Imprinting of male sex tissues by neonatal endogenous androgens in mice. Molecular alterations following exposure to cyproterone acetate

This study was conducted to evaluate the growth and biochemical responsiveness of the epididymis, vas deferens and seminal vesicles of adult mice exposed to cyproterone acetate during the first 10 days of life. Results indicate that the weight and protein content of sex accessory organs were significantly depressed, testosterone and dihydrotestosterone concentrations were unaffected or increased, the number of cytosolic androgen-binding sites was slightly or significantly reduced. The efficiency of exogenous testosterone in promoting growth and protein synthesis in target organs of castrated adult males was significantly lowered by neonatal cyproterone acetate treatment. It is concluded that a deficient androgenic stimulation during neonatal life induces a limited response of sex target organs to endogenous or exogenous androgens in adulthood.     

Antiandrogenic and antirenotropic effect of spironolactone.

Spironolactone given to male mice in dose 1.5 mg for 21 days produced significant changes not only in the highly androgen dependent seminal vesicles but also in the kidneys. Spironolactone blocked the action of endogenous testosterone in intact mice and led to further significant decrease in seminal vesicles and kidney weight in castrated mice. Such an effect could be due to a blocking of androgenic action of adrenal steroids.     

Effect of neonatal injections of estradiol, testosterone and cryproterone acetate on plasma and testicular testosterone and on the genital system in adult male mice]

On day old male mice received a single injection of oestradiol benzoate, testosterone propionate or cyproterone acetate in order to study their action on testicular development, particularly testosterone secretion. Oestrogenization of newborn males leads, when the animals mature, to a high proportion or cryptorchidism, to atrophy of testes and seminal vesicles, and inhibition of spermatogenesis. Testosterone levels were reduced in the plasma. Testosterone propionate produced moderate reduction of testicular weight but spermatogenesis was not impaired. Plasma testosterone level was reduced. Cyproterone acetate increased significantly testicular testosterone level.     

Further regression of seminal vesicles of castrated guinea pig by administration of cyproterone acetate

It has been established that a low level of secretory activity persisted in seminal vesicles of guinea pigs long after castration and that this may be due to a higher extratesticular androgen level in this animal. A RIA study revealed that the normal serum testosterone concentration of the guinea pigs was comparable to that of the rats, but the basal serum testosterone level after castration was ten times higher than rats under a similar condition. It was also shown that cyproterone acetate did not significantly lower the basal serum testosterone concentration in the castrated guinea pigs. The higher basal serum testosterone level is believed to be responsible for the slow and incomplete regression of this gland in the guinea pigs. There was a significant reduction in wet weight of the seminal vesicles after the treatment of castrated guinea pigs with cyproterone acetate. Ultrastructural study showed that there were both qualitative and quantitative changes in the cytoplasmic organelles. The Golgi apparatus further reduced in size and in the number of associated vesicles and vacuoles. There was a marked decrease in the number and size of secretory granules and lysosomes and an increase in the degree of undulation of the basement membrane. Accumulation of lipid droplets and glycogen was commonly observed. All these morphological evidences showed that further regression of the castrated guinea pig seminal vesicles can be achieved by cyproterone acetate treatment.     

The additive effects of progestins on testosterone-stimulated hepatic ethylmorphine metabolism and cytochrome P-450 content

The actions of several progestins on mouse liver were studied in terms of their inherent potency and for their ability to modify the biologic activity of testosterone. When hepatic ethylmorphine demethylase activity and cytochrome P-450 content were used as end points, the biological potency of progestins was ranked as follows: cyproterone acetate>progesterone>medroxyprogesterone acetate>hydroxyprogesterone caproate controls. The induced alterations of these parameters were, therefore, unrelated to reported progestational (cyproterone acetate medroxyprogesterone acetate>>hydroxyprogesterone caproate>progesterone) or androgenic (medroxyprogesterone acetate>cyproterone acetate = hydroxyprogesterone caproate = progesterone) actions of these steroids. A similar conclusion was reached when hepatic weight and microsomal protein content were used as end points.

When progestins (0.1–10 mg/day) were administered with testosterone (0.1 mg/day), the effect of both steroids were additive. This is in contrast to their actions on other tissues such as kidney and sub-maxillary gland where progestins potentiate and inhibit androgen action. We conclude from these studies that the mechanism of action of progestins on the liver differs from that on other tissues.     

Androgen dynamics in vitro in the human prostate gland. Effect of cyproterone and cyproterone acetate

Hyperplastic and adenocarcinomatous human prostatic tissue was superfused in vitro with radioactively labelled androst-4-ene-3,17-dione, testosterone and 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one), with and without addition of the anti-androgens cyproterone and cyproterone acetate. Cyproterone competitively inhibited the entry of the androgens into the majority of the tissues, whereas cyproterone acetate increased this entry. These findings indicated that transport of androstenedione, testosterone and 5alpha-dihydrotestosterone into prostatic tissue is performed by a specific mechanism, possibly involving a carrier situated in the cell membrane. The extent of metabolism of the three androgens was also modified: formation of 5alpha-dihydrotestosterone from testosterone, and of the latter from androstenedione, was decreased by cyproterone and increased by the acetate. Acetate was more effective than cyproterone in decreasing the ;uptake' of the perfused androgens by the tissue; at the same time, it increased the androgen clearance from the tissue. As cyproterone acetate is the more potent of the two anti-androgens, the possibility that these findings in vitro are related to the different anti-androgenic potency exhibited by the two compounds in vivo is discussed. ;Uptake' of the two anti-androgens and the response to their action on androgen dynamics were similar in adenocarcinomatous and hyperplastic glands.     

Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.     

Testosterone action in the rat ventral prostate. The effects of diethylstilboestrol and cyproterone acetate on the metabolism of [3H]testosterone and the retention of labelled metabolites by rat ventral prostate in vivo and in vitro

Recent reports have indicated that the prior metabolism of testosterone by the secondary sexual tissues may be necessary for its androgenic effect. The effects of two anti-androgens, diethylstilboestrol and cyproterone acetate (17alpha-acetoxy-6-chloro-1,2alpha-methylenepregna-4,6-diene-3,20-dione) used in the chemotherapy of human prostatic carcinoma, have been examined on both the metabolism of testosterone and the retention of its metabolites by the rat ventral prostate gland. Cyproterone acetate was found to inhibit the retention of labelled metabolites of [(3)H]-testosterone by prostatic nuclei, both in vivo and in vitro. This inhibition appeared to be competitive. In contrast with its effect on nuclear retention of metabolites of testosterone, cyproterone acetate had no significant effect on the metabolism of [(3)H]testosterone by rat ventral prostate tissue. Diethylstilboestrol similarly had little effect on the metabolism of [(3)H]testosterone by prostatic tissue, although it did appear partially to inhibit its initial metabolism in all the incubation systems used. Diethylstilboestrol inhibited the nuclear retention of dihydrotestosterone when both [(3)H]testosterone and diethylstilboestrol were injected intraperitoneally in vivo, but had no effect on dihydrotestosterone retention when both testosterone and diethylstilboestrol were supplied directly to the prostate either in vivo or in vitro. It was concluded that if diethylstilboestrol has an anti-androgenic effect at the level of the target organ as distinct from its effect on androgen production by the testes, then it is probably due to a mechanism differing from that of cyproterone acetate.     

Demonstration and partial characterization of cytosol receptors for testosterone.

Androgen uptake was investigated in several peripheral organs after administration of (1,2,6,7 minus -3H)testosterone to castrated male rats. The animals were killed after 30 min, the organs were taken out, and the radioactivity was determined after tissue combustion. A relatively high accumulation of androgen was found in pancreas, adrenals, spleen, thigh muscle, kidneys, and liver in addition to the classical androgen target organs coagulation glands, seminal vesicles, prostate, preputial glands, and harderian glands. In a second serier of experiments, nuclear and cytosol fractions were prepared from prostate, seminal vesicles, coagulation glands, preputial glands, spleen, submaxillary glands, kidneys, and pancreas from castrated male rats give (1,2,6,7 minus -3H)testosterone, and these fractions were then characterized by thin-layer and radio-gas chromatography with respect to their patterns of labeled steroids. Only prostate and seminal vesicles were found to contain significant amounts of nuclear 5alpha-(-3H)dihydrotestosterone. The major nuclear androgen was (-3H)testosterone that was the only detectable labeled steroid in coagulation glands, preputial glands, and spleen and that constituted 70% or more of the nuclear radioactivity in seminal vesicles, submaxillary glands, kidneys, and pancreas. These results indicate that testosterone itself may be the predominant active androgen principle in vivo in most androgen target organs and that conversion to 5alpha-dihydrotestosterone is generally not a prerequisite for androgen activity. Using an ultrasensitive micromodification of isoelectric focusing (cf. M. Katsumata and A. S. Goldman (1974), Biochem. Biophys. Acta 359, 112. It was possible to show that cytosol from kidney; submaxillary gland, thigh muscle, and levator ani muscle and nuclei from kidney and submaxillary gland contained androgen-binding proteins with pI's in the region 4.6-5.1 ("4.6 minus 5.1 Complex"). This complex also formed in vitro after incubation of (1,2,6,7 minus -3H)testosterone with cytosol from kidney and submaxillary gland. (1,2,6,7 minus -3H)Testosterone was bound with high affinity to receptor proteins in cytosol from both kidney, submaxillary gland, and thigh muscle with dissociation constants of 5.0 x 10 minus -12 M (kidney), 3.3 x 10 mi;nus -11 M and 4.1 x 10 minus -10 M (two types of binding sites, submaxillary gland), 2.4 x 10 minus -12 M (thigh muscle) and 1.9 x 10 minus -12 M (levator ani muscle). The number of binding sites was in all cases between 1 and 20 fmol/mg of protein. On the basis of these results the hypothesis is presented that a common class of testosterone receptors is present in most organs and that these receptors can be detected both in vivo and in vitro provided methods sensitive enough are utilized.     

Estradiol-17 beta inhibition of androgen uptake, metabolism and binding in epididymis of adult male rats in vivo: a comparison with cyproterone acetate

The effects of estradiol-17 beta on androgen uptake, metabolism and binding were studied in rat epididymis in vivo in comparison with cyproterone acetate. Steroids (250 ug/100 g body weight) were injected 5 min prior to 3H-testosterone in castrate rats. Estradiol-17 beta inhibited 3H-testosterone uptake into epididymal cytosol by 58% as compared to 38% by cyproterone acetate. 3H-Testosterone uptake into epididymal nuclei was inhibited 95% by estradiol-17 beta and 83% by cyproterone acetate. Total bound radioactivity in cytosol fractions was reduced to a greater extent by estradiol-17 beta than cyproterone acetate when either 3H-testosterone or 3H-dihydrotestosterone was injected. Binding of 3H-dihydrotestosterone to nuclear receptors was completely abolished by estradiol-17 beta; whereas approximately 20% binding remained in the nuclear extract after cyproterone acetate treatment. Metabolism of 3H-testosterone in vivo was also altered by estradiol-17 beta, resulting in diminished conversion to 3H-dihydrotestosterone. Cyproterone acetate, on the other hand, did not affect 3H-testosterone metabolism. Estradiol-17 beta and cyproterone acetate inhibited in vitro binding of 3H-dihydrotestosterone to the intracellular cytoplasmic receptor, but not the intraluminal androgen binding protein (ABP). These data suggest that estradiol-17 beta may have a more potent antiandrogenic effect on the epididymis than cyproterone acetate due to inhibition of 5 alpha reduction of testosterone as well as binding to the androgen receptor.     

Androgen receptor in nuclei of rat testis.

Testis nuclei of hypophysectomized rats selectively accumulate labeled testosterone and 5alpha-dihydrotestosterone following the injection of tritiated testosterone in vivo. Testosterone and 5alpha-dihydrotestosterone are bound to macromolecules in nuclei and can be extracted with 0.5 M KCl. Accumulation of protein bound radioactive androgens in nuclei of isolated seminiferous tubules is similar to that of whole testis. The relative amounts of testosterone and dihydrotestosterone in purified nuclei were similar to the relative amounts bound to cytoplasmic receptors, suggesting that cytoplasmic androgen-receptor complexes may be transported into the nuclei. Binding of labeled androgen is saturable and inhibited by prior injection of unlabeled testosterone or cyproterone acetate. Nuclear binding sites are destroyed by the proteolytic enzyme pronase, but not by DNase. Like the cytoplasmic androgen-receptor complexes in rat testis, nuclear androgen-protein complexes are heat labile and dissociate slowly at 0 degrees C. androgens fail to accumulate in testis nuclei of the Stanley-Gumbreck androgen insensitive rat, a species lacking cytoplasmic androgen receptors in testis and other androgen target tissues.     

Immunocytochemical detection of prolactin or prolactin-like immunoreactivity in epididymis of mature male mouse

Prolactin (PRL) binds to the testis of mice and rats where it increases the number of luteinizing hormone receptors, increases the binding of human chorionic gonadotropin (hCG) to LH receptors, and enhances testosterone synthesis and secretion. PRL also binds to the prostate and seminal vesicles of rats and humans where it increases organ weight and stimulates growth and uptake of testosterone. PRL binds to the epididymis of rats but the effect of PRL on this organ is unknown. In the present study, a standard immunoperoxidase (PAP) technique was used to detect the binding of endogenous and exogenous PRL or PRL-like peptides to the epididymis of the mature mouse. Throughout the epididymal duct, a positive reaction for peroxidase, suggesting PRL or PRL-like binding, occurred in the Golgi area of principal cells. In segment 1, positive reactions were also visualized in the perinuclear area and in the region located between the Golgi area and the apical surface of the principal cells (supra-Golgi area). In the corpus and cauda epididymidis, scattered entire principal cells were also positive. Throughout the epididymal duct, the reactions indicating the binding of exogenous PRL were slightly stronger than those testing for binding of endogenous peptides. The significance of such binding to the epididymis is uncertain but PRL may perform the same functions in epididymal principal cells as it does in the testis, prostate, and seminal vesicles.     

Immunocytochemical detection of prolactin or prolactin-like immunoreactivity in epididymis of mature male mouse

Summary Prolactin (PRL) binds to the testis of mice and rats where it increases the number of luteinizing hormone receptors, increases the binding of human chorionic gonadotropin (hCG) to LH receptors, and enhances testosterone synthesis and secretion. PRL also binds to the prostate and seminal vesicles of rats and humans where it increases organ weight and stimulates growth and uptake of testosterone. PRL binds to the epididymis of rats but the effect of PRL on this organ is unknown. In the present study, a standard immunoperoxidase (PAP) technique was used to detect the binding of endogenous and exogenous PRL or PRL-like peptides to the epididymis of the mature mouse. Throughout the epididymal duct, a positive reaction for peroxidase, suggesting PRL or PRL-like binding, occurred in the Golgi area of principal cells. In segment 1, positive reactions were also visualized in the perinuclear area and in the region located between the Golgi area and the apical surface of the principal cells (supra-Golgi area). In the corpus and cauda epididymidis, scattered entire principal cells were also positive. Throughout the epididymal duct, the reactions indicating the binding of exogenous PRL were slightly stronger than those testing for binding of endogenous peptides. The significance of such binding to the epididymis is uncertain but PRL may perform the same functions in epididymal principal cells as it does in the testis, prostate, and seminal vesicles.     

Androgenic regulation of luteinizing hormone secretion: relationship to androgen binding in sheep pituitary

Castrated ram lambs (wethers) were investigated for sensitivity to androgen feedback and to determine whether this feedback inhibition of luteinizing hormone (LH) was associated with changes in pituitary androgen receptors. Administration of Silastic capsules containing either dihydrotestosterone or testosterone was found to produce dose-dependent inhibitory effects on serum LH levels in wethers. Physiological dosages of these androgens (i.e., those that produce serum levels of dihydrotestosterone [0.24 ng/ml] or testosterone [2.1 ng/ml] similar to those of intact rams) resulted in differential inhibition of serum LH and LH content of the anterior pituitary. Whereas the inhibitory effect of dihydrotestosterone on pituitary LH content was much more dramatic than that seen with testosterone, the high dosage of testosterone also produced a substantial decrease in pituitary LH content. Responses of the pituitary to changes in serum androgen were compared to responses of the seminal vesicle, which served as a control androgen target organ. Androgen levels were positively correlated with seminal vesicle weights, but pituitary weights were unaffected by castration and/or androgen replacement. Treatments with dihydrotestosterone were associated with decreased cytosol androgen binding activity (i.e., receptors) in pituitary and seminal vesicle, suggesting that both of these tissues were sites of androgen action. Although testosterone inhibited serum LH levels, pituitary cytosol androgen receptors were not affected by changes in serum testosterone. We conclude from these data that dihydrotestosterone is a physiological regulator of pituitary LH secretion in the ram and that further study is needed to investigate the complex actions of testosterone and its metabolites on pituitary function.     

Effect of cyproterone acetate on the pregnancy-blocking ability of male mice and the possible chemical nature of the pheromone

Even though castration abolished the ability of alien males to induce implantation failure (the Bruce effect) in newly inseminated females, treatment of intact alien males with the steroidal antiandrogen, cyproterone acetate, for 14 days (short term) did not significantly depress their ability to induce the Bruce effect. However, prolonged treatment (42 days) with cyproterone acetate suppressed the pregnancy-blocking ability of alien males to some extent, possibly due to the antigonadotrophic properties of the drug. Ovariectomized alien females treated with implants of testosterone (androgenized females) exhibited the ability to block implantation in newly inseminated females, but concurrent treatment of androgenized females with cyproterone acetate did not depress this ability. The results strongly suggest that the pheromone involved in the male-induced implantation failure is not the product of an androgen-dependent tissue, but is likely to be a product of androgen metabolism.     

Glucocorticoids and aspirin inhibit and cyproterone acetate enhances the androgenic response in mouse kidney

Glucocorticoids and aspirin antagonize the androgenic response in mouse kidney, but not in ventral prostate or seminal vesicles. These agents impeded the testosterone-mediated increase in kidney weight, cytochrome c oxidase, and lysosomal hydrolases and urinary excretion of lysosomal hydrolases and proteins. They also attenuated the testosterone-induced decrease in enzyme latency and membrane stability of kidney lysosomes. In contrast, the antiandrogen cyproterone acetate is weakly androgenic in kidney and potentiates testosterone-induced lysosomal enzymuria and proteinuria (synandrogenic effect).     

Androgen control of the secretion in the sebaceous-like preen gland

Uropygial gland of quails was previously proved to have androgen receptors. The waxes secreted by this gland were shown to consist mainly of linear saturated fatty acids esterified by linear saturated diols. In adult male quails, the relative concentration of the C12 diol was found to be correlated to the androgen levels of the birds. Furthermore, in castrated quails implanted with testosterone propionate capsules, 6 days were required for the C12 diol to reach the level found in sexually active birds. These testosterone effects related to the C12 diol were nullified by cyproterone acetate administration. Consequently the C12 diol is a reliable specific marker for androgenicity in the quails' preen gland, a new and accurate model for the study of androgen action in sebaceous glands.     

Gamma-glutamyl transpeptidase of rat seminal vesicles; effect of orchidectomy and hormone administration on the transpeptidase in relation to its possible role in secretory activity.

γ-Glutamyl transpeptidase was prepared from rat seminal vesicles by two methods and was found to be similar to rat kidney γ-glutamyl transpeptidase with respect to substrate specificity, stimulation of “glutaminase” activity by maleate, and apparent molecular weight. Histochemical studies demonstrated that γ-glutamyl transpeptidase is concentrated in the secretory epithelium of the seminal vesicle. Like the epithelium itself, the enzyme responds to the presence or absence of testosterone. The content and specific activities of γ-glutamyl transpeptidase and γ-glutamyl cyclotransferase in rat seminal vesicles are low in orchidectomized animals, an effect which is reversed by administration of testosterone but accentuated by estradiol administration. These enzymes may be involved in the secretory functions of the seminal vesicles.     

Effects of steroidal and non-steroidal antiandrogens on the androgen binding properties of the rat ventral prostate androgen receptor

Steroidal (cyproterone acetate) and non-steroidal (RU23908 and hydroxyflutamide) antiandrogens are able to block testosterone-induced increases in nuclear androgen receptor (AR) in the prostate of 1-day orchidectomized rats, but when given alone, RU23908 and hydroxyflutamide increase nuclear AR (RU23908 greater than hydroxyflutamide) in the same animal model. The increases in nuclear AR induced by antiandrogen alone or with testosterone alone are blocked by cycloheximide 1 h after administration, suggesting that androgen or antiandrogens induce de novo AR synthesis. Concomitant to nuclear AR accumulation, testosterone is able to induce depletion of cytosol and microsomal AR. Blockade of testosterone-induced depletion of microsomal AR, but not of cytosol AR, occurs in the presence of antiandrogens. Cyproterone acetate has a higher relative binding affinity (RBA) for microsomal AR and cytosol AR than RU23908 or hydroxyflutamide. This phenomenon is in good agreement with the degree of inhibition by these compounds of the association rate of androgen for the microsomal AR. This correlation between RBA and inhibition of the initial rate of hormone binding to the receptor is not found for cytosol AR. The results show that antiandrogens are not 'pure' antagonists of androgen action and they are potent agonists in the absence of testosterone. Furthermore, testosterone alone or antiandrogens per se regulate AR levels acutely by protein-synthesis dependent mechanisms of action, in rat ventral prostate.     

The action of 5 alpha-dihydrotestosterone and antiandrogens on the activities of 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the pituitary gland of gonadectomized rats

In gonadectomized rats of either sex s.c. administration of 5 alpha-dihydrotestosterone (DHT) reversed, in a dose dependent manner, effects brought about by gonadectomy: it decreased pituitary wet weight and serum levels of LH and FSH and suppressed microsomal enzyme activities involved in testosterone and progesterone metabolism in the pituitary gland, NADPH-linked 5 alpha-reductase and NADH-linked 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH). Concomitantly administered nonsteroidal antiandrogen, flutamide (5 mg/day), antagonized some of the suppressive effects induced by a 14-day treatment of gonadectomized rats with high dose (1 mg/day) of DHT. It completely blocked DHT action on pituitary 5 alpha-reductase activity in the female rat and, in the male, inhibition was found to be 30-35%. In male, but not female rats, it completely blocked DHT suppression of serum FSH level whereas it slightly, but significantly inhibited DHT suppression of serum LH in rats of either sex. However, flutamide did not prevent DHT suppression of pituitary wet weight or NADH-linked 3 alpha-HSDH activity. Concomitantly administered progestational antiandrogen, cyproterone acetate (5 mg/day), inhibited DHT-induced weight increase of seminal vesicles by 50-55% and completely blocked the weight decrease of pituitary gland but did not antagonize DHT suppression of serum gonadotropins or pituitary enzyme activities. The results obtained with flutamide suggest that DHT-induced suppression of pituitary NADPH-linked 5 alpha-reductase, but not NADH-linked 3 alpha-HSDH activity, might involve an androgen receptor mechanism.