Hum. Genet. 52, 143-147 (1979) © by Springer-Verlag 1979

Short Communications A Highly Sensitive Peroxidase-Antiperoxidase Method for Detection of H-Y Antigen on Cultivated Human Fibroblasts U. Mtiller 1. and K. Bross 2 Institut ft~r Humangenetik und Anthropologie der Universit~t, Albertstr. 11, D-7800 Freiburg, Federal Republic of Germany "Medizinische Universitgtsklinik, Hugstetterstr. 55, D-7800 Freiburg, Federal Republic of Germany

Summary. A peroxidase-anti-peroxidase method for the detection of H-Y antigen at the single cell level is described. The efficiency of the test was examined in cultivated fibroblasts derived from control subjects and from XX males and a true hermaphrodite. For comparison, H-Y antigen was determined in blood cells of the same probands using the cytotoxic test. The finding of H-Y positive fibroblasts in the intersex patients has implications for the origin of these disorders.

Introduction Several techniques have been elaborated for the detection of H-Y antigen. The most common one is the cytotoxicity assay using H-Y antisera raised in isogenic rats or mice. Anti-H-Y antiserum is tested for cytotoxicity on sperm (Goldberg et al., 1971), male rodent tail epidermal ce'lls (Sche!d et al., 1972) or Raji lymphoma cells (Fellous et al., 1978) after absorption with the cells or tissues under study. Using these techniques, H-Y antigen has been found in all male mammalian tissues (Mtiller et al., 1978a) apart from diploid male germ cells (Zenzes et al., 1978a). However, this method does not permit localization of the antigen at a cellular level. For doing this, various techniques have been developed, e.g., mixed hemadsorption-hybrid antibody rosette formation (Wachtel et al., 1974), fluorescence staining (Galbraith et al., 1978), or a protein A-method (Tokuda et al., 1977). These assays, however, either are rather complex and laborious or not sensitive enough. We describe here a peroxidase-anti-peroxidas~ method for the detection of HY antigen on single cells, using anti-H-Y ant'isera and the peroxidase-antiperoxidase immunocomplex for labelling. This p~ocedure is easy to deal with and very sensitive, as is demonstrated by "titration" of H-Y antigen on fibroblast cell surfaces of normal and XX sex-reversed males. * To whom offprint requests should be sent

0340-6717/79/0052/0143/$ 01.00

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U. Miiller and K. Bross

Materials and Methods Anti-H-Y antisera were raised in highly inbred female Lewis rats as described earlier (Miiller et al., 1978a). All the other antisera were purchased from Cappel Laboratories (Cochranville, USA). Anti-H-Y antiserum and rabbit-anti-rat antiserum were absorbed with female fibroblasts. Antisera were diluted with 10% IgG-free fetal calf serum (FCS) containing NKH buffer (8.5g NaC1, 0.4 KC1/1 buffered with Hepes, pH 7.4). Immunocytochemical staining was performed on fibroblasts attached to poly-L-lysine coated glass slides as described by Bross et al. (1978). All procedures were carried out in a wet chamber on ice. Cells were successively incubated with different dilutions of anti-H-Y antiserum, rabbit-anti-rat IgG (Y400),goat-antirabbit IgG (1/10), and peroxidase-anti-peroxidase immunocomplex (PAP) produced in rabbit (J/50) for 15 min each. Every incubation step was followed by washing with NKH 0.1% gelatin. Staining was performed with diaminobenzidine(DAB containing hydrogene peroxide, followed by fixation with 1.33% OsO4 and covered with glycerine and cover glass) (for further details see Bross et al., 1978). To evaluate the reaction, the slides were read under a light microscope at a magnification of 400-fold. In addition, a cytotoxicity assay was performed (according to Scheid et al., 1972) to test for antigen concentration on blood cells. Buffy coat prepared from 12ml of blood was absorbed with 50 ~tl of 1/4diluted anti-H-Y antiserum and tested for cytotoxicity on rat male epidermal cells. Fibroblasts and blood cells of two male and two female healthy donors and three XX sexreversed patients were checked for H-Y antigen. Two of the patients were XX males (human XX male syndrome): no chromosome mosaic was found in cultivated lymphocytes and fibroblasts in either of them 1. The third patient was a true x x hermaphrodite. No chromosome mosaic was found in cultures of the blood, the right and left gonad, or the mammary gland. Histological examination revealed a dysgenetic left testis and an ovotesticular right testis (Fraccaro et al., 1979).

Results and Discussion A b o u t 90% of n o r m a l male fibroblasts were intensively stained, a n d m a n y of them showed c a p p i n g p h e n o m e n a (Fig. lc). F e m a l e fibroblasts showed n o or only very faint staining, a n d a few cells reacted s o m e w h a t more strongly (Fig. 1 a). The intensity of reaction o n fibroblasts of X X males was in the range of n o r m a l male controls, while fibroblasts of the true h e r m a p h r o d i t e showed weaker staining t h a n those of 'normal males, b u t clearly more t h a n fibroblasts from female controls (Fig. 1 b). I n Table 1 staining intensity is evaluated according to a scoring code for P A P (see legend to Table 1). The m e a n intensity of the reaction of 300 cells analyzed is shown. I n the respective p r o b a n d s , the a m o u n t of H-Y a n t i g e n detected o n fibroblasts by this staining reaction corresponds well to the r e m a i n i n g activity of a n t i - H - Y a n t i s e r u m in the cytotoxic test after a b s o r p t i o n with white b l o o d cells (Fig. 2). I n the latter test, again the h e r m a p h r o d i t e cells expressed less H-Y a n t i g e n t h a n do n o r m a l male controls or XX males. H-Y antigen was s h o w n to induce g o n a d a l cells to become a testis ( O h n o et al., 1978; Zenzes et al., 1978b; Mfiller et al., 1978b). However, testicular o r g a n i z a t i o n is only complete if a threshold of H-Y antigen c o n c e n t r a t i o n is reached, otherwise a n ovotestis develops. Reduced titers of H-Y antigen have been f o u n d in h e r m a p h r o d i t e s of various species such as goat (Wachtel et al., 1978), dog (Selden et al., 1978) a n d m a n (Wachtel et al., 1976; Fraccaro et al., 1 Cytogenetic studies were performed by Prof. Passarge of Essen and Dr. Stengel-Rutkowski of Munich

Detection of H-Y Antigen on Cultivated Human Fibroblasts

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Fig. la--c. Fibroblasts, labeled for H-Y antigen using an anti-H-Y antiserum (I/4) and the peroxidase-anti-peroxidase immunocomplex (a) fibroblasts from a normal female donor; (b) fibroblasts from a true XX hermaphrodite; (e) fibroblasts from a normal male donor

Table L Intensity of peroxidase staining for H-Y antigen on fibroblastsa Dilution of anti-H-Y antiserum

~4

1/8

1/16

~32

Normal male

6

7

6

5

Normal female

4

4

3

3

XX male 1

6

7

6

6

XX male 2

6

6

5

5

XX true hermaphrodite

5

5

5

4

Fibroblasts were judged according to a scoring code from 1 to 8:1 = negative; 2 = doubtful negative; 3 = doubtful positive (somewhat stronger reaction than in negative controls); 4 = weak positive (clear difference from negative controls); 5 = positive (cells covered with single granules); 6 = positive (granules or caps); 7 = positive (more granules or caps); 8 = strongest reaction (cells dark). For each code number at least 300 cells were analyzed

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U. Mfiller and K. Bross

60

40

20

i 114 dilution

i 1/8

I 1/16

of o n t i - H - Y

I /~ 1/32 antiserum

i C

Fig. 2. Cytotoxicity of different dilutions of anti-H-Y antiserum after absorption with blood cells (bnffy coat of 12ml blood) of normal male and female controls, two XX males and a true hermaphrodite. (/,) normal male; (e) XX male 1; (A) XX male 2; (n) true hermaphrodite; (o) normal female; (n) cytotoxicity of different dilutions of anti-H-Y antiserum; ( i ) complement control

1979) (for discussions on the genetic basis of disturbed gonadal development, see Wolf, 1979; Chapelle, 1972; Chapelle et al., 1978; Selden et al., 1978; Fraccaro et al., 1979). These data were obtained using a cytotoxic test after absorption of H-Y antiserum by blood cells or fibroblasts. These findings are confirmed here at the cellular level using the peroxidase-anti-peroxidase method. Corresponding to the development of an ovotestis, reduced expression of H-Y antigen was found in the true hermaphrodite, as is shown on fibroblasts using peroxidase-staining and on blood cells using anti-H-Y antiserum absorption. In the XX males tested here, the amount of H-Y antigen was in the range found in normal males, corresponding to the complete testicular development in this syndrome. To explain the development of testes without any apparent Y-chromosome material, three different hypotheses have been proposed (according to de la Chapelle, 1972): (1) The gene theory, presuming mutation(s) of postulated sex-determining genes; (2) The interchange theory, assuming (a) the translocation of a chromosome fragment on an autosome, or (b) the exchange of terminal segments between X and Y chromosomes during meiotic association; and (3) The mosaicism or eliminated Y-chromosome theory. Due to our finding of H-Y antigen on fibroblasts derived from XX males, cultivated for several passages in vitro, the hypothesis of a latent or preexisting mosaic as the cause of testicular differentiation must be abandoned, at least in the three patients tested here.

Acknowledgments. We are grateful to Prof. Ulrich Wolf for his continued encouragement and many helpful discussions. We are grateful to Mrs. M. Lindig for skillful technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 46 and Si 185/2).

References Bross, K. J., Pangalis, G. A., Staatz, C. G., Blume, K. G.: Demonstration of cell surface antigens and their antibodies by the peroxidase-antiperoxidase method. Transplantation 25, 331--334 (1978)

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Chapelle, A. de la: Nature and origin of males with XX sex chromosomes. Am. J. Hum. Genet. 24, 71--105 (1972) Chapelle, A. de la, Koo, G. C., Wachtel, S. S.: Recessive sex determining genes in human XX male syndrome. Cell 15, 837--842 (1978) Fellous, M., Gfinther, E., Kemler, R., Wiels, J., Berger, R., Guenet, J. L., Jakob, H., Jacob, F.: Association of the H-Y male antigen with fl2-microglobulin on human lymphoid and differentiated mouse teratocarcinoma cell lines. J. Exp. Med. 148, 58--70 (1978) Fraccaro, M., Tiepolo, L., Zuffardi, O., Chiumello, G., Di Natale, E., Gargantini, G., Wolf, U.: Familial XX true hermaphroditism and the H-Y antigen. Hum. Genet. 48, 45--52 (1979) Galbraith, G. M. P., Galbraith, R. M., Faulk, W. P., Wachtel, S. S.: Detection of H-Y antigen by fluorescence microscopy. Transplantation 26, 25--27 (1978) Goldberg, E. H., Boyse, E. A., Bennett, D., Scheid, M., Carswell, E. A.: Serological demonstration of H-Y (male) antigen on mouse sperm. Nature 232, 478--480 (1971) Mfiller, U., Aschmoneit, I., Zenzes, M. T., Wolf, U.: Binding studies of H-Y antigen in rat tissues. Indications for a gonad-specific receptor. Hum. Genet. 43, 151--157 (1978 a) Mfiller, U., Zenzes, M. T., Bauknecht, T., Wolf, U., Siebers, J. W., Engel, W.: Appearance ofhCG receptor after conversion of newborn ovariancells into testicular structures by H-Y antigen in vitro. Hum. Genet. 45, 203--207 (1978b) Ohno, S., Nagai, Y., Ciccarese, S.: Testicular cells lysostripped of H-Y antigen organize ovarian follicle-like aggregates. Cytogenet. Cell Genet. 20, 351--364 (1978) Scheid, M., Boyse, E. A., Carswell, E. A., Old, L. J.: Serologically demonstrable alloantigens of mouse epidermal cells. J. Exp. Med. 135, 938--955 (1972) Selden, J. R., Wachtel, S. S., Koo, G. C., Haskins, M. E., Patterson, D. F.: Genetic basis of XX male syndrome and XX true hermaphroditism: evidence in the dog. Science 201,644--646 (1978) Tokuda, S., Arrington, T., Goldberg, E. H., Richey, J.: Simpler technique for serological detection of H-Y antigen on mouse lymphocytes. Nature 267, 433--434 (1978) Wachtel, S. S., Basrur, P., Koo, G. C.: Recessive male-determining genes. Cell I5, 279--281 (1978) Wachtel, S. S., Koo, G. C., Breg, W. R., Thaler, H. T., Dillard, G. M., Rosenthal, I. M., Dosik, H., Gerald, P. S., Saenger, P., New, M., Lieber, E., Miller, O. J.: Serologic detection of a Y-linked gene in XX males and XX true hermaphrodites. N. Engl. J. Med. 295, 750--754 (1976) Wachtel, S. S., Koo, G. C., Zuckerman, E. E., HSmmerling, U., Scheid, M. P., Boyse, E.: Serological cross-reactivity between H-Y (male) antigens of mouse and man. Proc. Natl. Acad. Sci. USA 71, 1215--1218 (1974) Wolf, U.: XY gonadal dysgenesis and the H-Y antigen. Report on 12 cases. Hum. Genet. 47, 269--277 (1979) Zenzes, M. T., Mt~ller, U., Aschmoneit, I., Wolf, U.: Studies on H-Y antigen in different cell fractions of the testis during pubescence. Immature germ cells are H-Y antigen negative. Hum. Genet. 45, 297--303 (1978a) Zenzes, M. T., Wolf, U., Gt~nther, E., Engel, W.: Studies on the function of H-Y antigen: Dissociation and reorganization experiments on rat gonadal tissue. Cytogenet. Cell Genet. 20, 365--372 (1978b) Received May 9, 1979

A highly sensitive peroxidase-antiperoxidase method for detection of H-Y antigen on cultivated human fibroblasts.

Hum. Genet. 52, 143-147 (1979) © by Springer-Verlag 1979 Short Communications A Highly Sensitive Peroxidase-Antiperoxidase Method for Detection of H-...
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