A filter disc assay for the measurement and characterization of androgen-binding protein in unconcentrated media from Sertoli cell-enriched cultures

As easy, rapid and sensitive assay which permits measurements of androgen binding protein (ABP) in unconcentrated spent media from Sertoli cell cultures is described. The method is based on the adsorption of the 5 alpha-dihydrotestosterone-ABP complex onto DEAE-cellulose filter paper discs at pH 8.5. It is demonstrated that this method permits the characterisation of ABP (ligand specificity, kinetic properties) and represents a useful tool for the study of the effects of various hormones on ABP production by cultured Sertoli cells. When added on day 5 of culture, FSH and other agents that raise intracellular cAMP increase ABP secretion up to 3 times. Natural and synthetic androgens provoke a 1.5-1.8-fold increase. The effects of androgens and FSH are additive and cyproterone acetate blocks the effects of androgens in the presence as well as in the absence of FSH.     

Steroid control of sexual behavior and brain aromatase in the dove: effects of nonaromatizable androgens, methyltrienolone (R1881), and 5 alpha-dihydrotestosterone

This study examines the effects of nonaromatizable androgens, methyltrienolone (R1881) and 5 alpha-dihydrotestosterone (DHT) on aggressive courtship and vocal behavior in the male ring dove. Since androgens may influence behavior by increasing the formation of estrogen in the brain, the effects of R1881 and DHT on brain aromatase activity were also studied using an in vitro microassay. Under conditions in which testosterone induced aggressive courtship patterns, the nonaromatizable androgens were ineffective. But DHT and R1881 induced vocal behavior with equal efficiency, indicating that androgens can influence mechanisms of vocal behavior without conversion to estrogens. The behavioral effectiveness of both hormones was reduced (approximately 50%) when the period between castration and treatment was doubled. Testosterone propionate increased formation of E2 from 3H-testosterone in both the preoptic (POA) and anterior hypothalamic areas. Neither of the nonaromatizable androgens affected POA aromatase activity. The results suggest that only the aromatizable androgen, testosterone, which is also required specifically for male courtship, increases preoptic formation of estrogen.     

Differential endocrine regulation of genes enriched in initial segment and distal caput of the mouse epididymis as revealed by genome-wide expression profiling

We have performed genome-wide expression profiling of endocrine regulation of genes expressed in the mouse initial segment (IS) and distal caput of the epididymis by using Affymetrix microarrays. The data revealed that of the 15 020 genes expressed in the epididymis, 35% were enriched in one of the two regions studied, indicating that differential functions can be attributed to the IS and the more distal caput regions. The data, furthermore, showed that 27% of the genes expressed in the IS and/or distal caput epididymidis are under the regulation of testicular factors present in the duct fluid, while bloodborne androgens can regulate for 14% of them. This is in line with the high testis dependency of epididymal physiology. We then focused on genes with moderate or strong expression, showing strict segment enrichment and strong dependency on testicular factors. Analyses of the 59 genes, including upregulated and downregulated genes, fulfilling the criteria indicated that the expression of 18 (17 downregulated genes; 1 upregulated gene) of 19 gonadectomy-responsive genes enriched in the IS was not maintained by the androgen treatment, whereas the expression of all six downregulated genes enriched in the distal caput and the majority of those with no strict segment enrichment of expression (28 of 34; consisting of 23 downregulated and 5 upregulated genes) were maintained by androgens. Hence, it is evident that testicular factors other than androgens are important for the expression of IS-enriched genes, whereas the expression of distal caput-enriched genes is typically regulated by androgens. Identical data were obtained by independent clustering analyses performed for the expression data of 3626 epididymal genes. Several novel genes with putative involvement in epididymal sperm maturation, such as a disintegrin and metallopeptidase domain 28 (Adam28) and a solute carrier organic anion transporter family, member 4C1 (Slco4c1), were identified, indicating that this approach is successful for identifying novel epididymal genes.     

Contraception masculine hormonale

The activities of male and female gonades depend on the same pituitary hormones FSH and LH. In the field of contraception, the situations are very different: it is rather easy to disturb maturation then rupture of the dominant follicle, whereas it is much more difficult to inhibit the daily production of more than one hundred millions spermatozoa. Therefore, even in case of efficient inhibition of gonadotrophins, azoospermia is not systematically obtained, and the problem is to appreciate the risk of pregnancy corresponding to a given value of sperm concentration. Molecules used for hormonal male contraception can be classified in two groups: steroids and GnRH analogs. The inhibitory effect of supra-physiological doses of testosterone on sperm production has been well known for a long time. In a preliminary study conducted by World Health Organisation Task Force, 271 volunteers received 200 mg of testosterone enanthate IM once a week: 65% became azoospermic within a 6 months period; the mean sperm concentration in the non azoospermic subjects was 3 millions/ ml. Asiatic men were better responders than caucasian. In a second study recently published, the efficiency of male contraception was tested in 349 men having less than 3 millions/ml: 4 pregnancies occured, corresponding to an overall pregnancy rate of 1.4 per 100 person-years, which reached 8.1% if azoospermic subjects were excluded. The administration during a long period of androgens at a supra-physiological level is not devoid of potential risks. The association of progestagens and androgens allows to give androgens at physiological doses as a supplementation treatment, since the main contraceptive effect is due to the progestagens. Medroxy-progesterone acetate has been widely studied, but others molecules, as Nor-ethisterone or levonorgestrel, are more powerful inhibitors or gonadotrophins and are good candidates for male contraception. On the contrary, all the trials testing the efficiency of GnRH analogs were very disappointing, with or without androgens supplementation. Recent works using GnRH antagonists as Nalglu look promising, since almost all men became azoospermic, however several problems must be resolved (histamine-like side effects; high coast of the treatment). Since androgens are involved in all protocols, it is very important, in order to promote hormonal male contraception, to find some gallenic forms of androgens which allow to avoid injections.     

Target neurons of endogenous androgens immunocytochemically localized in male rat hypothalamus

A polyclonal antiserum against androgens, i.e., testosterone, 5alpha-dihydrotestosterone and androstenedione, was tested to reveal target neurons of endogenous androgens in the hypothalamus of both intact and castrated male rats. Paraffin sections of hypothalamus and testis were immunostained by using Avidin-Biotin Complex method and 3-3' diaminobenzidine to visualize the immunoperoxidase complex. Conventional control experiments for method and antiserum specificity were performed. The antiserum proved to be specific for androgens, i.e., testosterone, 5alpha-dihydrotestosterone and androstenedione. The nuclear labeling observed in tissues stained by this procedure is consistent with the hypothesis that the labeled neurons contained DHT, which is the main testosterone metabolite active in the cell nucleus. The antiserum was effective in staining not only the hypothalamic neurons of intact males with normal serum levels of testosterone but also the hypothalamic neuron of castrated males with very low serum levels of testosterone. Evidence is presented indicating that the immunostaining technique represents a more specific and sensitive method to identify target neurons of endogenous androgens than autoradiography.     

Androgen deficiency in women

Androgens are defined as the steroids having a binding affinity of the androgen receptor. In the reproduction age a daily production of testosterone is equally divided between the ovaries and adrenal and local tissue conversion of androstenedione and DHEA. After menopause the 80% of testosterone is produced in ovaries, but majority of precursors for peripheral conversion is adrenal origin. Androgen receptors are present throughout in the body; over 200 cellular actions of androgens have been described. Androgenic action is determined by quantitative level of the androgen present in the circulation, its degree of binding to proteins, the degree of interconversion to other androgens and estrogens, and the biological potency and androgen receptor binding affinity of the androgen. The most common clinical symptoms of androgen deficiency are the reduction of sex motivation, sex fantasy, sex enjoyment, sex arousal, vaginal vasocongestion, but also reduction of pubic hair, bone mass, muscle mass, worsening of quality of life (mood, affect, energy), more frequent vasomotors symptoms, insomnia, depression, headache. All these signs and symptoms can be multifactorial. Most common conditions associated with hypoandrogenism in women are hypothalamic-pituitary abnormalities, lack or insufficiency of ovaries, adrenal insufficiency, glucocorticoid therapy, exogenous estrogen administration. Besides the clinical picture the free testosterone measuring is important for diagnosis. The method of choice of this measure is equilibrium dialysis assay. Despite of clinical importance of androgen insufficiency in women, none of methods of androgen substitution is approved by FDA.     

Simultaneous determination of plasma estrogens, androgens, and progesterone during the human menstrual cycle

A method is described for the purification and quantitation of estradiol-17β (E₂), estrone (E₁), testosterone (T), androstenedione (A) and progesterone (P) from a single plasma sample extraction. A combination of Sephadex LH-20 column chromatography and thin layer chromatography is used to separate the phenolic and neutral steroids; quantitation of the purified steroids is by radioimmunoassay (E₂, E₁) and competitive protein binding (T,A,P) techniques. The precision and accuracy of this method is comparable with that reported by others. A preliminary investigation of the temporal relationship between estrogens androgens, progesterone and serum LH during five normal and one short luteal phase menstrual cycle is presented.     

Mesenchymal mechanisms in prostate organogenesis

Abstract The development of the prostate is dependent upon androgens and stromal–epithelial interactions. Understanding the molecules and mechanisms by which androgens control prostate organogenesis has been a considerable challenge over the past few decades. Similarly, identifying the molecular signals passing between stromal and epithelial cells has been difficult, and consequently understanding how androgens and stromal–epithelial signalling interact is poorly understood. There remains significant uncertainty regarding how androgens control the growth of the prostate, although several pathways have been identified that are required for prostate development or which alter prostate growth. This review will summarize past findings relating to the pathways that might mediate the effects of androgens as well as molecules that act as stromal to epithelial signals in the prostate. It will also examine the approaches used to identify pathways of importance and the historical concepts that have informed these studies. In particular, the question of which mechanisms might be involved in early prostate organogenesis as well as anatomic aspects of organ induction will be described. Finally, models of prostatic development will be proposed and discussed.     

Synthesis of 16α-3H androgen and estrogen substrates for 16α-hydroxylase

The synthesis of 16α-³H androgens and estrogens is described. 1-(³H)-Acetic acid in the presence of zinc dust reacts with 16α-bromo-17-ketosteroids to produce 16α-³H-17-ketosteroids. This chemical reaction was used to prepare 16α-³H-dehydroepiandrosterone (I) and 16α-³H-estrone acetate (XI) from 16α-bromo-dehydroepiandrosterone (X) and from 16α-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16α-³H-androstenedione (II) and 16α-³H-estradiol-17β (VII). 16α-³H-Estrone (VI) was obtained by the chemical hydrolysis of 16α-³H-estrone acetate. The label distribution as determined by microbiological 16α-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16α position. These substrates can be used for measuring the 16α hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX).     

Skeletal effects of androgen withdrawal

Hypogonadism is considered to be one of the major risk factors for osteoporosis in men. Therefore, it is an important goal for skeletal research to improve our understanding of the skeletal effects of androgens. Androgen deficiency during growth is associated with a failure to acquire normal peak bone mass, and there is good evidence that the effects of androgens on skeletal growth and the development of a male skeletal phenotype are mediated through the androgen receptor. In adult men, acute withdrawal of androgens by surgical or chemical castration induces high turnover bone loss. Similarly, orchidectomy of aged, non-growing male rats is associated with a pronounced and sustained increase in bone turnover and with true loss of cancellous and cortical bone. Interestingly, the changes in bone turnover induced by orchidectomy are paralleled by a concomitant increase in B lymphopoiesis in bone marrow of rats and mice. Although there is firm evidence that male bone metabolism can be influenced by androgens and estrogen, a variety of clinical and animal experimental data have strongly suggested that, under physiological circumstances, the maintenance of cancellous bone mass in males involves the skeletal action of estrogen derived from aromatization of androgens. Aged male rats appear to closely mimic the conditions induced by androgen withdrawal in adult humans, and this animal model may be used 1) to elucidate further the role of muscle as a mediator of the actions of androgens on bone, 2) to explore the regulatory functions of androgens and estrogens in the male skeleton and the immune system, and 3) to find new treatment strategies for the prevention and treatment of osteoporosis in men.     

Long-acting contraceptive agents: testosterone esters of unsaturated acids

The synthesis of 13 new esters of testosterone is described, with the esterifying acids bearing acetylenic, olefinic, or polyunsaturated functions in the chain, for evaluation as long-acting androgens.     

Steroids and receptors in canine mammary cancer

The aims of this study were to investigate the serum and tissue content of androgens and estrogens in canine inflammatory mammary carcinomas (IMC) as well as in non-inflammatory malignant mammary tumors (MMT), and assessed the immunoexpression of estrogen and androgen receptors using immunohistochemistry. Profiles for the androgens dehydroepiandrosterone (DHEA), androstenedione (A4), and testosterone (T), and for the estrogens 17beta estradiol (E2) and estrone-sulphate (SO4E1) were measured both in tissue homogenates and in serum of MMT and IMC by EIA techniques in 42 non-inflammatory malignant mammary tumors (MMT) and in 14 inflammatory mammary carcinomas (IMC), prospectively collected from 56 female dogs. Androgen receptor (AR) and estrogen receptor alpha (ERalpha) and beta (ERbeta) expression was studied using immunohistochemistry (strepavidin-biotin-peroxidase method) in samples of 32 MMT and 14 IMC, and counted by a computer image analyzer. IMC serum and tissue levels of androgens were significantly higher than MMT levels. Tissue content of estrogens was also significantly higher in IMC than in MMT. Serum values of SO4E1 were significantly higher in IMC, but serum levels of E2 were significantly lower in IMC compared to MMT cases. Medium-high androgen receptor intensity was observed in 64.28% of IMC and 40.62% of MMT. No important differences were found between ERalpha expression in IMC (100% negative) and MMT (90% negative). ERbeta and AR were intensely expressed in highly malignant inflammatory mammary carcinoma cells. To our knowledge, this is the first report relative to AR immunohistochemistry in canine mammary cancer and to estrogens or androgens in serum of dogs with benign or malignant mammary tumors.     

Analysis of androgen-sensitivity in rat prostate X mouse kidney cell hybrids

Variant androgen-sensitive cell lines were produced by fusing freshly isolated epithelial cells from the rat ventral prostate with a line of murine renal tumor (RAG) cells. The properties of the cloned lines of the prostate X RAG hybrids can be summarized as follows: (1) the modal chromosome number of the hybrid cell lines ranged from 68 to 176; (2) the cells had doubling times of 7.6-49.5 h; and (3) epitheloid, ameboid and intermediate morphologies were observed among the various lines. The proliferative response of various hybrid lines to treatment with 10 nM 5 alpha-dihydrotestosterone was used to classify the hybrids as either very sensitive (greater than 40% reduction in cell doubling time), sensitive (greater than 10% reduction in doubling time) to androgens, or insensitive (less than 10% reduction in doubling time) to androgens. There was no direct relationship between the androgen-sensitivity of the cells and their androgen receptor content, suggesting that these variant cell lines may be useful for the study of the genetic factors involved in cellular responses to androgens.     

The androgenic regulation of the activities of enzymes engaged in the synthesis of deoxyribonucleic acid in rat ventral prostate gland.

The restoration of mitosis and growth of the prostate gland of castrated animals by androgens provides a favourable experimental system for studying the hormonal regulation of enzymes engaged in DNA replication. 2. Many DNA polymerase activities were identified in the prostate gland, but only a 9S form with a particular preference for denatured DNA as template was conspicuously enhanced by androgenic stimulation. 3. Thymidine kinase also provided a sensitive indicator of the hormonal regulation of DNA replication, and on electrophoretic criteria, one discrete form of the enzyme appeared precisely with the onset of mitoris. 4. Evidence is presented to support the view that DNA ligase activity is intimately associated in the process of DNA replication in the prostate gland. 5. A spectrum of deoxyribonuclease activities is present in the prostate gland, but only one form (pI7.0) can safely be said to be implicated in the process of DNA replication. 6. Androgenic stimulation of the prostate gland leads to the appearance of a component capable of denaturing or unwinding prostate DNA. This component is seemingly distinct from RNA or DNA polymerase activities on the basis of several distince physicochemical characteristics. 7. The conspicuous feature of all the changes in enzyme activities evoked by androgens in the prostate gland is their acute tissue- and steroid-specificity. Such changes could not be mimicked in liver or spleen and the regulatory role of androgens could not be simulated by other classes of steroid hormones. Particularly on the basis of studies with the anti-androgen cyproterone acetate, it is concluded that the changes are initially mediated by the androgen-receptor system and the high-affinity binding of 5alpha-dihydrotestosterone in the prostate gland. 8. The results are discussed in the context of the mechanism of action of androgens.     

Androgenic modulation of progesterone metabolism by rat granulosa cells in culture

Effects of androgens on progesterone accumulation, utilization of exogenous progesterone and accumulation of [4-14C]progesterone metabolites by rat granulosa cells in culture were studied. Androgen increased progesterone accumulation in cultures without exogenous progesterone and slowed the overall decline of progesterone concentration in cultures supplemented with exogenous progesterone. Both aromatizable testosterone and nonaromatizable 5 alpha-dihydrotestosterone decreased [4-14C]progesterone utilization by granulosa cells by 12 to 30%. This effect was observed irrespective of whether the cells were continuously exposed to androgens or only pre-exposed. In he same experiments, androgens decreased conversion of radiolabeled progesterone to 20 alpha-hydroxy-4-pregnen-3-one by 11 to 50% and to 5 alpha-pregnane-3 alpha, 20 alpha-diol by 26 to 49%. Accumulation of 3 alpha-hydroxy-5 alpha-pregnan-20-one was not altered in 3 h incubations and was increased by up to 43% in 24 h incubations by androgen treatment. It is suggested that androgens alter progesterone catabolism by granulosa cells by decreasing 20 alpha-hydroxysteroid dehydrogenase activity and that this effect may contribute to overall stimulatory action of androgens on progesterone accumulation.     

Studies on the regulation of the concentration of androgens and androgen receptors in nuclei of prostatic cells.

Experiments were performed to assess the effect of intracellular androgen metabolism and the availability of cytoplasmic receptors on the concentration of androgens and androgen receptors in nuclei of prostatic cells. It was found that androgens are incorporated into the nucleus by a regulated, selective process which appears to limit the type and amount of androgen transported across the nuclear membrane. The metabolic conversion of testosterone to dihydrotestosterone which takes place in cytoplasm does not reduce transport and, very likely, affects only the ratio of testosterone and dihydrotestosterone transferred into the nucleus. In vivo, when the intranuclear concentration of androgens approaches 250 nM (8 pmol per mg DNA), an apparent concentration ceiling is reached even in the presence of a downward concentration gradient that would be expected to promote further transport across the nuclear membrane. This finding strongly suggests that in vivo the nuclear membrane acts as a barrier to the passage of androgens and, therefore, mitigates against the possibility that passive diffusion is an important mechanism of afferent transport of androgens into the nucleus. The ability of the nucleus to concentrate testosterone and dihydrotestosterone was clearly demonstrated in vivo when cytoplasmic concentrations of androgens of approximately 20 nM were accompanied by intranuclear concentrations in the vicinity of 250 nM. Since the measured concentration of testosterone and dihydrotestosterone in prostate of several species fall within the 5-20 nM range, it is evident that androgen concentrations in the nucleus as high as 250 nM may be typical of the physiological steady state. At the latter concentration the nucleus contains 60 000 androgen molecules: in approximate terms one third of this total is bound to a large molecular weight component of the nucleus, one third is bound to a 3.3 S receptor and one third is free or loosely bound. Since 60 000 androgen molecules and 20 000 receptor molecules appear in the nucleus before transport stops, it seems that the quantity of 4.4 S cytoplasmic receptor estimated at 174 plus or minus 24 pmol per mg protein (equivalent to about 8000 molecules per cell) is insufficient to account for the total influx of androgens and androgen receptors into the nucleus. Thus, although these results support the view that cytoplasmic receptors and the capacity to transport androgens are closely linked phenotypic markers of intracellular steroid hormone action, they suggest that the control of androgen concentration in the nucleus is achieved in a more intricate fashion than simply through a dependence on the presumed translocation of 4.4 S androgen-receptor complex into the nucleus.     

Regularities in androgen reception in rat liver and its role in realizing direct androgenous effects

The content of androgen receptors (AR) in the cytosol and nuclear fractions of rat liver cells was studied by using the radioligand method. It was found that the nuclear AR content depends on the sex of animals as well as on the initial level of AR in the cytosol fraction. Elevation of AR concentration (p less than 0.05) in liver nuclei of different groups of animals was noted one hour after injection of 100 micrograms of methyltrienolone (R-1881). This elevation correlated positively with the initial level of AR in the cytosol. The accumulation of AR in liver nuclei was more pronounced in intact males than in females. A direct positive correlation between the initial level of cytosol AR and the final regulatory effect of androgens on the hepatic AR content (r = 0.955, n = 6, p less than 0.02) and on the content of the hepatic unusual estrogen-binding protein (UEBP) (r = 0.957, n = 9, p less than 0.001) were found. It is concluded that androgens induce in liver nuclei the accumulation of AR whose level correlates with that of the initial cytosolic AR. The latter reflects the efficiency of the direct effect of androgens on liver cells.     

Androgens inhibit the formation of catechol estrogens by estrogen 2-hydroxylase present in rat liver microsomal preparations

The inhibition of estrogen 2-hydroxylase by androgens was demonstrated in screening assays and has been further investigated under initial velocity conditions. The ability of testosterone, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone to block the conversion of estradiol to 2-hydroxyestradiol by male rat liver microsomal preparations was determined using two radiotracer methods--the conversion of [4-14C]estradiol to [4-14C]2-hydroxyestradiol and the release of 3H2O from [2-3H]estradiol. The apparent Ki's for the androgens ranged from 12.0 to 14.0 microM, with the apparent Km for the substrate estradiol in these assays of 2.08 microM. Multiple inhibition studies with the androgens and 2-bromoestradiol, an effective estrogen inhibitor, in male rat liver microsomes resulted in Dixon plots consisting of a series of nonparallel, intersecting lines. Thus, the androgens and 2-bromoestradiol are non-exclusive inhibitors, i.e. the binding of one compound to the enzyme does not interfere with the binding of the other. These interactions of androgens suggest that the steroid hormonal environment be considered in the examination of the physiological role(s) of the estrogen 2-hydroxylase and the catechol estrogen products.