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.     

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.     

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

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.     

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.     

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.     

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.     

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.     

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.     

Abnormal ovarian morphogenesis in Drosophila melanogaster after injection of embryos with conditioned media from the Daudi cell line

Summary Drosophila melanogaster embryos were injected before the blastoderm stage with conditioned media from several male Burkitt's lymphoma human cell lines and the Daudi cell line. Such injections do not have any effect on the male genital apparatus or on the female tract. The Daudi conditioned medium modifies the ovarian morphogenesis of the flies and the rudimentary ovaries obtained look like nymphal gonads. Moreover, they have a drastically reduced number of germ cells. The ovaries that looked functional contain numerous necrotic germ cells and the mean number of ovarioles per fly is significantly smaller than that of the controls. The abnormalities observed resemble the results of experimental and genetic lack of germ cells. They disappear at very high dilution (1×10^–6).     

The Identification of Human H-Y Antigen and Testicular Transformation Induced by its Interaction with the Receptor Site of Bovine Fetal Ovarian Cells

beta 2m(-), HLA (-) Daudi human male Burkitt lymphoma cells excreted a group of protein subunits that shared three distinctive characteristics; their conspicuously longer half-lives compared to more hydrophilic Daudi excreted proteins, their tendency to form progressively larger polymers by means of interchain disulfide bridges, and the extreme hydrophobicity of these polymers. The plasma membrane of extragonadal somatic cells absorbed 1.2 to 2.8% of these hydrophobic proteins. The unoccupied H-Y receptor sites residing on the plasma membrane of bovine fetal ovarian cells, on the other hand, selectively absorbed polymers of 18,000 mol. wt. subunits, and this antigen-receptor interaction, if allowed to continue for five days, induced the formation of tunica albuginea and seminiferous tubules in bovine XX embryonic indifferent gonads. In this manner, human H-Y antigen excreted by Daudi cells has functionally been identified as a series of polymers derived from 18,000 mol. wt. subunits. While, the H-Y antigenic determinants were retained even by the largest polymeric form that became irreversibly water insoluble, the receptor binding activity was shown only by 36.8% of the available polymeric forms of 18,000 mol. wt. subunits, at the most. Nevertheless, once bound to the receptor site, these polymers were rapidly reduced to the monomeric form on the plasma membrane of bovine fetal ovarian cells. Accordingly, the 18,000 mol. wt. monomer might actually represent the functional form of H-Y antigen.     

In vitro rearing of Edovum puttleri,an egg parasitoid of the Colorado potato beetle,on artificial diets: Effects of insect cell line‐conditioned medium

Effects of conditioned media prepared from cell lines derived from 11 insect species (six families, three orders) on the in vitro growth and development of the egg parasitoid Edovum puttleri were investigated. The parasitoid exhibited significantly different responses to the various insect cell line‐conditioned media that were incorporated into the artificial diets. When cell lines were derived from embryos, higher percentages of 3rd instars and prepupae were observed than when cell lines were derived from fat body or ovaries. Medium conditioned with cell line IPLB‐CPB2 derived from the embryos of the Colorado potato beetle produced the best result. Preconditioning time was important. In general, 5 days of preconditioning appeared to be optimal. The growth‐ and development‐promoting effect may have resulted from growth factors or growth‐supporting factors produced/ released by the insect cell lines into the culture medium. Upon storage at 0–4°C for 7–14 days, the ability of cell line‐conditioned medium to promote development beyond the second instar was greatly reduced (approximately 10–55%). Our studies demonstrated that to support the in vitro growth and development of E. puttleri, insect hemolymph could be successfully replaced with insect cell line‐conditioned medium. These findings should facilitate the development of a cost‐effective mass‐rearing system for E. puttleri and/or other parasitoids. Arch. Insect Biochem. Physiol. 40:173–182, 1999. Published 1999 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

A protein from Daudi cell line conditioned medium induces ovarian dysgenesis in Drosophila melanogaster.

From a medium in which Daudi cells had been grown, we isolated by HPLC a protein that caused ovarian abnormalities in adult females of Drosophila melanogaster when injected into preblastoderm embryos. This protein, whose apparent M(r) is between 30,000 and 50,000, was found to be a moderately polar compound which is heat stable and whose activity is destroyed by acidification. The protein is characteristic of medium conditioned from Daudi cells.     

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.     

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 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.     

Does H-Y antigen induce the heterogametic ovary?

To determine whether phylogenetically conservative H-Y antigen plays any part in gonadal differentiation among the nonmammalian vertebrates, we studied expression and binding of H-Y in the frog, Xenopus laevis. Soluble H-Y obtained from mouse testis and soluble H-W from chicken ovary bound specifically to cells of the ZZ testis from normal Xenopus males. In addition, H-Y (H-W) appeared selectively in the ovaries of ZZ genetic males that had been induced to become functional females by exposure to estradiol. Our observations suggest that H-Y (H-W) antigen may be involved in differentiation of the ZW ovary, and also that synthesis of H-Y may be regulated by sex steroids in the primitive $\text{ZW}\text{ZZ}$ species.     

Conservatism of the H-Y/H-W receptor

Summary Soluble H-Y antigen is taken up by cells of the homogametic gonad of cattle, dog, chicken and South African clawed frog. After in vitro exposure to mouse testis supernatant or male fetal calf serum, XX ovary cells or ZZ testis cells, which are normally H-Y^-, acquire the H-Y⁺ (H-W⁺) phenotype and absorb mouse H-Y antibody in standard serological assays. In addition, H-Y antigens of the different species can compete for attachment to target cells of a single species. In a new competitive binding radioassay, uptake of tritiated human H-Y is blocked in XX bovine fetal ovarian cells exposed to non-labeled H-Y of mouse or fetal bull. Because H-Y antigens of the different species are cross-reactive serologically, positive reaction of H-Y from one species with gonadal cells of another signifies structural conservatism of the H-Y/H-W gonadal receptor. It follows that establishment of the H-Y/H-W-receptor complex is a common and critical early event in primary sex differentiation of the vertebrates, directing the initially indifferent embryonic gonad towards the heterogametic mode, which may be testicular or ovarian, depending on the species.     

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.     

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.