Nuclear Progesterone Receptors Are Up-Regulated by Estrogens in Neurons and Radial Glial Progenitors in the Brain of Zebrafish

In rodents, there is increasing evidence that nuclear progesterone receptors are transiently expressed in many regions of the developing brain, notably outside the hypothalamus. This suggests that progesterone and/or its metabolites could be involved in functions not related to reproduction, particularly in neurodevelopment. In this context, the adult fish brain is of particular interest, as it exhibits constant growth and high neurogenic activity that is supported by radial glia progenitors. However, although synthesis of neuroprogestagens has been documented recently in the brain of zebrafish, information on the presence of progesterone receptors is very limited. In zebrafish, a single nuclear progesterone receptor (pgr) has been cloned and characterized. Here, we demonstrate that this pgr is widely distributed in all regions of the zebrafish brain. Interestingly, we show that Pgr is strongly expressed in radial glial cells and more weakly in neurons. Finally, we present evidence, based on quantitative PCR and immunohistochemistry, that nuclear progesterone receptor mRNA and proteins are upregulated by estrogens in the brain of adult zebrafish. These data document for the first time the finding that radial glial cells are preferential targets for peripheral progestagens and/or neuroprogestagens. Given the crucial roles of radial glial cells in adult neurogenesis, the potential effects of progestagens on their activity and the fate of daughter cells require thorough investigation

Interference with zinc homeostasis and oxidative stress induction as probable mechanisms for cadmium-induced embryo-toxicity in zebrafish

International audienceThe present study was conducted to provide new insights into the mechanisms that may be responsible for cadmium (Cd)-induced toxicity in zebrafish larvae as well as the role of the trace element zinc (Zn) in reversing Cd harmful effects. For this purpose, zebrafish eggs were exposed to Cd or/and Zn for 96 h. The effects on morphological aspect; mortality rate; Cd, Zn, and metallothionein (MT) levels; oxidative stress biomarkers; as well as molecular expression of some genes involved in Zn metabolism (Zn-MT, ZIP10, and ZnT1) and in antioxidant defense system (Cu/Zn-SOD, CAT and GPx) were examined. Our results showed that Cd toxicity was exerted, initially, by an interference with Zn metabolism. Thus, Cd was able to modify the expression of the corresponding genes so as to ensure its intracellular accumulation at the expense of Zn, causing its depletion. An oxidative stress was then generated, representing the second mode of Cd action which resulted in developmental anomalies and subsequently mortality. Interestingly, significant corrections have been noted following Zn supplementation based, essentially, on its ability to interact with the toxic metal. The increases of Zn bioavailability, the improvement of the oxidative status, as well as changes in Zn transporter expression profile are part of the protection mechanisms. The decrease of Cd-induced MTs after Zn supplement, both at the protein and the mRNA level, suggests that the protection provided by Zn is ensured through mechanisms not involving MT expression but which rather depend on the oxidative status

Expression of kisspeptins and kiss receptors suggests a large range of functions for kisspeptin systems in the brain of the european sea bass

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts. © 2013 Escobar et al.Peer Reviewe

Nuclear progesterone receptors are up-regulated by estrogens in neurons and radial glial progenitors in the brain of zebrafish

In rodents, there is increasing evidence that nuclear progesterone receptors are transiently expressed in many regions of the developing brain, notably outside the hypothalamus. This suggests that progesterone and/or its metabolites could be involved in functions not related to reproduction, particularly in neurodevelopment. In this context, the adult fish brain is of particular interest, as it exhibits constant growth and high neurogenic activity that is supported by radial glia progenitors. However, although synthesis of neuroprogestagens has been documented recently in the brain of zebrafish, information on the presence of progesterone receptors is very limited. In zebrafish, a single nuclear progesterone receptor (pgr) has been cloned and characterized. Here, we demonstrate that this pgr is widely distributed in all regions of the zebrafish brain. Interestingly, we show that Pgr is strongly expressed in radial glial cells and more weakly in neurons. Finally, we present evidence, based on quantitative PCR and immunohistochemistry, that nuclear progesterone receptor mRNA and proteins are upregulated by estrogens in the brain of adult zebrafish. These data document for the first time the finding that radial glial cells are preferential targets for peripheral progestagens and/or neuroprogestagens. Given the crucial roles of radial glial cells in adult neurogenesis, the potential effects of progestagens on their activity and the fate of daughter cells require thorough investigation

5-hydroxymethylcytosine marks postmitotic neural cells in the adult and developing vertebrate central nervous system

International audienceThe epigenetic mark 5-hydroxymethylcytosine (5hmC) is a cytosine modification that is abundant in the central nervous system of mammals and which results from 5-methylcytosine oxidation by TET enzymes. Such a mark is suggested to play key roles in the regulation of chromatin structure and gene expression. However, its precise functions still remain poorly understood and information about its distribution in non-mammalian species is still lacking. Here, the distribution of 5hmC was investigated in the brain of adult zebrafish, African claw frog, and mouse in a comparative manner. We show that zebrafish neurons are endowed with high levels of 5hmC, whereas quiescent or proliferative neural progenitors show low to undetectable levels of the modified cytosine. In the brain of larval and juvenile Xenopus, 5hmC is also detected in neurons, while ventricular proliferative cells do not display this epigenetic mark. Similarly, 5hmC is enriched in neurons compared to neural progenitors of the ventricular zone in the mouse developing cortex. Interestingly, 5hmC colocalized with the methylated DNA binding protein MeCP2 and with the active chromatin histone modification H3K4me2 in mouse neurons. Taken together, our results show an evolutionarily conserved cerebral distribution of 5hmC between fish and tetrapods and reinforce the idea that 5hmC fulfills major functions in the control of chromatin activity in vertebrate neurons. J. Comp. Neurol. 525:478-497, 2017. © 2016 Wiley Periodicals, Inc

Are kisspeptins driving the onset of puberty in fish?

Trabajo presentado en el AQUA2012 ("Aquaculture Europe" y "World Aqauculture"), celebrado en Praga (República Checa), del 1 al 5 de septiembre de 2012Reproduction is the critical physiological function assuring the survival of the species. Most fish are ectotherm animals, whose reproduction, as well as other rhythmic biological processes, are influenced by the environmental conditions. As a result, to obtain new offspring at the most favourable time, fish need to synchronize their hormonal cascades though the brain-pituitary-gonad axis, with seasonal and daily photoperiod cycles. To date, the mechanisms controlling the reproductive axis have not been completely unravelled. In fact before the discovery of kisspeptins, GnRH was acknowledged in all vertebrates as the major initiator of the hormonal cascade modulating the reproductive axis. Nowadays, kisspeptins have become fascinating actors in the neuroendocrine regulation of reproduction at least in mammals, acting upstream of the hypophysiotrophic GnRH neurons. Several evidences suggest kisspeptins as the responsible of the onset of puberty in mammals. Recent phylogenetical analyses provided evidences that the number of kiss genes and kiss receptors varies from one class of vertebrate to the other. Most fish have two kiss (kiss1 and kiss2) and two kiss receptors genes (kiss1r and kiss2r)

BDNF Expression in Larval and Adult Zebrafish Brain: Distribution and Cell Identification

International audienceBrain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has emerged as an active mediator in many essential functions in the central nervous system of mammals. BDNF plays significant roles in neurogenesis, neuronal maturation and/or synaptic plasticity and is involved in cognitive functions such as learning and memory. Despite the vast literature present in mammals, studies devoted to BDNF in the brain of other animal models are scarse. Zebrafish is a teleost fish widely known for developmental genetic studies and is emerging as model for translational neuroscience research. In addition, its brain shows many sites of adult neurogenesis allowing higher regenerative properties after traumatic injuries. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the distribution of bdnf mRNAs in the larval and adult zebrafish brain and to characterize the phenotype of cells expressing bdnf mRNAs by means of double staining studies. Our results showed that bdnf mRNAs were widely expressed in the brain of 7 days old larvae and throughout the whole brain of mature female and male zebrafish. In adults, bdnf mRNAs were mainly observed in the dorsal telencephalon, preoptic area, dorsal thalamus, posterior tuberculum, hypothalamus, synencephalon, optic tectum and medulla oblongata. By combining immunohistochemistry with in situ hybridization, we showed that bdnf mRNAs were never expressed by radial glial cells or proliferating cells. By contrast, bdnf transcripts were expressed in cells with neuronal phenotype in all brain regions investigated. Our results provide the first demonstration that the brain of zebrafish expresses bdnf mRNAs in neurons and open new fields of research on the role of the BDNF factor in brain mechanisms in normal and brain repairs situations

Expression of kisspeptins in the brain and pituitary of the european sea bass (Dicentrarchus labrax)

Kisspeptins are now considered key players in the neuroendocrine control of puberty and reproduction, at least in mammals. Most teleosts have two kiss genes, kiss1 and kiss2, but their sites of expression are still poorly documented. As a first step in investigating the role of kisspeptins in the European sea bass, a perciform fish, we studied the distribution of kiss1 and kiss2-expressing cells in the brain of males and females undergoing their first sexual maturation. Animals were examined at early and late in the reproductive season. We also examined the putative expression of estrogen receptors in kiss-expressing cells and, finally, we investigated whether kisspeptins are expressed in the pituitary gland. We show that kiss1-expressing cells were consistently detected in the habenula and, in mature males and females, in the rostral mediobasal hypothalamus. In both sexes, kiss2-expressing cells were consistently detected at the level of the preoptic area, but the main kiss2 mRNA-positive population was observed in the dorsal hypothalamus, above and under the lateral recess. No obvious sexual differences in kiss1 and kiss2 mRNA expression were detected. Additional studies based on confocal imaging clearly showed that most kiss1 mRNA-containing cells of the mediobasal hypothalamus strongly express ERα and slightly express ERβ2. At the pituitary level, both sexes exhibited kiss1 mRNA expression in most FSHβ-positive cells and never in LHβ-positive cells. © 2012 Wiley Periodicals, Inc.Grant sponsor: EU Project LIFECYCLE; Grant number: FP7-222719-1 (to O.K., S.Z.); Grant sponsor: JAE-Predoc (CSIC, Spain; to S.E.).Peer Reviewe

Progenitor radial cells and neurogenesis in pejerrey fish forebrain.

International audienceThe central nervous system of adult teleost fish is peculiar because of the following features: (1) the persistence of radial glial cells, (2) an important neurogenic activity and (3) a high aromatase expression by radial cells. In this study, the proliferative zones of the forebrain were described using bromodeoxyuridine (BrdU) treatment in the brain of the pejerrey, an Acanthopterygian teleost fish. These cells were shown to have morphological and immunoreactive characteristics of radial cells and to express aromatase. Three different progenitor populations were identified based on the mobility and proliferation capacity 6 weeks after BrdU treatment: transit amplifying progenitors, slowly proliferating stem cells, and cells remaining in the proliferative zones showing no signs of mitotic activity. The proliferative cells were always located in the ventricular zone and were never observed in the brain parenchyma; however, 3 weeks later they were found away from these proliferative zones and displayed acetylated tubulin immunoreactivity. Other BrdU-positive cells showed astrocyte morphology and were immunoreactive to the S100 glial marker. These results show that in this fish, radial cells are true progenitors generating neurons and possibly astrocytes

Sexual dimorphism in the brain aromatase expression and activity, and in the central expression of other steroidogenic enzymes during the period of sex differentiation in monosex rainbow trout populations.

International audienceUsing genetic monosex male and female rainbow trout populations, the potential sex differences in the central expression of estrogen receptors (esr1, esr2a, esr2b), brain aromatase (cyp19a1b) and some other steroidogenic enzymes was studied over the period of sex differentiation (from 35 to 63 dpf: days post-fertilization) using quantitative polymerase chain reaction (q-PCR). In addition, aromatase activity was evaluated during this period. The results indicated that brain aromatase (cyp19a1b) expression and activity showed a clear and significant sexually dimorphic pattern with higher levels in male brain between 35 and 53 dpf before the time of gonad morphological differentiation. At that time the expression of a key enzyme involved in the conversion of cholesterol into steroids, the cyp11a1 (p450scc), as well as the estrogen receptors were also sexually dimorphic. The dimorphism was lost from 56 dpf onwards. Transcription factors such as nr5a1b (sf1) and nr0b1 (dax1), but not foxl2a were also higher in males than in females. These results demonstrate that, before or during the early period of morphological gonad differentiation, the brain exhibits a clear sexual dimorphism with respect to the expression and activity of aromatase as well as of certain enzymes and factors involved in steroid synthesis as p450scc and sf1. The results suggest a higher potentiality to produce estrogens by male brains during sex differentiation time