DIHYDROTESTOSTERONE CAUSES REVERSIBLE INFERTILITY IN MALE RATS

Transcription

1 . FERTILITY AND STERIIJTY Copyright c 1981 The American Fertility Society Vol. 36, No.6, June 1981 Printed in U.SA. DIHYDROTESTOSTERONE CAUSES REVERSIBLE INFERTILITY IN MALE RATS WOLFGANG LOTZ, PH.D.* ROBERT KRAUSE, M.D. Pkarma Research DilJision, F. Hoffmann-La Roche & Company, Ltd., Basel, Switzerland Dihydrotestosterone released continuously from a subcutaneously implanted Silastic elastomer was more potent than testosterone in reducing circulating gonadotropin and androgen concentrations and in inducing testicular atrophy and infertility despite normal ejaculatory function as judged by the presence of vaginal plugs in cage mates. All effects were reversible. Thus quantities of dihydrotestosterone which reduce circulating gonadotropin concentrations suffice to maintain the weights of accessory sex organs but not spermatogenesis. That the latter is possible with higher doses even in the complete absence of luteinizing hormone and follicle-stimulating hormone (state after hypophysectomy) has been shown by others. Fertil Steril35:691, 1981 The possibility of contraception in men by pharmacologic means has received little consideration in comparison with the enormous efforts devoted to this approach in women. One of the reasons for this disparity is undoubtedly the absence in the male organism of a simple phenomenon compar-. able to ovulation in the female which can be manipulated with reasonable directness. Another, perhaps equally decisive, aspect is that the technique of inhibition of gonadotropin release, so effective in inducing functional infertility in women, is associated with the unacceptable effect of potency reduction in men. Since the demonstration that low doses of testosterone or its esters hinder spermatogenesis in rats,1 rabbits,2 and man,3 attempts have been made in this direction by combining androgens with gonadotropin release inhibitors such as estradiol,4-6 gestagens (including the antiandrogen, cyproterone acetate),6-9 and danazo Reports that the 5a-reduced form of testosterone is a more potent gonadotropin release inhibitor and more potent androgen than testos- Received April 20, 1980; revised October 15, 1980; revised and accepted December 2,1980. *Reprint requests: Wolfgang Lotz, Ph.D., Pharma Research Division, F. Hoffmann-La Roche & Company, Ltd., Grenzacherstrasse 124, CH-4002, Basel, Switzerland. 691 terone prompted us to examine this androgen in male rats. The studies reported here show that dihydrotestosterone is more effective than testosterone as a reversible antifertility agent in male rats. MATERIALS AND METHODS Sexually experienced male rats of the Fiillinsdorf albino strain (Ibm: ROROrISPF) were maintained under controlled conditions. They received a subcutaneous implant of Silastic elastomer (Dow Corning, medical grade no. 382; 30-mg strip) impregnated with 3 mg of homogeneously distributed micronized steroid. The control animals received empty elastomer implants. Ejaculation capacity and fertility of the males were tested by pairing them with proestrous females. If a vaginal plug or spermatozoa were present the following morning, that day was designated day 1 of pregnancy. Fifteen days thereafter the females were killed and pregnancy data were recorded. After 3 weeks the implants were replaced and after 6 weeks the animals were either decapitated or freed of their implants to permit observation after a period of recovery. Determinations ofhormone concentrations were made in serum derived from blood which was drawn from the jugular

2 692 LOTZ AND KRAUSE June 1981 vein under light ether anesthesia or collected from the trunk. Serum was kept deep-frozen until assayed with specific radioimmunoassays (kits obtained from the National Institutes of Health, Bethesda, Md.). Results are expressed as nanograms of National Institute of Arthritis, Metabolism and Digestive Diseases RP1 per milliliter. Testosterone/dihydrotestosterone concentrations were measured by radioimmunoassay with antibodies raised in rabbits against testosterone-7 a carboxymethy lthioether coupled to bovine serum albumin (cross-reactivity: dihydrotestosterone 45%, androstenedione 2.1%, and androstanediol 0.7%; intra-assay coefficient of variation in duplicates: 4.8% to 7.7%; inter-assay coefficient of variation: 13% to 15%; sensitivity 5 pg/tube.) The tracer was [1,2,6,7(n)_3H]testosterone (Radiochemical Center, Amersham, England). Organ weights were recorded after decapitation, and the testes were fixed in formalin-sublimate for histologic evaluation. Student's t-test was used for statistical analysis except in the case of mean number of fetuses per pregnant animal. Because we could not know the nature of the numerical distribution of these data, we made use of the nonparametric Kolmogorov-Smimov test for that analysis. RESULTS The results obtained after 6 weeks of treatment with subcutaneous implants of elastomer impregnated with testosterone or dihydrotestosterone or devoid of added material (controls) are listed in Table 1. The fertility of the dihydrotestosteronetreated group was lost completely, although all animals ejaculated normally. Serum luteinizing hormone (LH) concentrations were almost halved in both treated groups after 3 and 6 weeks, whereas follicle-stimulating hormone (FSH) concentrations were reduced only in the dihydrotestosterone-treated group. In this group, testosterone/dihydrotestosterone concentrations were markedly reduced as opposed to the elevated concentrations in the testosterone-treated group. Testicular atrophy was observed in both groups, but it was more pronounced in the dihydrotestosterone-treated group; prostatic hypertrophy developed in both groups. In order to check these findings in older animals of proven fertility and to determine whether these effects were reversible, we conducted a second experiment. The results obtained after 3 and 6 weeks of treatment with the two androgens are compiled in Table 2. No effect on fertility as measured by number of pregnant rats and number of viable fetuses could be demonstrated after 3 weeks, despite a definite decrease in circulating LH and FSH concentrations. After 6 weeks of treatment, fertility in the dihydrotestosteronetreated group was reduced but not abolished. At autopsy of eight animals of each group, the testes of both treated groups showed marked (dihydrotestosterone) or moderate (testosterone) atrophy. Histologic examination of the testes revealed marked Leydig cell atrophy in both treated groups, associated with arrest of spermatogenesis at the spermatid level in the animals given dihydrotestosterone. Although mature spermatozoa were present in occasional tubules in some animals, they were rare. The tubules of the testosterone-treated animals displayed the full gamut TABLE 1. Fertility, Serum Hormone Concentrations, and Organ Weights After Six Weeks of Treatment with Dihydrotestosterone- or Testosterone-Impregnated Silastic Implants in Male Rats Mating Pregnant Fetuses/preg- LH Testosterone Subetance N rats rats at nant rat FSH," dihydrotsstosfn) sacrifice fn) (mean:t SEM) 3wk 6wk 6wk terone, o 6 wk ng/ml ng/ml ng/ml Control ± 2 44 ± 6 26 ± ± ± 0.3 Dihydrotes ± 2* 12 ± 1** 288 ± 18*** 0.3 ± 0.02*** tosterone Testosterone ± 2 28 ± 3* 14 ± 2* 376 ± 52t 4.5 ± 0.8* Body wt. Ventral Seminal Musculus N Testes" prostate" vesicleso levator ani B gm mg mg mg mg Control ± ± ± ± ± ± 10 Dihydrotestos ± ± ± 68*** 323 ± 13* 232 ± ± 13 terone Testosterone ± ± ± 114*** 340 ± 32* 350 ± 18*** 231 ± 13 "Values are means ± standard error of the mean. Significance by Student's t-test: *p < 0.05; **p < 0.01; ***p < 0.001; tnot significant.

3 Vol. 35, No.6 DIHYDROTESTOSTERONE CAUSES REVERSIBLE INFERTILITY 693 TABLE 2. Fertility, Serum Hormone Concentrations After Three and Six Weeks, and Organ Weights After Six Weeks of Treatment with Dihydrotestosterone- or Testosterone-Impregnated Silastic Implants in Male Rats N Mati~rats Pregnant rats at sacrifice CN> 3 wk of treatment Fetuses/pregnant rat (mean ± SEM) LH" FSH" Control ± ± ± 50 Dihydrotestosterone ± ± 1*** 188 ± 19* Testosterone ± ± 1*** 281 ± 24 6 wk of treatment N Mat~rats Pregnant rats Fetuses/pregnant LH" FSH" at sacrifice CN> rat (mean ± SEM) Control ± ± ± 36 Dihydrotestosterone ± 1.7 b 17 ± 1*** 178 ± 16** Testosterone ± ± 1*** 224 ± 18 6 wk of treatment N Body wt." Testes" Ventral. Seminal Musculus prostate" vesicles(1 levator ania gm rng rng rng mg Control ± ± ± ± ± ± 13 Dihydrotestosterone ± ± ± 30*** 381 ± ± ± 12 Testosterone ± ± ± 320* 383 ± ± 11* 310 ± 11 avalues are means ± standard error of the mean. Significance by Student's t-test; *p < 0.05; **p < 0.01; ***p < bkolmogorov-smirnov test: P = I of the spermatogenetic process, but the absolute numbers of secondary spermatocytes, spermatids, and mature spermatozoa were reduced. Serum LH concentrations were lowered in both treated groups and were comparable to the results obtained after 3 weeks; FSH levels were reduced only in the dihydrotestosterone-treated group. Mter 3 weeks of recovery from treatment, none of the dihydrotestosterone-treated animals was fertile (Table 3), despite normal ejaculatory function. Serum LH concentrations had returned to normal levels, but serum FSH and testosterone/ dihydrotestosterone concentrations in the dihydrotestosterone-treated group were increased to a statistically significant degree. After an additional recovery period of 3 weeks, the fertility of all animals was almost normal, as were all other variables except the FSH levels and the prostate weight in the dihydrotestosterone-treated group. Histologic inspection of the testes showed a fully normal picture in all animals, without differences between groups. DISCUSSION If it is assumed that both steroids were released from the Silastic elastomer implants at the same rate, it appears that, after 6 weeks of treatment, dihydrotestosterone reduced fertility more effectively than did testosterone. This may be deduced from its greater reduction of circulating FSH concentrations. The reductions of LH concentrations in both groups were similar to those seen in hypophysectomized rats. 15 The changes in testosterone/dihydrotestosterone concentrations measured after 6 weeks of treatment are difficult to interpret. From earlier experiments I5 we know that castrated rats treated with dihydrotestosterone-impregnated Silastic elastomer implants for 3 weeks had circulating androgen levels of 0.36 ± 0.03 ng/ml (castrated controls: 0.23 ± 0.04 ng/ml). Consequently the immunoreactive material (0.3 ± 0.02 ng/ml, Table 1) in intact dihydrotestosterone-treated rats could hardly have come from the testes. However, this amount seems to suffice for negative feedback at the hypophysial level and for maintenance of the weight of the seminal vesicles, and even hypertrophy of the ventral prostate. Similar analyses of androgen concentrations in the testosterone-treated group are impossible because of the lack of data in castrates. If it is supposed that steroidogenesis in this group is suppressed to the same extent as in the dihydrotestosterone-treated group, the higher metabolic :

4 694 LOTZ AND KRAUSE June 1981 TABLE 3. Fertility, Serum Hormone Concentrations After Three.and Six Weeks, and Organ Weights After Six Weeks cif Recovery from a Preceding Treatment with Dihydrotestosterone- or Testosterone-Impregnated Silastic Implants in Male Rats over a Period of Six Weeks 6 wk of treatment and 3 wk of recovery N Mati Pregnant rats Fetuses/pregnant LH" FSH Testosteronel rats <ill at sacrifice fn) rat (mean ± SEM) dihydrotestoeterone Control ± fr.9 21 ± ± ± 0.3 Dihydrotestosterone ± ± 47** 5.1 ± 0.7** Testosterone ± ± ± 68t 3.3 ± wk of treatment and 6 wk of recovery N Matift, Pregnant rats Fetuses/pregnant LH" FSH Testosteronel rats ( at sacrifice fn) rat (mean ± SEM) dihydrotestoeterone Control ± ± ± ± 0.7 Dihydrotestosterone ± l.4 b 19 ± ± 16* 2.4 ± 0.6 Testosterone ± ± ± ± wk of treatment and 6 wk of recovery N.Body wt" Testes Ventral Seminal Musculus prostate vesicleso levator &ni G gm mg mg mg mg Control ± ± ± ± ± ± 18 Dihydrotestosterone ± ± ± ± 6* 389 ± ± 24 Testosterone ± ± ± ± ± ± 13 avalues are means ± standard error of the mean. Significance by Student's t-test: *p < 0.05; **p < 0.01; ***p < 0.001; tnot significant. bkolmogorov-smirnov test: not significant. clearance rate of dihydrotestosterone as opposed to testosterone (87.2 versus 17.5 liters/24 hours/100 gm body weight 16 ) cannot account for the 15-fold difference in androgen levels. We therefore assume that a considerable portion of the androgen in circulation is contributed by the testes despite their atrophic condition. The increase in weight of the seminal vesicles evoked by testosterone and the lack of effect of dihydrotestosterone on this organ confirm previous observations. 14 This experiment was repeated with older animals at the same dose. The dose per kilogram of body weight was thus lower and the onset of infertility was delayed. Although hormone concentrations and organ weights were comparable to those of the first experiment after 6 weeks of treatment, 9 of 12 animals which mated in the dihydrotestosterone-treated group were fertile despite the arrest of spermatogenesis, probably owing to residues of mature spermatozoa in the ejaculatory tract. Three weeks after the end of treatment, all of the remaining dihydrotestosterone-treated animals were infertile despite normal ejaculations as judged by the presence of vaginal plugs in cage mates. Both FSH and testosterone/ dihydrotestosterone concentrations in these animals were increased to a statistically significant degree, whereaslh concentrations were normal. In contra,st to LH, release of which is immediately responsive to modulatory influences, FSH reacts to these influences sluggishly and its suppression during 6 weeks of treatment was followed by a prolonged spillover. Increased FSH concentrations could also reflect the positive feedback of androgens 17 or reduced inhibin production in the recovering testes. In summary, these results show that dihydrotestosterone was more potent than testosterone in reducing circulating gonadotropin and androgen concentrations and in inducing testicular atrophy and infertility despite normal ejaculations as judged by the presence of vaginal plugs in cage mates. All effects were reversible. Thus quantities of dihydrotestosterone which reduce circulating gonadotropin concentrations suffice to maintain the weight of accessory sex organs but not spermatogenesis. That the latter is possible with higher doses even in the complete absence of LH and FSH (state after hypophysec. tomy) has been shown by several groups

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