Quail Preoptic Area Research Articles

Sex Differences in the Rapid Control of Aromatase Activity in the Quail Preoptic Area

Adult male quail show high levels of aromatase activity in the preoptic area-hypothalamus (POA-HYP), which parallels the high number of aromatase-immunoreactive cells and elevated mRNA concentrations detected in this brain region by in situ hybridisation. Interestingly, females display considerably lower aromatase activity than males but have almost equal numbers of aromatase-immunoreactive cells and express similar levels of aromatase mRNA. Aromatase activity in the male POA-HYP can be rapidly regulated by calcium-dependent phosphorylations, in the absence of changes in enzyme concentration. In the present study, we investigated whether aromatase activity is differentially regulated by phosphorylations in males and females. A linear increase in accumulation of aromatisation products was observed in both sexes as a function of time but the rate of conversion was slower in females. Saturation analysis confirmed the lower maximum velocities (V(max) ) in females but indicated a similar affinity (K(m) ) in both sexes. Aromatase activity in females reacted differentially to manipulations of intracellular calcium. In particular, chelating calcium with ethylene glycol tetraacetic acid (EGTA) resulted in a larger increase of enzymatic activity in males than in females, especially in the presence of ATP. A differential reaction to kinase inhibitors was also observed between males and females (i.e. a larger increase in aromatase activity in females than in males after exposure to specific inhibitors). These findings suggest that the nature of aromatase is conserved between the sexes, although the control of its activity by calcium appears to be different. Additional characterizations of intracellular calcium in both sexes would therefore be appropriate to better understand aromatase regulation.

Dopamine Activates Noradrenergic Receptors in the Preoptic Area

Dopamine (DA) facilitates male sexual behavior and modulates aromatase activity in the quail preoptic area (POA). Aromatase neurons in the POA receive dopaminergic inputs, but the anatomical substrate that mediates the behavioral and endocrine effects of DA is poorly understood. Intracellular recordings showed that 100 microm DA hyperpolarizes most neurons in the medial preoptic nucleus (80%) by a direct effect, but depolarizes a few others (10%). DA-induced hyperpolarizations were not blocked by D1 or D2 antagonists (SCH-23390 and sulpiride). Extracellular recordings confirmed that DA inhibits the firing of most cells (52%) but excites a few others (24%). These effects also were not affected by DA antagonists (SCH-23390 and sulpiride) but were blocked by alpha2-(yohimbine) and alpha1-(prazosin) noradrenergic receptor antagonists, respectively. Two dopamine-beta-hydroxylase (DBH) inhibitors (cysteine and fusaric acid) did not block the DA-induced effects, indicating that DA is not converted into norepinephrine (NE) to produce its effects. The pK(B) of yohimbine for the receptor involved in the DA- and NE-induced inhibitions was similar, indicating that the two monoamines interact with the same receptor. Together, these results demonstrate that the effects of DA in the POA are mediated mostly by the activation of alpha2 (inhibition) and alpha1 (excitation) adrenoreceptors. This may explain why DA affects the expression of male sexual behavior through its action in the POA, which contains high densities of alpha2-noradrenergic but limited amounts of DA receptors. This study thus clearly demonstrates the existence of a cross talk within CNS catecholaminergic systems between a neurotransmitter and heterologous receptors.

Distribution of DARPP-32 immunoreactive structures in the quail brain: anatomical relationship with dopamine and aromatase

We recently demonstrated that dopamine (DA) as well as different DA receptor agonists and antagonists are able to decrease within a few minutes the aromatase activity (AA) measured in vitro in homogenates or in explants of the quail preoptic area — hypothalamus. In addition, DA also appears to regulate AA, in vivo presumably by modifying enzyme synthesis. The cellular mechanisms and the anatomical substrate that mediate these controls of AA by DA are poorly understood. Tyrosine hydroxylase-immunoreactive (TH-ir) fibers and punctate structures have been previously observed in close vicinity of aromatase-immunoreactive (ARO-ir) cells in the quail medial preoptic nucleus (POM) and bed nucleus striae terminalis (BST) but these fibers could reflect a noradrenergic innervation. We also do not know whether aromatase cells are dopaminoceptive. The main goal of the present study was therefore to bring more information on the anatomical relationships between aromatase expressing neurons and the dopaminergic system in the quail brain. The visualization by immunocytochemistry of DA and of the D1 receptor associated protein DARPP-32 was used to address these questions. DA-ir fibers were observed in the quail forebrain and overlapped extensively with nuclei that contain high densities of ARO-ir cells such as the POM and BST. This confirms that the previously reported TH-ir innervation of ARO-ir cells is, at least in part, of dopaminergic nature. DARPP-32-immunoreactive cells were found in periventricular position throughout the hypothalamus. DARPP-32-ir cells were also observed in telencephalic and mesencephalic areas (hyperstriatum accessorium, paleostriatum, nucleus intercollicularis, optic tectum). DARPP-32-ir fibers were widespread in tel-, di-, and mes-encephalic areas. The highest densities of immunoreactive fibers were detected in the lobus parolfactorius, paleostriatum augmentatum and substantia nigra/area ventralis of Tsai. In double-labeled sections, appositions between DARPP-32 fibers and ARO-ir cells were present in the dorsolateral POM and BST but DARPP-32 immunoreactivity was not detected in the ARO-ir perikarya (no colocalization). These data confirm the presence of a dopaminoceptive structures within the main cell clusters of ARO-ir cells in the quail brain but provide no evidence that these ARO-ir cells are themselves dopaminoceptive. Because DARPP-32 is not present in all types of cells expressing DA receptors, the presence of DA receptors that would not be associated with DARPP-32 in ARO-ir cells still remains to be investigated

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.

Mating-induced Fos and aromatase are not co-localized in the preoptic area.

Male sexual behavior is determined by the interaction of endocrine and environmental stimuli originating from the female, yet it is unknown how and where these stimuli are integrated within the brain. Activation of copulatory behavior by testosterone is limited by its central aromatization into an estrogen in the preoptic area. We investigated whether mating-induced neuronal activation as identified by the expression of the immediate early gene Fos occurs in aromatase-immunoreactive (ARO-ir) cells of the male quail preoptic area. Fos-immunoreactive (ir) cells were observed within and lateral to these ARO-ir cells groups but few ARO-ir cells contained Fos-ir indicating that mating-related stimuli do not directly affect estrogen-synthesizing cells.

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 [1β- 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 K m of 2.8 nM and a V max 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 (α1-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 ( K i's of 59 and 84 μM, 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.

Localization of testosterone-sensitive and sexually dimorphic aromatase-immunoreactive cells in the quail preoptic area

The distribution of aromatase-immunoreactive cells was studied in the medial preoptic nucleus of male and female quail that were sexually mature and gonadally intact, or gonadectomized, or gonadectomized and treated with testosterone. The study first confirmed the existence of a significant difference in the number of aromatase-immunoreactive cells between males and females (males > females) and the marked effect of castration and testosterone treatment which, respectively, decrease and restore the number of these cells. An analysis of the distribution in space of this neurochemically defined cell population was also carried out. This study revealed that castration does not uniformly decrease the density of aromatase-immunoreactive cells, but local increases are observed in an area directly adjacent to the third ventricle. A number of sex differences in the organization of the medial preoptic nucleus and its population of aromatase cells have, in addition, been identified. The density of aromatase-immunoreactive cells is not higher in males than in females throughout the nucleus, but a higher density of immunoreactive cells is present in the ventromedial part of the nucleus in females as compared to males. In addition, the cross-sectional area of the nucleus as defined by the population of aromatase-immunoreactive cells is larger in males than in females in its rostral part and its shape is more elongated in the dorso-ventral direction in females than in males. Some of these differences (e.g. higher density of ARO-ir cells in the ventromedial part of the female POM, shape of the nucleus) appear to be organizational in nature, because they are still present in birds exposed to the same endocrine conditions during adult life (e.g. gonadectomized and treated with a same dose of testosterone). This conclusion should now be tested by experiments manipulating the endocrine environment of quail embryos. The anatomical heterogeneity of the medial preoptic nucleus revealed by this study also suggests a functional heterogeneity and the specific roles of the medial and lateral parts of the nucleus should also be investigated.

Testosterone effects on neurotensin-immunoreactive cells in the quail preoptic area.

In the quail preoptic area (POA), neurotensin-immunoreactive (NT-ir) cells are mostly located in the sexually dimorphic medial preoptic nucleus (POM). They are more numerous in females than in males and, in females, their number varies during the ovulatory cycle. This suggests a control by steroids of NT expression in the POA. This idea was tested by comparing the NT-ir populations in gonadectomized males and females treated or not with testosterone. No sex differences in the NT-ir cell number was present in gonadectomized birds. Testosterone treatment increased the number of immunoreactive perikarya. This effect was anatomically specific and the increase was observed in the area caudal to the anterior commissure but not in POM itself.

Aromatase-immunoreactive cells in the quail brain: Effects of testosterone and sex dimorphism

We previously demonstrated that testosterone (T) increases aromatase activity (AA) and that AA is sexually dimorphic (males > females) in the quail preoptic area (POA). The precise anatomical localization of these effects is, however, impossible to obtain by biochemical assays even when samples are dissected by the Palkovits punch technique. We were recently able to set up an immunocytochemical (ICC) procedure that permits visualization of aromatase-immunoreactive (ARO-ir) cells in the quail brain. This showed that the ARO-ir cells of the quail POA actually outline the sexually dimorphic medial preoptic nucleus (POM). This ICC technique was used here to analyze the sex dimorphism of the quail preoptic aromatase and the localization of T effects on ARO-ir cells. In Experiment 1, the number of ARO-ir cells was counted in one section every 100 μm throughout the rostral to caudal extent of the POM of castrated birds that had been treated with increasing doses of T (5, 10, or 20 mm long Silastic implants). These T-treatments produced a dose-related increase in the sexual behavior of the birds and they increased the number of ARO-ir cells in POM, in the septal regions, and in the bed nucleus of the stria terminalis (BNST). The effect had a particularly large amplitude in the part of the POM located under the anterior commissure (AC). In Experiment 2, the same procedure was used to reanalyze the sex difference of the preoptic aromatase system. This showed that the POM of adult males contains more stained cells than the POM of females but only in a restricted region located just under and rostral to the AC. No significant sex difference was observed in the septum or in the BNST. In Experiment 3, the number of ARO-ir cells was determined in the POM of males and females that had been gonadectomized and treated with a same dose of T (40 mm implants). No sex difference in the number of ARO-ir cells could be detected in these conditions. This suggests that the sex difference in AA that had been previously observed in T-treated birds results either from a difference in aromatase concentration or activity in a similar number of positive cells or from a difference in the number of ARO-ir cells that is very discrete from the anatomical point of view. These experiments also indicate that ARO-ir cells in the region of POM located just under and rostral to AC are sexually dimorphic and T-sensitive, which suggests their involvement in the control of male sexual behavior. This conclusion is also supported but our recent studies based on electrolytic lesion and stereotaxic implantation of T in POM.

Implication of testosterone metabolism in the control of the sexually dimorphic nucleus of the quail preoptic area

In quail, testosterone (T) activates male copulation and affects the volume and cytoarchitectonic organization of the medial preoptic nucleus (POM). T metabolism (especially its aromatization) is critical for the production of these behavioral effects. We wondered whether T metabolism is also playing a role in the induction of the morphological changes in POM. We compared the effects of T and of its metabolites in this nucleus. To obtain an independent evaluation of the role played by aromatase, morphological effects of T associated or not with the aromatase inhibitor R76713 were also assessed. As previously observed, T increased the POM volume and the cross-sectional area of the neurons in the lateral part of the nucleus. The effects of T on the neurons in the lateral POM were mimicked in part by the combined treatment with estradiol and 5α-dihydrotestosterone. They were also blocked by the aromatase inhibitor. This suggests that T aromatization plays a critical role in the mediation of the cytoarchitectonic effects of T. A specific role for androgens alone remains to be established.

Neuroanatomical specificity in the autoregulation of aromatase-immunoreactive neurons by androgens and estrogens: an immunocytochemical study

Testosterone (T) increases brain aromatase activity (AA) in quail and other avian and mammalian species. It was shown both in quail and in rat that this enzymatic induction results from a synergistic action of androgens and estrogens. These studies provide little information on possible anatomical or cellular specificity of the effect. Using a polyclonal antiserum against human placental aromatase, we have previously identified aromatase-immunoreactive (ARO-ir) neurons in the quail brain and demonstrated that T increases the number of ARO-ir cells in the quail preoptic area (POA) supporting previous evidence that T increases AA in the brain. However, which T metabolites are involved, the actual mechanism of regulation and the possibility of anatomical specificity for these effects are not yet clear. In the present study, we disassociated the effects of androgens and estrogens in aromatase induction by comparin ARO-in neurons of quail treated with T alone or T in the presence of a potent aromatase inhibitor (R76713), which has been shown to depress AA levels and to suppress T-activated copulatory behavior. T increased the number of ARO-ir cells in POA, bed nucleus striae terminalis (BNST) and tuberal hypothalamus (Tu). The T effect was inhibited by concurrent treatment with aromatase inhibitor in Tu, but not in POA and BNST. The differential effect of the aromatase inhibitor fits in very well with our previous studies of the co-localization of aromatase and estrogen receptors. The T effect was blocked by R76713 in areas where ARO-ir and estrogen receptor-ir are generally co-localized (Tu) and was not affected in areas with mainly ARO-ir positive, estrogen receptor-ir negative cells (POA, BNST). This suggests anatomical differences in the expression or clearance of aromatase which may be differentially sensitive to androgens and dependent upon the presence of sex steroid receptors.

Copulatory behavior is controlled by the sexually dimorphic nucleus of the quail POA

The medial preoptic nucleus (POM) of the quail preoptic area is sexually dimorphic and testosterone sensitive. Stereotaxic implantation of needles filled with crystalline testosterone demonstrated that the POM is a critical site of steroid action in the control of Copulatory behavior. Only implants located in the POM reliably restored the behavior in castrated birds. Implants around the nucleus weakly activated the behavior; those which were distant by more than 200 μm were totally inactive. Electrolytic lesions confirmed the role of the POM in the control of Copulatory behavior. The percentage of the POM which was lesioned was highly correlated to the behavioral deficit while the absolute size of the lesion was not. Electrolytic lesions in or around POM also significantly decreased the volume of the nucleus suggesting that the afferents and efferents of the nucleus are required for its full development. The total volume of the POM was correlated with the sexual behavior of the birds. The morphological changes in POM observed following exposure to testosterone probably represent the signature of the behavioral effects of the steroid. The sexually dimorphic testosterone-sensitive POM is therefore an excellent animal model to study the brain-steroid interactions which mediate the activation of male reproductive behavior.

Cytoarchitectural analysis of the quail preoptic area. Evidence for a sex‐related dimorphism in the medial preoptic nucleus

Abstract Studies using the Nissl method on thick paraffin sections of the preoptic region of the Japanese quail hypothalamus reveal a complex cytoarchitecture with several differentiated nuclei. The most prominent nucleus is the medial preoptic nucleus whose staining and volume appear sexually dimorphic. A higher number of intensely stainable neurons are present in males than in females, probably reflecting different metabolic rates in the two sexes. The volume of the medial preoptic nucleus is also larger (40Z) in males than in females. Present results represent the first description of a sex‐related dimorphism in the hypothalamus of non‐mammalian vertebrates.

A sexually dimorphic nucleus in the quail preoptic area

The cytoarchitectural analysis of the preoptic-anterior hypothalamic region of the Japanese quail reveals a sexual dimorphism in the total volume of the medial preoptic nucleus (significantly larger in males than in females). Different nuclei of the region (dorsal preopticus, suprachiasmaticus) do not show any statistically significant difference. The sex-related difference is more consistent comparing the distribution of dark volume. This last is due to a larger number of cells containing high amount of Nissl's substance in male than in female. Present findings represent the first example of sexual dimorphism in the avian hypothalamus.