(Received 16th October 1969)

Transcription

1 EFFECT OF AN ANTI-ANDROGEN ON THE DIFFERENTIATION OF THE INTERNAL GENITAL ORGANS IN DOGS H. STEINBECK, F. NEUMANN and W. ELGER Main Laboratory of Schering AG, Berlin, West Germany (Received 16th October 1969) Summary. From experiments with anti-androgens, it can be concluded which processes in the sex differentiation of dogs are androgen\x=req-\ dependent and which are not. In contrast to rats but as in rabbits, retrogression of the Wolffian ducts can be induced in the treated members of this species compared to their controls, but retrogression of the M\l=u"\llerianducts is uninfluenced in both treated and control animals. From this, it is concluded that the latter process is not androgen-dependent. INTRODUCTION The most potent known anti-androgen was discovered by its ability to feminize male rat foetuses (Hamada, Neumann & Junkmann, 1963). It was found in subsequent experiments that this compound is capable of inhibiting effects of endogenously produced as well as exogenously administered androgens (Junk mann & Neumann, 1964; Neumann, Richter & Günzel, 1965; Neumann, 1966). Cyproterone acetate proved to be a valuable tool for the exploration of the biological rôle of androgens, especially when the surgical approach is diffi cult or even impossible, e.g. in investigations regarding the effects of testosterone within the testis itself (Neumann & von Berswordt-Wallrabe, 1965, 1966) or in foetal sex differentiation processes (Elger & Neumann, 1966; Neumann & Elger, 1966; Neumann, Elger & Kramer, 1966; Neumann & Kramer, 1964). The first species in which the entire genital tract was studied was the rabbit (Elger, 1966). In addition to feminization of the external genital organs, the following results were of special importance in male foetuses : the gonads were differentiated and descended normally; retrogression of the Müllerian ducts could not be prevented; above a critical dose level (5 mg/kg/day i.m.), involu tion of the Wolffian ducts was almost complete. This result was considered to be a proof of Jost's theory postulating that, besides androgens, other factors of the male gonad a play rôle in sex differentia tion (Müllerian duct inhibiting hormone). Initial studies on the rat foetus were confined to the differentiation of the feminization was found external genital organs, where more or less complete (Hamada et al., 1963; Junkmann & Neumann, 1964). In subsequent studies, 223

2 224 H. Steinbeck et al. the results obtained in rabbits after relatively low doses of cyproterone acetate could never be achieved in rats, although excessive doses were administered (Jost, 1967; Forsberg, Jacobsohn & Norgren, 1968; Elger, Neumann & von Berswordt-Wallrabe, personal communication). In spite ofmaximal feminization of the external genitalia, retrogressed Wolffian ducts were never found. The reason for these species differences is unknown and an extension of the seemed desirable in order to find out whether one experiments to other species or other reaction is the rule or the exception. MATERIAL AND METHODS Two beagle bitches were used as experimental animals. The bitches were mated once daily on 3 consecutive days, the middle day being regarded as Day 1 of pregnancy, when this condition had subsequently been established. Cyproterone acetate was injected i.m. as an oily solution (benzyl benzoate/ castor oil 1: 10) in a daily dose of 10 mg/kg from the 23rd to the 44th day of pregnancy. The foetuses were removed from the uterus by Caesarean section were embedded in on the 45th day. After fixing in Bouin's solution, they paraffin and serial sections were made at mm intervals, every twentieth section being mounted on microscope slides. Thus, two successive sections a represented distance of 0-1 mm. RESULTS Three male foetuses were examined from each of the two litters. As controls, two male and two female untreated foetuses of the same age were used. The internal genital organs of treated males and untreated controls are shown schematically in Text-fig. 1, after reconstruction from serial sections. Gonads In treated animals as well as in male and female controls, the gonads were still located in the immediate vicinity of the caudal kidney pole. No abnormal gonads were found. Müllerian ducts In all males, whether treated or not, the Müllerian ducts had retrogressed to minute epithelial and mesenchymal traces. These remnants, however, were found almost constantly throughout the whole length of the genital tract (PI. 1, Fig. 1). Only in the close proximity of the urogenital sinus did a caudal remnant (PI. 1, Fig. 2), which ended blindly in the forepart, persist in three treated males (967/1, 967/4, 722/2). Wolffian ducts All treated foetuses had degenerated Wolffian ducts, with the exception of one foetus (967/1) in which the ducts were not affected by the anti-androgen. In the remaining five foetuses, the Wolffian ducts had retrogressed either unilaterally or bilaterally and short sections of discontinuity were also found, particularly in the immediate vicinity of the urogenital sinus (722/4 and 722/7).

3 PLATE 1 Fig. 1. Mesogenital folds (MGF) of a male dog foetus whose mother had been treated rectum. Both Müllerian and Wolffian ducts have with cyproterone acetate. R retrogressed. The previous position of both ducts is marked by mesenchymal condensa tions. Fig. 2. Genital cord of a feminized male dog foetus in the vicinity of the urogenital sinus. A segment of a Müllerian vagina (MV) can be seen between the neck of the urinary bladder (NUB) and the rectum (R). Wolffian ducts are definitely absent at this level. Fig. 3. Feminized male dog foetus. Partition of the urogenital sinus into a smaller ventral (U, urethra!) and a wider dorsal (V, vaginal) portion. The prostate development is almost completely suppressed. RPB rudimentary prostatic bud. = = (Facing p. 224)

4 An Text-fig. 1. Scale dog foetuses. anti-androgen and the canine reproductive tract drawings of the internal reproductive tract of anti-androgen-treated 225

5 226 H. Steinbeck et al. Urogenital sinus In all treated male foetuses, a separation of the anterior part of the sinus, resulting in the formation of a (sinus-) vagina (PI. 1, Fig. 3), was observed, as in the female controls. The prostatic development was also suppressed to some extent (PI. 1, Figs. 2 and 3). External genital organs The external genital organs had reached a similar stage of development in the treated males as in female foetuses of the same age. Genital cord In the feminized male foetuses, the distance from the fusion of the urogenital folds to the sinus orifice of the genital ducts was relatively long compared with the male controls. The small number of controls, however, does not allow any definite conclusions to be drawn. DISCUSSION Under the influence of cyproterone acetate, the sexual differentiation in dogs is essentially similar to the pattern already seen in rabbits, thus confirming the androgen-dependence of the Wolffian ducts, the prostate and other structures and differentiation processes. Since the high degree of feminization of the male genital tract, including retrogression of the Wolffian ducts, was not accom panied by a substantial influence on the Müllerian ducts, these experiments further support Jost's theory of sexual differentiation. It can be presumed that the suppression of the Müllerian ducts by a specific testicular activity is the rule in mammals, but in our own experiments in rats, no retrogression of the Wolffian ducts was obtained by anti-androgen treatment. There are also in consistent results in vitro with cultures of the genital tract where spontaneous retrogression of the Müllerian ducts in the absence of the foetal gonad was observed (Price, Ortiz & Zaaijer, 1967). In recent experiments, we were able to stabilize the Wolffian ducts in female rat foetuses by methyl testosterone treatment of pregnant rats, an effect which could be completely abolished by concomitant cyproterone acetate treatment (Elger, Steinbeck, Cupceancu & Neumann, 1970). It seems, therefore, that those hormonal factors (androgens?) which physiologically stabilize the Wolffian ducts in rats differ from synthetic androgens with regard to their biological action. At this time, it is not possible to give a sound general interpretation of the hormonal dependence of the Müllerian and Wolffian ducts. It can only be stated that androgens are not involved in Müllerian duct retrogression (Jost, 1967) and that anti-androgens, consequently, do not prevent this process. REFERENCES Elger, W. (1966) Die Rolle der fetalen Androgene in der Sexual-differenzierung des Kaninchens und ihre Abgrenzung gegen andere hormonale und somatische Faktoren durch Anwendung eines starken Antiandrogens. Archs Anat. microse Morph. exp. 55, 657. Elger, W. & Neumann, F. (1966) The role of androgens in the differentiation of the mammary gland in male mouse fetuses. Proc. Soc. exp. Biol. Med. 123, 637.

6 An anti-androgen and the canine reproductive tract 227 Elger, W., Steinbeck, H., Cupceancu, B. & Neumann, F. (1970) Experiments with synthetic andro gens and anti-androgens on Wolffian duct differentiation in rat foetuses. J. Endocr. (In press). Forsberg, J.-G., Jacobsohn, D. & Norgren, A. (1968) Development of the urogenital tract in male offspring of rats injected during pregnancy with a substance with antiandrogenic properties (cyproterone). Z Anat. EntwGesch. 126, 320. Hamada, H., Neumann, F. & Junkmann, K. (1963) Intrauterine antimaskuline Beeinflussung von Rattenfeten durch ein stark gestagen wirksames Steroid. Ada endocr., Copenh. 44, 380. Jost, A. (1967) Steroids and sex differentiation of the mammalian fetus. Proceedings of the Und Int. Congress on Hormonal Steroids, Milan Excerpta med. Int. Congr. Ser. 132, 74. Junkmann, K. & Neumann, F. (1964) Zum Wirkungsmechanismus von an Feten antimaskulin wirk samen Gestagenen. Ada endocr., Copenh. Suppl. 90, 139. Neumann, F. (1966) Methods for evaluating antisexual hormones. In: Proceedings of the Int. Symposium Methods in Drug Evaluation, Milan, 1965, pp North Holland Pubi. Company, Amsterdam. Neumann, F. & von Berswordt-Wallrabe, R. (1965) Effects of a new anti-androgen, l,2a-methylene- 6-chlor-A6-17a-hydroxy-progesterone-17-acetate (SH 714), on the testicular structure of adult testosteronepropionate (TP)-treated hypophysectomized rats. Ada endocr., Copenh. Suppl. 100, 42. Neumann, F. & von Berswordt-Wallrabe, R. (1966) Effects of the androgen antagonist cyproterone acetate on the testicular structure, spermatogenesis and accessory sexual glands of testosteronetreated adult hypophysectomized rats. J. Endocr. 35, 363. Neumann, F. & Elger, W. (1966) The effect of the anti-androgen l,2a-methylene-6-chloro-a4'6 pregnadiene-17o;-ol-3,20-dione-17a-acetate (cyproterone acetate) on the development of the mammary glands of male foetal rats. J. Endocr. 36, 347. Neumann, F., Elger, W. & Kramer, M. (1966) Development of a vagina in male rats by inhibiting androgen receptors with an anti-androgen during the critical phase of organogénesis. Endocrino logy, 78, 628. Neumann, F. & Kramer, M. (1964) Antagonism of androgenic and anti-androgenic agents in their action on the rat fetus. Endocrinology, 75, 428. Neumann, F., Richter, K.-D. & Günzel, P. (1965) Wirkungen von Antiandrogenen. Zenlbl. VetMed., Reihe A, 12, 171. Price, D., Ortiz, E. & Zaaijer, J. P. (1967) Organ culture studies of hormone secretion in endocrine glands of fetal guinea pigs. III. The relation of testicular hormone to sex differentiation of the reproductive ducts. Anat. Ree 157, 27.