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Development 102, 251-258 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 Review Article 251 Genetics of sex determination in man and mouse P. N. GOODFELLOW1 and S. M. DARLING2 xHuman Molecular Genetics Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK Mount Sinai Hospital Research Institute, 600 University Avenue, Toronto, Ontario M5C 1X5, Canada 2 The cytological evidence has revealed a visible mechanical basis for the production of males and females in equal numbers and irrespective of external conditions (Wilson, 1909). Introduction In mammals, the sex of an individual is the result of an orderly cascade process initiated by genetic controls and propagated by endocrine mechanisms. This process results in the production of males and females which differ in reproductive function, anatomy, physiology and behaviour. Males and females also differ in sex chromosome constitution: males have a single X chromosome and a Y chromosome; females have two X chromosomes. Using DrosophUa species as a model, it was assumed that sex in mammals would depend on X-chromosome dosage; however, with improved techniques for karyotyping, investigation of individuals with abnormal sex chromosome constitutions refuted this hypothesis. Individuals with a single X chromosome develop as females (Ford et al. 1959; Welshons & Russell, 1959); individuals with a normal Y chromosome develop as males, independent of the presence of supernumerary X chromosomes (Jacobs & Strong, 1959; Cattanach, 1961). It was concluded that sex in mammals is determined chromosomally by the presence or absence of the Y chromosome. In 1930, Fisher proposed two alternative mechanisms for Y-chromosome control of sex: either all the genes encoding male traits are Y-linked or the Y chromosome encodes a control gene which regulates the expression of genes elsewhere in the genome. All the evidence is consistent with the second alternative and the current working hypothesis is that a Y-encoded gene induces the development of testes. Subsequent hormone production by the testes induces the male phenotype (Jost et al. Key words: sex determination, X chromosome, Y chromosome, TDF, Tdy, genetic map. 1973). As the male phenotype derives from the presence of the testis, the Y-encoded gene has been named testis-determining factor (TDF) in man and testis-determininggene-Ychromosome (Tdy) in mouse. In the absence of the Y chromosome, ovary-controlling genes induce the production of ovaries (Eicher & Washburn, 1986) and, in the absence of male hormones, the female phenotype develops (Jost et al. 1973). Key regulatory genes that are responsible for choosing between different developmental genetic programmes have been described in several animals (e.g. DrosophUa species, Akam, 1987). Identification of these regulatory genes in mammals has proved to be more problematic. However, the genes responsible for the choice between male and female phenotypes must be developmental regulatory genes. Isolation of these genes will not only answer fundamental questions about the phenomenon of sex but may also provide a general model for the genetic control of mammalian development. In this review, we will explore the genetic mechanisms that underlie the formation of the two sexes in mouse and man. This review covers much of the same ground as the recent Supplement to Development and readers desiring a more detailed treatment should consult that issue. Sex determination and sex differentiation The pivotal event in determining the sex of an individual is the specialization of the gonads. Once formed, the gonads control the subsequent sexual phenotype (Jost et al. 1973). For the purposes of this review, we will define sex determination as the process that results in either testis or ovary formation. The process subsequent to gonad formation we will define as sex differentiation. Obviously as our knowledge improves it may be possible to define more precisely the sex determination step prior to the formation of the specialized gonads. 252 P. N. Goodfellow and S. M. Darling The process of sex differentiation is well understood at the anatomical and physiological levels. In males, the development of the testis as an organ is associated with cellular development of male germ cells, Sertoli cells and Leydig cells. The Leydig cells produce testosterone, and high levels of this hormone induces Wolffian duct development leading to the formation of the epididymis, vas deferens and seminal vesicles. Testosterone also induces the bipotential primordia of the external genitalia to develop into penis shaft, scrotum and glans penis. The Sertoli cells produce anti-Mullerian hormone (AMH, also known as Miillerian-inhibiting factor, substance or hormone; MIF, MIS, MIH) which causes regression of the Miillerian ducts (Josso et al. 1977). The formation of ovaries in females is associated with the development of the female phenotype: the production of oviducts, uterus and cervix from the Miillerian duct; regression of the Wolffian duct and differentiation of the external genital primordia to produce female genitals. Surgical removal of the gonads from a fetus results in the subsequent formation of female internal and external genitalia, implying that the fetal ovaries are not directly required for differentiation of the female phenotype (Jost et al. 1973). The description of sex differentiation outlined above has been deduced largely from experiments designed to change the hormonal status of the fetus and the conclusions have been confirmed by analysis of individuals in whom gonadal and phenotypic sex have been dissociated by genetic mutation. A particularly illustrative mutation is testicular feminization (Tfm) which has been described in mouse, rat and man (Ohno, 1979). In the afflicted individual, testis formation and secretion of androgens and AMF is normal; however, the external phenotypic appearance is female with normal female external genitalia. The defect is due to a deficiency of androgen receptors in the target tissues (Attardi & Ohno, 1974; Migeon etal. 1981). Thus, differentiation proceeds as if testosterone is absent, with regression of the Wolffian duct and normal development of female external genitalia. The AMH secreted by the testes causes Miillerian duct regression so that internal female genitalia fail to develop. Similarly, several other mutations interfering with androgen metabolism cause dissociation between male sex determination and subsequent differentiation (Polani, I981a,b; Lyon etal. 1981). The detailed knowledge of the morphology and endocrinology of sexual differentiation makes this an ideal model system for studying control of organ formation at the cellular and molecular level. Compared with sex differentiation, information about the biochemical basis of sex determination is rudimentary. Nevertheless, genetic analysis of individuals with abnormal sex determination and the application of techniques of molecular biology have provided a starting point for understanding sex determination in mammals. The biology of the Y chromosome The mammalian Y chromosome is composed of two separate regions with distinct functions; both regions are essential for the Y chromosome's role in sex determination (Darlington, 1937). The first region is shared with the X chromosome and is responsible for pairing between the morphologically dissimilar X and Y chromosomes in male meiosis. It is assumed that pairing is essential for the correct meiotic segregation of sex chromosomes which forms the basis for chromosomal determination of sex. Maintenance of sequence homology is achieved by recombination within the shared region (Bengtsson & Goodfellow, 1987). Sequences and genes that exchange between the sex chromosomes fail to show classical 'sex linkage' and this type of inheritance has been termed pseudoautosomal (Burgoyne, 1982). The region shared by the sex chromosomes is frequently referred to as the pseudoautosomal region (PAR). In man, the PAR is at the ends of the X- and Y-chromosome short arms (Pearson & Bobrow, 1970; Chen & Falek, 1971); in mouse, the PAR is assumed to be at the end of the apparently single arm of the X and Y chromosomes. The second region on the Y chromosome encodes genes that must not recombine with the X chromosome. The paradigms for this behaviour are TDF and Tdy; if these sex-determining genes were to become X-linked the chromosomal basis of sex determination in man and mouse would break down. Genetic definition of TDF in man Several individuals have been found who have visible deletions- and translocations of the Y chromosome (Buhler, 1980; Davis, 1981; Goodfellow et at. 1985a). Correlation of sexual phenotype with these Ychromosome structural abnormalities suggested that TDF was located in the region around the centromere. These studies were hampered by the small size of the Y chromosome which made it difficult to define the limits of deletions and translocations. A second problem was the frequent occurrence of mosaicism and chimaerism which can result in a different karyotype in the tissue tested, usually blood or skin, compared with the gonad. More precise definition of the chromosomal location of TDF has been made possible by the construction of detailed maps of the human Y Genetics of sex determination chromosome. Although the Y chromosome is about one third the size of the X chromosome, it is remarkable in being almost completely devoid of genes. Whereas over 100 genes have been mapped to the X chromosome, only four or five genes are known to be encoded by the Y chromosome and one of these is pseudoautosomal (Goodfellow et al. 1985b). This paucity of genetic markers inhibited the construction of genetic maps until the introduction of molecular techniques allowed the isolation of DNA sequences, which provided an unlimited supply of markers (reviewed in Goodfellow etal. 1985a). Three types of genetic map have been constructed and all three have contributed to the definition of TDF. The first map is a meiotic map of the PAR. In each male meiosis, a single obligate recombination occurs within the PAR and the telomeric sequence DXYS14 (Cooke et al. 1985) shows 50 % recombination between the sex chromosomes (Rouyer et al. 1986). MIC2, a pseudoautosomal gene, recombines between the X and Y chromosomes with a frequency of 2% (Goodfellow et al. 1986). Between these extremes other markers show intermediate recombination rates. Recombination in the PAR of man has several features of unique biological interest. (1) The PAR has been estimated to be less than 5xl0 6 bp in size (Mondello et al. 1987; Rouyer et al. 1986); this means that the 50 % recombination in this region seen in male meiosis, occurs at a rate ten times higher than the genome average. (2) Double recombinants have not been found in the PAR (Rouyer et al. 1986; Goodfellow et al. 1986). One explanation is that interference is a function of physical size and not of recombination fraction. (3) Recombination in female meiosis in the PAR is only 4 - 5 % , approximately tenfold lower than in male meiosis (Rouyer et al. 1986; Goodfellow et al. 1986). The second type of map is a deletion map based on the sequences present in the genomes of XX males. About 1 in 20000 new-born males has a female karyotype with two X chromosomes (de la Chapelle, 1981). The majority of XX males have a normal phenotype, with the exception of the testes which are small; all XX males are sterile. In 1960, FergusonSmith and colleagues suggested that XX males might be caused by the inheritance of undetected Ychromosome sequences (Ferguson-Smith etal. 1960); this hypothesis was expanded by the proposal that an abnormal meiotic exchange between the X and Y chromosomes could lead to the formation of XX males (Ferguson-Smith, 1966). This suggestion has proved to be correct. About 80 % of XX males have inherited Y-derived sequences (Guellaen et al. 1984; Page, 1986; Pritchard, 1987) and these sequences are found at the tip of the X-chromosome short arm, 253 consistent with an XX interchange model (Andersson et al. 1986; Buckle et al. 1987). The amount of Yderived sequences transferred in different XX males is variable and this has allowed the construction of a deletion map of the Y chromosome (Vergnaud et al. 1986; Page, 1986). From this map, TDF has been placed in the distal region of the Y-specific part of the short arm, adjacent to the pseudoautosomal region. This implies that the pseudoautosomal gene MIC2 is a flanking marker for TDF, as is DXYS5, the mostdistal Y-specific marker described by Vergnaud et al. (1986). The region between MIC2 and DXYS5 on the Y chromosome contains all of the genetic information required for male sex determination. Indirect estimates and preliminary physical mapping by longrange restriction enzyme analysis both suggest that this region is not bigger than a few million basepairs (G. Rappold, personal communication). Any theory for the mode of action of TDF that requires multiple copies of the gene (Ohno, 1967; Chandra, 1985) must be constrained by these observations. The third map is a physical map utilizing long-range restriction mapping and pulsed field gel electrophoresis. Using DNA probes for MIC2 and DNA probes for several Y-specific sequences, we have constructed a map which spans the boundary between the PAR and the Y-specific regions of the Y chromosome (Pritchard et al. 1987). From this map, we have identified sequence motifs, known as HTF islands (Bird, 1986), which are frequently associated with the promoter regions of genes; one HTF island is Yspecific and lies close to the PAR boundary. This HTF island may define TDF. However, it should be stressed that not all genes are marked by the association with an HTF island (Bird, 1986) and TDF may fall into this class. Alternative candidate genes for the defined HTF island might include genes associated with Turner's syndrome. This syndrome is found in females with a single X chromosome and consists of streak gonads, due to premature loss of oocytes, and a variety of physical stigmata, including short stature (Turner, 1938). Sex-reversed females with an XY karyotype occur with a frequency of about 1 in 100 000 new-born females (Polani, 1981a). These females also have streak gonads and occasionally have other components of Turner's syndrome. The X-Y interchange model can be invoked to explain the generation of XY females since the reciprocal product of an abnormal X-Y recombination event would consist of a Y chromosome deleted for Y sequences including TDF and containing X-chromosomal sequences. Deletion of Y-specific sequences has been found in several XY females (Disteche etal. 1986; Mulleref al. 1986; Page, 1986). Two explanations are possible for the variable 254 P. N. Goodfellow and S. M. Darling expression of the nongonadal components of Turner's syndrome in XY females. The Y chromosome and/or pseudoautosomal region could encode genes the deletion of which results in Turner's syndrome (Rosenfeld et al. 1979); in this case, the HTF island we have identified may indicate the presence of these genes. Alternatively, the variable amount of X sequences transferred to the deleted Y chromosome may encode the Turner's syndrome genes. Genetic definition of Tdy in mouse Analysis of sex-reversed syndromes in mice has led to the localization of Tdy. In 1971, Cattanach et al. described a dominant mutation that causes sex reversal of female mice (Cattanach et al. 1971). Males carrying this mutation, called sex reversed (Sxr), produce four different types of offpsring when mated to normal females; normal XX females, normal XY males, Xtr-carrier XY males and sterile Sxr XX individuals with a male phenotype (XX Sxr males). Sxr was postulated to be either a constitutive mutation of an autosomal sex-determining gene that is normally under Y control, or the transposition of a strongly sex-determining Y region to an autosome. Evidence to support either of these hypotheses was lacking since attempts to map Sxr cytologically and by linkage studies had failed (Cattanach et al. 1971; Cattanach, 1975; Winsor et al. 1978; Chandley & Fletcher, 1980; Evans et al. 1980). Discovery of the chromosomal localization of Sxr came from an unexpected source. Singh et al. (1980) isolated a femalespecific satellite DNA sequence from the banded krait snake (Bkm, banded fcrait minor satellite). In these snakes, females are the heterogametic sex and males the homogametic sex. Using the Bkm DNA probe in Southern blot analysis of mouse DNAs, a hybridizing fragment was observed that was specific to male DNA. XX Sxr male DNA when analysed with the same probe showed the male-specific pattern. In addition, Singh & Jones (1982) were able to demonstrate by in situ hybridization to mouse chromosomes that there is a concentration of Bkmrelated sequences on the normal mouse Y chromosome at its centromere. XX Sxr males had a concentration of Bkm-related sequences localized at the distal end of a large chromosome which they identified as probably being the X chromosome. Singh & Jones (1982) went on to propose that the Sxr mutation results from this transposition of a fragment of the Y chromosome containing Tdy and a concentration of Bkm-related sequences onto another chromosome, most probably the X. Burgoyne (1982), Eicher (1982) and Hansmann (1982) have proposed a model to explain the location and transmission of Sxr. The consensus suggestion is that an extra copy of the Y- linked Tdy is present on the distal tip of the Y chromosome in the XY Sxr carrier male. The distal tip area of the Y chromosome is proposed to be the pseudoautosomal region and both the gene STS (Keitges et al. 1985) and a viral integration site (Habers et al. 1986) are known to be inherited pseudoautosomally in mice. During male meiosis, the duplicated Sxr fragment is transferred onto the X chromosome by an obligate pseudoautosomal recombination event, inheritance of this X chromosome results in XX Sxr males. Cytological confirmation of the presence of the Sxr fragment at the distal end of the Y chromosome in XY Sxr carriers and at the distal end of one X chromosome in XX Sxr males was shown by Evans et al. (1982). It has been suggested that Bkm sequences may be directly related to sex determination either by encoding a gene product (Jones, 1983; Epplen et al. 1982) or by binding an autosomal repressor of the male phenotype (Chandra, 1985). This type of hypothesis is very difficult to refute, however; the distribution of Bkm sequences throughout the mouse genome (Kiel-Metzger & Erickson, 1984) and the absence of a marked concentration of Bkm sequences on the Y chromosome in other species (Kiel-Metzger et al. 1985; and unpublished observations) are not entirely consistent with the hypothesis. Cytological investigation suggests that Sxr is about 5 x 106 bp in size and this must define the limits of Tdy. Recently, Sxr', a derivative of Sxr, has been found which has lost genetic information required for both H-Y antigen expression (McLaren et al. 1984) and early spermatogenesis (Burgoyne et al. 1986). The precise size of Sxr' is not known. The failure of Sxr' to express H-Y antigen and similar failure of human XX males to express H-Y antigens (Simpson et al. 1987) argues strongly that the H-Y antigen is not directly involved in sex determination. Autosomal and X-linked genes involved in sex determination It is part of the paradigm we are following that TDF and Tdy interact with other genes to induce the production of testes. Mutations in the 'responder' genes could therefore result in abnormal sex determination. Evidence for 'responder ' gene mutations has been obtained in man, mouse and several other species. However, it should be stressed that a loss-offunction mutation in a 'responder' gene would have the same phenotype as a gain-of-function mutation making the production of ovaries dominant to the production of testes. 20 % of XX males have no detectable Y-derived sequences. Three explanations for these males can be proposed (de la Chapelle, 1987). Genetics of sex determination (1) Mosaicism such that the gonads have a different genetic constitution to the tested tissues. (2) Inheritance of Y-derived sequences for which DNA probes are not available. (3) Mutation in 'responded genes. Several XX males have been described in which no Y-derived sequences have been found and the proximal pseudoautosomal region, inherited from the father, has been derived from the X chromosome (Petit et al. 1987). These males cannot have been derived by an X-Y interchange and one explanation is a loss of function mutation in a responder gene(s). Similarly, clustering of XX males in families may imply mutation in non-Y-linked genes (de la Chapelle, 1987). However, a proportion of these familial XX males are known to be due to X-Y interchange (Page etal. 1985). There are several examples in the mouse of autosomal loci disrupting normal male development in XY individuals. When transferring the Y chromosome from Mus domesticus poschiavinus, to the genome of the C57BL/6J inbred strain, Eicher et al. (1982) noticed there was a breakdown of the normal testis-determination mechanism resulting in partial or complete sex reversal of XY individuals. Similar XY females have been noted when crossing Y chromosomes from other Mus domesticus strains onto the C57BL/6J background (reviewed in Eicher & Washburn, 1986). These findings have led Eicher and her colleagues to postulate that the C57BL/6J strain contains an autosomal sequence which does not interact correctly with the Tdy locus or its product encoded by the Y chromosome from M. domesticus. This implies that Tdy is different on the two Y chromosomes and that C57BL/6J carries an allele different from that of M. domesticus at an autosomal locus involved in testis determination. This locus, designated Tda-1, probably resides on either chromosome 2 or 4 (Eicher & Washburn, 1986). Another autosomal sex determination locus known as T-associated sex reversal (Tas) is on chromosome 17 located within the hairpin-tail deletion (Washburn & Eicher, 1983). The Tas mutation causes sex reversal in XY mice on the C57BL/6J background but not when crossed onto a C3H/HEJ background. Again ovaries and ovotestes are found in the XY individuals. Washburn & Eicher (1983) and Eicher & Washburn (1986) have proposed that the Tas locus could function as the ovary-determining gene. Ovarian development would be initiated by the normal allele in the XX mouse but this allele would be inactive in XY individuals when Tdy initiates testis formation. If Tas is active in XY individuals before or simultaneously with Tdy then ovaries or ovotestes would develop. Alternatively, Tas could be defined 255 by loss-of-function mutations which fail to respond to Tdy. In both cases, Tdy function would be dominant to Tas function in normal sex determination. The Polled mutation in goats is an autosomal recessive and produces the male phenotype in karyotypically XX individuals (Hamerton et al. 1969). This puzzling mutation could be a weak constitutive mutant in a responder gene. The wood lemming (Myopus schisticolor) has a very complex sex-determining system (Schempp et al. 1985). In addition to the usual X and Y chromosomes, a modified X chromosome, X*, is also found. X* can be karyotypically distinguished from the normal X chromosome by G banding. Remarkably, X*Y animals are fertile females, implying that a 'responder' gene on the X chromosome is needed for testis formation and that this gene is mutated to a nonresponsive form on the X* chromosome. Although the results are reminiscent of rare human pedigrees in which sex reversal of XY individuals appears to be inherited as an X-linked trait (Simpson et al. 1981), the gross chromosomal differences between X and X* makes it unwise to draw a simple conclusion about sex determination based on the wood lemmings. Mode of action of testis-determining genes The biochemical products of TDF and Tdy have not been defined; nevertheless, it is possible to pose the question of where these genes are expressed. The gonads of both sexes are derived from two distinct tissues: somatic mesenchymal tissues which form the matrix of the gonad and primordial germ cells which migrate into and colonize the matrix. The germ cells eventually form the gametes. Two mutations in the mouse, Steel and White spotting, cause the number of germ cells colonizing the genital ridges to be reduced to a very low number without affecting formation of the gonad in either sex (McLaren, 1985). In addition, XY germ cells can undergo oogenesis and XO germ cells can reach the early stages of spermatogenesis (the presence of a second X chromosome inhibits spermatogenesis in both XX and XXY animals). These results suggest that germ cells are irrelevant for sex determination and that germ cells themselves respond to environmental signals (McLaren, 1985). Recent experiments suggest that the Sertoli cell may be the site of action of the testis-determining genes. In XX-XY chimaeric mice, where both XX and XY cells are found in a gonad, it is uncommon to find an ovotestis with separate ovarian and testicular regions (McLaren, 1985). Testicular development in chimaeras is usually found in the adults that have 25-30 % or more XY cells and ovarian development 256 P. N. Goodfellow and S. M. Darling in those with 15-20 % or less XY cells. Ovotestes are associated with the intermediate range. These results could be explained by the XY cells producing a diffusible inducing factor causing both somatic and germ cells to undergo testicular differentiation. Burgoyne et al. (1986) have proposed an alternative interpretation. In a series of transplantation experiments, juxtaposition of male and female fetal gonads failed to induce male development in the female gonads, although profound oocyte loss was noted. To explain the apparently contradictory results from chimaeras and transplantation, it was suggested that a diffusible substance produced by the male gonads inhibits the development of ovaries. A good candidate for this factor is AMH which has recently been found to induce atrophy of oocytes (Vigier et al. 1987). As Sertoli cells produce AMH and are present at the earliest stages of testis formation, it was proposed that these cells are the site of action of Tdy. This hypothesis was tested by reinvestigating the gonads of XX-XY chimaeras (P. Burgoyne, personal communication). It was found that Sertoli cells always had an XY chromosomal constitution, but the germ cells and Leydig cells in fetal testes could be either XX or XY. Sertoli cells never had an XX chromosomal contribution. This result suggests that a cell must have a Y chromosome to be a Sertoli cell in a developing gonad. The simplest but not the only explanation is that Tdy acts in a cell autonomous manner to produce Sertoli cells. Conclusions The Y chromosome is intimately associated with male sex determination in mammals. Although the biochemical basis of sex determination is not clear the precise chromosomal localization of TDFand Tdy can be used as the starting point for cloning these genes by reverse genetic methods (see Development 101 Supplement). 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