Dataset Information

Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation.

ABSTRACT: BACKGROUND: The vertebrate brain plays a critical role in the regulation of sexual maturation and reproduction by integrating environmental information with developmental and endocrine status. The European eel Anguilla anguilla is an important species in which to better understand the neuroendocrine factors that control reproduction because it is an endangered species, has a complex life cycle that includes two extreme long distance migrations with both freshwater and seawater stages and because it occupies a key position within the teleost phylogeny. At present, mature eels have never been caught in the wild and little is known about most aspects of reproduction in A. anguilla. The goal of this study was to identify genes that may be involved in sexual maturation in experimentally matured eels. For this, we used microarrays to compare the gene expression profiles of sexually mature to immature males. RESULTS: Using a false discovery rate of 0.05, a total of 1,497 differentially expressed genes were identified. Of this set, 991 were expressed at higher levels in brains (forebrain and midbrain) of mature males while 506 were expressed at lower levels relative to brains of immature males. The set of up-regulated genes includes genes involved in neuroendocrine processes, cell-cell signaling, neurogenesis and development. Interestingly, while genes involved in immune system function were down-regulated in the brains of mature males, changes in the expression levels of several receptors and channels were observed suggesting that some rewiring is occurring in the brain at sexual maturity. CONCLUSIONS: This study shows that the brains of eels undergo major changes at the molecular level at sexual maturity that may include re-organization at the cellular level. Here, we have defined a set of genes that help to understand the molecular mechanisms controlling reproduction in eels. Some of these genes have previously described functions while many others have roles that have yet to be characterized in a reproductive context. Since most of the genes examined here have orthologs in other vertebrates, the results of this study will contribute to the body of knowledge concerning reproduction in vertebrates as well as to an improved understanding of eel biology.

SUBMITTER: Churcher AM

PROVIDER: S-EPMC4175612 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation.

Churcher Allison M AM Pujolar Jose Martin JM Milan Massimo M Hubbard Peter C PC Martins Rute S T RS Saraiva João L JL Huertas Mar M Bargelloni Luca L Patarnello Tomaso T Marino Ilaria A M IA Zane Lorenzo L Canário Adelino V M AV

BMC genomics 20140917

<h4>Background</h4>The vertebrate brain plays a critical role in the regulation of sexual maturation and reproduction by integrating environmental information with developmental and endocrine status. The European eel Anguilla anguilla is an important species in which to better understand the neuroendocrine factors that control reproduction because it is an endangered species, has a complex life cycle that includes two extreme long distance migrations with both freshwater and seawater stages and ...[more]

PMID: 25230743

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Project description:Gene expression analyses have been performed on brain tissue of sexually mature and immature males using microarrays. 60 eels were transferred to two independent temperature controlled recirculation water units connected to two 500 L cylindro-conical tanks (30 fish per tank) where the fish were acclimated to seawater (35 PSU salinity) over a 2 week period.Eel males in one of the seawater units were injected intramuscularly every week over a 140 day period with 2000 IU hCG/kg (human chorionic gonadotropin, Sigma–Aldrich Chemical) dissolved in 0.9% saline to induce sexual maturation. Eel males in the other recirculation unit were injected weekly over the same period with 0.9% NaCl (vehicle).At the end of the experiment, eels were anesthetized in a solution of 0.1mg/L tricaine methanesulfonate (MS-222, Sigma Aldrich) and blood samples collected into heparinized syringes. Tissues (brain, including the olfactory bulbs, telencephalon, diencephalon and mesencephalon, and gonads) were collected from sexually immature (n=12) and sexually mature males (n=12).RNA was extracted from brain tissue of a total of 24 (12 mature and 12 immature) males. For each individual, RNA was extracted separately for olfactory bulb, telencephalon and diencephalon. Because RNA concentrations of olfactory bulb, telencephalon and diencephalon were below the amount needed for microarray analysis, a separate experiment for each part of the brain was not possible. Therefore, we combined equal concentrations of olfactory bulb, telencephalon and diencephalon as a single brain sample for each individual.Microarray analysis was conducted using an European eel-specific array consisting of a total of 14,913 probes based on a large collection of high-throughput transcriptomic sequences (Pujolar et al. 2012). Probe sequences and further details on the microarray platform can be found on the GEO database under accession number GPL15124. Data was normalized using a quantile normalization procedure using R (http://www.r-project.org)

Project description:Gene expression analyses have been performed on liver tissue of sexually mature and immature males using microarrays. 60 eels were transferred to two independent temperature controlled recirculation water units connected to two 500 L cylindro-conical tanks (30 fish per tank) where the fish were acclimated to seawater (35 PSU salinity) over a 2 week period.Eel males in one of the seawater units were injected intramuscularly every week over a 140 day period with 2000 IU hCG/kg (human chorionic gonadotropin, Sigma–Aldrich Chemical) dissolved in 0.9% saline to induce sexual maturation. Eel males in the other recirculation unit were injected weekly over the same period with 0.9% NaCl (vehicle).At the end of the experiment, eels were anesthetized in a solution of 0.1mg/L tricaine methanesulfonate (MS-222, Sigma Aldrich) and blood samples collected into heparinized syringes. Tissues (brain, liver and gonads) were collected from sexually immature (n=12) and sexually mature males (n=12).RNeasy Mini Kit (Qiagen) was used to extract RNA from the livers. Tissue samples were pooled and, therefore, each of the biological replicates (n= 4 sexually immature, n=4 sexually mature) contains tissue from three fish. Total RNA concentration was determined using the Nanodrop ND-100 spectrophotometer (NanoDrop Technologies) and sample integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Microarray analysis was conducted using an European eel-specific array consisting of a total of 14,913 probes based on a large collection of high-throughput transcriptomic sequences (Pujolar et al. 2012). Probe sequences and further details on the microarray platform can be found on the GEO database under accession number GPL15124. Data was normalized using a quantile normalization procedure using R (http://www.r-project.org) A comparative analysis of gene expression was conducted between sexually mature and immature males. Sample labelling and hybridization were conducted following the details in Pujolar et al. (2012). Hybridized slides were scanned at 5 µm resolution using an Agilent DNA microarray scanner. Slides were scanned at two different sensitivity levels (XDR Hi 100% and XDR Lo 10%) and the two linked images generated were analysed together. Data were extracted and background subtracted using the standard procedure in Agilent Feature Extraction (FE) software v. 9.5.1. Hybridization success was evaluated using flag values, excluding those intensities not equal to 1.

Project description:Many enigmas surround different aspects of freshwater eel biology and life cycle. In the same way different hypothesis about why eels are disappearing from European continental waters have been proposed. One such proposal defends that poor fat accumulation of eels, due to pollution in continental waters, might be stopping eels from reproductive migration to the Sargasso Sea. Thus, habitat deterioration could be blamed for the decline in the catchment potential of the European rivers. In this context, and with the aim to study the mode of action of environmentally relevant chemicals in eels, the multi tissue transcriptome was sequenced (454 Titanium Roche) in order to design a high density custom oligonucleotide microarray (eArray, Agilent). To validate this tool a laboratory experiment was carried to analyze the gene trasncrition profiles related to chemical compounds released from pulp and paper mills; 100 μg/L mercury and 150 μg/L β-sitosterol (only Hg data is presented here). 20 yellow European eel (Anguilla anguilla) elvers (11.7±5.35 g) obtained from a local farm (Acuivas SL, Usurbil, Gipuzkoa) were exposed for 3, 6 and 9 days. Pyrosequencing allowed the design and construction of a 60K microarray platform containing 3923 gene signatures identified through BlastN analysis and 7212 sequences annotated through BlastX coupled to Blast2Go analysis. Additional 226 sequences were incorporated from NCBI databases and 3551 from the information available in EeelBase in 2011. Two probes were generated per sequence and they were spotted twice in the array. Hepatic gene expression profiling of the exposed eels indicated that Hg significantly down-regulated (LIMMA, adj. p<0.005) only gene signatures related to selenoprotein W-1 (SeW), something typically described in mammals exposed to methyl-mercury. Increasing the adj. p value to <0.05, 116 genes were significantly regulated (38 down-regulated and 79 up-regulated). Among them, we found additional selenoproteins such as ROS metabolism related genes; glutathione peroxidases (gpx1 & gpx4b) and thioredoxine which were up-regulated. In addition, complement system genes (C3 & C4b) were also up-regulated. Studying enriched Go pathways (p<0.005) and in relation with lipid homeostasis we observed that the following pathways were enriched after exposure to Hg: fatty acids degradation and metabolism of arachidonic acid, linoleic acid, ether lipids, alpha linolenic acid, and glycerophospholipids. In addition, among the top 10 significantly enriched KEGG pathways, p53 signalling, apoptosis, and MAPK signalling were present, suggesting possible effects on cell cycle regulation. In summary, transcriptome pyrosequencing and subsequently designed microarray provided the molecular tools to successfully study the gene transcription profiles of toxic chemical compounds such as Hg in European eel tissues. In addition to study the molecular modes of action of specific chemical compounds, the developed gene expression microarray will be useful in active monitoring of the quality of freshwater environments using caged sentinel eels. This study was funded by Basque Government (SAIOTEK S-PE09UN32; Consolidated Research Groups IT810-13) and UPV/EHU (UFI 11/37). Technical and human support provided by SGIker (UPV/EHU) is acknowledged.

Project description:Assisted propagation of the European eel will lead to a closed production cycle supplying the aquaculture industry with juvenile glass eels. Females require long-term weekly treatment with pituitary extract (PE), which is stressful and causes abnormalities in oogenesis. We tested the effects of 17α-methyltestosterone (17 MT), as potent androgen activating the androgen receptor, and 17β-estradiol (E2), as an inducer of vitellogenesis, to shorten the duration of PE treatment.Four groups of feminized eels were subjected to a simulated migration and subsequent injection with implants containing 17 MT (17 MT-group), E2 (E2-group) or 17 MT plus E2 (17 MT + E2-group) to test for synergistic effects, or without any steroids as controls (C-group). The effects of a 2-months treatment were investigated by determining the eye index (EI), hepatosomatic and gonadosomatic index (HSI and GSI, respectively), plasma steroid concentrations by liquid chromatography mass spectrometry (LCMS), gonadal histology, expression of androgen receptors a and b (ara, arb); estrogen receptor 1 (esr1); FSH receptor (fshr); vitellogenin receptor (vtgr) and aromatase (cyp19), and the required number of weekly PE injections to fully mature. For many parameters, both the 17 MT and E2 groups showed an increase vs. controls, with the 17 MT + E2 group showing a synergistic effect, as seen for EI, GSI (3.4 for 17 MT and for E2, 6.6 for 17 MT + E2), oocyte diameter and ara, arb and esr1 expression. Concentrations of almost all focal steroids decreased with simulated migration and steroid treatment. Only eels of the 17 MT-group showed increased expression of cyp19 and of fshr, while fshr expression increased 44-fold in the 17 MT + E2 group, highlighting that co-implantation is most effective in raising fshr mRNA levels. Specific for eels of the E2 groups were vitellogenesis-associated changes such as an increase of HSI, plasma E2, and presence of yolk in the oocytes. Steroid treatments reduced the duration of PE treatment, again synergistically for co-implantation. In conclusion, E2 is necessary to start vitellogenesis, but 17 MT has specific effects on cyp19 and fshr expression. The combination is necessary for synergistic effects and as such, steroid implants could be applied in assisted reproduction protocols for European eel to improve oocyte quality leading to the production of more vital larvae.

Dataset Information

Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation.

Publications

Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation.

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