Evidence for a gonad-specific receptor for H-Y antigen: binding of exogenous H-Y antigen to gonadal cells is independent of beta 2-microglobulin.

This report addresses the question whether two different types of binding exist for the reaction of H-Y antigen with the cell surface. Anti-H-Y antiserum in the presence of complement was cytotoxic only for gonadal cells expressing their own H-Y antigen, but not to ovarian cells loaded with H-Y antigen. H-Y antigen was co-redistributed with beta 2--microglobulin on newborn testicular cells, but some residual H-Y activity was found on similarly treated testis cells from 15 day old rats. After beta 2--microglobulin redistribution, testis cells maintained their binding capacity for exogenous H-Y antigen prepared from epididymal fluid or Daudi cell culture supernatants. This result suggests that exogenous H-Y antigen is bound via a gonad-specific receptor which is independent of beta 2--microglobulin and that this type of binding for H-Y antigen is different from the beta 2--m-associated expression of H-Y antigen on the cell surface.     

Organization in vitro of ovarian cells into testicular structures

Summary While it has been shown previously (Zenzes et al., 1978; Ohno et al., 1978) that when dissociated testicular cells are exposed to anti-H-Y antiserum in vitro they are prevented from reorganizing into testicular structures, forming ovarian follicular structures instead, the most conclusive evidence for the action of H-Y antigen would be the conversion of ovarian cells into testicular organization. Testing for H-Y antigen of the medium collected from cultivated testicular cells revealed a positive reaction. Dissociated ovarian cells of newborn rats cultivated in this medium reorganize into testicular structures. It is concluded that H-Y antigen is responsible for this histomorphologic change.     

H-Y antigen negative patients with testicular tissue and 46,XY karyotype

Summary H-Y antigen could not be detected on lymphocytes from two male pseudohermaphrodites with 46,XY karyotypes and testicular tissue. One of the patients had additional assays performed on fibroblasts grown from the skin, and the gonadal ridge—these were also negative. The H-Y antiserum was raised in rats, with Raji cells the target of cytotoxicity tests. In these patients, the substance that promoted testicular differentiation does not have serologic H-Y antigen detectable by the assay used. It appears that H-Y antigen that is commonly measured in neutralization reactions may not be the only form of testicular organizing factor present.     

Testicular cells lysostripped of H-Y antigen organize ovarian follicle-like aggregates.

A suspension of free testicular cells were obtained by mild trypsin treatment from newborn BALB/c testes, and their plasma membrane H-Y antigen sites were blocked (lysostripped) by an excess of H-Y antibody of proven specificity and potency (45 min in ice). Upon 16 h of the Moscona-type rotation culture, these treated testicular cells yielded primarily spherical aggregates, more than half of which demonstrated a strong resemblance to ovarian follicles. The resemblance was particularly striking between the smallest testicular folliculoids and primordial ovarian follicles that abound in the newborn female gonad. Under the same condition, control serum-treated testicular cells primarily yielded cylindrical tubular structures that can be very long. Over a critical range, concentrations of H-Y antibody apparently influenced the frequency of testicular folliculoid formation. The above directly supports the proposed testis-organizing function of H-Y antigen and is certainly compatible with the genetic situation encountered in the wood lemming (Myopus schisticolor), that in the functional absence of H-Y antigen, XY gonadal cells readily organize an ovary.     

Genetic, biochemical and functional study of the H-Y antigen in man

H-Y antigen, first described as a male minor transplantation antigen, is unvarying associated with testicular differentiation, more than the presence of Y chromosome. The weak reactivity of anti H-Y serum needs quantitative and very sensitive tests to detect presence or absence of H-Y. This antigen may act as an hormone, to induce testicular differentiation of target cells, bearing a specific receptor at their surface. The relationship between an H-Y molecule immunologically defined by its antigenicity and H-Y factor defined by its function to induce testicular organogenesis must be determined.     

H-Y binding in the gonad: inhibition by a supernatant of the fetal ovary.

H-Y antigen, the product of mammalian testis-determining genes, is released in the free state by testicular cells. Molecules of free H-Y antigen are bound in vitro by dispersed cells of the adult ovary. The binding reaction is inhibited by specific H-Y antibody. It is also inhibited by a diffusible factor of the newly differentiated fetal ovary. These observations favor the view that testicular organogenesis depend upon dissemination and binding of H-Y molecules by cells of the undifferentiated gonad (XY or XX, both having H-Y receptors), and raise the question of whether ovarian organogenesis may be promoted by a "female" molecule corresponding to H-Y of the male.     

H-Y antigen in 46,XY gonadal dysgenesis

Summary Presence of H-Y antigen has been correlated with testicular differentiation, and absence of H-Y with failure of testicular differentiation, in a variety of mammalian species. To determine more precisely the relationship between expression of H-Y antigen and development of the testis, we studied the cells of phenotypic females with the 46,XY male karyotype. Blood leukocytes were typed H-Y⁺ in five XY females with gonadal dysgenesis, although in other studies blood leukocytes from XY females with gonadal dysgenesis were typed H-Y^-. Thus mere presence of H-Y antigen is not sufficient to guarantee normal differentiation of the testis. In the present paper we review evidence for an additional factor in gonadal organogenesis, the H-Y antigen receptor. We infer that testicular development requires engagement of H-Y and its receptor. It follows that XY gonadal dysgenesis is the consequence of functional absence of the H-Y testis inducer as in the following conditions: failure of synthesis of H-Y or failure of specific binding of H-Y.     

Evidence for an H-Y Crossreactive Antigen in Invertebrates

A sex specific antigen which crossreacts with the mammalian H-Y antigen has been identified on the cell surface of hemocytes from the lobster ( Homarus americanus ) and the gonadal cells of three insect species. The hemocytes from the male lobster, the testicular cells from the male beetle ( P. cornutus ), and the ovarian cells from two Orthopteran species ( L. maderae and D. punctata ) specifically absorbed H-Y antibodies. The specificity of H-Y antibody absorptions by cells from only one sex, suggest that an ancestral H-Y-like antigen may be present in invertebrates which could be engaged in sexual (cellular) recognition events.     

Immunological and functional aspects of H-Y antigen

Summary Male-specific H-Y antigen may be defined by graft rejection, killer cell action or antibodies. Most commonly H-Y antigen is detected in assays using H-Y antisera. In these tests errors may arise from various causes: 1) Auto- and heteroantibodies cross-reacting with target cells. 2) Restriction phenomena. 3) MHC-dependent modification of the amount of H-Y antigen present on different tissues. 4) Modification of cell surface antigens by bacteria or viruses.Regarding the third definition of H-Y antigen, four different states can be distinguished in the mammalian male. H-Y occurs (1) as an integral part of the plasma membrane; (2) unspecifically attached to the membrane of human erythrocytes; (3) free in solution; (4) bound to its gonad-specific receptor.Redistribution experiments suggest that H-Y and ₂-m are associated on the cell membrane. Coredistribution is not found of H-Y and MHC antigens. An antibody blocking technique demonstrates association of H-Y and H-2D antigens on unfixed lymphoid, but not on testicular cells. Human erythrocytes lacking ₂-m do not integrate H-Y antigen into the cell membrane. Male erythrocytes, however, absorb H-Y antigen from the serum. The origin of H-Y antigen in the serum is not clear. It may be shed from cell membranes, derive from the testis which actively secretes H-Y antigen, or both.H-Y antigen is bound by a gonad-specific receptor. This receptor is present in the gonads of both sexes. H-Y antigen is supposed to mediate testis differentiation via this receptor. Reaggregation experiments in vitro using dissociated gonads of the newborn rat demonstrate that ovarian cells reorganize into testicular structures in the presence of H-Y antigen. The assumption cannot be confirmed that addition of H-Y antiserum to testicular cells results in ovarian structures. This finding, however, does not conflict with the view that H-Y antigen is involved in testis differentiation, e.g. by inducing testis cell-specific functions via the gonad-specific receptor.     

Primary sex determination: genetics and biochemistry

Summary On the basis of widespread phylogenetic conservatism, it has been propose'd that serologically-defined H-Y antigen is the inducer of primary sex differentiation in mammals, causing the initially indifferent gonad to become a testis rather than an ovary. The proposal has withstood extensive testing in a variety of biological circumstances: XX males have testes and are H-Y⁺ and fertile XY females lack testicular tissue and are H-Y; soluble H-Y antigen induces testicular organogenesis in XX indifferent gonads of the fetal calf in culture; H-Y antibody blocks tubular reaggregation of dispersed XY testicular cells, causing them to organize follicular clusters.There is a gonadal receptor for H-Y antigen: fetal ovarian cells that have been exposed to soluble H-Y (released for example by testicular Sertoli cells) take up the molecule and acquire the H-Y⁺ phenotype; they absorb H-Y antibody in serological tests. Specific uptake of soluble H-Y does not occur in the extra-gonadal tissues.It may be inferred that H-Y antigen is disseminated during embryogenesis and bound by specific receptors in cells of the primordial gonad, and that reaction of H-Y and its receptor signals a program of testicular differentiation, regardless of karyotype. The several anomalies of primary sexual differentiation manifest in such conditions as the XX male, the XX true hermaphrodite, and the XY female can thus reasonably be viewed as specific errors of synthesis, dissemination, and binding of H-Y antigen.H-Y is secreted by Daudi cells, cultured from a human XY Burkitt lymphoma. The Daudi-secreted moiety is a single hydrophobic protein of 18,000 molecular weight. Early attempts to characterize H-Y secreted by testicular Sertoli cells have yielded two molecules, one of 16,500 MW (corresponding to the Daudi-secreted 18,000 MW protein), and one of 31,000 MW. It remains to be ascertained whether both are in fact H-Y antigens, and if so, whether one is a polymer of the other, or whether each represents the product of genes with discrete testis-determining functions.     

Mammalian Cross-Reactive H-Y Antigen Induces Sex Reversal in vitro in the Avian Testis

Testes of either newborn rats or newly hatched chickens, dissociated into single cell suspensions, reorganize in vitro into their histotypic structures. In birds, the heterogametic female sex is H-Y antigen positive, and not the male as in mammals. Cocultivation of rat and chicken testicular cells results in the reorganization of an ovotestis. A similar result is obtained after cultivation of chicken testicular cells in the supernatant medium of cultured human male Burkitt lymphoma Daudi cells. Rat testicular Sertoli cells as well as Daudi cells are a source of H-Y antigen. The simultaneous application of H-Y antigen and anti-H-Y antiserum prevents ovotestis formation. It is concluded that H-Y antigen which is known to be testis-organizing in mammals, is the ovary-organizing factor in birds.     

In vitro studies of gonadal organogenesis in the presence and absence of H-Y antigen.

In a very strict sense, the primary (gonadal) sex of mammals is determined not so much by the presence or absence of the Y but the expression or nonexpression of the evolutionary extremely conserved plasma membrane H-Y antigen. The central somatic blastema of embryonic indifferent gonads contains one cell lineage characterized by the possession of S-F differentiation antigen that differentiates into testicular Sertoli cells in the presence of H-Y and into ovarian follicular (granulosa) cells in its absence. This cell lineage appears to play the most critical role in gonadal differentiation. Whether or not testicular Leydig cells and ovarian theca cells are similarly derived from the common cell lineage has not been determined. Nevertheless, if given H-Y antigen, presumptive theca-cell precursors of the fetal ovary acquire hCG (LH?)-receptors-the characteristic of fetal Leydig cells.     

H-Y antigen expression in a case of mixed gonadal dysgenesis

Summary H-Y antigen expression was studied on leukocytes and gonad-derived fibroblasts from a patient affected by mixed gonadal dysgenesis. Blood leukocytes and fibroblasts derived from the testis were typed H-Y positive, but the fibroblasts derived from the streak gonad were H-Y negative. Although the patient's karyotype was a mosaic, 45,XO/46,X+mar, as detected in-peripheral blood cells and testis-derived fibroblasts, all the fibroblasts derived from the streak gonad were 45,XO. These data suggests that the marker chromosome was in fact a Y-derived chromosome. Moreover, they showed that, at the gonadal level, a minority of H-Y positive 46,X+mar cells were able to organize a testis. Nevertheless, a large number of XO cells probably did not receive the testicular forming influence of the H-Y antigen and of the other masculinizing factors.     

H-Y antigens

H-Y antigen is defined as a male histocompatibility antigen that causes rejection of male skin grafts by female recipients of the same inbred strain of rodents. Male-specific, or H-Y antigen(s), are also detected by cytotoxic T cells and antibodies. H-Y antigen appears to be an integral part of the membrane of most male cells. In addition, H-Y antibodies detect a soluble form of H-Y that is secreted by the testis. The gene (Smcy/SMCY) coding for H-Y antigen detected by T cells has been cloned. It is expressed ubiquitously in male mice and humans, and encodes an epitope that triggers a specific T -cell response in vitro. Additional epitopes coded for by different Y-chromosomal genes are probably required in vivo for the rejection of male grafts by female hosts. The molecular nature of H-Y antigen detected by antibodies on most male cells is not yet known. Testis-secreted, soluble H-Y antigen, however, was found to be identical to Müllerian-inhibiting substance (MIS). MIS cross-reacts with H-Y antibodies and identical findings were obtained for soluble H-Y antigen and MIS, i.e., secretion by testicular Sertoli and, to a lesser degree, ovarian cells, binding to a gonad-specific receptor, induction of gonadal sex reversal in vitro and, in cattle, in vivo. H-Y antisera also detect a molecule or molecules associated with the heterogametic sex in nonmammalian vertebrates. Molecular data on this antigen or antigens are not yet available.     

Production of H-Y antibody in the ascites fluid of mouse and localization of the antigen on cells and tissues

A procedure is described for the production of large amounts of ascites fluid containing specific H-Y antibody. The distribution of H-Y antigen on mouse epididymal spermatozoa, thymocytes, and splenocytes was carried out using this specific antibody in the microcytotoxicity test and ELISA. Employing the indirect immunofluorescent technique, the H-Y antigen was localized on the acrosomal membrane of mouse epididymal and washed ejaculated human spermatozoa and on the entire membrane of mouse splenocytes and thymocytes. Immunohistochemical localization of the antigen in the testicular section indicated its presence in the cytoplasm of Leydig cells and on the membrane of Sertoli cells and sperm heads.     

The testis as a secretory organ for H-Y antigen

Summary After cultivation of dissociated rat testicular tissues, H-Y antigen is detectable in the medium; this is not the case if nongonadal male tissues are incubated. Release of H-Y antigen by testis cells is inhibited by the addition of cycloheximide. All tissues still type H-Y positive after culture. It is assumed that the testis actively secretes H-Y antigen. This assumption is supported by the finding that the amount of H-Y antigen in the epididymal fluid increases with the age of the animals.     

H-Y antigen and sexual dysgenesis in man

H-Y antigen is a surface component associated with the heterogametic sex of various species and supposed to induce testicular differentiation. Genes controlling directly or not the expression of H-Y antigen and testicular differentiation have been localized on Y as well as on X chromosome and even autosomal chromosome. However the genetical localization of the H-Y structural gene remains unknown. We analysed the expression of H-Y antigen in three types of sexual dysgenesis (males bearing XX caryotype, testicular feminization syndrome and one case of hermaphroditism) to clarify the function and the genetics of this antigen.     

An in vitro model of gonad differentiation in the chicken. Estradiol-induced sex-inversion results in the occurrence of serological H-Y antigen

Dissociated cells from the gonads and mesonephros of 8-day-old chicken embryos were reorganized in rotation culture. The aggregates obtained from gonadal cells exhibited specific morphologic and histologic sex differences. In the presence of estradiol, aggregates from testicular cells showed characteristics similar to control ovarian aggregates, while in ovarian aggregates under estradiol treatment the female organization became more pronounced. Determination of serological H-Y antigen revealed that male aggregates of gonads and mesonephros were negative for H-Y and those of female embryos were positive for H-Y. Administration of estradiol did not change the H-Y findings in female aggregates. In contrast, in the male, gonadal cultures became H-Y positive while mesonephros cultures remained negative. It is assumed that estradiol induces the occurrence of H-Y antigen in the gonads.     

In vitro studies of gonadal organogenesis in the presence and absence of H-Y antigen

Summary In a very strict sense, the primary (gonadal) sex of mammals is determined not so much by the presence or absence of the Y but the expression or nonexpression of the evolutionary extremely conserved plasma membrane H-Y antigen. The central somatic blastema of embryonic indifferent gonads contains one cell lineage characterized by the possession of S−F differentiation antigen that differentiates into testicular Sertoli cells in the presence of H-Y and into ovarian follicular (granulosa) cells in its absence. This cell lineage appears to play the most critical role in gonadal differentiation. Whether or not testicular Leydig cells and ovarian theca cells are similarly derived from the common cell lineage has not been determined. Nevertheless, if given H-Y antigen, presumptive theca-cell precursors of the fetal ovary acquire hCG (LH?)-receptors—the characteristic of fetal Leydig cells. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. This work was supported by Contract NO1-CB-33907, and Grants No. 1 RO1 AG00042 and No. 5 RO1 CA16952 from the National Institutes of Health.     

Two plasma membrane antigens of testicular sertoli cells and H-2-restricted versus unrestricted lysis by female T cells.

Sertoli cell-only seminiferous tubules of sterile XX,Sxrl-male mice served as an excellent source of pure Sertoli cells. When H-2-compatible female mice were immunized 3 times with these Sertoli cells, resulting antibodies recognized two antigens on the plasma membrane of testicular Sertoli cells. They were male-specific, but ubiquitously expressed H-Y antigen and the cell lineage-specific antigen which Sertoli cells shared with ovarian follicular cells. Doubly primed (2 or 3 times in vivo, and once in vitro) cytotoxic T cells from these females lysed target Sertoli cells in both H-2-restricted and nonrestricted manners. While H-2-restricted killings were attributable to H-Y antigen, further work is needed to identify the Sertoli follicular cell lineage-specific antigen as the cause of H-2-nonrestricted killings.