Rapid changes in production and behavioral action of estrogens.

It is well established that sex steroid hormones bind to nuclear receptors, which then act as transcription factors to control brain sexual differentiation and the activation of sexual behaviors. Estrogens locally produced in the brain exert their behavioral effects in this way but mounting evidence indicates that estrogens also can influence brain functioning more rapidly via non-genomic mechanisms. We recently reported that, in Japanese quail, the activity of preoptic estrogen synthase (aromatase) can be modulated quite rapidly (within minutes) by non-genomic mechanisms, including calcium-dependent phosphorylations. Behavioral studies further demonstrated that rapid changes in estrogen bioavailability, resulting either from a single injection of a high dose of estradiol or from the acute inhibition of aromatase activity, significantly affect the expression of both appetitive and consummatory aspects of male sexual behavior with latencies ranging between 15 and 30 min. Together these data indicate that the bioavailability of estrogens in the brain can change on different time-scales (long- and short-term) that match well with the genomic and non-genomic actions of this steroid and underlie two complementary mechanisms through which estrogens modulate behavior. Estrogens produced locally in the brain should therefore be considered not only as neuroactive steroids but they also display many (if not all) functional characteristics of neuromodulators and perhaps neurotransmitters.

Effects of calmodulin on aromatase activity in the preoptic area.

Oestrogens derived from the neural aromatisation of testosterone play a key role in the activation of male sexual behaviour in many vertebrates. Besides their slow action on gene transcription mediated by the binding to nuclear receptors, oestrogens have now been recognised to have more rapid membrane-based effects on brain function. Rapid changes in aromatase activity, and hence in local oestrogen concentrations, could thus rapidly modulate behavioural responses. We previously demonstrated that calcium-dependent kinases are able to down-regulate aromatase activity after incubations of 10-15 min in phosphorylating conditions. In the present study, in quail hypothalamic homogenates, we show that Ca2+ or calmodulin alone can very rapidly change aromatase activity. Preincubation with 1 mM EGTA or with a monoclonal antibody raised against calmodulin immediately increased aromatase activity. The presence of calmodulin on aromatase purified by immunoprecipitation and electrophoresis was previously identified by western blot and two consensus binding sites for Ca2+-calmodulin are identified here on the deduced amino acid sequence of the quail brain aromatase. The rapid control of brain aromatase activity thus appears to include two mechanisms: (i) an immediate regulatory process that involves the Ca2+-calmodulin binding site and (ii) a somewhat slower phosphorylation by several protein kinases (PKC, PKA but also possibly Ca2+-calmodulin kinases) of the aromatase molecule.

Interactions between kinases and phosphatases in the rapid control of brain aromatase.

Aromatization of testosterone into oestradiol plays a key role in the activation of male sexual behaviour in many vertebrate species. Rapid changes in brain aromatase activity have recently been identified and the resulting changes in local oestrogen bioavailability could modulate fast behavioural responses to oestrogens. In quail hypothalamic homogenates, aromatase activity is down-regulated within minutes by calcium-dependent phosphorylations in the presence of ATP, MgCl2 and CaCl2 (ATP/Mg/Ca). Three kinases (protein kinases A and C and calmodulin kinase; PKA, PKC and CAMK) are potentially implicated in this process. If kinases decrease aromatase activity in a reversible manner, then it would be expected that the enzymatic activity would increase and/or return to baseline levels in the presence of phosphatases. We showed previously that 0.1 mM vanadate (a general inhibitor of protein phosphatases) significantly decreases aromatase activity but specific protein phosphatases that could up-regulate aromatase activity have not been identified to date. The reversibility of aromatase activity inhibition by phosphorylations was investigated in the present study using alkaline and acid phosphatase (Alk and Ac PPase). Unexpectedly, Alk PPase inhibited aromatase activity in a dose-dependent manner in the presence, as well as in the absence, of ATP/Mg/Ca. By contrast, Ac PPase completely blocked the inhibitory effects of ATP/Mg/Ca on aromatase activity, even if it moderately inhibited aromatase activity in the absence of ATP/Mg/Ca. However, the addition of Ac PPase was unable to restore aromatase activity after it had been inhibited by exposure to ATP/Mg/Ca. Taken together, these data suggest that, amongst the 15 potential consensus phosphorylation sites identified on the quail aromatase sequence, some must be constitutively phosphorylated for the enzyme to be active whereas phosphorylation of the others is involved in the rapid inhibition of aromatase activity by the competitive effects of protein kinases and phosphatases. Two out of these 15 putative phosphorylation sites occur in an environment corresponding to the consensus sites for PKC, PKA (and possibly a CAMK) and, in all probability, represent the sites whose phosphorylation rapidly blocks enzyme activity.

Rapid decreases in preoptic aromatase activity and brain monoamine concentrations after engaging in male sexual behavior.

In Japanese quail, as in rats, the expression of male sexual behavior over relatively long time periods (days to weeks) is dependent on the local production of estradiol in the preoptic area via the aromatization of testosterone. On a short-term basis (minutes to hours), central actions of dopamine as well as locally produced estrogens modulate behavioral expression. In rats, a view of and sexual interaction with a female increase dopamine release in the preoptic area. In quail, in vitro brain aromatase activity (AA) is rapidly modulated by calcium-dependent phosphorylations that are likely to occur in vivo as a result of changes in neurotransmitter activity. Furthermore, an acute estradiol injection rapidly stimulates copulation in quail, whereas a single injection of the aromatase inhibitor vorozole rapidly inhibits this behavior. We hypothesized that brain aromatase and dopaminergic activities are regulated in quail in association with the expression of male sexual behavior. Visual access as well as sexual interactions with a female produced a significant decrease in brain AA, which was maximal after 5 min. This expression of sexual behavior also resulted in a significant decrease in dopaminergic as well as serotonergic activity after 1 min, which returned to basal levels after 5 min. These results demonstrate for the first time that AA is rapidly modulated in vivo in parallel with changes in dopamine activity. Sexual interactions with the female decreased aromatase and dopamine activities. These data challenge established views about the causal relationships among dopamine, estrogen action, and male sexual behavior.

Relationships between aromatase activity in the brain and gonads and behavioural deficits in homozygous and heterozygous aromatase knockout mice.

The present study was carried out to determine whether aromatase knockout (ArKO) mice are completely devoid of aromatase activity in their brain and gonads and to compare aromatase activity in wild-type and ArKO mice, as well as in heterozygous (HET) mice of both sexes that were previously shown to display a variety of reproductive behaviours at levels intermediate between wild-type and ArKO mice. Aromatase activity was extremely low, and undetectable by the tritiated water assay, in homogenates of the preoptic area-hypothalamus of adult wild-type mice, but was induced following a 12-day treatment with testosterone. The induction of aromatase activity by testosterone was significantly larger in males than in females. Even after 12 days exposure to testosterone, no aromatase activity was detected in the brain of ArKO mice of either sex whereas HET mice showed intermediate levels of activity between ArKO and wild-type. Aromatase activity was also undetectable in the ovary of adult ArKO females but was very high in the wild-type ovary and intermediate in the HET ovary. In wild-type mice, a high level of aromatase activity was detected on the day of birth even without pretreatment with testosterone. This neonatal activity was higher in males than in females, but females nevertheless appear to display a substantial level of oestrogen production in their brain. Aromatase activity was undetectable in the brain of newborn ArKO males and females and was intermediate between wild-type and ArKO in HET mice. In conclusion, the present study confirms that ArKO mice are unable to synthesize any oestrogens, thereby validating the ArKO mouse as a valuable tool in the study of the physiological roles of oestradiol. In addition, it demonstrates that the intermediate behaviour of HET mice presumably reflects the effect of gene dosage on aromatase expression and activity, that aromatase activity is sexually differentiated in mice during the neonatal period as well as in adulthood and, finally, that the neonatal female brain produces substantial amounts of oestrogens that could play a significant role in the sexual differentiation of the female brain early in life.

The neuroendocrinology of reproductive behavior in Japanese quail.

Sex steroid hormones such as testosterone have widespread effects on brain physiology and function but one of their best characterized effects arguably involves the activation of male sexual behavior. During the past 20 years we have investigated the testosterone control of male sexual behavior in an avian species, the Japanese quail (Coturnix japonica). We briefly review here the main features and advantages of this species relating to the investigation of fundamental questions in the field of behavioral neuroendocrinology, a field that studies inter-relationship among hormones, brain and behavior. Special attention is given to the intracellular metabolism of testosterone, in particular its aromatization into an estrogen, which plays a critical limiting role in the mediation of the behavioral effects of testosterone. Brain aromatase activity is controlled by steroids which increase the transcription of the enzyme, but afferent inputs that affect the intraneuronal concentrations of calcium also appear to have a pronounced effect on the enzyme activity through rapid changes in its phosphorylation status. The physiological significance of these slow genomic and rapid, presumably non-genomic, changes in brain aromatase activity are also briefly discussed.

Calcium-dependent phosphorylation processes control brain aromatase in quail.

Increased gene transcription activated by the binding of sex steroids to their cognate receptors is one important way in which oestrogen synthase (aromatase) activity is regulated in the brain. This control mechanism is relatively slow (hours to days) but recent data indicate that aromatase activity in quail preoptic-hypothalamic homogenates is also rapidly (within minutes) affected by exposure to conditions that enhance Ca2+-dependent protein phosphorylation. We demonstrate here that Ca2+-dependent phosphorylations controlled by the activity of multiple protein kinases including PKC, and possibly also PKA and CAMK, can rapidly down-regulate aromatase activity in brain homogenates. These phosphorylations directly affect the aromatase molecule itself. Western blotting experiments on aromatase purified by immunoprecipitation reveal the presence on the enzyme of phosphorylated serine, threonine and tyrosine residues in concentrations that are increased by phosphorylating conditions. Cloning and sequencing of the quail aromatase identified a 1541-bp open reading frame that encodes a predicted 490-amino-acid protein containing all the functional domains that have been previously described in the mammalian and avian aromatase. Fifteen predicted consensus phosphorylation sites were identified in this sequence, but only two of these (threonine 455 and 486) match the consensus sequences corresponding to the protein kinases that were shown to affect aromatase activity during the pharmacological experiments (i.e. PKC and PKA). This suggests that the phosphorylation of one or both of these residues represents the mechanism underlying, at least in part, the rapid changes in aromatase activity.

Seasonal variation in androgen-metabolizing enzymes in the diencephalon and telencephalon of the male European starling (Sturnus vulgaris).

In seasonally breeding songbirds, seasonal fluctuations occur in serum testosterone (T) concentrations and reproductive behaviours. Many T-dependent behaviours are regulated by the activity of androgenic and oestrogenic metabolites within specific brain regions. Male European starlings breed in spring when circulating T concentrations peak. T and its metabolites act within portions of the diencephalon to regulate the pituitary-gonadal axis and to activate courtship and copulation. Song in male starlings is critical for mate attraction during the breeding season and is regulated by steroid-sensitive nuclei in the telencephalon and diencephalon. Outside the breeding season, T is undetectable, however, males continue to sing at high levels. This suggests that singing outside of the breeding season might not be T-dependent as it appears to be in the spring. Alternatively, singing when T is low might continue to be regulated by T due to increased sensitivity of the brain to the action of the steroid. This increased sensitivity could be mediated by changes in intracellular T metabolism leading to increased production of active or decreased production of inactive metabolites. To explore the relationship between T-metabolism and reproductive behaviour, we analysed seasonal changes in the activity of four brain T-metabolizing enzymes: aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSDH), 5alpha-reductase (all three convert T into active metabolites) and 5beta-reductase (converts T into an inactive metabolite) in the diencephalon and telencephalon. In the anterior and posterior diencephalon, the highest aromatase was observed in spring when this region is critical for courtship and copulation. In the telencephalon, aromatase was highest and 5beta-reductase was lowest throughout the winter months well prior to the reproductive season and these enzymes presumably maximize T-activity within this region. Although these data do not indicate whether the metabolic changes occur specifically within song nuclei, these findings are compatible with the idea that singing in male starlings outside the breeding season may be regulated by steroids despite the presence of low serum T concentrations. Overall, seasonal changes in T-metabolizing enzymes appear to play a significant role in seasonal changes in behaviour and reproductive physiology.

The control of preoptic aromatase activity by afferent inputs in Japanese quail.

This review summarizes current knowledge on the mechanisms that control aromatase activity in the quail preoptic area, a brain region that plays a key role in the control of reproduction. Aromatase and aromatase mRNA synthesis in the preoptic area are enhanced by testosterone and its metabolite estradiol, but estradiol receptors of the alpha subtype are not regularly colocalized with aromatase. Estradiol receptors of the beta subtype are present in the preoptic area but it is not yet known whether these receptors are colocalized with aromatase. The regulation by estrogen of aromatase activity may be, in part, trans-synaptically mediated, in a manner that is reminiscent of the ways in which steroids control the activity of gonadotropic hormone releasing hormone neurons. Aromatase-immunoreactive neurons are surrounded by dense networks of vasotocin-immunoreactive and tyrosine hydroxylase-immunoreactive fibers and punctate structures. These inputs are in part steroid-sensitive and could therefore mediate the effects of steroids on aromatase activity. In vivo pharmacological experiments indicate that catecholaminergic depletions significantly affect aromatase activity presumably by modulating aromatase transcription. In addition, in vitro studies on brain homogenates or on preoptic-hypothalamic explants show that aromatase activity can be rapidly modulated by a variety of dopaminergic compounds. These effects do not appear to be mediated by the membrane dopamine receptors and could involve changes in the phosphorylation state of the enzyme. Together, these results provide converging evidence for a direct control of aromatase activity by catecholamines consistent with the anatomical data indicating the presence of a catecholaminergic innervation of aromatase cells. These dopamine-induced changes in aromatase activity are observed after several hours or days and presumably result from changes in aromatase transcription but rapid non-genomic controls have also been identified. The potential significance of these processes for the physiology of reproduction is critically evaluated.

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 (ERalpha) are not normally co-localized with aromatase in this brain area. Estradiol receptor beta (ERbeta) has been recently cloned in quail and localized in POM but we do not yet know whether ERbeta 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.

Rapid and reversible inhibition of brain aromatase activity.

Many actions of androgens require their conversion via the enzyme aromatase into oestrogens. Changes in brain aromatase activity are thought to take place via changes in enzyme concentration mediated by effects of sex steroids on aromatase transcription. These changes are relatively slow which fits in well with the fact that oestrogens are generally viewed as slow-acting messengers that act via changes in gene transcription. More recently, fast actions of oestrogens, presumably at the level of the cell membrane, have been described both in the female brain and in the male brain after the conversion of testosterone to oestradiol. It is difficult to reconcile the slow regulation of oestrogen synthesis (that occurs via changes in aromatase concentration) with a rapid action at the membrane level. Even if fast transduction mechanisms are available, this will not result in rapid changes in brain function if the availability of the ligand does not also change rapidly. Here, we report that aromatase activity in neural tissue of male Japanese quail (Coturnix japonica) is rapidly downregulated in the presence of Mg(2+), Ca(2+) and ATP in hypothalamic homogenates and in brain explants exposed to high Ca(2+) levels following a K(+)-induced depolarization or the stimulation of glutamate receptors. The K(+)-induced inhibition of aromatase activity is observed within minutes and reversible. Given that aromatase is present in presynaptic boutons, it is possible that rapidly changing levels of locally produced oestrogen are available for nongenomic regulation of neuronal physiology in a manner more akin to the action of a neuropeptide than previously hypothesized.

Ontogeny of aromatase and tyrosine hydroxylase activity and of aromatase-immunoreactive cells in the preoptic area of male and female Japanese quail.

The aromatization of testosterone into oestrogens plays a key role in the control of many behavioural and physiological aspects of reproduction. In the quail preoptic area (POA), aromatase activity and the number of aromatase-immunoreactive (ARO-ir) cells are sexually differentiated (males > females). This sex difference is implicated in the control of the sexually dimorphic behavioural response of quail to testosterone. We analysed the ontogenetic development of this sex difference by measuring aromatase activity and counting ARO-ir cells in the POA of males and females from day 1 post hatch to sexual maturity. We investigated in parallel another enzyme: tyrosine hydroxylase, the rate limiting step in catecholamine synthesis. Between hatching and 4 weeks of age, aromatase activity levels were low and equal in males and females. Aromatase activity then markedly increased in both sexes when subjects initiated their sexual maturation but this increase was more pronounced in males so that a marked difference in aromatase activity was present in 6 and 8 week-old subjects. Tyrosine hydroxylase activity progressively increased with age starting immediately after hatching and there was no abrupt modification in the slope of this increase when birds became sexually mature. No sex difference was detected in the activity of this enzyme. The number of ARO-ir cells in the POA progressively increased with age starting at hatching. No sex difference in ARO-ir cell numbers could be detected before subjects reached full sexual maturity. The analysis of the three-dimensional organization of ARO-ir cells in the POA revealed that, with increasing ages, ARO-ir cells acquire a progressively more lateral position: they are largely periventricular in young birds but they are found at higher density in the lateral part of the medial preoptic nucleus in adults. These data indicate that aromatase activity differentiates sexually when birds reach sexual maturity presumably under the activating effects of the increased testosterone levels in males. The number of ARO-ir cells, however, begins to increase in a non sexually differentiated manner before the rise in plasma testosterone in parallel with the increased tyrosine hydroxylase activity. Whether this temporal coincidence results from a general ontogenetic pattern or from more direct causal links remains to be established.

Localization and controls of aromatase in the quail spinal cord.

In adult male and female Japanese quail, aromatase-immunoreactive cells were identified in the spinal dorsal horns from the upper cervical segments to the lower caudal area. These immunoreactive cells are located mostly in laminae I-III, with additional sparse cells being present in the medial part of lamina V and, at the cervical level exclusively, in lamina X around the central canal. Radioenzyme assays based on the measurement of tritiated water release confirmed the presence of substantial levels of aromatase activity throughout the rostrocaudal extent of the spinal cord. Contrary to what is observed in the brain, this enzyme activity and the number of aromatase-immunoreactive cells in five representative segments of the spinal cord are not different in sexually mature males or females and are not influenced in males by castration with or without testosterone treatment. The aromatase activity and the numbers of aromatase-immunoreactive cells per section are higher at the brachial and thoracic levels than in the cervical and lumbar segments. These experiments demonstrate for the first time the presence of local estrogen production in the spinal cord of a higher vertebrate. This production was localized in the sensory fields of the dorsal horn, where estrogen receptors have been identified previously in several avian and mammalian species, suggesting an implication of aromatase in the modulation of sensory (particularly nociceptive) processes.

Distribution of aromatase activity in the brain and peripheral tissues of passerine and nonpasserine avian species.

Many behavioral effects of testosterone on hypothalamic and limbic brain areas are mediated by the action, at the cellular level, of estrogens derived from local testosterone aromatization. Aromatase activity and cells containing the aromatase protein and mRNA have accordingly been identified in the brain areas involved in the control of behavior. The presence of an unusually high level of aromatase activity has been detected in the telencephalon of one songbird species, the zebra finch (Taeniopygia guttata), and it is suspected that this high telencephalic aromatase may be a specific feature of songbirds but this idea is supported only by few experimental data. The distribution of aromatase activity in the brain of zebra finches and of one nonsongbird species, the Japanese quail (Coturnix japonica), was compared with the distribution of aromatase activity in the brain of four species of free-living European songbirds, the chaffinch (Fringilla coelebs, Fringillidae), willow warbler (Phylloscopus trochilus, Sylviidae), great tit (Parus major, Paridae), and pied flycatcher (Ficedula hypoleuca, Muscicapidae). High levels of enzyme activity were observed in the diencephalon of all species. The high levels of aromatase activity that had been observed in the zebra finch telencephalon and were thought to be typical of songbirds were also present in the four wild oscine species but not in quail. None of these songbird species had, however, a telencephalic aromatase activity as high as that in the zebra finch, which may represent an extreme as far as the activity of this enzyme in the telencephalon is concerned. Measurable levels of aromatase activity were also detected in all songbird species in the liver and in the three other brain areas that were assayed, the optic lobes, cerebellum, and brain stem, with the exception of the cerebellum in willow warblers and quail, but no detectable activity was observed in the testes, muscle, and adrenals of all species. Additional studies will be needed to identify the functional significance of estrogen synthesis in areas that are not classically known to be implicated in the control of reproduction. Within a given species, the birds that had the highest plasma testosterone levels also displayed the highest levels of diencephalic aromatase activity and the interspecies differences in the two variables were positively related. This raises the possibility that the absolute level of diencephalic aromatase represents a species-specific characteristic under the control of plasma testosterone levels. There was, in contrast, no correlation between the aromatase activity in the telencephalon and the plasma testosterone levels but the enzyme activity was correlated with the plasma levels of luteinizing hormone. These data bring additional support to the idea that the diencephalic and telencephalic aromatases are controlled by independent mechanisms.

Regional distribution and control of tyrosine hydroxylase activity in the quail brain.

Tyrosine hydroxylase (TH) activity, the rate-limiting step in the synthesis of catecholamines, was quantified in the preoptic area-hypothalamus of adult male Japanese quail by a new assay measuring the tritiated water production from 3,5-[3H]-L-tyrosine. Maximal levels of activity were observed at a 20-25 microM concentration of substrate, with more than 50% inhibition of the activity being recorded at a 100 microM concentration. TH activity was linear as a function of the incubation time during the first 20 min and maximal at a pH of 6.0. TH was heterogeneously distributed in the quail brain with highest levels of activity being found (in decreasing order) in the mesencephalon, diencephalon, and telencephalon. Given the large size of the telencephalon, this is the brain area that contains, as a whole, the highest level of enzyme activity. TH inhibitors that have been well-characterized in mammals, such as 3-iodo-L-tyrosine and L-alpha-methyl-p-tyrosine (AMPT) completely inhibited the enzyme activity at a 100 microM concentration. In mammals, the accumulation of catecholamines exerts a negative feedback control on TH activity. Similar controls were observed in the quail brain. Two inhibitors of the DOPA decarboxylase that should lead to accumulation of DOPA depressed TH activity by 60% or more, and the inhibitor of the dopamine beta-hydroxylase, fusaric acid that should cause an accumulation of dopamine, suppressed 90% of the TH activity. The addition of exogenous DOPA, dopamine, or norepinephrine to the brain homogenates also strongly inhibited TH activity, independently confirming the feedback effects of the enzyme products on the enzyme activity. These data demonstrate that TH activity in the quail brain is heterogeneously distributed and acutely regulated, as it is in mammals, by the accumulation of its products and of the derived catecholamines.

Neuroanatomical distribution and variations across the reproductive cycle of aromatase activity and aromatase-immunoreactive cells in the pied flycatcher (Ficedula hypoleuca).

The anatomical distribution and seasonal variations in aromatase activity and in the number of aromatase-immunoreactive cells were studied in the brain of free-living male pied flycatchers (Ficedula hypoleuca). A high aromatase activity was detected in the telencephalon and diencephalon but low to negligible levels were present in the optic lobes, cerebellum, and brain stem. In the diencephalon, most aromatase-immunoreactive cells were confined to three nuclei implicated in the control of reproductive behaviors: the medial preoptic nucleus, the nucleus of the stria terminalis, and the ventromedial nucleus of the hypothalamus. In the telencephalon, the immunopositive cells were clustered in the medial part of the neostriatum and in the hippocampus as previously described in another songbird species, the zebra finch. No immunoreactive cells could be observed in the song control nuclei. A marked drop in aromatase activity was detected in the anterior and posterior diencephalon in the early summer when the behavior of the birds had switched from defending a territory to helping the female in feeding the nestlings. This enzymatic change is presumably controlled by the drop in plasma testosterone levels observed at that stage of the reproductive cycle. No change in enzyme activity, however, was seen at that time in other brain areas. The number of aromatase-immunoreactive cells also decreased at that time in the caudal part of the medial preoptic nucleus but not in the ventromedial nucleus of the hypothalamus (an increase was even observed), suggesting that differential mechanisms control the enzyme concentration and enzyme activity in the hypothalamus. Taken together, these data suggest that changes in diencephalic aromatase activity contribute to the control of seasonal variations in reproductive behavior of male pied flycatchers but the role of the telencephalic aromatase in the control of behavior remains unclear at present.

Anatomical relationships between aromatase and tyrosine hydroxylase in the quail brain: double-label immunocytochemical studies.

The activation of male sexual behavior in Japanese quail (Coturnix japonica) requires the transformation of testosterone to 17beta-estradiol by the enzyme aromatase (estrogen synthetase). There are prominent sex differences in aromatase activity that may be regulated in part by sex differences in catecholaminergic activity. In this study, we investigate, with double-label immunocytochemistry methods, the anatomical relationship between the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) and aromatase (ARO) in the quail brain. The immunoreactivity observed for each antigen generally matched the previously described distribution. One exception is the observation that cells weakly labeled for aromatase were found widely distributed throughout the telencephalon. The presence of telencephalic aromatase was confirmed independently by radioenzymatic assays. There was an extensive overlap between the distribution of the two antigens in many brain areas. In all densely labeled aromatase-immunoreactive (ARO-ir) cell groups, including the preoptic medial nucleus, nucleus of the stria terminalis, mediobasal hypothalamus, and paleostriatum ventrale, ARO-ir cells were found in close association with TH-ir fibers. These TH-ir fibers often converged on an ARO-ir cell, and one or more TH-ir punctate structure(s) were found in close contact with nearly every densely labeled ARO-ir cell. In the telencephalon (mostly the neostriatum), all TH-ir fibers were found to be part of fiber groups that surrounded weakly immunoreactive aromatase cells. The few cells exhibiting an intracellular colocalization were detected in the anteroventral periventricular nucleus. These results are consistent with the hypothesis that catecholaminergic inputs regulate brain aromatase.

A direct dopaminergic control of aromatase activity in the quail preoptic area.

In the quail preoptic area (POA) anatomical and pharmacological data suggest that catecholamines may be implicated in the control of testosterone (T) aromatization into estrogens. The biochemical mechanism(s) mediating this control of the enzyme activity is (are) however unexplored. The present studies were carried out to investigate whether the catecholamines, dopamine (DA) and norepinephrine (NE) are able to directly affect aromatase activity (AA) measured during in vitro incubations of POA homogenates. AA was quantified in the POA-hypothalamus of adult male Japanese quail by measuring the tritiated water production from [1beta-3H]-androstenedione. Enzyme activity was linear as a function of the incubation time and of the protein content of homogenates. It exhibited a typical Michaelis-Menten kinetics, with an apparent Km of 2.8 nM and a Vmax of 266.6 fmol h(-1) mg wet weight(-1). AA was then measured at a substrate concentration of 25 nM in the presence of catecholamines and some of their receptor agonists or antagonists, at two concentrations, 10(-3) and 10(-6) M. Norepinephrine and prazosin (alpha1-adrenergic antagonist) had no or very limited effects on AA at both concentrations. In contrast, DA and some D1 and/or D2 receptor agonists (apomorphine[D1/D2], SKF-38393 [D1] and RU-24213 [D2]) depressed AA by 40 to 70% at the 10(-3) M concentration. One D2 receptor antagonist also produced a major inhibition of AA (sulpiride) while other antagonists either had no significant effect or only produced moderate decreases in enzyme activity (SCH-23390 [D1], spiperone [D2], pimozide [D2]) as did two DA indirect agonists, amfonelic acid and nomifensine. The inhibitory effect of the agonists was not antagonized by the less active antagonists, SCH-23390 [D1] or spiperone [D2]. Taken together these results suggest that the inhibitory effects do not involve specific binding of DA or its agonists/antagonists to dopaminergic receptors mediating changes in cAMP concentration. This conclusion is also supported by the observation that addition of dibutyryl cAMP did not change brain AA. It appears more likely that DA and dopaminergic drugs inhibit AA by a direct effect on the enzyme, as suggested by the competitive nature of DA and SKF-38393 inhibition of AA (Ki's of 59 and 84 microM, respectively). The functional significance of this effect should still be demonstrated but this mechanism may represent an important physiological pathway through which neurotransmitters could rapidly affect steroid-dependent processes such as the neural synthesis of estrogens. This would provide a mean by which environmental stimuli could affect reproductive behavior and physiology.

Uptake of (28)Mg by duodenal and jejunal brush border membrane vesicles in the rat.

Mg uptake was investigated with (28)Mg by a rapid filtration procedure in rat duodenal and jejunal brush border membrane (BBM) vesicles, prepared by CaCl(2)a or MgCl(2)b differential precipitation. At 1 mM Mg, 10 s uptakes were lower in jejunal vesicles (3.5(a) or 5.5(b) nmol/10 s per mg protein) than in duodenal vesicles (11.4(a) or 13.5(b) nmol/10 s per mg protein). The equilibrated 60 min uptakes were also lower in jejunum (11.0(a) or 26.6(b) nmol/60 min per mg protein) than in duodenum (l8.8(a) or 26.6(b) nmol/60 min per mg protein). The influence of medium osmolarity on 10s and 60 min uptakes of Mg indicated that Mg was 'transported' into osmotically active spaces. The effect of Mg concentration on the 10 s uptake suggested the existence of one single mechanism of transport in the duodenum, with an apparent K(T) of 1 mM, and of two mechanisms in the jejunum, with apparent K(T) values of 0.2 and 2-5 mM. Despite different amounts of calcium and magnesium in CaCl(2) and MgCl(2) precipitated vesicles, there were no large differences in magnesium uptakes depending on the mode of preparation of the vesicles. In contrast, calcium uptakes. measured with (45)Ca, were six to nine times higher in MgCl(2) prepared jejunal vesicles, and were always much higher than magnesium uptakes measured under the same conditions. At 0.1 mM calcium concentration, calcium uptake was depressed by 0.025 mM verapamil (50 percent) and by 0.1 mM ZnCl(2)(40-75 percent), while Mg uptakes were unaffected. L-leucine or L-phenylalanine (5 mM), two inhibitors of intestinal alkaline phosphatase, decreased Mg uptake by 30 to 40 percent at 1 mM Mg, but had no significant effect at 0.1 mM, and did not affect calcium uptakes at all. A possible involvement of alkaline phosphatase in magnesium uptake was ascertained in jejunal BBM vesicles treated with phosphatidylinositol-specific phospholipase C, which partially released alkaline phosphatase from the BBM. Calcium uptakes were unaffected by the treatment, while magnesium uptakes were significantly decreased at 1 mM Mg. These results confirm that magnesium and calcium are transported by distinct mechanisms in the jejunum.

Characterization of two mechanisms of (28)Mg uptake in rat jejunal brush border membrane vesicles.

Uptakes of (28)Mg at 10 s were measured at 0.1 and 1mM MgCl(2), to mainly represent one or other of the two uptake mechanisms earlier shown to be present in rat jejunal brush border membrane vesicles, one with an apparent KT of 0.2 mM, the other in the millimolar range. Both mechanisms had an optimal temperature close to 28 degrees C, inactivation at 37 degrees C being more acute for the low affinity mechanism (55 percent, P < 0.01). Both mechanisms were equally stimulated by an electrical potential difference (negative inside the vesicles) imposed by a potassium gradient and not affected by the nature of the anion accompanying magnesium. At 0.1 mM MgCl(2), the uptake was increased by an outwardly directed proton gradient, pH 8.2 outside and 7.4 inside (38 percent, P < 0.05), but not depressed when the gradient was in the opposite direction, pH 6.6 outside and 7.4 inside. It was trans-stimulated by magnesium, strongly inhibited by amiloride and to a smaller extent by furosemide, but uninfluenced by 0.1 mM NaCl or by 100 mM NaCl, NaSCN or KCl. A slight but significant inhibition (20-30 per cent) was recorded in the presence of 0.1 mM CoCl(2), NlCl(2) or BaCl(2). At 1 mM MgCl(2), the uptake was not influenced by pH gradient, was not trans-stimulated by Mg and was not affected by furosemide. A 40 percent inhibition by amiloride was, however, recorded. Also 100 mM NaCl or KCl doubled the uptake, while 1 mM NaCl or 100 mM NaSCN did not affect it. In contrast, all the divalent cations tested produced an inhibition (from 60 to 12 percent) in the following order: Co > or = Mn > Ca > or = Ni> Ba > Sr, when used at the same concentration as magnesium. The results showed that cobalt and calcium were not true competitors. In conclusion, two distinct mechanisms would operate magnesium entry at the brush border: (1) an electrogenic high affinity Mg/Mg,H exchange, sensitive to amiloride and furosemide, and (2) an electrogenic low affinity mechanism, inhibited by the presence of several divalent cations and dependent on the presence or activity of alkaline phosphatase.