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.     

H-Y antigen in X,i(Xq) gonadal dysgenesis: Evidence of X-linked genes in testicular differentiation

Summary Three years ago, we detected H-Y antigen in the white blood cells of a phenotypic female with several of the stigmata of Turner's syndrome, and the mosaic karyotype: 45,X/46,X,i(Xq). We surmised at the time that the isochromosome, i(Xq), may have contained occult Y-chromosome-derived material. We have now confirmed the presence of H-Y in this patient and we have obtained evidence for the presence of H-Y in four of five other similar patients, all of whom are notable for carrying at least a single cell line with the karyotype 46,X,i(Xq). Although we cannot categorically exclude the presence of Y-chromosomal genes in the cells of these patients, there is no cytogenetic evidence of structural rearrangement involving the Y in any of the cases. Expression of H-Y antigen in association with i(Xq) thus implies that H-Y structural genes are X-situated, or alternatively that they are autosomal and X-regulated. It would follow that the H-Y⁺ cellular phenotype per se is not a valid marker for the Y-chromosome, and that H-Y genes that have been mapped to the pericentric region of the Y may be regulatory.     

Sex determination and phenotype in wood lemmings with XXY and related karyotypic anomalies

Summary The wood lemming, Myopus schisticolor, possesses a unique sex determining system comprising both XX and XY females. Normal female development in the presence of XY is guaranteed by a mutation on the X, apparently associated with a structural rearrangement in Xp. This mutation inactivates the testis-inducing and male-determining factor on the Y and distinguishes X^* from X, and X^*Y females from XY males. Normal fertility of X^*Y females is ensured by a mitotic (double) nondisjunction mechanism which, at an early fetal stage, eliminates the Y from the germ line and replaces it by a copy of the X^*.Numerical sex chromosome aberrations are not infrequent and the trisomics XXY and X^*XY are relatively common. XXY individuals are sterile males with severe suppression of spermatogenesis. Among X^*XY animals, both males and females, as well as a true lateral hermaphrodite have been observed. Primary deficiency of germ cells, impairment of spermatogenesis and sterility are characteristic traits of the X^*XY males, whereas X^*XY females have normal oogenesis and are fertile. Both these extremes (except female fertility) coexist in the true hermaphrodite described in the present study. These apparently contradictory observations are explainable under the assumption that X^* and X in X^*XY individuals are inactivated non-randomly or that the cells are distributed unequally. Inactivation of the X or X^* determines whether or not the H-Y antigen will be expressed. When comparing conditions in Myopus and in man, an additional assumption has to be made in relation to the gene(s) involved in sex determination, located in Xp:In Myopus they do not escape inactivation, whereas in man they have been claimed to remain active.     

H-Y antigen and sexual dysgenesis in man

H-Y antigen is a surface component associated with the heterogametic sex of various species and supposed to induce testicular differentiation. Genes controlling directly or not the expression of H-Y antigen and testicular differentiation have been localized on Y as well as on X chromosome and even autosomal chromosome. However the genetical localization of the H-Y structural gene remains unknown. We analysed the expression of H-Y antigen in three types of sexual dysgenesis (males bearing XX caryotype, testicular feminization syndrome and one case of hermaphroditism) to clarify the function and the genetics of this antigen.     

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.     

Cytological identification of two X-chromosome types in the wood lemming (Myopus schisticolor)

In the wood lemming (Myopus schisticolor) three genetic types of sex chromosome constitution in females are postulated: XX, X^*X and X^*Y (X^*=X with a mutation inactivating the male determining effect of the Y chromosome). Males are all XY. It is shown in the present paper that the two types of X chromosomes, X and X^*, exhibit differences in the G-band patterns of their short arms. In addition, it was demonstrated in unbanded chromosomes that the short arm in X^* is shorter than in X. The origin of these differences is still obscure; but they allow to identify and to distinguish the individual types of sex chromosome constitution, as of XX versus X^*X females and of X^*Y females versus XY males, on the basis of G-banded chromosome preparations from somatic cells.     

Recessive male-determining genes

The autosomal dominant gene polled (P) causes hornlessness in goats. Chromosomal females (XX) that are P/P homozygotes develop testes or ovotestes. Thus with respect to its testis-determining properties, P or a closely linked gene acts as an autosomal recessive.Polled intersex goats are H-Y⁺. This finding is consistent with the view that there may be a cluster of testis-determining H-Y genes on the Y chromosome, and that translocation of a subcritical portion of these genes may generate a recessive mode of sex determination.     

H-Y antigen in Turner's syndrome patients with different sex chromosome constitutions

Summary H-Y antigen was determined in seven XO-, nine XO/XX patients, in one patient with i(Xq), and in one patient with a mosaic XO/XYqh-. It turned out that all patients are H-Y antigen positive, confirming the results of earlier investigations of H-Y antigen in patients with Turner's syndrome. The results in XO/XX mosaics clearly demonstrate that the XO-cell is H-Y antigen positive and support the view of a regulatory gene for H-Y antigen gene expression which is located on the X chromosome.     

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.     

Genetic aspects of H-Y antigen

Summary While it remains to be clarified what detection of H-Y antigen by current methods means, the existence of a factor governing testicular differentiation of the indifferent gonadal anlage seems to be well established. There are various kinds of evidence that H-Y antigen as a biologically meaningful factor has a complex genetical basis. There is the contribution of the Y chromosome which, independent of the number of other chromosomes, especially of X chromosomes, leads to a male phenotype. The X chromosome must be involved also because structural aberrations of its distal short arm influence the expression of the H-Y structural gene. Due to examples of autosomal inheritance of various forms of sex reversal, an autosomal gene is assumed to be involved as well. Arguments are presented favoring the assumption that the structural H-Y gene is autosomal, while genes on the X and Y chromosomes have a controlling function.This genetic control mechanism for H-Y antigen seems to have evolved secondary to placentation in mammals. In non-mammalian vertebrates, H-Y antigen is controlled by other factors, e.g. steroid hormones. While the functional role of H-Y antigen in directing differentiation of the heterogametic gonad appears to have been preserved during evolution, the mechanism of its control has changed. This latter mechanism is only poorly understood.     

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.     

Correlation between the number of sex chromosomes and the H-Y antigen titer

Summary H-Y antigen was studied serologically on blood cells and cultured fibroblasts of patients with numerical aberrations of the sex chromosomes. As compared with normal males, patients with the karyotypes 48,XXXY and 49,XXXXY have reduced H-Y antigen titrs; a tendency toward reduced titers can also be detected in the 47,XXY Klinefelter syndrome. The existence of an intermediary titer was further substantiated by a quantitative absorption test applied to cells with the 49,XXXXY karyotype. It appears that in the presence of one Y chromosome, the H-Y antigen titer decreases with an increasing number of X chromosomes. In contrast, the H-Y antigen titer is increased if, at a given number of X chromosomes, the number of Y chromosomes is increased, as in the 47,XYY male. Consequently, patients with 48,XXYY chromosomes are in the male control range. The findings are interpreted under the hypothesis of a controlling or modifying influence of the sex chromosomes on the titer of H-Y antigen.     

The process of a Y-loss event in an XO/XO mammal, the Ryukyu spiny rat

The Ryukyu spiny rat, Tokudaia osimensis, has an XO/XO sex chromosome constitution, lacking a Y chromosome and the mammalian sex-determining gene SRY. To investigate the Y-loss event, we traced three proto-Y-linked genes, RBMY1A1, EIF2S3Y, and KDM5D, in the genome. The original Y-linked RBMY1A1 was lost as well as SRY, and the remaining RBMY1A1 was a processed pseudogene on autosome. In contrast, EIF2S3Y and KDM5D were conserved in genomes of both sexes as a result of their translocation from the Y chromosome to the X chromosome and/or autosomes. Furthermore, these genes were expressed in gonads and brains of both sexes. Our study indicated a loss of Y-linked genes with important male functions to be necessary for the Y chromosome to disappear. These functions might have been retained through the acquisition of new genes, and therefore, the Y-loss has had no harmful effect on the maintenance of this species.     

H-Y antigen expression in a case of mixed gonadal dysgenesis

Summary H-Y antigen expression was studied on leukocytes and gonad-derived fibroblasts from a patient affected by mixed gonadal dysgenesis. Blood leukocytes and fibroblasts derived from the testis were typed H-Y positive, but the fibroblasts derived from the streak gonad were H-Y negative. Although the patient's karyotype was a mosaic, 45,XO/46,X+mar, as detected in-peripheral blood cells and testis-derived fibroblasts, all the fibroblasts derived from the streak gonad were 45,XO. These data suggests that the marker chromosome was in fact a Y-derived chromosome. Moreover, they showed that, at the gonadal level, a minority of H-Y positive 46,X+mar cells were able to organize a testis. Nevertheless, a large number of XO cells probably did not receive the testicular forming influence of the H-Y antigen and of the other masculinizing factors.     

The role of the Y-chromosome in sex determination.

The basic plan of gonadal development in both sexes is female unless testes are induced by factor(s) of the Y chromosome, known as testis determining factor(s) (TDF). It is not clearly established whether the Y chromosome control is autonomous or under the control of a gene on the X chromosome or autosomes. A gene for the H-Y antigen (Histocompatibility-Y antigen) has been postulated to be the factor determining testicular differentiation. Recent studies have demonstrated that the gene for testis determination and the H-Y determinant are two separate entities. Although earlier cytogenetic observations localized TDF on the pericentric region of the short arm of the Y chromosome, subsequent findings by high-resolution chromosome banding and molecular analysis localise TDF to the distal part of the short arm of the Y chromosome, adjacent to the pseudoautosomal region. A candidate for TDF, the ZFY, was localised within the 140 kb interval where the position of TDF was defined, and considered as the TDF gene. However, a smaller gene sequence of 35 kb, the SRY, situated outside the 140 kb ZFY region, has recently been isolated and proved to be the only and the smallest part of the Y chromosome necessary for male sex determination.     

Genetic control of H-Y synthesis. A hypothesis

Summary In view of the claimed serological H-Y positivity observed in patients with ovarian dysgenesis (for example, 45,X) and in XO mice (neither of whom have a Y chromosome), it is suggested that genetic control is exercised over the H-Y system by structural genes on the pairing segments of the X and Y chromosomes, acting on an autosomally coded H-Y precursor.     

The incidence of sex-chromosome anomalies following irradiation of mouse spermatogonia with single or fractionated doses of x-rays

Attempts to recover XO offspring resulting from 600 R irradiation of spermatogonia proved negative. X-Rays were administered either in a single dose or in 100+ 500 R fractions separated by 24 h, and controls were strictly comparable in all respects. Altogether 14016 offspring were scored, including a small group derived from irradiated mature spermatozoa. The breeding scheme allowed phenotypic detection of paternal or maternal sex-chromosome loss, paternal nondisjunction, and certain translocations. All phenotypically exceptional mice were examined cytologically and through breeding tests. Similar tests of the mothers of presumed O/X^p exceptionals revealed that in 9 of 14 cases there was a pre-existing XO condition, indicating the importance of performing such a test. Two of the 3a X^m/O-appearing mice were probably X^m/O///X^m/X^p mosaics, with the integument predominantly XO and the germinal tissue predominantly X/X.The frequency of paternal sex-chromosome loss was 2.4 · 10^?3 in the controls and 2.0 · 10^?3 in the two irradiated groups, where X^m/O's must therefore be assumed to be of spontaneous origin. Since translocation experiments provide evidence that chromosome breaks are induced by irradiation of spermatogonia, the failure to recover XO's is explained in one of two possible ways. (1) Breakage in spermatogonia does not lead to recoverable single-chromosome loss, either because no sister-chromatid joining occurs, or, if it does, because this leads to cell-division failure. (2) Alternatively, sex chromosome loss does occur, but resulting X/O and O/Y cell progeny is inviable in the testis—a suggestion supported by evidence from natural and artificial mosaics.The results of the present experiment lend further support to our earlier suggestion that most spontaneously occurring X^m/O mice are the result of events occurring after sperm entry into the egg. The spontaneous frequency of paternal sex-chromosome loss has ranged over two orders of magnitude in various reports. On the other hand, the frequencies of spontaneous X^m loss (O/X^p daughters of X/X females/total daughters) and of paternal nondisjunction (2 · X^m/X^p/Y frequency)     

Aberrant chromosomal sex-determining mechanisms in mammals, with special reference to species with XY females

Both mouse and man have the common XX/XY sex chromosome mechanism. The X chromosome is of original size (5-6% of female haploid set) and the Y is one of the smallest chromosomes of the complement. But there are species, belonging to a variety of orders, with composite sex chromosomes and multiple sex chromosome systems: XX/XY1Y2 and X1X1X2X2/X1X2Y. The original X or the Y, respectively, have been translocated on to an autosome. The sex chromosomes of these species segregate regularly at meiosis; two kinds of sperm and one kind of egg are produced and the sex ratio is the normal 1:1. Individuals with deviating sex chromosome constitutions (XXY, XYY, XO or XXX) have been found in at least 16 mammalian species other than man. The phenotypic manifestations of these deviating constitutions are briefly discussed. In the dog, pig, goat and mouse exceptional XX males and in the horse XY females attract attention. Certain rodents have complicated mechanisms for sex determination: Ellobius lutescens and Tokudaia osimensis have XO males and females. Both sexes of Microtus oregoni are gonosomic mosaics (male OY/XY, female XX/XO). The wood lemming, Myopus schisticolor, the collared lemming, Dirostonyx torquatus, and perhaps also one or two species of the genus Akodon have XX and XY females and XY males. The XX, X*X and X*Y females of Myopus and Dicrostonyx are discussed in some detail. The wood lemming has proved to be a favourable natural model for studies in sex determination, because a large variety of sex chromosome aneuploids are born relatively frequently. The dosage model for sex determination is not supported by the wood lemming data. For male development, genes on both the X and the Y chromosomes are necessary.     

Univalent sex chromosomes in spermatocytes of Sxr-carrying mice

Pachytene configurations of the sex chromosomes were studied in whole-mount, silver-stained preparations of spermatocytes in mice with XY,Sxr, XX,Sxr, XO,Sxr, XO,Sxr+5¹² and T(X;4)37H,YSxr chromosomes, and non-Sxr-carrying controls. XY,Sxr males showed an increased number of X and Y univalents and of self-synapsed Y chromosomes. In T(X;4)37H,YSxr males an increased proportion of trivalent+Y configurations was also accompanied by higher numbers of self-paired Y univalents; the proportion of trivalent+X⁴ was not increased, but that of self-synapsed X⁴ univalents was. There was more selfsynapsis in cells containing one univalent than in cells containing two univalents. Spermatocytes of XX,Sxr mice contained single univalent X, which was never seen to be self-synapsed, but self-synapsis of the X occurred in a proportion of cells in XO,Sxr males. There were no self-paired X chromosomes in the XO,Sxr+5¹² mouse although lowlevel pairing of the 5¹² chromosome occurred. All four XX,Sxr and XO,Sxr males contained testicular sperm, and testicular sperm were also present in one T(X;4)37H male, while another such male had sperm in the caput. It is concluded that (1) self-synapsis of univalents is affected by variable conditions in the cell as well as by the DNA sequences of the chromosome, and (2) that the level of achievable spermatogenesis is not always rigidly predetermined by a chromosome anomaly but can be modulated by the genetic background.     

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.