Impaired 3H-testosterone uptake and metabolism in the accessory sex glands of diabetic castrated male rats.

The uptake and retention of 1,2-3H-testosterone in accessory sex glands, muscle and liver of streptozotocin diabetic castrated male rats, insulin-treated diabetic castrated rats and non-diabetic castrated control rats were studied at various time intervals after an intravenous injection. Diabetes reduced the retention of 3H-testosterone in the prostate, the preputial gland and the epididymis. Exogenous insulin slightly increased the retention of 3H-testosterone in these tissues of diabetic rats. No significant differences in the radioactivity in the rectus abdominis muscle, the coagulating glands and the seminal vesicles were found between the various experimental groups. Ventral prostate homogenates obtained from diabetic and control rats were incubated with 3H-testosterone in vitro. The steroids were extracted and thin-layer chromatographs were scanned for radioactivity. In prostatic homogenates taken from diabetic rats, testosterone transformation to dihydrotestosterone was reduced. The results indicate that the impaired function and androgen retention of the accessory sex glands of diabetic male rats is at least partly due to the reduced formation of dihydrotestosterone from testosterone.     

Genetic endocrine and ethological mechanisms of differential reproduction. I. A comparative genetic analysis of the basal testosterone level in the blood plasma and the relative weight of the testicles and accessory sex glands in male laboratory mice

Comparative genetic study of the basal plasma testosterone level, relative weights of testicles, seminal vesicles, and preputial glands in 13 strains of laboratory mice showed significant interstrain differences in all indices studied. Under the relative rest condition, a significant positive interstrain (genotypic) correlation was found between basal blood testosterone level and the weight of preputial glands and testicles. At the same time, no intrastrain (paratypic) correlations between hormonal and morphometric indices under study were found. An important positive and highly significant paratypic correlative dependence was seen between the relative weights of seminal vesicles and preputial glands.     

Carcinogenesis in accessory sex organs of rats induced by 3,2′-dimethyl-4-aminobiphenyl: response to androgen deprivation

 It is generally accepted that early human prostate cancers reveal higher androgen dependency than do advanced ones. In the present study, we examined whether the animal model of prostate cancer has already lost androgen dependency at the early stages of carcinogenesis. At experimental week 46, androgen deprivation was induced in rats and the incidences of atypical hyperplasia and cancer were examined in the ventral, dorsolateral prostate, coagulating glands, and seminal vesicles. Androgen deprivation significantly lowered the incidence of atypical hyperplasia in all four organs. As for the incidence of cancer, no significant differences were observed in the coagulating glands and seminal vesicles. Regarding atypical hyperplasia, androgen deprivation significantly decreased the proliferative cell nuclear antigen labeling index in the coagulating gland and seminal vesicles. The presence of cancer was also decreased in the coagulating gland but not in the seminal vesicles. With control group specimens, more intense staining of androgen receptor was observed in atypical hyperplasias than in cancers. Compared with the atypical hyperplasias, the cancers revealed low androgen dependency at the early stages of carcinogenesis. The cancers in the seminal vesicles also revealed higher androgen independency than did those in the coagulating gland. Accepted: 6 May 1997     

Effect of antiandrogen cyproterone acetate on the action of testosterone on mouse kidney.

The loss of endogenous testosterone in castrated male mice leads to a marked decrease in seminal vesicle and kidney tissue weight. 21 days' administration of exogenous testosterone abolished the effect of castration on the seminal vesicles and kidney tissue. The antiandrogen cyproterone acetate produced significant changes in the target tissue for androgens, i.e. in the seminal vesicles. In every case it blocked the action of both exogenous and endogenous testosterone on the seminal vesicles, but failed to block the "renotropic" action of testosterone, expressed as relative kidney weight. Contrary to its effect on the seminal vesicles, it did not influence relative kidney weight in normal animals. It likewise did not block the effect of exogenous testosterone on kidney tissue. The mechanism of the action of cyproterone acetate in androgen-dependent tissues is known to consist in inhibition of androgen binding to specific cell receptors in the target tissues. Some of the specific androgen receptors in mouse kidney are evidently different in character from those in the accessary sex glands, that being the reason why cyproterone acetate has an antiandrogenic, but not an antirenotropic effect. In agreement with experiments on rats, adrenal weight also decreases in mice after the administration of cyproterone acetate.     

Testosterone and dihydrotestosterone levels in epididymis, vas deferens, seminal vesicle and preputial gland of mice after hCG injection

Temporal changes of testosterone (T) and dihydrotestosterone (DHT) levels were measured by RIA in epididymis, vas deferens, seminal vesicle and preputial gland of adult male mice after a single injection of hCG. The response of circulating T to hCG stimulation was rapid and persisted over a period of 48 h. The temporal changes of androgen content of target organs paralleled the modifications of circulating T. In all organs the high androgen levels attained at 1 or 4 h plateaued until 24 h, decreased thereafter and returned to basal values at 72 h. The concentration of T by sex accessory organs was more accelerated by hCG injection than its conversion into DHT.     

A study of the androgenic function of the epididymis

Rats were subjected to bilateral orchidectomy or orchido - epididymidectomy and maintained on either 500 micrograms testosterone or testosterone propionate daily. The ventral lobes of the prostate were subsequently excised and examined for androgen receptors in terms of the total present in the cytosol and the nucleus, the proportion unoccupied by endogenous androgen and the relative populations that were nuclease excisable or nuclease resistant in the two groups of animals. A further group of animals was subjected to unilateral deferential venotomy and the same parameters examined in the ipsi- and contra-lateral lobes of the ventral prostate and the seminal vesicles. In the absence of the epididymides there was a reduction in the number of receptors per prostatic cell and an increase in the proportion that were unoccupied. The nuclei from these glands contained fewer receptors than did those from the animals in which the epididymides had not been excised. The effect of unilateral deferential venotomy was to bring about a relative increase in the number of cytoplasmic receptors in the ipsilateral lobes of the ventral prostate with a much greater proportion unoccupied compared with the lobes contra-lateral to the ligation. There was again an increase in the proportion of nuclease-sensitive receptors in the nuclei ipsilaterally. The conclusions are that the absence of the epididymides in androgen-maintained rats or deferential venotomy induces a relative androgen- deficiency of the prostate and seminal vesicles as reflected in the androgen receptor populations of these organs.     

Sensitivity and specificity of the bioassay of estrogenicity in mammary gland and seminal vesicles of male mice

Young intact (18 days old) and adult castrated males of CBA and C3H/Di mice were used for measuring the estrogenicity on the basis of growth response of mammary epithelial structures and the weight of seminal vesicles. It was demonstrated that heavier young males had disproportionally heavier seminal vesicles (sex steroid-responsive organs) than small animals at day 33 of age (that is on the day when experimental animals were killed and organs dissected). However, the weight of the spleen (sex steroid-nonresponsive organ) was proportionally related to body weight. To minimize variability in hormone responsiveness, all animals were weighed at the age of 18 days and only males weighing 8+/-1 g were used for hormone treatment. The percentage area of mammary fat pad occupiedby mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.01 microg x d(-1). The maximum effective dose of estradiol was 0.1 microg x d(-1) and dose 10 microg x d(-1) of estradiol decreased mammary size to control level (inverted-U-shaped dose-response curve). Progesterone alone stimulated mammary growth only in high doses (500 microg x d(-1) and higher) in young intact males, but had no effect on mammary growth in adult castrated animals. In young intact males, estradiol alone, or progesterone alone decreased the weight of seminal vesicles. No such inhibitory effect of these hormones was noted in adult castrated males. Progesterone acted synergistically with estradiol to produce higher mammary growth compared to that in males treated with estradiol alone. In the presence of progesterone seminal vesicles weight was decreased by estradiol given in such low doses as 0.001 microg x d(-1) of estradiol, which is 10 times lower than that effective in animals treated with estradiol alone. On the other hand, in the adult castrated males a combination of estradiol plus progesterone stimulated seminal vesicles weight. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate (a synthetic steroid exhibiting progestantial and estrogenic activities) and inhibited by both testosterone and cortisol. Estradiol, progesterone, norethindrone acetate, or testosterone did not affect spleen weight and size of mammary lymph nodes.However, cortisol significantly decreased not only spleen weights but also size of mammary lymph nodes. These results showthat simultaneous evaluation of mammary gland growth, seminal vesicles, and the spleen weight in the same animal is suitable for bioassay of estrogenicity as well as for detection of androgenic and antiandrogenic activities.     

Androgen glucuronides: I. Direct formation in rat accessory sex organs

A simple one-step procedure is described on the isolation of androgen glucuronides from various rat tissues. This procedure uses polyacrylamide gel electrophoresis, and permits a quantitative isolation of a single band containing the total androgen glucuronides without the contamination of free androgens and androgen sulfates. This procedure was used to determine the ability of various tissues of the rat to form androgen glucuronides directly when they were incubated with 1,2-[³H]-testosterone (0.1 μM) $\text{in}$$\text{vitro}$. Of eleven organs studied, only the accessory sex organs (ventral prostate, seminal vesicle, and coagulating gland), liver, and kidney were capable of forming androgen glucuronides. At the end of a one-hour incubation period, approximately 1% of the total radiolabeled steroids in the prostatic tissue minces were in the form of glucuronide conjugates. The predominant androgen glucuronide formed in the accessory sex organs was 5α-androstane-3α,17β-diol 17β-d-glucuronide. This is in contrast to the rat liver and kidney in which testosterone glucuronide was the predominant conjugate.A similar amount of labeled glucuronide conjugates was formed from either [³H]-testosterone, [³H]-dihydrotestosterone or [³H]-androstenedione, whereas negligible amount of steroid conjugates was formed from [³H]-cortisol. The formation of androgen glucuronides requires metabolically active tissues; furthermore, the conjugation process was inhibited by the antiandrogen, cyproterone acetate, or by metabolic inhibitors, such as oligomycin or N-ethylmaleimide.     

The influence of aging on androgen dynamics in the male rat

Androgen assimilation was investigated in a variety of accessory sex organs (seminal vesicles and anterior, dorsal, lateral, and ventral prostates) and in several nonaccessory sex organs in male Wistar rats. After administration of a pulse dose of [³H]testosterone in vivo to intact young (3–4 months old) rats, [³H]testosterone was the primary radioactive steroid recovered from most organs examined, except for the secondary sex glands where the reduced metabolites, [³H]5α-dihydrotestosterone (DHT) and [³H]5α-androstanediol(s), predominated. At longer postinjection times, [³H]DHT was preferentially retained in the accessory sex glands, presumably reflecting intracellular metabolism of [³H]testosterone to this compound and subsequent specific binding of [³H]DHT to receptor proteins. At the longest postinjection interval investigated, the ventral prostate retained greater concentrations of [³H]DHT than the lateral prostate which in turn had a higher [³H]DHT concentration than the seminal vesicles or anterior or dorsal prostates. The latter three glands retained approximately equal concentrations of [³H]DHT. Scatchard plot analyses of cytosol binding in 24-h castrates indicated that with one exception, the level of high affinity DHT binding sites was generally correlated with the retention of [³H]DHT in vivo in intact rats. Specifically, while the affinity for DHT binding in all accessory sex organs was the same, the number of high affinity binding sites per mg wet tissue weight was on the order of ventral prostate > anterior prostate ≥ seminal vesicles ≥ dorsal prostate > lateral prostate. Studies of the influence of aging to 22–26 months revealed no apparent differences in the affinity of the DHT receptor for its ligand in any of the accessory sex glands from 24-h castrates when the receptors were present in levels sufficiently high to quantify. The concentration of available DHT receptors with advancing age remained constant in the anterior and dorsal prostates, increased in the seminal vesicles, and declined in the ventral and lateral prostates. The decreases observed in the ventral prostate were only partial, but the receptors of the lateral prostate declined to nondetectable levels.     

Demonstration of an androgen receptor in rat pancreas.

[1,2,6,7-3H]Testosterone (250 muCi) was administered to castrated male rats; after 30 min a labelled testosterone-receptor protein complex with a pI of 5.1 was recovered from the pancreatic cytosol. A labelled testosterone-receptor complex with an identical pI was also extracted from the nuclear fraction of rat pancreas after incubation of minced pancreatic tissue with 0.1 muM-]1,2,6,7-3H]testosterone for 30 min at 37 degrees C. Studies in vitro showed that [1,2,6,7-3H]testosterone was bound to a receptor protein focusing at a pI of 5.1 and with a Kd of 2 nM and a number of binding sites of 4.7 fmol/mg of protein in castrated male rats. The testosterone-receptor complex sedimented at 3.5 S in high-salt sucrose-density gradients, was excluded from Sephadex G-200 and Ultragel ACA-34, was stable towards treatment with dextran-coated charcoal, was relatively sensitive to heat, and was stable to treatment with deoxyribonuclease and ribonuclease, but was sensitive to treatment which proteinase. It is suggested that the pancreatic androgen receptor, which was also present in castrated female rats, may play a role in sex-steroid regulation of pancreatic function.     

Localization,metabolism, and binding of estrogens in the male rat

Estrogen assimilation by male Wistar rats was examined in these studies in several accessory sex organs (seminal vesicles and anterior, dorsal, lateral, and ventral prostates) as well as in a variety of nonaccessory sex organs. When [³H]estradiol was injected into intact 3- to 4-month-old rats in a pulse dose, no selective accumulation of radioactivity recovered as estradiol was found in the accessory sex glands when compared to other organs. This was due at least in part to the metabolism of estradiol to estrone and to the relatively low concentration of high affinity estrophilic molecules in the accessory sex organs. The order for the rate of formation of estrone from estradiol in tissues obtained from intact animals was ventral prostate > lateral and dorsal prostate > anterior prostate and seminal vesicles. Steroid specificity studies for cytosol estradiol binding by the ventral prostate and seminal vesicles revealed that estrophilic molecules exist in these organs. Based on Scatchard plot analyses in 24-h castrates, the number of available estradiol binding sites was too low in the ventral prostate to quantify accurately, but the seminal vesicles contained distinctly more estrophilic activity than the ventral prostate. The affinity for the seminal vesicle cytosol estradiol-estrophile binding exceeded that quantified for the seminal vesicle dihydrotestosterone-androphile reaction while the number of estradiol binding sites was less than that quantified for dihydrotestosterone. In relation to the accessory sex organs of other species, the rat seminal vesicles have a relatively small amount of cytosol estrophile. The findings that the seminal vesicles catabolize less estradiol and contain significantly more estrophilic activity than the ventral prostate is consistent with and offers insight into the noted estrogenic sensitivity of the seminal vesicles and lack thereof in the rat ventral prostate. With aging of the rat from 3–4 months to 22–26 months, the affinity of the seminal vesicle estradiol-estrophile interaction was unchanged but the number of binding sites increased significantly.     

Enhanced differentiation of sexually dimorphic organs in L-thyroxine treated Tfm mice

Summary In the present study we examined the effects of L-thyroxine on the differentiation of three sexually dimorphic organs in mice affected by the testicular feminization mutation (Tfm): the submaxillary salivary glands, the kidneys, and the adrenal glands. In the salivary gland we compared the effects of L-thyroxine and testosterone in normal and Tfm mice on the morphology of the intercalated tubules and on the NGF content of this gland. In the kidney we examined thyroid hormone effects on the sexually dimorphic proximal convoluted tubules. In the adrenal gland we studied the effects of the same hormone on the weight of the organ and on the structure and ultrastructure of the zona reticularis in untreated and L-thyroxine treated Tfm mice. The results show that the thyroid hormone enhances differentiation of the dimorphic structures in all these organs and increases the secretory activity of the salivary and adrenal glands. The stimulatory effects of L-thyroxine are more pronounced on the weight of the adrenal than of the salivary gland and of the kidney. Our results raise the question whether the enhanced differentiation elicited by L-thyroxine in the three sexually dimorphic organs of Tfm mutants is due to a direct hormonal effect, or whether it is fully, or in part, mediated through the hypothalamo-pituitary-thyroid axis.This investigation was supported by the Progetto Finalizzato Biologia della Riproduzione of the Italian National Council of Research (CNR)     

Testosterone concentration in testicular venous blood of adult rats and seminal citric acid and fructose concentration as well as weight of accessory sex organs.

In 31 adult rats of Wistar strain (body weight 231 +/- 14g) the relationships between testosterone concentration in testicular venous blood of an individual rat and weight of accessory sex organs (testes, seminal vesicles, ventral prostate, levator ani muscle) and citric acid and fructose concentrations in seminal vesicles were investigated. No direct relationship was found between testosterone concentration and the above parameters. The correlation coefficient varied from 0.042 to 0.394.     

Epitestosterone--an endogenous antiandrogen?

Epitestosterone (17 alpha-hydroxy-4-androsten-3-one), a normal constituent of human plasma and urine, prevents the testosterone induced changes in body weight and in organ weights of seminal vesicles and kidney of castrated male mice. It competes with methyltrienolone in the binding to rat prostate cytosol (Ki = 29.8 nmol.l-1. It inhibits also the activity of 5 alpha-reductase from rat prostate pellet (Ki = 1.2 mumols.l-1). Epitestosterone can be considered as a weak antiandrogen in the term of displacement of androgen from receptor binding and as an efficient inhibitor of 5 alpha-reductase.     

The metabolism of testosterone and dihydrotestosterone in an androgen-dependent tumour. A possible correlation between dihydrotestosterone and tumour growth in vivo

The effects of dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) and testosterone on the growth of the androgen-dependent Shionogi SC-115 tumour in mice have been compared and the metabolites in the tumour arising from each steroid have been identified. After the transfer of SC-115 tumour cells to castrated male mice, treatment of the recipients with dihydrotestosterone produced a striking proliferative response that enabled earlier tumour detection and led to a higher tumour incidence than obtained with testosterone. At short intervals after the intravenous injection of 200muCi of [1,2-(3)H]testosterone the amounts of radioactivity in tumour, muscle and seminal vesicles were almost equal. The metabolism of [1,2-(3)H]testosterone in tumour and muscle was slight in comparison with the extensive metabolism in seminal vesciles. Whereas up to 7% of the total neutral steroid recovered from whole tumour tissue and isolated nuclei was in the form of [1,2-(3)H]dihydrotestosterone, the amount of this compound in the corresponding preparations from seminal vesciles was several times greater. When the metabolism of [1,2-(3)H]dihydrotestosterone in tumour tissue was studied, it was found that more than 60% of the total neutral steroid in both cytoplasm and nuclei consisted of [1,2-(3)H]dihydrotestosterone. Thus much higher intracellular concentrations of dihydrotestosterone occurred with the administration of this steroid than with testosterone. Tumour cell proliferation was suppressed by oestradiol and the amount of androgen in nuclei was significantly decreased by high doses of this hormone.     

The stress effect in the prenatal period on sexual excitation and sexual orientation of the mice males

Estrus female behind holed transparent partition produced sexual motivation and sexual arousal in males. It was manifested in behavioral changes (an increase in time spent near the partition) and the testosterone level augmentation in blood. Female mice were exposed to stress (1 h/day restraint) in the last week of gestation. Prenatal stress was shown to decrease the blood corticosterone level as well as to diminish sexual motivation and sexual arousal in adult male mice. Estrus female exposure produced a lesser behavioral response and a lesser testosterone level augmentation. No changes in weight of testicles, seminal vesicles or adrenal glands were found, but preputial gland weight increased. In prenatally stressed males, a female preference decrease and a male preference increase were revealed in the partner preference test. These data suggest that prenatal stress decreases sexual motivation in males and leads to clear predisposition to homosexuality, although it does not produce complete inversion of sexual orientation.     

Male accessory sex glands produce heparin-binding proteins that bind to cauda epididymal spermatozoa and are testosterone dependent

Heparin binds to bovine sperm and stimulates capacitation in vitro. Seminal plasma alters the ability of epididymal sperm to bind heparin, and several heparin-binding proteins (HBPs) have been identified in bull seminal plasma. This study had three objectives: 1) to identify production sites of seminal plasma HBPs, 2) to determine which HBPs bound to cauda epididymal sperm, and 3) to determine whether presence of HBPs was testosterone dependent. Proteins from bull or rat seminal vesicles, prostates, and bulbourethral glands were separated by heparin affinity high-performance liquid chromatography. HBPs were found in all accessory glands of rats and bulls, but the major source of bovine seminal plasma HBPs appeared to be seminal vesicles. Between 25% and 50% of the protein from each gland bound to the heparin column, and NaCl concentrations required to elute proteins ranged from 0.15 to 1.4 M. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that major HBPs were relatively small, with molecular weights between 13 and 31 kDa, but some HBPs also exhibited higher molecular weights, between 40 and 100 kDa. Radioiodinated HBPs from each bovine gland were incubated with epididymal sperm. Labeled HBPs binding to sperm exhibited molecular weights of 14, 16, 24, and 30 kDa as determined by SDS-PAGE and autoradiography. The HBP content of the accessory sex glands decreased significantly in castrated rats and was restored to levels of sham-operated controls by testosterone replacement. Heparin-binding proteins may play a role in fertilization by attaching to sperm surfaces, enabling heparin-like glycosaminoglycans in the female reproductive tract to induce capacitation.     

Modifications of the gonadal function in the adult rat after fetal exposure to spironolactone

The effects of fetal exposure to spironolactone (SPL), an aldosterone antagonist with weak antiandrogen and gestagen properties, upon the pituitary-gonadal axis were studied in the offspring of rats that had been treated daily from gestation day 14 to day 20 with 10 or 20 mg SPL or the solvent vehicle (for controls). At 70-80 days of age, SPL-exposed rats showed no alterations in external genitalia or in body weight. However, males displayed a dose-dependent decrease in the weights of the ventral prostate and seminal vesicles. Whereas basal and gonadotropin-releasing hormone (GnRH)-induced plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, and 5 alpha-dihydrotestosterone levels were similar to controls, basal plasma and pituitary prolactin (Prl) levels were reduced (SPL-exposed 6.8 +/- 1.0 vs. controls 15.8 +/- 2.8 ng/ml and 6.1 +/- 1.2 vs. 11.6 +/- 1.8 microgram/anterior pituitary gland; mean +/- SEM). Cytosolic androgen receptors in ventral prostate were nonsignificantly decreased, but they increased after GnRH in contrast to controls. Nuclear androgen receptors were normal. Females displayed normal estrous cycles. Basal and GnRH-induced plasma FSH, Prl, estradiol, and progesterone concentrations were similar to controls, whereas plasma LH was elevated. Estrogen receptors in uterine cytosol were low and increased after GnRH. Ovaries and uteri were enlarged. The present study demonstrates that in utero exposure to SPL leads to endocrine dysfunctions that persist into adulthood. They are characterized in males by hypoprolactinemia, reduced weights of accessory sex organs, and a suggestion of functional modifications of androgen receptors. In females they are characterized by increased LH secretion, increased ovarian and uterine weights, and decreased uterine cytosol estrogen receptors, suggesting enhanced estrogenic action.     

Synergistic effect of testosterone and of a luteinizing hormone-releasing hormone agonist on androgen receptor content in the ventral prostate of castrated rats

The aim of the present experiment was that of studying the effect of an LHRH agonist analog on the prostatic content of cytosol and nuclear salt-extractable and salt-resistant androgen receptors (AR). Castrated rats were treated for six days with the LHRH agonist WY 40972 (A), with testosterone enanthate (T) or with A plus T. Intact adult male rats and castrated rats treated with the vehicle served as controls. The animals were sacrificed 18 h after the last subcutaneous injection. The ventral prostates were quickly removed and submitted to subcellular fractionation for the determination of cytosol and nuclear AR content. In addition, the weights of the prostates and of the seminal vesicles were recorded, and serum levels of LH and FSH were evaluated by radioimmunoassay. The dissociation constants (Kd) of cytosol and nuclear AR, on the order of 1 x 10(-9) M, were not affected by the various treatments. Conversely, the combined treatment with T and A induced a significant increase of nuclear AR in the prostatic tissue, when compared to the levels found in castrated rats treated with T alone and in intact rats. The treatment with T was able to restore the reproductive organs to their normal weights. The treatment with A inhibited the hypersecretion of gonadotropins induced by castration. The results show that, under the conditions of the present experiment, A exhibits a synergistic effect with T on nuclear AR content in the rat ventral prostate. The results also suggest that A acts directly on this androgen-dependent structure.