H-Y antigen: No evidence for alleles in wild strains of mice

H-Y antigen(s) coded or controlled by the Y chromosome in a variety of wild mouse strains have been compared with those of the inbred laboratory strains C57BL/6 (B6) and C57BL/10 (B10). H-Y antigen(s) were detected by H-2-restricted cytotoxic T cells from B6 and B10 female mice primed in vivo and boosted in vitro with syngeneic male spleen cells: There was no difference in the degree of H-Y specific lysis of male cells from the C57BL strains and of F₁ hybrids or B6 congenic mice carrying the Y chromosome from the wild mouse strains examined. This result indicated that at the level of target cell specificity the H-Y antigen(s) from wild and laboratory strains were indistinguishable. H-Y antigen(s) were also found to be indistinguishable at the level of the in vitro induction of the anti H-Y cytotoxic response: F₁ female mice, primed in vivo and boosted in vitro with homologous F₁ male cells, all made H-Y-specific responses and where it could be examined, the target cell specificity of the anti-H-Y cytotoxic cells showed that B10 male cells as well as the homologous F₁ male cells (where the Y chromosome was derived from the wild strain) were good targets. Finally, possible differences in H-Y transplantation antigens between the wild strains and the B10 laboratory strain were examined by grafting F₁ male mice, the progeny of B10 females, and wild strain males with B10 male skin. These grafts were not rejected during an observation period of more than 9 months. Taken together, neither the cytotoxic data nor the skin graft data provide any evidence for allelism of H-Y even though the mouse strains examined were collected from widely disparate geographical locations.     

Effects of H-Y Antigen on Morphologic and Endocrine Differentiation of Gonads in Mammals

The effects of H-Y antigen, released in a soluble state by male human Burkitt lymphomas and mouse teratomas, were studied in the fetal gonads from 13 to 21-day-old rats and 98-day-old calves, using histologic techniques, radioimmunoassays for testosterone, and bioassays for Müllerian-inhibiting substance (MIS). Whereas no effect was detected in the younger gonads, some structural changes were observed in bovine and 21-day-old rat ovaries when cultured for 5 days with Daudi and male teratoma supernatants, but there was no synthesis of testosterone or MIS. These observations raise questions concerning the critial period of sensitively to H-Y antigen of female gonads, and the dissociation between morphologic and biochemical events.     

The male minor transplantation antigen preferentially activates recipient CD4+ T cells through the indirect presentation pathway in vivo

To evaluate the priming and trafficking of male Ag-reactive CD4(+) T cells in vivo, we developed an adoptive transfer model, using Marilyn (Mar) TCR transgenic T cells that are specific for the H-Y minor transplantation Ag plus I-A(b). By manipulating donor and recipient strain combinations, we permitted the Mar CD4(+) T cells to respond to the H-Y Ag after processing and presentation by recipient APCs (indirect pathway), or to the male Ag as expressed on donor APCs (direct pathway). Mar CD4(+) T cells responding through the indirect pathway specifically proliferated and expressed activation markers between days 2 and 4 posttransplant, migrated to the graft 2-3 days before cessation of graft heartbeat, and were detected in close proximity to transplant-infiltrating recipient APCs. Intriguingly, adoptively transferred Mar T cells did not respond to male heart or skin grafts placed onto syngeneic MHC class II-deficient female recipients, demonstrating that activation of Mar T cell preferentially occurs through cognate interactions with processed male Ag expressed on recipient APCs. The data highlight the potency of indirect processing and presentation pathways in vivo and underscore the importance of indirectly primed CD4(+) T cells as relevant participants in both the priming and effector phases of acute graft rejection.     

Induction of allograft tolerance to the H-Y antigen in adult C57BL/6 mice: differential effects on delayed-type hypersensitivity and cytolytic T-lymphocyte activity

This study describes the induction of allograft tolerance to the "male-specific," minor histocompatibility antigen, H-Y, in adult C57BL/6 female mice, and the effects of this tolerance induction on two immune parameters associated with graft rejection: delayed-type hypersensitivity (DTH) and cytolytic T-lymphocytes (CTL). B6 females tolerized to H-Y, by a single iv injection of C57BL/6 male lymphocytes, exhibited prolonged or permanent survival of B6 male tail skin grafts. Graft-induced DTH against H-Y antigen was reduced or abrogated in tolerized females. Delayed onset of graft rejection in partially tolerant females correlated with delayed onset of DTH, and eventual rejection of grafts was accompanied by an increase in H-Y-specific DTH. In contrast, H-Y-specific CTL activity was not consistent with graft status. These data demonstrate a correlation between H-Y-specific DTH and rejection of male skin grafts by B6 female mice and are most consistent with a major effector role for DTH in chronic graft rejection.     

H-Y antigen in the study of sex determination and control of sex ratio

It has been proposed, on the basis of widespread phylogenetic conservation, that H-Y antigen is the inducer of primary sex, causing the undifferentiated XY gonad to become a testis in male heterogametic species such as the human and bovine. That proposition has withstood extensive testing in vivo and in vitro. Freemartin gonads are H-Y⁺, for example, and masculinization of the freemartin gonad has been attributed to soluble H-Y, borne and transmitted in the serum of the bull twin, and bound in ovarian receptors of the female. We have applied monoclonal H-Y antibodies to the identification of gender in embryos of the bovine. Our preliminary results imply presence of H-Y in bovine embryos of the morula and blastocyst stages recovered at about 6–12 days of gestation. Assignment of H-Y phenotype -- positive in males and negative in females -- allows selective implantation of male and female during embryo transfer. Thus in an early study, we correctly identified gender in 6 of 7 calves born healthy at term, after transfer of 8 blastocysts.     

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     

Review lecture. Immunology of H-Y antigen and its role in sex determination

H-Y was originally discovered as a transplantation antigen that caused female mice of certain inbred strains to reject skin from otherwise identical males. The ability to make the skin graft rejection response and, in vitro, cytotoxic T cell responses against H-Y is controlled by genes within the major histocompatibility complex, H-2, and by non-H-2 genes. H-Y belongs to a class of weak transplantation antigens characterized by an inability to elicit responses under many conditions. Although genetic factors are very important in determining responsiveness, their action can be modified by immunization procedures. H-Y has been proposed as the differentiation signal that causes the formation of the testes from the undifferentiated gonad in the developing embryo. This hypothesis has been explored by using a series of mice whose karyotype and phenotypic sex are paradoxical.     

H-Y transplantation antigen in human XO females

Summary When sensitized with human cultured fibroblasts of the XY and XO, but not XX, sex chromosomal types C57BL/6 female mice reject syngeneic male grafts accelerated (second set graft reaction). These findings demonstrate that the antigenic determinants of H-Y antigen of man and mouse are homologous and that XO females (at least those tested) carry the H-Y transplantation antigen. The results are discussed in the light of the question of differences between the H-Y antigen as defined by grafting and serology and the chromosomal localization of the H-Y structural gene(s).     

鼠源性抗雄性特异性抗原噬菌体Fab抗体的制备及分析

利用噬菌体抗体库筛选技术获得抗雄性特异性抗原的噬菌体Fab抗体,首次采用雄鼠脾细胞对鼠源性抗雄性特异性抗原噬菌体Fab抗体库进行3轮亲和富集和2轮雌鼠脾细胞吸附,对筛选后特异性噬菌体Fab抗体进行ELISA分析,重组率鉴定及基因测序分析。结果显示,5次筛选后的15个菌落中有9个能产生抗雄性特异性抗原特异性噬菌体抗体,噬菌体Fab抗体的基因重组率为60%,E5克隆的重链、轻链可变区序列分别属于VH1和VκⅣ基因家族,这为挑选出高亲和力的抗雄性特异性抗原噬菌体Fab抗体奠定了实验基础,将推进雄性特异性抗原及其抗体的研究进程,并为性别控制研究开创新途径。     

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.     

Serology of H-Y antigen

Summary Anti-H-Y antiserum is generally obtained from female inbred mice or rats that have been hyperimmunized with syngeneic male cells. The specificity of such antiserum is defined by its reactivity for male but not female cells. A number of conventional serological assays have been used to measure that reactivity. However, H-Y is a weak antigen, evidently represented sparingly on the surfaces of cells other than sperm, epidermal cells and brain cells; thus the srological assays for H-Y are technically difficult. Yet H-Y serology has enabled significant progress toward the understanding of primary sex differentiation.A recent advance in H-Y serology is the establishment of monoclonal anti-H-Y antisera which promise to facilitate analysis and clarification of the H-Y system.     

Differential expression of H-Y antigen on lymphocyte subsets: analysis by flow cytometry

Expression of H-Y antigen in human white blood cells was measured using flow cytometry with monoclonal antibodies. In this system, lymphocytes were stained preferentially in the male, and to a lesser extent in the female. Analysis of the lymphocyte subsets with biotinylated H-Y antibody conjugated with streptavidin-fluorescein isothiocyanate (FITC) and subset-specific antibody conjugated with phycoerythrin derivative (RD1) revealed differential expression of H-Y among the subsets of the male. In samples from eight men, 41.1% +/- 21.7% of B cells (B1) were stained, compared with 20.7% +/- 12.8% of cytotoxic-suppressor T cells (T8) and 5.4% +/- 3.0% of helper-inducer T cells (T4). In samples from seven women, 12.4% +/- 10.9% of B cells were stained, but staining of T cells was negligible.     

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.     

H-Y Antigen Expression in Temperature Sex-Reversed Turtles (Emys orbicularis)

H-Y antigen has been used as a marker for the heterogametic sex and is assumed to be an organizing factor for the heterogametic gonad. In the turtle Emys orbicularis , H-Y antigen is restricted to the female cells, indicating a female heterogamety (ZZ/ZW) sex-determining mechanism. Moreover, the sexual differentiation of the gonads is temperature sensitive, and complete sex reversal can be obtained at will. In this framework the relationships between H-Y antigen, temperature, and gonadal phenotype were studied. Mouse H-Y antiserum was absorbed with blood and gonadal cells of control wild male and female adults, and with blood and gonadal cells from three lots of young turtles from eggs incubated at 25–26°C (100% phenotypic males), at 30–30.5°C (100% phenotypic females), or at 28.5–29°C (majority of females with some males and intersexes). The residual activity of H-Y antiserum was then estimated using an immunobacterial rosette technique. In adults, both blood cells and gonadal cells were typed as H-Y negative in males and as H-Y positive in females. In each of the three lots of young, blood cells were H-Y negative in some individuals and H-Y positive in others. The proposed interpretation is that the H-Y negative individuals were genotypic males (ZZ) and the H-Y positive were genotypic females (ZW). The gonads of these animals were then pooled in different sets according to their sexual phenotype and to the presumed genotypic sex (i.e., blood H-Y phenotype). Testicular cells were typed as H-Y negative in genotypic males as well as in the presumed sex-reversed genotypic females; likewise, ovarian cells were typed as H-Y positive in genotypic females as well as in the presumed sex-reversed genotypic males. These results provide additional evidence that H-Y antigen expression is closely associated with ovarian structure in vertebrates displaying a ZZ/ZW sex-determining mechanism.     

Frequency and cross-reactivity od cytolytic T lymphocyte precursors reacting against male alloantigens

It is well established that cytotoxic T lymphocytes (CTL) specific for the male minor histocompatibility antigen (H-Y) are generated by restimulation in vitro of in vivo primed spleen cells from C57BL/6 (H-2b) female mice with syngeneic male spleen cells. When tested on target cells from H-2 different strains, the male-specific C57BL/6 CTL populations exhibited significant lysis of DBA/2 (H-2d), A (H-2a), but not C3H (H-2k), male and female target cells. In an attempt to document this cross-reactivity further at the clonal level, a sensitive technique of limiting dilution analysis was used to determine the specificity of C57BL/6 individual CTL precursors (CTL-P) reactive against the male antigen. The mean frequency of anti-H-Y CTL-P in spleens of primed female mice was about 1/3500. Between one-third to one-tenth of these CTL-P produced a progeny that cross-reacted with H-2d (allogeneic) female target cells. These findings were confirmed by the analysis of the reactivity pattern exhibited by male-specific CTL clones derived by limiting dilution. Of 99 clones tested, 13 were found to cross-react with female DBA/2 target cells. These results thus indicate that a relatively large proportion (greater than 10%) of H-2b CTL-P directed against the H-Y antigen cross-react with target cells expressing H-2d alloantigens in the absence of H-Y antigen.     

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.     

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.     

H-Y antigen as a tool for the determination of the heterogametic sex in Amphibia

H-Y antigen was investigated in three amphibian species with different degrees of sex-chromosome differentiation: Bufo bufo, Triturus vulgaris, and Pyxicephalus adspersus. No heteromorphic sex chromosomes were found in B. bufo, but an examination of the progeny of hermaphrodites (Ponse, 1942) indicated that the female of this species was heterogametic (ZW). Sex chromosomes differing only by a very small heterochromatic region at their telomeres were found in the male of T. vulgaris (XY). Pyxicephalus adspersus revealed high differentiated ZW sex chromosomes. The results of the H-Y antigen studies on these three species indicate that H-Y antigen is expressed only in the heterogametic sex, irrespective of differences in morphological differentiation of the sex chromosomes. Therefore, H-Y antigen could be a valuable tool in determining the heterogametic sex, not only in Amphibia but possibly also in other vertebrate species that have either evolved no heteromorphic sex chromosomes or where sex-reversal experiments are not possible.     

Increased H-Y antigen levels associated with behaviorally induced, female-to-male sex reversal in a coral-reef fish

It has been proposed that H-Y antigen is the synthetic product of sex-determining genes, and that H-Y antigen controls ontogenetic differentiation of the heterogametic sex throughout vertebrates. The coral-reef fish Anthias squamipinnis is a protogynous hermaphrodite in which all individuals mature initially as females. Males result when adult females change sex as a consequence of alterations in behavioral interactions within social groups. Three assay methods were used to measure H-Y antigen levels in the spleens, gonads, and epidermal tissue of 16 adult females and in 16 males that had been induced to change sex from a prior female phase by the removal of a pre-existing male from each of 16 social groups. In 15 male-female pairs, the H-Y antigen levels were higher in male than in female spleen, gonad, and epidermis tissues. The precise temporal relationship between the onset of sex change and the increase in the H-Y antigen level was not examined. If, as we strongly suspect, the temporal relationship proves to be close, the inference will be that the behavioral cues inducing sex change also influence the synthetic activity of genes controlling H-Y antigen production.