Turner syndrome patients are H-Y positive

Summary H-Y antigen was examined in six patients exhibiting the characteristic features of Turner syndrome. Five of the patients were of the karyotype 45,X, and one was a mosaic 45,X/46,Xi(Xq). H-Y antigen was detected in all of them, however, compared to male controls, their antigen titer was reduced. Within the intermediate range between female and male controls, considerable interindividual variation was detected among the patients which could be due at least in part to biological variation. The findings permit the inference that the H-Y structural gene is not Y-linked, and support the assumptions of an X-linked gene escaping inactivation and of it controlling the expression of the H-Y structural gene. It is probable that the structural gene itself is autosomal. The results also suggest that male gonadal differentiation is dependent on a threshold level of H-Y antigen concentration.Supported by the Deutsche Forschungsgemeinschaft (SFB 46)     

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.     

A gene controlling H-Y antigen on the X chromosome

Summary The existence of a strict correlation between presence of testicular tissue and presence of H-Y antigen in mammals and man leads to the conclusion that H-Y antigen is an essential differentiation factor in testicular morphogenesis. Presence of low titers of this differentiation antigen even in fertile females indicates that its morphogenetic effect depends on a threshold. Here, studies on H-Y antigen in female individuals with various deletions of the X-chromosome are reported. It turns out that deletion of Xp results in the synthesis of reduced amounts of H-Y antigen, while deletion of Xq does not. In a fertile female with only Xp223 deleted due to an X/Y translocation, including the distal Yq, presence of a reduced H-Y titer allows for the tentative assignment of a controlling gene repressing the H-Y structural gene. From the cases studied, it follows that the H-Y structural gene is autosomal and under the control of X- and Y-linked genes. The conception emerges that interaction between X- and Y-linked genes or their products results in variation of the H-Y antigen titer. The fate of the indifferent gonadal anlage to differentiate into the male or the female direction will depend on the titer of H-Y antigen reached by the action or interaction of the controlling genes involved.Supported by the Deutsche Forschungsgemeinschaft (SFB 46)     

T-cell and antibody typing of a mouse population segregating for Sxr and H-2 haplotype

During investigation of the frequency of recombination of the testis determining gene, Tdy, and the minor histocompatibility antigen gene Hya on the Sxr segment in an outbred mouse stock, we identified two fertile males, one XY and the other XYSxr, which typed H-2k positive using the H-2b anti-H-2k monoclonal antibody HB50, but whose cells failed either to stimulate H-Y specific H-2k restricted T-cell clones, or to be killed by anti-H-2k or anti-H-2k restricted H-Y specific cytotoxic T cells. We investigated these two mice and their existing relatives, using H-2 and H-Y typing methods. The progeny of their test matings with H-2b homozygous C57BL/6 females were also investigated. The results indicate that the transmission of the Hya gene on the Y chromosomes from both mice, and the additional Hya gene on the Sxr segment of the carrier male, allowed for the expression of the H-Y antigen and its detection in the presence of an H-2 haplotype for which we had H-2 restricted H-Y specific typing cells (H-2b and H-2k). Furthermore, we identified the haplotype of the two original males as expressed in the H-2 homozygous and heterozygous F2 progeny as H-2q and discovered an unexpected cross reactivity of the monoclonal anti KkDk antibody HB13 with half the cells of H-2q homozygotes, but not qb heterozygotes.     

H-Y antigen in Swyer syndrome and the genetics of XY gonadal dysgenesis

Summary The H-Y antigen is a plasma membrane antigen involved in the organogenesis of the mammalian testis. Its expression on human cells is determined by a Y-linked gene. Phenotypic females affected by 46,XY gonadal dysgenesis (Swyer's syndrome) can be either H-Y-positive or H-Y-negative. In this paper we report H-Y antigen and endocrine studies in a sibship with three affected sisters. Immunological studies were performed on two of the patients, and a clearly positive expression was detected in both cases. Endocrine studies consisted in the investigation of the hypothalamic-pituitary-gonadal axis, which revealed that gonadal hormone insufficiency is the only endocrine abnormality associated with the syndrome. A new genetic interpretation and classification of XY gonadal dysgenesis is proposed.     

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.     

H-Y antigen in transsexuality,and how to explain testis differentiation in H-Y antigen-negative males and ovary differentiation in H-Y antigen-positive females

Summary H-Y antigen was determined in eight transsexual patients. Two of the four male-to-female transsexual patients typed as H-Y antigen-negative, while the other two typed as expected from their phenotypic and gonadal sex, namely H-Y antigen-positive. Of the four female-to-male transsexual patients, three typed as H-Y antigen-positive and one was H-Y antigen-negative, as expected. The presence of normal testes in H-Y antigen-negative males is assumed to result from a mutation of nucleotide sequences of the H-Y structural gene for antigenic determinants. Thus, an H-Y is produced with normal receptor-binding activity which can sustain the testis determination of the bipotent gonadal anlage. In the case of H-Y antigen-positive females with normal ovaries a deletion of the autosomally located H-Y structural gene is assumed. This deletion should affect sequences for repressor-binding (as was suggested for H-Y antigen-positive XX-males) and for receptor-binding activity of the H-Y antigen molecule. The resulting H-Y antigen is unable to bind to the gonadal receptor of the bipotent gonadal anlage. Thus an ovary is determined. The relevance of H-Y antigen for the aetiology of transsexualism is discussed.     

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.     

Survival after transfer of “sexed” mouse embryos exposed to H-Y antisera

Immunological means were used to determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Antisera to histocompatibility-Y (H-Y) antigen were prepared in adult C57BL/6 female mice by repeated intraperitoneal injections of spleen cells from males of the same strain. Eight-to 16-cell embryos were cultured in BMOC-3 alone or BMOC-3 without bovine serum albumin to which one of the following had been added: H-Y antiserum and normal guinea pig serum (NGPS), NGPS alone, normal mouse serum alone or normal mouse serum and NGPS. After 24 hr of culture, embryos were classified as either affected or unaffected. An embryo was classified as affected if degeneration of the embryo or breakdown of one or more cells was observed. A total of 1000 embryos were cultured in BMOC-3 with H-Y antiserum and NGPS (treated embryos). Two hundred and fifty embryos were cultured in each of the other four media (control embryos). Eighty-seven (9%) of the control embryos and 479 (48%) of the treated embryos were classified as affected after culture. Unaffected embryos, approximately 12 each, were transferred to pseudopregnant recipients. One-hundred forty control embryos (17%) survived to term with 67 females (48%) and 73 males (52%) born. Fifty-eight treated embryos (14%) survived to term, producing 50 females (86%) and 8 males (14%). Percentage of females from embryos cultured in antiserum was greater than for embryos cultured in any other media (P<0.001). These results demonstrate that detection of H-Y antigen on preimplantation embryos may be a useful and effective method of determining sex of an embryo prior to transfer.     

On the selective elimination of Y-bearing sperm

The potential use of antibodies that selectively recognize either X-bearing or Y-bearing sperm is self-evident. Thus our attention was directed to the fact that under optimal conditions, H-Y antibody lyses 50% of mouse spermatozoa. Accordingly, we asked whether expression of H-Y antigen is haploid in spermatozoa from XY male mice heterozygous for the autosomal dominantSxr gene, for if H-Y expression were haploid, H-Y antibody would be expected to kill 75% of spermatozoa derived from these XY,Sxr/- males. However, maximal lysis remained at the 50% level, which indicates that haploid expression of H-Y antigen and the potential immunoselection of Y-(or X-) bearing spermatozoa are unlikely.     

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).     

Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired in mice lacking HS1 protein, a substrate of the antigen-receptor-coupled tyrosine kinases.

HS1, an intracellular protein expressed specifically in hematopoietic cells, is rapidly tyrosine phosphorylated after cross-linking of antigen receptors on B and T lymphocytes, implicating involvement of this molecule in the signal transduction pathways from the antigen receptors as a substrate of membrane-associated tyrosine kinase(s). The development of lymphoid cells in HS1-deficient mice, generated through gene targeting, appeared normal. However, antibody production to T-independent antigen and proliferative responses of splenic B and T cells after cross-linking of the antigen receptors were impaired in these mutant mice. Furthermore, B cells in the peritoneal cavity of the mutant mice were resistant to multivalent cross-linking of the antigen receptor, which causes apoptosis of such cells in normal mice. Crossing the HS1-deficient mice with the mice harboring transgenes encoding alpha and beta chains of T-cell antigen receptor against a male H-Y antigen resulted in a progeny that demonstrated a significantly impaired ability of thymic negative selection. These results indicate that HS1 is a novel molecule involved in the antigen-receptor-derived signaling pathways and plays important roles not only in clonal expansion, but also in clonal deletion of B and T cells.     

Expression of the H-Y Antigen on thymus cells and skin: Differential genetic control linked toK end ofH-2

The strength of the H-Y antigen on thymus cells and on skin was compared in differentH-2-congenic mouse strains using a host-versus-graft reaction popliteal lymph node assay, and skin grafts from males of parental strains grafted to F₁ hybrid females. The results revealed considerable differences in the strength of the H-Y antigen among different congenic strains; these differences demonstrate the effect of theH-2-linked gene on the expression of the H-Y antigen. The linkage withH-2 was also confirmed in tests with segregating F₂ generations. In the strains bearing recombinantH-2 haplotypes, the strength of the H-Y antigen is similar to that of parental strain from which the recombinant received itsK end, and the responsible gene (or genes) map to the left ofI-C. The effect of theH-2-linked gene(s) on thymus cells and skin is different. The gene linked to theK end ofH- 2^b determines a strong H-Y antigen on thymus cells, but a relatively weak H-Y antigen on skin. The gene linked to theK end ofH- 2^k determines a weak H-Y antigen on thymus cells, but a strong H-Y antigen on skin. The gene linked to theK end ofH- 2^d determines a weak H-Y antigen on both thymus cells and skin. Our observations raise the possibility that the structural gene for the H-Y antigen is linked toH-2. Alternative (but not exclusive) explanations invoke regulatory effects ofH-2 on the expression of the H-Y antigen, possibly by means of the control of the cellular andogen receptors.     

Testis-organizing H-Y antigen as a discrete protein; Its MHC restricted immune recognition and the genomic environment in which H-Y gene operates

Summary Human H-Y antigen in solution demonstrates the high affinity, low capacity binding affinity to its specific receptor exclusively residing on the plasma membrane of gonadal somatic elements, and this interaction induced precocious testicular differentiation in XX embryonic indifferent gonads. This H-Y polypeptide is hydrophobic and made of 160 or so amino acid residues to which no more than 5 glucosamine residues are attached. Surprising similarities to interferons are noted with interest.The male determing part of the mouse Y chromosome DNA was recently identified by Singh et al. (in press) on sex reversed XX,Sxr/-male mice. We found this part to contain a 30 KBP long stretch in which neither a single Hae III restriction site GGCC nor a single Alu I restriction site AGCT resides. By contrast, the coding sequence for H-Y antigen should contain one ot two Hae III sites and three or four Alu I sites per 0.5 KBP. Thus, the Y-linked male determining gene is believed to represent a regulatory element rather than the H-Y antigen structural gene.We are thus forced to consider genetic regulatory mechanisms as they operate in mammals. In this respect, it is of utmost importance to realize the oft neglected peculiarity of the mammalian genome in which genes (coding sequences) are out-numbered nearly 50-to-1 by senseless junk or selfish DNA sequences. Even in the euchromatic region of chromosomes, the average distance between neighboring genes was estimated as 35 KBP. Being junk, these long stretches of intergenic spacers can not rely upon natural selection to eliminate deleterious consequences of randomly drifting mutational base changes; i.e., inadvertent generation of RNA polymerase II promotor sites as well as nonsense coding sequences in the midst of spacers. Intergenic spacers avoid these deleterious consequences by apparently starting as repeats of a specific short base sequence, such as the primordial sequence of 20 BP: (AGCTG) (AGCTG) (AGCTG) (GGGTG). Thus, repeated sequences within intergenic spacers do not appear to be concerned with regulation of genes downstream. On the other hand, the primordial sequence above is particularly vulnerable to frequent and inadvertent generation of the RNA polymerase III promotor sequence AGCAGGGT. Consequently, short RNAs are often transcribed from intergenic spacers. The regulatory significance of such short RNA is also doubtful.Evolutionary reasons as to why the immune system chooses to recognize not H-Y antigen per se but (H-Y + altered self MHC) antigen complexes are also discussed. Mistaken identification of certain other antigens as H-Y is inherent in this associative recognition.     

H-Y antigen expression in a case of XX true hermaphroditism

Summary Cells from an XX true hermaphrodite expressed a reduced amount of H-Y antigen when compared with normal XY cells and with cells from his father, who had an XY/XX chromosomal constitution. His mother had a normal karyotype and was H-Y negative. The four brothers of the patient were clinically and karyotypically normal. An X-Y interchange followed by random inactivation of the X chromosome is proposed to explain the H-Y antigen titer found in the patient.     

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.     

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.     

Sharing of an HLA-B27-restricted H-Y antigen between rat and mouse

The purpose of this work was twofold: 1 to learn whether rats transgenic for HLA-B27 and the human ₂-microglobulin gene HB2M can mount B27-restricted cytolytic T lymphocyte (CTL) responses to the male H-Y antigen, and 2 to learn whether such CTLs would recognize both rat and mouse H-Y in the context of HLA-B27. Female rats of the B27/HB2M transgenic line 21-4L were primed in vivo with cells from males of the same line. CTL effectors were generated from lymph node cells of these females following culture with irradiated antigen-presenting cells from either male 21-4L rats or male mice of the B27/HB2M transgenic 56-3 line. The CTLs showed male-specific, B27-specific lysis of both rat and mouse targets. Lysis of B27 targets was inhibitable by monoclonal antibodies specific for B27 or rat CD8. Specific lysis of male B27 rat and mouse targets was inhibitable equally by either rat or mouse male B27 cold targets, but not significantly by female or nontransgenic cold targets. The B27-restricted CTLs neither recognized nor were inhibited by B27⁺ or B27^- male or female human targets. These results demonstrate that CD8⁺, B27-restricted, anti-H-Y CTLs recognize and evolutionarily conserved H-Y peptide antigen in both rats and mice. In addition, they establish the transgenic rat as a model system for examining the T-cell response to antigen presented by class I HLA molecules. Correspondence to: J. D. Taurog.     

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.