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.     

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.     

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.     

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.     

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 specific interactions of Sertoli-cell-secreted proteins with antibodies directed against H-Y antigen

Radiolabeled proteins secreted into the medium by rat Sertoli cells in primary culture have been examined for specific interactions with polyclonal and monoclonal antibodies directed against serologically detectable H-Y antigen(s). None of the proteins secreted by Sertoli cells reacted specifically with H-Y antibodies, as determined with immunoprecipitation procedures and immunoabsorbent affinity chromatography, followed by SDS gel electrophoresis. Radioactivity profiles of proteins obtained after reaction with H-Y antibodies were similar to those observed after treatment with nonimmune sera or with irrelevant antibodies. We obtained comparable findings with proteins secreted by the mouse cell line TM4, which is of presumptive Sertoli cell origin, and with proteins present in ram rete testis fluid. These and other findings presented do not support the contention that Sertoli cells secrete a protein having the properties of serologically detectable H-Y antigen as previously described.     

A quantitative radioimmunoassay for membranous and soluble H-Y antigen typing

Summary Two sensitive and quantitative methods for membranous or soluble H-Y antigen typing using rat anti-H-Y immune sera and ¹²⁵I labelled protein A were carried out.These techniques were used to study H-Y antigen expression in human cell lines, and to refine the hypothesis that ( ₂m) serves as an anchorage point for H-Y antigen.     

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.     

Identification of a male-specific histocompatibility protein on preimplantation porcine embryos

An indirect immunofluorescence assay was used to detect the presence of male-specific protein(s) on various stages of preimplantation porcine embryos. Embryos were collected at slaughter from the reproductive tracts of day?2.5, ?4, ?5, ?6, and ?8 (day 0 = first day of estrus) sows and gilts. Embryos were placed in medium containing an anti-male primary antibody, washed, and transferred to culture drops containing a fluorescein isothiocyanate (FITC)-labeled secondary antibody. Embryos were classified as either fluorescent (H-Y positive) or nonfluorescent (H-Y negative), transferred to coded drops, and karyotyped to examine sex chromosomes. A total of 91 eight-cell to blastocyst stage embryos were evaluated; of these, 46% were classified as fluorescent and 54% as nonfluorescent. Of readable metaphase spreads (65%) from these embryos, 81% (48 of 59, P < 0.005) were correctly sexed by immunological detection of the male-specific antigen. Although 13 % (2/15)of four-cell embryos evaluated were classified as fluorescent, the accuracy with which embryos at this stage were sexed by detection of H-Y antigen was not different from 50%. Fifty percent of eight-cell embryos were classified as H-Y positive with 78% of embryos correctly sexed. It was concluded that the eight-cell embryo is the earliest stage of development for which there is evidence for expression of H-Y antigen. Detection of the male-specific protein was difficult at the expanded blastocyst stage.     

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.     

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.     

Expression of a male-specific factor on various stages of preimplantation bovine embryos

Four-cell to blastocyst stage bovine embryos were collected from superovulated donors and cultured for 90 min in Ham's F-10 medium (HF-10) containing 10% (V/V) absorbed anti-histocompatibility (H)-Y antiserum. Embryos were then washed 3 times and placed in HF-10 supplemented with 10% (V/V) fluorescein isothiocynate (FITC)-conjugated goat anti-mouse gamma globulin. After an additional wash, embryos were placed in fresh drops of HF-10, individually evaluated at 200 X magnification, and classified as either fluorescent (H-Y-positive) or nonfluorescent (H-Y-negative). Embryos were then placed in drops of HF-10 containing 14% vinblastin and cultured for 4-6 h. Embryos were coded and individually karotyped, and the sex chromosomes were identified. H-Y antigen was detected as early as the eight-cell stage, but not at the four-cell stage. Seventy-nine percent of fluorescent embryos and 89% of nonfluorescent embryos were XY and XX, respectively. Another experiment was carried out in which H-Y antigen was detected on intact inner cell masses (ICM) isolated by immunosurgery from expanded blastocysts that also had been assayed for H-Y antigen. Eighty-eight and 92%, respectively, of ICM classified as fluorescent or nonfluorescent had been scored the same as intact blastocysts. It is concluded from these data that H-Y antigen can be detected on eight-cell to blastocyst stage bovine embryos. There appears to be a localization of detectable antigen in the area of the ICM at the expanded blastocyst stage. Detection of H-Y antigen is an effective, noninvasive method for identification of the sex of preimplantation bovine embryos.     

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.     

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.     

Binding of anti-H-Y monoclonal antibodies to separated X and Y chromosome bearing porcine and bovine sperm

Studies designed to answer the question whether or not H-Y antigen is preferentially expressed on Y chromosome bearing sperm have resulted in conflicting results. This is probably due to the absence of reliable methods for estimating the percentage of X and Y chromosome bearing sperm in fractions, enriched or depleted for H-Y antigen positive sperm. In recent years a reliable method for separating X and Y chromosome bearing sperm has been published. With this method, separation is achieved by using a flow cytometer/cell sorter, which detects differences in DNA content. This technique provided the first opportunity for testing anti-H-Y antibody binding to fractions enriched for X and Y chromosme bearing sperm, directly. A total of 7 anti-H-Y monoclonal antibodies were tested using sorted porcine sperm and in one experiment also sorted bovine sperm. All monoclonal antibodies bound only a fraction of the sperm (20 to 50%). However, no difference in binding to the X and Y sperm enriched fractions was found. Therefore, the present experiments do not yield evidence that H-Y antigen is preferentially expressed in Y chromosome bearing sperm. © 1993 Wiley-Liss, Inc.     

Recovery of intestinal membrane binding sites for K88 E. coli from pig mucosal organ cultures

Summary Putative receptors for K88+ E. coli from piglet intestinal epithelium were released into the organ culture medium and were demonstrated by direct binding with K88+ E. coli through the utilization of an in vitro binding procedure or by immunoprecipitation with K88 antigen.Incorporation of ¹⁴C-glucosamine by newborn to day old and 3-week to 6-week old piglet jejunal and ileal mucosa, in organ culture, occurred throughout the 24 hr culture period. Uptake in both age groups and both areas of the intestine was similar with a somewhat greater incorporation by the older age group.Secretion of ¹⁴C-glucosamine-labeled components into the culture medium was demonstrated by gel filtration of the concentrated medium. Some large molecular weight components eluted in the void volume in excess of 2 x 10⁶ daltons. A second peak of activity was spread from approximately 690K to 25K daltons. All eluted fractions demonstrated binding to K88+ E. coli.Antibodies to purified brush borders from susceptible pigs produced prominent precipitation bands following double diffusion with concentrated organ culture media which confirmed that the organ culture media contained labeled proteins of brush border origin.Immunoprecipitation of the intestinal mucosal organ culture media with K88+ pili and pilus antisera, followed by electrophoresis with SDS and reduced conditions, demonstrated a subunit of approximately 35K daltons.     

Variable spread of X inactivation affecting the expression of different epitopes of the Hya gene product in mouse B-cell clones

Cloned B-cell lines from a female T16H/XSxr mouse in which Tdy expression was suppressed due to X inactivation and from a male X/XSxr mouse, both of the (kxb)F₁ haplotype, were examined for H-Y expression. This was determined both by their ability to act as targets for H-2^k and H-2^b-restricted H-Y-specific cytotoxic T cells and by their ability to stimulate the proliferation of H-2K^k, H-2D^b (class I) and A^b (class II)-restricted T-cell clones. In B-cell clones from the T16H/XSxr mouse, expression of H-Y/D^b exhibited partial X inactivation and only a proportion ( 30%) of the cells were targets for or stimulated H-2D^b-restricted H-Y-specific T cells. In contrast, H-Y eiptopes restricted by H-2^k (H-Y/K^k, H-Y/D^k) and A^b (H-Y/A^b) exhibited no X inactivation. Furthermore, no inactivation of H-Y/D^b, H-Y/A^b, or H-Y^k was observed in the male X/XSxr mouse. These results indicate that the T16H/XSxr female is a mosaic, as a result of the variable spread of X inactivation into the Sxr region. They further suggest that the H-Y antigen recognized in association with H-2^k and H-2D^b class I molecules and A^b class II molecules may be the product of more than one gene.     

H-2 effects on cell-cell interactions in the response to single non-H-2 alloantigens

Individual mice were tested for their proliferative T-cell response to H-Y- and H-3-incompatible stimulator cells in secondary mixed lymphocyte culture. Responders expressing the H-2 ^bhaplotype were restricted in their response to stimulators presenting H-Y and H-3 in the context of H-2 ^b. Lymphocytes from individual B10 females proliferated in response to H-Y presented with I-A ^band D ^b. The ratio of I-A ^b/D ^b-restricted responses varied between individual responders, indicating significant qualitative variation between genetically identical responders. The majority of the proliferative response in all tested mice was restricted to the entire H-2 ^bhaplotype suggesting complementation of I-A ^b- and D ^b-region genes in presenting the H-Y antigen. Similar observations were made in the response of individual B10.LP mice to the H-3 antigen. H-3-specific, proliferating T cells were restricted to H-3 antigen presented with K ^bA^band D ^bwith significant variation between individuals in their preference for H-3 plus K ^bA^band D ^b. In contrast to the response to H-Y, the proliferative response to H-3 plus H-2 ^bcould be accounted for by the summation of the proliferative responses to H-3. plus K ^bA^band D ^b. These observations demonstrate that the proliferative response to non-H-2 H antigens in the context of I-region determinants is not a sine qua non for the T-cell response to these antigens. Further, the individual qualitative and quantitative variation observed with individual genetically identical mice has strong implications for our knowledge of intrastrain variation in immune responsiveness and the characterization of inbred strains for immune responsiveness.     

The male antigen. II. Regulation of the primary and secondary responses to H-Y by H-2 associated genes

The primary response of females of ten inbred mouse strains to the male antigen (H-Y) was investigated by transfer of peritoneal exudate cells (PEC). Three distinct classes of reactivity were seen. Early primary response to H-Y was associated with H-2 haplotypes b and s; intermediate response with H-2 haplotypes, k, d, i, and h; and late or absent response with H-2 haplotypes a and f. The failure of A.CA (H-2^f) females to mount a detectable primary response against syngeneic male cells was not due to the lack of the antigen from A.CA male cells. The ability of (A.CA × B10)F₁ hybrid females to respond to the male antigen demonstrated the dominant nature of the B10 (H-2^b) response and excluded the possibility that A.CA females possess a dominant self-antigen cross-reactive with H-Y. The secondary response of eight inbred strains was investigated; at least three distinct levels of reactivity were apparent. The speed of the secondary response was associated with the various H-2 haplotypes in the same way as the primary response. The implications of differential strain reactivity, background effect, and the association of Ir-1 with response to H-Y are discussed.