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1 ) The antifertility effects of a number of simple alkane sulphonic esters have been examined in the male rat by a combination of serial mating of treated animals and histological examination of the testes. The results are compared and contrasted with those obtained with Busulphan (Myleran), dimethylmyleran and various derivatives of ethyleneimine.(2) A spectrum of results on various stages of spermatogenesis has been obtained, which is difficult to reconcile with a common mode of action due to alkylation of cellular materials.(3) Certain simple esters produce strikingly different effects. Thus, methylethanesulphonate and methylmethanesulphonate cause early sterility due to interference with spermatids and spermatozoa. Isopropyl alkane sulphonates, on the other hand, cause sterility only after a delay of several weeks.(4) These esters are effective by mouth and their action is cumulative. Methylmethanesulphonate and methylethanesulphonate in small daily doses produce predictable periods of sterility, according to the dose rate given, which are completely reversible. There is no indication of any action on libido, nor evidence so far of toxic effects from these courses of treatment.(5) With the series of compounds examined, it has been possible to move the focus of drug action from spermatogonia to spermatids and spermatozoa with reduction of side effects. It is concluded that this work demonstrates that selective interference with fertility in the male is a distinct possibility.

The effects of tumour inhibitory doses of tretamine (triethylenemelamine), busulphan, and melphalan on the fertility of male rats have been examined. The aromatic nitrogen mustard, melphalan, was inactive, but busulphan has a highly selective action on spermatogenesis which contrasts strikingly with that of tretamine. The main action of tretamine was exerted upon spermatocytes or spermatids, but, with increasing dose, the effects spread to involve a wide range of spermatogenic cells including mature sperm, so that infertility could be induced very rapidly. Busulphan, however, interfered with the development of spermatogonia for several weeks, although other germinal cells were unaffected and continued to develop into mature spermatozoa. This accounted for the continuation of normal fertility for 7 weeks after a dose, before sterility suddenly developed. The antifertility activity of tretamine could be simulated by a variety of other ethyleneimino compounds, potency being greatest in trifunctional and least in monofunctional compounds. The latter were, however, very destructive to the seminiferous epithelium with increasing dose. In the rat, there appeared to be no definite relationship between the ability of alkylating substances to interfere with the activity of normal and pathological proliferating tissues, as represented by the germinal epithelium, haematopoietic, and tumour tissue. Although carcinogenicity was a biological property of alkylating agents, other chemical types of carcinogen did not interfere with fertility.In small doses, tretamine (triethylenemelamine) produces selective effects on spermatogenesis in rats, as revealed by fertility studies (Bock and Jackson, 1957). It appears that the drug can interfere with various stages of this complex process so that the sperm produced, although capable of reaching and penetrating ova, are incapable of promoting further development. Tretamine is well known for its cytotoxic effects due, it is thought, to some specific action on dividing cells. Because of its similarity to radiation, tretamine is classified as a radiomimetic drug. The present work describes the comparative actions on male rat fertility of tretamine, busulphan (Myleran), and melphalan as representative of three chemical types of alkylating radiomimetic substances. In addition the activities of a number of tri-, di-, and monofunctional ethyleneimino compounds have been examined. METHODS AND MATERIALSThe animals used were an inbred strain of Wistar rat of American origin, maintained on a standard diet provided by the Scottish N.E. Agricultural Society. The method of investigating the effect of a drug on male rat fertility has been described previously (Bock and Jackson, 1957). Briefly, it consisted of pairing treated male rats with females of established fertility, and replacing the latter at intervals of 1 week for as long as considered necessary. The date of insemination was determined by examination of a vaginal smear each morning. Because of the number of females required in each experime...

1. rRNA was isolated from rat liver at short intervals after the intraperitoneal injection ofThese doses were chosen to minimize the effects of toxicity. 2. The following methods of hydrolysis of [14C]methylated rRNA were employed: enzymic digestion to nucleosides at pH8; alkaline hydrolysis and conversion into nucleosides; acid hydrolysis to bases. 3. The methylation products were analysed by chromatography on columns of Dowex-50 (HI form) and Dowex-50 (NH4+ form). 4. With both methylating agents the principal product of methylation was 7-methylguanine. Differences were obtained, however, in the molar proportions of the minor bases 3-methylcytosine, 1-methyladenine and 7-methyladenine. Methylation at the 0-6 position of guanine was a significant feature of rRNA obtained from the NN-di[14C]methylnitrosamine-treated animals but was not detected in rRNA after treatment with [14C]methyl methanesulphonate.

Summary.-The reaction of the hepatocarcinogen N,N-dimethylnitrosamine has been compared with that of methyl methanesulphonate, a methylating agent which is not a liver carcinogen. Consistent differences have been observed in the reaction of rat liver DNA in vivo with these agents; 06-alkylation and the production of unidentified acid-labile products were distinctive features of the reaction with the carcinogenic nitroso compound but were undetectable or in low yield, respectively, after reaction with the alkyl sulphonate. Evidence has been obtained for the excision of these reaction products in animals treated with the hepatocarcinogen and the significance of their relative stabilities is discussed.

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Serial sperm counts have been made in rabbits treated with alkylating agents. After single injections of Busulphan, Tretamine and isopropyl methanesulphonate, aspermia developed during the 10th and 11th weeks. Other simple monofunctional esters of methane and ethane sulphonic acids, which induced sterility in rats as a result of their action on spermatozoa and late spermatids, did not depress the sperm count or interfere with sperm motility in the rabbit. The results suggest that the duration of spermatogenesis in the rabbit is about 10 weeks and indicate that in this species, as in rats and mice, a steady turnover of spermatozoa occurs. Fertility tests in male rabbits after administration of Tretamine showed that small doses readily induced sterility which could be maintained by continued administration.

1. RNA was isolated from rat liver at selected times after the intraperitoneal injection of either [(14)C]methyl methanesulphonate (50mg/kg) or [(14)C]dimethylnitrosamine (2mg/kg). These doses were chosen to minimize effects due to toxicity. 2. Two methods of extraction and purification of RNA were used and an analysis of the radioactivity present was made by column chromatography of acid hydrolysates of the purified RNA. 3. The extent of methylation of guanine, the principal site of alkylation in rat liver RNA, was determined at times up to 14 days after injection. Although dimethylnitrosamine is a potent liver carcinogen and methyl methanesulphonate is not carcinogenic to rat liver, the rate of disappearance of 7-methylguanine from RNA was similar for both compounds, with a half-life of about 3.5 days. 4. An estimate of the biological half-life of rRNA was made by using [(3)H]orotic acid. A half-life of 5 days was obtained and this was not affected by injecting animals with unlabelled methyl methanesulphonate at the same dosage of 50mg/kg used in the studies of RNA methylation. 5. After administration of labelled orotic acid, reutilization of labelled RNA degradation products probably results in an overestimation of the biological half-life for rRNA. It is suggested that non-toxic doses of methylating agents such as methyl methanesulphonate and dimethylnitrosamine may prove to be a more effective way of accurately estimating the biological turnover of RNA species.