Induction of male-typical aggression by androgens but not by estrogens in adult female mice

Ovariectomized adult CF-1 female mice were implanted with silastic capsules containing either testosterone (T), dihydrotestosterone (DHT), methyltrienolone (R1881), estradiol (E2), diethylstilbestrol (DES), or oil vehicle and were tested for aggressive behavior. The androgenic treatments (T, DHT, R1881) were highly effective in promoting male-like aggression while the estrogens (DES, E2) were completely ineffective. Subsequent receptor-binding studies confirmed assumptions about the specificity of DES, DHT, and R1881 binding to estrogen and androgen receptors in mouse hypothalamus.     

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.     

Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied $\text{in vitro}$. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10^?9?10^?6 M) of 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone; DHT), 5α-androstane-3α, 17β-diol (3α-diol), or the synthetic androgen 17β-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20α-hydroxy-4-pregnen-3-one(20α-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20α-OH-P production in a dose-related manner (R1881 > 3α-diol > DHT). In the presence of an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), the FSH-stimulated pregnenolone (3β-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3α-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3β-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20α-hydroxysteroid dehydrogenase(20α-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20α-OH-P, but involves an enhancing action upon 3β-HSDΔ⁵, Δ⁴-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.     

A comparison of the effects of methyltrienolone (R 1881) and 5 alpha-dihydrotestosterone on sexual behavior of castrated male rats.

The synthetic steroid methyltrienolone (R 1881) binds specifically with high affinity to intracellular androgen receptors and is not metabolized to androstanediol. Administration of R 1881 (1 mg/day) to castrated male rats facilitated intromission in significantly more animals than did 5α-dihydrotestosterone (DHT) (1 mg/day); however, the percentage of animals ejaculating and the pattern of behavior displayed were equivalent in the two groups. Combined administration of estradiol benzoate (EB) (2 μg/day) plus either R 1881 or DHT further facilitated males' sexual performance to levels previously seen in castrated male rats of the same strain when given testosterone propionate (TP). The results suggest that conversion of DHT to 3α- or 3β-androstanediol neither detracts from nor contributes to its ability to activate sexual behavior in the male rat.     

Human placenta contains a high affinity R1881 binding site that is not the androgen receptor

Human placental cytosol was shown to contain a species that binds the synthetic androgen, methyltrienolone (R1881) with high affinity (Kd 6.5 nM). Major differences were found between this placental androgen binding species and the classical androgen receptor found in human foreskin cytosol. Competitive binding assays in the placental cytosol using [3H]R1881 as tracer showed a 200-fold excess of testosterone to compete poorly, while dihydrotestosterone and the synthetic androgen mibolerone did not compete at all. The placental R1881 binding component was found not to bind to hydroxylapatite, although all classes of steroid receptors are reported to do so. Temperature studies showed that the placental binding site is stable at elevated temperatures with no loss of binding after 4 h at 45 degrees C. Ion exchange chromatography showed that the placental R1881 binding site eluted from DEAE cellulose at a lower salt concentration than foreskin androgen receptors. These results show that R1881 is not entirely specific for androgen receptors and that human placenta contains an androgen binding site that is not the classical androgen receptor.     

Characterization of unoccupied (R) and occupied (RA) androgen binding components of the hyperplastic human prostate.

Saturation protocols were developed for measurement of unoccupied (R) and steroid-occupied (RA) androgen binding components of human hyperplastic prostate. The concentration of unoccupied cytoplasmic binding sites (2 hr incubation at 2 degrees C) for the synthetic androgen R1881 (17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) and the synthetic progestin R5020 (17alpha,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) respectively was 10.7 +/- 1.4 and 14.3 +/- 3.2 fmoles per mg cytosol protein and the apparent steroid affinity respectively was 9.6 +/- 0.8 and 1.6 +/- 0.4 x 10(8) M-1. Steroid specificity of the unoccupied cytoplasmic R1881 and R5020 binding sites was similar. When R1881 and R5020 were employed as probes of total, R plus RA, cytoplasmic binding components (20-24 hr incubation at 15 degrees C) saturable binding of R5020 was not detectable. Total cytoplasmic R1881 binding site concentration and apparent affinity for R1881 were 51.7 +/- 3.3 fmoles per mg cytosol protein and 2.7 +/- 0.6 x 10(7) M-1. R5020 was a poor inhibitor of R1881 binding to total cytoplasmic R1881 binding components.     

Androgens and FSH affect androgen receptor and aromatase distribution in the porcine ovary

In the present study the authors investigated whether androgens could interact with FSH to induce aromatase and androgen receptor expression in porcine granulosa cells. Dissected whole porcine follicles (small, medium, and large) were incubated for 8 hours in M199 medium supplemented with testosterone (10(-7) M), FSH (100 ng/ml) or both those hormones. After incubation, the follicles were fixed and immunostained to visualise androgen receptor and aromatase. In cultures of granulosa cells isolated from small and large follicles, oestrogen secretion was measured by appropriate RIA. Incubation of follicles with testosterone and FSH increased aromatase immunoreactivity in preantral and early antral (i.e. small) follicles. The immunostaining for androgen receptor was slightly higher in medium follicles, while such hormonal stimulation had no effect on small and large follicles. Moreover, granulosa cells isolated from small follicles cultured with both testosterone and FSH produced more estradiol than control cultures (40 pg vs. 100 pg/10(5) cells). The level was relatively close to that obtained in the culture of control granulosa cells isolated from large preovulatory follicles (105 pg/10(5) cells). These results indicate that testosterone acts synergistically with FSH to increase aromatase expression in the small porcine follicles.     

Binding of methyltrienolone to various androgen-dependent and androgen-responsive tissues in four animal species.

Comparative sucrose gradient studies of the in vitro binding of dihydrotestosterone (DHT) and of a synthetic androgen, methyltrienolone (R 1881), have been done with the cytosols of various tissues of the rat, mouse, cock and man. With rat prostate cytosol, the amount of R 1881 and DHT binding in the 8-9S region of the gradient was found to be comparable. Specific 8-9S peaks of R 1881 were also found in rat levator ani/bulbocavernosus and skeletal muscles and in the mouse kidney. Only 4-5S peaks could be demonstrated in the cock's comb while DHT under the same conditions showed both 8-9S and 4-5S binding. Binding of R 1881 to the cytosol of the hyperplastic prostate was polydispersed, and showed evidence of the presence of aggregates. Evidence was also found that R 1881 could bind to the progesterone receptor in rat uterus. Our study supports the theory that in a given species the androgen receptors are similar if not identical in all the tissues. The synthetic androgen R 1881 appears to be a useful tool for androgen receptor studies in various animal species provided that the tissue under study contains no progesterone receptor.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Characterization of androgen receptors after photoaffinity labelling with [3H]methyltrienolone (R1881)

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in rat prostate, in a human transplantable prostatic adenocarcinoma (PC-82) and in calf uterus. Androgen receptors preparations were partially purified either via differential chromatography on 2',5'-ADP-Sepharose (rat prostate), via anion exchange fast protein liquid chromatography (rat prostate and PC-82) or via DNA-cellulose chromatography (calf uterus). Purification factors obtained with the three different methods were: 245, 75 and 40 respectively. Photolabelling of receptor preparations was performed via irradiation with a high pressure mercury lamp either before or after partial purification. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of approx 95 kD. The covalent attachment of [3H]R1881 to the 95 kD protein could be completely suppressed by a 200-fold molar excess of dihydrotestosterone. In rat prostate cytosol an androgen receptor with a molecular mass of approx 50 kD could be photoaffinity labelled with R1881. A similar size was found for the androgen receptor in the human prostatic adenocarcinoma. Our results show that photoaffinity labelling of androgen receptors with [3H]R1881 as ligand can be applied for characterization of partial purified androgen receptor preparations.     

Analysis of androgen action on pituitary gonadotropin and prolactin secretion in ewes

To study the role of androgens in the control of gonadotropin and prolactin secretion in ther ewe, we have characterized androgen receptors in pituitary cytosol, and investigated the effect of androgens on pituitary hormone release in vivo and in vitro. High affinity, low capacity receptors, with an affinity for methyltrienolone (R1881) greater than 5 alpha-dihydrotestosterone (5 alpha-DHT) greater than testosterone (T) much greater than androstenedione (A4), estradiol-17 beta (E2) and progesterone (P), were identified in pituitary cytosol. Addition of 1 nM 5 alpha-DHT, but not A4, inhibited luteinizing hormone (LH) release from pituitary cells in vitro, induced by 10(10) to 10(-7) M luteinizing hormone releasing hormone (LHRH). The release of follicle-stimulating hormone (FSH) with 10(-9) M LHRH was inhibited when cells were incubated with 1 nM 5 alpha-DHT. 5 alpha-DHT had no effect when higher or lower doses of LHRH were used. In ovariectomized ewes, neither an i.v. injection of 1 mg, nor intracarotid injections of up to 1 mg, 5 alpha-DHT affected plasma LH, FSH or prolactin levels, despite dose-related increases in plasma 5 alpha-DHT levels. Daily or twice daily i.m. injections of 5 mg 5 alpha-DHT in oil did not affect LH or FSH levels, but daily injections of 20 mg significantly reduced plasma LH levels within 4 days and plasma FSH levels within 6 days. Thus, despite the presence of androgen receptors in the ewe pituitary, we conclude that androgens per se are of minimal importance in the regulation of pituitary LH, FSH and prolactin secretion in the ewe. The low binding affinity of A4 and the lack of its effect on hormone secretion in vitro suggests that A4 may act as an estrogen precursor rather than an androgenic hormone. The function of the pituitary androgen receptor remains to be established.     

Time-courses of the appearance/disappearance of nuclear androgen + receptor complexes in the brain and adenohypophysis following testosterone administration/withdrawal to castrated male rats: relationships with gonadotropin secretion

We characterized the temporal dynamics of brain and pituitary cell nuclear androgen receptor binding and serum androgen and gonadotropin levels associated with the implantation and removal of testosterone (T)-filled Silastic capsules into performed s.c. flank pouches of castrated, awake male rats. These capsules produced serum T levels in the physiologic range. The number of cell nuclear androgen + receptor complexes, as measured in an exchange assay using [3H]R1881, increased 15-fold at 0.5 h after capsule insertion in the HPAS (combined hypothalamus, preoptic area, amygdala and septum) and anterior pituitary gland, but then showed a second progressive rise within the next 8 h. This pattern suggests that T exerts an initial action in the tissues to alter the affinity and/or number of available androgen receptors. There was a lag time of 2-4 h to the first indication of negative feedback suppression of LH secretion. Serum LH levels declined only slightly at 4 h after capsule insertion but continued to fall thereafter, reaching undetectable values by 24 h. In contrast, serum FSH levels declined only slightly after 24 h of T exposure. After removal of the T capsules, serum T levels declined to castrate values within 2 h at which time the level of androgen + receptor complexes had fallen to 60% in the brain and pituitary. Serum LH and FSH concentrations were unchanged at 2 h after capsule removal, but rose significantly within the next 2 h. The data indicate that the occupation of androgen receptors rapidly changes in response to variations in circulating T in a fashion that implicates their involvement in the expression of this steroid's negative feedback actions on gonadotropin secretion.     

Sexual differentiation of androgen-sensitive and estrogen-sensitive regulatory systems for aggressive behavior

CF-1 female mice were treated with either testosterone (T), diethylstilbestrol (DES), or methyltrienolone (R1881) on the day of birth and were subsequently tested for their responsiveness to the aggression-promoting property of androgen or estrogen during adulthood. The results showed that neonatal exposure to androgen enhanced subsequent sensitivity to androgenic stimulation but did not alter responsiveness to estrogens. Neonatal estrogen treatment established the capacity to exhibit aggression in response to estrogenic stimulation in adulthood but had little effect on responsiveness to androgens. These data indicate that the androgenic and estrogenic metabolites of T have distinct roles in masculinization of the neural substrate for aggressive behavior.     

Steroid hormone regulation of prostatic acid phosphatase expression in cultured human prostatic carcinoma cells

We have investigated the modulation of prostatic acid phosphatase expression in the human prostatic cancer cell line LNCaP in response to the natural androgens testosterone and dihydrotestosterone, the female sex steroid estradiol and the synthetic androgen R1881 (methyltrienolone). Testosterone and dihydrotestosterone at 1 microgram/ml enhance the acid phosphatase synthesis by a factor of 3.5, while a hundred-fold lower concentration of the synthetic androgen R1881 induces an almost five-fold increase in the expression of this enzyme. The stimulation by all androgens tested and estradiol was dose-dependent. The synthetic glucocorticoid triamcinolone acetonide does not modulate the prostatic acid phosphatase expression in LNCaP cells, neither alone nor in combination with R1881.     

Androgen receptor in human placental villi

In studies with a synthetic androgen, R 1881, an androgen-binding component was found in the cytosol of human placental villi. Kinetic analysis indicated that the Kd value of this component was 1.4 nM at 0-4 degrees C and that binding of R 1881 amounted to 277 +/- 73 fmol/mg protein. glycerol density gradient ultracentrifugation showed a peak of binding activity in the 8S region in a medium of low ionic strength, but in the 4.5S region in a medium containing 9.5 M KCl. The R 1881-binding component was inactivated by mild heat- or trypsin-treatment, but not by treatment with DNase or RNase. Most of the R 1881-binding activity was sedimented at 20 to 40% saturation of ammonium sulfate. These findings indicate that the R 1881-binding component in human placental cytosol is quite similar in its characteristics to androgen receptors, which are present in various androgen-responsive organs. Testosterone was a more potent competitor of R 1881-binding than DHT or cyproterone acetate. Scatchard plots indicated that the binding site of testosterone was identical with that of R 1881. These findings suggest that the androgen receptor in placental cytosol is specific for testosterone. The Kd value for testosterone was calculated to be 3.2 nM.     

Demonstration of a nuclear androgen receptor in erythroblasts obtained by culture of human bone marrow

The new techniques of culture of bone marrow have shown that androgens and 5 beta steroids exert a direct effect on erythroid precursor cells from human or animal bone marrow. By contrast, the mechanisms of the intracellular actions of those compounds are poorly understood. Tritiated methyltrienolone (R 1881), a synthetic androgen that highly binds to androgen receptor, has been used for the study of a binding activity in nuclear extracts of cultured erythroblasts from human bone marrow. The nuclear extracts contain binding sites saturable at low concentrations of 3H-R 1881 (20-30 nM). Scatchard analysis revealed that the R 1881-nuclear complex has a dissociation constant (Kd) of 25-50 nM, and the number of binding sites was 235-370 fmoles/mg protein. The complex sedimented on 5-20% sucrose gradient in the 3.9 S region and 5 beta dihydrotestosterone compete strongly with R 1881 for binding sites. This binding component has characteristics of high affinity, low-capacity, sedimentation behaviour, and specificity commonly attributed to "androgen receptors".     

Mechanism of androgen-receptor augmentation. Analysis of receptor synthesis and degradation by the density-shift technique

The ductus deferens smooth muscle tumor cell line (DDT1MF-2) contains receptors for, and is stimulated by, androgens. Cells cultured in the absence of androgens maintain a basal level of androgen receptors. Following incubation with various concentrations of the synthetic androgen methyltrienolone (R1881) for 1-6 h, the concentration of these receptors increased from 6.0 to 12.2 fmol/micrograms of DNA, while the equilibrium dissociation constant (Kd) of 0.5 nM for this steroid remained unchanged. The steroid-induced increase in androgen receptor levels was specific for androgens and dependent upon protein synthesis. The mechanism of receptor augmentation was examined by utilization of isotopically dense amino acids to determine rates of receptor appearance and degradation in the presence or absence of [3H]R1881. In the absence of androgens, the half-life of the androgen receptor was 3.1 h, with a rate constant (kD) of 0.22/h. In the presence of 1 nM [3H]R1881, however, the half-life was 6.6 h, with kD = 0.11/h. The rate constant for receptor synthesis (ks) in the absence or presence of [3H]R1881 was calculated to be 1.35 and 2.23 fmol/micrograms of DNA/h, respectively. Thus, androgen-induced androgen-receptor augmentation is explained by an increase both in receptor half-life and in rates of receptor synthesis.     

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well.     

Photoaffinity labelling of androgen receptors with 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preparations.