Cell, Vol. 7, 315-321,
March
1976,
Copyright”
1976 by MIT
Major Regulatory Genes for Mammalian Sexual Development Susumu Ohno Department of Biology City of Hope National Medical Duarte, California 91010
Center
The principal regulatory elements concerned with the genetic basis of sex determination and differentiation in mammals appear to be comparatively simple (Ohno, 1971; Lyon, 1974). A gene or set of genes on the Y chromosome causes the indifferent embryonic gonad to develop as a testis, an ovary forming in the absence of the Y. Thus the Y-linked gene or genes determine the primary or gonadal sex. It appears, however, that this Y-linked testisdetermining gene makes no further direct contribution to subsequent sexual differentiation of mammalian embryos. Thus in the presence of testosterone, either an XY or an XX embryo undergoes masculine differentiation, feminine differentiation occurring in its absence. Since the testis organized by the Y normally serves as the only significant source of fetal androgen, concordance between the gonadal sex and the phenotypic sex is usually achieved. It follows then that the minimum requirement for sexual development of mammals is the presence of two major regulatory genes: one for the gonadal sex determination, which should be on the Y, and the other for the phenotypic sexual differentiation, whichcan beeitherx-linkedorautosomallyinherited. The Y-Linked Testis Determining Gene and H-Y Antigen Development of the indifferent gonad into a testis under the influence of the Y chromosome begins as soon as the migration of primordial germ cells from the yolk sac to the gonadal ridge is completed. In the XY gonadal ridge, many of the germ cells move deeper into the central gonadal blastema, while in the XX gonadal ridge, germ cells remain in the periphery and multiply until they form a thick layer (Gropp and Ohno, 1966). Consequently, the direct contact between migrant primordial germ cells and resident somatic elements of the central blastema is made considerably earlier in the male gonad than in the female gonad, and there is little doubt that this earlier contact leads to the formation of seminiferous tubules instead of ovigerous cords. In fact, a classical experiment by Moscona revealed that such direct cell-cell recognition among different cell types at their membrane surface is the basis of organogenesis (Moscona, 1957). Inasmuch as the Y chromosome directs the organogenesis of a testis, and since migrant XY primordial germ cells and resident XY somatic elements appear to attract each other to make direct contact, it follows that the Y-linked testis-determining gene or genes seem
Review
very likely to specify a plasma membrane protein or proteins. By serological methods, those proteins (or the sugar moieties of glycoprotein) which reside in the plasma membrane and are accessible to antibody can be recognized as the surface antigens of intact viable ceils. In fact, one such antigen specified by the Y chromosome has been known in the mouse since 1955 (Eichwald and Silmser, 1955). Rejection of male skin grafts by females within a highly inbred strain of mice must be due to the Y-linked plasma membrane protein (H-Y antigen) or proteins, because individual differences should not exist with regard to any of the autosomally inherited as well as X-linked plasma membrane proteins. This H-Y antigen is ubiquitously expressed in various cell types of all the males, but absent from those of normal females (Gasser and Silvers, 1972). Using antimouse H-Y antibody raised in C57BL female mice that received repeated grafts of syngeneic male cells, Wachtel, Koo, and Boyse (1975a) recently demonstrated that the cell surface component recognized as H-Y antigen has been highly conserved in mammalian evolution. Male cells, but not female cells, of other mammalian species, including man, possessed a component indistinguishable from the mouse H-Y antigen on their plasma membrane. Such an extreme evolutionary conservation is unusual for cell surface components, and is reminiscent of the conservatism exhibited by the histone IV gene locus. The H-Y antigen must have performed an invariant function throughout the evolution of mammals, and that invariant function seems almost certain to be the induction of a testis. For this and other reasons, we have proposed that H-Y antigen is the long sought after product of the Ylinked testis-determining gene of mammals (Wachtel et al., 1975b). The proposed identity of H-Y antigen and the testis-determining gene product can best be tested on exceptional individuals whose gonadal sex does not agree with their chromosomal sex. Any individual who possesses testes in spite of the apparent absence of the Y must somehow have acquired the testis-determining gene. Thus his cells should type as H-Y antigen positive. Conversely, the testis-determining gene of any individual whose gonads had not undergone testicular differentiation, in spite of the apparent presence of the Y, must have somehow been rendered inert. Thus her cells should type as H-Y antigen negative. A subsequent series of tests performed on a considerable variety of such exceptional individuals, humans as well as mice and other mammals, yielded no exception from the above expectation (S. S. Wachtel, personal communication).
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Can the proposed identity of H-Y antigen and the Y-linked testis-determining gene product explain other pertinent observations made on testicular development of mammals? In random fusions between two blastocysts 50% of the cases should involve heterosexual pairs, thus producing experimentally XX/XY mosaic mice. The curious fact is that these XX/XY mosaic mice show a much greater tendency to develop as males than as females (Mintz, 1969; McLaren, 1971), since true hermaphroditism is a very rare event (Tarkowski, 1970). In the case of man too, at least some of the socalled XX males appear to be cryptic XX/XY mosaics, harboring some XY cells in their testes (De La Chapelle, 1972). Testicular development seen in the cow’s gonad that was born co-twin with the bull might also be due to the presence of migrant XY cells in that gonad (Ohno et al., 1962; Goodfellow, Strong, and Stewart, 1965). It would appear that in all these cases, a minority, if a substantial one, of XY cells in the mosaic gonad was able to entice the majority of XX cells to participate in testicular organization. These results suggest that differentiating XY cells of the fetal gonad produce a hormone-like enticing substance. Its existence and very limited range of effectiveness have been demonstrated in experiments in which a heterosexual pair of fetal rat gonads (sixteenth day of gestation) was transplanted to a subcapsular position in the kidney of castrated adult rats. The testis was able to destroy the ovarian cortex and induce seminiferous tubule development in the ovarian medulla, provided that the two were in direct contact with each other. When a testis and an ovary were separated by a distance of 8 mm, no appreciable effect by the former was seen on the latter (Macintyre et al., 1960). We might consider the role of H-Y antigen in this context. Although the expression of H-Y antigen is ubiquitous, it appears to play a sexual role only in the fetal gonad. Thus nongonadal cells of testicular feminized TfmlY mice still typed as H-Y antigen positive, although no masculine development beyond the formation of testes occurs in these mutant XY mice (Bennett et al., 1975). The uniqueness of gonadal XY cells as opposed to all other XY cells of the male body may reside in the possession of specific receptors for H-Y antigen in addition to H-Y antigen itself. In fact, such coexistence should be a prerequisite for direct cell-cell contact recognition between migrant germ cells and resident somatic elements which lead to the formation of a testis. What if XX gonadal cells, while lacking in H-Y antigen, are also endowed with specific H-Y antigen receptors? An analogous situation has been found in androgen target cells of the normal female. Although these XX cells are not normally exposed to
endogenous testosterone, they are nevertheless equipped with the same amount of specific nuclearcytosol androgen receptor protein as the homologous XY cells, as is discussed below. This, in fact, is the very reason that XX fetuses readily masculinize in response to exogenously administered androgen. Similarly, the constitutive presence of specific H-Y antigen receptors on the membrane surface of gonadal cells might be the very reason that XX cells can be enticed by neighboring XY cells to participate in testicular organization. When these gonadal XX cells become coated with disseminated H-Y antigen, they can indeed become an equal partner to gonadal XY cells in the organization of the testis. In the immune system, a single species of antibody molecules, anchored on the membrane surface, serves as the receptors of each committed B cell, the antigen antibody binding at the surface triggering the increased production and subsequent secretion of that antibody. In the same manner, the establishment of direct contact between gonadal XY cells through their H-Y antigen and its receptors might trigger the increased production and subsequent dissemination of H-Y antigen which then can coat gonadal XX cells in the same mosaic gonad. If this is the mechanism, disseminated H-Y antigen may provide the hormonelike substance mentioned earlier, which has an extremely short range of effectiveness (Figure 1). The experiment which unequivocally tests the postulated transference of H-Y antigen from XY cells to XX cells in the mosaic gonad is technically difficult. Nevertheless, we may already have obtained the evidence of H-Y antigen transference on lymphocytes of human XX males (Wachtel et al., 1975b). When informative with regard to the Xlinked blood group antigen X,, these XX males type as though both their Xs are of the maternal derivation; they are X,-negative in spite of having X,-positive fathers. Two explanations are possible. First, the reciprocal exchange between the X and the Y in the meiotic process of the father transferred the testis-determining gene from the Y to the X, and the X, gene from the X to the Y (Ferguson-Smith, 1966). Second, they started as XY zygotes, and the XX cell line subsequently arose by mitotic nondisjunctions. Although their lymphocytes and cultured fibroblasts consist exclusively of the XX line, the original XY line remains in the gonads and other inaccessible organs (De La Chapelle, 1972). The manner of meiotic pairing between the X and the Y speaks rather strongly against the first possibility. It then follows that the source of H-Y antigen found on XX lymphocytes of these males has to be the XY cells that lurk in nonhemopoietic organs of their bodies.
Genetic 317
Regulation
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Development
All in all, the proposal determining gene specifies explains most, if not all, tions that have been made
that the Y-linked testisH-Y antigen satisfactorily of the pertinent observaon mammalian testicular
organized testicular cells
Figure 1. Ubiquitous Expression of Enticement in the Gonad
of H-Y Antigen
and
the
Nature
Assuming H-Y antigen (solid black column with one rectangular and one rounded edge) to be the Y-linked, testis-determining gene product, this figure schematically depicts its ubiquitous presence in all the cells of the male and its total absence in the female, as well as its proposed mode of action in organogenesis of the testis. Although endowed with H-Y antigen, nongonadal XY cells lack specific H-Y antigen receptors on their plasma membrane (top row). Their female counterparts may be equipped with a number of anchorage sites (slightly round indentations on the cell surface). Thus they can be coated with disseminated H-Y antigen in the XX/XY mosaic situation (see text for discussion of human XX males). The plasma membrane of gonadal XY cells (germ cells as well as somatic elements) are unique, since they have both H-Y antigen and its specific receptors (columnar indentations on the cell surface), as shown in the middle row. Direct contacts between these gonadal XY cells established through their H-Y antigen and its specific receptors result in organogenesis of the testis. The established contact may trigger these XY cells to produce more H-Y antigen, which is disseminated to their immediate surroundings (bottom row). If so, disseminated H-Y antigen is a hormone-like substance with an extremely short range of effectiveness. The presence of such a hormone-like substance was shown by the experiments of Macintyre et al. (1960). Gonadal XX cells may have anchorage as well as specific receptor sites for H-Y antigen (middle and bottom rows). Thus when they are coated by H-Y antigen in the XX/XY mosaic gonad, they can participate in organogenesis of the testis as full partners.
differentiation. Genes for the postpubertal function of germ cells and gametes are not the concern of this paper. The X-Linked Tfm Locus as the Regulatory Locus of Secondary Sexual Characteristics The testis induced by the regulatory locus on the Y above mentioned then secretes testosterone, which induces male development of the accessory glands and ducts. Regardless of their sex chromosome constitutions, mammalian embryos have the inherent tendency to develop female secondary sex characteristics. Thus if deprived of the endogenous testosterone source by castration, XY embryos manifest the female phenotype, whereas the exogenous administration of testosterone readily masculinizes the XX fetus and gives it the complete set of male secondary sex characteristics (Jost, 1970; Price, 1970). It would appear that a gene or a set of genes which confers the androgen responsiveness to appropriate cell types of the mammalian body is the regulatory locus which governs the development of secondary sexual characteristics. The masculinizing action of testosterone on its target cells is mediated by the product of a gene on the X chromosome. This product activates all the genes required for manifestation of the male phenotype in response to circulating testosterone (Ohno, 1971). Evidence for this crucial involvement of a single X-linked gene in development of secondary sexual characteristics comes from a mutation of the relevant gene in the mouse (Tfm), resulting in complete failure to respond to testosterone. A chromosomally XY animal with this mutant gene develops testes, because the normal Y chromosome is present and therefore is H-Y positive, but shows no further male differentiation, thus exhibiting the syndrome known as “testicular feminization” (Lyon and Hawkes, 1970). Unlike peptide hormones, possibly because of their lipophilic nature, steroid hormones appear to penetrate readily the plasma membrane of cells. Thus their specific receptors are found not on the plasma membrane of cells but in the cytosol (supernatant) fraction of the cytoplasma. Such so-called nuclear-cytosol steroid receptor proteins have all the characteristics of being regulatory proteins. These receptors show very high binding affinity to specific steroids: Kd of the order of 1 O-9-1 O-10 M. The act of binding to a specific steroid creates the secondary binding site (acceptor-binding site) on a receptor protein via allosteric effect. Steroidbound receptors then move into the nucleus and associate with the chromatin to induce the synthesis of a specific set of structural gene products. There now exists a consensus that the androgen insensitivity exhibited by all the target cell types of
Cell 318
affected testicular feminized XY individuals is due to a mutational deficiency of the nuclear-cytosol androgen receptor protein, not only in mice (Gehring, Tomkins, and Ohno, 1971; Attardi and Ohno, 1974; Gehring and Tomkins, 1974) but also in rats (Bardin et al., 1973) as well as in man (Keenan et al., 1974). Thus the remainder of this review paper is devoted to discussing several crucial problems in association with the proposition that the X-linked Tfrn locus is the one and only regulatory locus which governs the development of male secondary sex characteristics. One or Two Different Kinds of Androgen Receptors? In some, but not all, of the androgen target organs, 5a-reductase efficiently converts testosterone (TES) to 5a-dihydrotestosterone (DHT) (Wilson and Walker, 1969). Thus there was a time when the human testicular feminization syndrome was thought to be caused by 5a-reductase deficiency (Heinrichs et al., 1969; Northcutt, Island, and Liddle, 1969). Subsequently, however, 5a-reductase deficiency was recognized in man as an independent autosomal recessive trait. Unlike Tfm individuals, XY individuals, deficient in this enzyme, show a considerable degree of masculinization at birth, and further masculinization occurs after puberty (Imperato-McGinley et al., 1974). Nevertheless, the fact that there appeared to be two types of androgen target organs, one rich in 5a-reductase, the other not having this enzyme, prompted a number of investigators to suggest that mammals might possess two different kinds of androgen receptors: DHT receptor and TES receptor. The mouse kidney is one of a few androgen target organs that persist in mutant. Tfm/Y individuals. Bardin et al. (1973) believed that the mouse kidney normally contains not DHT receptor but TES receptor, and what is deficient in androgen nonresponsive TfmlY kidney is TES receptor. If this is true, the Tfm mutation must cause deficiency of both DHT and TES receptors, since the embryonic Wolffian duct and urogenital sinus of Tfm/Y totally fail to masculinize even under the influence of exogeeously administered DHT (Goldstein and Wilson, 1972). It then follows that since a single locus can specify only one protein, the X-linked Tfm locus must not be an androgen receptor locus, but a regulatory locus of a higher hierarchy which regulates the expression of two androgen receptor loci-one for DHT and the other for TES. Our studies, on the other hand, have consistently demonstrated that all the target organs of androgen have the same receptor, which shows the highest binding affinity to DHT but also binds to TES with a respectable affinity. This one and only androgen receptor might best be called DHT-TES receptor, and what is deficient
in the kidney, submaxillary salivary glands, and brain of Tfm/Y is this DHT-TES receptor (Gehring et al., 1971; Attardi and Ohno, 1974; Gehring and Tomkins, 1974). Those androgen target organs which are relatively deficient in 5a-reductase, such as the mouse kidney, are rich in the second enzyme which converts DHT further to 5a-androstandiol (Bardin et al., 1973). Thus unless a precaution is taken, 3H-DHT, added to the cytosol fraction, is rapidly metabolized, giving falsely low receptor-bound counts on sucrose or glycerol gradient profiles. Such organs as the mouse kidney or levator ani muscle must normally make do with TES, because its intracellular conversion to DHT is negligible. Nevertheless, the mouse kidney responds better to administered DHT than to TES (Ohno, Dofuku, and Tettenborn, 1971; Bardin et al., 1973) reflecting the fact that DHT-TES receptor protein present in this organ also has a higher binding affinity to DHT than TES. All in all, the androgen responsiveness of all the divergent target organs appears to be mediated by a single species of DHT-TES receptor protein. Thus there is no need to invoke a higher regulatory locus which controls two separate receptor loci. Constitutive Production of Androgen Receptor Even if the Tfm locus specified the one and only androgen receptor protein, if this protein itself is inducible, the need still exists to identify a higher regulatory locus which controls the expression of this gene. Fortunately, a number of organs present in the adult mouse body of both sexes are normally androgen-responsive. They are the brain, kidney, submaxillary salivary glands, preputial glands, and levator ani muscle, among others. Our previously mentioned studies revealed that no sexual difference exists in the amount of DHT-TES receptor protein present in those bisexual androgen target organs. Thus within each target organ, the production of androgen receptor protein is constitutive, not influenced either by the presence of the Y chromosome or by different circulating testosterone levels. The observed constitutivity in each target organ, of course, was expected, for the classical embryological studies have shown that XX embryos masculinize readily and fully in response to exogenously administered androgen. The problem still remained, however, that until very recently, it was believed that only specific target organs or cell types of the body contain a receptor protein specific for each steroid hormone. The organ-specific occurrence of androgen receptor protein again calls for the presence of a higher regulatory gene which turns on the Tfm locus only in androgen-responsive cell types. It now appears that this belief in organ-specific occurrence of steroid receptor protein is incorrect. One can hardly regard cultured fibroblasts as an-
Genetic 319
Regulation
of Sexual
Development
drogen target cells, for no appreciable response can be monitored after their exposure to a reasonable concentration of androgen, that is, 10-a M. Although a very high concentration of TES (>lO-6 M) is toxic to cultured fibroblasts, there is no need to invoke the presence of a high affinity receptor protein for such toxic effect. Yet Keenan et al. (1974) were able to demonstrate a mutational deficiency of DHT-TES receptor protein in cultured fibroblasts obtained from human testicular feminized patients, since similarly cultured fibroblasts from normal males and females contained a very respectable amount of this receptor protein. It has indeed been found that permanent mouse fibroblast L-cell lines, as well as muscles in general, also contain receptor proteins for androgen and other steroid hormones (E. Baulieu, personal communication). The mouse kidney, while quite responsive to androgen, shows no appreciable response to estrogen (Ohno et al., 1971). Yet it contains not only androgen receptor, but also a good amount of estradiol receptor (our unpublished data). It would now appear that DHT-TES receptor protein and possibly other steroid receptor proteins are ubiquitously expressed in all the cell types of the body in the same manner as H-Y antigen. Thus when considering the genetic regulatory mechanism of mammalian sexual development, there appears to be no need to invoke a higher regulatory locus which controls the X-linked Tfm locus. induction of Different Sets of Structural Gene Products in Different Target Organs Inasmuch as DHT-TES receptors appear to be present ubiquitously in nearly all the cell types of the body, a difference between target and nontarget cells cannot now be attributed to the presence and absence of androgen receptor protein. Furthermore, the fact is that although apparently mediated by the single species of DHT-TES receptor protein, androgen does not provoke a uniform response from different target cell types. In some, cell proliferation is an androgen-dependent process. Thus those target cell types which constitute the embryonic Wolffian duct, for example, die off if they are not continuously stimulated by androgen. In other cell types, such as proximal tubule cells of the mouse kidney, androgen does not cause waves of mitotic activity. Instead, it merely causes marked hypertrophy of individual cells which comprise a static population. This indicates that divergent sets of structural gene products are induced by androgen in different target organs. This was indeed found to be so. For example, androgen induces a 30-100 fold increase in activities of supernatant alcohol dehydrogenase and lysosomal/3-glucuronidase in the mouse kidney (Dofuku, Tettenborn, and Ohno, 1971 a), whereas in the mouse submaxillary salivary
glands, neither of the above enzymes is inducible by androgen. Instead, a marked induction of epithelial growth factor (peptide hormone) occurs (Lyon, Hendry, and Short, 1973). Yet both target organs of affected Tfm/Y are noninducible by androgen. Any group of structural genes that is under the control of the same regulatory protein must necessarily possess similar recognition signals encoded in base sequences, which we shall loosely define as operators. Whether the regulatory protein recognizes these operator base sequences on DNA or on its fresh transcripts (heterogenous nuclear RNAs) is still a debatable point. Although a dramatic increase in specific species of cytoplasmic messenger RNAs has been repeatedly observed in a variety of steroid hormone target organs, this can be due to either an increased transcription or the stabilization and more efficient processing of nuclear messenger RNA precursors (heterogenous nuclear RNAs). There is little doubt that each structural gene inducible by androgen is endowed with an operatorlike recognition signal. A mutation of the autosomally inherited ,&glucuronidase locus of the mouse rendered this gene insensitive to the inducing effect of androgen in the kidney of wild-type males and females, while this mutation did not affect its constitutive levels in nontarget organs (Dofuku, Tettenborn, and Ohno, 1971a, 1971 b). Subsequently, it was shown that a markedly reduced inducibility of a mutant /3-glucuronidase is not attributable to a reduced half-life of increasingly synthesized enzyme (Swank, Paigen, and Ganschow, 1973), thus supporting the notion that this and a similar mutation affected the operator-like recognition signal of the ,&glucuronidase gene. Furthermore, the observation that in the presence of the wild-type allele at the X-linked Tfm locus, such mutation of the pglucuronidase locus did not affect the inducibility of other enzymes in the mouse kidney confirmed the obvious point that unlinked autosomally inherited structural genes that are under the control of the X-linked Tfm locus in androgen target organs are equipped with individual operator-like signals. Within the context of there being only one species of DHT-TES receptors in the mammalian body, organ-specific responses provoked by androgen can be explained by the following alternatives. First, all the structural genes that are androgen-inducible in any part of the body are equipped with similar operator-like signals that are designed to be recognized directly by the acceptor binding site of DHTTES receptor protein. In nontarget cell types, all these operator-like signals remain inaccessible to DHT-TES receptor protein, since these structural genes come under the control of other regulatory proteins not pertinent to sex. This is predicted in
Cell 320
the model proposed by Britten and Davidson (1969). In each target cell type, on the other hand, operator-like signals of only a specific set of structural genes become accessible to androgen receptor protein. Thus organ specificity is the function of other cell type-specific regulatory systems upon which the regulatory system for sexual differentiation is superimposed. Second, only a specific set of structural genes that are androgen-inducible in a particular target cell type share similar operatorlike signals. These signals are designed to be recognized, not directly by DHT-TES receptor protein, but by an androgen-inducible organ or cell type-specific regulatory protein, and it is only this organ-specific regulatory gene that possesses an operator-like signal that can be recognized directly by the acceptor binding site of DHT-TES receptor protein. These two alternatives are schematically illustrated in Figure 2. I favor the first alternative because
the second is unnecessarily redundant and uneconomical. According to the second alternative, there have to be as many organ- or cell type-specific secondary regulatory loci as there are numbers of different androgen target cell types, and all these regulatory loci should be equipped with similar operator-like signals that are designed to be recognized by the acceptor binding site of DHT-TES receptor protein. It follows then that the accessibility to these operator-like signals of a variety of secondary regulatory genes should again be regulated by another organ-specific regulatory system. Furthermore, the second alternative predicts that there should be a mutational deficiency of a secondary regulatory protein which should render only one particular androgen target organ nonresponsive to androgen. If such a mutation exists, it should have already been recognized in human as well as veterinary clinics. An Apparent Androgen Response Which May Be an Estrogen Response Androstenedione serves as a natural precursor of estrone in the female gonad. Similarly, testosterone can be aromatized to become estradiol in certain organs of the body, whereas the reverse conversion of estradiol to testosterone never occurs. Thus regarding those androgen target organs that are rich in aromatizing enzymes, a danger exists in mistaking a response induced by in situ converted estradiol as a part of the androgen-induced responses. One would expect that such false androgen responses, if they exist, would not be totally abolished by the Tfm mutation. A prime candidate is the neonatal masculinization process of the central nervous system. This subject, however, is currently too controversial to merit a thorough discussion. Acknowledgments
Figure 2. Two Alternative Androgen Receptor Protein
Mechanisms
of Gene
Activation
by
Although apparently mediated by a single species of DHT-TES receptor protein, androgen elicits divergent responses from different target cell types. This scheme illustrates two alternative explanations for such divergent responses. The first alternative (1) does not require a subset of organ-specific regulatory loci. All the structural genes (A, B. and C) that are androgen-inducible in one or another target cell type are equipped with similar operator-like recognition signals (arrow heads) that will be recognized by the acceptor binding site of androgen-bound receptor. In nontarget organs, these recognition signals of A and B, as well as of C, remain inaccessible, whereas in the target cell type illustrated here, only that of A remains inaccessible. Thus in this target cell, the products of structural genes B and C are androgen-inducible. In the second alternative (2), not the structural genes A, B, and C. but a subset of target cell type-specific regulatory genes a and /J’ possess arrowhead recognition signals for the acceptor binding site of androgen-bound receptor. In this target cell type, only the P-gene recognition site remains accessible, thus only p regulatory protein is induced, which in turn can recognize only structural genes B and C, but not A. Since both alternatives must postulate cell typespecific masking of certain genes, the second alternative only serves to introduce an unnecessary complication.
This work was supported by a contract and grants and also by the Edwin G. Roberts Research Fund.
from
the NIH,
Bardin, C. W., Bullock, L. P., Sherins, R. J., Mowszowicz, Blackburn. W. R. (1973). Rec. Prog. Horm. Res. 29. 65.
I., and
Attardi.
B., and Ohno,
Bennett, D., Boyse, M., and Yanagisawa, Britten,
S. (1974).
E. A., Lyon, M. F., Mathieson, K. (1975). Nature 257, 236.
R. J., and Davidson,
De La Chapelle,
Cell 2, 205.
A. (1972).
E. H. (1969). Am. J. Hum.
B. J., Scheid,
Science Genet.
765, 349.
24, 71.
Dofuku, R., Tettenborn, 232, 5.
U., and Ohno,
S. (1971a).
Nature
New Biol.
Dofuku, R., Tettenborn. 234, 259.
U.. and Ohno,
S. (1971 b). Nature
New Biol.
Eichwald,
E. J., and Silmser,
Ferguson-Smith, Gasser,
C. R. (1955).
M. A. (1966).
D. L., and Silvers,
Lancet
W. K. (1972).
Transplant.
Bull. 2, 154.
ii, 475. Adv.
Immunol.
75, 215.
Genetic 321
Regulation
Gehring,
of Sexual
U., and Tomkins,
Gehring. U., Tomkins, Biol. 232, 106. Goldstein,
Gropp,
G. M. (1974).
G. M., and
J. L., and Wilson,
Goodfellow, cet i, 1040.
S. A., Strong,
A., and Ohno,
Heinrichs, Clin. Res.
Development
Jost,
J. 0. (1972).
S. (1966).
W. J., Karszinin, 77, 143.
A. (1970).
Sot.
Lyon,
M. F. (1974).
M. F., and Hawkes,
Proc.
Lyon, M. F., Hendry. 58, 357.
Roy.
Sot.
Macintyre, M. N., Hunter, Rec. 136, 137.
8259,
Peterson,
119.
A. J., Jones, H. W., and Metab. 38, 1143.
8787,
243.
Nature
Short,
Lan-
W. L. (1969).
T., and
Lond.
S. G. (1970).
J., and
51, 1647.
74, 505.
Ft., and Herman, L., Gautier,
Roy.
New
J. S. S. (1965).
2. fur Zellforschung.
R., Wyss,
Phil. Trans.
Nature
J. Clin. Invest.
B. S., Meyer, W. J., Hadjian, C. J. (1974). J. Clin. Endocrinol.
Lyon,
S. (1971).
S. J., and Steward,
Imperato-McGinley, J., Guerreso, Ft. E. (1974). Science 786, 1213. Keenan, Migeon,
Cell 3, 59.
Ohno,
227, 1217.
R. V. (1973).
J. E., and Morgan,
J. Endocrinol.
A. H. (1960).
Anat.
McLaren. A. (1971). In Edinburgh Symposium on the Genetics of the Spermatozoan, R. A. Beatty and S. Gluecksohn Waelsch, eds. (Edinburgh), p. 313. Mintz, B. (1969). In First Conference on the Clinical Delineation of Birth Defects, 5, D. Bergsma and V McKusick, eds. (National Foundation), p. 11. Moscona,
A. (1957).
Proc.
Nat. Acad.
Northcut, R. C., Island, D. P., and Endocrinol. Metab. 29, 422. Ohno,
S. (1971).
Ohno, S., 2, 128.
Nature
Dofuku,
Tettenborn.
S.. Trujillo, J., Stenius, Cytogenetics 1, 258.
Price,
D. (1970).
Tarkowski,
Phil. Trans. Paigen.
U. (1971).
C., Christain, Roy.
Sot.
J. Clin.
A. K. (1970).
Phil. Trans. G. C.,
Wachtel, S. S., Ohno, Nature 257, 235.
S., Koo,
J. D., and Walker,
and
8259,
Roy.
Sot.
Lond.
8259,
E. Z. (1975a).
G. C.. and
Genet.
Boyse,
R.
133.
R. E. (1973).
Boyse,
J. D. (1969).
Clin.
L. C., and Teplitz,
Lond.
K., and Ganschow,
Wachtel, S. S., Koo, 254, 270.
Wilson,
C. W. (1969).
234, 134.
R.. and
Ohno, (1962).
Swank, R. T., Biol. 81, 225.
Sci. USA 43, 184. Liddle,
J. Mol. 107. Nature
E. A. (1975b).
J. Clin. Invest.
48, 381.
March
1976,
Copyright”
1976 by MIT
Major Regulatory Genes for Mammalian Sexual Development Susumu Ohno Department of Biology City of Hope National Medical Duarte, California 91010
Center
The principal regulatory elements concerned with the genetic basis of sex determination and differentiation in mammals appear to be comparatively simple (Ohno, 1971; Lyon, 1974). A gene or set of genes on the Y chromosome causes the indifferent embryonic gonad to develop as a testis, an ovary forming in the absence of the Y. Thus the Y-linked gene or genes determine the primary or gonadal sex. It appears, however, that this Y-linked testisdetermining gene makes no further direct contribution to subsequent sexual differentiation of mammalian embryos. Thus in the presence of testosterone, either an XY or an XX embryo undergoes masculine differentiation, feminine differentiation occurring in its absence. Since the testis organized by the Y normally serves as the only significant source of fetal androgen, concordance between the gonadal sex and the phenotypic sex is usually achieved. It follows then that the minimum requirement for sexual development of mammals is the presence of two major regulatory genes: one for the gonadal sex determination, which should be on the Y, and the other for the phenotypic sexual differentiation, whichcan beeitherx-linkedorautosomallyinherited. The Y-Linked Testis Determining Gene and H-Y Antigen Development of the indifferent gonad into a testis under the influence of the Y chromosome begins as soon as the migration of primordial germ cells from the yolk sac to the gonadal ridge is completed. In the XY gonadal ridge, many of the germ cells move deeper into the central gonadal blastema, while in the XX gonadal ridge, germ cells remain in the periphery and multiply until they form a thick layer (Gropp and Ohno, 1966). Consequently, the direct contact between migrant primordial germ cells and resident somatic elements of the central blastema is made considerably earlier in the male gonad than in the female gonad, and there is little doubt that this earlier contact leads to the formation of seminiferous tubules instead of ovigerous cords. In fact, a classical experiment by Moscona revealed that such direct cell-cell recognition among different cell types at their membrane surface is the basis of organogenesis (Moscona, 1957). Inasmuch as the Y chromosome directs the organogenesis of a testis, and since migrant XY primordial germ cells and resident XY somatic elements appear to attract each other to make direct contact, it follows that the Y-linked testis-determining gene or genes seem
Review
very likely to specify a plasma membrane protein or proteins. By serological methods, those proteins (or the sugar moieties of glycoprotein) which reside in the plasma membrane and are accessible to antibody can be recognized as the surface antigens of intact viable ceils. In fact, one such antigen specified by the Y chromosome has been known in the mouse since 1955 (Eichwald and Silmser, 1955). Rejection of male skin grafts by females within a highly inbred strain of mice must be due to the Y-linked plasma membrane protein (H-Y antigen) or proteins, because individual differences should not exist with regard to any of the autosomally inherited as well as X-linked plasma membrane proteins. This H-Y antigen is ubiquitously expressed in various cell types of all the males, but absent from those of normal females (Gasser and Silvers, 1972). Using antimouse H-Y antibody raised in C57BL female mice that received repeated grafts of syngeneic male cells, Wachtel, Koo, and Boyse (1975a) recently demonstrated that the cell surface component recognized as H-Y antigen has been highly conserved in mammalian evolution. Male cells, but not female cells, of other mammalian species, including man, possessed a component indistinguishable from the mouse H-Y antigen on their plasma membrane. Such an extreme evolutionary conservation is unusual for cell surface components, and is reminiscent of the conservatism exhibited by the histone IV gene locus. The H-Y antigen must have performed an invariant function throughout the evolution of mammals, and that invariant function seems almost certain to be the induction of a testis. For this and other reasons, we have proposed that H-Y antigen is the long sought after product of the Ylinked testis-determining gene of mammals (Wachtel et al., 1975b). The proposed identity of H-Y antigen and the testis-determining gene product can best be tested on exceptional individuals whose gonadal sex does not agree with their chromosomal sex. Any individual who possesses testes in spite of the apparent absence of the Y must somehow have acquired the testis-determining gene. Thus his cells should type as H-Y antigen positive. Conversely, the testis-determining gene of any individual whose gonads had not undergone testicular differentiation, in spite of the apparent presence of the Y, must have somehow been rendered inert. Thus her cells should type as H-Y antigen negative. A subsequent series of tests performed on a considerable variety of such exceptional individuals, humans as well as mice and other mammals, yielded no exception from the above expectation (S. S. Wachtel, personal communication).
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Can the proposed identity of H-Y antigen and the Y-linked testis-determining gene product explain other pertinent observations made on testicular development of mammals? In random fusions between two blastocysts 50% of the cases should involve heterosexual pairs, thus producing experimentally XX/XY mosaic mice. The curious fact is that these XX/XY mosaic mice show a much greater tendency to develop as males than as females (Mintz, 1969; McLaren, 1971), since true hermaphroditism is a very rare event (Tarkowski, 1970). In the case of man too, at least some of the socalled XX males appear to be cryptic XX/XY mosaics, harboring some XY cells in their testes (De La Chapelle, 1972). Testicular development seen in the cow’s gonad that was born co-twin with the bull might also be due to the presence of migrant XY cells in that gonad (Ohno et al., 1962; Goodfellow, Strong, and Stewart, 1965). It would appear that in all these cases, a minority, if a substantial one, of XY cells in the mosaic gonad was able to entice the majority of XX cells to participate in testicular organization. These results suggest that differentiating XY cells of the fetal gonad produce a hormone-like enticing substance. Its existence and very limited range of effectiveness have been demonstrated in experiments in which a heterosexual pair of fetal rat gonads (sixteenth day of gestation) was transplanted to a subcapsular position in the kidney of castrated adult rats. The testis was able to destroy the ovarian cortex and induce seminiferous tubule development in the ovarian medulla, provided that the two were in direct contact with each other. When a testis and an ovary were separated by a distance of 8 mm, no appreciable effect by the former was seen on the latter (Macintyre et al., 1960). We might consider the role of H-Y antigen in this context. Although the expression of H-Y antigen is ubiquitous, it appears to play a sexual role only in the fetal gonad. Thus nongonadal cells of testicular feminized TfmlY mice still typed as H-Y antigen positive, although no masculine development beyond the formation of testes occurs in these mutant XY mice (Bennett et al., 1975). The uniqueness of gonadal XY cells as opposed to all other XY cells of the male body may reside in the possession of specific receptors for H-Y antigen in addition to H-Y antigen itself. In fact, such coexistence should be a prerequisite for direct cell-cell contact recognition between migrant germ cells and resident somatic elements which lead to the formation of a testis. What if XX gonadal cells, while lacking in H-Y antigen, are also endowed with specific H-Y antigen receptors? An analogous situation has been found in androgen target cells of the normal female. Although these XX cells are not normally exposed to
endogenous testosterone, they are nevertheless equipped with the same amount of specific nuclearcytosol androgen receptor protein as the homologous XY cells, as is discussed below. This, in fact, is the very reason that XX fetuses readily masculinize in response to exogenously administered androgen. Similarly, the constitutive presence of specific H-Y antigen receptors on the membrane surface of gonadal cells might be the very reason that XX cells can be enticed by neighboring XY cells to participate in testicular organization. When these gonadal XX cells become coated with disseminated H-Y antigen, they can indeed become an equal partner to gonadal XY cells in the organization of the testis. In the immune system, a single species of antibody molecules, anchored on the membrane surface, serves as the receptors of each committed B cell, the antigen antibody binding at the surface triggering the increased production and subsequent secretion of that antibody. In the same manner, the establishment of direct contact between gonadal XY cells through their H-Y antigen and its receptors might trigger the increased production and subsequent dissemination of H-Y antigen which then can coat gonadal XX cells in the same mosaic gonad. If this is the mechanism, disseminated H-Y antigen may provide the hormonelike substance mentioned earlier, which has an extremely short range of effectiveness (Figure 1). The experiment which unequivocally tests the postulated transference of H-Y antigen from XY cells to XX cells in the mosaic gonad is technically difficult. Nevertheless, we may already have obtained the evidence of H-Y antigen transference on lymphocytes of human XX males (Wachtel et al., 1975b). When informative with regard to the Xlinked blood group antigen X,, these XX males type as though both their Xs are of the maternal derivation; they are X,-negative in spite of having X,-positive fathers. Two explanations are possible. First, the reciprocal exchange between the X and the Y in the meiotic process of the father transferred the testis-determining gene from the Y to the X, and the X, gene from the X to the Y (Ferguson-Smith, 1966). Second, they started as XY zygotes, and the XX cell line subsequently arose by mitotic nondisjunctions. Although their lymphocytes and cultured fibroblasts consist exclusively of the XX line, the original XY line remains in the gonads and other inaccessible organs (De La Chapelle, 1972). The manner of meiotic pairing between the X and the Y speaks rather strongly against the first possibility. It then follows that the source of H-Y antigen found on XX lymphocytes of these males has to be the XY cells that lurk in nonhemopoietic organs of their bodies.
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Development
All in all, the proposal determining gene specifies explains most, if not all, tions that have been made
that the Y-linked testisH-Y antigen satisfactorily of the pertinent observaon mammalian testicular
organized testicular cells
Figure 1. Ubiquitous Expression of Enticement in the Gonad
of H-Y Antigen
and
the
Nature
Assuming H-Y antigen (solid black column with one rectangular and one rounded edge) to be the Y-linked, testis-determining gene product, this figure schematically depicts its ubiquitous presence in all the cells of the male and its total absence in the female, as well as its proposed mode of action in organogenesis of the testis. Although endowed with H-Y antigen, nongonadal XY cells lack specific H-Y antigen receptors on their plasma membrane (top row). Their female counterparts may be equipped with a number of anchorage sites (slightly round indentations on the cell surface). Thus they can be coated with disseminated H-Y antigen in the XX/XY mosaic situation (see text for discussion of human XX males). The plasma membrane of gonadal XY cells (germ cells as well as somatic elements) are unique, since they have both H-Y antigen and its specific receptors (columnar indentations on the cell surface), as shown in the middle row. Direct contacts between these gonadal XY cells established through their H-Y antigen and its specific receptors result in organogenesis of the testis. The established contact may trigger these XY cells to produce more H-Y antigen, which is disseminated to their immediate surroundings (bottom row). If so, disseminated H-Y antigen is a hormone-like substance with an extremely short range of effectiveness. The presence of such a hormone-like substance was shown by the experiments of Macintyre et al. (1960). Gonadal XX cells may have anchorage as well as specific receptor sites for H-Y antigen (middle and bottom rows). Thus when they are coated by H-Y antigen in the XX/XY mosaic gonad, they can participate in organogenesis of the testis as full partners.
differentiation. Genes for the postpubertal function of germ cells and gametes are not the concern of this paper. The X-Linked Tfm Locus as the Regulatory Locus of Secondary Sexual Characteristics The testis induced by the regulatory locus on the Y above mentioned then secretes testosterone, which induces male development of the accessory glands and ducts. Regardless of their sex chromosome constitutions, mammalian embryos have the inherent tendency to develop female secondary sex characteristics. Thus if deprived of the endogenous testosterone source by castration, XY embryos manifest the female phenotype, whereas the exogenous administration of testosterone readily masculinizes the XX fetus and gives it the complete set of male secondary sex characteristics (Jost, 1970; Price, 1970). It would appear that a gene or a set of genes which confers the androgen responsiveness to appropriate cell types of the mammalian body is the regulatory locus which governs the development of secondary sexual characteristics. The masculinizing action of testosterone on its target cells is mediated by the product of a gene on the X chromosome. This product activates all the genes required for manifestation of the male phenotype in response to circulating testosterone (Ohno, 1971). Evidence for this crucial involvement of a single X-linked gene in development of secondary sexual characteristics comes from a mutation of the relevant gene in the mouse (Tfm), resulting in complete failure to respond to testosterone. A chromosomally XY animal with this mutant gene develops testes, because the normal Y chromosome is present and therefore is H-Y positive, but shows no further male differentiation, thus exhibiting the syndrome known as “testicular feminization” (Lyon and Hawkes, 1970). Unlike peptide hormones, possibly because of their lipophilic nature, steroid hormones appear to penetrate readily the plasma membrane of cells. Thus their specific receptors are found not on the plasma membrane of cells but in the cytosol (supernatant) fraction of the cytoplasma. Such so-called nuclear-cytosol steroid receptor proteins have all the characteristics of being regulatory proteins. These receptors show very high binding affinity to specific steroids: Kd of the order of 1 O-9-1 O-10 M. The act of binding to a specific steroid creates the secondary binding site (acceptor-binding site) on a receptor protein via allosteric effect. Steroidbound receptors then move into the nucleus and associate with the chromatin to induce the synthesis of a specific set of structural gene products. There now exists a consensus that the androgen insensitivity exhibited by all the target cell types of
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affected testicular feminized XY individuals is due to a mutational deficiency of the nuclear-cytosol androgen receptor protein, not only in mice (Gehring, Tomkins, and Ohno, 1971; Attardi and Ohno, 1974; Gehring and Tomkins, 1974) but also in rats (Bardin et al., 1973) as well as in man (Keenan et al., 1974). Thus the remainder of this review paper is devoted to discussing several crucial problems in association with the proposition that the X-linked Tfrn locus is the one and only regulatory locus which governs the development of male secondary sex characteristics. One or Two Different Kinds of Androgen Receptors? In some, but not all, of the androgen target organs, 5a-reductase efficiently converts testosterone (TES) to 5a-dihydrotestosterone (DHT) (Wilson and Walker, 1969). Thus there was a time when the human testicular feminization syndrome was thought to be caused by 5a-reductase deficiency (Heinrichs et al., 1969; Northcutt, Island, and Liddle, 1969). Subsequently, however, 5a-reductase deficiency was recognized in man as an independent autosomal recessive trait. Unlike Tfm individuals, XY individuals, deficient in this enzyme, show a considerable degree of masculinization at birth, and further masculinization occurs after puberty (Imperato-McGinley et al., 1974). Nevertheless, the fact that there appeared to be two types of androgen target organs, one rich in 5a-reductase, the other not having this enzyme, prompted a number of investigators to suggest that mammals might possess two different kinds of androgen receptors: DHT receptor and TES receptor. The mouse kidney is one of a few androgen target organs that persist in mutant. Tfm/Y individuals. Bardin et al. (1973) believed that the mouse kidney normally contains not DHT receptor but TES receptor, and what is deficient in androgen nonresponsive TfmlY kidney is TES receptor. If this is true, the Tfm mutation must cause deficiency of both DHT and TES receptors, since the embryonic Wolffian duct and urogenital sinus of Tfm/Y totally fail to masculinize even under the influence of exogeeously administered DHT (Goldstein and Wilson, 1972). It then follows that since a single locus can specify only one protein, the X-linked Tfm locus must not be an androgen receptor locus, but a regulatory locus of a higher hierarchy which regulates the expression of two androgen receptor loci-one for DHT and the other for TES. Our studies, on the other hand, have consistently demonstrated that all the target organs of androgen have the same receptor, which shows the highest binding affinity to DHT but also binds to TES with a respectable affinity. This one and only androgen receptor might best be called DHT-TES receptor, and what is deficient
in the kidney, submaxillary salivary glands, and brain of Tfm/Y is this DHT-TES receptor (Gehring et al., 1971; Attardi and Ohno, 1974; Gehring and Tomkins, 1974). Those androgen target organs which are relatively deficient in 5a-reductase, such as the mouse kidney, are rich in the second enzyme which converts DHT further to 5a-androstandiol (Bardin et al., 1973). Thus unless a precaution is taken, 3H-DHT, added to the cytosol fraction, is rapidly metabolized, giving falsely low receptor-bound counts on sucrose or glycerol gradient profiles. Such organs as the mouse kidney or levator ani muscle must normally make do with TES, because its intracellular conversion to DHT is negligible. Nevertheless, the mouse kidney responds better to administered DHT than to TES (Ohno, Dofuku, and Tettenborn, 1971; Bardin et al., 1973) reflecting the fact that DHT-TES receptor protein present in this organ also has a higher binding affinity to DHT than TES. All in all, the androgen responsiveness of all the divergent target organs appears to be mediated by a single species of DHT-TES receptor protein. Thus there is no need to invoke a higher regulatory locus which controls two separate receptor loci. Constitutive Production of Androgen Receptor Even if the Tfm locus specified the one and only androgen receptor protein, if this protein itself is inducible, the need still exists to identify a higher regulatory locus which controls the expression of this gene. Fortunately, a number of organs present in the adult mouse body of both sexes are normally androgen-responsive. They are the brain, kidney, submaxillary salivary glands, preputial glands, and levator ani muscle, among others. Our previously mentioned studies revealed that no sexual difference exists in the amount of DHT-TES receptor protein present in those bisexual androgen target organs. Thus within each target organ, the production of androgen receptor protein is constitutive, not influenced either by the presence of the Y chromosome or by different circulating testosterone levels. The observed constitutivity in each target organ, of course, was expected, for the classical embryological studies have shown that XX embryos masculinize readily and fully in response to exogenously administered androgen. The problem still remained, however, that until very recently, it was believed that only specific target organs or cell types of the body contain a receptor protein specific for each steroid hormone. The organ-specific occurrence of androgen receptor protein again calls for the presence of a higher regulatory gene which turns on the Tfm locus only in androgen-responsive cell types. It now appears that this belief in organ-specific occurrence of steroid receptor protein is incorrect. One can hardly regard cultured fibroblasts as an-
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Development
drogen target cells, for no appreciable response can be monitored after their exposure to a reasonable concentration of androgen, that is, 10-a M. Although a very high concentration of TES (>lO-6 M) is toxic to cultured fibroblasts, there is no need to invoke the presence of a high affinity receptor protein for such toxic effect. Yet Keenan et al. (1974) were able to demonstrate a mutational deficiency of DHT-TES receptor protein in cultured fibroblasts obtained from human testicular feminized patients, since similarly cultured fibroblasts from normal males and females contained a very respectable amount of this receptor protein. It has indeed been found that permanent mouse fibroblast L-cell lines, as well as muscles in general, also contain receptor proteins for androgen and other steroid hormones (E. Baulieu, personal communication). The mouse kidney, while quite responsive to androgen, shows no appreciable response to estrogen (Ohno et al., 1971). Yet it contains not only androgen receptor, but also a good amount of estradiol receptor (our unpublished data). It would now appear that DHT-TES receptor protein and possibly other steroid receptor proteins are ubiquitously expressed in all the cell types of the body in the same manner as H-Y antigen. Thus when considering the genetic regulatory mechanism of mammalian sexual development, there appears to be no need to invoke a higher regulatory locus which controls the X-linked Tfm locus. induction of Different Sets of Structural Gene Products in Different Target Organs Inasmuch as DHT-TES receptors appear to be present ubiquitously in nearly all the cell types of the body, a difference between target and nontarget cells cannot now be attributed to the presence and absence of androgen receptor protein. Furthermore, the fact is that although apparently mediated by the single species of DHT-TES receptor protein, androgen does not provoke a uniform response from different target cell types. In some, cell proliferation is an androgen-dependent process. Thus those target cell types which constitute the embryonic Wolffian duct, for example, die off if they are not continuously stimulated by androgen. In other cell types, such as proximal tubule cells of the mouse kidney, androgen does not cause waves of mitotic activity. Instead, it merely causes marked hypertrophy of individual cells which comprise a static population. This indicates that divergent sets of structural gene products are induced by androgen in different target organs. This was indeed found to be so. For example, androgen induces a 30-100 fold increase in activities of supernatant alcohol dehydrogenase and lysosomal/3-glucuronidase in the mouse kidney (Dofuku, Tettenborn, and Ohno, 1971 a), whereas in the mouse submaxillary salivary
glands, neither of the above enzymes is inducible by androgen. Instead, a marked induction of epithelial growth factor (peptide hormone) occurs (Lyon, Hendry, and Short, 1973). Yet both target organs of affected Tfm/Y are noninducible by androgen. Any group of structural genes that is under the control of the same regulatory protein must necessarily possess similar recognition signals encoded in base sequences, which we shall loosely define as operators. Whether the regulatory protein recognizes these operator base sequences on DNA or on its fresh transcripts (heterogenous nuclear RNAs) is still a debatable point. Although a dramatic increase in specific species of cytoplasmic messenger RNAs has been repeatedly observed in a variety of steroid hormone target organs, this can be due to either an increased transcription or the stabilization and more efficient processing of nuclear messenger RNA precursors (heterogenous nuclear RNAs). There is little doubt that each structural gene inducible by androgen is endowed with an operatorlike recognition signal. A mutation of the autosomally inherited ,&glucuronidase locus of the mouse rendered this gene insensitive to the inducing effect of androgen in the kidney of wild-type males and females, while this mutation did not affect its constitutive levels in nontarget organs (Dofuku, Tettenborn, and Ohno, 1971a, 1971 b). Subsequently, it was shown that a markedly reduced inducibility of a mutant /3-glucuronidase is not attributable to a reduced half-life of increasingly synthesized enzyme (Swank, Paigen, and Ganschow, 1973), thus supporting the notion that this and a similar mutation affected the operator-like recognition signal of the ,&glucuronidase gene. Furthermore, the observation that in the presence of the wild-type allele at the X-linked Tfm locus, such mutation of the pglucuronidase locus did not affect the inducibility of other enzymes in the mouse kidney confirmed the obvious point that unlinked autosomally inherited structural genes that are under the control of the X-linked Tfm locus in androgen target organs are equipped with individual operator-like signals. Within the context of there being only one species of DHT-TES receptors in the mammalian body, organ-specific responses provoked by androgen can be explained by the following alternatives. First, all the structural genes that are androgen-inducible in any part of the body are equipped with similar operator-like signals that are designed to be recognized directly by the acceptor binding site of DHTTES receptor protein. In nontarget cell types, all these operator-like signals remain inaccessible to DHT-TES receptor protein, since these structural genes come under the control of other regulatory proteins not pertinent to sex. This is predicted in
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the model proposed by Britten and Davidson (1969). In each target cell type, on the other hand, operator-like signals of only a specific set of structural genes become accessible to androgen receptor protein. Thus organ specificity is the function of other cell type-specific regulatory systems upon which the regulatory system for sexual differentiation is superimposed. Second, only a specific set of structural genes that are androgen-inducible in a particular target cell type share similar operatorlike signals. These signals are designed to be recognized, not directly by DHT-TES receptor protein, but by an androgen-inducible organ or cell type-specific regulatory protein, and it is only this organ-specific regulatory gene that possesses an operator-like signal that can be recognized directly by the acceptor binding site of DHT-TES receptor protein. These two alternatives are schematically illustrated in Figure 2. I favor the first alternative because
the second is unnecessarily redundant and uneconomical. According to the second alternative, there have to be as many organ- or cell type-specific secondary regulatory loci as there are numbers of different androgen target cell types, and all these regulatory loci should be equipped with similar operator-like signals that are designed to be recognized by the acceptor binding site of DHT-TES receptor protein. It follows then that the accessibility to these operator-like signals of a variety of secondary regulatory genes should again be regulated by another organ-specific regulatory system. Furthermore, the second alternative predicts that there should be a mutational deficiency of a secondary regulatory protein which should render only one particular androgen target organ nonresponsive to androgen. If such a mutation exists, it should have already been recognized in human as well as veterinary clinics. An Apparent Androgen Response Which May Be an Estrogen Response Androstenedione serves as a natural precursor of estrone in the female gonad. Similarly, testosterone can be aromatized to become estradiol in certain organs of the body, whereas the reverse conversion of estradiol to testosterone never occurs. Thus regarding those androgen target organs that are rich in aromatizing enzymes, a danger exists in mistaking a response induced by in situ converted estradiol as a part of the androgen-induced responses. One would expect that such false androgen responses, if they exist, would not be totally abolished by the Tfm mutation. A prime candidate is the neonatal masculinization process of the central nervous system. This subject, however, is currently too controversial to merit a thorough discussion. Acknowledgments
Figure 2. Two Alternative Androgen Receptor Protein
Mechanisms
of Gene
Activation
by
Although apparently mediated by a single species of DHT-TES receptor protein, androgen elicits divergent responses from different target cell types. This scheme illustrates two alternative explanations for such divergent responses. The first alternative (1) does not require a subset of organ-specific regulatory loci. All the structural genes (A, B. and C) that are androgen-inducible in one or another target cell type are equipped with similar operator-like recognition signals (arrow heads) that will be recognized by the acceptor binding site of androgen-bound receptor. In nontarget organs, these recognition signals of A and B, as well as of C, remain inaccessible, whereas in the target cell type illustrated here, only that of A remains inaccessible. Thus in this target cell, the products of structural genes B and C are androgen-inducible. In the second alternative (2), not the structural genes A, B, and C. but a subset of target cell type-specific regulatory genes a and /J’ possess arrowhead recognition signals for the acceptor binding site of androgen-bound receptor. In this target cell type, only the P-gene recognition site remains accessible, thus only p regulatory protein is induced, which in turn can recognize only structural genes B and C, but not A. Since both alternatives must postulate cell typespecific masking of certain genes, the second alternative only serves to introduce an unnecessary complication.
This work was supported by a contract and grants and also by the Edwin G. Roberts Research Fund.
from
the NIH,
Bardin, C. W., Bullock, L. P., Sherins, R. J., Mowszowicz, Blackburn. W. R. (1973). Rec. Prog. Horm. Res. 29. 65.
I., and
Attardi.
B., and Ohno,
Bennett, D., Boyse, M., and Yanagisawa, Britten,
S. (1974).
E. A., Lyon, M. F., Mathieson, K. (1975). Nature 257, 236.
R. J., and Davidson,
De La Chapelle,
Cell 2, 205.
A. (1972).
E. H. (1969). Am. J. Hum.
B. J., Scheid,
Science Genet.
765, 349.
24, 71.
Dofuku, R., Tettenborn, 232, 5.
U., and Ohno,
S. (1971a).
Nature
New Biol.
Dofuku, R., Tettenborn. 234, 259.
U.. and Ohno,
S. (1971 b). Nature
New Biol.
Eichwald,
E. J., and Silmser,
Ferguson-Smith, Gasser,
C. R. (1955).
M. A. (1966).
D. L., and Silvers,
Lancet
W. K. (1972).
Transplant.
Bull. 2, 154.
ii, 475. Adv.
Immunol.
75, 215.
Genetic 321
Regulation
Gehring,
of Sexual
U., and Tomkins,
Gehring. U., Tomkins, Biol. 232, 106. Goldstein,
Gropp,
G. M. (1974).
G. M., and
J. L., and Wilson,
Goodfellow, cet i, 1040.
S. A., Strong,
A., and Ohno,
Heinrichs, Clin. Res.
Development
Jost,
J. 0. (1972).
S. (1966).
W. J., Karszinin, 77, 143.
A. (1970).
Sot.
Lyon,
M. F. (1974).
M. F., and Hawkes,
Proc.
Lyon, M. F., Hendry. 58, 357.
Roy.
Sot.
Macintyre, M. N., Hunter, Rec. 136, 137.
8259,
Peterson,
119.
A. J., Jones, H. W., and Metab. 38, 1143.
8787,
243.
Nature
Short,
Lan-
W. L. (1969).
T., and
Lond.
S. G. (1970).
J., and
51, 1647.
74, 505.
Ft., and Herman, L., Gautier,
Roy.
New
J. S. S. (1965).
2. fur Zellforschung.
R., Wyss,
Phil. Trans.
Nature
J. Clin. Invest.
B. S., Meyer, W. J., Hadjian, C. J. (1974). J. Clin. Endocrinol.
Lyon,
S. (1971).
S. J., and Steward,
Imperato-McGinley, J., Guerreso, Ft. E. (1974). Science 786, 1213. Keenan, Migeon,
Cell 3, 59.
Ohno,
227, 1217.
R. V. (1973).
J. E., and Morgan,
J. Endocrinol.
A. H. (1960).
Anat.
McLaren. A. (1971). In Edinburgh Symposium on the Genetics of the Spermatozoan, R. A. Beatty and S. Gluecksohn Waelsch, eds. (Edinburgh), p. 313. Mintz, B. (1969). In First Conference on the Clinical Delineation of Birth Defects, 5, D. Bergsma and V McKusick, eds. (National Foundation), p. 11. Moscona,
A. (1957).
Proc.
Nat. Acad.
Northcut, R. C., Island, D. P., and Endocrinol. Metab. 29, 422. Ohno,
S. (1971).
Ohno, S., 2, 128.
Nature
Dofuku,
Tettenborn.
S.. Trujillo, J., Stenius, Cytogenetics 1, 258.
Price,
D. (1970).
Tarkowski,
Phil. Trans. Paigen.
U. (1971).
C., Christain, Roy.
Sot.
J. Clin.
A. K. (1970).
Phil. Trans. G. C.,
Wachtel, S. S., Ohno, Nature 257, 235.
S., Koo,
J. D., and Walker,
and
8259,
Roy.
Sot.
Lond.
8259,
E. Z. (1975a).
G. C.. and
Genet.
Boyse,
R.
133.
R. E. (1973).
Boyse,
J. D. (1969).
Clin.
L. C., and Teplitz,
Lond.
K., and Ganschow,
Wachtel, S. S., Koo, 254, 270.
Wilson,
C. W. (1969).
234, 134.
R.. and
Ohno, (1962).
Swank, R. T., Biol. 81, 225.
Sci. USA 43, 184. Liddle,
J. Mol. 107. Nature
E. A. (1975b).
J. Clin. Invest.
48, 381.
