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.     

Immunoprecipitation of human H-Y antigen

Summary In the absence of beta-2-microglobulin and MHC-determined cell surface antigens, cultured cells of the Burkitt lymphoma, Daudi, secrete testis-inducing H-Y antigen into the surrounding medium. We have precipitated Daudi-secreted H-Y antigen by two methods, one using mouse H-Y antibody and goat anti-mouse Ig, and the other using mouse H-Y antibody and Sepharose beads coated with protein A. The estimated molecular weight of the specific immunoprecipitate was 15,000–18,000 Daltons.     

Association of HL-A antigens andβ 2-microglobulin at the cellular and molecular level

Surfaces of cultured human lymphoid cells RPMI 1788, RPMI 4098, RPMI 8866, Raji, and WI-L2 were found to contain bothβ ₂-microglobulin (β ₂-μ) and HL-A determinants when tested by direct complement-dependent cytotoxicity andquantitative absorption with different cytotoxic antiβ ₂-μ antisera and specific HL-A alloantisera. The same antigenic specificities were found in 3M KCl extracts of these cultured cells with a sensitiveβ ₂-μ radioimmunoassay and an HL-A antigen blocking assay. Daudi cells provided a contrast, since noβ ₂-μ or HL-A determinants were found on their surfaces or in 3 M KCl extracts prepared from them. Results from specific antibody blocking tests suggest a close association betweenβ ₂-μ and HL-A determinants on plasma membranes of cultured human lymphoid cells. A solid state immunoadsorbent containing antiβ ₂-μ antibodies effectively removed all detectable HL-A antigenic activity from some 3M KCl extracts of cultured human lymphoid cells as well as from some sera. Adsorption of HL-A antigens to these immunoadsorbents was specific since it was blocked only by prior addition ofβ ₂-μ. Once on the antiβ ₂-μ immunoadsorbents, HL-A antigens still reacted specifically with HL-A alloantibodies in quantitative absorption experiments. HL-A antigens andβ ₂-μ could be eluted from antiβ ₂-μ immunoadsorbents with a variety of chaotropic reagents and detergents, but thus far potassium bromide and sodium dodecyl sulfate (SDS) appear to be the most effective. SDS-PAGE of these eluates indicated that HL-A antigens were considerably purified by adsorption to antiβ ₂-μ immunoadsorbents and that two major molecular size fragments were distinguishable, i.e., ∼33,000 for HL-A and ∼ 12,000 forβ ₂-μ.     

Presence of an abnormal β 2-microglobulin mRNA in Daudi cells: Induction by interferon

Human α and β interferons increase the amount of class I human histocompatibility messenger RNA HLA-A, B, C and β₂-microglobulin in most human cells studied to date. This report concerns the effect of interferons on the Burkitt lymphoma-derived cell line Daudi, which does not express HLA-A, B, C antigens or β₂-microglobulin on its membrane. HLA-A, B, C messenger RNA present in Daudi cells is increased by both α and β interferons. Furthermore, we have shown that although it was not possible to detect mature β₂-microglobulin protein in the cytoplasm or on the cell membrane of Daudi cells, a poly A⁺ messenger RNA is present in Daudi cells, which hybridizes with a cDNA clone specific for human β²-microglobulin. This abnormal messenger RNA is, however, increased normally by interferon. These effects were also observed with human interferon β on a variant of Daudi cells characterized by a markedly reduced sensitivity to anti-proliferative and anti-cellular effects of human interferon α.[/p]     

Induction of HLA expression in Daudi cells after cell fusion

The Daudi cell line, established from a Burkitt lymphoma, has recently been found to be HLA- andΒ ₂-microglobulin-negative, although it expresses B lymphocyte alloantigens. This report is concerned with the reexpression of HLA-A10, B38, and B17 on the Daudi cell, after cell fusion with another human cell line (Raji) or with mouse fibroblasts. In the latter fusion, the same HLA specificities are re-expressed, but not humanΒ ₂-microglobulin while mouseΒ ₂-microglobulin andH-2 could be detected. No such reexpression was observed when Daudi was fused with the F9 mouse teratocarcinoma, which lacks mouseΒ ₂-m andH-2. No HLA activity (alloantigenic and xenogenic activity) was detected in the membrane or cytoplasm of Daudi, using salt extraction and sonication. Therefore we postulate thatΒ ₂-microglobulin could be necessary for the expression and possible synthesis of the HLA antigen.     

In vitro synthesis of beta2-microglobulin by human fetal tissues.

Human fetal tissues were cultured in the presence of ¹⁴C-labeled amino acids and the culture fluids analyzed by radioimmunoelectrophoresis for the presence of radioactive β₂-microglobulin. Synthesis of the protein was demonstrated in all the tissues studied. Using the Ouchterlony method, β₂-microglobulin was also detected in culture media from serially transferred cell cultures of fetal tissues and established fetal cell strains.     

Rat testicular germ cells and sertoli cells release different types of bioactive transforming growth factor beta in vitro

Several in vivo studies have reported the presence of immunoreactive transforming growth factor-β's (TGF-β's) in testicular cells at defined stages of their differentiation. The most pronounced changes in TGF-β₁ and TGF-β₂ immunoreactivity occurred during spermatogenesis. In the present study we have investigated whether germ cells and Sertoli cells are able to secrete bioactive TGF-β's in vitro, using the CCl64 mink lung epithelial cell line as bioassay for the measurement of TGF-β. In cellular lysates, TGF-β bioactivity was only observed following heat-treatment, indicating that within these cells TGF-β is present in a latent form. To our surprise, active TGF-β could be detected in the culture supernatant of germ cells and Sertoli cells without prior heat-treatment. This suggests that these cells not only produce and release TGF-β in a latent form, but that they also release a factor which can convert latent TGF-β into its active form. Following heat-activation of these culture supernatant's, total TGF-β bioactivity increased 6- to 9-fold. Spermatocytes are the cell type that releases most bioactive TGF-β during a 24 h culture period, although round and elongated spermatids and Sertoli cells also secrete significant amounts of TGF-β. The biological activity of TGF-β could be inhibited by neutralizing antibodies against TGF-β₁ (spermatocytes and round spermatids) and TGF-β₂ (round and elongating spermatids). TGF-β activity in the Sertoli cell culture supernatant was inhibited slightly by either the TGF-β₁ and TGF-β₂ neutralizing antibody.These in vitro data suggest that germ cells and Sertoli cells release latent TGF-β's. Following secretion, the TGF-β's are converted to a biological active form that can interact with specific TGF-β receptors. These results strengthen the hypothesis that TGF-β's may play a physiological role in germ cell proliferation/differentiation and Sertoli cell function.     

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.     

The absence of β 2-microglobulin in Daudi cells: Active gene but inactive messenger RNA

The Daudi cell line is characterized by an absence of HLA antigen on its surface. This has been attributed to a lack of beta 2-microglobulin (beta 2m) while the heavy chain of HLA is present intracellularly. Karyotype analysis of Daudi cells has shown a deletion involving one of the beta 2-microglobulin alleles. It was generally believed that the absence of beta 2-microglobulin in that cell line resulted from an absence of expression of the remaining gene. We report here the unexpected finding of a normal amount of beta 2-microglobulin messenger RNA in Daudi cells. This was demonstrated by "Northern blot" hybridization with cDNA plasmid clones as a probe. This mRNA, however, when purified by hybridization-selection with beta 2-microglobulin plasmid DNA, is unable to function as messenger in protein synthesis and is therefore an inactive mRNA. The finding of a translationally inactive beta 2-microglobulin mRNA provides a new explanation for the absence of beta 2-microglobulin and therefore of HLA antigens in Daudi cells.     

In situ nick translation of metaphase chromosomes with biotin-labeled d-UTP

Summary Conditioned culture medium from Daudi cells was used as a source of soluble H-Y antigen. Concentrated culture medium was labeled with ¹²⁵I and then fractionated by gel filtration. Column fractions were assayed for the presence of H-Y antigen by urease-ELISA. H-Y antigen-containing fractions were then pooled and subjected to an improved immunoprecipitation protocol. Three predominant H-Y antigenic proteins were identified with estimated molecular weights of above 200,000, 50,000, and 20,000.     

Isolation and characteristics of a rabbit β2-microglobulin: Comparison with human β2-microglobulin

A low molecular weight β₂-globulin has been isolated from urine of rabbits treated with sodium chromate. Several findings indicate that this protein is the rabbit counterpart of human β₂-microglobulin which is known to occur linked to the major histocompatibility (HL-A) antigens of man. The rabbit β₂-globulin and human β₂-microglobulin are very similar in amino acid composition, molecular size, molecular weight, electrophoretic mobility, and also in the presence of apparently analogous disulfide loops. More than half of the amino acids in the two proteins seem to be present in identical numbers. The urinary excretion of both proteins is increased by agents which induce renal tubular damage.     

Possible relationship between H-Y antigen and female gonadogenesis in the Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Summary

The Daudi cell line, a male human line from Burkitt's lymphoma, possesses the peculiarity of releasing H-Y antigen into its culture medium. Daudi-conditioned medium was injected into Colorado potato beetles either during the blastoderm stage, when individualization of the pole cells occurs, or later, during gonadal differentiation. Ovarian and testicular sections examined at hatching showed that only ovarian differentiation was affected by the Daudi conditioned medium, which, irrespective of the day of injection, reduced the number of the terminal filaments of the future lateral ovarioles. Furthermore, in some cases sexual differentiation was blocked altogether. When H-Y antigen was precipitated from the conditioned medium with specific H-Y antisera, the effectiveness of Daudi-conditioned medium was partially destroyed. These results suggest that mammalian H-Y antigen inhibits morphogenetic events leading to ovarian differentiation in the Colorado potato beetle.     

The HLA-dependent expression of testis- organizing H-Y antigen by human male cells.

The proposal that the stable expression of organogenesis-directing plasma membrane antigens, such as testis-organizing H-Y antigen, requires beta2-microglobulin-MHC antigen dimers as anchorage sites was tested on Daudi human Burkitt lymphoma cells [46, XY, 15q-, 14q+, beta2-m(-), HLA(-)]. The H-Y antigen level of Daudi was only 20% of that of Raji and Ramos, two human male pseudodiploid Burkitt lymphoma lines that were beta2-m(+), HLA(+). When Daudi is hybridized with beta2-m(+), HLA(+) cell lines, beta2-microglobulin, supplied by the latter, is known to restore the expression of Daudi HLA antigens A10 and BW17. Such restoration of HLA antigen expression markedly elevated H-Y antigen levels in those somatic hybrids. Thus the H-Y antigen level of the Daudi x Raji 8A (male X male) hybrid became equal to that of TetraRaji--the colcemide-induced Raji tetraploid line. Two independently derived Daudi x Hela D98 (male x female) hybrids, DAD 1 and DAD 10, demonstrated even higher H-Y antigen levels comparable to that of normal male peripheral blood lymphocytes.     

Cellular cooperation in mitogen-induced human leukocyte inhibitory factor (LIF) synthesis.

Interactions between human T and B lymphocytes and between lymphocyte subpopulations and accessory cells in lymphokine synthesis were investigated. The cells were stimulated with leukoagglutinin (LA), concanavalin A (Con A), protein A (prot A) and anti-β₂-microglobulin (anti-β₂m). The presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose-migration method. The results indicated that monocytes augmented LIF synthesis of T cells but suppressed that of B cells. Monocyte-helper effect was mediated by both cell-cell contact and soluble factors. In addition, T lymphocytes were found to augment B-cell LIF production. B lymphocytes enhanced Con A- but suppressed LA-induced LIF production by T cells. T-cell/B-cell collaboration was based on a direct cell-cell contact and no soluble factors were found.     

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.     

Enhancement of tumor cell susceptibility to lymphokine-activated killer cells by treatment with the streptococcal preparation OK432

We investigated whether tumor cell lysis by LAK cells was augmented by treatment with OK432in vitro. NK and LAK activity against K562 cells was not enhanced by their treatment with OK432. In contrast, the susceptibility of OK432-treated Daudi and KATO-III cells to lysis by LAK cells was enhanced. Succinate dehydrogenase activity and RNA synthesis were impaired in Daudi and KATO-III cells by treatment with OK432, and moreover the expression of HLA Class I antigen and ₂-microglobulin was inhibited in OK432-treated KATO-III cells. Thus, it is suggested that the enhancement of the susceptibility of OK432-treated tumor cells with regard to succinate dehydrogenase activity, RNA synthesis, and HLA Class I antigen expression.     

Elaboration of leukocyte migration inhibitory factor by human lymphocyte subpopulations stimulated with mitogens.

This paper describes experiments to determine whether human lymphocyte sub-populations stimulated with a variety of mitogens, leucoagglutinin (LA), concanavalin A (con A), pokeweed mitogen (PWM), protein A (prot A), and anti-β₂-microglobulin (anti-β₂m), synthesize lymphokines. T and B lymphocytes as well as unseparated mononuclear cells were stimulated with the mitogens, and the presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by an agarose migration method. Culture supernatants stimulated with LA or prot A were also fractionated on Sephadex G-100 columns, and LIF-containing fractions were tested for heat stability and the effect of monosaccharides. The results indicated that LA and con A caused LIF synthesis only in T-cell populations, while PWM stimulated both T and B lymphocytes and prot A and anti-β₂mm were B-cell stimulants. Furthermore, LIF from LA-and prot-A-stimulated cultures behaved similarly upon physicochemical characterization.     

Delayed-type hypersensitivity responses to H-Y: Characterization and mapping ofIr genes

This paper examines the delayed-type hypersensitivity (DTH) response to male (H-Y) antigen(s). Female mice of theH?2 ^b haplotype developed delayed footpad reaction to syngeneic or allogenic male thymus and spleen cells after priming with syngeneic male thymus and spleen cells. The reaction peaks at 24 h, has classical DTH histology and is specific to H-Y antigen as it is not elicited with female cells. Cell transfer studies show that donor/recipient matching at theI?B ^b subregion is necessary for sucessful transfer of DTH and that the effective primed population is Thy-1⁺, Lyt-1⁺, 2^?. DTH response to H-Y antigen appears to be confined to mice of theH?2 ^b haplotype. There appears to be a lack of associative recognition between H-Y antigen and MHC-coded determinants in the effector phase of DTH, and macrophage processing of H-Y seems likely, since nonresponder haplotypes can elicit the DTH response. Studies withH?2 ^b recombinant mouse strains indicate that the dominantIr gene is located in theI?B region. Female F₁ hybrid mice derived from matings of strains not involvingH?2 ^b haplotype failed to develop DTH to H-Y. In summary, these data imply that a complete correlation exists between DTH to H-Y and the ability to reject male skin graft, suggesting that the effector mechanisms of skin-graft rejection may closely involve DTH cells.     

Luminescence catalyst immunoassay of β2-microglobulin with haemin as a chemically amplifiable label

Luminescence catalyst immunoassay, a nonradioactive and nonenzymatic immunoassay, has been applied to the determination of β₂-microglobulin (β₂-MG). The method is characterized by high sensitivity and simple handling, since the analytical method employs a catalytically amplifiable label and solid-phase sandwich technique, respectively. In the first stage, an antibody-immobilized plate is reacted with the analyte (β₂-MG). In the second stage, the bound β₂-MG undergoes successive binding with haemin-labelled antibody. In the last stage, the plate is placed in the luminol-H₂O₂ system to generate luminescence. A calibration curve with a β₂-MG concentration at the midpoint of 100 ng ml^?1 was obtained. The 10 and 90% response levels in the curve are 10 and 1000 ng ml^?1.     

Heterogeneity of β2-Microglobulin in Human Urine

Purification and characterization of β₂-microglobulin from human urine was performed. The yield was 30.1%, and 150.4 mg of β₂-microglobulin was obtained. The final preparation of β₂-microglobulin obtained showed three bands on disc gel electrophoresis at pH 9.5, and all of them have immunological activity. However, these three bands migrated as a single band on disc gel electrophoresis at pH 4.3. It is concluded that the three bands observed on disc gel electrophoresis at pH 9.5 were charge isomers. The isoelectric points of isomers were determined by isotachophoresis and two of them were 5.4 and 5.9 respectively, while the other one was not determined.