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J. MATOUŠEK

Summary.

The antigenicity of bull seminal ribonuclease (AS RNase) appears to depend on the species of the recipient.

Partly purified AS RNase formed haemagglutination and precipitation antibodies in rabbits against the original antigen and also against pancreatic RNase A. Absorption of these antisera by pancreatic RNase A demonstrated that the antisera to the seminal RNase were still present in the sera. Rabbit antibodies to bovine pancreatic RNase A reacted with its homologous antigen and AS RNase.

Antibodies did not prevent the action of AS RNase in vivo. Antigenantibody complexes injected (i) into the testes of adult mice caused degeneration, (ii) into pregnant mice caused embryonic mortality, and (iii) into 6-day-old mice resulted in death.

Production of antibodies to AS RNase in mice also caused testicular damage, embryonic death and degeneration of Crocker tumour cells. Egg production in hens was reduced by similar treatment.

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J. MATOUŠEK

Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, Liběchov, Czechoslovakia

(Received 7th October 1974)

A protein substance responsible for embryotoxic and aspermatogenic effects in mice was isolated from bull seminal vesicle fluid by Dostál & Matoušek (1973) and its ribonuclease activity was determined (Matoušek, Pavlok, Dostál & Grozdanovič, 1973). This enzyme also caused disturbances of embryonic development in guinea-pigs and rabbits (Matoušek, Fulka & Pavlok, 1973).

The present paper presents an account of the embryotoxic effect of the enzyme (AS RNase) in rats and the results of the study of absorption and binding of AS RNase by various embryonic and adult tissues.

Wistar rats weighing 250 to 270 g and 5 to 10 days pregnant were given a single subcutaneous injection of 40 mg pure AS RNase, together with 40, 80 or 120 mg partly purified ribonuclease designated AS RNase (BS) (Dostál & Matoušek, 1973) diluted in

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J. MATOUSEK

Summary.

The effect on spermatogenesis of intratesticular and subcutaneous immunization with genital tract fluids of bulls was studied in sexually mature guinea-pigs, rabbits and sheep.

Immunization with bovine seminal vesicle fluid produced a decrease in the index weight of the testes, a disturbance of spermatogenesis and, in more severe cases, a distortion and contraction of the seminiferous tubules. In all animals immunized subcutaneously, identical circulating antibodies were found in animals with damaged testes and in animals with normal testes. They did not cause spermagglutination, immobilization or spermatotoxic reactions. No circulating antibodies could be found in guinea-pigs and rabbits immunized by a single intratesticular injection.

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J. MATOUŠEK

Summary.

Subcutaneous injection of the seminal vesicle fluid of bulls into female mice which were subsequently mated resulted in a reduction in the number of embryos. The action of a number of different enzymes on the seminal vesicle fluid did not remove its antifertilizing activity.

The supernatant resulting from precipitation of the seminal vesicle fluid with acetic acid and ammonium sulphate had considerable antifertilizing activity. Injection of this fraction before and after mating demonstrated the antiembryonic character of the antifertilizing substance. Neither the number of ovulated eggs nor their fertilization was affected by the antiembryonic substance and there was no adverse effect on lactation, but the body weight of young, and even of adult, animals declined.

The antiembryonic substance affects the embryo at an early stage and continues to affect the organism for a long time. Necrotic changes can be observed in embryos as early as the 3rd day after the first injection. The number of leucocytes decreases for a short time after injection of the antiembryonic substance.

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J. DOSTÁL and J. MATOUŠEK

Damage to spermatogenesis in mice, rats, guinea-pigs, rabbits and rams after the intramuscular or subcutaneous administration of bull seminal vesicle fluid was reported by Matoušek (1966, 1969). Matoušek & Petrovská (1969) also observed an inhibitory effect of bull seminal vesicle fluid on the fertility of female mice and hens. The present report deals with the separation and purification of a substance that causes aspermatogenesis in mice.

Seminal vesicle fluid obtained from mature bulls at slaughter was diluted 2·5-fold with 2% acetic acid and mixed for 30 min at room temperature. The precipitate was separated from the supernatant fluid by centrifugation at 7000 g for 45 min. The aspermatogenic substance remained in the supernatant fluid. The bulk of the protein was then precipitated by 3 m-ammonium sulphate and separated from the supernatant by further

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J. DOSTÁL and J. MATOUŠEK

Summary.

Aspermatogenic substance (AS) was isolated from bull seminal vesicle fluid by precipitation with acetic acid and ammonium sulphate, and subjected to CM Sephadex C-50 and Sephadex G-100 column chromatography. The molecular weight of the monomer of AS was 22,000 and of the dimer was 44,000 as determined by gel filtration. The purity of the AS was controlled by disc electrophoresis in acrylamide gel, starch-gel electrophoresis, immunoelectrophoresis in agar gel, by peak homogeneity at the last step of separation and by the ultracentrifugation pattern. The N-terminal amino acid was lysine. The absorbance of 0·1% AS at 280 nm in pH 5·5 buffer was 0·510. The s value obtained for 0·5% AS solution was 2·7.

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J. MATOUŠEK and EVA PETROVSKÁ

The finding of the aspermatogenic effect of the seminal vesicle fluid of bulls (SVFB) in heterologous species (Matoušek, 1966, 1969) led us to preliminary experiments to examine its effect on females.

Mice (C57BL) were injected s.c. with a mixture of SVFB fluids in four doses of 0·05 ml without adjuvant administered in the course of 8 days. Hens (Rhode Island Red) were injected i.v. with the same fluid on three subsequent days at 1 ml per dose. Two days after the last injection, the mice were mated within 10 days to males of the same strain and the mating controlled by copulation plugs. Twelve days after mating the mice were killed and the number of implanted embryos determined.

The hens were observed for egg laying 3 weeks before the experiment and 3 weeks after the first

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J. MATOUŠEK, R. STANĚK and L. VESELSKÝ

Summary.

The biosynthesis of the aspermatogenic substance in the seminal vesicle fluid of bulls is dependent on the androgen level. The aspermatogenic substance is not present in sexually immature bulls; its synthesis can be evoked, however, by means of testosterone propionate. Castration is followed by a loss of libido and a decline in the production of fluids from the accessory genital glands; injections of testosterone propionate restore libido, the secretion of fluids and their aspermatogenic effect.

The aspermatogenic substance is thermostable, it is not digested by a number of enzymes including proteolytic enzymes, but it must evidently be bound to a protein complex in order to induce aspermatogenesis.

Rabbits developed several antibodies against bull vesicular fluid treated with enzymes, as judged by double diffusion, immunoelectrophoresis and haemagglutination tests. The number and character of the precipitation and haemagglutination antibodies in immunized animals did not differ whether the testes were damaged or undamaged. The fluorescence technique did not detect any binding of antibodies either to epididymal spermatozoa or to histological sections of the testes of rabbits and bulls.

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J. MATOUŠEK, A. PAVLOK, J. DOSTÁL and J. GROZDANOVIČ

Summary.

Injection of mouse testes with aspermatogenic substance (AS) isolated from bull seminal vesicle fluid inhibited sperm production and caused a decrease in the weight of the testes. Subcutaneous injections of AS also caused a decrease in testis weight, but spermatogenesis was only slightly affected.

Administration of AS (2 to 2·5 mg) to female mice 1 to 13 days after mating resulted in a highly significant decrease in the number of embryos. When concentrations of 12·5 μg AS/ml, or more, were present in the medium in which two-blastomere mouse embryos were incubated, further development was arrested in 100% of the embryos. In vivo, however, death of the embryos occurred only after nidation. Injection of AS into prepubertal males or females did not provoke any visible disturbances at the time, or later when sexual maturity was reached.

The presence of AS in the diluent had no effect on the sperm motility or sperm survival time of bull, ram, boar and rabbit spermatozoa or on the fructolysis of bull and ram spermatozoa.

Samples of AS showed no proteolytic, DNase, esterase, alkaline phosphatase, acid phosphatase or lactate dehydrogenase activities but had high RNase activity and AS was therefore named AS RNase. Incorporation of [2-14C]orotic acid into male mice injected with AS RNase made it possible to detect a significant reversible decrease of pyrimidine synthesis in testicular tissue. No decrease of RNA in liver and kidney tissue could be detected.