Rapid modulation of gene expression profiles in the telencephalon of male goldfish following exposure to waterborne sex pheromones.

Sex pheromones rapidly affect endocrine physiology and behaviour, but little is known about their effects on gene expression in the neural tissues that mediate olfactory processing. In this study, we exposed male goldfish for 6h to waterborne 17,20ßP (4.3 nM) and PGF2α (3 nM), the main pre-ovulatory and post-ovulatory pheromones, respectively. Both treatments elevated milt volume (P=0.001). Microarray analysis of male telencephalon following PGF2α treatment identified 71 unique transcripts that were differentially expressed (q<5%; 67 up, 4 down). Functional annotation of these regulated genes indicates that PGF2α pheromone exposure affects diverse biological processes including nervous system functions, energy metabolism, cholesterol/lipoprotein transport, translational regulation, transcription and chromatin remodelling, protein processing, cytoskeletal organization, and signalling. By using real-time RT-PCR, we further validated three candidate genes, ependymin-II, calmodulin-A and aldolase C, which exhibited 3-5-fold increase in expression following PGF2α exposure. Expression levels of some other genes that are thought to be important for reproduction were also determined using real-time RT-PCR. Expression of sGnRH was increased by PGF2α, but not 17,20ßP, whereas cGnRH expression was increased by 17,20ßP but not PGF2α. In contrast, both pheromones increase the expression of glutamate (GluR2a, NR2A) and γ-aminobutyric acid (GABAA γ2) receptor subunit mRNAs. Milt release and rapid modulation of neuronal transcription are part of the response of males to female sex pheromones.

Meta-type analysis of dopaminergic effects on gene expression in the neuroendocrine brain of female goldfish.

Dopamine (DA) is a major neurotransmitter important for neuroendocrine control and recent studies have described genomic signaling pathways activated and inhibited by DA agonists and antagonists in the goldfish brain. Here we perform a meta-type analysis using microarray datasets from experiments conducted with female goldfish to characterize the gene expression responses that underlie dopaminergic signaling. Sexually mature, pre-spawning [gonadosomatic index (GSI) = 4.5 ± 1.3%] or sexually regressing (GSI = 3 ± 0.4%) female goldfish (15-40 g) injected intraperitoneally with either SKF 38393, LY 171555, SCH 23390, sulpiride, or a combination of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and α-methyl-p-tyrosine. Microarray meta-type analysis identified 268 genes in the telencephalon and hypothalamus as having reciprocal (i.e., opposite between agonism and antagonism/depletion) fold change responses, suggesting that these transcripts are likely targets for DA-mediated regulation. Noteworthy genes included ependymin, vimentin, and aromatase, genes that support the significance of DA in neuronal plasticity and tissue remodeling. Sub-network enrichment analysis (SNEA) was used to identify common gene regulators and binding proteins associated with the differentially expressed genes mediated by DA. SNEA analysis identified gene expression targets that were related to three major categories that included cell signaling (STAT3, SP1, SMAD, Jun/Fos), immune response (IL-6, IL-1ß, TNFs, cytokine, NF-κB), and cell proliferation and growth (IGF1, TGFß1). These gene networks are also known to be associated with neurodegenerative disorders such as Parkinsons' disease, well-known to be associated with loss of dopaminergic neurons. This study identifies genes and networks that underlie DA signaling in the vertebrate CNS and provides targets that may be key neuroendocrine regulators. The results provide a foundation for future work on dopaminergic regulation of gene expression in fish model systems.

Quantitative proteomics in teleost fish: insights and challenges for neuroendocrine and neurotoxicology research.

Neuroendocrine systems integrate both extrinsic and intrinsic signals to regulate virtually all aspects of an animal's physiology. In aquatic toxicology, studies have shown that pollutants are capable of disrupting the neuroendocrine system of teleost fish, and many chemicals found in the environment can also have a neurotoxic mode of action. Omics approaches are now used to better understand cell signaling cascades underlying fish neurophysiology and the control of pituitary hormone release, in addition to identifying adverse effects of pollutants in the teleostean central nervous system. For example, both high throughput genomics and proteomic investigations of molecular signaling cascades for both neurotransmitter and nuclear receptor agonists/antagonists have been reported. This review highlights recent studies that have utilized quantitative proteomics methods such as 2D differential in-gel electrophoresis (DIGE) and isobaric tagging for relative and absolute quantitation (iTRAQ) in neuroendocrine regions and uses these examples to demonstrate the challenges of using proteomics in neuroendocrinology and neurotoxicology research. To begin to characterize the teleost neuroproteome, we functionally annotated 623 unique proteins found in the fish hypothalamus and telencephalon. These proteins have roles in biological processes that include synaptic transmission, ATP production, receptor activity, cell structure and integrity, and stress responses. The biological processes most represented by proteins detected in the teleost neuroendocrine brain included transport (8.4%), metabolic process (5.5%), and glycolysis (4.8%). We provide an example of using sub-network enrichment analysis (SNEA) to identify protein networks in the fish hypothalamus in response to dopamine receptor signaling. Dopamine signaling altered the abundance of proteins that are binding partners of microfilaments, integrins, and intermediate filaments, consistent with data suggesting dopaminergic regulation of neuronal stability and structure. Lastly, for fish neuroendocrine studies using both high-throughput genomics and proteomics, we compare gene and protein relationships in the hypothalamus and demonstrate that correlation is often poor for single time point experiments. These studies highlight the need for additional time course analyses to better understand gene-protein relationships and adverse outcome pathways. This is important if both transcriptomics and proteomics are to be used together to investigate neuroendocrine signaling pathways or as bio-monitoring tools in ecotoxicology.

Evidence for alternative splicing of a dopamine D2 receptor in a teleost.

Previous attempts at identifying an alternatively spliced dopamine (DA) D2 receptor in teleosts have proven unsuccessful. We provide evidence of a splicing event of a goldfish D2 (gfD2b1) receptor in the neuroendocrine brain of adult goldfish that produces a spliced short isoform (gfD2b1S). We also identify an additional novel D2b paralog (gfD2b2) that does not appear to be alternatively spliced in adult fish during the reproductive cycle. Relatively high mRNA levels of gfD2b1S were observed in the neuroendocrine brain and pituitary of sexually immature fish compared with sexually regressing fish. Real-time RT-PCR revealed that intraperitoneal injection of either SCH 23390 or sulpiride-D1- or D2-specific antagonists, respectively-decreased mRNA levels of gfD2b1S by 3.9-fold without affecting the unspliced isoforms. We suggest that the expression of the spliced D2 receptor modulates the inhibitory tone of DA throughout the reproductive cycle. The deduced amino acid sequence of gfD2b1S lacks 29 amino acids in the same region as the short isoform of mammalian D2. We propose that the gfD2b1S splice variant is the teleost ortholog of mammalian D2S. The hypothesis that D2 receptor splicing is a relatively recent innovation in higher tetrapods is not supported by our results.

Lumiestrone is Photochemically Derived from Estrone and may be Released to the Environment without Detection.

Endocrine disrupting chemicals are adversely affecting the reproductive health and metabolic status of aquatic vertebrates. Estrone is often the dominant natural estrogen in urban sewage, yet little is known about its environmental fate and biological effects. Increased use of UV-B radiation for effluent treatments, and exposure of effluents to sunlight in holding ponds led us to examine the effects of environmentally relevant levels of UV-B radiation on the photodegradation potential of estrone. Surprisingly, UV-B-mediated degradation leads to the photoproduction of lumiestrone, a little known 13α-epimer form of estrone. We show for the first time that lumiestrone possesses novel biological activity. In vivo treatment with estrone stimulated estrogen receptor (ER) α mRNA production in the male goldfish liver, whereas lumiestrone was without effect, suggesting a total loss of estrogenicity. In contrast, results from in vitro ER-dependent reporter gene assays indicate that lumiestrone showed relatively higher estrogenic potency with the zebrafish ERß2 than zfERα, suggesting that it may act through an ERß-selectivity. Lumiestrone also activated human ERs. Microarray analysis of male goldfish liver following in vivo treatments showed that lumiestrone respectively up- and down-regulated 20 and 69 mRNAs, which was indicative of metabolic upsets and endocrine activities. As a photodegradation product from a common estrogen of both human and farm animal origin, lumiestrone is present in sewage effluent, is produced from estrone upon exposure to natural sunlight and should be considered as a new environmental contaminant.

Rapid dopaminergic modulation of the fish hypothalamic transcriptome and proteome.

BACKGROUND: Dopamine (DA) is a major neurotransmitter playing an important role in the regulation of vertebrate reproduction. We developed a novel method for the comparison of transcriptomic and proteomic data obtained from in vivo experiments designed to study the neuroendocrine actions of DA. METHODS AND FINDINGS: Female goldfish were injected (i.p.) with DA agonists (D1-specific; SKF 38393, or D2-specific; LY 171555) and sacrificed after 5 h. Serum LH levels were reduced by 57% and 75% by SKF 38393 and LY 171555, respectively, indicating that the treatments produced physiologically relevant responses in vivo. Bioinformatic strategies and a ray-finned fish database were established for microarray and iTRAQ proteomic analysis of the hypothalamus, revealing a total of 3088 mRNAs and 42 proteins as being differentially regulated by the treatments. Twenty one proteins and mRNAs corresponding to these proteins appeared on both lists. Many of the mRNAs and proteins affected by the treatments were grouped into the Gene Ontology categorizations of protein complex, signal transduction, response to stimulus, and regulation of cellular processes. There was a 57% and 14% directional agreement between the differentially-regulated mRNAs and proteins for SKF 38393 and LY 171555, respectively. CONCLUSIONS: The results demonstrate the applicability of advanced high-throughput genomic and proteomic analyses in an amendable well-studied teleost model species whose genome has yet to be sequenced. We demonstrate that DA rapidly regulates multiple hypothalamic pathways and processes that are also known to be involved in pathologies of the central nervous system.

Gene expression profiling of the fathead minnow (Pimephales promelas) neuroendocrine brain in response to pulp and paper mill effluents.

The toxicity of pulp and paper mill effluents (PPMEs) has been greatly decreased, yet some continue to negatively affect fish reproduction. We hypothesized that PPMEs are affecting the brain resulting in decreased reproductive performance. Our goal was to use gene expression profiling to test the hypothesis that PPMEs are having an effect on neural systems in the fathead minnow (FHM; Pimephales promelas) in vivo. Sexually mature male and female FHM were exposed to 100% final biotreated PPMEs from 5 different sources for 5 days. Using an oligo-array (15K genes) we examined the effect of PPMEs on gene expression in the hypothalamus of female fish. We validated selected genes (cholecystokinin, RevErbbeta2, and urotensin I) that were identified by microarray analysis using real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We compared the FHM microarray dataset to multiple microarray datasets from experiments conducted with goldfish injected with different dopaminergic pharmaceuticals to examine whether PPMEs could be affecting the dopamine system. Exposure of FHM to PPMEs resulted in varying degrees of spawning inhibition. Microarray analysis revealed surprisingly few genes in the brain that were commonly affected by the different PPMEs. Real-time RT-PCR confirmed the changes in expression for cholecystokinin, RevErbbeta2, and urotensin I. Comparison of the FHM and goldfish microarray datasets suggest that some PPMEs may be acting on the dopamine system. We show that PPMEs are neuroactive in fish and may be acting through some of the pathways in a manner similar to dopamine.

Profiling neuroendocrine gene expression changes following fadrozole-induced estrogen decline in the female goldfish.

Teleost fish represent unique models to study the role of neuroestrogens because of the extremely high activity of brain aromatase (AroB; the product of cyp19a1b). Aromatase respectively converts androstenedione and testosterone to estrone and 17beta-estradiol (E2). Specific inhibition of aromatase activity by fadrozole has been shown to impair estrogen production and influence neuroendocrine and reproductive functions in fish, amphibians, and rodents. However, very few studies have identified the global transcriptomic response to fadrozole-induced decline of estrogens in a physiological context. In our study, sexually mature prespawning female goldfish were exposed to fadrozole (50 mcirog/l) in March and April when goldfish have the highest AroB activity and maximal gonadal size. Fadrozole treatment significantly decreased serum E2 levels (4.7 times lower; P = 0.027) and depressed AroB mRNA expression threefold in both the telencephalon (P = 0.021) and the hypothalamus (P = 0.006). Microarray expression profiling of the telencephalon identified 98 differentially expressed genes after fadrozole treatment (q value <0.05). Some of these genes have shown previously to be estrogen responsive in either fish or other species, including rat, mouse, and human. Gene ontology analysis together with functional annotations revealed several regulatory themes for physiological estrogen action in fish brain that include the regulation of calcium signaling pathway and autoregulation of estrogen receptor action. Real-time PCR verified microarray data for decreased (activin-betaA) or increased (calmodulin, ornithine decarboxylase 1) mRNA expression. These data have implications for our understanding of estrogen actions in the adult vertebrate brain.

Defining global neuroendocrine gene expression patterns associated with reproductive seasonality in fish.

BACKGROUND: Many vertebrates, including the goldfish, exhibit seasonal reproductive rhythms, which are a result of interactions between external environmental stimuli and internal endocrine systems in the hypothalamo-pituitary-gonadal axis. While it is long believed that differential expression of neuroendocrine genes contributes to establishing seasonal reproductive rhythms, no systems-level investigation has yet been conducted. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, by analyzing multiple female goldfish brain microarray datasets, we have characterized global gene expression patterns for a seasonal cycle. A core set of genes (873 genes) in the hypothalamus were identified to be differentially expressed between May, August and December, which correspond to physiologically distinct stages that are sexually mature (prespawning), sexual regression, and early gonadal redevelopment, respectively. Expression changes of these genes are also shared by another brain region, the telencephalon, as revealed by multivariate analysis. More importantly, by examining one dataset obtained from fish in October who were kept under long-daylength photoperiod (16 h) typical of the springtime breeding season (May), we observed that the expression of identified genes appears regulated by photoperiod, a major factor controlling vertebrate reproductive cyclicity. Gene ontology analysis revealed that hormone genes and genes functionally involved in G-protein coupled receptor signaling pathway and transmission of nerve impulses are significantly enriched in an expression pattern, whose transition is located between prespawning and sexually regressed stages. The existence of seasonal expression patterns was verified for several genes including isotocin, ependymin II, GABA(A) gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays. CONCLUSIONS/SIGNIFICANCE: Using both theoretical and experimental strategies, we report for the first time global gene expression patterns throughout a breeding season which may account for dynamic neuroendocrine regulation of seasonal reproductive development.

Mimicking the natural doping of migrant sandpipers in sedentary quails: effects of dietary n-3 fatty acids on muscle membranes and PPAR expression.

Wild semipalmated sandpipers (Calidris pusilla) eat n-3 fatty acids to prime their muscles for long migrations. Sedentary bobwhite quails (Colinus virginianus) were used as a model to investigate the mechanisms for this natural doping. Our goal was to characterize the stimulating effects of n-3 eicosapentaenoic acid (EPA) and n-3 docosahexaenoic acid (DHA) on oxidative capacity. Mechanisms linked to changes in membrane composition and in gene expression for peroxisome proliferator-activated receptors (PPAR) were investigated. Dietary n-3 fatty acids stimulated the activities of oxidative enzymes by 58-90% (citrate synthase, cytochrome oxidase, carnitine palmitoyl transferase and hydroxyacyl dehydrogenase), and sedentary quails showed the same changes in membrane composition as sandpipers preparing for migration. EPA and DHA have the same doping effect. The substitution of n-6 arachidonic acid by n-3 EPA in membrane phospholipids plays an important role in mediating the metabolic effects of the diet, but results provide no significant support for the involvement of PPARs (as determined by changes in gene expression). The fatty acid composition of mitochondrial membranes and sarcoplasmic reticulum can be monitored by measuring total muscle phospholipids because all phospholipids are equally affected by diet. Only extreme regimes of endurance training can lead to increments in oxidative capacity matching those induced here by diet. As they prepare for long migrations, semipalmated sandpipers improve their physical fitness by eating! Choosing n-3 fatty acid doping over endurance training strikes us as a better strategy to boost aerobic capacity when rapid storage of energy is critical.

The goldfish (Carassius auratus) as a model for neuroendocrine signaling.

Goldfish (Carassius auratus) are excellent model organisms for the neuroendocrine signaling and the regulation of reproduction in vertebrates. Goldfish also serve as useful model organisms in numerous other fields. In contrast to mammals, teleost fish do not have a median eminence; the anterior pituitary is innervated by numerous neuronal cell types and thus, pituitary hormone release is directly regulated. Here we briefly describe the neuroendocrine control of luteinizing hormone. Stimulation by gonadotropin-releasing hormone and a multitude of classical neurotransmitters and neuropeptides is opposed by the potent inhibitory actions of dopamine. The stimulatory actions of gamma-aminobutyric acid and serotonin are also discussed. We will focus on the development of a cDNA microarray composed of carp and goldfish sequences which has allowed us to examine neurotransmitter-regulated gene expression in the neuroendocrine brain and to investigate potential genomic interactions between these key neurotransmitter systems. We observed that isotocin (fish homologue of oxytocin) and activins are regulated by multiple neurotransmitters, which is discussed in light of their roles in reproduction in other species. We have also found that many novel and uncharacterized goldfish expressed sequence tags in the brain are also regulated by neurotransmitters. Their sites of production and whether they play a role in neuroendocrine signaling and control of reproduction remain to be determined. The transcriptomic tools developed to study reproduction could also be used to advance our understanding of neuroendocrine-immune interactions and the relationship between growth and food intake in fish.

Microarray analysis in the zebrafish (Danio rerio) liver and telencephalon after exposure to low concentration of 17alpha-ethinylestradiol.

17alpha-ethinylestradiol (EE2) is detected in sewage effluent at concentrations that can disrupt normal reproductive function in fish. The objectives of this study were to identify novel genomic responses to EE2 exposure using microarray and real-time RT-PCR analysis in the liver and telencephalon of male zebrafish. Zebrafish were exposed to an environmentally relevant nominal concentration of 10ng/L EE2 for a 21-day period. In the liver, common biomarkers for estrogenic exposure such as vitellogenin 1 and 3 (vtg1; vtg3), estrogen receptor alpha (esr1), and apolipoprotein A1 (apoA1) mRNA were identified by microarray analysis as being differentially regulated. Real-time RT-PCR confirmed that vtg1 was induced approximately 700-fold, vtg3 was induced approximately 100-fold and esr1 was induced approximately 20-fold. As determined by microarray analysis, ATPase Na+/K+ alpha 1a.4 (atp1a1a.4) and ATPase Na+/K+ beta 1a (atp1b1a) mRNA were down-regulated in the liver. Gene ontology (GO) analysis revealed that there were common biological processes and molecular functions regulated by EE2 in both tissues (e.g. electron transport and cell communication) but there were tissue specific changes in gene categories. For example, genes involved in protein metabolism, carbohydrate metabolism were down-regulated in the liver but were induced in the telencephalon. This study demonstrates that (1) tissues exhibit different gene responses to low EE2 exposure; (2) there are pronounced genomic effects in the liver and (3) multi-tissue gene profiling is needed to improve understanding of the effects of human pharmaceuticals on aquatic organisms.

Metabolite production during transformation of 2,4,6-trinitrotoluene (TNT) by a mixed culture acclimated and maintained on crude oil-containing media.

Metabolites formed during 2,4,6-trinitrotoluene (TNT) removal by a mixed bacterial culture (acclimated and maintained on crude oil-containing medium and capable of high rates of TNT removal) were characterized. In resting cell experiments in the absence of glucose, 46.2 mg/l TNT were removed in 171 h (87.5% removal), with a combined total formation of 7.7 mg/l amino-4,6-dinitrotoluene (ADNT) and 0.3 mg/l 4,4'-azoxytetranitrotoluene and 2',4-azoxytetranitrotoluene, leaving 70% of the initial TNT unaccounted for. In the presence of glucose, resting cells removed 45.4 mg/l TNT in 49 h (95.5% removal), with 9.1 mg/l ADNT and 2.4 mg/l azoxy compounds being produced, leaving 70.3% of the TNT unaccounted for. Growing cells (glucose present) were capable of removing 44.2 mg/l TNT within 21 h (97.9% removal), with the concomitant formation of 1.8 mg/l ADNTs and 2.2 mg/l azoxy compounds. Denitrated TNT in the form of 2,6-dinitrotoluene was also produced in growing cells with a maximum amount of 1.31 mg/l after 28 h, followed by a slight decrease with time, leaving 88.5% of the initial TNT unaccounted for after 171 h. Radiolabeled (14)C-TNT studies revealed 4.14% mineralization after an incubation period of 163 days with growing cells.

High TNT-transforming activity by a mixed culture acclimated and maintained on crude-oil-containing media.

A mixed microbial culture originating from a petroleum-contaminated site and maintained on crude oil exhibited high 2,4,6-trinitrotoluene (TNT) transformation activity. Cultivation of the mixed culture in glucose-containing medium for 29 h resulted in almost complete transformation of 100 ppm TNT. TNT transformation was observed with both growing and resting cells. With subculturing, it was found that TNT could support growth of the mixed culture when supplied as sole carbon source, sole nitrogen source, or sole carbon and nitrogen source. The finding that a mixed microbial culture maintained on crude oil exhibited high TNT transformation activity without prior subculture on TNT-containing media is novel and may have potential practical applications in the bioremediation of munitions-contaminated soil and wastewater.