BACKGROUND
animal reproduction depends on the precise timing and coordination of behavioral and physiological processes.
for successful reproduction to occur, animals need to reach the appropriate developmental stage, locate and attract mates and respond appropriately to reproductively relevant signals.
the accuracy of each aspect of reproduction is essential to ensure that genetic material is passed on to the next generation.
a major function of the brain is to establish the appropriate developmental and endocrine status for reproduction, and then to co-ordinate this with aspects of behavior and sexual maturation.
in vertebrates, this coordination is established through the hypothalamus and pituitary gland  <cit> .
the pituitary gland secretes follicle-stimulating hormone  and luteinizing hormone  which are gonadotropins that act on the gonads to induce sex steroid synthesis and promote gametogenesis.
the pituitary gonadotropes are in the vicinity of neuron endings that release neuropeptides and neurotransmitters, such as gonadotropin releasing-hormone  and dopamine, which stimulate and inhibit gonadotropin secretion respectively.
following gonadotropin release, sex steroids produced by gonad feedback to the brain and pituitary provide information about reproductive status  <cit> .
brain and pituitary gene expression changes that are associated with reproduction have been observed in several teleosts.
these include changes in the pattern of gnrh expression at maturation in masu salmon oncorhynchus masou
<cit> , european sea bass dicentrarchus labrax
<cit> , red seabream pagrus major
<cit> , goldfish  <cit>  and european eel anguilla anguilla
<cit> .
changes in the expression levels of lhβ and fshβ gene subunits have also been observed in the pituitary of several species, including european sea bass and european eel  <cit> .
additionally, changes in the levels of kisspeptin system genes have also been observed in several teleosts  <cit> , as have changes in aromatase expression and activity  <cit> .
many of these gene expression changes occur in areas of the brain that are associated with the detection and processing of sensory information.
due to its complex life cycle and extreme long distance migrations, a. anguilla is an intriguing model in which to investigate reproduction related changes in the brain.
the european eel is believed to spawn in a single location in the sargasso sea  <cit> .
after spawning, larvae are transported by surface currents in the gulf stream to the shores of europe and north africa.
when larvae reach the continental shelf, they undergo metamorphosis into glass eels that migrate into fresh, brackish and coastal waters as yellow eels.
after a highly variable feeding period of 5– <dig> years, yellow eels metamorphose into silver eels that migrate from their widely dispersed foraging areas to the common spawning ground in the sargasso sea.
this migration is approximately  <dig> - <dig>  km long.
the changes that occur during metamorphosis into silver eels include an increase in eye size, a change in body coloration and a regression of the digestive system.
eels do not feed during the transatlantic adult migration and rely exclusively on fat content for energy.
after reaching the spawning grounds in the sargasso sea, eels reproduce once then die shortly thereafter  <cit> .
a. anguilla is also an important species to better understand the molecular aspects of reproduction because it is an endangered species, is of interest for aquaculture purposes and occupies a key position within the teleost phylogeny.
while much progress has been made over the last century, many aspects of eel reproduction remain poorly understood, including the physiological and molecular mechanisms responsible for sexual maturation and the coordination of reproductive events  <cit> .
european eels are sexually immature when they start their migration across the atlantic to the spawning grounds; it is not clear if they mature sexually during migration or after they arrive at sargasso sea.
maturation experiments through hormonal induction have shown dramatic changes at the physiological level  as well as changes in secondary sexual characteristics .
final maturation in eels does not appear to be triggered by swimming alone and is likely influenced by exposure to environmental cues as the eels swim across the atlantic  <cit> .
in addition, reproductive pheromones are believed to have a role in the regulation of final maturation and reproduction in eels  <cit> .
if sexual maturation in a. anguilla is triggered by environmental signals, then signal detection is expected to occur through sensory pathways and signal processing is expected to occur in the brain before the neuroendocrine response is activated.
at present, mature adult eels have never been caught in the wild and eels do not mature in captivity without treatment with gonadotropic hormones.
the inability to reach sexual maturation in captivity is due to a lack in sufficient pituitary gonadotropin production  <cit> .
in eels, maturation is under double neuroendocrine control which occurs in some teleosts  <cit>  and which can be reversed by gonadotropic hormone treatments  <cit> .
the dopaminergic inhibitory process leads to insufficient gnrh stimulation, and to the inhibition of pituitary gonadotropes fsh and lh, which exert direct and indirect control on gonad development by acting on steroid metabolism  <cit> .
the goal of the present gene profiling study was to identify changes in the brain that are involved in the coordination of reproductive events in the european eel, including those involved in neuroendocrine regulation of gametogenesis.
more specifically, genes involved in sexual maturation and reproduction are expected to be expressed at higher levels in the brain of sexually mature males compared to immature males.
additionally, since the coordination of reproductive events is influenced by environmental cues that must be integrated with the physiological and developmental status of the animal, it is expected that changes at the molecular level occur in the areas associated with the processing of sensory signals .
RESULTS
using a false discovery rate of  <dig> , a total of  <dig>  differentially expressed genes were identified.
of this set,  <dig> were expressed at higher levels in brains  of sexually mature males while  <dig> were expressed at lower levels relative to brains of sexually immature males.
when searched against the refseq proteins using blastx  <cit> , these transcripts retrieved  <dig> and  <dig> unique sequences, respectively.
functional annotation using david
to obtain a list of gene identifiers that could be used for the functional analysis of differentially expressed genes, the a. anguilla transcriptome assembly contigs that were used to design the microarray were used in a blastx search of swissprot proteins.
the result of this search revealed that  <dig>  of the  <dig>  transcripts had alignments with e-values of  <dig>  or less in swissprot.
when the contigs without hits in swissprot were searched against refseq proteins and then the nr database, alignments for additional  <dig>  and  <dig> sequences were retrieved, respectively.
using this approach, sequence identifiers for a total of  <dig>  contigs were retrieved.
for the analyses with david  <cit> , only differentially expressed transcripts with blast alignment e-values of  <dig>  or less were considered.
functional annotation analysis with david uncovered a number of significantly enriched terms within the list of up- and down-regulated genes .
figure  <dig> details the proportion of over-represented terms belonging to the following go categories: biological process, cellular component and molecular function.
for each of the three main go categories, terms were assigned to subcategories.figure  <dig>
proportion of differentially expressed terms belonging to the go categories biological process, molecular function and cellular component.
enriched terms from the lists of significantly up and down-regulated genes  were identified using david.
for each of the three main go categories, terms were assigned to subcategories.
cluster analysis with david identified eight significantly enriched clusters of up-regulated genes and five significantly enriched clusters of down-regulated genes .
for this analysis, only clusters with ease scores greater than or equal to  <dig>  were considered.
clusters of up-regulated genes include many that are involved in dna binding, calcium singling and nervous system development and differentiation.
clusters of down-regulated genes include genes for rna processing, immune system function and nucleotide binding.
differential expression: keyword analysis
to identify specific sets of differentially expressed genes, the protein descriptions obtained from the blastx search of the eukaryotic proteins in refseq were used.
in total,  <dig>  out of  <dig>  assembly contigs showed alignments with an e-value score of  <dig>  or better in the refseq proteins.
the complete list of differentially expressed genes can be found in additional file 4: table s <dig>  among this set, we identified  <dig> neuroendocrine genes that are differentially expressed in the brains of sexually mature male eels .table  <dig>
differentially expressed neuroendocrine genes in the brains of sexually mature and immature male eels
genes that have positive direction values were up-regulated in the brains of sexually mature male eels in comparison to sexually immature male eels while those that are negative were down-regulated in this group.
gene descriptions are based on blastx hits in refseq proteins.
a false discovery rate of  <dig>  was used to identify differentially expressed genes.
the results of the david analysis suggest that some re-organization of signaling pathways in the brain occurs at sexual maturation.
therefore, it seemed likely that there would also be changes in the expression levels for genes that are involved directly in cell-cell communication such as receptors and channels.
we therefore searched for up and down regulated channels and g protein-coupled receptors .
seven gpcrs were differentially expressed.
four of these belong to the glutamate family of gpcrs  <cit> , one belongs to the rhodopsin-like family and two belong to the secretin family .
one of the glutamate family receptors, an extracellular calcium sensing receptor-like gene, was expressed at higher levels in the brains of sexually mature males.
the other three were expressed at lower levels in the brains of sexually mature males compared with sexually immature males.
the receptor from the rhodopsin-like gpcr family was the serotonin receptor  which was expressed at higher levels in the brains of sexually mature males than in immature males .
the gpcr 64-like gene and the gpcr 125-like gene belong to the secretin family and were expressed at higher levels in the brains of sexually mature males.
additionally, there were eleven differentially expressed channels  seven of which were expressed at higher levels in the brains of sexually mature males in comparison to the brains of immature males.table  <dig>
differentially expressed gpcrs and channels in the brains of sexually mature male eels
g protein-coupled receptors  were assigned to families based on the grafs classification system  <cit> .
genes that have positive direction values were up-regulated in the brains of sexually mature male eels in comparison to immature males while those that are negative were down-regulated in this group.
gene descriptions are based on blastx hits in refseq proteins.
a false discovery rate of  <dig>  was used to identify differentially expressed genes.
quantitative polymerase chain reaction
eight differentially expressed genes were chosen for quantitative polymerase chain  using the same rna samples that were used for the microarray .
this list includes: galanin peptide , glutamate decarboxylase  <dig> , corticotropin releasing factor binding protein , c-type natriuretic peptide , gamma-aminobutyric acid receptor subunit δ , neuropeptide y , mammalian ependymin-related protein  and the iroquois-class homeodomain protein  <dig> .
the tata box binding protein  and elongation factor 1-alpha  <dig>  were used as controls.figure  <dig>
comparison of qpcr results with the microarray results for eight differentially expressed genes.
the expression levels for eight differentially expressed genes were determined by quantitative polymerase chain reaction  with the same rna pools that were used for the microarray.
these include the following differentially expressed genes: galanin peptide , glutamate decarboxylase  <dig> , corticotropin releasing factor binding protein , c-type natriuretic peptide , gamma-aminobutyric acid receptor subunit δ , neuropeptide y , mammalian ependymin-related protein  and the iroquois-class homeodomain protein  <dig> .
bars represent mean fold changes, error bars represent standard deviations and * represents significant  increases in the level of expression in the brains of sexually mature male eels in comparison to immature male eels.
to verify that the fold changes observed in the microarray experiments correlated with the fold changes observed in the qpcr experiments, spearman’s rank order correlation was used.
the results of this test indicate a positive relationship exists between the fold changes observed in the microarray and qpcr experiments .
the qpcr expression levels between immature and mature samples were further evaluated using student's t-tests.
galp, crfpb, cnp, merp and irx <dig> were expressed at significantly higher levels in the brains of mature males compared with mature  while the differences in expression of npy, gad <dig> and gabdr were below the statistical significance level.
discussion
this study shows that major changes in the gene expression profiles occur in the brains of male european eels at sexual maturity.
this shift includes an increase in the expression levels of genes involved in cell signaling, including receptor and channels, as well as genes involved in development and differentiation.
several neuroendocrine genes were also expressed at higher levels in the brains of mature males in comparison to the brains of immature males.
these genes are likely involved in gonadal maturation and development and act through the brain-pituitary-gonad axis.
genes involved in immune system function were down-regulated in the brains of sexually mature males, suggesting that a re-allocation of resources occurs at this stage.
interestingly, the expression levels of several receptors and channels change at sexual maturity suggesting that some neuronal rewiring occurs at this stage.
more transcripts for neuropeptide y  were observed in the brains of mature male eels in comparison to immature males.
npy is a short peptide whose expression pattern has been described in the brains of several teleosts.
for example, in goldfish, npy expression has been detected in the forebrain including the telencephalon, preoptic area, the olfactory bulb and thalamic regions  <cit> .
in the japanese eel anguilla japonica, npy is highly expressed in the telencephalon, optic tectum thalamus and hypothalamus  <cit> .
npy stimulates lh release in fish  <cit> , has a role in growth hormone  and gonadotropin hormone  secretion  and may coordinate growth, feeding and reproduction in fish  <cit> .
in goldfish, fasting and exposure to sex steroids increases npy mrna levels in the brain  <cit> .
furthermore, the npy response in goldfish depends on reproductive status  <cit> .
food deprivation in a. japonica also causes increases of npy mrna levels in the brain  <cit> .
changes in feeding behavior and regulation of energy expenditure are expected to accompany the maturation process in fish, particularly in species such as a. anguilla that allocate a lot of resources to reproduction  <cit> .
since the european eel does not feed at sexual maturity, it is possible that increased levels of npy are related to nutritional and developmental status.
genes involved in gamma-aminobutyric acid  signaling were also expressed at higher levels in the brains of mature males.
for example, glutamate decarboxylase  <dig> , an enzyme involved in the production of gaba from l-glutamic acid, was more abundant in the brains of sexually mature than immature males.
gaba is a neurotransmitter involved in the control of pituitary hormone secretion in teleosts  <cit> .
gaba has a stimulatory effect on lh by enhancing gnrh release and inhibiting dopamine turnover  <cit> .
the gaba a receptor subunit δ  was expressed at a higher level in the brains of sexually mature male eels than in immature males.
gaba a receptors are hetero-pentamer ligand gated channels that are gated by gaba binding.
it appears that different combinations of receptor subunits may confer functional specificity  <cit> .
in goldfish, four gaba a subunits  were expressed at higher levels in the brains of sexually mature females  <cit> .
similarly, the gaba a receptor γ  <dig> subunit was expressed at higher levels in the brains of goldfish exposed to prostaglandin-f2α  than in controls  <cit> .
in the current study, the δ subunit was up-regulated in sexually mature males; this is intriguing given that the δ subunit appears to be involved in neurosteroid modulation of the gaba a receptor  <cit> .
in the brains of sexually mature males, the 5-hydroxytryptamine  receptor 1b was expressed at a higher level than in immature males.
the brain serotonergic system is one of the mechanisms used to cope with the dramatic changes during maturation.
this system plays an important role in a complex neuroendocrine loop that functions in homeostasis and acclimation during physiological or environmental challenges  <cit> .
corticotropin releasing factor binding protein  was also expressed at a higher level in the brains of mature males than in immature males.
crfbp has an inhibitory role on crf which is a regulator of stress responses  <cit> .
in fish, acute stress results in an increase in crfbp expression in the hypothalamus  <cit> .
for european eels, reproduction is a stressful event because the spawning migration journey is very long .
in addition, it is presumably challenging for both sexes to coordinate the temporal and spatial aspects of spawning in the vast sargasso sea which is  <dig>  km wide and  <dig>  km long.
at the mature stage, european eels are likely to reduce their threshold for assessing risks and threats since they have a single opportunity to reproduce.
therefore the observed increases in crfbp may be part of the stress response and of changes in behavior that are associated with reproduction.
an increase in the levels of mammalian ependymin-related protein  in the brains of mature males was observed.
merps belong to a phylogenetically diverse clade that is related to ependymins  <cit>  which are glycoproteins that are found in abundance in the cerebrospinal fluid of fish.
ependymins are involved in synaptic changes during memory consolidation and regeneration of the optic nerve .
changes in the levels of ependymin-ii expression have been observed in the hypothalamus in goldfish at breeding season  <cit>  and in the telencephalon following exposure to a female pheromone  <cit> .
several genes involved in calmodulin  signaling were differentially expressed in the brains of sexually mature males .
for example, calm is involved in calcium signaling and nervous system function including memory consolidation  <cit>  and was up-regulated in the brains of sexually mature male eels.
in goldfish, calm levels also change with reproductive status.
during breeding season, calm and calcium/calmodulin-dependant protein kinase  <dig>  levels are highest in the hypothalamus in female goldfish  <cit>  and calm levels are highest in the telencephalon of males following exposure to pgf2α
<cit> .
sexual maturation alters the eel’s internal state and changes in the way the animal relates to its environment.
in the european eel, the memory circuitry is expected to be important for reproduction due to the long migration across the atlantic to the sargasso sea.
though cam signaling genes are likely involved in many processes in the brains of mature eels, it is possible that the changes observed here are also related to memory.
the microarray results showed a high positive correlation with those obtained in selected genes by qpcr thus providing a global validation of the microarray results.
npy, gad <dig> and gabdr did not seem to follow the same pattern; this could be related to the low expression levels of these genes  or to the existence of alternative transcripts that are differentially detected by the two methods.
however, these possibilities were not further investigated.
recent studies in fish show that specific cells in the brain respond to light and exert neuroendocrine control of testis development  <cit> .
in a. anguilla, behavioral evidence indicates that photoreceptor sites that are independent of the pineal exist in the brain  <cit> .
eels become more sexually mature after months of migration in deep waters while only surfacing at night.
in a. anguilla, spawning is believed to occur in the top  <dig> meters  <cit>  where there is more light than in deeper water.
the results of the current study show that several genes with roles in photosignal transduction and visual system development were up-regulated in the brains of mature males.
this list includes retinal rod rhodopsin-sensitive cgmp 3' 5'-cyclic phosphodiesterase subunit γ and cyclic nucleotide-gated cation channel α- <dig> which is involved in olfactory and visual signal transduction.
it also includes the calcium binding protein  <dig>  which has an important role in synaptic function in photoreceptor cells  <cit>  and the iroquois-class homeodomain protein .
irx <dig> is a transcription factor that is involved in cone cell differentiation and retinal, craniofacial and gonad development  <cit> .
the transcription factor hes <dig> was also up-regulated in the brains of sexually mature males.
hes <dig> belongs to a family of genes that have roles in cell differentiation and has a role in retina development  <cit> .
while it is possible that these genes have other roles in the brains of mature males, an intriguing avenue for future research would be to determine whether there is a link between neurohormone systems, gonad development and photoreceptors in the brains of a. anguilla like there is in masu salmon  <cit> .
several genes involved in neurogenesis, cellular differentiation and development were up-regulated in the brain of sexually mature males .
this set includes genes from the go categories such as go: <dig> , go: <dig> , go: <dig>  and go: <dig> .
in addition, many differentially expressed ion channels and gpcrs were differentially expressed  which suggests that some re-organization and/or re-growth occurs at maturation.
the calcium-activated potassium channel would tend to hyperpolarize  neurons in response to neuromodulators/neurotransmitters.
down-regulation of these channels suggests a less responsive, more ‘hard-wired’ brain, which may be advantageous for certain aspects of reproduction.
gnrh, aromatase, kisspeptin system genes and sex steroid receptors are also known to have roles in reproduction.
the microarray included a probe for one isoform of gnrh <dig> and no differences in expression were observed between immature and mature males.
the array did not contain probes for other gnrh genes or isoforms.
although gnrh <dig> controls gonadotrophin secretion in the pituitary  <cit> , the fact that the fish were artificially induced to mature using human chorionic gonadotrophin suggests that no positive or negative feedback mechanisms towards gnrh <dig> were occurring at the time of sampling.
the microarray also contained probes for transcripts that are similar to the estrogen-related receptor beta like  <dig> and the androgen receptor beta and no significant differences in expression were detected.
kisspeptin system genes and aromatase were not analyzed in this survey.
at present, mature eels have never been caught in the wild and the only way to obtain sexually mature animals is through artificial induction of maturation.
it is therefore not clear to what extent changes that occur at the molecular level in the brain are the result of specific gonadal feedback mechanisms.
the eyes and the olfactory epithelium change with sexual maturation in a. anguilla and the american eel anguilla rostrata
<cit>  suggesting that there is a change in the use of sensory modalities with reproduction.
as mentioned previously, the data from this study suggest that the forebrains of male eels undergo some sort of rewiring at sexual maturation.
as the areas of the brain tested here are associated with the processing and conversion of sensory input to physiological responses, it is possible that changes in the brain at sexual maturity are influenced by changes in sensory system requirements that occur at reproduction.
CONCLUSIONS
in this study, we used nearly  <dig>  microarray probes to identify gene expression changes in the brains of eels that occur at sexual maturation.
of the set of differentially expressed genes,  <dig> were expressed at higher levels in sexually mature males while  <dig> were expressed at lower levels than in immature males.
this finding shows that the brains of eels undergo major changes at the molecular level during reproduction.
the set of up-regulated genes includes genes that are involved in neuroendocrine processes which likely act through the brain-pituitary-gonad axis.
it also includes genes involved in cell-cell signaling, neurogenesis and development indicating that the brain is undergoing developmental changes that are associated with maturation and reproduction.
a major advantage to the approach employed here is that it allows many genes to be tested at the same time.
it also allows for the identification of genes not previously known to be involved in particular processes such as reproduction.
consequently, some of these differentially expressed genes identified here have roles that have been described in other studies while others have roles that have yet to be characterized.
furthermore, since the majority of the genes tested here have orthologs in other vertebrates, the results from this study can be used to understand reproduction across a diversity of vertebrate species.
most importantly however, is that we have defined a set of genes that appear to be important for sexual maturation in a. anguilla.
these findings therefore contribute to the body of knowledge concerning eel biology and will, in the future, contribute to aquaculture and conservation efforts.

 animal reproduction depends on the precise timing and coordination of behavioral and physiological processes. for successful reproduction to occur, animals need to reach the appropriate developmental stage, locate and attract mates and respond appropriately to reproductively relevant signals. the accuracy of each aspect of reproduction is essential to ensure that genetic material is passed on to the next generation. a major function of the brain is to establish the appropriate developmental and endocrine status for reproduction, and then to co-ordinate this with aspects of behavior and sexual maturation. in vertebrates, this coordination is established through the hypothalamus and pituitary gland. the pituitary gland secretes follicle-stimulating hormone and luteinizing hormone which are gonadotropins that act on the gonads to induce sex steroid synthesis and promote gametogenesis. the pituitary gonadotropes are in the vicinity of neuron endings that release neuropeptides and neurotransmitters, such as gonadotropin releasing-hormone and dopamine, which stimulate and inhibit gonadotropin secretion respectively. following gonadotropin release, sex steroids produced by gonad feedback to the brain and pituitary provide information about reproductive status. brain and pituitary gene expression changes that are associated with reproduction have been observed in several teleosts. these include changes in the pattern of gnrh expression at maturation in masu salmon oncorhynchus masou, european sea bass dicentrarchus labrax, red seabream pagrus major, goldfish and european eel anguilla anguilla. changes in the expression levels of lhβ and fshβ gene subunits have also been observed in the pituitary of several species, including european sea bass and european eel. additionally, changes in the levels of kisspeptin system genes have also been observed in several teleosts, as have changes in aromatase expression and activity. many of these gene expression changes occur in areas of the brain that are associated with the detection and processing of sensory information. due to its complex life cycle and extreme long distance migrations, a. anguilla is an intriguing model in which to investigate reproduction related changes in the brain. the european eel is believed to spawn in a single location in the sargasso sea. after spawning, larvae are transported by surface currents in the gulf stream to the shores of europe and north africa. when larvae reach the continental shelf, they undergo metamorphosis into glass eels that migrate into fresh, brackish and coastal waters as yellow eels. after a highly variable feeding period of 5– years, yellow eels metamorphose into silver eels that migrate from their widely dispersed foraging areas to the common spawning ground in the sargasso sea. this migration is approximately -  km long. the changes that occur during metamorphosis into silver eels include an increase in eye size, a change in body coloration and a regression of the digestive system. eels do not feed during the transatlantic adult migration and rely exclusively on fat content for energy. after reaching the spawning grounds in the sargasso sea, eels reproduce once then die shortly thereafter. a. anguilla is also an important species to better understand the molecular aspects of reproduction because it is an endangered species, is of interest for aquaculture purposes and occupies a key position within the teleost phylogeny. while much progress has been made over the last century, many aspects of eel reproduction remain poorly understood, including the physiological and molecular mechanisms responsible for sexual maturation and the coordination of reproductive events. european eels are sexually immature when they start their migration across the atlantic to the spawning grounds; it is not clear if they mature sexually during migration or after they arrive at sargasso sea. maturation experiments through hormonal induction have shown dramatic changes at the physiological level as well as changes in secondary sexual characteristics. final maturation in eels does not appear to be triggered by swimming alone and is likely influenced by exposure to environmental cues as the eels swim across the atlantic. in addition, reproductive pheromones are believed to have a role in the regulation of final maturation and reproduction in eels. if sexual maturation in a. anguilla is triggered by environmental signals, then signal detection is expected to occur through sensory pathways and signal processing is expected to occur in the brain before the neuroendocrine response is activated. at present, mature adult eels have never been caught in the wild and eels do not mature in captivity without treatment with gonadotropic hormones. the inability to reach sexual maturation in captivity is due to a lack in sufficient pituitary gonadotropin production. in eels, maturation is under double neuroendocrine control which occurs in some teleosts and which can be reversed by gonadotropic hormone treatments. the dopaminergic inhibitory process leads to insufficient gnrh stimulation, and to the inhibition of pituitary gonadotropes fsh and lh, which exert direct and indirect control on gonad development by acting on steroid metabolism. the goal of the present gene profiling study was to identify changes in the brain that are involved in the coordination of reproductive events in the european eel, including those involved in neuroendocrine regulation of gametogenesis. more specifically, genes involved in sexual maturation and reproduction are expected to be expressed at higher levels in the brain of sexually mature males compared to immature males. additionally, since the coordination of reproductive events is influenced by environmental cues that must be integrated with the physiological and developmental status of the animal, it is expected that changes at the molecular level occur in the areas associated with the processing of sensory signals.  using a false discovery rate of, a total of differentially expressed genes were identified. of this set, were expressed at higher levels in brains of sexually mature males while were expressed at lower levels relative to brains of sexually immature males. when searched against the refseq proteins using blastx, these transcripts retrieved and unique sequences, respectively. functional annotation using david to obtain a list of gene identifiers that could be used for the functional analysis of differentially expressed genes, the a. anguilla transcriptome assembly contigs that were used to design the microarray were used in a blastx search of swissprot proteins. the result of this search revealed that of the transcripts had alignments with e-values of or less in swissprot. when the contigs without hits in swissprot were searched against refseq proteins and then the nr database, alignments for additional and sequences were retrieved, respectively. using this approach, sequence identifiers for a total of contigs were retrieved. for the analyses with david, only differentially expressed transcripts with blast alignment e-values of or less were considered. functional annotation analysis with david uncovered a number of significantly enriched terms within the list of up- and down-regulated genes. figure  details the proportion of over-represented terms belonging to the following go categories: biological process, cellular component and molecular function. for each of the three main go categories, terms were assigned to subcategories.figure proportion of differentially expressed terms belonging to the go categories biological process, molecular function and cellular component. enriched terms from the lists of significantly up and down-regulated genes were identified using david. for each of the three main go categories, terms were assigned to subcategories. cluster analysis with david identified eight significantly enriched clusters of up-regulated genes and five significantly enriched clusters of down-regulated genes. for this analysis, only clusters with ease scores greater than or equal to were considered. clusters of up-regulated genes include many that are involved in dna binding, calcium singling and nervous system development and differentiation. clusters of down-regulated genes include genes for rna processing, immune system function and nucleotide binding. differential expression: keyword analysis to identify specific sets of differentially expressed genes, the protein descriptions obtained from the blastx search of the eukaryotic proteins in refseq were used. in total, out of assembly contigs showed alignments with an e-value score of or better in the refseq proteins. the complete list of differentially expressed genes can be found in additional file 4: table s among this set, we identified neuroendocrine genes that are differentially expressed in the brains of sexually mature male eels.table differentially expressed neuroendocrine genes in the brains of sexually mature and immature male eels genes that have positive direction values were up-regulated in the brains of sexually mature male eels in comparison to sexually immature male eels while those that are negative were down-regulated in this group. gene descriptions are based on blastx hits in refseq proteins. a false discovery rate of was used to identify differentially expressed genes. the results of the david analysis suggest that some re-organization of signaling pathways in the brain occurs at sexual maturation. therefore, it seemed likely that there would also be changes in the expression levels for genes that are involved directly in cell-cell communication such as receptors and channels. we therefore searched for up and down regulated channels and g protein-coupled receptors. seven gpcrs were differentially expressed. four of these belong to the glutamate family of gpcrs, one belongs to the rhodopsin-like family and two belong to the secretin family. one of the glutamate family receptors, an extracellular calcium sensing receptor-like gene, was expressed at higher levels in the brains of sexually mature males. the other three were expressed at lower levels in the brains of sexually mature males compared with sexually immature males. the receptor from the rhodopsin-like gpcr family was the serotonin receptor which was expressed at higher levels in the brains of sexually mature males than in immature males. the gpcr 64-like gene and the gpcr 125-like gene belong to the secretin family and were expressed at higher levels in the brains of sexually mature males. additionally, there were eleven differentially expressed channels seven of which were expressed at higher levels in the brains of sexually mature males in comparison to the brains of immature males.table differentially expressed gpcrs and channels in the brains of sexually mature male eels g protein-coupled receptors were assigned to families based on the grafs classification system. genes that have positive direction values were up-regulated in the brains of sexually mature male eels in comparison to immature males while those that are negative were down-regulated in this group. gene descriptions are based on blastx hits in refseq proteins. a false discovery rate of was used to identify differentially expressed genes. quantitative polymerase chain reaction eight differentially expressed genes were chosen for quantitative polymerase chain using the same rna samples that were used for the microarray. this list includes: galanin peptide, glutamate decarboxylase, corticotropin releasing factor binding protein, c-type natriuretic peptide, gamma-aminobutyric acid receptor subunit δ, neuropeptide y, mammalian ependymin-related protein and the iroquois-class homeodomain protein. the tata box binding protein and elongation factor 1-alpha were used as controls.figure comparison of qpcr results with the microarray results for eight differentially expressed genes. the expression levels for eight differentially expressed genes were determined by quantitative polymerase chain reaction with the same rna pools that were used for the microarray. these include the following differentially expressed genes: galanin peptide, glutamate decarboxylase, corticotropin releasing factor binding protein, c-type natriuretic peptide, gamma-aminobutyric acid receptor subunit δ, neuropeptide y, mammalian ependymin-related protein and the iroquois-class homeodomain protein. bars represent mean fold changes, error bars represent standard deviations and * represents significant increases in the level of expression in the brains of sexually mature male eels in comparison to immature male eels. to verify that the fold changes observed in the microarray experiments correlated with the fold changes observed in the qpcr experiments, spearman’s rank order correlation was used. the results of this test indicate a positive relationship exists between the fold changes observed in the microarray and qpcr experiments. the qpcr expression levels between immature and mature samples were further evaluated using student's t-tests. galp, crfpb, cnp, merp and irx were expressed at significantly higher levels in the brains of mature males compared with mature while the differences in expression of npy, gad and gabdr were below the statistical significance level. discussion this study shows that major changes in the gene expression profiles occur in the brains of male european eels at sexual maturity. this shift includes an increase in the expression levels of genes involved in cell signaling, including receptor and channels, as well as genes involved in development and differentiation. several neuroendocrine genes were also expressed at higher levels in the brains of mature males in comparison to the brains of immature males. these genes are likely involved in gonadal maturation and development and act through the brain-pituitary-gonad axis. genes involved in immune system function were down-regulated in the brains of sexually mature males, suggesting that a re-allocation of resources occurs at this stage. interestingly, the expression levels of several receptors and channels change at sexual maturity suggesting that some neuronal rewiring occurs at this stage. more transcripts for neuropeptide y were observed in the brains of mature male eels in comparison to immature males. npy is a short peptide whose expression pattern has been described in the brains of several teleosts. for example, in goldfish, npy expression has been detected in the forebrain including the telencephalon, preoptic area, the olfactory bulb and thalamic regions. in the japanese eel anguilla japonica, npy is highly expressed in the telencephalon, optic tectum thalamus and hypothalamus. npy stimulates lh release in fish, has a role in growth hormone and gonadotropin hormone secretion and may coordinate growth, feeding and reproduction in fish. in goldfish, fasting and exposure to sex steroids increases npy mrna levels in the brain. furthermore, the npy response in goldfish depends on reproductive status. food deprivation in a. japonica also causes increases of npy mrna levels in the brain. changes in feeding behavior and regulation of energy expenditure are expected to accompany the maturation process in fish, particularly in species such as a. anguilla that allocate a lot of resources to reproduction. since the european eel does not feed at sexual maturity, it is possible that increased levels of npy are related to nutritional and developmental status. genes involved in gamma-aminobutyric acid signaling were also expressed at higher levels in the brains of mature males. for example, glutamate decarboxylase, an enzyme involved in the production of gaba from l-glutamic acid, was more abundant in the brains of sexually mature than immature males. gaba is a neurotransmitter involved in the control of pituitary hormone secretion in teleosts. gaba has a stimulatory effect on lh by enhancing gnrh release and inhibiting dopamine turnover. the gaba a receptor subunit δ was expressed at a higher level in the brains of sexually mature male eels than in immature males. gaba a receptors are hetero-pentamer ligand gated channels that are gated by gaba binding. it appears that different combinations of receptor subunits may confer functional specificity. in goldfish, four gaba a subunits were expressed at higher levels in the brains of sexually mature females. similarly, the gaba a receptor γ subunit was expressed at higher levels in the brains of goldfish exposed to prostaglandin-f2α than in controls. in the current study, the δ subunit was up-regulated in sexually mature males; this is intriguing given that the δ subunit appears to be involved in neurosteroid modulation of the gaba a receptor. in the brains of sexually mature males, the 5-hydroxytryptamine receptor 1b was expressed at a higher level than in immature males. the brain serotonergic system is one of the mechanisms used to cope with the dramatic changes during maturation. this system plays an important role in a complex neuroendocrine loop that functions in homeostasis and acclimation during physiological or environmental challenges. corticotropin releasing factor binding protein was also expressed at a higher level in the brains of mature males than in immature males. crfbp has an inhibitory role on crf which is a regulator of stress responses. in fish, acute stress results in an increase in crfbp expression in the hypothalamus. for european eels, reproduction is a stressful event because the spawning migration journey is very long. in addition, it is presumably challenging for both sexes to coordinate the temporal and spatial aspects of spawning in the vast sargasso sea which is  km wide and  km long. at the mature stage, european eels are likely to reduce their threshold for assessing risks and threats since they have a single opportunity to reproduce. therefore the observed increases in crfbp may be part of the stress response and of changes in behavior that are associated with reproduction. an increase in the levels of mammalian ependymin-related protein in the brains of mature males was observed. merps belong to a phylogenetically diverse clade that is related to ependymins which are glycoproteins that are found in abundance in the cerebrospinal fluid of fish. ependymins are involved in synaptic changes during memory consolidation and regeneration of the optic nerve. changes in the levels of ependymin-ii expression have been observed in the hypothalamus in goldfish at breeding season and in the telencephalon following exposure to a female pheromone. several genes involved in calmodulin signaling were differentially expressed in the brains of sexually mature males. for example, calm is involved in calcium signaling and nervous system function including memory consolidation and was up-regulated in the brains of sexually mature male eels. in goldfish, calm levels also change with reproductive status. during breeding season, calm and calcium/calmodulin-dependant protein kinase levels are highest in the hypothalamus in female goldfish and calm levels are highest in the telencephalon of males following exposure to pgf2α. sexual maturation alters the eel’s internal state and changes in the way the animal relates to its environment. in the european eel, the memory circuitry is expected to be important for reproduction due to the long migration across the atlantic to the sargasso sea. though cam signaling genes are likely involved in many processes in the brains of mature eels, it is possible that the changes observed here are also related to memory. the microarray results showed a high positive correlation with those obtained in selected genes by qpcr thus providing a global validation of the microarray results. npy, gad and gabdr did not seem to follow the same pattern; this could be related to the low expression levels of these genes or to the existence of alternative transcripts that are differentially detected by the two methods. however, these possibilities were not further investigated. recent studies in fish show that specific cells in the brain respond to light and exert neuroendocrine control of testis development. in a. anguilla, behavioral evidence indicates that photoreceptor sites that are independent of the pineal exist in the brain. eels become more sexually mature after months of migration in deep waters while only surfacing at night. in a. anguilla, spawning is believed to occur in the top meters where there is more light than in deeper water. the results of the current study show that several genes with roles in photosignal transduction and visual system development were up-regulated in the brains of mature males. this list includes retinal rod rhodopsin-sensitive cgmp 3' 5'-cyclic phosphodiesterase subunit γ and cyclic nucleotide-gated cation channel α- which is involved in olfactory and visual signal transduction. it also includes the calcium binding protein which has an important role in synaptic function in photoreceptor cells and the iroquois-class homeodomain protein. irx is a transcription factor that is involved in cone cell differentiation and retinal, craniofacial and gonad development. the transcription factor hes was also up-regulated in the brains of sexually mature males. hes belongs to a family of genes that have roles in cell differentiation and has a role in retina development. while it is possible that these genes have other roles in the brains of mature males, an intriguing avenue for future research would be to determine whether there is a link between neurohormone systems, gonad development and photoreceptors in the brains of a. anguilla like there is in masu salmon. several genes involved in neurogenesis, cellular differentiation and development were up-regulated in the brain of sexually mature males. this set includes genes from the go categories such as go:, go:, go: and go:. in addition, many differentially expressed ion channels and gpcrs were differentially expressed which suggests that some re-organization and/or re-growth occurs at maturation. the calcium-activated potassium channel would tend to hyperpolarize neurons in response to neuromodulators/neurotransmitters. down-regulation of these channels suggests a less responsive, more ‘hard-wired’ brain, which may be advantageous for certain aspects of reproduction. gnrh, aromatase, kisspeptin system genes and sex steroid receptors are also known to have roles in reproduction. the microarray included a probe for one isoform of gnrh and no differences in expression were observed between immature and mature males. the array did not contain probes for other gnrh genes or isoforms. although gnrh controls gonadotrophin secretion in the pituitary, the fact that the fish were artificially induced to mature using human chorionic gonadotrophin suggests that no positive or negative feedback mechanisms towards gnrh were occurring at the time of sampling. the microarray also contained probes for transcripts that are similar to the estrogen-related receptor beta like and the androgen receptor beta and no significant differences in expression were detected. kisspeptin system genes and aromatase were not analyzed in this survey. at present, mature eels have never been caught in the wild and the only way to obtain sexually mature animals is through artificial induction of maturation. it is therefore not clear to what extent changes that occur at the molecular level in the brain are the result of specific gonadal feedback mechanisms. the eyes and the olfactory epithelium change with sexual maturation in a. anguilla and the american eel anguilla rostrata suggesting that there is a change in the use of sensory modalities with reproduction. as mentioned previously, the data from this study suggest that the forebrains of male eels undergo some sort of rewiring at sexual maturation. as the areas of the brain tested here are associated with the processing and conversion of sensory input to physiological responses, it is possible that changes in the brain at sexual maturity are influenced by changes in sensory system requirements that occur at reproduction.  in this study, we used nearly microarray probes to identify gene expression changes in the brains of eels that occur at sexual maturation. of the set of differentially expressed genes, were expressed at higher levels in sexually mature males while were expressed at lower levels than in immature males. this finding shows that the brains of eels undergo major changes at the molecular level during reproduction. the set of up-regulated genes includes genes that are involved in neuroendocrine processes which likely act through the brain-pituitary-gonad axis. it also includes genes involved in cell-cell signaling, neurogenesis and development indicating that the brain is undergoing developmental changes that are associated with maturation and reproduction. a major advantage to the approach employed here is that it allows many genes to be tested at the same time. it also allows for the identification of genes not previously known to be involved in particular processes such as reproduction. consequently, some of these differentially expressed genes identified here have roles that have been described in other studies while others have roles that have yet to be characterized. furthermore, since the majority of the genes tested here have orthologs in other vertebrates, the results from this study can be used to understand reproduction across a diversity of vertebrate species. most importantly however, is that we have defined a set of genes that appear to be important for sexual maturation in a. anguilla. these findings therefore contribute to the body of knowledge concerning eel biology and will, in the future, contribute to aquaculture and conservation efforts. 
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. 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. 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. the vertebrate brain plays a critical role in the regulation of sexual maturation and reproduction by integrating environmental information with developmental and endocrine status. for this, we used microarrays to compare the gene expression profiles of sexually mature to immature males. the set of up-regulated genes includes genes involved in neuroendocrine processes, cell-cell signaling, neurogenesis and development. here, we have defined a set of genes that help to understand the molecular mechanisms controlling reproduction in eels. of this set, were expressed at higher levels in brains of mature males while were expressed at lower levels relative to brains of immature males. 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. 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. using a false discovery rate of, a total of differentially expressed genes were identified.
 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.  using a false discovery rate of, a total of differentially expressed genes were identified. of this set, were expressed at higher levels in brains of mature males while 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.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords eelneuroendocrinebraingene expressionreproductionmicroarrayissue-copyright-statement© the author 2014 