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 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.     

H-Y binding in the gonad: inhibition by a supernatant of the fetal ovary.

H-Y antigen, the product of mammalian testis-determining genes, is released in the free state by testicular cells. Molecules of free H-Y antigen are bound in vitro by dispersed cells of the adult ovary. The binding reaction is inhibited by specific H-Y antibody. It is also inhibited by a diffusible factor of the newly differentiated fetal ovary. These observations favor the view that testicular organogenesis depend upon dissemination and binding of H-Y molecules by cells of the undifferentiated gonad (XY or XX, both having H-Y receptors), and raise the question of whether ovarian organogenesis may be promoted by a "female" molecule corresponding to H-Y of the male.     

Sex differentiation in mammals and tempo of growth: probabilities vs. switches

In the conventional model of sex differentiation in placental mammals, a switch is envisaged to steer the indifferent gonad into the path of either testicular or ovarian development. The immediate cause of the switch is thought to be the presence or absence of Sertoli cells, which in turn is controlled by the presence or absence of the testis-determining factor on the Y chromosome (TDF in humans, Tdy in mice). Quantitative investigations indicate, however, that the rate of growth of XY gonads is faster than that of XX gonads before the formation of Sertoli cells, and furthermore, that XY embryos develop faster than XX embryos long before the formation of gonadal ridges. Since the genetic constitution of the sex chromosomes appears to manifest itself from the earliest embryonic stages onwards, the concept of indifferent gonads being switched into alternate pathways becomes inappropriate. A model is proposed in which gonadal differentiation depends on developmental thresholds: the formation of Sertoli cells needs to occur by a particular stage in time in a sufficiently developed gonad, failing which the gonad will enter the ovarian pathway. While TDF is the principal factor enhancing the rate of gonadal growth, other factors which influence development rates can modulate the probability of a gonad becoming either a testis or an ovary.     

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

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

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.     

Testis-determining H-Y antigen in XO males of the mole-vole (Ellobius lutescens)

The XO sex chromosome constitution has been found in both sexes of the mole-vole (Ellobius lutescens) belonging to the rodent family Microtinae. This enigmatic species has apparently been enduring a 50% zygotic lethality. The current serological study revealed the presence in XO males and the absence from XO females of H-Y (histocompatibility Y) antigen. In all the mammalian species studied thus far, the expression of H-Y antigen strictly coincided with the presence of testicular tissue and not necessarily with the presence of the Y chromosome. The testis-organizing function of the H-Y gene appears to have been confirmed.In the mole-vole, X linkage of the testis-organizing H-Y gene is favored over its autosomal inheritance. Only X linkage of the H-Y gene creates a compelling evolutionary need to change the female sex chromosome constitution from XX to XO, and to abandon the dosage compensation by an X inactivation mechanism, so that the nonproductive X^H-YX zygote can be eliminated as an embryonic lethal. With regard to the electrophoretic mobilities of three X-linked marker enzymes, however, a genetic difference between the male-specific X^H-Y and the female-specific X was not detected. This might reflect a relatively recent speciation.     

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.     

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.     

Testicular cells lysostripped of H-Y antigen organize ovarian follicle-like aggregates.

A suspension of free testicular cells were obtained by mild trypsin treatment from newborn BALB/c testes, and their plasma membrane H-Y antigen sites were blocked (lysostripped) by an excess of H-Y antibody of proven specificity and potency (45 min in ice). Upon 16 h of the Moscona-type rotation culture, these treated testicular cells yielded primarily spherical aggregates, more than half of which demonstrated a strong resemblance to ovarian follicles. The resemblance was particularly striking between the smallest testicular folliculoids and primordial ovarian follicles that abound in the newborn female gonad. Under the same condition, control serum-treated testicular cells primarily yielded cylindrical tubular structures that can be very long. Over a critical range, concentrations of H-Y antibody apparently influenced the frequency of testicular folliculoid formation. The above directly supports the proposed testis-organizing function of H-Y antigen and is certainly compatible with the genetic situation encountered in the wood lemming (Myopus schisticolor), that in the functional absence of H-Y antigen, XY gonadal cells readily organize an ovary.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4+/+ and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

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.     

Aberrant testicular differentiation in 46,XY gonadal dysgenesis: Morphology,endocrinology, serology

Summary In an infant with gonadal dysgenesis and somatic anomalies, the internal and external genitalia were female but the gonads contained tubular structures suggesting male differentiation. The karyotype was 46,XY with no evidence of structural aberration or mosaicism. Hormonal metabolism and H-Y antigen expression were assayed in cultured gonadal cells. Although unable to synthesize testosterone, the cultured cells were able to convert it to dihydrotestosterone. H-Y antigen was present, perhaps at a level lower than that in cells from normal XY males. Our observations indicate that a modicum of testicular organogenesis may precede the involution that results in a streak gonad in some cases of gonadal dysgenesis.     

H-Y Antigen Negative Germ Cells in Gonadal Sex Organization in vitro

Dissociation-reorganization experiments were done with gonadal cells of newborn rats. Rotation cultures consisted of mixtures of somatic and germ cells of opposite sex. Somatic cells, ovarian or testicular, determined a female or male type respectively, of gonadal histomorphic organization. Germ cells did not affect the type of organization of somatic cells. Accordingly, suspensions containing somatic cells of one sex together with germ cells of both sexes, reorganized in rotation culture, into either a) follicles containing XX or XY germ cells, or b) tubules containing XX or XY or both types of germ cells. These results give morphological evidence for heterosexual germ-somatic cells interactions. Based on morphological and H-Y antigen studies, failure of germ cells to bind and express H-Y antigen is considered as a possible factor for this failure of germ cells to affect gonadal sex.     

46,XY gonadal dysgenesis: Isoncogenesis related to H-Y phenotype or breast development?

Summary Among women with 46,XY gonadal dysgenesis, there is a high incidence of gonadal tumors. Because of evidence of a connection between occurrence of those tumors, H-Y phenotype, and breast development, we surveyed 55 cases of 46,X gonadal dysgenesis and 12 related cases involving chromosomal and/or skeletal abnormalities. Our survey, including three new cases presented here, indicates that H-Y phenotype but not breast development may be related to the development of the gonadoblastoma-dysgerminoma. Thus among women with 46,XY gonadal dysgenesis, there are H-Y^– and H-Y⁺ classes, but gonadal tumors are found almost exclusively in the H-Y⁺ class. Yet one of our patients may represent an exception to the association of H-Y⁺ phenotype and gonadal tumors in this syndrome.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

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.     

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.     

Ovarian development in 46,XY gonadal dysgenesis

Summary In human the XY ovary is degenerative, there being scant evidence of persistence of that organ beyond the perinatal period. Here we describe indications of functional ovarian tissue in a 17-year-old female with male karyotype, H-Y⁺ cellular phenotype, and some signs of the Turner syndrome. Her gonads were removed after the onset of secondary amenorrhea. Histological examination revealed a degenerative right ovary devoid of germ cells and follicles, and a left streak gonad. There was no trace of testicular development in either side.     

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.