Christian Saligaut

The Relative Contribution Exerted by AF-1 and AF-2 Transactivation Functions in Estrogen Receptor α Transcriptional Activity Depends upon the Differentiation Stage of the Cell

The activity of the transactivation functions (activation function (AF)-1 and AF-2) of the estrogen receptor α (ERα) is cell-specific. This study aimed to decipher the yet unclear mechanisms involved in this differential cell sensitivity, with particular attention to the specific influence that cell differentiation may have on these processes. Hence, we comparatively evaluated the permissiveness of cells to either ERα AFs in two different cases: (i) a series of cell lines originating from a common tissue, but with distinct differentiation phenotypes; and (ii) cell lines that undergo differentiation processes in culture. These experiments demonstrate that the respective contribution that AF-1 and AF-2 make toward ERα activity varies in a cell differentiation stage-dependent manner. Specifically, whereas AF-1 is the dominant AF involved in ERα transcriptional activity in differentiated cells, the more a cell is de-differentiated the more this cell mediates ERα signaling through AF-2. For instance, AF-2 is the only active AF in cells that have achieved their epithelial-mesenchymal transition. Moreover, the stable expression of a functional ERα in strictly AF-2 permissive cells restores an AF-1-sensitive cell context. These results, together with data obtained in different ERα-positive cell lines tested strongly suggest that the transcriptional activity of ERα relies on its AF-1 in most estrogen target cell types.

Serotonin and dopamine turnover in the female rainbow trout (Oncorhynchus mykiss) brain and pituitary: Changes during the annual reproductive cycle

Brain serotonin (5HT) and dopamine (DA) turnover were studied at various stages of the reproductive cycle of the female rainbow trout by simultaneous determination by HPLC of neurotransmitters and major related metabolites. An increase of 5HT turnover in telencephalon and hypothalamus and a decrease of DA turnover in pituitary and hypothalamus were observed during the periovulatory period. Some changes also occurred during vitellogenesis: decreased 5HT metabolite in telencephalon and preoptic area and increased DA content in preoptic area. These data suggest that physiological fluctuations of biogenic amines could be involved in both ovarian recrudescence and ovulation, with major effects on the hypothalamo-hypophysial complex during the periovulatory period.

Brain aminergic systems in salmonids and other teleosts in relation to steroid feedback and gonadotropin release

Two distinct gonadotropins, GtH I and GtH II have been evidenced in most of the teleost species, especially in salmonids. GtH II release is controlled by a dopaminergic inhibitory tone. An oestrogenic activation of a dopaminergic preoptico-hypophysial pathway should be involved in the rainbow trout. GtH I release depends in salmonids upon a negative oestrogenic feedback, but a control by catecholamines is not evidenced. Some targets of sexual steroids on catecholaminergic neurones in fish are evoked.

Dynamic characteristics of serotonin and dopamine metabolism in the rainbow trout brain: a regional study using liquid chromatography with electrochemical detection

Aminergic metabolism was studied in discrete brain regions of the postovulated female rainbow trout using a liquid chromatography electrochemical detection method. 3 Methoxytyramine (3MT) was the major dopaminergic catabolite, suggesting that catechol-o-methyl transferase is the main dopamine (DA) catabolic enzyme. Two populations of brain regions were found: one with a high DA content and low 3MT/DA ratio (hypothalamus and telencephalon), suggesting that these regions could present a high density of DA perikarya; the other with a high 3MT/DA ratio (pituitary, preoptic area, myelencephalon and optic tectum) suggesting that these regions could present a high density of DA axonal endings. 5 Hydroxytryptamine (5HT) content differed, but an homogeneous distribution of monoamine oxidase was found in different brain regions. High 5HT content was found in the hypothalamus and telencephalon; 5HT was however not detectable in the pituitary.

Localization of tyrosine hydroxylase and its messenger RNA in the brain of rainbow trout by immunocytochemistry and in situ hybridization

This report describes the distribution of tyrosine hydroxylase (TH)‐expressing structures in the brain of rainbow trout (Oncorhynchus mykiss). TH neurons have been localized by the use of two complementary techniques, immunocytochemistry and in situ hybridization of TH messenger RNA. Results obtained from in situ hybridization and immunocytochemistry were in agreement. TH cells were observed in many areas of the brain, with a higher density at the level of the olfactory bulbs where TH‐positive neurons are abundant in the internal cell layer. In the telencephalon, two populations of TH neurons can be distinguished: one group is located in the area ventralis telencephali pars dorsalis, and the other group is located in the area ventralis telencephali pars ventralis and extends laterally in the area ventralis telencephali pars lateralis. Many labeled neurons are also seen in the preoptic area as well as in the hypothalamus, where several clusters of TH‐positive cells are observed. Some of these neurons located in the paraventricular organ grow a short cytoplasmic extension directed to the ventricular wall and are known to be cerebrospinal fluid‐contacting cells. The most caudal TH neurons are observed at the level of the locus caeruleus. At the level of the pituitary, TH‐positive fibers are observed in the neurohypophysis. The TH‐immunoreactive innervation at the level of the pituitary provides a neuroanatomic basis for the effects of dopamine and/or norepinephrine on the release of pituitary hormones in fish. J. Comp. Neurol. 449:374–389, 2002.

Tyrosine hydroxylase activity and dopamine turnover of rainbow trout (Oncorhynchus mykiss) brain: the special status of the hypothalamus

The dynamics of catecholamine (CA)-synthesis enzymes have been poorly studied in fish. Tyrosine hydroxylase (TH), the rate-limiting enzyme of CA synthesis has been only studied inin vitro conditions. In the present report thein vivo CA synthesis and the CA metabolism were studied in different regions of the forebrain of the rainbow trout. Levels of norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and the rate of accumulation of 3,4-dihydroxyphenylalanine (DOPA) were determined by HPLC following a treatment with hydroxybenzylhydrazine (NSD), a potential inhibitor of DOPA decarboxylase. Kinetics of the accumulation of DOPA and of the decline of DOPAC were in agreement with those found in rat, evidencing that the accumulation of DOPA following NSD can be used in trout to quantify thein vivo enzymatic activity of tyrosine hydroxylase. Experiments using treatment with NSD or with methyl-p-tyrosine reached a same conclusion: the DA neuronal activity in trout is much higher than NE neuronal activity. However, the hypothalamus had high DA levelsvs. lowin vitro andin vivo TH activities and exhibited a low CA turnover.

Effects of Estrogens and Endocrine-Disrupting Chemicals on Cell Differentiation–Survival–Proliferation in Brain: Contributions of Neuronal Cell Lines

Estrogens and estrogen receptors (ER) are key actors in the control of differentiation and survival and act on extrareproductive tissues such as brain. Thus, estrogens may display neuritogenic effects during development and neuroprotective effects in the pathophysiological context of brain ischemia and neurodegenerative pathologies like Alzheimers disease or Parkinsons disease. Some of these effects require classical transcriptional “genomic” mechanisms through ER, whereas other effects appear to rely clearly on “membrane-initiated mechanisms” through cytoplasmic signal transduction pathways. Disturbances of these mechanisms by endocrine-disrupting chemicals (EDC) may exert adverse effects on brain. Some EDC may act via ER-independent mechanisms but might cross-react with endogenous estrogen. Other EDC may act through ER-dependent mechanisms and display agonistic/antagonistic estrogenic properties. Because of these potential effects of EDC, it is necessary to establish sensitive cell-based assays to determine EDC effects on brain. In the present review, some effects of estrogens and EDC are described with focus on ER-mediated effects in neuronal cells. Particular attention is given to PC12 cells, an interesting model to study the mechanisms underlying ER-mediated differentiating and neuroprotective effects of estrogens.

Differential regulation of tyrosine hydroxylase and estradiol receptor expression in the rainbow trout brain.

In numerous fish species, dopamine has been found to strongly inhibit gonadotropin release. Among the enzymes that regulate dopamine turnover, tyrosine hydroxylase (TH), the rate-limiting anabolic enzyme, could be a target for endocrine feedback regulation. Since dopamine turnover is stimulated by estradiol in rainbow trout, we have investigated the effect of estradiol on TH and estradiol receptor expression. In situ hybridization was used to quantify mRNA levels in the brain of ovariectomized female rainbow trout implanted or not with estradiol pellets. We demonstrated that preoptic TH and estradiol receptor mRNA levels are greatly decreased by gonadectomy during vitellogenesis. For TH expression, this effect was reversed in part by estradiol supplementation. We have also confirmed the existence of an inhibitory gonadal feedback on FSH secretion, mediated by estradiol. The stimulating effect of estradiol on TH expression found in this study could be a pathway involved in gonadal feedback on gonadotropin release.

Absence of direct regulation of prolactin cells by estradiol-17β in rainbow trout (Oncorhynchus mykiss)

The effects of estradiol-17 beta (E2) implants on plasma prolactin (PRL) concentrations, pituitary PRL content and pituitary PRL mRNA levels were examined in rainbow trout (Oncorhynchus mykiss). Intact immature fish treated with 1 mg estradiol-17 beta did not show significant changes in both PRL mRNA levels and pituitary PRL content after 3 days of treatment. In a similar experiment, no changes were observed in plasma PRL levels followed during 7 days. Similarly, lack of estradiol-17 beta effect on plasma PRL levels and on final PRL pituitary content was observed in ovariectomized female rainbow trout treated during 48 days with 25 mg estradiol-17 beta and in mature male fish over a 3-week treatment period. Localization of estradiol receptor (ER) mRNAs in the pituitary was carried out by Northern blot analysis using a full-length rainbow trout estrogen receptor (rtER) cDNA as a probe. The rostral pars distalis of the pituitary which contained mostly PRL cells showed the lower amount of rtER mRNA when compared to other parts of the pituitary. Moreover, two mRNAs of different size (3.5 and 1.4 kb) were detected in different parts of the pituitary. Further hybridization experiments using probes containing part of the rtER cDNA (E domain or C and D domains) indicated that the small-sized mRNA (1.4 kb) probably encodes a truncated ER protein lacking hormone binding domain or an ER-related protein. Thus, only the 3.56 kb mRNA appeared to be involved in the regulation of pituitary function by estradiol. In situ hybridization analysis allowed a more precise localization of this rtER mRNA in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the potential activity of major dietary compounds as selective estrogen receptor modulators in two distinct cell models for proliferation and differentiation

ABSTRACT Estrogen receptors (ERs) &agr; and &bgr; are distributed in most tissues of women and men. ERs are bound by estradiol (E2), a natural hormone, and mediate the pleiotropic and tissue‐specific effects of E2, such as proliferation of breast epithelial cells or protection and differentiation of neuronal cells. Numerous environmental molecules, called endocrine disrupting compounds, also interact with ERs. Phytoestrogens belong to this large family and are considered potent therapeutic molecules that act through their selective estrogen receptor modulator (SERM) activity. Using breast cancer cell lines as a model of estrogen‐dependent proliferation and a stably ER‐expressing PC12 cell line as a model of neuronal differentiating cells, we studied the SERM activity of major dietary compounds, such as apigenin, liquiritigenin, daidzein, genistein, coumestrol, resveratrol and zearalenone. The ability of these compounds to induce ER‐transactivation and breast cancer cell proliferation and enhance Nerve Growth Factor (NGF) ‐induced neuritogenesis was assessed. Surprisingly, although all compounds were able to activate the ER through an estrogen responsive element reporter gene, they showed differential activity toward proliferation or differentiation. Apigenin and resveratrol showed a partial or no proliferative effect on breast cancer cells but fully contributed to the neuritogenesis effect of NGF. However, daidzein and zearalenone showed full effects on cellular proliferation but did not induce cellular differentiation. In summary, our results suggest that the therapeutic potential of phytoestrogens can diverge depending on the molecule and the phenotype considered. Hence, apigenin and resveratrol might be used in the development of therapeutics for breast cancer and brain diseases. HIGHLIGHTSSERM activity of dietary compounds on proliferation and differentiation is studied.All the dietary compounds tested transactivate estrogen receptors.Apigenin and resveratrol could be good candidates for future therapeutics.Daidzein and zearalenone are to be avoided to maintain human health.