Hum Genet (1981) 57:68-70

© Springer-Verlag 1981

Serological Evidence for H-Y Antigen in XO-Female Mice

Wolfgang Engel, Barba ra Klemme, and Annelo t te Ebrecht

Institut ffir Humangenetik der Universitfit G6ttingen, Nikolausberger Weg 5a, D-3400 GOttingen, Federal Republic of Germany

Summary. H-Y antigen was examined in XX-, XY-, and XO- mice using spleen, kidney, and liver cells of the animals for the absorp t ion of the anti-H-Y ant i serum produced in the rat. The cells of the XY- and XO-mice were found to be H-Y ant igen- positive while the cells of the XX-mice were negative. As in Turne r syndrome pat ients with 45,X, in the XO-female mice the H-Y ant igen titre was reduced as compared to normal XY-male mice; in termediate values between those of normal male and female mice were obtained. These results clearly indicate tha t as in man, in the mouse the s t ructural gene for H-Y antigen is not Y-linked hut is located on an autosome. Fur thermore , the con- cept of the regulat ion of the H-Y ant igen gene expression in the human (Wolf et al. 1980a, b) by an X-linked repressor gene, escaping X-inact ivat ion in the XX-female and an Y-linked inducer gene also seems to hold true in the mouse.

Introduction

Ohno (1979) ment ioned two viable al ternatives with respect to the localisation of the s tructural gene for H-Y antigen, namely the Y- and X-linkage. In the case of Y-linkage, the s t ructural gene for H-Y antigen is assumed to exist in multiple copies and its activity is modula ted by a regulatory gene located on the X- chromosome. In the case of X-linkage, the expression of the gene(s) should be regulated by a Y-linked activating gene and an autosomal repressing gene. Most au thors favour the Y- linkage of the H-Y structural gene and thus, in all H-Y antigen- positive pat ients and animals with testicular different iat ion but absence of Y-chromosomal material , the t rans locat ion of one or more copies of the H-Y structural gene was assumed. In 1978, Wolf proposed a genetic model for the regulat ion of H-Y antigen expression in which the expression of an autosomal ly localised s t ructural gene for H-Y ant igen is regulated by an X- linked repressor gene and a Y-linked inducer gene. This model is suppor ted by the findings of Wol f et al. (1980a, b) tha t patients with different deletions of Xp and pat ients lacking one X-chromosome (45,X; Turner ' s syndrome) are H-Y ant igen-

positive. We here repor t results of H-Y ant igen investigations in mice

with different sex ch romosome const i tut ions, namely XX, XY, and XO. While XX-mice are H-Y antigen-negative, XY- and XO-mice are H-Y antigen positive. Following the postulate of Ohno (1973) tha t the m a m m a l i a n X-linkage group is a "frozen accident in evolut ion", our f inding of H-Y ant igen in the XO- mouse supports Wolf ' s hypothesis (Wolf 1978).

Offprint requests to: W. Engel

Materials and Methods

XO-female mice and XY-male mice carrying the X-linked marker gene Tabby (Ta) were kindly supplied by Dr. Mary Lyon (MRC Radio- biology Unit, Harwell, England) for studies on ovarian differentiation. By selected breeding XO- and XX-females could be differentiated from each other through the expression of the gene Ta (Ta/O and Ta/+, respectively) (Lyon 1961). Three XX-, XO-, and XY-mice, each, were killed, exsanguinated, and spleen, kidney, and liver were prepared. The organs were minced into small pieces, washed several times with phosphate buffered saline (PBS) and then gently dounced in PBS in a Belco-homogenizer. The supernatant containing the dissociated cells was centrifuged at i000 rpm for 10 min at 4°C. The cell pellet obtained was resuspended in PBS and washed three to four more times in PBS. Subsequently, the number of cells was determined and using the trypan blue exclusion test, the number of dead cells was counted. The number of dead cells in all preparations was less than 5%. Six to eight x 10 6 cells of each preparation were used for the absorption of the anti-H-Y anti- serum.

Antiserum to H-Y antigen was raised in isogenic rats by six weekly intraperitoneal injections of 15-20 million male spleen cells into female animals. Blood was taken seven days after the last injection, the sera were inactivated at 56°C for 30 min and absorbed with an equal volume of human female A~B-erythrocytes and subsequently tested for cyto- toxic activity using the human male lymphoid cell line "Raft" and the human female lymphoid cell line "Bjab" in the presence of rabbit complement in appropriate dilution. Rabbit complement was selected according to low cytotoxicity on Raji- and Bjab-cells.

The presence or absence of H-Y antigen on spleen, kidney, and liver cells was determined using a cytotoxic test (Fellous et al. 1978). All cell suspensions were scored as coded samples. Thirty gl of the anti-H-Y antiserum absorbed with female A~B-erythrocytes were incubated with 6-8 x 106 cells for 60 rain on ice. After spinning down the cells, the anti- H-Y antiserum was transferred in four different dilutions (1 gl per hole) on micro-titreplates. One gl of Raji- and Bjab-cells (103 cells per gl) was used as H-Y antigen-positive and H-Y antigen-negative target cells, respectively (incubated for 45min at ambient temperature). Sub- sequently, 5 gl of appropriately diluted rabbit complement was added and incubated for an additional 45 min at ambient temperature. After removing the liquid (cells stuck to the plastic wall), 1 ~tt of 0.1% trypan blue-Hanks solution was added and iysed cells counted.

Results and Discussion

In each of the mice investigated, residual cytotoxicity of ant i -H- Y ant iserum was tested after absorp t ion with spleen, liver, and kidney cells, respectively. The results obta ined are depicted in Figs. 1 and 2. It can be inferred f rom the figures tha t the cells of the XY-male mice are H-Y antigen-posit ive while the cells of the XX-female mice are H-Y antigen-negative. After absorp t ion of the ant i -H-Y ant i serum with the cells of the XO-female mice,

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Fig. 1. Cytotoxicity ofanti-H-Y antiserum to Raji-cells after absorption with 6-8 × 106 spleen, kidney, or liver cells of(o) XX-, (e) XY-, and (A) XO-mice. The different cells of one mouse of each sex chromosome constitution were tested in parallel and three mice of each sex chromosome constitution were investigated in all. Identical results were obtained. Kidney cells of the nine mice were tested twice. In each experiment the absorbed anti-H-Y antiserum was tested with respect to its cytotoxicity to H-Y antigen-negative, female Bjab- cells in the presence of rabbit complement (A: real control

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Fig. 2. Cytotoxicity ofanti-H-Y antiserum to Raji-cells after absorption with 6-8 x 106 spleen, kidney, or liver cells of (o) one XX-, (e) one XY-, and (A) three different XO-mice. This experiment was done separately from that shown in Fig. 1 and the same cell type of the five mice was included in the same experiment. In each experiment the absorbed anti- H-Y antiserum was tested with respect to its cytotoxicity to H-Y antigen- negative, female Bjab-cells in the presence of rabbit complement (zx: real control)

titre courses in termediate between those for the XY- and XX- mouse cells were obta ined, indicat ing tha t XO-female mice are H-Y antigen-posit ive, bu t with a reduced a m o u n t of H-Y ant igen on the cell surface as compared to XY-male cells.

Recently, Wol f et al. (1980b) repor ted H-Y ant igen in five patients with Turner ' s syndrome, four of them had the karyo- type 45,X and one was 45 ,XX/46,Xi(Xq) . As in the XO-mice tested here, the ant igen titre in all pat ients was reduced compared to male controls. However, a considerable inter- individual var ia t ion of the titre was found. As can be seen f rom Fig. 2, in the three XO-female mice tested, the H-Y ant igen titres were very similar and also did not depend on the cell type used. Nevertheless, the differences between the results ob ta ined with X O - T u r n e r pat ients and in XO-female mice could be due to the cell types used for the investigations. While Wol f et al. (1980 b) used the freely circulating and ageing granulocytes and erythrocytes of the b lood for absorp t ion of the anti-H-Y ant i- serum, in the XO-female mice the cells of dissociated organs were used. Our assumpt ion is suppor ted by the observat ion that intermediate H-Y ant igen titre courses .obta ined with the leuco- cytes of female- to-male t ranssexual pat ients are not charac- teristic for o ther cell-types of the patients; testicular and muscle cells are as positive as those of no rma l male controls (own un- publ ished results). One o ther reason for the difference between the titre courses of XO-Turne r pat ients and XO-female mice

might be seen in differences of the cellular composi t ion of the body. While in the XO-mouse all cells lack one sex chromo- some, in the XO-Turner pat ients cryptic mosaics with X O / X X or X O / X Y canno t be excluded, even after careful cytogenetic investigations. Since it is generally assumed that surviving XO- zygotes are not the result of meiotic bu t of mitot ic non-dis junc- tion or of early ch romosome loss, many, if not all, surviving XO-concept ions may be mosaics (Ford 1969; G e r m a n 1970; Bou6 and Bou6 1973; W a r b u r t o n et al. 1980). Thus, the presence of an XX-cell line would lead to a reduction, an XY- cell line to an increase of the H-Y ant igen titre as compared to the intermediate levels expected in the pure XO-Turner pa- tients. The quest ion as to whether the inter individual var ia t ion of the H-Y ant igen titres in the XO-Turner pat ients reflects differences in the n u m b e r of H-Y antigen molecules per cell or is due to criptic mosaics can be solved by test ing H-Y ant igen of cloned XO-cells of different Turne r patients.

The implicat ions of H-Y ant igen-posi t ivi ty in Turner - syndrome pat ients for the ch romosomal localisation of the H-Y structural gene and for the regulat ion of its expression in man has been formula ted already by Wol f et al. (1980b). According to these au thors the s t ructural gene for H-Y antigen is localised on an au tosome and its expression is regulated by an X-l inked repressor gene and a Y-linked inducer gene. In XX-females the repressor gene should not undergo inact ivat ion (Wolf et al.

70

1980a, b). Our observat ion of an intermediate level of H-Y antigen in XO-female mice strongly indicates tha t Wolf ' s concept of an au tosomal localisation of the H-Y structural gene and of the regulat ion of the expression of this gene by two different control l ing genes on the X- and Y-chromosomes also holds true in the mouse. Thus, as in X X h u m a n males (Wachte l et al. 1976) H-Y antigen-posit ivi ty in X X Sxr /+ male mice (Bennett et al. 1977) could be the consequence of mu ta t ion of the autosomal ly localised H-Y structural gene ra ther than that of Y /au tosome or Y / X t rans lcoat ion as was suggested by Wachtel et al. (1977). Fur the rmore , it can be assumed tha t this regulatory system proposed by Wolf (1978) is realized in all mammals , and possibly also in lower vertebrates. It should be ment ioned here, tha t also in the chicken and in Xenopus the structural gene for H-Y ant igen is not localised on the W- chromosome (Maller et al. 1979; Wachtel et al. 1980).

The s tudy of H-Y ant igen in pat ients with different struc- tural abnormal i t ies of the Y-chromosome led some authors to suggest tha t the s tructural gene for H-Y ant igen is localised on the shor t or on the proximal long arm of the Y-chromosome (e.g. Koo et al. 1977; More i ra-Fi lho et al. 1979; Faggiano et al. 1980). However, the results of Wolf et al. (1980a, b) in pat ients With X p - and XO and of our own results in the XO-mouse , suggest tha t Yp and the proximal par t of the long arm of the Y- chromosome, respectively, were p robab ly the locality for an inducer gene ra ther than the structural .gene of the H-Y antigen.

The XO-condi t ion in the mouse, as in mos t mammals , is usually female (Ca t tanach 1974). In contras t to the h u m a n XO it does not show any phenotypic abnormal i t ies (Welshons and Russell 1959; Ca t t anach 1962; Morr is 1968) and is fertile, al- though its reproduct ive life-span is shor ter than normal (Lyon and Hawker 1973). In both , the H-Y ant igen activity observed (Wolf et ai. 1980b; our own results) seems to be below the threshold required for testicular de terminat ion of the b ipo ten t gonadal anlage in embryogenesis. However, it remains un- known why the fate of the embryonic gonad in XO-Turner syndrome pat ients is so different f rom tha t in XO-mice.

Acknowledgements. We would like to thank Dr. Mary F. Lyon, MRC Radiobiology Unit, Harwell, for supplying us with a breeding nucleus of XO-mice and Dr. M. Schmid for valuable suggestions. Mrs. Monika Pelz and Mr. Volker Schimkus are thanked for preparing the man- uscript. This work was supported by the Deutsche Forschungsgemein- schaft (En 84/12).

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Received October 2, 1980