Catherine Pasqualini

Acute Stimulatory Effect of Estradiol on Striatal Dopamine Synthesis

Abstract: The acute effect of physiological doses of estradiol (E2) on the dopaminergic activity in the striatum was studied. In a first series of experiments, ovariectomized rats were injected with 17α or 17β E2 (125, 250, or 500 ng/kg of body weight, s.c.), and in situ tyrosine hydroxylase (TH) activity (determined by DOPA accumulation in the striatum after intraperitoneal administration of NSD 1015) was quantified. A dose‐dependent increase in striatal TH activity was observed within minutes after 17β (but not 17α) E2 treatment. To examine whether E2 acts directly on the striatum, in a second series of experiments, anesthetized rats were implanted in the striatum with a push‐pull cannula supplied with an artificial CSF containing [3H]tyrosine. The extracellular concentrations of total and tritiated dopamine (DA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) were measured at 20‐min intervals. Addition of 10−9M 17β (but not 17α) E2 to the superfusing fluid immediately evoked an ∼50% increase in [3H]DA and [3H]DOPAC extracellular concentrations, but total DA and DOPAC concentrations remained constant. This selective increase in the newly synthesized DA and DOPAC release suggested that E2 affects DA synthesis rather than DA release. Finally, to determine whether this rapid E2‐induced stimulation of DA synthesis was a consequence of an increase in TH level of phosphorylation, the enzyme constant of inhibition by DA (Ki DA) was calculated. Incubation of striatal slices in the presence of 10−9M 17β (but not 17α) E2 indeed evoked an approximate twofold increase in the Ki DA of one form of the enzyme. It is concluded that physiological levels of E2 can act directly on striatal tissue to stimulate DA synthesis. This stimulation appears to be mediated, at least in part, by a decrease in TH susceptibility to end‐product inhibition, presumably due to phosphorylation of the enzyme. The rapid onset of this effect, and the fact that the striatum does not contain detectable nuclear E2 receptors, suggest a nongenomic action of the steroid.

Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty

Abstract In various adult teleost fishes, LH ovulatory peak is under a dual neurohormonal control that is stimulatory by GnRH and inhibitory by dopamine (DA). We investigated whether DA could also be involved in the inhibitory control of LH at earlier steps of gametogenesis by studying the model of the European eel, Anguilla anguilla, which remains at a prepubertal stage until the oceanic reproductive migration. According to a protocol previously developed in the striped bass, eels received sustained treatments with GnRH agonist (GnRHa), DA-receptor antagonist (pimozide), and testosterone (T) either alone or in combination. Only the triple treatment with T, GnRHa, and pimozide could trigger dramatic increases in LH synthesis and release as well as in plasma vitellogenin levels and a stimulation of ovarian vitellogenesis. Thus, in the prepubertal eel, removal of DA inhibition is required for triggering GnRH-stimulated LH synthesis and release as well as ovarian development. To locate the anatomical support for DA inhibition, the distribution of tyrosine hydroxylase (TH) in the brain and pituitary was studied by immunocytochemistry. Numerous TH-immunoreactive cell bodies were observed in the preoptic anteroventral nucleus, with a dense tract of immunoreactive fibers reaching the pituitary proximal pars distalis, where the gonadotrophs are located. This pathway corresponds to that mediating the inhibition of LH and ovulation in adult teleosts. To our knowledge, this is the first demonstration of a pivotal role for DA in the control of LH and puberty in a juvenile teleost. These data support the view that DA inhibition on LH secretion is an ancient evolutionary component in the neuroendocrine regulation of reproduction that may have been partially maintained throughout vertebrate evolution.

Dopaminergic inhibition of reproduction in teleost fishes: ecophysiological and evolutionary implications.

Abstract: In many teleosts, dopamine (DA) exerts direct inhibitory control on gonadotropes, counteracting the stimulatory effect of gonadotropin‐releasing hormone (GnRH) on gonadotropin release. This dual control by GnRH and DA has been demonstrated in various adult teleosts and has major implications for aquaculture. Because of its unique life cycle, the European eel has provided a powerful model for demonstrating the key role of DA in the control of puberty. Data from tetrapods suggest that the inhibitory role of DA on reproduction is not restricted to the teleosts. Thus, DA inhibitory control could represent an ancient evolutionary component in the neuroendocrine regulation of reproduction that may have been differentially maintained throughout vertebrate evolution. The intensity of DA inhibition, its main site of action, and its involvement in the control of puberty, seasonal reproduction, ovulation, spermiation, or even sex change may differ among classes of vertebrates, as well as within smaller phylogenetic units such as teleosts or mammals. An inhibitory role for DA has been reported also in some invertebrates, indicating that neuronal DA pathways may have been recruited in various groups of metazoa to participate in the control of reproduction. In addition to the incontestable GnRH neurons, the recruitment of DA neurons for the neuroendocrine control of reproduction provides an additional brain pathway for the integration of various species‐specific, internal, and environmental cues. In teleosts, the plasticity of the DA neuroendocrine role may have contributed to their large diversity of biological cycles and to their successful adaptation to various environments.

Neuroendocrine control by dopamine of teleost reproduction

While gonadotropin-releasing hormone (GnRH) is considered as the major hypothalamic factor controlling pituitary gonadotrophins in mammals and most other vertebrates, its stimulatory actions may be opposed by the potent inhibitory actions of dopamine (DA) in teleosts. This dual neuroendocrine control of reproduction by GnRH and DA has been demonstrated in various, but not all, adult teleosts, where DA participates in an inhibitory role in the neuroendocrine regulation of the last steps of gametogenesis (final oocyte maturation and ovulation in females and spermiation in males). This has major implications for inducing spawning in aquaculture. In addition, DA may also play an inhibitory role during the early steps of gametogenesis in some teleost species, and thus interact with GnRH in the control of puberty. Various neuroanatomical investigations have shown that DA neurones responsible for the inhibitory control of reproduction originate in a specific nucleus of the preoptic area (NPOav) and project directly to the region of the pituitary where gonadotrophic cells are located. Pharmacological studies showed that the inhibitory effects of DA on pituitary gonadotrophin production are mediated by DA-D2 type receptors. DA-D2 receptors have now been sequenced in several teleosts, and the coexistence of several DA-D2 subtypes has been demonstrated in a few species. Hypophysiotropic DA activity varies with development and reproductive cycle and probably is controlled by environmental cues as well as endogenous signals. Sex steroids have been shown to regulate dopaminergic systems in several teleost species, affecting both DA synthesis and DA-D2 receptor expression. This demonstrates that sex steroid feedbacks target DA hypophysiotropic system, as well as the other components of the brain-pituitary gonadotrophic axis, GnRH and gonadotrophins. Recent studies have revealed that melatonin modulates the activity of DA systems in some teleosts, making the melatonin-DA pathway a prominent relay between environmental cues and control of reproduction. The recruitment of DA neurons for the neuroendocrine control of reproduction provides an additional brain pathway for the integration of various internal and environmental cues. The plasticity of the DA neuroendocrine role observed in teleosts may have contributed to their large diversity of reproductive cycles.

Biochemical and functional alterations of central GABA receptors during chronic estradiol treatment

The characteristics of GABA and benzodiazepine receptors were examined in the hippocampus, striatum and cerebral cortex of female rats at various times (up to 9 months) after the subcutaneous implantation of an estradiol pellet (10 mg). A significant decrease in the Bmax of the high-affinity binding of [3H]muscimol to membranes from these 3 regions was detected as soon as one week after the implantation. Although the characteristics of the high-affinity binding of [3H]flunitrazepam remained unaffected during the whole treatment, the stimulatory effect of GABA (and muscimol) on this binding was significantly reduced by estrogenization. The changes in GABA receptor binding appeared functionally relevant since the elevation of striatal acetylcholine levels normally induced by the peripheral administration of muscimol (5 mg/kg) was significantly lower in estradiol-treated than in control female rats. In contrast to that observed in intact female rats, the implantation of estradiol in hypophysectomized animals did not affect the characteristics of [3H]muscimol binding to hippocampal, striatal and cortical membranes. [3H]muscimol binding was also unchanged in female rats implanted with estradiol and treated chronically with bromocriptine for 3 weeks. Since both hypophysectomy and the chronic administration of bromocriptine suppressed the hyperprolactinemia normally induced by estrogenization, the down-regulation of central GABA receptors very likely involved prolactin in intact animals implanted with 17-beta-estradiol.

Stage- and Region-Specific Expression of Estrogen Receptor α Isoforms during Ontogeny of the Pituitary Gland1

The expression time course of estrogen receptor α (ERα) was analyzed by RT-PCR in fetal and newborn rat pituitaries. In addition to the classical ERα messenger RNA (mRNA), three shorter transcripts were detected and subsequently cloned. Sequence analysis showed that they corresponded to ERα mRNAs lacking exon 3 (which encodes a zinc finger in the DNA-binding domain), exon 4 (which encodes the nuclear localization signal and part of the steroid-binding domain), or both exons 3 and 4. As analyzed by RT-PCR and ribonuclease protection assay, the respective expression levels of the different transcripts varied dramatically during pituitary development; short forms appeared 4 days before full-length ERα mRNA. On Western blots from rat pituitaries of different ages, an ERα-specific antiserum labeled four protein bands of the expected molecular weights, revealing that all four ERα mRNAs are translated in vivo. Immunocytochemistry, using the same antiserum, showed the ERα to be present first in the cytosol of int...

Inhibitory Actions of Acute Estradiol Treatment on the Activity and Quantity of Tyrosine Hydroxylase in the Median Eminence of Ovariectomized Rats

The effects of acute estradiol (E2) treatment on both the activity of tyrosine hydroxylase (TH) in the median eminence and the serum level of prolactin (PRL) were investigated. Twelve‐day‐ovariectomized rats were injected with 17β‐E2 (25μg sc) at 1100 h and sacrificed hourly from 1200 to 2300 h. TH activity was quantified by measuring the amount of exogenous tyrosine converted to L‐DOPA in vitro by aliquots of median eminence homogenates. Serum PRL levels were evaluated by radioimmunoassay. A biphasic response of TH activity to treatment was observed: an immediate decrease occurred—preceding and accompanying a rise in serum PRL—followed by an increase beyond control levels 2 h after the maximal release of PRL. The increase in TH activity could be prevented by the pretreatment of rats with a specific rat PRL antiserum, suggesting it was not due to E2 per se but rather mediated by the E2‐induced PRL elevation. To pin‐point the process underlying the E2‐induced decrease in TH activity, we evaluated the kinetic parameters of TH in the median eminence as well as its quantity (by Western blot analysis) in the median eminence and arcuate nucleus. Finally, we used a sensitive dot‐blot assay to quantify specific TH messenger ribonucleic acid in the arcuate nucleus. The decrease in TH activity after E2 treatment paralleled an immediate decrease in the affinity of TH for its pterin cofactor (6‐MPH4), while Vmax remained unchanged. A decrease in the amount of TH protein in the arcuate nucleus and median eminence as well as in the TH messenger ribonucleic acid level in the arcuate nucleus was also observed, but the latency of these effects precluded a major involvement in the immediate decline of TH activity. Therefore, when observed separately from those of PRL, E2 effects on TH in tuberoinfundibular dopaminergic neurons are clearly inhibitory consisting of a ‘deactivation’ of the enzyme together with a reduction of its synthesis.

Anterior pituitary dopamine receptors during the rat estrous cycle. A detailed analysis of proestrus changes.

The binding of [3H]-spiperone to partially purified rat anterior pituitary plasma membranes was quantified throughout the estrous cycle in relation with the serum prolactin (PRL) levels. Receptor affinity remained unchanged throughout the cycle (Kd:0.08-0.16 nM). The number of receptors was constantly high from diestrus I 10.30 h to proestrus 10.30 h, as long as serum PRL remained low. Between 10.30 and 17.30 h on proestrus, there was a rapid and marked decrease in receptor numbers (Bmax, from 180 +/- 50 to 48 +/- 10 fmol/mg protein: means +/- SEM of three independent determinations), which coincided with the preovulatory PRL surge. Subsequently, [3H]-spiperone binding gradually increased (from 48 +/- 10 to 110 +/- 21 fmol/mg protein, at 21.30 h), while PRL returned to basal levels. On the afternoon of estrus, the number of dopamine receptors was also negatively correlated with the increase in serum PRL. These results show that, while receptor affinity is constant, the number of dopamine binding sites changes significantly and rapidly on the afternoon of proestrus. A rapid decrease in receptor content is temporally correlated with the onset of the preovulatory PRL surge. Therefore, the number of [3H]-spiperone binding sites may be regulated during the estrous cycle. In addition, the proestrus decrease in the number of these receptors might be a decisive component in the initiation or maintenance of the preovulatory PRL surge in the rat.

Two distinct dopamine D2 receptor genes in the European eel: molecular characterization, tissue-specific transcription, and regulation by sex steroids.

Two full-length cDNA encoding putative dopamine D2-like receptors were cloned from the brain of female European eel. The deduced protein sequences, termed D2A- and D2B-R, exhibit closer phylogenetic relationships to vertebrate D2 receptors compared with D3 and D4 or D1 receptors. The two protein sequences share 100% identity within the transmembrane domains containing the highly conserved amino acids involved in dopamine binding. Accordingly, an apparent single population of sites on eel brain membranes bound [(3)H]spiperone, a D2-R-specific antagonist, with a K(d) of 0.2 +/- 0.04 nM. However, D2A- and D2B-R significantly differ within the amino terminus and the third intracellular loop. As analyzed by quantitative PCR and in situ hybridization, both receptor transcripts were found, with different relative abundance, in the majority of brain areas and in the pituitary, whereas in the retina, olfactory epithelium, spinal cord, and adipose tissue, only D2A-R gene was expressed. Because sex steroid hormones recently have been shown to regulate eel brain dopamine systems, we analyzed the effect of steroids on the amount of D2-R transcripts by quantitative PCR and in situ hybridization. In eels treated with testosterone, the gene expression of the D2B-R, but not D2A-R, was increased in a region-dependent manner. The effect of testosterone on D2B-R transcript levels was mimicked by dihydrotestosterone, a nonaromatizable androgen, whereas estradiol had no stimulatory action, evidencing an androgen receptor-dependent mechanism. Although functionality of the two receptors awaits determination of D2-R proteins, we hypothesize that differences in the tissue expression pattern and hormonal regulation of eel D2A- and D2B-R gene expression could represent selective forces that have contributed to the conservation of the duplicated D2-R.

Dopamine inhibits reproduction in female zebrafish (Danio rerio) via three pituitary D2 receptor subtypes.

In many teleosts, the stimulatory control of gonadotrope axis by GnRH is opposed by an inhibitory control by dopamine (DA). The functional importance of this inhibitory pathway differs widely from one teleostean species to another. The zebrafish (Danio rerio) is a teleost fish that has become increasingly popular as an experimental vertebrate model. However, the role of DA in the neuroendocrine control of its reproduction has never been studied. Here the authors evaluated in sexually regressed female zebrafish the effects of in vivo treatments with a DA D2 receptor (D2-R) antagonist domperidone, or a GnRH agonist, alone and in combination, on the pituitary level of FSHβ and LHβ transcripts, the gonadosomatic index, and the ovarian histology. Only the double treatment with GnRH agonist and domperidone could induce an increase in the expression of LHβ, in the gonadosomatic index, and a stimulation of ovarian vitellogenesis, indicating that removal of dopaminergic inhibition is required for the stimulatory action of GnRH and reactivation of ovarian function to occur. Using double immunofluorescent staining on pituitary, the authors showed in this species the innervation of LH cells by tyrosine-hydroxylase immunoreactive fibers. Finally, using in situ hybridization and immunofluorescence, the authors showed that the three subtypes of zebrafish DA D2-R (D2a, D2b, and D2c) were expressed in LH-producing cells, suggesting that they all may be involved in mediating this inhibition. These results show for the first time that, in zebrafish, DA has a direct and potent inhibitory action capable of opposing the stimulatory effect of GnRH in the neuroendocrine control of reproduction.