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.     

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.     

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.     

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.     

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.     

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.     

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.     

应用生殖免疫方法制作性别化冷冻精液对奶牛性别控制的研究

本实验在种公牛站精液生产过程中应用生殖免疫技术方法 ,制作性别化冷冻精液 ,结合人工授精技术进行奶牛性别控制的研究实验。根据精子DNA含量存在的差异 ,利用荧光染料与精子DNA结合 ,通过蔗糖溶液密度梯度离心法 ,分离出和鉴别出牛精液中的X与Y精子作为抗原。经与小鼠免疫后 ,制成H Y抗血清IgG ,再经酶联免疫吸附法 (ELISA)析测表明 ,获得了具有一定纯度和工作效价的阳性抗Y精子的H Y抗血清IgG ,H Y抗血清对性别化精液冷冻前 ,解冻后活力的影响与对照组无显著差异。配种受胎试验结果表明 ,性别化冷冻精液在获得与正常冷冻精液相似的情期受胎率的同时 ,对后代性别比率有显著影响 ,奶牛产母犊率可达 60 7% ,比自然产母犊性比率理论值提高 1 0 7个百分点 (P <0 0 5 )。     

Dicentric Y chromosome in a patient with gonadal dysgenesis and seminoma

Summary A male patient with ambiguous external genitalia developed a seminoma in the left inguinal region; his internal genitalia included a streak gonad on the right and a small uterus.Cytogenetic studies demonstrated a dicentric Y chromosome with unstable behavior during cell division, which resulted in 45,X/46,X,dic(Y)/47,X,dic(Y),dic(Y) mosaicism.Immunogenetic studies allowed the identification of the male-determining H-Y antigen on both leukocytes and red cells of the patient.The significance of these results is discussed with respect to recent data on the genetic control of H-Y antigen.This work was supported in part by CNR Centro di studio per l'Immunogenetica e l'Istocompatibilità     

A protocol for detection of H-Y antigenic variants

A skin grafting protocol is described for finding H-Y antigenic variants. The method is applicable regardless of the location of the structural gene(s) for this antigen (X, Y, or autosomal). Use of this protocol revealed no evidence for H-Y antigenic variation between C57BL/6J and strains 129/J, A.BY/SnJ, C3H.SW/SnJ, and LP/J.     

Do X and Y spermatozoa differ in proteins?

This article reviews the current knowledge about X- and Y-chromosomal gene expression during spermatogenesis and possible differences between X- and Y-chromosome-bearing spermatozoa (X and Y sperm) in relation to whether an immunological method of separation of X and Y spermatozoa might some day be feasible. Recent studies demonstrated that X- and Y-chromosome-bearing spermatids do express X- and Y-chromosomal genes that might theoretically result in protein differences between X and Y sperm. Most, if not all, of these gene products, however, are expected to be shared among X and Y spermatids via intercellular bridges. Studies on aberrant mouse strains indicate that complete sharing might not occur for all gene products. This keeps open the possibility that X and Y sperm may differ in proteins, but until now, this has not been confirmed by comparative studies between flow-cytometrically sorted X and Y sperm for H-Y antigen or other membrane proteins.     

A gene controlling H-Y antigen on the X chromosome

Summary The existence of a strict correlation between presence of testicular tissue and presence of H-Y antigen in mammals and man leads to the conclusion that H-Y antigen is an essential differentiation factor in testicular morphogenesis. Presence of low titers of this differentiation antigen even in fertile females indicates that its morphogenetic effect depends on a threshold. Here, studies on H-Y antigen in female individuals with various deletions of the X-chromosome are reported. It turns out that deletion of Xp results in the synthesis of reduced amounts of H-Y antigen, while deletion of Xq does not. In a fertile female with only Xp223 deleted due to an X/Y translocation, including the distal Yq, presence of a reduced H-Y titer allows for the tentative assignment of a controlling gene repressing the H-Y structural gene. From the cases studied, it follows that the H-Y structural gene is autosomal and under the control of X- and Y-linked genes. The conception emerges that interaction between X- and Y-linked genes or their products results in variation of the H-Y antigen titer. The fate of the indifferent gonadal anlage to differentiate into the male or the female direction will depend on the titer of H-Y antigen reached by the action or interaction of the controlling genes involved.Supported by the Deutsche Forschungsgemeinschaft (SFB 46)     

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.     

Recessive sex-determining genes in human XX male syndrome

Maleness is normally inherited as a dominant trait (a single copy of the Y chromosome induces testicular differentiation of the embryonic gonad), but our genealogic study of three XX males in one pedigree indicated an autosomal recessive mode of male inheritance. Subsequent study revealed the presence of H-Y antigens in the three XX males and in their mothers, and suggested that excess H-Y may be found in the fathers. Inasmuch as H-Y loci have been mapped to the human Y chromosome, these data favor the view that H-Y structural loci comprise a family of testis-determining genes, and that Y autosome (or Y-X) translocation can generate either dominant or recessive modes of XX sex reversal, depending upon the particular portion of H-Y genes transferred.     

Fractionation of bovine spermatozoa for sex selection: A rapid immunomagnetic technique to remove spermatozoa that contain the H-Y antigen

A study was conducted to rapidly fractionate bovine spermatozoa on the basis of cell-surface H-Y antigen (i.e., Y chromosome-bearing spermatozoa). A novel, rapid immunomagnetic method was developed for removal of spermatozoa that bound to anti-H-Y IgG. Fluorescent labeling and flow cytometry were used to measure the efficiency with which spermatozoa binding to anti-H-Y were removed by the immunomagnetic technique. Washed bovine spermatozoa (n=7 bulls) were treated with a mouse monoclonal IgG antibody to H-Y antigen (MoAb 12/49). Fluorescent labeled goat antibody against mouse IgG was added to label those spermatozoa with cell-surface H-Y antigens. Supermagnetized polymer beads coated with an anti-antibody to the MoAb 12/49 were then added to the spermatozoa. After 20 min of incubation, spermatozoa were exposed for 2 min to a magnet, causing the magnetized particles to adhere to the sides of the tube. Nonmagnetized spermatozoa in the supernatent were aspirated and analyzed for fluorescent label by flow cytometry. Approximately 50% of spermatozoa not subjected to immunomagnetic separation were fluorescent labeled, and about one-half of the spermatozoa were observed microscopically to be bound to the magnetized polymer beads prior to magnetic separation (P<0.05). Following magnetic separation, only 1.2% (P<0.05) of the spermatozoa in the magnetic supernatent were fluorescent labeled. Assuming that only Y chromosome-bearing spermatozoa have cell-surface H-Y antigens, the present immunomagnetic fractionation removed almost all of the Y chromosome-bearing spermatozoa, leaving a population that was greater than 98% X chromosome-bearing spermatozoa.     

Deletion mapping of H-Y antigen to the long arm of the human Y chromosome

A gene encoding or controlling the expression of the H-Y transplantation antigen was previously mapped to the human Y chromosome. We now report the sublocalization of this gene on the long arm of the human Y chromosome. Eight patients with Y-chromosomal abnormalities were examined with a series of existing and new DNA markers for the Y chromosome. The resulting deletion map was correlated with H-Y antigen expression. We conclude that the H-Y antigen gene maps to a portion of deletion interval 6 that is identified by specific DNA markers.     

Pairing of X and Y chromosomes,non-inactivation of X-linked genes,and the maleness factor

Summary In this paper observations are summarized and speculations discussed, and it is suggested that some loci on the distal short arm of the X chromosome (Xp) are not randomly inactivated in the female, because they are within the proximal part of the pairing segment between Xp and Yp. This peculiarity of gene expression may be a remnant of the evolutionary history of the sex chromosomes, the pairing segment of which may involve at least 27% of Xp and 95% of Yp. Crossing over seems to occur mostly in the terminal third of the X/Y pairing segment. However, crossing-over inhibition control may lapse, or may be on the X and Y (e.g. Xg, H-Y, STS, and perhaps others) might cross over with a variable frequency which is proportional to their distances from the telomeres of the short arms. It is postulated that the DNA of the pairing segment is composed in a way which may also permit unequal crossing over to occur between the X and the Y, thereby giving rise to exceptions to X-or Y-linked inheritance. The peculiarities of behaviour and the position of other loci on the sex chromosomes are also discussed briefly.     

The etiology of maleness in XX men

Summary Information relating to the etiology of human XX males is reviewed. The lesser body height and smaller tooth size in comparison with control males and first-degree male relatives could imply that the patients never had any Y chromosome. Neither reports of occasional mitoses with a Y chromosome, nor of the occurrence of Y chromatin in Sertoli cells are convincing enough to support the idea that low-grade or circumscribed mosaicism is a common etiologic factor. Reports of an increase in length of one of the X chromosomes in XX males are few and some are conflicting. Nor is there any evidence to support the idea of loss of material. However, absence of visible cytogenetic alteration does not rule out the possibility of translocations, exchanges or deletions.A few familial cases are known. Mendelian gene mutations may account for a number of instances of XX males, similar genes being well known in several animal species. The existing geographical differences in the prevalence of human XX males could be explained by differences in gene frequency. But if gene mutation were a common cause of XX maleness there would be more familial cases.Any hypothesis explaining the etiology of XX males should take into account the following facts. There are at least 4 examples of XX males who have inherited the Xg allele carried by their fathers, and at least 9 of such males who have not. The frequency of the Xg phenotype among XX males is far closer to that of males than to that of females, while the absence of any color-blind XX males (among 40 tested) resembles the distribution in females. Furthermore, H-Y antigen is present in XX males, often at a strength intermediate between that in normal males and females. Finally, in a pedigree comprising three independently ascertained XX males, the mothers of all three are H-Y antigen-positive, and the pattern of inheritance of the antigen in two of them precludes X-chromosomal transmission.Many of the data are consistent with the hypothesis that XX males arise through interchange of the testic-determining gene on the Y chromosome and a portion of the X chromosome containing the Xg gene. However, actual evidence in favor of this hypothesis is still lacking, and the H-Y antigen data are not easy to explain. In contrast, if recent hypotheses on the mechanisms controlling the expression of H-Y antigen are confirmed, a gene exerting negative control on testis determination would be located near the end of of the short arm of the X chromosome. This putative gene is believed not to be inactivated in normal females, for at least two other genes located in the same region, i.e. Xg and steroid sulfatase, are not. Deletion or inactivation of these loci would explain how XX males arise and would be consistent with most, but not all, the facts.There is yet no single hypothesis that by itself can explain all the facts accumulated about XX males. While mosaicism appears very unlikely in most cases, Mendelian gene mutation, translocation, X-Y interchange, a minute deletion or preferential inactivation of an X chromosome, or part thereof, remain possible. The etiology of XX maleness may well be heterogeneous.     

H-Y antigen: localization of the H-Y gene

Testicular development in a patient with deletion of the distal (fluorescent) segment of the Y chromosome is described. The presence of a normal dose of H-Y antigen was demonstrated by Goldberg's cytotoxicity test. It is concluded that the distal fluorescent segment of the Y chromosome is void of genes regulating H-Y antigen activity.     

G6PD-Puerto Limón: A new deficient variant of glucose-6-phosphate dehydrogenase associated with congenital nonspherocytic hemolytic anemia

Summary H-Y antigen was examined in eight male patients with X polysomies, namely four patients with 47,XXY, one patient with 48,XXXY, two patients with 49,XXXXY, and one patient with the mosaic 47,XXY/49,XXXXY. In all patients the H-Y antigen titers were lower than in normal 46,XY males. However, a linear correlation between the number of additional X chromosomes and the reduction of H-Y antigen titers could not be demonstrated. Such a correlation would be expected if the gene for the repressor of H-Y antigen expression is active also on the additional X chromosomes.