Cell, Vol. 16. 691-695,
April 1979,
Copyright
0 1979
by MIT
lmmunogenetic Aspects Sexual Differentiation Stephen S. Wachtel Memorial Sloan-Kettering Cancer 1275 York Avenue New York, New York 10021 and Department of Pediatrics New York HospitalCornell Medical Center New York, New York 10021
of Abnormal
Center
In mammals, testicular differentiation and subsequent male or intersexual development may occur in the apparent absence of the Y chromosome. Thus, for example, XX males and XX true hermaphrodites, the latter exhibiting a combination of ovarian and testicular tissue, have been documented in several mammalian species including goat (Hamerton et al., 1969) dog (Hare, 1976) mouse (Mittwoch and Buehr, 1973) and human (de la Chapelle, 1972). There are reports of XX males and XX true hermaphrodites in the same sibship in man (Berger et al., 1970; Kasdan et al., 1973) and recently Selden et al. (1978) discovered ovotestes in the XX mother of an XX male in a family of cocker spaniels. Both dogs were H-Y’ in serological tests, and both carried an apparent Y-to-autosome translocation inherited from the father of the true hermaphrodite (grandfather of the XX male). These findings favor the view that XX true hermaphroditism and the XX male syndrome are alternative manifestations of a common event associated with abnormal inheritance of Y chromosomal (H-Y) genes. Yet why should related individual animals with identical sex chromosomes and identical H-Y’ phenotypes exhibit the different modes of sexual differentiation and development represented by the XX male with testes, on the one hand, and the XX true hermaphrodite with ovotestes, on the other? The inherent tendency of the mammalian embryo to develop as a female is countered by genes on the Y chromosome which induce the undifferentiated gonadal rudiment to become a testis instead of an ovary. Testosterone, secreted by the newly formed testis, induces the secondary sexual characteristics of the male (Jost, 1970). Thus the pivotal role of the Y chromosome in mammalian sex determination is limited to the induction of the testis. Much evidence points to H-Y antigen as the Y-determined factor responsible for this induction (Wachtel and Ohno, 1979; Ohno et al., 1979; also see below). Phylogenetic Conservation of H-Y Antigen Differentiation of testis is associated with the presence of H-Y antigen in all mammals and indeed in all male heterogametic (XV) vertebrates studied thus far, regardless of apparent karyotype or secondary sex phenotype (Wachtel, 1977). Hence H-Y antibody is absorbed not only by cells of the male rat or mouse, but also by cells from males representing species as
Review
divergent as pig and platyfish (Fellous et al., 1978; P. Pechan, S. Wachtel and R. Reinboth, manuscript in preparation). As noted above, XX males and XX true hermaphrodites of the dog-are H-Y+. The same is true of XX males and XX true hermaphrodites of the goat (Wachtel. Basrur and Koo, 1978) and human (Wachtel et al., 1976a), and XY females with testicular feminization syndrome (Bennett et al., 1975; Koo et al., 1977b); fertile XY females of the wood lemming, however, are H-Y- (see below). To the extent that H-Y antigen is specified by genes in the pericentric (maledetermining) region of the human Y chromosome (Koo et al., 1977a; Wachtel and Ohno, 1979) its occurrence in XX subjects implies the presence of at least a portion of the Y. In the mouse, the autosomal dominant gene Sxr (‘sex-reversed”) causes expression of H-Y and differentiation of testis in embryos with the XX karyotype (Bennet et al., 1977). Yet if Sxr represents a translocation of the Y, the translocated segment is so minute as to be thus far undetectable cytologically. It follows that Sxr and H-Y genes are either very closely linked or identical. A Hormone-like Inductive Role of H-Y Antigen It has been suggested that the mammalian testis is induced by a diffusible substance released from XY cells (Ford, 1970) and the following considerations indicate that H-Y, in common with other components of the plasma membrane, is in fact released in the free state: H-Y occurs on XX cells within the XX/XV testes of the chimeric male mouse (Ohno et al., 1978a). It is abundant in the masculinized gonads of the bovine freemartin embryo (Ohno et al., 1976) even when only a small proportion of XY cells are present. It would follow that disseminated H-Y can be found by XY and XX target cells, both having H-Y receptors, and that irreversible commitment to testicular development is the usual consequence of this engagement, assuming a certain minimal threshold of H-Y production. Indeed, Muller et al. (1978) have now demonstrated binding of “exogenous” H-Y (from rat epididymal fluid) by cells of the XX fetal rat gonad, and Ohno et al. (1979) report induction of testicular tubules in XX presumptive ovaries of the fetal calf with H-Y antigen released from cultured Daudi cells. XY Ovaries In the absence of H-Y antigen, XY gonadal cells of the mammal organize an ovary. Dispersed testicular Sertoli cells (XV) that have been lysostripped of H-Y antigen reaggregate in vitro to form ovarian follicles in mice (Ohno, Nagai and Ciccarese, 1978b) and rats (Zenzes et al., 1978) and as indicated above, failure of H-Y synthesis leads to development of ovaries in fertile XY females of the wood lemming, Myopus schisticolor (Wachtel et al., 1976b). It is not surprising that XY cells within the gonads of XX/XV chimeric mice can also be induced to participate in ovarian differ-
Cell 692
entiation. These XY cells may even become oocytes (Ford et al., 1975; Evans, Ford and Lyon, 1977). Since ovarian differentiation in XY cells of the wood lemming in vivo and of the mouse and rat in vitro always goes hand in hand with the absence of H-Y antigen, it follows that H-Y should be absent from XY as well as XX cells of the XX/XV chimeric ovary. But can ovarian differentiation be attributed wholly to the absence of H-Y? In that case, we should expect differentiation of ovotestes in XX/XV chimeric gonads with a paucity of XY cells, for example; yet true hermaphrodites are rare among chimeric mice, whereas XX/XV females with grossly normal ovaries are common (McLaren, 1976). [An example of hermaphroditic development in regular association with sex-chromosomal mosaicism may be found in XO/XY and XO/XY/XYY mice of the BALB/cWt strain. Presence of the aneuploid cell line notwithstanding, there is evidence that the high incidence of hermaphroditism in this strain (3%) is due at least in part to presence of a deficient BALB/cWt Y chromosome (Beamer, Whitten and Either, 19791.1 An Ovary-Inducing Antigen? The rarity of ovotestes in XX/XV chimeric mice as compared with the far more commonly definitive and unequivocal differentiation of testes or ovaries could be explained if the testis-organizing H-Y molecule were competing with another molecule capable of blocking occupation of H-Y receptors. Since ovarian differentiation is the normal consequence of reaction of this second molecule, the latter might be aptly viewed as an inducer of ovarian rather than testicular differentiation. As to the mechanism, it could be envisioned that there are nonspecific receptors on the surfaces of both XX and XY cells and that the differentiative response depends upon which of the two inducers is engaged by these receptors (Figure I), or that there are specific receptors having alternative and exclusive differentiative responses (Figure 2). In the XX/XV chimeric gonad, the two inducers would be placed in competition among the receptors of the cell surface, and the predominance of one or the other would lead to unambiguous testicular or ovarian differentiation. Why then do most XX/XV chimeric gonads become testes? It has been argued that this is because testicular tissue differentiates earlier than ovarian tissue (Tarkowski, 1961) (although testicular differentiation in the freemartin occurs after ovarian differentiation in the normal cow). It could also be argued, however, that during the development of the normal XY rodent, H-Y is disseminated in large quantities to compete successfully with the ovary inducer. In the normal XX embryo there would be no need for a corresponding dissemination of the ovary inducer, because there is no competition from Y-determined testis inducer. Thus in the chimeric or mosaic gonad, H-Y antigen would
be likely to dominate ably be masculine.
and differentiation
would
prob-
Competition in the XX Ovotestis In man (but not to the same degree in mice), two X chromosomes are evidently needed to sustain normal ovarian differentiation. This is true to the extent that two X chromosomes are necessary for survival of female germ cells (Short, 1978). Embryos with the 45X0 karyotype initially develop ovaries, but these degenerate and are later represented by “dysgenetic” gonads lacking definitive ovarian structure and characterized instead by undifferentiated stroma. It follows that in H-Y+ human embryos with the 46,Xx karyotype (XX’), factors favoring ovarian versus testicular induction are more obviously in competition. If general dominance of H-Y leads to development of testes, then true hermaphroditism could be viewed as the outcome of inconclusive competition, which might be expected with a certain degree of probability in XX/XX’ mosaics. Thus, for example, regional dominance of H-Y leads to development of one testis and a contralateral ovary (“alternating hermaphroditism”), whereas local dominance within a single gonad or in both gonads leads to development of ovotestis [versus ovary (“unilateral hermaphroditism”) or bilateral ovotestes (“bilateral hermaphroditism”); see Figure 31.
Figure
1. Nonspecific
Receptors
for Gonad-Organizing
Proteins
Molecules of H-Y antigen (small Ys) and ovary inducer (small OS) are disseminated (perhaps by different cells and at different stages) and bound by nonspecific receptors on target cells (1) of XX/XV for XX’) fatal gonad. Excess of H-Y causes displacement of ovary inducer, thereby promoting organization of testis.
Sexual 693
Differentiation
Simultaneous differentiation of ovarian and testicular architecture in such embryos must reflect the presence of H-Y in presumptive testis and the absence of H-Y from presumptive ovary, and this implies that the true hermaphrodite is an H-Y+/H-Ymosaic regardless of karyotype. This implication is borne out in a study performed by Winters et al. (1979). The subject of this study appeared to be a male with first degree hypospadias and a 46,Xx karyotype, but exploration of the scrotum revealed a bilobed gonad comprising both ovarian and testicular elements. Serological tests showed that cells cultured from the testicular portion of the ovotestis were H-Y’, whereas cells cultured from the ovarian portion were H-Y-. In view of the above discussion, this could denote XX/XX’ mosaicism heavily favoring an XX cell line in the fetal gonad or, alternatively, the presence of a mosaic cell line bearing receptors with low affinity for disseminated H-Y.
tured male lymphomas that have retained /3*m-HLA. But when Daudi HLA antigens are restored by hybridization of Daudi cells with female cells of the HeLa D98 line, normal expression of H-Y is also restored (Beutler et al., 1978; but see Fellous et al.. 1978). On the basis of these observations, and in view of evidence for the dissemination of H-Y, it has been proposed that cell surface antigens determined by the major histocompatibility complex (H-2 in the mouse, HLA in man) act in association with Pn-microglobulin as the nonspecific cell membrane anchorage sites of organogenesis-directing proteins in general (Ohno, 1977). It follows that molecules of H-Y antigen and the putative ovary inducer should be disseminated at a particular stage of embryogenesis and bound by MHC-determined anchorage sites or “carriers,” and that once saturated with a particular inducer, cells of the primordial gonad should be impelled to follow a particular pathway of differentiation.
A Role of HLA in Sex Determination? In mice, the reaction against H-Y incompatible transplants is influenced by major histocompatibility complex (MHC) antigens of the donor, so that H-2k male skin grafts are rejected more quickly than H-2b male skin grafts by H-2b/H-2k hybrid females (Silvers and Billingham, 1967; Wachtel, Gasser and Silvers, 1973; Kralova and Demant, 1976); in rats, male cells carrying the Ag-B’ haplotype most strongly sensitize female mice with respect to mouse H-Y antigen (Silvers and Yang, 1973); and in vitro the reaction of cytotoxic T cells to H-Y-incompatible target cells is MHC-restricted (much as the reaction of cytotoxic T cells to viral-infected or viral-transformed target cells is MHCrestricted), not only in rodents (Simpson and Gordon, 1977) but also in man (Goulmy et al., 1977). These observations point to a functional relationship between H-Y molecules and components of the MHC. There is now direct evidence of a physical association between H-Y and ,&-microglobulin-HLA in the human male: cultured “Daudi” cells from a male Burkitt lymphoma that has lost P*rn (and thereby HLA) absorb considerably less H-Y antibody than cells from cul-
A Gonad-Specific Receptor All normal cells are endowed with MHC antigens, and thus an essential difference between somatic and gonadal tissues must lie in possession by the latter of specific receptors (as well as nonspecific anchorage sites) for the inducers of testicular and ovarian differentiation (Figure 2). Certainly MHC-determined carriers are present in XX and XY cells. The same must be true for gonad-specific receptors of both H-Y (Muller et al., 1978) and the ovary inducer, since as we have seen, XX gonads can become testes and XY gonads can become ovaries. Whereas H-Y and the ovary inducer are bound by the same nonspecific carrier, the gonad-specific receptor should engage only the active site of its particular substrate-in the case of H-Y, the phylogenetically conserved moiety identified by antibody from male-grafted female mice. No doubt the specific active site of the disseminated testis inducer is the same H-Y antigen borne “nonspecifically” in skin, blood and other somatic tissues, and also in gonadal tissues of the normal male. Thus sexual differentiation of the primordial gonad presumably involves not only dissemination of an inducer molecule and its binding to MHC-determined carriers, but also its engagement by specific receptors which may or may not be MHC-associated. If in fact /%m-HLA components serve as nonspecific carriers for the inducers of gonadal differentiation in man, a critical difference between XX true hermaphrodites and XX males may reside in the peculiar affinity of certain HLA haplotypes for an ovarian versus testicular determinant. Furthermore, if dissemination of a testis inducer were a conditio sine qua non of testicular differentiation as all available evidence indicates, then certain forms of pure gonadal dysgenesis could also be HLA-associated. On the other hand, general failure of testicular differentiation in H-Y’ subjects with the 46,XY karyotype could result from lack
Figure
2. Specific
Receptors
for Gonad-Organizing
Proteins
Uncommitted cell (1) of the XX/XY chimeric or mosaic fetal gonad possessing specific receptors for H-Y antigen and ovary-inducing “antigen” may follow alternative and mutually exclusive pathways of differentiation: ovarian (a) or testicular(b). Occupation of one receptor by its respective inducer blocks occupation of adjacent receptor via steric interference or changes in membrane topography, for example.
Cell 694
local
General
Dominance
Testis
restis
Ford, C. E.. Evans, E. P.. Burtenshaw, M. D., Clegg, H. M.. Tuffrey. M. and Barnes, R. D. (1975). A functional “sex-reversed” oocyte in the mouse. Proc. Roy. Sot. Lond. B 190. 187-l 97. Goulmy, E.. Termijtelen, A., Bradley, B. A. and van Rood, J. J. (1977). Y-antigen killing by T cells of women is restricted by HLA. Nature 266, 544-545. Hamerton. J. L.. Dickson, J. M.. Pollard, Short, R. V. (1969). Genetic intersexuality (Supplement) 7. 25-51.
ReDional Dominance
Dominance
Hare, 15.
Ovotestls
Ovotestls
Figure 3. Modalities XX/XX’ Fetal Gonad
ovary
of Testicular
Differentiation
TestIs in the
XX”
or
Black dots denote areas of H-Y saturation. General Dominance: prevalence of H-Y’ leads to development of unambiguous testes in “XX” male. Regional Dominance: H-Y structural genes in one gonad but not the other cause “alternating hermaphroditism.” This condition could also result from mosaicism of receptor determinants. Local Dominance: differentiation of ovotestis (the most common gonad in XX true hermaphroditism) reflects focus of cells bearing H-Y structural determinants or, alternatively, a local mosaicism of receptor determinants.
of affinity of disseminated H-Y for a particular gonadspecific receptor, and any fitful attempts to organize a testis could be attributed to indigenous H-Y molecules which constitute a more “stable” portion of the cell membrane in all male tissues. Acknowledgments This work was supported Received
November
by grants
6. 1978;
revised
References Beamer. W. G.. Whitten. W. K. and Either, E. M. (1979). Spontaneous sex mosaicism in BALB/cWt mice. In Symposium on Genetic Mosaics and Chimeras in Mammals, 1978, in press. Bennett, D.. Boyse. E. A., Lyon, M. F.. Mathieson, B. J.. Scheid, and Yanagisawa. K. (1975). Expression of H-Y (male) antigen phenotypically female Jfm/Y mice. Nature 257, 236-238.
M. in
Bennett, D., Mathieson, B. J., Scheid. M., Yanagisawa, K.. Boyse, E. A., Wachtel, S. S. and Cattanach. B. M. (1977). Serological evidence for H-Y antigen in Sxr. XX sex-reversed phenotypic male?.. Nature 265, 255-257. J. (1970). Rev. Eur.
Beutler, B., Nagai. Y.. Ohno. S.. Klein, G. and Shapiro, I. M. (1978). The HLA-dependent expression of testis-organizing H-Y antigen by human male cells. Cell 73, 509-513. de la Chapelle, A. (1972). with XX sex chromosomes.
Analytic review: Am, J. Human
nature and origin of males Genet. 24, 71-I 05.
Evans, E. P., Ford, C. E. and Lyon, M. F. (1977). Direct evidence of the capacity of the XY germ cell in the mouse to become an oocyte. Nature 267, 430-431. Fellous, M.. Gunther, E.. Kemler. R., Wiels. J., Berger. R.. Guenet, J. L.. Jakob. H. and Jacob, F. (1978). Association of the H-Y male antigen with fin-microglobulin on human lymphoid and differentiated mouse teratocarcinoma cell lines. J. Exp. Med. 147. 58-70. Ford, C. E. (1970). Cytogenetics and sex determination mammals. J. Biosoc. Sci. Suppl. 2, 7-30.
in the dog. Can. Vet. J. 17. 7-
Jest. A. (1970). Hormonal factors in the sex differentiation mammalian foetus. Phil. Trans. Roy. Sot. Lond. B 259, 119-I
of the 30.
Kasdan, R.. Nankin, H. R.. Troen, P., Wald, N., Pan, S. and Yanihara. T. (1973). Paternal transmission of maleness in XX human beings. N. Eng. J. Med. 288.539-545. Koo. G. C., Wachtel. S. S., Krupen-Brown, K.. Mittl. L. R., Breg, W. R.. Genel, M.. Rosenthal, I. M.. Borgaonkar, D. S.. Miller, D. A.. Tantravahi. R.. Schreck. R. R.. Erlanger, B. F. and Miller, 0. J. (1977a). Mapping the locus of the H-Y gene on the human Y chromosome. Science 198.940-942. Koo. G. C.. Wachtel, S. S., Saenger. P. S., New, M. I.. Dosik. H., Amarose. A. P., Dorus, E. and Ventruto, V. (1977b). H-Y antigen: expression in human subjects with the testicular feminization syndrome. Science 7 96, 655-656. Kralova. J. and Demant, P. (1976). Expression of the H-Y antigen on thymus cells and skin: differential genetic control linked to K end of H-2. Immunogenetics 3, 583-594. McLaren. A. (1976). New York: Cambridge
Mammalian Chimeras. University Press).
Mittwoch, U. and Buehr. M. L. (1973). Gonadal sex reversed mice. Differentiation 1, 219-224.
(Cambridge, growth
London,
in embryos
of
Binding specific
Ohno. S. (1977). The original function of MHC antigens as the general plasma membrane anchorage site of organogenesis-directing proteins. Immunol. Rev. 33, 59-69.
1 I, 1979
Berger, R.. Abonyi, D.. Nodot, A., Vialatte. J. and Lejeune. Hermaphrodisme vrai et “garcon XX” dans une fratrie. Etudes Clin. Biol. 15, 330-333.
Intersexuality
Muller. U.. Aschmoneit. I.. Zenzes, M. T. and Wolf, U. (1978). studies of H-Y antigen in rat tissues. Indications for a gonad receptor. Human Genet. 43. 151-l 57.
from the NIH. January
W. C. D. (1976).
C. E.. Grieves, S. A. and in goats. J. Reprod. Fertil.
in man and
Ohno. S.. Christian, L. C.. Wachtel, S. S. and Koo. G. C. (1976). Hormone-like role of H-Y antigen in bovine freemartin gonad. Nature 261,
597-599.
Ohno. S.. Ciccarese. S., Nagai. Y. and Wachtel, S. S. (1978a). H-Y antigen in testes of XX (BALB)/XY (C3H) chimaeric male mouse. Arch. Androl. 7, 127-l 33. Ohno. S.. Nagai. Y. and Ciccarese, S. (1978b). Testicular cells lysostripped of H-Y antigen organize ovarian follicle-like aggregates. Cytogenet. Cell Genet. 20. 351-364. Ohno, S.. Nagai. Y.. Ciccarese, S. and Iwata. H. (1979). Testisorganizing H-Y antigen and the primary sex-determining mechanism of mammals. Recent Prog. Hormone Res.. in press. Selden. J. R., Wachtel. S. S.. Koo. G. C.. Haskins. M. E. and Patterson, D. F. (1978). Genetic basis of XX male syndrome and XX true hermaphroditism: evidence in the dog. Science 201, 644-646. Short, R. V. (1978). Sex determination and differentiation mammalian gonad. Internat. J. Androl. Suppl. 2, 21-28. Silvers, W. K. and Billingham. expressivity of histocompatibility
R. E. (1967). Genetic background and genes. Science 158, 118-l 19.
Silvers, W. K. and Yang, S.-L. (1973). Male-specific ogy in mice and rats. Science 781, 570-572.
antigen:
Simpson, E. and Gordon, R. D. (1977). Responsiveness antigen. Ir gene complementation and target cell specificity. Rev. 35. 59-75. Tarkowski. A. K. (1961). Mouse eggs. Nature 790. 857-860. Wachtel, S. S. (1977). Rev. 33, 33-58.
of the
H-Y antigen:
chimaeras genetics
developed and serology.
homolto H-Y Immunol.
from
fused
Immunol.
Sexual 695
Differentiation
Wachtel. S. S. and Ohno. S. (1979). The immunogenetics development. Prog. Med. Genet.. in press.
of sexual
Wachtel, S. S., Gasser, D. L. and Silvers, W. K. (1973). Male-specific antigen: modification of potency by the H-2 locus in mice. Science 161, 0624363. Wachtel, S. S., Basrur. P. and Koo. G. C. (1978). determining genes. Cell 7.5, 279-281.
Recessive
male-
Wachtel. S. S.. Koo. G. C.. Breg. W. Ft., Thaler. H. T., Dillard, G. M., Rosenthal, I. R.. Dosik, H., Gerald, P. S.. Saenger. P., New. M., Lieber. E. and Miller, 0. J. (1976a). Serologic detection of a Y-linked gene in XX males and XX true hermaphrodites. N. Eng. J. Med. 295. 750-754. Wachtel. S. S.. Koo, G. C.. Ohno, S.. Gropp. A., Dev. V. G.. Tantravahi, R.. Miller, D. A. and Miller, 0. J. (1976b). H-Y antigen and the origin of XY female wood lemmings (Myopus schisficolor). Nature 264, 638-639. Winters, S. J., Wachtel. S. S., White, B. J.. Koo. G. C.. Javadpour, N.. Loriaux. D. L. and Sherins. R. J. (1979). H-Y antigen mosaicism in the gonad of a 46,Xx true hermaphrodite. N. Eng. J. Med. 300. 745-749. Zenzes, M. T.. Wolf, U., Gunther, E. and Engel, W. (1978). Studies on the function of H-Y antigen: dissociation and reorganization experiments of rat gonadal cells. Cytogenet. Cell Genet. 20, 365-372.
April 1979,
Copyright
0 1979
by MIT
lmmunogenetic Aspects Sexual Differentiation Stephen S. Wachtel Memorial Sloan-Kettering Cancer 1275 York Avenue New York, New York 10021 and Department of Pediatrics New York HospitalCornell Medical Center New York, New York 10021
of Abnormal
Center
In mammals, testicular differentiation and subsequent male or intersexual development may occur in the apparent absence of the Y chromosome. Thus, for example, XX males and XX true hermaphrodites, the latter exhibiting a combination of ovarian and testicular tissue, have been documented in several mammalian species including goat (Hamerton et al., 1969) dog (Hare, 1976) mouse (Mittwoch and Buehr, 1973) and human (de la Chapelle, 1972). There are reports of XX males and XX true hermaphrodites in the same sibship in man (Berger et al., 1970; Kasdan et al., 1973) and recently Selden et al. (1978) discovered ovotestes in the XX mother of an XX male in a family of cocker spaniels. Both dogs were H-Y’ in serological tests, and both carried an apparent Y-to-autosome translocation inherited from the father of the true hermaphrodite (grandfather of the XX male). These findings favor the view that XX true hermaphroditism and the XX male syndrome are alternative manifestations of a common event associated with abnormal inheritance of Y chromosomal (H-Y) genes. Yet why should related individual animals with identical sex chromosomes and identical H-Y’ phenotypes exhibit the different modes of sexual differentiation and development represented by the XX male with testes, on the one hand, and the XX true hermaphrodite with ovotestes, on the other? The inherent tendency of the mammalian embryo to develop as a female is countered by genes on the Y chromosome which induce the undifferentiated gonadal rudiment to become a testis instead of an ovary. Testosterone, secreted by the newly formed testis, induces the secondary sexual characteristics of the male (Jost, 1970). Thus the pivotal role of the Y chromosome in mammalian sex determination is limited to the induction of the testis. Much evidence points to H-Y antigen as the Y-determined factor responsible for this induction (Wachtel and Ohno, 1979; Ohno et al., 1979; also see below). Phylogenetic Conservation of H-Y Antigen Differentiation of testis is associated with the presence of H-Y antigen in all mammals and indeed in all male heterogametic (XV) vertebrates studied thus far, regardless of apparent karyotype or secondary sex phenotype (Wachtel, 1977). Hence H-Y antibody is absorbed not only by cells of the male rat or mouse, but also by cells from males representing species as
Review
divergent as pig and platyfish (Fellous et al., 1978; P. Pechan, S. Wachtel and R. Reinboth, manuscript in preparation). As noted above, XX males and XX true hermaphrodites of the dog-are H-Y+. The same is true of XX males and XX true hermaphrodites of the goat (Wachtel. Basrur and Koo, 1978) and human (Wachtel et al., 1976a), and XY females with testicular feminization syndrome (Bennett et al., 1975; Koo et al., 1977b); fertile XY females of the wood lemming, however, are H-Y- (see below). To the extent that H-Y antigen is specified by genes in the pericentric (maledetermining) region of the human Y chromosome (Koo et al., 1977a; Wachtel and Ohno, 1979) its occurrence in XX subjects implies the presence of at least a portion of the Y. In the mouse, the autosomal dominant gene Sxr (‘sex-reversed”) causes expression of H-Y and differentiation of testis in embryos with the XX karyotype (Bennet et al., 1977). Yet if Sxr represents a translocation of the Y, the translocated segment is so minute as to be thus far undetectable cytologically. It follows that Sxr and H-Y genes are either very closely linked or identical. A Hormone-like Inductive Role of H-Y Antigen It has been suggested that the mammalian testis is induced by a diffusible substance released from XY cells (Ford, 1970) and the following considerations indicate that H-Y, in common with other components of the plasma membrane, is in fact released in the free state: H-Y occurs on XX cells within the XX/XV testes of the chimeric male mouse (Ohno et al., 1978a). It is abundant in the masculinized gonads of the bovine freemartin embryo (Ohno et al., 1976) even when only a small proportion of XY cells are present. It would follow that disseminated H-Y can be found by XY and XX target cells, both having H-Y receptors, and that irreversible commitment to testicular development is the usual consequence of this engagement, assuming a certain minimal threshold of H-Y production. Indeed, Muller et al. (1978) have now demonstrated binding of “exogenous” H-Y (from rat epididymal fluid) by cells of the XX fetal rat gonad, and Ohno et al. (1979) report induction of testicular tubules in XX presumptive ovaries of the fetal calf with H-Y antigen released from cultured Daudi cells. XY Ovaries In the absence of H-Y antigen, XY gonadal cells of the mammal organize an ovary. Dispersed testicular Sertoli cells (XV) that have been lysostripped of H-Y antigen reaggregate in vitro to form ovarian follicles in mice (Ohno, Nagai and Ciccarese, 1978b) and rats (Zenzes et al., 1978) and as indicated above, failure of H-Y synthesis leads to development of ovaries in fertile XY females of the wood lemming, Myopus schisticolor (Wachtel et al., 1976b). It is not surprising that XY cells within the gonads of XX/XV chimeric mice can also be induced to participate in ovarian differ-
Cell 692
entiation. These XY cells may even become oocytes (Ford et al., 1975; Evans, Ford and Lyon, 1977). Since ovarian differentiation in XY cells of the wood lemming in vivo and of the mouse and rat in vitro always goes hand in hand with the absence of H-Y antigen, it follows that H-Y should be absent from XY as well as XX cells of the XX/XV chimeric ovary. But can ovarian differentiation be attributed wholly to the absence of H-Y? In that case, we should expect differentiation of ovotestes in XX/XV chimeric gonads with a paucity of XY cells, for example; yet true hermaphrodites are rare among chimeric mice, whereas XX/XV females with grossly normal ovaries are common (McLaren, 1976). [An example of hermaphroditic development in regular association with sex-chromosomal mosaicism may be found in XO/XY and XO/XY/XYY mice of the BALB/cWt strain. Presence of the aneuploid cell line notwithstanding, there is evidence that the high incidence of hermaphroditism in this strain (3%) is due at least in part to presence of a deficient BALB/cWt Y chromosome (Beamer, Whitten and Either, 19791.1 An Ovary-Inducing Antigen? The rarity of ovotestes in XX/XV chimeric mice as compared with the far more commonly definitive and unequivocal differentiation of testes or ovaries could be explained if the testis-organizing H-Y molecule were competing with another molecule capable of blocking occupation of H-Y receptors. Since ovarian differentiation is the normal consequence of reaction of this second molecule, the latter might be aptly viewed as an inducer of ovarian rather than testicular differentiation. As to the mechanism, it could be envisioned that there are nonspecific receptors on the surfaces of both XX and XY cells and that the differentiative response depends upon which of the two inducers is engaged by these receptors (Figure I), or that there are specific receptors having alternative and exclusive differentiative responses (Figure 2). In the XX/XV chimeric gonad, the two inducers would be placed in competition among the receptors of the cell surface, and the predominance of one or the other would lead to unambiguous testicular or ovarian differentiation. Why then do most XX/XV chimeric gonads become testes? It has been argued that this is because testicular tissue differentiates earlier than ovarian tissue (Tarkowski, 1961) (although testicular differentiation in the freemartin occurs after ovarian differentiation in the normal cow). It could also be argued, however, that during the development of the normal XY rodent, H-Y is disseminated in large quantities to compete successfully with the ovary inducer. In the normal XX embryo there would be no need for a corresponding dissemination of the ovary inducer, because there is no competition from Y-determined testis inducer. Thus in the chimeric or mosaic gonad, H-Y antigen would
be likely to dominate ably be masculine.
and differentiation
would
prob-
Competition in the XX Ovotestis In man (but not to the same degree in mice), two X chromosomes are evidently needed to sustain normal ovarian differentiation. This is true to the extent that two X chromosomes are necessary for survival of female germ cells (Short, 1978). Embryos with the 45X0 karyotype initially develop ovaries, but these degenerate and are later represented by “dysgenetic” gonads lacking definitive ovarian structure and characterized instead by undifferentiated stroma. It follows that in H-Y+ human embryos with the 46,Xx karyotype (XX’), factors favoring ovarian versus testicular induction are more obviously in competition. If general dominance of H-Y leads to development of testes, then true hermaphroditism could be viewed as the outcome of inconclusive competition, which might be expected with a certain degree of probability in XX/XX’ mosaics. Thus, for example, regional dominance of H-Y leads to development of one testis and a contralateral ovary (“alternating hermaphroditism”), whereas local dominance within a single gonad or in both gonads leads to development of ovotestis [versus ovary (“unilateral hermaphroditism”) or bilateral ovotestes (“bilateral hermaphroditism”); see Figure 31.
Figure
1. Nonspecific
Receptors
for Gonad-Organizing
Proteins
Molecules of H-Y antigen (small Ys) and ovary inducer (small OS) are disseminated (perhaps by different cells and at different stages) and bound by nonspecific receptors on target cells (1) of XX/XV for XX’) fatal gonad. Excess of H-Y causes displacement of ovary inducer, thereby promoting organization of testis.
Sexual 693
Differentiation
Simultaneous differentiation of ovarian and testicular architecture in such embryos must reflect the presence of H-Y in presumptive testis and the absence of H-Y from presumptive ovary, and this implies that the true hermaphrodite is an H-Y+/H-Ymosaic regardless of karyotype. This implication is borne out in a study performed by Winters et al. (1979). The subject of this study appeared to be a male with first degree hypospadias and a 46,Xx karyotype, but exploration of the scrotum revealed a bilobed gonad comprising both ovarian and testicular elements. Serological tests showed that cells cultured from the testicular portion of the ovotestis were H-Y’, whereas cells cultured from the ovarian portion were H-Y-. In view of the above discussion, this could denote XX/XX’ mosaicism heavily favoring an XX cell line in the fetal gonad or, alternatively, the presence of a mosaic cell line bearing receptors with low affinity for disseminated H-Y.
tured male lymphomas that have retained /3*m-HLA. But when Daudi HLA antigens are restored by hybridization of Daudi cells with female cells of the HeLa D98 line, normal expression of H-Y is also restored (Beutler et al., 1978; but see Fellous et al.. 1978). On the basis of these observations, and in view of evidence for the dissemination of H-Y, it has been proposed that cell surface antigens determined by the major histocompatibility complex (H-2 in the mouse, HLA in man) act in association with Pn-microglobulin as the nonspecific cell membrane anchorage sites of organogenesis-directing proteins in general (Ohno, 1977). It follows that molecules of H-Y antigen and the putative ovary inducer should be disseminated at a particular stage of embryogenesis and bound by MHC-determined anchorage sites or “carriers,” and that once saturated with a particular inducer, cells of the primordial gonad should be impelled to follow a particular pathway of differentiation.
A Role of HLA in Sex Determination? In mice, the reaction against H-Y incompatible transplants is influenced by major histocompatibility complex (MHC) antigens of the donor, so that H-2k male skin grafts are rejected more quickly than H-2b male skin grafts by H-2b/H-2k hybrid females (Silvers and Billingham, 1967; Wachtel, Gasser and Silvers, 1973; Kralova and Demant, 1976); in rats, male cells carrying the Ag-B’ haplotype most strongly sensitize female mice with respect to mouse H-Y antigen (Silvers and Yang, 1973); and in vitro the reaction of cytotoxic T cells to H-Y-incompatible target cells is MHC-restricted (much as the reaction of cytotoxic T cells to viral-infected or viral-transformed target cells is MHCrestricted), not only in rodents (Simpson and Gordon, 1977) but also in man (Goulmy et al., 1977). These observations point to a functional relationship between H-Y molecules and components of the MHC. There is now direct evidence of a physical association between H-Y and ,&-microglobulin-HLA in the human male: cultured “Daudi” cells from a male Burkitt lymphoma that has lost P*rn (and thereby HLA) absorb considerably less H-Y antibody than cells from cul-
A Gonad-Specific Receptor All normal cells are endowed with MHC antigens, and thus an essential difference between somatic and gonadal tissues must lie in possession by the latter of specific receptors (as well as nonspecific anchorage sites) for the inducers of testicular and ovarian differentiation (Figure 2). Certainly MHC-determined carriers are present in XX and XY cells. The same must be true for gonad-specific receptors of both H-Y (Muller et al., 1978) and the ovary inducer, since as we have seen, XX gonads can become testes and XY gonads can become ovaries. Whereas H-Y and the ovary inducer are bound by the same nonspecific carrier, the gonad-specific receptor should engage only the active site of its particular substrate-in the case of H-Y, the phylogenetically conserved moiety identified by antibody from male-grafted female mice. No doubt the specific active site of the disseminated testis inducer is the same H-Y antigen borne “nonspecifically” in skin, blood and other somatic tissues, and also in gonadal tissues of the normal male. Thus sexual differentiation of the primordial gonad presumably involves not only dissemination of an inducer molecule and its binding to MHC-determined carriers, but also its engagement by specific receptors which may or may not be MHC-associated. If in fact /%m-HLA components serve as nonspecific carriers for the inducers of gonadal differentiation in man, a critical difference between XX true hermaphrodites and XX males may reside in the peculiar affinity of certain HLA haplotypes for an ovarian versus testicular determinant. Furthermore, if dissemination of a testis inducer were a conditio sine qua non of testicular differentiation as all available evidence indicates, then certain forms of pure gonadal dysgenesis could also be HLA-associated. On the other hand, general failure of testicular differentiation in H-Y’ subjects with the 46,XY karyotype could result from lack
Figure
2. Specific
Receptors
for Gonad-Organizing
Proteins
Uncommitted cell (1) of the XX/XY chimeric or mosaic fetal gonad possessing specific receptors for H-Y antigen and ovary-inducing “antigen” may follow alternative and mutually exclusive pathways of differentiation: ovarian (a) or testicular(b). Occupation of one receptor by its respective inducer blocks occupation of adjacent receptor via steric interference or changes in membrane topography, for example.
Cell 694
local
General
Dominance
Testis
restis
Ford, C. E.. Evans, E. P.. Burtenshaw, M. D., Clegg, H. M.. Tuffrey. M. and Barnes, R. D. (1975). A functional “sex-reversed” oocyte in the mouse. Proc. Roy. Sot. Lond. B 190. 187-l 97. Goulmy, E.. Termijtelen, A., Bradley, B. A. and van Rood, J. J. (1977). Y-antigen killing by T cells of women is restricted by HLA. Nature 266, 544-545. Hamerton. J. L.. Dickson, J. M.. Pollard, Short, R. V. (1969). Genetic intersexuality (Supplement) 7. 25-51.
ReDional Dominance
Dominance
Hare, 15.
Ovotestls
Ovotestls
Figure 3. Modalities XX/XX’ Fetal Gonad
ovary
of Testicular
Differentiation
TestIs in the
XX”
or
Black dots denote areas of H-Y saturation. General Dominance: prevalence of H-Y’ leads to development of unambiguous testes in “XX” male. Regional Dominance: H-Y structural genes in one gonad but not the other cause “alternating hermaphroditism.” This condition could also result from mosaicism of receptor determinants. Local Dominance: differentiation of ovotestis (the most common gonad in XX true hermaphroditism) reflects focus of cells bearing H-Y structural determinants or, alternatively, a local mosaicism of receptor determinants.
of affinity of disseminated H-Y for a particular gonadspecific receptor, and any fitful attempts to organize a testis could be attributed to indigenous H-Y molecules which constitute a more “stable” portion of the cell membrane in all male tissues. Acknowledgments This work was supported Received
November
by grants
6. 1978;
revised
References Beamer. W. G.. Whitten. W. K. and Either, E. M. (1979). Spontaneous sex mosaicism in BALB/cWt mice. In Symposium on Genetic Mosaics and Chimeras in Mammals, 1978, in press. Bennett, D.. Boyse. E. A., Lyon, M. F.. Mathieson, B. J.. Scheid, and Yanagisawa. K. (1975). Expression of H-Y (male) antigen phenotypically female Jfm/Y mice. Nature 257, 236-238.
M. in
Bennett, D., Mathieson, B. J., Scheid. M., Yanagisawa, K.. Boyse, E. A., Wachtel, S. S. and Cattanach. B. M. (1977). Serological evidence for H-Y antigen in Sxr. XX sex-reversed phenotypic male?.. Nature 265, 255-257. J. (1970). Rev. Eur.
Beutler, B., Nagai. Y.. Ohno. S.. Klein, G. and Shapiro, I. M. (1978). The HLA-dependent expression of testis-organizing H-Y antigen by human male cells. Cell 73, 509-513. de la Chapelle, A. (1972). with XX sex chromosomes.
Analytic review: Am, J. Human
nature and origin of males Genet. 24, 71-I 05.
Evans, E. P., Ford, C. E. and Lyon, M. F. (1977). Direct evidence of the capacity of the XY germ cell in the mouse to become an oocyte. Nature 267, 430-431. Fellous, M.. Gunther, E.. Kemler. R., Wiels. J., Berger. R.. Guenet, J. L.. Jakob. H. and Jacob, F. (1978). Association of the H-Y male antigen with fin-microglobulin on human lymphoid and differentiated mouse teratocarcinoma cell lines. J. Exp. Med. 147. 58-70. Ford, C. E. (1970). Cytogenetics and sex determination mammals. J. Biosoc. Sci. Suppl. 2, 7-30.
in the dog. Can. Vet. J. 17. 7-
Jest. A. (1970). Hormonal factors in the sex differentiation mammalian foetus. Phil. Trans. Roy. Sot. Lond. B 259, 119-I
of the 30.
Kasdan, R.. Nankin, H. R.. Troen, P., Wald, N., Pan, S. and Yanihara. T. (1973). Paternal transmission of maleness in XX human beings. N. Eng. J. Med. 288.539-545. Koo. G. C., Wachtel. S. S., Krupen-Brown, K.. Mittl. L. R., Breg, W. R.. Genel, M.. Rosenthal, I. M.. Borgaonkar, D. S.. Miller, D. A.. Tantravahi. R.. Schreck. R. R.. Erlanger, B. F. and Miller, 0. J. (1977a). Mapping the locus of the H-Y gene on the human Y chromosome. Science 198.940-942. Koo. G. C.. Wachtel, S. S., Saenger. P. S., New, M. I.. Dosik. H., Amarose. A. P., Dorus, E. and Ventruto, V. (1977b). H-Y antigen: expression in human subjects with the testicular feminization syndrome. Science 7 96, 655-656. Kralova. J. and Demant, P. (1976). Expression of the H-Y antigen on thymus cells and skin: differential genetic control linked to K end of H-2. Immunogenetics 3, 583-594. McLaren. A. (1976). New York: Cambridge
Mammalian Chimeras. University Press).
Mittwoch, U. and Buehr. M. L. (1973). Gonadal sex reversed mice. Differentiation 1, 219-224.
(Cambridge, growth
London,
in embryos
of
Binding specific
Ohno. S. (1977). The original function of MHC antigens as the general plasma membrane anchorage site of organogenesis-directing proteins. Immunol. Rev. 33, 59-69.
1 I, 1979
Berger, R.. Abonyi, D.. Nodot, A., Vialatte. J. and Lejeune. Hermaphrodisme vrai et “garcon XX” dans une fratrie. Etudes Clin. Biol. 15, 330-333.
Intersexuality
Muller. U.. Aschmoneit. I.. Zenzes, M. T. and Wolf, U. (1978). studies of H-Y antigen in rat tissues. Indications for a gonad receptor. Human Genet. 43. 151-l 57.
from the NIH. January
W. C. D. (1976).
C. E.. Grieves, S. A. and in goats. J. Reprod. Fertil.
in man and
Ohno. S.. Christian, L. C.. Wachtel, S. S. and Koo. G. C. (1976). Hormone-like role of H-Y antigen in bovine freemartin gonad. Nature 261,
597-599.
Ohno. S.. Ciccarese. S., Nagai. Y. and Wachtel, S. S. (1978a). H-Y antigen in testes of XX (BALB)/XY (C3H) chimaeric male mouse. Arch. Androl. 7, 127-l 33. Ohno. S.. Nagai. Y. and Ciccarese, S. (1978b). Testicular cells lysostripped of H-Y antigen organize ovarian follicle-like aggregates. Cytogenet. Cell Genet. 20. 351-364. Ohno, S.. Nagai. Y.. Ciccarese, S. and Iwata. H. (1979). Testisorganizing H-Y antigen and the primary sex-determining mechanism of mammals. Recent Prog. Hormone Res.. in press. Selden. J. R., Wachtel. S. S.. Koo. G. C.. Haskins. M. E. and Patterson, D. F. (1978). Genetic basis of XX male syndrome and XX true hermaphroditism: evidence in the dog. Science 201, 644-646. Short, R. V. (1978). Sex determination and differentiation mammalian gonad. Internat. J. Androl. Suppl. 2, 21-28. Silvers, W. K. and Billingham. expressivity of histocompatibility
R. E. (1967). Genetic background and genes. Science 158, 118-l 19.
Silvers, W. K. and Yang, S.-L. (1973). Male-specific ogy in mice and rats. Science 781, 570-572.
antigen:
Simpson, E. and Gordon, R. D. (1977). Responsiveness antigen. Ir gene complementation and target cell specificity. Rev. 35. 59-75. Tarkowski. A. K. (1961). Mouse eggs. Nature 790. 857-860. Wachtel, S. S. (1977). Rev. 33, 33-58.
of the
H-Y antigen:
chimaeras genetics
developed and serology.
homolto H-Y Immunol.
from
fused
Immunol.
Sexual 695
Differentiation
Wachtel. S. S. and Ohno. S. (1979). The immunogenetics development. Prog. Med. Genet.. in press.
of sexual
Wachtel, S. S., Gasser, D. L. and Silvers, W. K. (1973). Male-specific antigen: modification of potency by the H-2 locus in mice. Science 161, 0624363. Wachtel, S. S., Basrur. P. and Koo. G. C. (1978). determining genes. Cell 7.5, 279-281.
Recessive
male-
Wachtel. S. S.. Koo. G. C.. Breg. W. Ft., Thaler. H. T., Dillard, G. M., Rosenthal, I. R.. Dosik, H., Gerald, P. S.. Saenger. P., New. M., Lieber. E. and Miller, 0. J. (1976a). Serologic detection of a Y-linked gene in XX males and XX true hermaphrodites. N. Eng. J. Med. 295. 750-754. Wachtel. S. S.. Koo, G. C.. Ohno, S.. Gropp. A., Dev. V. G.. Tantravahi, R.. Miller, D. A. and Miller, 0. J. (1976b). H-Y antigen and the origin of XY female wood lemmings (Myopus schisficolor). Nature 264, 638-639. Winters, S. J., Wachtel. S. S., White, B. J.. Koo. G. C.. Javadpour, N.. Loriaux. D. L. and Sherins. R. J. (1979). H-Y antigen mosaicism in the gonad of a 46,Xx true hermaphrodite. N. Eng. J. Med. 300. 745-749. Zenzes, M. T.. Wolf, U., Gunther, E. and Engel, W. (1978). Studies on the function of H-Y antigen: dissociation and reorganization experiments of rat gonadal cells. Cytogenet. Cell Genet. 20, 365-372.
