EFFECT OF CYPROTERONE ACETATE ON PUBERTY IN RATS. Experimental Research Pharma, Schering AG, period. Certain advantages of antiandrogen

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1 EFFECT OF CYPROTERONE ACETATE ON PUBERTY IN RATS H. STEINBECK and F. NEUMANN Experimental Research Pharma, Schering AG, Department of Endocrinology, 1 Berlin 651 {Received I5tk August 1970) Summary. Cyproterone acetate treatment delayed the ability of adolescent male rats to inseminate and impregnate female rats. Within 6 weeks of the cessation of treatment, full fertility was achieved. After a 9-week recovery period, the accessory sexual gland weights were still less than those of controls. From experiments on adult rats, this might have been expected because the recovery time was too short. Body weights remained below those of controls throughout period. Certain advantages of antiandrogen medication in cases of male precocious puberty are suggested. the observation treatment over other steroid INTRODUCTION Puberty has been defined as the stage of development at which the ability to reproduce sexually is achieved, i.e. when germ cells are released (Asdell, 1946; Critchlow & Bar-Sela, 1967). In male rats, it is not possible to advance this time because of the length of the spermatogenic cycle which already covers most of the period between birth and puberty. A further problem is to attempt to delay the onset of puberty without interference with later reproductive life. Paedia tricians are confronted with this problem in cases of precocious puberty. Medroxyprogesterone acetate has been the main steroid used clinically in boys and girls (Kupperman & Epstein, 1962; Bierich & Nowak, 1965; Laron & Rumney, 1965; Thamdrup, 1965; van der Werff ten Bosch, 1965; Schoen, 1966), the principal objective of treatment being to prevent androgen-mediated premature epiphysial closure and resultant dwarfism. The effective principle of medroxyprogesterone acetate action is not a direct androgen antagonism but rather an inhibition of gonadotrophin release which, in turn, leads to decreased androgen secretion (see Neumann, 1969). The use of antiandrogens in the treatment of precocious puberty has, therefore, been proposed (Laron, 1968; Helge, Weber, Neumann & Hammerstein, 1969; Steinbeck, von Berswordt- Wallrabe, Elger, Hahn & Neumann, 1969) as a more specific form oftreatment. This study deals with the effects of peripubertal cyproterone acetate treat ment on the reproductive performance of male rats. The influence of this treat ment on bone growth has been published elsewhere (Hertel, Kramer & Neumann, 1969). 59

2 H. Steinbeck and F. Neumann MATERIAL AND METHODS Thirteen male Sprague-Dawley rats were given a daily subcutaneous injection of 5-0 mg cyproterone acetate/100 g body weight from the 25th to 80th day of life. Commencing at the 40th day of life, fertility tests were performed at weekly intervals until a 100% impregnation rate was achieved in three successive tests. The body weight was also recorded every week. To evaluate insemination and impregnation rates, each male was placed overnight with two pro-oestrous females. The following morning, the females were separated from the males and checked for vaginal plugs and the presence of spermatozoa in vaginal smears. Eight days later, the females were killed and implantation sites were counted. At the end of the experiments, the males were killed and the weights of the following organs were recorded: testes, prostates, seminal vesicles, levator ani muscles, preputial glands and adrenals. A solvent-treated group of fourteen rats from the same batch served as controls for all measurements. RESULTS The spermatogenic cycle in male rats commences at 4 to 6 days of age and lasts 40 days to the completion of spermatogenesis (Clermont & Perey, 1957; Roosen-Runge, 1962). Another 13 to 14 days are required for transport of spermatozoa to the vas deferens and their final maturation to become fertile at the age of 57 to 60 days (Clegg, 1960). As can be seen from Text-fig. 1, this was the time at which puberty was reached in the control group as evaluated by insemination and impregnation rates. Within 2 weeks, all rats in this group were able to reproduce. 100 Treatment period 80 % Days of age Text-fig. 1. Influence of peripubertal cyproterone acetate treatment on the reproductive performance of male rats ( ). O, Controls.-, Insemination;-, Impregna tion. By contrast, only a very small percentage of the cyproterone-acetate-treated males inseminated females during the treatment period and none of these matings was fertile. Whereas the first rats of the control group began to copulate at the 54th day, treated rats did not copulate until the 61st day, by which time all the controls had inseminated females with a 93 % impregnation rate.

3 Effect of cyproterone acetate on puberty in rats 61 The first fertile mating was seen 16 days after cessation of treatment, but it was not until 28 days that all treated rats achieved the ability to reproduce. The growth rate was significantly reduced in cyproterone-acetate-treated rats from the 28th day of treatment, and their body weights were still below those of the controls 64 days after the cessation of treatment (Text-fig. 2). Days of age Text-fig. 2. Influence of peripubertal cyproterone acetate treatment on the body weight development of male rats ( ). o, Controls. Table 1 organ weights at the 144th day of life after cyproterone acetate treatment (5-0 mg/100 g b.wt) from the 25th to 80th day of life No. of animals Adrenal Testis Seminal vesicle Prostate Levator ani muscle Cyproterone acetate Controls ± ± All weights expressed in mg/100 g b.wt, means + S.D. All weight differences are statistically significant at the 99% level in the U-test of Mann & Whitney (1947). Although the testicular weights in treated animals were higher than the control weights after a 64-day recovery period, the weights of the accessory sexual glands were still considerably depressed (Table 1). There was, however, no difference in adrenal weights at the time of autopsy.

4 62 H. Steinbeck and F. Neumann DISCUSSION To our knowledge, this is the first report to show that peripubertal antiandrogen treatment does not interfere with later reproductive performance. Within 6 weeks of the cessation of treatment, fertile matings were achieved by all rats although their accessory sexual gland weights were still significantly depressed after a 2-month recovery period. Full restoration of these organs could not, however, be expected by this time because studies on adult rats with comparable cyproterone acetate doses revealed that full restoration of the accessory glands is not achieved until 12 to 15 weeks after the end of treatment (Steinbeck, Mehring & Neumann, 1971). The body growth rate decreased during treatment, presumably due to the antianabolic effect of the compound (Junkmann & Neumann, 1964; Neumann, von Berswordt-Wallrabe, Elger, Steinbeck & Hahn, 1968). Because the rate of growth only returned to normal levels about 5 weeks after the cessation of treatment and did not increase, the body weight of the treated group remained significantly smaller than that of the controls. Presumably the body weight development reflects not only the antianabolic effects but also skeletal changes since it has been shown that similar cyproterone acetate treat ment delays bone maturation and alters bone growth (Hertel et al., 1969). As has been demonstrated with medroxyprogesterone acetate in humans, cyproterone acetate delays the time at which the ability to reproduce is achieved in male rats. This effect common to both compounds is achieved by completely different mechanisms. Whereas cyproterone acetate inhibits androgen effects acetate interferes with andro mainly at the receptor sites, medroxyprogesterone gen synthesis by gonadotrophin inhibition. This raises some theoretical objec tions against the use of medroxyprogesterone acetate. It is known that, after prolonged gonadotrophin suppression, hypothalamic regulatory centres become more and more insensitive to the suppressing agent (Hohlweg, 1956). Therefore, increasing amounts of gonadotrophins might escape central suppression and stimulate androgen synthesis unless the suppressor dose is increased. These androgens and also androgens from other sources, e.g. from the adrenals, are not counteracted and might be sufficient to exert undesirable effects. Cyproterone acetate, on the other hand, not only antagonizes androgens from all sources but also exerts some inhibitory action on the adrenals, pre sumably by acth depression (Domenico & Neumann, 1967). Gonadotrophin secretion, and hence gonadal function, is not, or is only slightly, affected (Steinbeck et al, 1971). With due reservations, it might therefore be assumed that antiandrogen treatment has some advantage over mere gonadotrophin-secretion-inhibitory treatment in cases of male precocious puberty. REFERENCES Asdell, S. A. (1946) Patterns ofmammalian reproduction. Comstock, Ithaca, New York. Bierich, J. R. & Nowak, A. H. (1965) Investigations on precocious puberty. Acta endocr., Copenh. Suppl. 101, 20. Clegg, E. J. (1960) The age at which male rats become fertile. J. Reprod. Fert. 1, 119.

5 Effect of cyproterone acetate on puberty in rats 63 Clermont, Y. & Perey, B. (1957) Quantitative study of the cell population of the seminiferous tubules in immature rats. Am. J. Anat. 100, 241. Critchlow, V. & Bar-Sela, M. E. (1967) Control ofthe onset ofpuberty. In: Neuroendocrinology, vol. II, chap. 20, p Eds. L. Martini and W. F. Ganong. Academic Press, New York. Domenico, A. & Neumann, F. (1967) Wirkung von antiandrogen wirksamen Steroiden auf die Funktion und Morphologie der Nebennieren von Ratten. In: 12. Symp. Dtsch. Ges. für Endokrinologie, p Wiesbaden Springer, Berlin. Helge, H, Weber, B., Neumann, F. & Hammerstein, J. (1969) Idiopathic precociouspuberty : indication for therapeutic use of cyproterone acetate, an antigonadotropic and antiandrogenic substance? In: Proc. Seventh Ann. Conference of the European Society for Paediatric Endocrinology, Malmö. (In press). Hertel, P., Kramer, M. & Neumann, F. (1969) Einfluss eines Antiandrogens (Cyproteronacetat) auf Knochenwachstum und Knochenreifung männlicher Ratten. Arzneimittel-Forsch. 19, Hohlweg, W. (1956) Über die Bedeutung der Regulation der peripheren Hormondrüsen im Hinblick auf eine praktische Hormontherapie. Dte. GesundhWes. 11, 245. Junkmann, K. & Neumann, F. (1964) Zum Wirkungsmechanismus von an Feten antimaskulin wirksa men Gestagenen. Ada endocr., Copenh. Suppl. 90, 139. Kupperman, H. S. & Epstein, J. A. (1962) Medroxyprogesterone acetate in the treatment of constitu tional sexual precocity. J. clin. Endocr. Metab. 22, 456. Laron, A. (1968) The Eighth Ann. Conference of the European Society for Paediatric Endocrinology, Vienna. Cited by Hertel, P. (1969) Einfluss eines Antiandrogens auf Knochenwachstum und Knochen reifung männlicher Ratten. Doctoral thesis, Berlin. Laron, A. & Rumney, G. (1965) Treatment of precocious sexual development by medroxyprogesterone acetate. Ada endocr., Copenh. Suppl. 101, 27. Mann, H. B. & Whitney, D. R. (1947) On a test of whether one of two random variables is statistically larger than the other. Ann. math. Statist. 18, 50. Neumann, F. (1969) Wirkung von Gestagenen auf äusseres Genitale, Cervix, Uterus, Tube, Ovar und Hoden. In: Gestagene, Handbuch der experimentellen Pharmakologie, vol. 22/2, chap. IX-B/I, p. 50. Ed.. Junkmann. Springer-Verlag, Berlin. Neumann, F., Von Berswordt-Wallrabe, R., Elger, W., Steinbeck, H. & Hahn, J. D. (1968) Effects of antiandrogens. Excerpta med. Int. Congr. Ser. 184, 823. Roosen-Runge, E. C. (1962) The process of spermatogenesis in mammals. Biol. Rev. 37, 343. Schoen, E. J. (1966) Treatment ofidiopathic precocious puberty in boys. J. clin. Endocr. Metab. 26, 363. Steinbeck,., Mehring, M. & Neumann, F. (1971) Comparison of the effects of cyproterone, cypro terone acetate and oestradiol on testicular function, accessory sexual glands and fertility in a long-term study on rats. J. Reprod. Fert. 26, 65. Steinbeck,., von Berswordt-Wallrabe, R., Elger, W., Hahn, J. D. & Neumann, F. (1969) Actions of androgen antagonists. Fundación para Investigaciones Hormonales, Simposio Esteroides Sexuales, Bogotá, Saladruck, Berlin. Thamdrup, E. (1965) Trials with progestational agents on the treatment of precocious puberty. Ada endocr., Copenh. Suppl. 101, 28. van der Werff ten Bosch, J. J. (1965) Idiopathic true sexual precocity in girls, and effects of treatment with 6a-methyl, 17-a-hydroxyprogesterone. Ada endocr., Copenh. Suppl. 101, 29.