Sites of aromatization of [H]testosterone in forebrain of male, female and androgen receptor-deficient Tfm mice: an autoradiographic study

The distribution of radioactivity after injection of [3H]testosterone was studied in the forebrain of adult mice by thaw-mount autoradiography. Nuclear labeling was high in neurons in the dorsal part of the medial nucleus of the amygdala and in the dorsocaudal part of the bed nucleus of the stria terminalis. Low nuclear uptake occurred in the medial preoptic nucleus, in mediobasal hypothalamic nuclei and in the ventromedial amygdala. Nuclear concentration of radioactivity was not influenced by competition with dihydrotestosterone and was present in androgen receptor deficient Tfm mice. It was totally abolished by competition with estradiol. This indicates that in the brain [3H]testosterone is converted to estrogenic metabolites which bind to estrogen receptors. Nuclear labeling after [3H]testosterone was restricted to a few of the brain nuclei, known to contain estrogen receptors indicating that aromatization occurs only in select regions. The results suggest that testosterone acts on the brain via estrogen receptors.     

The interaction of testosterone with the brain of the orchidectomized primate fetus

At certain times during gestation, the testes of the fetal macaque produce plasma levels of testosterone (T) that are similar to those of adults. It is thought that testosterone acts on the brain via estrogen and androgen receptors to organize the development of sexually dimorphic neural structures that underlie sex differences in behavior. To test the proposition that there are male-female differences in the occupation of steroid receptor binding sites during fetal development in the cynomolgus macaque, we have compared the uptake of [³H]T and its metabolites in: (1) 5 intact males (plasma T 571.2 ± 215.5ng/100ml); (2) 5 intact females (33.8 ± 25.2 ng/100 ml); (3) in 5 males orchidectomized in utero (14.6 ± 5.7ng/100ml). About 1 week after fetal gonadectomy or sham-operation, all fetuses were given 500 μ Ci [³H]T s.c. and were then delivered 60 min later by Cesarian section. Brains were removed and dissected into blocks containing the hypothalamus and preoptic area, amygdala, hippocampus, and midbrain. Samples of cerebral and cerebellar cortex were also obtained. Purified nuclear pellets were prepared by centrifugation through 2 M sucrose and were extracted into ether and analyzed by high performance liquid chromatography. Hypothalamic nuclear concentrations of [³H]E₂ in intact males (847 ± 195dpm per mg DNA) were significantly lower than those in sham-operated females (2147 ± 542dpm per mg DNA) (p < 0.05), but those in orchidectomized males (2233 ± 345dpm per mg D DNA) were similar to concentrations in females. Thus, fetal gonadectomy abolished the differences between males and females, supporting the hypothesis that the occupation of estrogen receptors is greater in intact male than in intact female fetuses at this stage of gestation. This differences might account for some of T's organizational effects on primate physiology and behavior.     

Hypothalamic testosterone increase in the male rat at birth

In the male rat, a dramatic increase in serum testosterone occurs during the first 2 h of postnatal life. Since the hypothalamus is known to be an important site for sexual differentiation of the brain, this early testosterone surge was a good model to use to study the transfer of serum testosterone to the hypothalamus and cerebral cortex. Endogenous testosterone was measured by radioimmunoassay in the hypothalamus and the cerebral cortex of the foetus and newborn rats during the first 6 h following birth. In the male, hypothalamic testosterone increased between 0 h in utero and 2 h; in the males gonadectomized at 0 h in utero and killed at the age of 2 h, the testosterone surge was abolished, clearly indicating the testicular origin of this hormone in the neonate. The small testosterone increase in the cerebral cortex compared with that in the hypothalamus reflects a preferential uptake of this hormone by the hypothalamus of the newborn. In the female, hypothalamic testosterone slightly decreased between 0 h in utero and 6 h. These results are in agreement with the view that hypothalamic modifications form the basis for some behavioral and physiological changes attributed to the effect of perinatal hormonal stimulation.     

ICV testosterone induces Fos in male Syrian hamster brain

Previous studies in our laboratory have demonstrated intracerebroventricular (ICV) self-administration of testosterone in hamsters. This suggests that androgens are reinforcing, independent of their anabolic effects. Furthermore, pharmacologic testosterone acts as a depressant: ICV testosterone infusion acutely reduces respiration, locomotion and body temperature. However, with chronic exposure, males develop tolerance. To understand mechanisms for androgen action, we looked for Fos expression after acute and chronic ICV infusion of testosterone or vehicle. 32 castrated males with chronic physiologic testosterone replacement (n=8/group) were infused ICV for 4h/day with 40 microg testosterone or 40 microl vehicle. Half of the males were perfused after the first day of ICV infusion; the remaining males were perfused after 15 days of ICV infusion. 60 microm coronal brain slices were cut. Every fourth section was stained for Fos. Additional sections were stained for androgen receptors (AR) and estrogen receptors (ER). Testosterone infusion induced Fos above control in the posteromedial bed nucleus of the stria terminalis (BSTPM), posteromedial amygdala (MeP), lateral habenula (LHb), median raphe (MnR), lateral pontine nucleus (Pn), and ventral tegmental area (VTA). In particular, Fos was elevated in the BSTPM, MeP, LHb, and VTA only after 1 day of testosterone. 15 days of testosterone enhanced Fos expression above control in the MnR and lateral Pn. Importantly, Fos was not present in all brain areas involved in reward or in those with dense steroid receptors. Furthermore, AR and ER immunostaining was unaltered by testosterone infusion. We conclude that pharmacologic testosterone activates select steroid-sensitive brain regions, as well as midbrain areas involved in reinforcement of commonly-abused drugs.     

Immunocytochemical localization of androgen receptors in the male songbird and quail brain.

The distribution of androgen receptors was studied in the brain of the Japanese quail (Coturnix japonica), the zebra finch (Taeniopygia guttata), and the canary (Serinus canaria) by immunocytochemistry with a polyclonal antibody (AR32) raised in rabbit against a synthetic peptide corresponding to a sequence located at the N-terminus of the androgen receptor molecule. In quail, androgen receptor-immunoreactive cells were observed in the nucleus intercollicularis and in various nuclei of the preoptic-hypothalamic complex, namely, the nucleus preopticus medialis, the ventral part of the nucleus anterior medialis hypothalami, the nucleus paraventricularis magnocellularis, the nucleus ventromedialis hypothalami, and the tuberal hypothalamus. In the two songbird species, labeled cells were also observed in various nuclei in the preoptic-hypothalamic region, in the nucleus taeniae, and in the nucleus intercollicularis. Additional androgen receptor-immunoreactive cells were present in the androgen-sensitive telencephalic nuclei that are part of the song control system. These immunoreactive cells filled and outlined the boundaries of the hyperstriatum ventrale, pars caudalis, nucleus magnocellularis neostriatalis anterioris (both in the lateral and medial subdivisions), and nucleus robustus archistriatalis. The immunoreactive material was primarily present in cell nuclei but a low level of immunoreactivity was also clearly detected in cytoplasm in some brain areas. These studies demonstrate, for the first time, that androgen receptors can be detected by immunocytochemistry in the avian brain and the results are in general agreement with the binding data obtained by autoradiography with tritiated dihydrotestosterone. Immunocytochemical methods offer several advantages over autoradiography and their use for the study of the androgen receptor will greatly facilitate the analysis of steroid-sensitive systems in the avian brain.     

Valproate inhibits the conversion of testosterone to estradiol and acts as an apoptotic agent in growing porcine ovarian follicular cells

PURPOSE: Long-term valproate (VPA) treatment has been associated with hyperandrogenism and polycystic ovaries in women with epilepsy. The exact mechanisms of action of the drug on sex steroid hormone function are still unsettled. The aim of the present study was to investigate the action of VPA on basal and gonadotropin-stimulated steroid secretion in porcine ovarian follicular cells and to measure the conversion of testosterone to estradiol. Second, the action of VPA on proliferation and apoptosis of follicular cells was investigated. METHODS: Small and medium follicles were obtained from pig ovaries on days 8-10 and 14-16 of the estrus cycle. Both follicular compartments, theca and granulosa cells, were cultured as a coculture resembling follicles in vivo. VPA in concentrations of 100 and 250 micrg/ml was added to the control or gonadotropin-stimulated cultures. RESULTS: VPA caused a significant increase in basal and luteinizing hormone (LH)-stimulated testosterone secretion from small follicles, whereas in medium follicles, an increased basal but decreased LH-stimulated testosterone secretion was found. VPA caused decreased basal and follicle-stimulating hormone (FSH)-stimulated estradiol secretion by small follicles, whereas only the higher concentration decreased estradiol secretion in medium follicles. The conversion of testosterone to estradiol by small follicles was decreased under the influence of VPA in testosterone-alone and in testosterone-plus-FSH-stimulated cultures, whereas this was seen at only the higher VPA concentration in medium follicles. VPA had no effect on cell proliferation and viability, whereas in a dose-dependent manner, VPA increased caspase-3 activity. CONCLUSIONS: VPA affected steroidogenesis in both unstimulated and gonadotropin-stimulated porcine ovarian follicular cells and inhibited the conversion of testosterone to estradiol. In addition, VPA may act as an apoptotic agent in both small and medium-sized follicles.     

Estrogen binding and the actions of testosterone in the brain of the male rhesus monkey

Autoradiography and high performance liquid chromatography (HPLC) were used to determine where metabolites of testosterone interact with estrogen binding sites in the brain of the male primate. Three days after castration, animals received a subcutaneous injection of either estradiol benzoate (EB, 200 micrograms/kg, n = 4) or oil vehicle (controls, n = 4). Three hours later, 5 mCi [3H]testosterone was administered as an intravenous bolus. At 60 min, brains were rapidly removed, left halves were used for autoradiography and right halves were dissected into 14 samples for HPLC of nuclear and supernatant fractions. In control males, labeled neurons were observed in preoptic area, hypothalamus and amygdala. In EB-pretreated males, the number of labeled neurons was reduced by 35% in the anterior hypothalamus and ventromedial nucleus, and by 65% in the cortical and accessory basal amygdaloid nuclei, but was not significantly reduced in other brain regions. In hypothalamus, preoptic area and amygdala, EB-pretreatment reduced nuclear concentrations of [3H]estradiol to 37-55% of control levels, but reduced neither the nuclear concentrations of [3H]testosterone nor the supernatant concentrations of [3H]estradiol and [3H]testosterone. The data suggest that the actions of testosterone in regions such as the arcuate nucleus and lateral septal nucleus primarily involve unchanged testosterone or dihydrotestosterone, while in regions such as the amygdala, aromatization and interaction with estrogen receptors is involved also.     

Agaridoxin: A fungal catecholamine which acts as an alpha 1 agonist of mammalian hypothalamic adenylate cyclase

Agaridoxin, a catecholamine isolated from mushrooms, and 4 synthetic analogues cause activation of adenylate cyclase in the presence of guanylyl imidodiphosphate (Gpp(NH)p) in membrane particles prepared from rat hypothalamus. These compounds also activate adenylate cyclase preparations from rat kidney, liver and cerebral cortex. In the presence of tyrosinase, these compounds are readily oxidized to quinones which lack agonist activity. Studies with selective adrenergic blockers suggest that agaridoxin acts at an alpha 1-type receptor. Agaridoxin-mediated adenylate cyclase stimulation is most effectively antagonized by WB-4101 and phenoxybenzamine, while propranolol and yohimbine are without inhibitory effect. Agaridoxin and the alpha 1 agonist methoxamine inhibited the binding of [3H]WB-4101 in rat hypothalamic and cerebral cortical membranes. The values of Ki for both compounds are lower than that of norepinephrine. The agaridoxin analogue, 4-aminocatechol hydrochloride, is a more effective and potent adenylate cyclase activator than agaridoxin or methoxamine.     

Progestins affect reproductive behavior and androgen receptor dynamics in male guinea pig brain

The present study extends previous studies of ours by comparing the anti-androgenic effect of a progestin agonist (R5020) with progesterone (P). Intact male guinea pigs treated with P (1 and 10 mg/day) and R5020 (100 micrograms/day) had greater latency to mount and lower numbers of mounts and intromissions compared to controls. Ejaculation and plasma testosterone concentration were not affected. Specific brain regions were analyzed for androgen receptor (AR) content. Progestins produced fewer (P less than 0.01) nuclear AR in hypothalamus-preoptic area and pituitary without associated changes in cytosolic AR. These data are best interpreted by postulating an effect of P on AR dynamics mediated through the P receptor and not by competition for androgen binding to its receptor.     

Neuroanatomical distribution of androgen and estrogen receptor-immunoreactive cells in the brain of the male roughskin newt

Immunohistochemistry was used to investigate the neuroanatomical distribution of androgen and estrogen receptors in brains of adult male roughskin newts, Taricha granulosa, collected during the breeding season. Immunoreactive cells were found to be widely distributed in specific brain areas of this urodele amphibian. Androgen receptor-immunoreactive (AR-ir) cells were observed in the olfactory bulbs, habenula, pineal body, preoptic area, hypothalamus, interpeduncular nucleus, area acusticolateralis, cerebellum, and motor nuclei of the medulla oblongata. Estrogen receptor-immunoreactive (ER-ir) cells were found in the lateral septum, amygdala pars lateralis, pallium, preoptic area, hypothalamus, and dorsal mesencephalic tegmentum. This immunocytochemical study of the newt brain reveals AR-ir and ER-ir cells in several regions that have not been previously reported to contain androgen and estrogen receptors in non-mammalian vertebrates. Additionally, the distribution of AR-ir and ER-ir cells in the newt brain, in general, is consistent with previous studies, suggesting that the distribution of sex steroid receptor-containing neurons in some brain regions is relatively conserved among vertebrates. © 1996 Wiley-Liss, Inc.     

Neopterin acts as an endogenous cognitive enhancer

Neopterin is found at increased levels in biological fluids from individuals with inflammatory disorders. The biological role of this pteridine remains undefined; however, due to its capacity to increase hemeoxygenase-1 content, it has been proposed as a protective agent during cellular stress. Therefore, we investigated the effects of neopterin on motor, emotional and memory functions. To address this question, neopterin (0.4 and/or 4 pmol) was injected intracerebroventricularly before or after the training sessions of step-down inhibitory avoidance and fear conditioning tasks, respectively. Memory-related behaviors were assessed in Swiss and C57BL/6 mice, as well as in Wistar rats. Moreover, the putative effects of neopterin on motor and anxiety-related parameters were addressed in the open field and elevated plus-maze tasks. The effects of neopterin on cognitive performance were also investigated after intraperitoneal lipopolysaccharide (LPS) administration (0.33 mg/kg) in interleukin-10 knockout mice (IL-10^−/−). It was consistently observed across rodent species that neopterin facilitated aversive memory acquisition by increasing the latency to step-down in the inhibitory avoidance task. This effect was related to a reduced threshold to generate the hippocampal long-term potentiation (LTP) process, and reduced IL-6 brain levels after the LPS challenge. However, neopterin administration after acquisition did not alter the consolidation of fear memories, neither motor nor anxiety-related parameters. Altogether, neopterin facilitated cognitive processes, probably by inducing an antioxidant/anti-inflammatory state, and by facilitating LTP generation. To our knowledge, this is the first evidence showing the cognitive enhancer property of neopterin.     

Nuclear and cytosolic androgen receptor levels in the limbic brain of neonatal male and female rats

Nuclear and cytosolic androgen receptors in the limbic brain were measured in neonatal male and female rat pups. There were no sex differences in cytosolic receptor concentrations during the neonatal period in any of the regions studied (hypothalamus, amygdala, preoptic area and septum). Receptor concentrations in all 4 regions increase gradually over the first 10 days of life, with no change in the affinity for 5-alpha-dihydrotestosterone. Nuclear receptor levels in intact pups, measured using an exchange assay, are highest between days 4 and 8 of life. In general, nuclear receptor levels are higher in males than in females; however, this sex difference is most consistently seen in the amygdala. These results are discussed in relation to sex differences in circulating testosterone levels and with respect to the contribution of androgens to the sexual differentiation of behavior.     

Neuropeptide Y acts within the rat testis to inhibit testosterone secretion

The factors that influence Leydig cell activity currently include peptides such as neuropeptide Y (NPY). In this work we investigated the ability of this compound, injected directly into the testes of adult male rats, to alter testosterone (T) release into the general circulation. At a 5 μg/kg dose administered 1 h prior to challenge with human chorionic gonadotropin (hCG, 1.0 U/kg, iv), NPY significantly (P < 0.01) blunted the T response to this gonadotropin. The inhibitory effect of NPY was observed in animals pretreated with an antagonist to gonadotropin-releasing hormone or not, indicating that the decrease in plasma T found was most likely independent of pituitary luteinizing hormone. However, testicular levels of steroidogenic acute regulatory (STAR) protein or translocator protein (TSPO) in the Leydig cells did not exhibit consistent changes, which suggested that other mechanisms mediated the blunted T response to hCG. We therefore used autoradiography and immunohistochemistry methodologies to identify NPY receptors in the testes, and found them primarily located on blood vessels. Competition studies further identified these receptors as being Y₁, a subtype previously reported to modulate the vasoconstrictor effect of NPY. The absence of significant changes in STAR and TSPO levels, as well as the absence of Y₁ receptors on Leydig cells, suggest that NPY-induced decreases in T release is unlikely to represent a direct effect of NPY on these cells. Rather, the very high expression levels of Y₁ found in testicular vessels supports the concept that NPY may alter gonadal activity, at least in part, through local vascular impairment of gonadotropin delivery to, and/or blunted T secretion from, Leydig cells.     

In vitro measurement of cytosol and cell nuclear androgen receptors in male rat brain and pituitary

An in vitro assay procedure is described for measuring androgen receptor binding in cytosol and cell nuclei of brain and pituitary tissue using [³H]R1881 as ligand. The cell nuclear assay uses the exchange method, which permits assessment of endogenous occupancy of androgen receptors in brain and pituitary. Competition and saturation analysis indicated that [³H]R1881 binding has the specificity and nanomolar affinity expected of an androgen receptor.Moreover, we demonstrated that androgen receptor binding predominated in cytosol from castrated rats and in cell nuclei of male rats treated in vivo with testosterone. Furthermore, as expected, testicular feminized male rats showed low levels of putative androgen receptors in both cytosol and cell nuclei.