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The global modification of mammalian and plasmid DNAs by the novel platinum compounds cis-[PtCl(2)(isopropylamine)(1-methylimidazole)] and trans-[PtCl(2)(isopropylamine)(1-methylimidazole)] and the reactivity of these compounds with reduced glutathione (GSH) were investigated in cell-free media using various biochemical and biophysical methods. Earlier cytotoxicity studies had revealed that the replacement of the NH(3) groups in cisplatin by the azole and isopropylamine ligands lowers the activity of cisplatin in both sensitive and resistant cell lines. The results of the present work show that this replacement does not considerably affect the DNA modifications by this drug, recognition of these modifications by HMGB1 protein, their repair, and reactivity of the platinum complex with GSH. These results were interpreted to mean that the reduced activity of this analog of cisplatin in tumor cell lines is due to factors that do not operate at the level of the target DNA. In contrast, earlier studies had shown that the replacement of the NH(3) groups in the clinically ineffective trans isomer (transplatin) by the azole and isopropylamine ligands results in a radical enhancement of its activity in tumor cell lines. Importantly, this replacement also markedly alters the DNA binding mode of transplatin, which is distinctly different from that of cisplatin, but does not affect reactivity with GSH. Hence, the results of the present work are consistent with the view and support the hypothesis systematically tested by us and others that platinum drugs that bind to DNA in a fundamentally different manner from that of conventional cisplatin may have altered pharmacological properties.

Human neurodegenerative and neuromuscular disorders are associated with a class of gene mutations represented by expansion of trinucleotide repeats. DNA testing is important for the diagnosis of these diseases because clinical discrimination is complicated by their late onset and frequently overlapping symptomatology. However, detection of pathologic alleles expanded up to several thousand trinucleotides poses a challenge for the introduction of rapid, fully automatic, and simple DNA diagnostic procedures. Here we propose a simple two-step polymerase chain reaction (PCR) protocol for rapid molecular diagnostics of myotonic dystrophy, Huntington's disease, and possibly also other triplet expansion diseases. Standard PCR amplification with target repeat flanking primers is used for the detection of alleles of up to 100 repeats; next, triplet-primed PCR is applied for detection of larger expansions. Automated capillary electrophoresis of amplicons allows rapid discrimination between normal, premutated and expanded (CTG/CAG)(n) alleles. Using the suggested protocol, the expanded allele was successfully detected in all test DNA samples with known genotypes. Our experience demonstrates that the suggested two-step PCR protocol provides high sensitivity, specificity, and reproducibility; is significantly less time-consuming; is easier to perform; and provides a better basis for automation than previous methods requiring Southern analysis. Therefore, it can be used for confirmation of uncertain clinical diagnoses, for prenatal testing in at-risk families, and, generally in research on these diseases.

Background: Human genome dynamic mutations are a new class of gene mutations represented by an unstable number of trinucleotide repeats and causing severe human hereditary neuromuscular and neurodegenerative diseases. The identification of pathological expanded alleles on the molecular level is important for clinical diagnostics., Methods and Results: For the molecular diagnostics of expanded tandem repeat trinucleotide sequences we have introduced a fast and efficient TP-PCR fluorescent method according to Warner et al. (1996). We have modified this TP-PCR method for a rapid detection of expanded CTG alleles of the DMPK gene (myotonic dystrophy, MD) into a two-level protocol; first, the heterozygote sample DNAs were selected using P1/P2 primers flanking repeat tracts and, second, the TP-PCR protocol used was focused above all on the identification of a pathological allele. A fluorescent-labelled specific primer in TP-PCR was used for the exact determination of the number of CAG repeats of the gene IT-15 (Huntington's disease--HD) in the diagnostically important region of the grey zone (35 to 39 CAG). The reproducibility of the PCR results was demonstrated on control DNA samples with the known genotype and, in the case of MD, also by Southern blot analysis. We have especially shown the possibility of a cheaper PCR-P1/P2 and TP-PCR protocol, which can be used, with silver staining of separated PCR products on polyacrylamide gels., Conclusions: Our experience with introducing the above-mentioned PCR methods into laboratory practice clearly documents the possibilities of their general applicability in the molecular diagnostics of hereditary diseases characterised by instability of the trinucleotide repeat tracts.

Complex diagnosis of muscular dystrophies including clinical, bioptical and molecular genetic approaches has been provided in a limited extent in this country. Our group of neurologists, pathologists and geneticists has examined approximately 240 patients suspected of having muscular dystrophies, mostly coming from Southern and Northern Moravia. The patients were sent to the examination most often from departments of neurology and clinical genetics, and less frequently from departments of internal medicine. According to the final diagnosis, the patients were divided into groups: with dystrophinopathies and carriers of dystrophinopathies (DMD/BMD), merosin deficient form of congenital muscular dystrophy, and Emery-Dreifuss muscular dystrophy including the carriers of this disease. Some relatives of patients with dystrophinopathies were also examined using the methods of segregation analysis. High proportion of the DMD/BMD patients can be detected by the methods of molecular genetics. Analysis of mRNA using RT PCR and PTT enables the detection of deletions, duplications, and point mutations in dystrophin gene and encompasses a larger diagnostic scope in comparison with examinations of DNA level by the multiplex PCR method from the peripheral blood which enables only deletion detections. Immunophenotyping of the dystrophin protein plays an important role especially using antibodies against carboxyterminal (DYS2) and rod domain (DYS1) of dystrophin. Deficient sarcolemmal expression of DYS2 and DYS1 reveals unambiguously a pathological dystrophin. On the other hand, less pronounced deficiencies in dystrophin expression in BMD patients and DMD/BMD carriers may not always be detected in muscle biopsies. In this case, it is necessary to supplement the examination by Western blotting and genotype analysis. The examination of patients with clinically diagnosed muscular dystrophy should start with a muscle biopsy which enables the estimation of presence and degree of structural changes. Application of antibodies against the components of DGC and emerin may reveal a deficiency in expression of these proteins. Immunohistochemical examination completed by Western blotting leads to the subsequent molecular genetic analysis of DNA or mRNA. Secondary deficiencies in expression of other DGC proteins are often revealed in muscle biopsies of dystrophinopathies and this fact must be taken into account in the evaluation of immunohistochemical findings. There is a possibility of replacement of invasive muscle biopsy by skin biopsy or buccal mucosal smears in cases of merosin and emerin deficiencies. Commercially available antibodies against merosin, emerin, calpain and sarcoglycans enable extensive identification and detailed classification of muscular dystrophies. Screening of the patients based on the application of methods described and discussed in this report is the task of the forthcoming period.

No Abstract

Complex of osmium tetroxide with 1,10-phenanthroline (Os,phen) reacts with double-stranded B-DNA in contrast to osmium tetroxide, pyridine and other osmium structural probes which show a strong preference for single-stranded DNA (ssDNA) (Palecek, E. in Abelson, J.N., and Simon, M.I. (eds), Lilley, D.M.J., and Dahlberg, J.E., (volume eds.), Methods in Enzymology, Vol. 212, DNA Structures, part B., Academic Press, 139-155 (1992)). Modification of negatively supercoiled DNA (scDNA) with Os,phen changes the DNA electrophoretic mobility inducing the DNA relaxation at lower degrees of modification followed by formation of positive supercoils at higher modification extents. Electrophoretic mobility of the Os,phen-modified DNA fragments in agarose gel is almost unchanged while a strong retardation of the same fragments is observed in polyacrylamide gels. Os,phen-modified DNA is hypersensitive to nuclease S1. Cleavage of this DNA by restriction enzymes is selectively inhibited showing a preference of Os,phen for TA and AT dinucleotide steps. DNA modification by Os,phen is inhibited by low and moderate concentrations of MgCl2. The covalent binding of Os,phen to double-stranded DNA (dsDNA) is preceded by noncovalent interactions (probably intercalation) inducing DNA structural changes; the shape of the Os,phen-modified DNA molecule appears to be severely deformed.

Immunohistochemical method detecting dystrophin in muscle biopsies was introduced and applied in 121 cases with a large scale of neuromuscular diseases. A monoclonal antibody NCL-DYS 2 (Novocastra) was used for the detection of C-terminal domain of dystrophin. Normal, i.e. sarcolemmal, localization of dystrophin was found in controls, in inactivity atrophy, neurogenic lesions and congenital myopathies. A similar situation except regenerating fibres was found in myositis and progressive muscular dystrophies different from Duchenne (DMD) and Becker (BMD) types, DMD cases showed a complete or nearly complete loss of sarcolemmal reaction product, whereas a partial loss of dystrophin in membrane was found in BMD cases as well as in transmitter females. Fibres splitting during neurogenic and myogenic lesions had dystrophin in newly produced sarcolemmal parts. Sarcolemmal immunoreactivity starting as early as in the 10th-12th week of gestation was found in human fetuses.

Repeated intraperitoneal administration of F9 teratocarcinoma stem cells (first dose of 3.8 X 10(8) + 3 doses of 7.5 X 10(8) cells at two-week intervals) to guinea pigs starting from birth prevented in more than one third of them (in 10 out of 28) the induction of autoimmune aspermatogenesis by subsequent immunization with spermatogenic cells emulsified with FCA. Cytotoxic and immunofluorescent activities against spermatogenic cells were similar in groups of males after tolerance induction and subsequent immunization and of males immunized only. However, these two groups differed substantially in serological activities against F9 cells which were significantly higher in the former group. The results are discussed in connection with the establishment of the blood-testis barrier which may have resulted in the absence of autotolerance towards spermatogenic cell antigens.

The synthetic steroid antiandrogen, cyproterone acetate, and the androgen, testosterone isobutyrate, were injected subcutaneously into adult male and female guinea pigs of two outbred strains (laboratory coloured and albino Pirbright-Harley stocks). Cyproterone acetate was given as 1, 10 and 20 daily 5-mg doses (body-weight matched with immunosuppressively effective doses in mice) and as 30 doses of 15 micrograms/100 g b.w. (body-weight matched with those used in the long-term human male contraception), testosterone as 1 and 10 doses of 5 mg/100 g b.w. The guinea pigs of the two stocks used showed different sensitivity: with the same doses of CA albino guinea pigs compared to the coloured ones declined in weight, had lower viability, reduced thymus weight, diminished capacity for SRBC haemolysin formation, a lower testes weight and inhibited spermatogenesis. Both stocks reacted by similar lymphopoenia, however. Testosterone neither inhibited haemolysin formation nor produced lymphopoenia but induced a more marked reduction of thymus weight than did CA and, in addition, produced greater depletion of cortex lymphocytes in albino guinea pigs. The thymus of albino guinea pigs was more sensitive to testosterone than to cyproterone acetate. Sexual dimorphism was apparent in some criteria in albino guinea pigs following cyproterone acetate and testosterone. Cyproterone acetate in the lowest doses had no inhibitory effect on thymus or SRBC formation but also did not affect spermatogenesis in albino guinea pigs. The hitherto accepted classification of animal species according to which guinea pigs (and also man) belong to steroid-resistant species is discussed and questioned, and the potential risks of application of sexual steroids in male contraception are stressed.

Mice immunized with teratocarcinoma F9 cells or human blood group substances A, B or H exhibited significant inhibition of spermatogenesis comparable to the inhibition induced by immunization with testicular cells. All these immunization schemes resulted in the production of antibodies which recognize antigens common to F9 and spermatogenic cells. In addition to these antigens, both anti-F9 and anti-ABH sera also recognize antigens which are specific for F9 cells. One of them was identified as SSEA-1. These results support the hypothesis that oncofetal F9 antigens are carbohydrate structures, which may play an important role in spermatogenic cell differentiation.

The anti-F9 and anti-spermatogenic cell sera with known cytotoxic and immunofluorescent activities were produced in albino guinea pigs immunized with F9 or spermatogenic cells emulsified with Freund's complete adjuvant. Short-term administration of the globulin fractions (3 x 50 mg at 24-h intervals + 1 x FCA on the first day) obtained from these antisera to adult albino guinea pigs resulted in a significant inhibition of spermatogenesis and appearance in the sera of marked cytotoxic activity against F9 cells. These results together with the impairment of fertility in male and female mice immunized with teratocarcinoma stem cells described by other authors are interpreted as supporting the hypothesis that the F9 antigens may play an essential role in spermatogenesis and function of spermatozoa.

Monoclonal antibodies TEC-01, TEC-02, and TEC-03, which define three developmentally regulated antigens TEC-1 (SSEA-1-like), TEC-2, and TEC-3, have been used to isolate and characterize teratocarcinoma stem cell mutants with altered expression of surface glycoconjugates. Mutants lacking TEC-1 antigen have been isolated by exposing mutagenized P19S1801A1 cells to TEC-01 antibody, which was conjugated to the toxin from Ricinus communis. None of the mutants exhibits significant changes in the expression of TEC-3 antigen, but some are defective in the expression of TEC-2 antigen. Analysis of the expression of TEC-1,2,3 antigens in different lectin-resistant F9 and OTF9-63 cell lines has shown that all express TEC-1 antigen, but some lectin-resistant phenotypes exhibit reduction in the expression of TEC-2 and/or TEC-3 antigens. Mutational events in genes regulating the expression of specific glycosyltransferases or glycosidases appear to be the biochemical mechanism regulating the expression of TEC-1 and TEC-2 antigens.

Androgen receptors were found both in cytosol prepared from intact thymuses of the adult castrated B10. A male and in cytosol from thymuses of the castrated males that had been previously given whole-body irradiation with 6.0 Gy (60Co). Histologically, these thymuses were represented by the reticuloepithelial component, no lymphocytes were found in the cortex and small numbers of lymphocytes remained in the medulla. The 3H-dihydrotestosterone-receptor complex sedimented in the 4S region, as revealed by the 5-20% sucrose gradient centrifugation in buffer containing 0.4 M KC1. Free androgen and non-specifically bound androgen were removed by specific antibody coupled to CNBr-activated Sepharose-4B.

A side-effect of the administration of cyproterone acetate, an antiandrogenic steroid, to newborn, juvenile or adult male mice (in doses comparable to those used clinically) was found in a marked reduction of the white pulp of the spleen and reduced weight or even absence of the thymus.

Progestin (medroxyprogesterone acetate, trade name Depo-Provera, Upjohn, Belgium) in 6 body-weight-matched doses with those used in women, i.e., 0.05 mg/mouse, given intramuscularly at 4-5-day intervals (duration of one ovulation) induced a marked reduction of fertility with precocious atresia of Graafian follicles, minimal numbers of growing follicles and an excess of corpora lutea; the lymphoid system remained morphologically and functionally normal. A combination of progestin and androgen (testosterone isobutyrate, trade name Agovirin Depot Biotika, Czechoslovakia) in a total dose of 1 mg + 0.9 mg (body-weight-matched with that used in men) or one order higher and divided into 6 doses given at 17-day intervals, i.e. roughly one half of the duration of spermatogenesis, had no inhibitory effect on spermatogenesis, whereas higher doses damaged the lymphoid system. This damage was reflected in a markedly reduced thymus weight with great depletion of cortex lymphocytes and diminished capacity for antibody formation to sheep red blood cells. The significance of the immunosuppressive effect for estimating the risks involved in the administration of sex steroids in human contraception is discussed.

It has previously been shown that osmium tetroxide, pyridine (Os,py) and osmium tetroxide, 2,2'-bipyridine (Os,bipy) are powerful probes of the DNA structure. To increase the possibilities of the detection of osmium-modified DNAs polyclonal antibodies against DNA modified with Os,py and Os,bipy were elicited in rabbits. Specificity of these sera or purified IgG was tested by ELISA and retardation of the DNA electrophoretic mobility in agarose gels. Antibodies against DNA-Os,py (anti-DNA-Os,py) reacted with single-stranded and double-stranded DNA-Os,py but they did not react with unmodified DNA; with DNA-Os,bipy only a weak reaction was observed. The specificity of the anti-DNA-Os,bipy was similar. Competition experiments with anti-DNA-Os,py showed a weak reaction with RNA-Os,py but no reaction with osmium-modified proteins and unmodified proteins and RNA. The results suggest that anti-DNA-Os,py may become an important tool in studies of DNA structure in situ.

Mice homozygous for tAE5, a recessive allele at the complex T-locus, are characterized by their unique short-tailed phenotype as well as by runting and low fertility. Histological and histochemical studies of the lymphoid and reproductive systems disclosed structural changes in the mutant spleen resembling those found in autoimmune conditions. Involution of the mutant thymus was greatly accelerated compared to normal. Necrotic changes occurred during spermiogenesis whereas ovarian structure was normal in mutants. The possible mechanisms of the mutant effects are discussed in the framework of other similar syndromes and the mode of action of alleles at the complex T-locus.

Androgen derivatives appeared to have different biological activities in vivo and in vitro. Testosterone-17-isobutyrate given in three doses of 50 or 200 microgram increased significantly the weight of seminal vesicles and reduced thymus weight in castrated males, whereas testosterone-17-hemisuccinate, testosterone-D-beta-glucoside and testosterone-3-(O-carboxymethyl)-oxime had no such effects. Similarly, ten 1-mg doses of testosterone-17-isobutyrate, unlike testosterone-17-hemisuccinate, resulted in a marked reduction of thymus weight in non-castrated males and in a significant inhibitory effect on the activity of spermatogenesis. On the other hand, all three androgen derivatives, which had appeared inactive in vivo, had similar effects in vitro (as had active testosterone) as demonstrated by inhibition of Concanavalin A-induced lymphocyte activation expressed by 14C thymidine incorporation and inhibition of cell agglutination. These results seem to suggest that as for the regulation of androgen-dependent organs and functions (such as the size of seminal vesicles, activity of spermatogenesis, thymus size), hormonally active androgens are also involved in certain immunosuppressive effects in vivo. On the other hand, in vitro immunological effects are produced by both hormonally active and non-active androgen derivatives as well as by other steroid hormones, the common denominator being the steroid structure.

In the presence of pyridine and other ligands osmium tetroxide binds covalently to pyrimidine bases in DNA. Properties of osmium-modified native and denatured calf thymus DNA, and plasmid Co1E1 DNA were investigated by means of differential pulse polarography, absorption spectrophotometry, circular dichroism, agarose gel electrophoresis, and nuclease S1 digestion. A great difference in the reaction kinetics of native and denatured DNAs with osmium, pyridine was observed. On the ground of the slow stepwise reaction kinetics of native DNA in the initial stage of its modification by osmium it has been suggested that the primary reaction sites do not include bases contained in the intact double helix. Osmium binding to sporadic primary reaction sites (represented e.g. by bases in the vicinity of a single-strand break) in native calf thymus DNA resulted in local changes in DNA conformation limited to a close neighbourhood of the binding site. At higher osmium/nucleotide ratios disordering of the DNA structure over a region extending beyond the immediate binding site was observed. With denatured DNA the same type of structure disordering was detected already in the initial stage of the reaction at osmium/nucleotide ratios as low as 0.01. Osmium binding to the supercoiled Co1E1 DNA resulted in its relaxation without nicking and it increased its sensitivity to linearization by cleavage with nuclease S1. The behaviour of Co1E1 DNA has been explained by the formation of a denatured region in the molecule (accompanied by a coupled loss of duplex and superhelical turns). It has been suggested that osmium can be used to label and to visualize distorted regions in the DNA double helix.

Significant inhibition of spermatogenesis and production of antibodies against membrane antigens of spermatogenic and F9 teratocarcinoma cells were observed in mice of the strain 129/Sv after immunization with human blood group substances from saliva of A, B or H secretors. Absorption of the mouse anti-ABH sera with appropriate human erythrocytes did not change their reactivity with testicular and F9 cells, whereas absorption with F9 cells eliminated the reactivity with both F9 and spermatogenic cells. This pattern of reactivity, together with higher binding of the anti-ABH sera to the cells expressing stage-specific embryonic antigen 1 (SSEA-1), suggests that these antisera contain antibodies against developmentally regulated carbohydrate antigens. SSEA-1 was found in the blood group substances used for immunization. The results support the hypothesis that the oncofetal F9 antigens and spermatogenic differentiation antigens are similar carbohydrate structures.

A method has been developed for the isolation of plasmid DNA suitable for physical and physicochemical measurements. The procedure is based on the deproteinization of the cleared lysate of bacterial cells (after amplification of plasmids by chloramphenicol) by phenol at pH 8.0 and subsequent removal of chromosomal DNA by means of phenol at pH 4.0 and separation of RNA on a hydroxyapatite column at higher temperature. ColE1 DNA sample was compared with samples of the same DNA prepared by three thus far used methods. Samples obtained by means of the latter methods were contaminated with chromosomal DNA, RNA, or ethidium bromide. The presence of ethidium bromide in the DNA sample was a factor interfering in the electrochemical analysis, chromosomal DNA and RNA were disturbing in the use of other methods. DNA separated by the method devised by us was free of any detectable contaminants and fulfilled the high requirements for sample purity of differential pulse polarography. Measurements performed by means of differential pulse polarography showed that the content of single-stranded segments in superhelical ColE1 DNA is less than 0.15% (i.e. less than 20 bases per molecule). This is in keeping with the notion that a cruciform is formed in this DNA (as a result of tension due to supercoiling) in the region of inverted repeat sequence, containing only 5 bases in the single-stranded loop region.

Two monoclonal IgG3 (kappa) antibodies against murine Thy-1.2 antigen produced by murine 1B5 and 1aG4/C5 hybridomas were partially characterized. The 1aG4/C5 antibody has slightly higher affinity for the Thy-1.2 antigen in binding tests and more efficiently kills the Thy-1.2+ thymocytes in cytotoxicity assays as compared to the 1B5 antibody. The latter, in addition, reacts significantly with the Thy-1.1 antigen (the allelic form of Thy-1 antigen expressed on the cells of the donor of the immune cells. Both monoclonal antibodies exhibit some characteristic properties of IgG3 of myeloma origin, e.g. a tendency to aggregation, high pI and interaction with protein A. Our monoclonal antibodies are sensitive to pepsin digestion, resistant to trypsin, their disulphide bonds are rapidly cleaved by sulphitolysis and reduction by dithiothreitol. They possess characteristic acidic peptides bearing the disulphide bonds between the heavy chains. These antibodies, however, differ to some extent from each other in some properties (precipitation with staphylococcal protein A, solubility, pI, electrophoretic behaviour of the light chains). They possess different heavy chain peptides bearing the interchain disulphide bonds and thus they probably differ in the hinge region. This structural difference may be associated with different sensitivity of these two antibodies to sulphitolysis and proteolysis.

The synthetic steroid antiandrogen cyproterone acetate (CA) was administered subcutaneously to adult male mice of the inbred strains B10, B10.D2, B10.BR and C57BL/6 either as a single dose of 1 mg or as 10 daily doses of 0.5 mg. At 48 h after a single dose of CA, the thymus and spleen weights were markedly reduced and the morphology of the thymus was changed (depletion of cortex lymphocytes) in some strains. At 6 h and at 12 days after the 10th dose of CA, thymuses in males of all strains were markedly diminished with a histologically apparent involution affecting the cortex, as well as the medulla and reticuloepithelial tissue. Spleen weights in males of some strains were also significantly lower. SRBC haemolysin titres at 12 days after the 10th dose and spermatogenesis at 6 h and at 12 days after the 10th dose were significantly decreased in males of all strains; in none of the males was sterility produced by the CA doses used, however, Indications of the strain differences could only be seen in the onset and duration of the damage to the spleen and the corresponding decrease in haemolysin formation. The importance of the consequences of short-term administration of CA is discussed in terms of potential risks for long-term administration of CA in the regulation of male fertility.

Covalent binding of osmium tetroxide to negatively supercoiled DNA in vitro initially induces its relaxation, accompanied by a formation of a single denaturation "bubble" per molecule. Binding of further osmium results in DNA overwinding and the appearance of positive supercoils as demonstrated by gel electrophoresis and electron microscopy.

Local structure of the homopurine.homopyrimidine tract in a supercoiled plasmid pEJ4 was studied using chemical probes at single-nucleotide resolution. The conformation of the homopyrimidine strand was probed by osmium tetroxide, pyridine (Os,py) while that of the homopurine strand was tested by diethyl pyrocarbonate (DEPC), i.e. by probes reacting preferentially with single-stranded DNA. At weakly acidic pH values, a strong Os,py attack on three nucleotides at the centre of the (dC-dT)16 block and a weaker attack on two nucleotides at the end of the block were observed. DEPC modified adenines in the 5'-half of the homopurine strand. Os,py modification at the centre of the block corresponded to the loop of the hairpin formed by the homopyrimidine tract, while DEPC modification corresponded to the unstructured half of the homopurine strand in the model of protonated triplex H form of DNA.

Partial absorption of oligospecific reagents by the particulate membrane fraction prepared from isolated interstitial cells grown in culture (the harvested cell population contained about 80% Leydig cells)suggests that membranes of Leydig cells carry antigenic specificities H-2K (11,25) and H-2D (4) but not antigens controlled by the I region. The results of absorption experiments have been confirmed by the methods at the level of individual cells; the native Leydig cells gave a positive reaction in the dye exclusion cytotoxic and immunofluorescent tests with he polyvalent regent B10D2 and B10 (directed against antigens of the regions H-2K through H-2I-E).

The effects of a prolonged subcutaneous administration of SCH 13521 dissolved in 0.3% hydroxypropyl cellulose (2-8 weeks in daily doses of 0.2 or 1.0 mg amounting to an estimated equivalent of experimental and curative doses used by others in laboratory animals and men) were studied in males of the mouse inbred strain C57BL/6. Following the treatment, the activity of spermatogenesis (expressed as the mean number of seminiferous tubules containing mature sperm and epididymal sperm count) was inhibited while the testis weight was not reduced, obviously due to an absolute increase of the interstitial tissue which was a marked histological feature of the testes, particularly following the higher doses of SCH 13521. Lower doses and shorter-lasting administration of the compound seem to inhibit the activity more effectively because after a prolonged administration reparatory processes tend to be triggered via a stimulatory effect on the synthesis of testosterone in Leydig cells. The solvent alone, hydroxypropyl cellulose, had some inhibitory effect on spermatogenesis. The lymphoid system remained both morphologically and functionally unaffected by SCH 13521 unlike the steroidal antiandrogen cyproterone actetate.

A specific antiandrogenic steroid cyproterone acetate was administered daily to mice of three different inbred strains starting from the day of birth until the age of 30 days. The total dose per mouse was 17.2 mg. This treatment resulted in developmental retardation which was manifested in a number of ways: at the age of 30 days, the weight of the body was well as spleen, testes and particularly thymus was significantly reduced; histologically, the normal proportion of the red and white pulp in the spleen was changes; spermatogenesis (but not oogenesis) was markedly retarded corresponding to the age of 12-15 days in normal males; also skin displayed a persisting immaturity as reflected by an abundance of mast cells. Minor signs of toxic changes were seen in the liver. Skin grafts from CA-pretreated donors had a subnormal immunogenicity; when transplanted across the MSA-barrier, they survived significantly longer than control grafts and about 23% took. Significantly prolonged survival was also observed with H-3 incompatible skin grafts from CA-pretreated donors, particularly from male donors. Across the barrier dicated, but did not reach a level of significance. The present study extends our previous observations concerning the androgen dependence of a normal immunogenic expression of H-antigens. The antiandrogenic effect of CA is comparable to the more complex effect of neonatal orchiectomy in terms of the subnormal immunogeneity of MSA-incompatible skin grafts from 30-day-old males which seems to be arrated at a stage typical for 1-2-day-old normal males.