androgen on the seminal vesicles it had neither a blocking effect on the penile

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1 MORPHOLOGICAL AND BEHAVIOURAL EFFECTS OF AN 'ANTIANDROGEN' IN MALE RATS F. A. BEACH and W. H. WESTBROOK Department of Psychology, University of California, Berkeley, California 94720, U.S.A. (Received 23 February 1968) SUMMARY Male rats injected with 10 mg. cyproterone acetate daily for 28 days were given weekly mating tests with receptive females. At the conclusion of testing the animals were killed and the testes and seminal vesicles were weighed. These organs and the glans penis were then prepared for microscopic analysis. The experimental treatment had no detectable effect upon copulatory behaviour nor upon the testis or the cornified papillae in the integument of the glans penis. The seminal vesicles of treated males appeared to be non-secretory and were lighter than those of control rats but heavier than the vesicles of untreated castrates. It is concluded that although cyproterone acetate partially blocked the action of endogenous androgen on the seminal vesicles it had neither a blocking effect on the penile papillae nor on the central nervous mechanisms which mediate mating responses. INTRODUCTION Compounds which prevent testicular hormone from exerting its normal effects on the organism but do not interfere with the secretion of the hormone have been called 'antiandrogens'. One substance alleged to possess such properties is cypro terone acetate (CA) which has the following formula: l,2a-methylene-6-chloro-a6-17a-hydroxyprogesterone acetate. According to Neumann & Elger (1966) male rats given CA very early in development show anatomical feminization and female-like sexual responses. At the same time the masculine mating reactions are incomplete. Another report from the same laboratory states that adult males given 3-0 mg. or 0-3 mg. CA/day for 3 weeks were 'sexually inactive' for 10 days after cessation of treatment (Investigator's Manual, Schering Co., 1966). In an attempt to repeat part of the latter findings Zucker (1966) administered CA to male rats and guinea-pigs and found that standardized, quantitative tests of copulatory behaviour (not reported by Neumann and his associates) revealed no effect of the treatment. Whalen & Edwards (cited in Whalen, 1968) injected a control group of castrated male rats with testosterone propionate and an experimental group with testosterone propionate plus CA. The groups did not differ with respect to copulatory performance, but the seminal vesicles were significantly lighter in males receiving CA than in those receiving androgen alone. 25 Endoc. 42, 3

2 380 F. A. Beach and W. H. Westbrook The present study was conducted for two purposes : (1) to repeat and extend earlier work on possible behavioural effects of CA and (2) to discover whether this substance interferes with the stimulant action of endogenous androgen on the cornified papillae in the outermost layer of the integument of the glans penis. These spike-shaped structures are greatly reduced or eliminated within 3-4 weeks after castration but can be maintained or restored by treating castrated males with testosterone propionate (Lyons, Berlin & Friedlander, 1942). The amount of TP necessary to maintain the number of genital papillae at precastration level is approximately equal to the amount needed to maintain normal copulatory performance (Beach & Levinson, 1950). Tissue lying immediately beneath the cornified papillae is liberally supplied with encapsulated nerve endings and it has been suggested that during coitus the papillae function as accessory sensory structures much as a hair, acting as a lever, serves to excite neural activity in the endings at the base of the follicle (Beach & Levinson, 1950). MATERIALS AND METHODS Adult male rats of the Long-Evans strain were used which had been given extensive copulatory experience before the present investigation. Nine males were first observed in two standardization tests with receptive females. The apparatus and techniques employed have been standardized and used in our laboratory for many years ; they yield highly consistent results (Beach & Jordan, 1956). Treatment was then begun and for 4 weeks five rats received daily i.m. injections of 10 mg. CA in 0-2 ml. sesame oil, while four control animals were injected with the same volume of inert oil. To prepare the active material, crystalline cyproterone acetate was dissolved in the smallest possible volume of CP benzyl benzoate and the resulting solution was diluted with sesame oil to a concentration of 50 mg./ml. While treatment was in progress, all males were given formal mating tests at weekly intervals. Their sexual behaviour was thus measured after the experimental animals had received a total of 70, 140, 210 and 280 mg. of CA. Within 24 hr. after the final test the rats were killed, the seminal vesicles were weighed (dry) and they and the glans penis were fixed in formalin. Subsequently samples of tissue from the vesicles and glans of each animal were sectioned and stained for histological examina tion. RESULTS Twenty-eight daily injections of 10 mg. CA had no detectable effect upon any of the standard measures customarily recorded during mating tests. These included the speed with which copulation was begun, the frequency and timing of the intro missions which preceded ejaculation, and the length of the post-ejaculatory interval before copulatory activity was resumed. In none of these indices of sexual responsive ness and ability did the experimental and control groups differ significantly, nor was there any consistent trend or change in the course of the experiment. Four weeks of CA treatment did exert clear-cut effects on the seminal vesicles, but not on the testes. Testis weight expressed as % of body weight was {R = ) for the control group and (R = ) for the experi mental rats. Microscopic examination of sample sections of testes from CA-treated

3 Effects of an antiandrogen 381 males indicated that secretory function was unimpaired. The effects of CA injection on the seminal vesicles were qualitatively similar to but quantitatively less than those produced by castration. They were revealed in part by weight records shown in Table 1 which includes values for normal males and untreated castrates not used in this study. According to the Mann-Whitney?7-test the difference between untreated normal and cyproterone control groups is not statistically significant, but the difference between control and experimental groups is significant and so is that between the experimental group and the untreated castrates. As shown in the Table, there is no overlap between any two of these three groups. Weight of seminal vesicles as % of body weight and average number of cornified papillae of one cross-section of the glans penis of the rat Table 1. Seminal vesicles Weight Number -'-, of Group n Mean Range papillae Normal untreated rats Controls (inert oil) Cyproterone acetate Castrated rats, untreated Examination of the histological material showed that CA treatment had almost totally suppressed secretory activity in the vesicles. Photomicrographs reproduced in the Plate illustrate this effect. The cells lining the lumen in vesicles of control males were tall, and the abundant cytoplasm was full of secretory granules. Appre ciable amounts of secretion had accumulated in the lumen. In the seminal vesicles of rats treated with CA the lumina contained no extracellular secretory material. The cells of the lumen walls were clearly inactive, being relatively flat and consisting primarily of a nucleus but very little cytoplasm. In contrast to the seminal vesicles, the integument of the glans penis appeared to be unaffected by 4 weeks of CA treatment. Cross-sections through the glans of control and experimental animals showed no differences. As shown in the Plate, cornified papillae were as large in the treated males as in those which had received only control injections of sesame oil. The relative density of papillae was estimated by counting the number present in three cross-sections of the glans taken from males in each group. Mean values are shown in Table 1. DISCUSSION The results of this investigation agree with those of Zucker (1966) and of Whalen & Edwards (Whalen, 1968) in showing that, at the concentration and for the length of treatment here employed, CA does not affect the male rat's copulatory responses to receptive females. From this finding it can be tentatively concluded that the experimental treatment did not interfere with the action of endogenously produced androgen on the CNS. In contrast, CA prevented the circulating androgen from exerting its normal stimulative effects upon the seminal vesicles. It should be noted, however, that this 25-2

4 382 F. A. Beach and W. H. Westbrook inhibition of androgenic action was not complete, for the seminal vesicles of experi mental males were significantly heavier than those of untreated castrates. Apparently the androgen-dependent papillae of the glans penis are less sensitive to CA than are the vesicles because treatment which clearly depressed the secretory activity of the vesicles had no noticeable effect on the size or number of penile papillae. The fact that the seminal vesicles were affected by the experimental treatment but the glans penis and the CNS were not is open to at least three interpretations : (1) CA may not interfere with the action of testosterone on the glans and the nervous system. (2) The vesicles may be more sensitive to androgen and larger doses of CA might have inhibited androgenic stimulation of the glans and nervous system. (3) The vesicles may react more promptly to a decrease in androgenic activity, and more prolonged administration of the same concentration of CA might eventually have induced regressive changes in the glans and in behaviour. Evaluation of these alternative explanations must await further investigation. This study was supported in part by Public Health Service Research Grant MH from the National Institute of Mental Health. Dr Martin Friedrichs of Berlin Cyproterone acetate was generously supplied by Laboratories, Inc., New York, N.Y. REFERENCES Beach, F. A. & Jordan, L. (1956). Sexual exhaustion and recovery in the male rat. Q. Jl exp. Psychol. 8, Beach, F. A. & Levinson, G. (1950). Effects of androgen on the glans penis and mating behaviour of castrated male rats. J. exp. Zool. 72, Lyons, W. R., Berlin, I. & Friedlander, S. (1942). Cornification of the balanopreputial epithelium in normal rats and in castrated rats treated with testosterone propionate. Endocrinology 31, Neumann, F. & Elger, W. (1966). Permanent changes in gonadal function and sexual behaviour as a result of early feminization of male rats by treatment with an anti-androgen steroid. Endokrinologie 50, Whalen, R. E. (1968). Differentiation of the neural mechanisms which control gonadotrophin secretion and sexual behaviour. In Perspectives in reproduction and sexual behaviour : A memorial to William C. Young. Ed. M. Diamond. Bloomington: Indiana University Press. (In press.) Zucker, I. (1966). Effects of an anti-androgen on the mating behaviour of male guinea-pigs and rats. J. Endocr. 35, DESCRIPTION OF PLATE Seminal vesicles ( x 400) and glans penis ( x 100) of normal (A), cyproterone acetate-injected (B) and castrated (C) rats.

5 Journal of Endocrinology, Vol. 42, No. 3 Vesicle Plate Glans A B c F. A. BEACH and W. H. WESTBROOK (Facing p. 382)