Effects of aromatase inhibitors on in vitro steroidogenesis by Atlantic salmon (Salmo salar) gonadal and brain tissue

In order to assess the efficacy of selected aromatase inhibitors on Atlantic salmon (Salmo salar) ovarian and brain tissue, in vitro systems were developed for measuring 17beta-estradiol (E(2)) production by these tissues. Isolated vitellogenic follicles, or homogenised whole brains were incubated at 10 degrees C in complete Cortlands solution for 18 or 42 h respectively, and E(2) levels in the medium were determined by RIA. The addition of testosterone to the medium increased E(2) production in all preparations. E(2) production by whole brain homogenate was reduced by co-incubation with the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD), 4-androstene-4-ol-3,17-dione (OHA), aminoglutethimide, fadrozole or miconazole. Fadrozole, ATD, and OHA reduced E(2) production by vitellogenic follicles at a medium concentration of 0.1 microg mL(-1), whereas miconazole was only effective at 10 microg mL(-1). This study demonstrates a simple and rapid screening method for assessing the efficacy of aromatase inhibitors on fish tissues, and that the aromatase inhibitors ATD, OHA and fadrozole are potent inhibitors of both brain and gonadal aromatase in vitro, in Atlantic salmon.     

Aggressive behavior in birds: an experimental model for studies of brain-steroid interactions

1. Although testosterone (T) stimulates aggressiveness in males of many vertebrate species, it is now known that the full expression of T actions in the central nervous system requires aromatization to estradiol (E2) and subsequent binding of formed E2 to its receptor. 2. We have recently confirmed these as rate-limiting steps in the control of sex-related and individual differences in aggressiveness of the Japanese quail (Coturnix coturnix japonica). 3. In this review, we describe some of the neuroendocrine factors which control aggression with a focus on our recent studies in quail.     

Brain aromatase activity and behavioral consequences in the male rat treated in utero with 4-hydroxy-androstenedione

Pregnant female rats were given daily injections of a potent aromatase inhibitor, 4-hydroxy-delta 4-androstenedione (4OHA), throughout the latter half of the pregnancy (days 11 to 22; the day of insemination was designated as Day O) and male fetuses and pups were obtained. Control animals were male offspring of mothers treated with oil vehicle. When measured by the tritium-water method, significant reductions in the aromatase activity were detected in the hypothalamic and preoptic continuum (HPOA) of the male fetuses and pups, over a period from day 16 of the pregnancy until a day after birth. All parturitions in the experimental as well as control animals occurred on day 22 of the pregnancy. Behavioral and anatomical consequences of the prenatal treatment were examined in adulthood. When mounted by stud male rats, the male litters of the 4OHA-treated mother showed lordosis at a significantly higher frequency both in terms of the number of positive test sessions (each consisted of a 20-min period in which the subject showed lordosis at least once) and the lordosis quotient (percent lordosis occurrence per 10 mounts). When placed with receptive female rats, the experimental animals were no less active in mounting or ejaculating than the control. No significant difference existed between the experimental and control animals in the weight of the testes or the accessorial genitalia, or the serum testosterone levels. Partial but significant intervention of behavioral defeminization of the brain was associated with decreased HPOA aromatase activity during the prenatal period.     

Neuroanatomical specificity in the co-localization of aromatase and estrogen receptors

The relative distributions of aromatase and of estrogen receptors were studied in the brain of the Japanese quail by a double-label immunocytochemical technique. Aromatase immunoreactive cells (ARO-ir) were found in the medial preoptic nucleus, in the septal region, and in a large cell cluster extending from the dorso-lateral aspect of the ventromedial nucleus of the hypothalamus to the tuber at the level of the nucleus inferioris hypothalami. Immunoreactive estrogen receptors (ER) were also found in each of these brain areas but their distribution was much broader and included larger parts of the preoptic, septal, and tuberal regions. In the ventromedial and tuberal hypothalamus, the majority of the ARO-ir cells (over 75%) also contained immunoreactive ER. By contrast, very few of the ARO-ir cells were double-labeled in the preoptic area and in the septum. More than 80% of the aromatase-containing cells contained no ER in these regions. This suggests that the estrogens, which are formed centrally by aromatization of testosterone, might not exert their biological effects through binding with the classical nuclear ER. The fact that significant amounts of aromatase activity are found in synaptosomes purified by differential centrifugation and that aromatase immunoreactivity is observed at the electron microscope level in synaptic boutons suggests that aromatase might produce estrogens that act at the synaptic level as neurohormones or neuromodulators.     

Characterization of estrogen receptor in estrogen-dependent transplantable rat pituitary tumor MtT/F84

Properties of nuclear and cytosolic estrogen receptors (ERs) were examined in a new transplantable rat pituitary tumor designated as MtT/F84, of which growth is stimulated by estrogen. The optimal incubation conditions of both nuclear and cytosolic exchange were found to be at 37 degrees C for 15 min and at 25 degrees C for 2 hr, respectively. Molybdate increased a specific binding of estradiol (E2) as determined by [3H]E2-binding assay. Sucrose density gradient analyses of crude cytosol revealed specific peaks of radioactivity in both 4-5S and 8-10S areas. However, only a single 5S peak was present in 0.4M KCl-extractable nuclear ER. Molybdate also enhanced the stability of cytosolic 8-10S receptor in density gradient sedimentation behavior. Scatchard plot analysis for nuclear ER yielded a single class of binding sites with a dissociation constant (Kd) of 0.317 nM and the maximum number of binding sites (NBSmax) of 25.4 fmol/mg protein. Saturation analysis of [3H]estrogen binding to cytosolic ER also yielded a straight line with a Kd of 0.146 nM and NBSmax of 58.5 fmol/mg protein. The effect of E2 administration on the intracellular distribution of ER was also examined. A marked disappearance in the ER binding in cytosol with a concomitant increase in binding in nuclear fraction was found after the administration of the unlabeled E2 in vivo, whereas the total number of ER did not change. Thus, it is concluded that properties of ER in the MtT/F84 were very similar to those in other target organs such as uterus and pituitary gland.     

Characterization of estrogen receptors in the hamster brain

Although the hamster is frequently used as an experimental animal for studying reproductive neuroendocrinology and sex behavior, estrogen receptors (ER) in the central nervous system have not been fully characterized. Using Sephadex LH-20 gel filtration and DNA-cellulose affinity chromatography, estrogen binding macromolecules having the physicochemical properties of classical ER were identified in cytosolic and nuclear extracts of brain tissues. These receptors exhibited high affinity for estradiol (Kd = 10(-9) M), limited capacity (30-50 fmol/g tissue), and estrogen specificity; however, competition studies indicate that brain and uterine ER have different binding kinetics. The neuroanatomic distribution of ER was similar in males and females with highest levels in the limbic brain and consistently low levels in remaining forebrain and mid/hindbrain. No sex differences in receptor number or other binding parameters were evident. Sucrose gradient centrifugation showed that cytosolic ER sedimented in the 7-8S region of a 5-20% linear gradient (no salt), whereas nuclear ER had a sedimentation coefficient of 5S under high ionic strength. On DNA-cellulose affinity columns, these receptors had an elution maximum of 0.18 M NaCl. After a single injection of estradiol, nuclear ER increased and cytosolic ER were depleted. The lower estradiol binding affinity and receptor levels in hamster brain as compared to the rat are consistent with observed species differences in neural sensitivity to estrogen. We expect these data in hamsters, a markedly photosensitive species, to provide a basis for future studies examining the role of receptors in mediating the effects of day-length on steroid dependent feedback and behavioral responses.     

Effects of the nonsteroidal inhibitor R76713 on testosterone-induced sexual behavior in the Japanese quail (Coturnix coturnix japonica)

A new triazole derivative, R76713 (6-[4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H- benzotriazole), was recently shown to inhibit aromatase selectively without affecting other steroid-metabolizing enzymes and without interacting with estrogen, progestin, or androgen receptors. This compound was tested for its capacity to intefere with the induction of copulatory behavior by testosterone (T) in castrated Japanese quail (Coturnix coturnix japonica). In a first experiment, R76713 inhibited (range 0.01 to 1 mg/kg) the activation of sexual behavior by T silastic implants and hypothalamic aromatase activity in castrated male quail in a dose-dependent manner. The 5 alpha- and 5 beta- reductases of T were not systematically affected. Stereotaxic implantation of R76713 in the medial preoptic area similarly blocked the behavior activated by systemic treatment with T, demonstrating that central aromatization of androgen is implicated in the activation of behavior. These inhibiting effects of R76713 on behavior were observed when implants were placed in the medial part of the nucleus preopticus medialis, confirming the implication of this brain area in the control of male copulatory behavior. Finally, the behavioral inhibition produced by R76713 could be reversed by simultaneous treatment with a dose of estradiol, which was not behaviorally effective by itself. This suggests that the behavioral deficit induced by the inhibitor was specifically due to the suppression of estrogen production. This also shows that the activation of copulatory behavior probably results from the interaction of androgens and estrogens at the brain level, as the two treatments separately providing these hormonal stimuli (T with the aromatase inhibitor on one hand and a low dose of estradiol on the other hand) had almost no behavioral effects but they synergized to activate copulation when given concurrently. These data confirm the critical role of preoptic aromatase in the activation of reproductive behavior and demonstrate that R76713 is a useful tool for the in vivo study of estrogen-dependent processes.     

In vivo aromatization of [3H]testosterone in high and low mating lines of Japanese quail

Japanese quail selected bidirectionally for adult mating frequency were utilized to study in vivo aromatization of testosterone (T) in relation to masculine copulatory behavior. Functionally castrated high (HM) and low mating (LM) line quail were injected with 75 microCi of [3H]T. One hour after the injection, all radioactivity recovered in telencephalic-diencephalic brain tissue was in the form of T, dihydrotestosterone (DHT), or estradiol (E2). Neither the total 3H nor the [3H]T metabolite radioactivity differed between the two genetic lines. Of all [3H]T metabolic radioactivity, [3H]E2 represented 45 +/- 6 % in the HM line and 46 +/- 6% in the LM line, indicating that the line difference in mating frequency was not due to a corresponding difference in aromatase activity. Inasmuch as both the HM and LM line birds actively converted T to E2, these results implicate a neural mechanism involving E2-receptor interactions as the cause of the behavioral differences between the HM and LM lines.     

Specific activation of estrogen receptor alpha and beta enhances male sexual behavior and neuroplasticity in male Japanese quail

Two subtypes of estrogen receptors (ER), ERα and ERβ, have been identified in humans and numerous vertebrates, including the Japanese quail. We investigated in this species the specific role(s) of each receptor in the activation of male sexual behavior and the underlying estrogen-dependent neural plasticity. Castrated male Japanese quail received empty (CX) or testosterone-filled (T) implants or were daily injected with the ER general agonist diethylstilbestrol (DES), the ERα-specific agonist PPT, the ERβ-specific agonist DPN or the vehicle, propylene glycol. Three days after receiving the first treatment, subjects were alternatively tested for appetitive (rhythmic cloacal sphincter movements, RCSM) and consummatory aspects (copulatory behavior) of male sexual behavior. 24 hours after the last behavioral testing, brains were collected and analyzed for aromatase expression and vasotocinergic innervation in the medial preoptic nucleus. The expression of RCSM was activated by T and to a lesser extent by DES and PPT but not by the ERβagonist DPN. In parallel, T fully restored the complete sequence of copulation, DES was partially active and the specific activation of ERα or ERβ only resulted in a very low frequency of mount attempts in few subjects. T increased the volume of the medial preoptic nucleus as measured by the dense cluster of aromatase-immunoreactive cells and the density of the vasotocinergic innervation within this nucleus. DES had only a weak action on vasotocinergic fibers and the two specific ER agonists did not affect these neural responses. Simultaneous activation of both receptors or treatments with higher doses may be required to fully activate sexual behavior and the associated neurochemical events.     

Regulation of steady-state luteinizing hormone messenger ribonucleic acid levels, de novo synthesis, and release by sex steroids in primary pituitary cell cultures of male African catfish, Clarias gariepinus

Primary pituitary cell cultures from sexually mature adult male African catfish, Clarias gariepinus, were used to study the regulation of LH biosynthesis by sex steroids. The cell cultures were exposed to testosterone (T), estradiol (E(2)), or 5alpha-dihydrotestosterone (DHT), a nonaromatizable analogue of T, and to the likewise nonaromatizable 11-ketotestosterone (KT) and 11beta-hydroxyandrostenedione (OHA), physiologically relevant androgens in fish. Both T and E(2) elevated glycoprotein alpha (GPalpha) and LHbeta steady-state mRNA levels (quantified by RNase protection assay), de novo synthesis (metabolic incorporation of radioactive amino acids and subsequent immune precipitation of LH), and release of preferentially newly synthesized LH, while DHT had no effect. Inhibiting the aromatase activity abolished the stimulatory effects of T. The effects of E(2) on LH mRNA levels and de novo synthesis were dose dependent. Incubation with 10 ng/ml KT elevated GPalpha and LHbeta mRNA levels, while other concentrations of KT or all concentrations of OHA tested had no effect. The amount of newly synthesized LH, on the other hand, was decreased dose-dependently by OHA but not by KT. Since this OHA-induced decrease did not change the specific activity (dpm immune precipitable [(3)H]-LH/ng immune-reactive LH) of LH, we hypothesize that OHA exerted its effect by activating a crinophagic breakdown of secretory granules in catfish gonadotrophs. Electron microscopic examination of gonadotrophs after in vitro exposure to 50 ng OHA/ml revealed that breakdown organelles had increased in size significantly. We conclude that the balanced production of aromatizable (mainly stimulatory) and 11-oxygenated androgens (mainly inhibitory) may be an important factor in regulating the amounts of LH available for secretion in male African catfish.     

Estradiol down-regulation of the rat uterine estrogen receptor

We have previously shown that neonatal exposure of rats to pharmacologic doses of diethylstilbestrol via daily injections resulted in a significant decrease in the estrogen-binding capacity of the uterine estrogen receptor (ER). In this study, we examined the effects of physiologic and pharmacologic doses of estradiol (E2) administered to adult ovariectomized rats via Silastic implants. Two days after implantation, uteri were removed, weighted, and homogenized, and ER levels were determined in the supernatant (hydroxylapatite assay) and low-speed pellet (nuclear exchange assay). Implants containing E2 concentrations of 0.005 or 0.05 mg/ml increased cytosolic but not total ER-binding capacity, whereas 0.5 or 5.0 mg of E2/ml implants decreased the binding capacity of cytosol ER to 40% and total ER to 50% of control values. The 0.005-mg/ml dose increased cytosol ER without increasing uterine weight; all higher doses significantly increased uterine weight. Determination of ER protein by an ER radioimmunoassay showed the same extent of reduction of ER concentration as the binding assays, demonstrating that the loss in E2 binding capacity is homologous down-regulation. The down-regulation of ER was maximal at 24 hr and was completely reversible after implant removal, although the time required to recover from down-regulation was dose dependent. Uterine weight also returned to control levels slowly after implant removal. Neither the sedimentation rate of the down-regulated ER nor the Kd of the cytosolic ER changed following long-term implantation; however, the Kd of the nuclear ER decreased significantly. This is the first demonstration of in vivo homologous down-regulation of uterine ER. ER down-regulation may play a role in several biologic processes.     

Effect of the aromatase inhibitor, 4 hydroxyandrostenedione, on the endotoxin-induced changes in steroid hormones in male rats.

The increase in circulating estrogen concentrations that follows injection of Escherichia coli endotoxin (Endo) may be due to increased aromatase activity. We have therefore analysed the effect of the aromatase inhibitor, 4 hydroxyandrostenedione (4OHA) on the steroid hormone response of male rats, particularly the dramatic increase in estrogens and decrease in androgens, induced by Endo. The concentrations of corticosterone (B), progesterone (P4), 17 alpha hydroxyprogesterone (17 alpha OHP4), androstenedione (delta 4), testosterone (T), estrone (E1) and estradiol (E2) were determined 2 hours after injection of increasing doses of 4OHA with and without Endo. The increase in serum estrogen concentrations and drop in serum androgen levels in response to Endo were blocked by a single dose of 4OHA. The effect of 4OHA appeared to be dose dependent. Low doses (30 mg/kg and 50 mg/kg) induced significant changes in the estrogen and androgen responses, but the high dose (100 mg/kg) blocked all changes in sex steroids induced by Endo. 4OHA did not alter the Endo-induced changes in other steroids.     

The ontogeny of cytosol and nuclear progestin receptors in male rat brain and its male-female differences

The fetal and postnatal development of the progestin receptor systems in the intact male rat brain was investigated by means of the in vitro cytosol binding and the nuclear exchange assay using [³H]R5020 ([17α-methyl-³H]17α,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione). The cortical cytosol receptors, first detectable at day 0, rapidly increased at day 7, reaching a maximum at day 10, then gradually declined thereafter. The receptors in the HPOA appeared clearly at day 1, increased during the first 10 days, then remained constant at days 14–21. The postnatal developmental patterns of cytosol brain progestin receptors in males were essentially similar to those in females, but there were some differences between both sexes. The male HPOA at days 10–14 contained more receptors than the female one. Nuclear progestin binding was low in the neonatal male brain at days 1–3. Despite the low level of serum progesterone, the cortical nuclear binding suddenly increased at days 7–10, then remained high at days 14–21. A similar, though less pronounced, pattern was seen in the HPOA. The male pattern of nuclear binding, thus, essentially resembled the female one. However, lower binding in the cortex and, possibly, HPOA was found in males than in females at days 10–21. After progesterone injection postnatal male rats accumulated a lower concentration of progestin receptors in the cortex and, possibly, HPOA than similarly-treated females.It is concluded from these results that progestin receptors in male rat brain appear immediately after birth and develop differentially in the cortex and HPOA. The sudden onset of increased nuclear translocation of endogenous progestin receptor complexes may occur in the brain at around days 7–10. There is a marked sex difference in the nuclear progestin receptor system in the postnatal brain, particularly the cortex. Moreover, the postnatal male brain has lower capacities of nuclear receptor translocation than does the female one. The progestin receptor system in the cortex and, possibly, HPOA of rats in the early postnatal life might be involved with some processes in the mechanism of sexual differentiation of the brain.     

Regulation of estrogen-stimulated lordosis behavior and hypothalamic progestin receptor induction by antiestrogens in female rats

Two estrogen antagonists, CI-628 (CI) and tamoxifen (TX), were used to examine the relationship between estrogen priming of lordosis behavior and progestin receptor induction in the hypothalamus-preoptic area (HPOA) of ovariectomized female rats. Lordosis behavior was assessed by measuring lordosis quotients (LQ) in response to injection of 2 micrograms of estradiol benzoate (EB) followed 48 hr later by 500 micrograms of progesterone (P). Behavior testing began 4 hr after P injection. The effects of antiestrogens were assessed by injecting CI and TX (1-2 mg) from 0 to 48 hr prior to EB. Levels of cytosol progestin receptor in the HPOA were determined by quantifying the specific binding of 0.5 nM [3H]R5020 to cytosols from animals receiving the same EB and antiestrogen treatments used in behavioral testing. TX given concurrently with or CI given 2 hr before EB abolished both lordosis behavior and induction of HPOA progestin receptors. In contrast, CI given 12 hr prior to EB abolished lordosis but permitted a 95% elevation in the concentration of progestin binding sites in the HPOA. TX or CI given 48 hr before EB resulted in moderate levels of lordosis (mean LQs from 56 to 69) and induction of HPOA progestin receptors from 85 to 130% above noninjected controls. However, CI given 24 hr prior to EB produced less than a 40% increase in brain R5020 binding even though lordosis behavior was equivalent to that seen in the 48-hr animals (mean LQ = 53). These data indicate that the effects of antiestrogens on female sexual behavior and on the synthesis of brain progestin receptors depend on which antiestrogen is used and the time interval between administration of estrogen and antiestrogen. They also demonstrate that under some conditions estrogen induction of cytosol progestin receptors in the HPOA can be dissociated from estrogen priming of lordosis behavior in rats.     

Phosphorylation processes mediate rapid changes of brain aromatase activity

The enzyme aromatase (also called estrogen synthase) that catalyzes the transformation of testosterone (T) into estradiol plays a key limiting role in the action of T on many aspects of reproduction. The distribution and regulation of aromatase in the quail brain has been studied by radioenzyme assays on microdissected brain areas, immunocytochemistry, RT-PCR and in situ hybridization. High levels of aromatase activity (AA) characterize the sexually dimorphic, steroid-sensitive medial preoptic nucleus (POM), a critical site of T action and aromatization for the activation of male sexual behavior. The boundaries of the POM are clearly outlined by a dense population of aromatase-containing cells as visualized by both immunocytochemistry and in situ hybridization histochemistry. Aromatase synthesis in the POM is controlled by T and its metabolite estradiol, but estradiol receptors alpha (ER) are not normally co-localized with aromatase in this brain area. Estradiol receptor beta (ERβ) has been recently cloned in quail and localized in POM but we do not yet know whether ERβ occurs in aromatase cells. It is therefore not known whether estrogens regulate aromatase synthesis directly or by affecting different inputs to aromatase cells as is the case with the gonadotropin releasing hormone neurons. The presence of aromatase in presynaptic boutons suggests that locally formed estrogens may exert part of their effects by non-genomic mechanisms at the membrane level. Rapid effects of estrogens in the brain that presumably take place at the neuronal membrane level have been described in other species. If fast transduction mechanisms for estrogen are available at the membrane level, this will not necessarily result in rapid changes in brain function if the availability of the ligand does not also change rapidly. We demonstrate here that AA in hypothalamic homogenates is rapidly down-regulated by exposure to conditions that enhance protein phosphorylation (addition of Ca²⁺, Mg²⁺, ATP). This inhibition is blocked by kinase inhibitors which supports the notion that phosphorylation processes are involved. A rapid (within minutes) and reversible regulation of AA is also observed in hypothalamic explants incubated in vitro and exposed to high Ca²⁺ levels (K⁺-induced depolarization, treatment by thapsigargin, by kainate, AMPA or NMDA). The local production and availability of estrogens in the brain can therefore be rapidly changed by Ca²⁺ based on variation in neurotransmitter activity. Locally-produced estrogens are as a consequence available for non-genomic regulation of neuronal physiology in a manner more akin to the action of a neuropeptide/neurotransmitter than previously thought.