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.     

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.     

Inhibition of H-Y cell-mediated cytolysis by monoclonal H-Y-specific antibody

Assays of H-Y-specific, cell-mediated cytolysis (CMC) in vitro were carried out with B6 female effector cells and B6 male target cells. Monoclonal H-Y antibody was added to the lytic assay to test whether the antigenic determinant(s) involved in H-Y-specific CMC was distinct from the serologically detected H-Y antigen. Significant blocking was observed, suggesting that the H-Y antigen detectable serologically is similar to H-Y antigen recognized by cytotoxic T cells.Abbreviations used in this paper B6 C57BL/6 - BALB BALB/c - CMC cell-mediated cytolysis - E effector cells - T target cells     

On the secretion of H-Y antigen

It has been proposed that H-Y antigen secreted by cells of the Sertoli lineage is bound by receptors on these and other cells of the primordial gonad and thereby initiates formation of the testicular cords, and that H-Y is not an integral transmembrane component but a part of a ternary system with β₂-microglobulin and products of the MHC. It follows that cultured Daudi cells, which lack β₂-microglobulin and HLA, should secrete H-Y. This is consistent with evidence obtained with monoclonal H-Y antibody and an ELISA. By this method, free H-Y was demonstrable in the supernatant fluids of cultured Sertoli cells and Daudi cells. The assay provides a useful alternative to detection of H-Y in the complement-dependent cytotoxicity test.     

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.     

Studies on H-Y antigen in different cell fractions of the testis during pubescence

Summary Various cell types of the rat testis during pubescence, including germ, Sertoli, and Leydig cells, were partially enriched. The fractions were tested for the presence, binding, and secretion of H-Y antigen. The main results are: Immature germ cells are H-Y antigen-negative until the late diploid stages, and late primary spermatocytes or spermatids become positive; the somatic cells of the gonad are positive at all ages examined (18 days old to adulthood). Secretion of H-Y antigen is restricted to the Sertoli cell fraction. Binding of externally supplied antigen takes place on Leydig cells; the Sertoli cell surface will be saturated because of active secretion; there is no binding to germ cells. Thus, immature germ cells seem to be the only H-Y antigen-negative cells of the male organism, and the Sertoli cells seem to be the only ones to secrete H-Y antigen.     

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.     

Correlation between the number of sex chromosomes and the H-Y antigen titer

Summary H-Y antigen was studied serologically on blood cells and cultured fibroblasts of patients with numerical aberrations of the sex chromosomes. As compared with normal males, patients with the karyotypes 48,XXXY and 49,XXXXY have reduced H-Y antigen titrs; a tendency toward reduced titers can also be detected in the 47,XXY Klinefelter syndrome. The existence of an intermediary titer was further substantiated by a quantitative absorption test applied to cells with the 49,XXXXY karyotype. It appears that in the presence of one Y chromosome, the H-Y antigen titer decreases with an increasing number of X chromosomes. In contrast, the H-Y antigen titer is increased if, at a given number of X chromosomes, the number of Y chromosomes is increased, as in the 47,XYY male. Consequently, patients with 48,XXYY chromosomes are in the male control range. The findings are interpreted under the hypothesis of a controlling or modifying influence of the sex chromosomes on the titer of H-Y antigen.     

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.     

X-linked genes of the H-Y antigen system in the wood lemming (Myopus schisticolor)

Summary H-Y antigen was investigated in 18 specimens representing six different sex chromosome constitutions of the wood lemming (Myopus schisticolor). The control range of H-Y antigen was defined by the sex difference between normal XX females (H-Y negativeper definitionem) and normal XY males (H-Y positive, full titer). H-Y antigen titers of the X^*Y and X^*0 females were in the male control range, while in the X^*X and X0 females the titers were intermediary. Data were obtained with two different H-Y antigen assays: the Raji cell cytotoxicity test and the peroxidase-antiperoxidase (PAP) method. Fibroblasts, gonadal cells, and spleen cells were checked. Presence of full titers of H-Y antigen in the absence of testis differentiation is readily explained by the assumption of a deficiency of the gonadspecific receptor of H-Y antigen. Since sex reversal is inherited as an X-linked trait, genes for this receptor are most likely X-linked. The implications of our findings are discussed in connection with earlier findings concerning H-Y antigen in XY gonadal dysgenesis in man and the X0 situation in man and mouse.     

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.     

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.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Dissociation of alloantigen recognition from self major histocompatibility complex-restricted recognition of cytolytic T lymphocytes by monoclonal antireceptor antibodies

Two monoclonal antibodies (mAb) directed to the dual reactive cytolytic T lymphocyte clone OH8 (Db + H-Y and H-2d) were established. Analysis by cell surface staining and immunoprecipitation of radiolabeled surface molecules of OH8 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that both mAb recognized an identical heterodimeric, clonotypic structure on OH8 cells, i.e., T cell receptor. However, although the MR3-2 mAb inhibited the lysis of either Db + H-Y or H-2d targets by OH8, the MR3-6 mAb inhibited the lysis of H-2d target cells, but not that of Db + H-Y target cells. Modulation of T cell receptor by either MR3-2 or MR3-6 mAb rendered the OH8 cytolytic T lymphocyte incapable of killing both Db + H-Y and H-2d target cells. These findings suggest that different epitopes of OH8 T cell receptor were involved for the recognition of self + antigen and alloantigen.     

Mesenchymal entactin-1 (nidogen-1) is required for adhesion of peritubular cells of the rat testis in vitro

Epithelial-like Sertoli cells isolated from immature rat testis aggregate to form tubule-like structures when cultured on a monolayer of mesenchyme-derived peritubular cells. At the end of this morphogenetic process both cell types are separated by a basement membrane. In this study the gene expression of monocultures and direct cocultures of peritubular cells and Sertoli cells was examined using DD-RT-PCR. One of the isolated cDNA clones showed high homology to the cDNA encoding the basement membrane component entactin-1 (nidogen-1). Even though the entactin-1 (nidogen-1) gene is transcribed in peritubular cells, Sertoli cells, and in direct cocultures, the mRNA is translated only by the peritubular cells. No entactin-1 (nidogen-1) was detected in the Sertoli cells by Western blotting. Moreover, peritubular cell monocultures and cocultures showed the presence of one single band at 152 kDa in the supernatant, whereas in cell lysates two bands were detectable at 152 kDa and 150 kDa. Perturbation experiments using monoclonal antibodies directed against entactin-1 (nidogen-1) were performed with peritubular cells and Sertoli cells, respectively, and demonstrated loss of cell adhesion of the peritubular cells, while the Sertoli cells remained adherent. From these data we conclude that entactin-1 is exclusively produced and secreted by mesenchymal peritubular cells, and affects adhesion of peritubular cells in an autocrine manner.     

Priming for a cytotoxic response to minor histocompatibility antigens: antigen specificity and failure to demonstrate a carrier effect.

Spleen cells from normal mice do not give a detectable in vitro cytotoxic T cell (CTL) response to minor H antigens. Spleen cells from animals primed in vivo with minor H antigens give a strong CTL response when boosted in culture with the appropriate stimulating cells. Here I have studied the requirements for priming a CTL response to minor H antigens. It is shown that priming is just as antigen specific as is cytolytic effector function. That is, priming cells have to carry the same minor antigens as the challenge cells. Inducing a graft-vs-host reaction in vivo does not nonspecifically allow spleen cells to respond to minor H antigens in vitro. Using minor H congenic mice (congenic for H-Y and/or H-7) I have also tried, and failed, to demonstrate a carrier effect in priming. Female mice primed to H-Y were challenged in culture with cells bearing H-Y and H-7 antigens in the hope that a helper response to H-Y would augment a CTL response to H-7. This did not happen, however. Such primed and boosted cells gave a strong secondary CTL response to H-Y but none to H-7. It is concluded that in order to prime for a detectable in vitro response to minor antigens it is necessary to expose the CTL precursors to antigen in vivo. This either expands the size of the pool of precursors by cell division or changes them in some qualitative way.     

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.     

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.