Defining Global Neuroendocrine Gene Expression Patterns Associated with Reproductive Seasonality in Fish

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. gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays.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

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 ERβ2 than zfERα, suggesting that it may act through an ERβ-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

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 signalling 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 signalling. Sexually mature, pre-spawning (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 remodelling. 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 signalling (STAT3, SP1, SMAD, Jun/Fos), immune response (IL6, IL1β, TNFs, cytokine, NF-κB), and cell proliferation and growth (IGF1, TGFβ1). 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 signalling 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

Metabolite production during transformation of 2,4,6-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 14C-TNT studies revealed 4.14% mineralization after an incubation period of 163 days with growing cells.NRC publication: Ye

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.NRC publication: Ye

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.NRC publication: Ye

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

International audienceSex 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 6 h to waterborne 17,20 beta P (4.3 nM) and PGF(2 alpha) (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 PGF(2 alpha), treatment identified 71 unique transcripts that were differentially expressed (q < 5%; 67 up, 4 down). Functional annotation of these regulated genes indicates that PGF(2 alpha),1 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 PGF(2 alpha), 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 PGF(2 alpha), but not 17,20 beta P, whereas cGnRH expression was increased by 17,20 beta P but not PGF2a. In contrast, both pheromones increase the expression of glutamate (GluR2a, NR2A) and gamma-aminobutyric acid (GABA(A) gamma 2) receptor subunit mRNAs. Milt release and rapid modulation of neuronal transcription are part of the response of males to female sex pheromones. (C) 2013 Elsevier Inc. All rights reserved

The goldfish (Carassius auratus) as a model for neuroendocrine signaling. Molecular and Cellular Endocrinology 293

We would like to dedicate this article to the memory of Professor Richard E. Peter, a true pioneer in neuroendocrine signaling. He passed away suddenly and prematurely in March 2007. We respected his vision and benefited from his friendship and encouragement. Keywords: Goldfish Reproduction Neurotransmitters Brain Microarray Isotocin Activin a b s t r a c t 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 gonadotropinreleasing hormone and a multitude of classical neurotransmitters and neuropeptides is opposed by the potent inhibitory actions of dopamine. The stimulatory actions of ␥-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