Synaptic Long-Term Potentiation and Depression in the Rat Medial Vestibular Nuclei Depend on Neural Activation of Estrogenic and Androgenic Signals

Estrogenic and androgenic steroids can be synthesised in the brain and rapidly modulate synaptic transmission and plasticity through direct interaction with membrane receptors for estrogens (ERs) and androgens (ARs). We used whole cell patch clamp recordings in brainstem slices of male rats to explore the influence of ER and AR activation and local synthesis of 17β-estradiol (E2) and 5α-dihydrotestosterone (DHT) on the long-term synaptic changes induced in the neurons of the medial vestibular nucleus (MVN). Long-term depression (LTD) and long-term potentiation (LTP) caused by different patterns of high frequency stimulation (HFS) of the primary vestibular afferents were assayed under the blockade of ARs and ERs or in the presence of inhibitors for enzymes synthesizing DHT (5α-reductase) and E2 (P450-aromatase) from testosterone (T). We found that LTD is mediated by interaction of locally produced androgens with ARs and LTP by interaction of locally synthesized E2 with ERs. In fact, the AR block with flutamide prevented LTD while did not affect LTP, and the blockade of ERs with ICI 182,780 abolished LTP without influencing LTD. Moreover, the block of P450-aromatase with letrozole not only prevented the LTP induction, but inverted LTP into LTD. This LTD is likely due to the local activation of androgens, since it was abolished under blockade of ARs. Conversely, LTD was still induced in the presence of finasteride the inhibitor of 5α-reductase demonstrating that T is able to activate ARs and induce LTD even when DHT is not synthesized. This study demonstrates a key and opposite role of sex neurosteroids in the long-term synaptic changes of the MVN with a specific role of T-DHT for LTD and of E2 for LTP. Moreover, it suggests that different stimulation patterns can lead to LTD or LTP by specifically activating the enzymes involved in the synthesis of androgenic or estrogenic neurosteroids.     

Intracellular Ca2+ stimulates the binding to androgen receptors in platelets

In this study, we demonstrated that ADP-induced platelet aggregation activates the binding of testosterone (T) to its receptor. It is well known that binding of ADP to its receptors induced the release of Ca2+ ions from dense bodies into the cytosol of platelets. In this work, we compared the binding of testosterone or dihydrotestosterone to their receptors using cytosol obtained from ADP-treated and non-treated platelets. These experiments were repeated using EGTA (a calcium chelator) or U73122 (a phospholipase C enzymatic activity inhibitor) to the ADP-treated platelets. In addition, we also developed a competition analysis for the androgen receptors (AR) using [3H]DHT, non-radioactive T, DHT or cyproterone acetate from ADP-treated platelets cytosol. The results from this study indicate that the cytosol obtained from non-ADP-treated platelets did not show any binding to [3H]T or [3H]DHT, whereas cytosol from ADP-treated platelets binds to the radio-labeled androgens. Furthermore cytosol from ADP plus U73122-treated platelets did not show binding to [3H]T or [3H]DHT. These data suggest that intracellular Ca2+ ions stimulates the binding of androgens to their receptors in platelets cytosol. The competition analysis shows that T and DHT have high affinities for the androgen receptors with similar IC50 values, whereas cyproterone acetate shows a lower affinity. The results from these data clearly indicate the presence of androgen receptors in platelets.     

Specific morphogenetic events in mouse external genitalia sex differentiation are responsive/dependent upon androgens and/or estrogens

The objective of this study was to perform a comprehensive morphologic analysis of developing mouse external genitalia (ExG) and to determine specific sexual differentiation features that are responsive to androgens or estrogens. To eliminate sex steroid signaling postnatally, male and female mice were gonadectomized on the day of birth, and then injected intraperitoneally every other day with DES (200ng/g), DHT (1μg/g), or oil. On day-10 postnatal male and female ExG were dissected, fixed, embedded, serially sectioned and analyzed. We identified 10 sexually dimorphic anatomical features indicative of normal penile and clitoral differentiation in intact mice. Several (but not all) penile features were impaired or abolished as a result of neonatal castration. Those penile features remaining after neonatal castration were completely abolished with attendant clitoral development in androgen receptor (AR) mutant male mice (X(Tfm)/Y and X/Y AR-null) in which AR signaling is absent both pre- and postnatally. Administration of DHT to neonatally castrated males restored development of all 10 masculine features to almost normal levels. Neonatal ovariectomy of female mice had little effect on clitoral development, whereas treatment of ovariectomized female mice with DHT induced partial masculinization of the clitoris. Administration of DES to neonatally gonadectomized male and female mice elicited a spectrum of development abnormalities. These studies demonstrate that the presence or absence of androgen prenatally specifies penile versus clitoral identity. Differentiated penile features emerge postnatally and are sensitive to and dependent upon prenatal or pre- and postnatal androgen. Emergence of differentiated clitoral features occurs postnatally in either intact or ovariectomized females. It is likely that each penile and clitoral feature has a unique time-course of hormonal dependency/sensitivity.     

Identification of two nuclear androgen receptors in kelp bass (Paralabrax clathratus) and their binding affinities for xenobiotics: comparison with Atlantic croaker (Micropogonias undulatus) androgen receptors.

Two distinct nuclear androgen receptors (ARs) were identified in brain and ovarian tissues of kelp bass, Paralabrax clathratus, termed kbAR1 and kbAR2, which correspond to the two nuclear ARs we have previously characterized in Atlantic croaker, Micropogonias undulatus, termed acAR1 and acAR2. Scatchard analysis of nuclear fractions of whole brain tissue demonstrated that kbAR1 had a single class of high-affinity binding sites for testosterone (T; K(d) of 1. 8 nM and B(max) of 1.0 pmol/g tissue), whereas cytosolic fractions of kbAR2 ovarian tissue had a single class of high-affinity binding sites for dihydrotestosterone (DHT; K(d) of 0.1 nM and B(max) of 0.5 pmol/g tissue). Competition studies showed that both kbAR1 and kbAR2 were specific for androgens. However, kbAR1 bound only T with high affinity, whereas kbAR2 bound DHT, mibolerone, 17alpha-methyl-testosterone, T, and 11-ketotestosterone with high affinity. In addition, we examined the binding affinities of dichlorodiphenyltrichloroethane and its derivatives, several hydroxylated polychlorinated biphenyl (PCB) congeners, PCB mixtures, and the fungicide vinclozolin and its two metabolites M1 and M2 for the two ARs in Atlantic croaker ovarian, testicular, and brain tissues and in kelp bass ovarian and brain tissues. Only 4, 4'-PCB-3-OH and 2',5'-PCB-3-OH demonstrated greater than 50% displacement of [(3)H]testosterone from either acAR1 or kbAR1. In contrast, with the exception of vinclozolin, all of the xenobiotics examined demonstrated binding to acAR2 in testicular and ovarian tissues. The binding affinities were highest in the testicular tissue with M2, 2,2'5'-PCB-4-OH, and o,p'-DDD all binding with EC(50)s less than 10 microM. The binding affinities of xenobiotics to kbAR2 in ovarian tissue were similar to their binding affinities for ovarian acAR2. The finding that AR1 and AR2 possess different binding affinities for natural androgens and synthetic steroids, as well as for xenobiotics, suggests that the activities of androgens and of certain xenobiotics will depend upon the type of AR present within the target tissue.     

Differential effects of testosterone plus dihydrotestosterone on male courtship of castrated newts, Taricha granulosa

Sexually responsive Taricha granulosa males were castrated and implanted with testosterone (T), dihydrotestosterone (DHT), T plus DHT, or no steroid. The incidence of sexual behavior was highest in castrates implanted with T plus DHT. Newts implanted with T or DHT exhibited sexual behavior more frequently than did the untreated castrates, which exhibited none during the last 5 days of testing. A second experiment was conducted using sexually unresponsive males. The occurrence of sexual behavior remained low in castrates implanted with T plus DHT, untreated castrates, and intact control males. These results support the hypothesis that for this species of amphibian the presence of adequate levels of testicular androgen is a necessary, but not sufficient, condition for the manifestation of sexual behavior; the appearance of these behaviors requires, in addition, the presence of some nontesticular hormone.     

Detection of spatially regulated gene expression by hybridization histochemistry.

Steady state measurements of kidney androgen-regulated protein (KAP) mRNA suggested that KAP gene expression was unusually sensitive to low hormone-receptor levels. Two of the criteria used to reach this conclusion involved relative insensitivity to treatment with a competitive antiandrogen and a partial androgen response of the gene in Tfm/Y androgen receptor (AR) deficient mice. These data may indicate the ability of the KAP gene to respond to an extremely low level of androgen-AR complex or that the effect of androgens is, at least in part, indirect. Hybridization in situ revealed that KAP mRNA expression was restricted to proximal tubule epithelial cells in the juxtamedullary region of castrated animals rather than throughout the cortex as in intact males. Examination of sections of kidneys from Tfm/Y mice before and after testosterone (T) treatment revealed that only the juxtamedullary tubules expressed KAP mRNA and that T increased the level of KAP mRNA in these cells. Treatment of Tfm/Y animals with other steroids showed that beta-estradiol treatment mimicked the effect of T while dihydrotestosterone (DHT) had no effect. The facts that DHT and T both stimulate cortical expression of KAP mRNA in normal animals but DHT has no effect on the juxtamedullary cells in the Tfm/Y variant may indicate that the action of T is due to an estrogenic metabolite. Castrated, hypophysectomized males exhibited no KAP gene expression, while in the presence of T, expression was observed throughout the cortex as in intact males. These results clearly indicate the involvement of pituitary hormones in KAP gene expression in the juxtamedullary tubules. These studies have shown that the regulation of KAP gene expression in the mouse kidney is much more complex than originally believed. Future studies will further investigate the roles of estrogen and specific pituitary hormones in KAP gene expression.     

Effects of androgens on body weight, feeding, and courtship behavior in the pigeon

Adult male pigeons, some intact and some castrated in adulthood, were housed in individual cages kept in an isolated room with temperature and lighting controlled. Weekly measurements were made of ad lib. food intake and body weight for 4 mo after surgery. Castration was followed by a significant depression in body weight and by initially depressed but then progressively enhanced feeding. Food deprivation elicited an increase in food intake proportional to body weight loss, but castrates consumed less food at 100%, 90%, and 80% of ad lib. feeding weight than either intact birds or castrates treated daily with testosterone propionate (TP). Castrates gained weight and ate more than controls in response to daily treatments (im) with TP (6 mg/400 g) or 5a-dihydrotestosterone (DHT, 6 mg/400 g), while androstenedione (15 mg/400 g) and androsterone (15 mg/400 g) were ineffective. Administration of 100 mg DHT (sc) to castrates produced a significant enhancement of body weight without elevating the level of food intake. The biological potency of these diverse androgens on male courtship behavior was reciprocal to that for weight-promoting potency. The results suggest that the structural requirements of the androgen molecule for promoting body weight differ from those for stimulating sexual behavior.     

Androgen-induced neurite outgrowth is mediated by neuritin in motor neurones

In the brain, the spinal cord motor neurones express the highest levels of the androgen receptor (AR). Experimental data have suggested that neurite outgrowth in these neurones may be regulated by testosterone or its derivative 5alpha-dihydrotestosterone (DHT), formed by the 5alpha-reductase type 2 enzyme. In this study we have produced and characterized a model of immortalized motor neuronal cells expressing the mouse AR (mAR) [neuroblastoma-spinal cord (NSC) 34/mAR] and analysed the role of androgens in motor neurones. Androgens either activated or repressed several genes; one has been identified as the mouse neuritin, a protein responsible for neurite elongation. Real-time PCR analysis has shown that the neuritin gene is expressed in the basal condition in immortalized motor neurones and is selectively up-regulated by androgens in NSC34/mAR cells; the DHT effect is counteracted by the anti-androgen Casodex. Moreover, DHT induced neurite outgrowth in NSC34/mAR, while testosterone was less effective and its action was counteracted by the 5alpha-reductase type 2 enzyme inhibitor finasteride. Finally, the androgenic effect on neurite outgrowth was abolished by silencing neuritin with siRNA. Therefore, the trophic effects of androgens in motor neurones may be explained by the androgenic regulation of neuritin, a protein linked to neurone development, elongation and regeneration.     

Regulation of androgen receptor mRNA and protein level by steroid hormones in human mammary cancer cells.

The regulation of the human androgen receptor (AR) by steroid hormones in human mammary cancer cells was investigated using immunocytochemical and ligand binding assays for its protein and Northern blot analyses for the corresponding mRNA. MFM-223 cells contain high levels of ARs and are growth-inhibited by dihydrotestosterone (DHT). The AR protein is down-regulated to 57% of the control by 10 nM DHT after 24 h, and the corresponding mRNA is also reduced. The nonsteroidal antiandrogen hydroxyflutamide had no effect on the AR level, whereas after incubation with 1 microM cyproterone acetate a slight down-regulation was observed. The AR level was restored completely after release from a 7 day treatment with DHT. However, only 60% of the control level was restored, if the cells wer grown in the presence of DHT for 6 weeks. In androgen-pretreated cells the proliferation rate remained decreased even after the withdrawal of DHT. Concomitantly the distinct growth inhibition was lost. Transfection experiments demonstrated a reduced activity of the residual androgen receptor in these pretreated cells. In addition to the AR, EFM-19 cells also contain significant amounts of estrogen and progesterone receptors. EFM-19 cells are not growth inhibited by physiological concentrations of DHT. Autoregulation of AR was also found in this cell line. Additionally, reduced levels of AR protein and mRNA were found in EFM-19 cells after treatment with the synthetic progestin R5020. The maximum effect of R5020 was observed at the high concentration of 1 microM. Estrogen treatment with 10 nM 17 beta-estradiol for 3 days reduced the AR level only by 25%.     

Immunolocalization of androgen receptor in the epididymis of rats with dihydrotestosterone deficiency

Dihydrotestosterone (DHT), 5alpha-reduced metabolite of testosterone, is the most potent androgen in the epididymis. The conversion of T into DHT is carried out by 5alpha-reductase. The activity of 5alpha-reductase type 2, preferentially expressed in the epididymis can be inhibited by a finasteride (a steroid-based specific inhibitor of 5alpha-reductase type 2) which results in DHT deficiency. The aim of the study was to examine the morphology of epididymis and the immunolocalization of an androgen receptor (AR) in the initial segment, caput and cauda epididymis of rats treated with finasteride for 56 days. There were no morphological changes in the morphology of epididymal epithelium in the experimental rats. Immunostainable AR was localized in nuclei of epithelial cells, smooth muscle cells and mainly in the cytoplasm of interstitial cells in the epididymis of control rats. In the epididymis of experimental rats, AR immunostaining was noticed mainly in the cytoplasm of epithelial cells and interstitial cells. The single cells of the initial segment epithelium, basal cells and smooth muscle cells of cauda epididymis showed nuclear AR staining. In conclusion, finasteride affected the expression of the AR in the rat epididymis without changing the morphology of epididymal epithelium. Altered AR expression reflected the hormonal status within the epididymis.     

Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E₂), testosterone, and dihydrotestosterone (DHT). Mechanistically, E₂ and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E₂ signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E₂ enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E₂ treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E₂ and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E₂ and DHT membrane-associated PKC activity in costochondral cartilage cells.     

The presence of a hitherto undefined high-capacity androgen binding macromolecule in human ovarian cancer tissue

Sex hormone binding globulin (SHBG) is known to interfere in the quantitation of androgen receptors (AR) if dihydrotestosterone (DHT) is used. We used a monoclonal antibody to remove SHBG from cytosol. In cytosol of benign prostatic hyperplastic (BPH) tissue low capacity binding for DHT, but not for R1881, was found after removal of SHBG. AR were detected in 18 of 20 ovarian cancer cytosols. In the two AR-negative cases, non-saturable binding for DHT, testosterone and R1881 was observed. Incubation with anti-SHBG did not change this. An hitherto undefined androgen binding macromolecule(s), with high-capacity binding for natural and synthetic androgens, but not for estrogen and progesterone, seems to be present in these ovarian cancer tissues. The functionality of these androgen binding macromolecules in ovarian cancer is yet to be demonstrated.     

Testosterone-Dependent Interaction between Androgen Receptor and Aryl Hydrocarbon Receptor Induces Liver Receptor Homolog 1 Expression in Rat Granulosa Cells

Androgens play a major role in the regulation of normal ovarian function; however, they are also involved in the development of ovarian pathologies. These contrasting effects may involve a differential response of granulosa cells to the androgens testosterone (T) and dihydrotestosterone (DHT). To determine the molecular pathways that mediate the distinct effects of T and DHT, we studied the expression of the liver receptor homolog 1 (LRH-1) gene, which is differentially regulated by these steroids. We found that although both T and DHT stimulate androgen receptor (AR) binding to the LRH-1 promoter, DHT prevents T-mediated stimulation of LRH-1 expression. T stimulated the expression of aryl hydrocarbon receptor (AHR) and its interaction with the AR. T also promoted the recruitment of the AR/AHR complex to the LRH-1 promoter. These effects were not mimicked by DHT. We also observed that the activation of extracellular regulated kinases by T is required for AR and AHR interaction. In summary, T, but not DHT, stimulates AHR expression and the interaction between AHR and AR, leading to the stimulation of LRH-1 expression. These findings could explain the distinct response of granulosa cells to T and DHT and provide a molecular mechanism by which DHT negatively affects ovarian function.     

Antiproliferative actions of the synthetic androgen, mibolerone, in breast cancer cells are mediated by both androgen and progesterone receptors

Androgen signaling, mediated by the androgen receptor (AR), is a critical factor influencing growth of normal and malignant breast cells. Given the increasing use of exogenous androgens in women, a better understanding of androgen action in the breast is essential. This study compared the effects of 5alpha-dihydrotestosterone (DHT) and a synthetic androgen, mibolerone, on estradiol (E(2))-induced proliferation of breast cancer cells. DHT modestly inhibited E(2)-induced proliferation and mibolerone significantly inhibited proliferation in T-47D cells. The effects of both androgens could be reversed by an AR antagonist, suggesting that their actions were mediated, in part, by AR. Whereas high physiological doses (10-100nM) of DHT reduced E(2)-mediated induction of the estrogen-regulated gene progesterone receptor (PR) to basal levels, mibolerone at lower doses (1nM) eliminated PR expression, suggesting that mibolerone may also act via the PR. In the AR positive, PR-negative MCF-7 cells, mibolerone had modest effects on E(2)-induced proliferation, but was a potent inhibitor of proliferation in the AR positive, PR positive MCF-7M11 PRA cells. The effects of mibolerone in breast cancer cells were similar to those of the progestin, medroxyprogesterone acetate. Our results demonstrate that mibolerone can have both androgenic and progestagenic actions in breast cancer cells.     

The effects of androgens and estrogen on the external morphology and electric organ discharge waveform of Gnathonemus petersii (Mormyridae, Teleostei)

The effects of androgens and estrogen on the external morphology and electric organ discharge (EOD) waveform in Gnathonemus petersii, a weakly discharging electric fish, were investigated. Following preimplant data collection, juvenile and adult fish were gonadectomized and implanted with silastic capsules containing either high or low doses of testosterone (T), dihydrotestosterone (DHT), estradiol-17 beta (E2), or cholesterol. One group of fish was treated with high doses of DHT + E2. Radioimmunoassays revealed that low-dose implants resulted in plasma T levels comparable to and high-dose implants about sixfold greater than those found in adult males imported during breeding season. High-dose E2 implants resulted in higher plasma E2 levels in adults than those in juveniles. At either dose, both androgens induced male-like indentations in the dorsal margin of the anal fin of juveniles and adult females by 4 weeks postimplant. Both low and high doses of T decreased the peak power spectrum frequency (PPSF) of Fourier transformations of EODs and increased the durations of phases 2 and 3 of the EOD in juveniles and adults, but the high doses caused more rapid and profound effects. The two doses of T caused opposite effects on the durations of phases 1 and 4 juveniles. The low dose of T decreased the durations of phases 1 and 4, while the high dose increased them. In adults, the high dose of T increased the duration of phase 1, but had inconsistent effects on the duration of phase 4. Total EOD durations were increased by both doses of T in juveniles, while adults showed inconsistent effects possibly due to individual variability in hormone sensitivity. Compared to T, DHT exerted similar, but less dramatic effects on all measures, but only at high doses. E2 significantly increased adult PPSFs, the first such finding in a mormyrid species. E2 had no effects on juvenile PPSFs, or on adult or juvenile EOD phase durations. The effects of DHT + E2 on PPSF and phases 2 and 3 were similar to those of DHT alone. These findings demonstrate quantifiable steroid-dependent plasticity in the durations of individual phases of EODs in an electric fish and are the first to show that the external morphology in Gnathonemus petersii is androgen-dependent. The results are discussed with regard to methodological considerations and hormone studies involving sex differences in EODs reported for this and other species.     

Hormonal regulation of male reproductive behavior in the lizard Anolis sagrei: a test of the aromatization hypothesis

This study examined the hypothesis that aromatization of testosterone (T) to estradiol (E) is required to activate reproductive behavior in castrated male lizards (Anolis sagrei). Adult, reproductively active males were assigned to an intact control group or to one of four treatment groups. Treatment males were castrated and 1 week later three of the four castrated groups were implanted with subcutaneous pellets containing either 0.05 mg of E, 0.5 mg of T, or 0.5 mg of dihydrotestosterone (DHT). Two weeks after pellet implantation, males were tested with stimulus males, and 2 days later were tested with stimulus females. Behavioral tests were of 15-min duration and were videotaped. Significantly fewer E-treated castrates erected a crest in tests with stimulus males than did intact males. In tests with stimulus females, significantly fewer E-treated castrates displayed, neck-gripped, and intromitted than did intact males. Estradiol-treated castrates also showed significantly less display behavior than did intact males. However, aggressive and sexual behavior of DHT-treated castrates was not significantly different from that of intact males. The same was true for T-treated castrates with the exception that display behavior in tests with stimulus females was reduced compared to that of intact males. The results suggest that aromatization of T to E is not required for induction of androgen-dependent reproductive behavior in this lizard.