Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

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.     

Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia

To clarify the importance of endogenous estrogens during sex differentiation in a teleost fish, the Nile tilapia, we examined the target events for endogenous estrogens and their role during gonadal sex differentiation. The expression of CYP19a (P450arom) precedes any morphological gonadal sex differentiation. Further to these findings, the treatment of XX fry with non-steroidal aromatase inhibitor (AI), Fadrozole, from seven to 14 days after hatching caused complete sex reversal to functional males. The XX sex reversal induced by AI was rescued completely with simultaneous estrogen treatment. We also found that XY fry treated with estrogen, before the appearance of morphological sex differences, caused complete sex reversal from males to females. Taken together, these results suggest that endogenous estrogens are required for ovarian differentiation. To identify the down-stream gene products of estrogen during ovarian differentiation, we performed subtractive hybridization using mRNA derived from normal and estrogen treated XY gonads. Two out of ten gene products were expressed in germ cells, whereas the others were expressed in somatic 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.     

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.     

Sox9b/sox9a2-EGFP transgenic medaka reveals the morphological reorganization of the gonads and a common precursor of both the female and male supporting cells

We have established an enhanced green fluorescent protein (EGFP) transgenic medaka line that mimics the expression of sox9b/sox9a2 to analyze the morphological reorganization of the gonads and characterize the sox9b-expressing cells during gonadal formation in this fish. After the germ cells have migrated into the gonadal areas, a cluster of EGFP-expressing cells in the single gonadal primordium was found to be separated by the somatic cells along the rostrocaudal axis and form the bilateral lobes. We observed in these transgenic fish that EGFP expression persists only in the somatic cells directly surrounding the germ cells. As sex differentiation proceeds, dmrt1 and foxl2 begin to be expressed in the EGFP-expressing cells in the XY and the XX gonads, respectively. This indicates that the sox9b-expressing cells reorganize into two lobes of the gonad and then differentiate into Sertoli or granulosa cells, as common precursors of the supporting cells. Hence, our sox9b-EGFP medaka system will be useful in future studies of gonadal development.     

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.     

Sex inversion as a model for the study of sex determination in vertebrates

As a consequence of genetic sex determination, the indifferent gonadal blastema normally becomes either a testis or an ovary. This applies to mammals and to the majority of non-mammalian vertebrates. With the exception of placental mammals, however, partial or complete sex inversion can be induced in one sex by sexual steroid hormones of the opposite sex during a sensitive period of gonadogenesis. There is evidence that also during normal gonadogenesis in these species, in the XY/XX mechanism of sex determination testicular differentiation is induced by androgens, and in the ZZ/ZW mechanism, ovarian differentiation by oestrogens. In either case, the hormones may act via serological H-Y antigen as a morphogenetic factor. In contrast, in placental mammals including man, primary gonadal differentiation is independent of sexual steroid hormones, and factors directing differential gonadal development have not yet been conclusively identified. However, various mutations at the chromosome or gene level, resulting respectively in sex inversion or intersexuality, have provided clues as to some genes involved and their possible nature. In this context also, serological H-Y antigen is discussed as a possible factor acting on primordial gonadal cells and inducing differential growth or morphogenesis or both. The data available at present allow a tentative outline of the genetics of sex determination in placental mammals.     

Cell-autonomous and inductive signals can determine the sex of the germ line of drosophila by regulating the gene Sxl

To investigate the mechanism of sex determination in the germ line, we analyzed the fate of XY germ cells in ovaries, and the fate of XX germ cells in testes. In ovaries, germ cells developed according to their X:A ratio, i.e., XX cells underwent oogenesis, XY cells formed spermatocytes. In testes, however, XY and XX germ cells entered the spermatogenic pathway. Thus, to determine their sex, the germ cells of Drosophila have cell-autonomous genetic information, and XX cells respond to inductive signals of the soma. Results obtained with amorphic and constitutive mutations of Sxl show that both the genetic and the somatic signals act through Sxl to achieve sex determination in germ cells.     

H-Y Antigen and Sex-Determination in Turtles

H-Y antigen was investigated in 14 turtle species belonging to five different families. In 13 species the female was typed as H-Y antigen positive, only in Chinemys reevesi was the male H-Y antigen positive. Since in all vertebrate species studied till now, the expression of H-Y antigen is strictly correlated with the heterogametic sex, it can be assumed that in turtles both types of sex determining mechanisms are realized, namely the ZZ/ZW-and the XX/XY mechanism; both mechanisms are realized in species belonging to one and the same turtle family. However, most turtle species seem to be endowed with a ZZ/ZW sex determining mechanism.     

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.     

Meiotic germ cells antagonize mesonephric cell migration and testis cord formation in mouse gonads

The developmental fate of primordial germ cells in the mammalian gonad depends on their environment. In the XY gonad, Sry induces a cascade of molecular and cellular events leading to the organization of testis cords. Germ cells are sequestered inside testis cords by 12.5 dpc where they arrest in mitosis. If the testis pathway is not initiated, germ cells spontaneously enter meiosis by 13.5 dpc, and the gonad follows the ovarian fate. We have previously shown that some testis-specific events, such as mesonephric cell migration, can be experimentally induced into XX gonads prior to 12.5 dpc. However, after that time, XX gonads are resistant to the induction of cell migration. In current experiments, we provide evidence that this effect is dependent on XX germ cells rather than on XX somatic cells. We show that, although mesonephric cell migration cannot be induced into normal XX gonads at 14.5 dpc, it can be induced into XX gonads depleted of germ cells. We also show that when 14.5 dpc XX somatic cells are recombined with XY somatic cells, testis cord structures form normally; however, when XX germ cells are recombined with XY somatic cells, cord structures are disrupted. Sandwich culture experiments suggest that the inhibitory effect of XX germ cells is mediated through short-range interactions rather than through a long-range diffusible factor. The developmental stage at which XX germ cells show a disruptive effect on the male pathway is the stage at which meiosis is normally initiated, based on the immunodetection of meiotic markers. We suggest that at the stage when germ cells commit to meiosis, they reinforce ovarian fate by antagonizing the testis pathway.     

Analysis of sex differences in EGC imprinting

Sex-specific differences are apparent in the methylation patterns of H19 and Igf2 imprinted genes in embryonic germ cells (EGCs) derived from 11.5 or 12.5 days post coitum (dpc) primordial germ cells (PGCs). Here we studied whether these differences are associated either with the sex chromosome constitution of the EGCs or with the sex of the genital ridge (testis versus ovary) from which the PGCs were isolated. For this purpose we derived pluripotent EGC lines from sex-reversed embryos, either XY embryos deleted for Sry (XY(Tdym1)) or XX embryos carrying an Sry transgene. Southern blotting of the EGC DNA was used to analyze the differentially methylated regions of Igf2 and H19. The analysis revealed that both genes were more methylated in EGCs with an XY sex chromosome constitution than in those with an XX sex chromosome constitution, irrespective of the phenotypic sex of the genital ridge from which the EGCs had been derived. We conclude that the sex-specific methylation is intrinsic and cell-autonomous, and is not due to any influence of the genital ridge somatic cells upon the PGCs.     

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

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

The number of x chromosomes causes sex differences in adiposity in mice

Sexual dimorphism in body weight, fat distribution, and metabolic disease has been attributed largely to differential effects of male and female gonadal hormones. Here, we report that the number of X chromosomes within cells also contributes to these sex differences. We employed a unique mouse model, known as the "four core genotypes," to distinguish between effects of gonadal sex (testes or ovaries) and sex chromosomes (XX or XY). With this model, we produced gonadal male and female mice carrying XX or XY sex chromosome complements. Mice were gonadectomized to remove the acute effects of gonadal hormones and to uncover effects of sex chromosome complement on obesity. Mice with XX sex chromosomes (relative to XY), regardless of their type of gonad, had up to 2-fold increased adiposity and greater food intake during daylight hours, when mice are normally inactive. Mice with two X chromosomes also had accelerated weight gain on a high fat diet and developed fatty liver and elevated lipid and insulin levels. Further genetic studies with mice carrying XO and XXY chromosome complements revealed that the differences between XX and XY mice are attributable to dosage of the X chromosome, rather than effects of the Y chromosome. A subset of genes that escape X chromosome inactivation exhibited higher expression levels in adipose tissue and liver of XX compared to XY mice, and may contribute to the sex differences in obesity. Overall, our study is the first to identify sex chromosome complement, a factor distinguishing all male and female cells, as a cause of sex differences in obesity and metabolism.     

小鼠胚胎干细胞向生殖细胞分化的研究

小鼠胚胎干细胞(ESC)在体外可以分化为多种细胞类型,其中包括各阶段的生殖细胞,甚至精细胞和成熟卵母细胞。ESC向生殖细胞分化的效率受到包括生长因子、激素和体细胞等多种因素的影响,在体外形成的是雌性配子还是雄性配子与ESC是XX型还是XY型没有必然联系。简要综述了小鼠生殖细胞在体内外的分化发育、性别决定和增殖等,并总结和展望了ESC向生殖细胞分化研究面临的问题和应用前景。     

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.