to Class II genes as defined by Lahn and Page [1]. These Class II genes have no X homologue and hail from other regions of the genome. It is therefore particularly interesting to examine the origin of these genes. I will show here that at least some of them have homologues on the X chromosome and were therefore homegrown on the sex chromosomes. Some beautiful experiments on fruitflies [9] suggested that a gene already on the Y chromosome could evolve a selfish male-advantage/female-disadvantage function. In these experiments, an allele of an autosomal gene was artificially selected in females only. After only 30 generations, the region containing this allele was shown to have a detrimental effect on male viability and fertility.
The Wimp Y Chromosome
The third model proposes that the Y chromosome is merely a relic of a former autosome, and all that the genes that it bears are relics of genes that happened to be on the chromosome that accidentally became a Y chromosome. This model derives from the suggestion that the mammalian X and Y chromosomes differentiated from a homologous pair of ancient autosomes (proto-sex chromosomes) by the progressive degradation of the Y chromosome [10].
The Y chromosome is thought to have been originally defined by its acquisition of a male-determining gene [11]. Other genes with male-specific functions then accumulated nearby, and selection kept a male-specific package together by suppressing crossing over. Within this genetically isolated region, all kinds of genetic accidents occurred and could not be repaired because there was no crossing over between the X and Y chromosomes, so the region was rapidly degraded and deleted. The only genes that could survive on the Y chromosome were those that became indispensable in male reproduction.
Information on the possible origins of genes on the Y chromosome can help to distinguish between the models. Class I genes with homologues on the X chromosome are likely to be relics of ancient homology, as predicted by the wimp Y hypothesis. However, Class II genes that are testis specific and have no homologues on the X chromosome may have been recruited by a selfish Y.
THE ORIGIN AND EVOLUTION OF THE Y CHROMOSOME
These models and their predictions about the origin of genes on the Y chromosome can be examined by looking for evolutionary intermediates of Y degradation in the mammals most distantly related to humans. Comparisons of eutherians with marsupials (pouched mammals that diverged 130 million years ago) and monotremes (egg-laying mammals that diverged 170 million years ago) have provided a rich source of variation to study the origin and function of mammalian sex chromosomes and sex-determining genes.
Sex Chromosome Differentiation
The wimp Y model has its origins in a hypothesis put forward three decades ago to explain the peculiarities of snake sex chromosomes. The pattern in snakes is completely opposite from the pattern in mammals: snakes with a ZW karyotype are female and snakes with a ZZ karyotype are male. Like the mammalian X chromosome, the Z chromosome is large, containing about 6% of the genome in all snake families. The W chromosome is much more variable. Some snake families have a tiny, heterochromatic W chromosome, whereas others have a W chromosome that is much the same size as the Z chromosome. Ohno [10] suggested that these patterns represent stages in the gradual breakdown of the W chromosome, starting from a pair of equivalent proto-sex chromosomes.
Birds also have a ZZ male and ZW female system, and cross-species chromosome "painting" (in situ hybridization with a mixture of DNA sequences from an isolated Z chromosome) shows that even birds as distantly related as the chicken and the emu have a genetically identical Z chromosome [12]. As in snakes, the W chromosome in birds has different sizes in different families; it is large in ratite (flightless) birds and small in carinate birds. Chromosome painting with the chicken Z chromosome sequences shows that the W chromosome is largely homologous to the Z chromosome in the emu, but has become small and heterochromatic in the chicken. A process of W chromosome degradation has therefore taken place to different extents, independently in different bird and snake lineages.
The same sort of event is proposed to have occurred in mammals. Although the X and Y chromosomes are very different in size and gene content, there is excellent evidence that they were once homologous, and the Y chromosome has been largely degraded during 200 million years of mammalian evolution. Significantly, the human X and Y chromosomes share considerable homology. First, the PAR is homologous, and this region contains at least nine genes. Second, many genes on the differentiated region of the Y chromosome also have obvious homologues on the X chromosome—genes that share DNA sequence and code for similar protein products. Since comparative mapping shows no homology between the mammalian XY pair and the bird ZW pair, these two sex chromosome systems must have evolved independently.
The Ancient X and Y Chromosomes
The gene content of the ancient mammalian sex chromosomes can be deduced by comparing eutherian sex chromosomes with those of the distantly related marsupial and monotreme mammals and by defining the shared regions. Sex chromosomes have been compared in two groups of marsupials—macropodids (kangaroos and wallabies) and dasyurids (small rodent-like insectivores)—as well as in two of the three species of monotreme, the fabled platypus and its spiny cousin, the echidna.
Marsupials have a genome about the same size as that of eutherian mammals, but their DNA is packaged into a few very large chromosomes. However, the X and Y chromosomes are unusually small, the dasyurid X chromosome constituting only about 3% of the haploid genome, and the Y chromosome barely visible under the microscope as a tiny dot. Gene mapping reveals that these small marsupial X and Y chromosomes are minimal mammalian sex chromosomes, little changed from the ancient mammalian X and Y chromosomes in a common ancestor of all mammals 170 million years ago.
Comparative mapping of the marsupial X chromosome was accomplished by making hybrids between rodent and marsupial cells. The hybrids retained only one or two marsupial chromosomes in a background of rodent chromosomes, so it was possible to identify marsupial proteins in hybrids by isozyme typing and marsupial genes by Southern blotting. The presence or absence of a particular gene or gene product could then be correlated with the presence or absence of a particular marsupial chromosome. More recently, a number of marsupial homologues of human X-linked genes have been cloned and mapped by fluorescence in situ hybridization.
This gene mapping showed that much of the human X chromosome is homologous to the smaller marsupial X chromosome [13]. All of the genes on the long arm of the human X chromosome and on the region around the centromere were also found on the X chromosome in marsupials, implying that this region represented an ancient mammalian X chromosome at least 130 million years old. The same set of genes was also mapped to the X chromosome in monotremes, pushing back the age of this chromosomal region to 170 million years. This region (called the X conserved region, XCR) is equivalent to the ancient mammalian X chromosome.
The Y chromosome is also partially homologous between humans and marsupials, but represents only a tiny region. Four genes from the human Y chromosome have now been found to map to the tiny marsupial Y chromosome and must have been on the Y chromosome for at least 130 million years (personal communication with Waters). These genes, SRY, RPS4Y, SMCY, and RBMY, map to two small regions at either end of the human Y chromosome, which constitutes the Y conserved region (YCR). They were probably originally contiguous at the border of the large heterochromatic region, until an inversion separated them a few million years ago. The small conserved region of the human Y chromosome (constituting as little as 10 Mb) is all that is left of the original mammalian Y chromosome.
Regions Added to the Sex Chromosomes
Surprisingly, the genes on the rest of the short arm of the human X chromosome were all found on autosomes in marsupials and in monotremes. Most of them cluster on the short arm of chromosome 5 in the tammar wallaby and on chromosome 1 in monotremes. This pattern could mean either that the marsupial and monotreme X chromosomes both lost a large chunk after their divergence from eutherians, or that the human X chromosome gained a chunk. The latter possibility is favored, since the same piece of X chromosome is unlikely to have been lost independently from the marsupial and monotreme X chromosomes. This conclusion suggested that the human X chromosome contains an X added region (XAR), which is still located on autosomes in other mammals [13].
Recently, the conserved and added regions of the human X chromosome have been directly demonstrated by chromosome painting between the marsupial and human X chromosomes [14]. A DNA probe derived from a flow-sorted wallaby X chromosome was amplified and tagged with fluorescent dye, then hybridized in situ to human chromosomes. The probe bound to the long arm and pericentromeric region of the human X chromosome, identifying the XCR that represents the ancient X chromosome. It did not bind to the rest of the short arm that represents the added region, XAR (Fig. 2).
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The human Y chromosome contains an equivalent added region. The presence of this region was first obvious from a map of the X chromosome, since many genes on the human Y chromosome have partners on Xp within the XAR. They, too, must therefore have been added. More recently, the addition has been directly demonstrated by cloning and mapping the wallaby homologues of genes on the human Y chromosome (personal communication with Waters). Eight of these genes mapped to chromosome 5p in the wallaby, to the same position as the XAR genes. These genes define a Y added region (YAR), which shares homology with the XAR. The YAR forms the great majority of the human Y chromosome and includes the PAR.
How could a piece of an autosome be added to both the X and Y chromosomes? We know that a piece of autosome added to the differential region of the X chromosome could not be transferred to the Y chromosome, because the X and Y chromosomes do not pair over this region. Instead, a compound X chromosome is formed, which pairs with a Y1 (the original Y) region and a Y2 (the original autosome) region at meiosis. The addition is therefore likely to have occurred at the stage when the X and Y chromosomes were only partially differentiated. If a piece of autosome was added to the X chromosome above the PAR, it would cross over onto the Y chromosome at the next meiosis. Once the piece was on the X and Y chromosomes, it would enlarge the PAR [13]. There may have been two or more separate additions, since the XAR is present as at least two autosomal clusters in monotremes and marsupials.
Thus, most of the human Y chromosome derives from a relatively recent addition to the sex chromosomes. The original and added regions of the Y chromosome have both been greatly degraded in size and gene content, and the older YCR has all but disappeared.
The Y Chromosome—Going, Going ...
Thus, gene mapping shows that the human Y chromosome evolved from the proto-X/Y chromosome plus the added region, now represented by the human X chromosome, with which it was once homologous.
But what happened to all the genes on the original Y chromosome? More than 1000 genes have been cloned from the human X chromosome, compared with only about 30 on the Y chromosome, despite exhaustive screening. A clue to the genes' fate is given by the finding that several X-linked genes detect inactive homologues on the Y chromosome. For instance, the sex-linked steroid sulfatase (STS) gene on the X chromosome detects homologous sequences on the Y chromosome, but these belong to a partially deleted pseudogene [15]. The Y copy of UBE1X has disappeared altogether from the human Y chromosome, although bits of pseudogenes can be detected on the Y chromosome in monkeys; an active copy of the gene still resides on the mouse Y chromosome, and the gene is pseudoautosomal in the platypus [16]. Thus, the process of Y degradation continues.
Indeed, additions may have saved the human Y chromosome from disappearing altogether. In addition to the added autosomal segment, the Y chromosome has also been enlarged by the addition or amplification of repetitive-sequence DNA. Almost half of the human Y chromosome is composed largely of two simple repeats. In the kangaroo and wallaby, the ribosomal genes and associated heterochromatin have been added to both the X and Y chromosomes and have all but degenerated on the Y chromosome [17]. In other species, large aggregates of heterochromatin have been added solely to the Y chromosome. Maybe additions of heterochromatin act as a kind of ballast.
Is the Y chromosome therefore headed for extinction? Without further additions to the Y chromosome in dasyurid marsupials, the unfortunate Y chromosome has been whittled down to a minimal 10 Mb. This little package evidently contains a TDF needed to determine maleness, as well as sundry spermatogenesis genes, but little else. Indeed, there are species of marsupials that dispense with the Y chromosome during somatic growth [18] by eliminating it from the embryo. This process must mean that the Y chromosome no longer bears any genes required for general functions. There are two rodent species (mole voles) that have completely lost the Y chromosome [19]. Presumably, in these species, the Y chromosome first lost everything but the TDF, whose control function was then taken over by a new gene elsewhere on the genome.
If the Y chromosome is a relic of the original undifferentiated proto-X/Y chromosome that derived from a pair of autosomes with no sex-determining function, it follows that all of the genes on the Y chromosome evolved from genes now represented on the X chromosome. It is therefore of interest to examine the origins of genes on the Y chromosome, particularly those suspected of male-specific functions, like spermatogenesis and sex determination.
ORIGIN OF SPERMATOGENESIS GENES ON THE Y CHROMOSOME
There is evidence that one or more genes on the Y chromosome are essential for spermatogenesis. Deletion of parts of the human Y chromosome near the heterochromatic region have been found in men producing few or no sperm. Deletions of part of the tiny short arm of the mouse Y chromosome and of several parts of the long arm are associated with an absence of sperm or abnormal sperm. There are several genes on the Y chromosome that map within these regions, and these become candidate spermatogenesis genes. Their origin is of particular interest for evaluating models of the Y chromosome.
Spermatogenesis Genes from the Ancient Proto-X/Y Chromosome
The RBM gene (named for the 
NA-
inding 
otif that its protein product contains) is present in about 30 copies on the human Y chromosome, but only two of these copies are transcribed [20]. The RBM gene appeared to be a prototypical Class II gene: it is multicopy and testis specific, and apparently has no homologue on the X chromosome. Where did it come from, then? Was it an ancient, autosomal male-specific gene that found an appropriate spot on a selfish Y chromosome? A close homologue (called HNRPG) has been described on chromosome 6 and codes for one of a large family of ubiquitously expressed RNA-processing and transport proteins. The speculation was that a copy of this autosomal gene, introns and all, somehow made its way onto the Y chromosome and became modified and testis specific. It acquired a role in spermatogenesis and was then amplified in tandem on the human Y chromosome.
However, a spin-off from recent studies of the gene in marsupials shows that RBM is really a Class I gene after all. Both X-borne and Y-borne copies of this gene were detected, first in marsupials and then in eutherian mammals. When human RBM was used to detect homologous sequences in Southern blots of marsupial DNA, "dosed" fragments (with twice the intensity in females as in males) were observed, as well as a male-specific band. An RBM homologue with a sequence identical to that of HNRPG was then isolated from the human X chromosome; in fact, the supposedly autosomal HNRPG turned out to be an X-borne gene after all, and the chromosome 6 homologue was exposed as an intronless pseudogene that could not be translated into a sensible protein [21]. Both X and Y copies of Rbm were also found in the mouse [22]. Thus, the Y-borne RBMY (as it is now called) has a homologue on the X chromosome, RBMX, just as do all the ubiquitously expressed Class I genes on the human Y chromosome. Like these genes, RBMY must have evolved from genes on the original proto-X/Y chromosome. The RBMY gene is therefore a relic of the conserved ubiquitous RBMX.
How could a gene coding for a generic RNA-processing protein be molded into a spermatogenesis gene? The sequence similarity between RBMX and RBMY implies that their functions in RNA processing are likely to be similar. The most obvious sequence differences between RBMX and RBMY are the internal amplification of an intron containing a "SRGY box" containing serine-arginine-glycine-tyrosine, but since this amplification is not present in the mouse or marsupial gene, it is likely that the major difference lies in its testis-specific expression. Perhaps its product wields post-transcriptional control over other genes specifically in the testis.
Spermatogenesis Genes from the Added Region
Two genes within the YAR on the Y chromosome may also have a function in spermatogenesis. Both ZFY and DFFRY have copies on the X chromosome, from which the Y-borne genes were obviously derived. Since their marsupial homologues are autosomal, they must derive from genes on the autosomal region that were added recently to the eutherian X and Y chromosomes. ZFY and ZFX are both ubiquitously expressed in humans and code for a zinc finger protein that resembles a transcription factor. In mice, Zfx is expressed ubiquitously, but Zfy is testis specific and lies in a critical chromosomal region for spermatogenesis [23]. Perhaps human Zfy retains the original housekeeping function that it shares with Zfx, but Zfy has recently acquired a specific function in spermatogenesis in rodents.
The DFFRY gene has a similar origin in the YAR, but may have already had an ancient function in germ-cell differentiation, before it was relocated to the sex chromosomes. Like ZFY, this gene is ubiquitously expressed in man, but testis specific in the mouse [24]. Significantly, mutations in DFFRY have been detected in sterile men [25]. Even more telling is the observation that the gene has a homologue in Drosophila that affects ovarian function, as well as eye development (indeed, its name derives from Drosophila fat facets). Evidently, then, this gene has an ancient function in the development of germ cells, as well as in other tissues, and its relocation on the mammalian Y chromosome allowed it to specialize in sperm development in males. Both DFFRX and DFFRY belong to a family of genes that affect protein stability, so DFFRY may be another testis-specific, posttranscriptional regulator that evolved from a gene with an ancient function in germ-cell development in both sexes.
The three candidate spermatogenesis genes RBMY, ZFY, and DFFRY, would all be classified as Class I genes with their origin in homologous genes on the X chromosome, either within the conserved or the added region. Are there, after all, any Class II genes that were relocated from other chromosomes to a selfish Y chromosome? Two candidate spermatogenesis genes, which apparently have no X chromosome homologues, may point to alternative origins for Y-borne spermatogenesis genes.
Genes Relocated to a Selfish Y Chromosome?
The 
eleted in 
oospermia (DAZ) gene was also cloned from a deletion interval near the heterochromatin on the human Y chromosome and is deleted in many sterile men [26]. This gene, too, codes for an RNA-binding protein, though one in a different family from RBMY. The DAZ gene also exists in multiple copies and is expressed only in the testis. However, there appears to be no X chromosome homologue, and the only obvious source for the gene is an autosomal homologue, DAZLA, on human chromosome 3. In mice, there is an autosomal Dazla homologue, but no Y-borne sequence, and in marsupials, only autosomal homologues can be detected [27]. The DAZLA gene is also gonad specific. These observations suggest that a copy of the sequence somehow moved from its autosomal location early in primate evolution. Since the gene possesses introns, it could not have moved by retrotransposition (i.e., by integration of a DNA copy of the RNA transcript), unless the relocation occurred via an unprocessed transcript. Significantly, DAZ is homologous to the gonad-specific boule gene in Drosophila, mutations of which cause meiotic block and sterility. Thus, DAZ, too, has an ancient function in germ-cell differentiation, and a copy of this handy gene was somehow moved from an autosome onto a selfish Y chromosome.
The recently discovered CDY gene on the human Y chromosome has an even more recent origin. This gene is testis specific and multicopy, and has no X chromosome homologue (i.e., it is a Class II gene). There is no Y chromosome homologue in any nonprimate or even in prosimians, suggesting that it moved onto the primate Y chromosome only recently. There is a homologue, CDYL, on chromosome 6 that is transcribed ubiquitously, but in the mouse, the autosomal homologue Cdyl is alternately transcribed into ubiquitous and testis-specific products. The autosomal homologue contains introns, but the Y-specific CDY does not, so it evidently moved onto the Y chromosome by retroposition [28].
Both DAZ and CDY therefore do appear to be dinkum (real) Class II genes with an autosomal origin. At least DAZ appears to have capitalized on an ancient function in germ-cell differentiation, even before it somehow moved onto a selfish Y chromosome.
Multiple Origins of Spermatogenesis Genes on the Y Chromosome
Thus, the candidate spermatogenesis genes represent at least four different origins for genes on the Y chromosome. Some, like RBMY, were present on the original proto-X/Y chromosome, and others, like DFFRY, were acquired along with a large autosomal addition to the proto-X/Y chromosome. They all became male specific after the X and Y chromosomes differentiated. Other genes were relocated to the Y chromosome from autosomal copies, one by retroposition, and another by movement of a genomic copy or an unprocessed transcript. Some candidate spermatogenesis genes, like DFFRY and DAZ, evidently had an ancient function in germ-cell differentiation, whereas others, like RBM and ZFY, were evidently innocent housekeeping genes that were remodeled to fill a male-specific role.
How does this information help us to evaluate the different models of Y chromosome origin and function? It is evident that many or most of the genes on the Y chromosome, including three with suspected functions in spermatogenesis, have copies on the X chromosome from which they were derived, as proposed by the wimp Y model. However, at least two bona fide Class II genes must have arisen by relocation of genes from autosomes, and at least one of these had an ancient function in germ-cell differentiation, as proposed by the selfish Y hypothesis.
THE ORIGIN AND FUNCTION OF TDF
The origin of the male-dominant TDF itself is of special interest, since it is the properties of this gene that have given the Y chromosome its undeservedly macho image.
Cloning the TDF Gene
The TDF gene on the Y chromosome was pinpointed using DNA from patients having only parts of a Y chromosome (deletion mapping). Females with an XY karyotype have been described, in whom most of the Y chromosome was present, but obviously the TDF gene was missing. Conversely, males with an XX karyotype have been described, in whom a tiny piece of Y chromosome was added to one X chromosome [29]. Running down the small piece of the Y chromosome that harbored TDF and cloning the SRY gene from this region [30], was a major triumph of this "positional cloning" strategy.
The SRY gene was confirmed to be the long-sought TDF by mutation analysis in humans. Several female patients have been described with an XY karyotype and single base changes within the SRY gene [31]. The equivalent gene, SRY in the mouse, was shown to be sex determining; insertion of this gene into XX embryos produced transgenic males [32]. Other species of eutherian mammals had an equivalent gene on the Y chromosome, and marsupials also had a Y-borne SRY homologue [33] (a criterion that eliminated an earlier candidate gene, ZFY).
The SRY gene proved to be one of a large family of genes that code for architectural proteins containing an HMG box, a short stretch of amino acids that binds to DNA and bends it through a specific angle. Presumably, this bending brings sequences on either side of the binding sequence, or the proteins bound to them, into juxtaposition. Other members of this SOX gene family have been shown to code for factors that turn other genes on in development. It was therefore expected that SRY, too, would act as a transcriptional activator, accounting for its male-dominant role.
The Origin of SRY
The SRY gene, the all-important male-determining switch, might be expected to be a classic Class II gene: a gene unique to the Y chromosome and expressed only in the genital ridge. It was therefore a shock to find that SRY, too, has a homologue on the X chromosome. The presence of this homologue was first noted in marsupials, when the human SRY detected both male-specific bands (denoting a Y-borne gene) and bands that showed clear dosage differences between males (one dose) and females (two doses) on Southern blots, which revealed an X-borne homologue [34]. This SOX3 gene was eventually also localized to the X chromosome in humans and mice, and is expressed in the central nervous system (CNS), as well as in the genital ridge [35]. Thus, SRY is really a Class I gene that, like most of the other genes on the human Y chromosome, evolved from a counterpart on the X chromosome.
How did the CNS-determining SOX3 gene evolve into the testis-determining SRY? Sequence comparisons between species suggest that SRY is a degraded relic of SOX3 with quite a different (and perhaps opposite) action. It may even have different modes of action in different species. It is poorly conserved—not what you would expect of a gene critical for reproduction. Even within the critical HMG box, the amino acid sequence is only about 80% identical between the human, mouse, and marsupial SRY genes, and outside the box, the sequences cannot even be lined up. Strangely, it appears that the mouse Sry gene contains an activating domain, essential for sex determination, that is absent in SRY genes of other species [36]. Another surprise is that the transcription pattern of this gene is inconsistent. As we would expect, it is transcribed in the genital ridge in the mouse, just before testis determination; but it is expressed more widely in the human embryo and is virtually ubiquitous in marsupials. The angle of bending, too, appears to be different in mouse and human. Is it possible that this critical gene has different actions in different species?
The Action of SRY
Is SRY a transcriptional activator, as we would expect from its male-dominant action? Strangely, its properties look more like those of an inhibitor, and it has been suggested that it may operate not by turning on testis-differentiating genes, but by turning off a testis inhibitor (perhaps SOX3) [37]. Another related gene, SOX9, was found to be involved in testis determination in mammals and other vertebrates [38–40] and may interact with related genes to effect testis determination. This idea has been supported by the recent finding that mice with an insertional mutation upstream of SOX9 are male, even in the absence of SRY, suggesting failure to bind an inhibitor at an upstream control site (Bishop et al., unpublished communication).
The SRY gene need not necessarily be the original mammalian sex-determining gene. Indeed, there is no direct evidence that SRY is sex determining in marsupials, and no SRY has yet been discovered in monotremes. A possible forerunner to SRY is a mutation of SOX3 that no longer inhibits SOX9. The SOX3 gene may have originally worked in a dosage-sensitive manner, requiring two doses of the normal SOX3 to inhibit SOX9 and produce a female [37].
There may have been an even earlier mammalian sex-determining gene, perhaps represented by a new candidate sex-determining gene, ATRY, that was recently cloned from the marsupial Y chromosome. The ATRY gene is expressed appropriately in the marsupial embryo and is testis specific, whereas SRY is ubiquitous. The ATRY gene, too, has an X homologue from which it obviously evolved. In humans and mice, there is an X homologue, ATRX, and mutations in this gene causes XY female sex reversal. However, the ATRY gene has evidently been lost from the Y chromosome in eutherian mammals. Perhaps SOX3 first took over from ATRY in eutherians, then SRY later took over from SOX3. In rodents, Sry has evidently acquired a new function, and in mole voles, it has been superseded yet again with the loss of the entire Y chromosome.
Thus, even the mighty TDF is a wimp—a degraded relic of a normal CNS-determining gene that just gets in the way of another gene (SOX3?) in repressing SOX9. The SRY gene probably was not the first sex-determining gene in mammals and will not be the last, if mole voles are a portent of the future.
CONCLUSIONS
Of the three models of the human Y chromosome presented—the dominant Y chromosome, the selfish Y chromosome, and the wimp Y chromosome—the last model has the most explanatory power. Homology of the human Y chromosome with the X chromosome within the PAR and outside of the PAR suggests an origin as an ancient autosome pair. The genetic paucity and high content of repeated sequences and pseudogenes of the Y chromosome tells a tale of genetic degradation. Homology of a small part of the human Y chromosome with the tiny marsupial Y chromosome reveals the last vestiges of the ancient mammalian Y chromosome, a region of only a few megabases, containing only four known genes. Homology of the bulk of the human Y chromosome with autosomes in marsupials reveals a relic of a recent autosomal addition.
The ancient and added regions of the human Y chromosome may be represented on a map (Fig. 3). There is practically nothing left of the ancient mammalian Y chromosome (blue), and most of the chromosome is derived from the autosomal addition (red), which itself is degrading fast. This degraded Y chromosome is a wimp by any measure. It is deficient at every locus within the differentiated region. From its beginning as a gene-rich autosome, equivalent in gene content to the X chromosome, it has lost most of its 3000 or 4000 genes, as they were progressively mutated and deleted. A few pathetic relics remain as inactive pseudogenes. A mere handful of genes have clung to survival because of mutations that allowed them to adopt a male-specific function essential for sex determination or spermatogenesis.
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However, there are at least a few genes on the human Y chromosome that do not seem to originate from the ancient proto-X/Y chromosome or from autosomal addition. These genes must have been incorporated quite recently into the primate Y chromosome, by integration either of the DNA copy of an RNA transcript, or of a genomic copy of an autosomal gene. These genes would have to be colored green on our map (Fig. 3). So the Y chromosome is not only a wimp, it is a selfish wimp.
Perhaps, then, it is time to reassess the adage that maleness is dominant, and femaleness is a default condition. The view of females as some sort of deficient, mutant males arises from observations that an embryo with an XY karyotype will develop into a female if anything goes wrong with the male sex-determining pathway, from the absence of a functional SRY gene to the failure to bind and use testosterone. In genetic terms, however, it is the Y chromosome—the genetic element that defines maleness—that is the mutant element.
This wimp Y chromosome is disappearing fast, and, indeed, its future looks grim. In marsupials, there is only a tiny remnant left, and this remnant can be dispensed with in somatic tissues of some species. Even in eutherians, animals in which the Y chromosome has been salvaged by the addition of a large bit of autosome and some heterochromatic ballast, the Y chromosome is small and degenerate, and has few essential functions left. If we come back in 10 or 100 million years, we may well find that humans, like mole voles, have dispensed with the Y chromosome entirely. Perhaps they will have evolved an entirely new pair of sex chromosomes, beginning with an autosomal gene that took over a sex-determining function from SRY, the last in a long line of apparently disposable sex-determining genes.
FOOTNOTES
1 Supported by Australian Research Council grant A00000786 and National Health and Medical Research Council (Australia) grant 980990. 
2 Correspondence: Jennifer M. Graves, Department of Genetics, Building NW7, La Trobe University, Kingsbury Drive, Bundoora, Melbourne, Victoria 3093, Australia. FAX: 613 9479 2480; j.graves{at}gen.latrobe.edu.au
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