The Pituitary Gland of the European Eel Reveals Massive Expression of Genes Involved in the Melanocortin System
et al. (2013) The Pituitary Gland of the European Eel Reveals
Massive Expression of Genes Involved in the Melanocortin System. PLoS ONE 8(10): e77396. doi:10.1371/journal.pone.0077396
The Pituitary Gland of the European Eel Reveals Massive Expression of Genes Involved in the Melanocortin System
Eirill Ager-Wick 0
Ron P. Dirks 0
Erik Burgerhout 0
Rasoul Nourizadeh-Lillabadi 0
Danille L. de Wijze 0
Herman P. Spaink 0
Guido E. E. J. M. van den Thillart 0
Katsumi Tsukamoto 0
Sylvie Dufour 0
Christiaan V. Henkel 0
Juan Fuentes, Centre of Marine Sciences & University of Algarve, Portugal
0 1 Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science , Oslo, Norway, 2 ZF-screens B.V., Leiden , The Netherlands , 3 Institute of Biology, Leiden University , Leiden , The Netherlands , 4 Atmosphere and Ocean Research Institute, the University of Tokyo , Kashiwa, Chiba, Tokyo , Japan , 5 Research Unit BOREA, Biology of Aquatic Organisms and Ecosystems, CNRS 7208, Museum National d Histoire Naturelle , Paris , France
Hormones secreted from the pituitary gland regulate important processes such as development, growth and metabolism, reproduction, water balance, and body pigmentation. Synthesis and secretion of pituitary hormones are regulated by different factors from the hypothalamus, but also through feedback mechanisms from peripheral organs, and from the pituitary itself. In the European eel extensive attention has been directed towards understanding the different components of the brain-pituitary-gonad axis, but little is known about the regulation of upstream processes in the pituitary gland. In order to gain a broader mechanistic understanding of the eel pituitary gland, we have performed RNA-seq transcriptome profiling of the pituitary of prepubertal female silver eels. RNA-seq reads generated on the Illumina platform were mapped to the recently assembled European eel genome. The most abundant transcript in the eel pituitary codes for pro-opiomelanocortin, the precursor for hormones of the melanocortin system. Several genes putatively involved in downstream processing of pro-opiomelanocortin were manually annotated, and were found to be highly expressed, both by RNA-seq and by qPCR. The melanocortin system, which affects skin color, energy homeostasis and in other teleosts interacts with the reproductive system, has so far received limited attention in eels. However, since up to one third of the silver eel pituitary's mRNA pool encodes pro-opiomelanocortin, our results indicate that control of the melanocortin system is a major function of the eel pituitary.
Funding: This work was supported by the Norwegian School of Veterinary Science and the Research Council of Norway (184851), by Centre National de
la Recherche Scientifique and LAgence Nationale de la Recherche (08-BLAN-0173), and by private resources from ZF-screens B.V., Leiden University
and The University of Tokyo. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: We have the following interests. HPS and GEEJMvdT are founders and shareholders of ZF-screens B.V. CVH, RPD, and DLdW are
(or have been) employees of ZF-screens B.V. ZF-screens B.V. supported this study. Passie voor Vis BV and Nijvis-Holding BV provided farmed eels.
There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing
data and materials, as detailed online in the guide for authors.
The European eel (Anguilla anguilla) has a long and complex
life cycle. It is now listed as a critically endangered species ,
leading to an urgent need to learn more about its biology and
reproduction. Spawning of the European eel occurs in unknown
areas of the Sargasso Sea . Larvae drift to the European
coasts following oceanic currents, where they metamorphose
into glass eels and migrate into continental habitats. They can
stay in brackish water or in rivers for years as juvenile yellow
eels before they develop into prepubertal silver eels . Silver
eels are still sexually immature when they leave the continental
habitats and migrate back to the sea. They remain blocked at
the prepubertal stage as long as the reproductive migration is
prevented . Therefore, maturation needs to occur during the
oceanic migration or at their spawning grounds . The
prepubertal silver eels are the last known stage of the eel life
cycle in natural conditions. Although spawning European eels
have never been caught in the wild, the spawning site of the
Japanese eel (Anguilla japonica) was recently discovered close
to the West Mariana Ridge . Adults and newly hatched
larvae were found in close proximity to the first collection of
Japanese eel eggs.
Attempts to promote spontaneous maturation,
gametogenesis and spawning of eels in aquaculture have so
far been unsuccessful. In order to accomplish successful
artificial reproduction, there is a need for development of
strategies to control the secretion of hormones from the
pituitary gland. To achieve this goal the endocrinological
changes in the pituitary should be studied during maturation
both at the transcriptome and proteome level. The pituitary
comprises the neurohypophysis (posterior pituitary) and the
adenohypophysis (anterior pituitary), both regulated by the
hypothalamus. The adenohypophysis of teleosts is a major
endocrine organ and is organized into different compartments,
i.e. rostral and proximal pars distalis and pars intermedia,
where different hormones are produced in their respective cell
types . The two gonadotropins, follicle-stimulating
hormone and luteinizing hormone, directly control gonadal
development. Other pituitary hormones, such as growth
hormone and thyroid-stimulating hormone, regulate other
physiological systems, but also play a role in reproduction.
Adrenocorticotropic hormone, - and -melanocyte stimulating
hormone and -endorphin derive from a common precursor
hormone, pro-opiomelanocortin. These hormones are
important components of the melanocortin system, which is
involved in the regulation of different physiological processes,
and, in teleosts, possibly also in reproduction .
In order to get closer to understanding important molecular
mechanisms at work in the European eel pituitary we have
utilized high throughput RNA sequencing (RNA-seq) to profile
the global gene expression in the pituitary of prepubertal silver
eels. RNA-seq provides the opportunity to study the
transcriptome in a specific organism, tissue or cell type by
sequencing millions of short fragments simultaneously. The
number of reads produced is a function of the abundance of a
transcript, and thus the read density is used to quantify gene
expression . In addition to providing information about
gene expression levels, RNA-seq also enables the discovery of
new genes and transcripts and can reveal alternative splice
The draft genomes of the European and the Japanese eel
have recently been published [15,16], enabling for the first time
reliable gene expression profiling using RNA-seq. However,
curated gene annotations and expression data are still scarce
for eels, and therefore the included gene predictions for the eel
genome are primarily based on generic gene models .
In this study, we have therefore used RNA-seq evidence to
manually improve the annotations of the most abundantly
expressed genes in the eel pituitary.
Materials and Methods
Animals and experimental design
Female European eels (Anguilla anguilla) obtained in the
Netherlands were used in this study. Four prepubertal eels
(one wild and three farmed) in the silvering transition period
were sampled for RNA-seq. To get closer to a complete picture
of the European eel transcriptome we included one sample
from two other developmental stages: a farmed immature
yellow eel and a wild artificially matured eel. For qPCR
validation of key findings we used pituitary material from five
farmed silver eels, of which three were the same samples used
for RNA-seq. Permission for capturing eels during the migration
season was obtained from the Netherlands' Ministry of
Agriculture and Fisheries. Experiments were approved by the
animal ethical commission of Leiden University (DEC #08112
and #11093). The farmed animals (one yellow and three silver
eels, samples 24) were transported from the farms to the
University (1.52 h) in buckets with some water, and the
pituitaries were sampled immediately upon arrival. The wild
silver eel (sample 1) was kept for one week at the University in
a seawater recirculation system prior to sampling. The
artificially matured eel received weekly injections of salmon
pituitary extract (20 mg) for 17 weeks and ovulation was
induced by injection with 17, 20-dihydroxy-4-pregnen-3-one
(DHP) (2 mg kg-1) as recently published . The matured eel
was sampled just after ovulation. As an estimate of the
reproductive status the gonadosomatic index (GSI) was
calculated. Calculation of silver index, eye index and other
morphological and physiological measurements were
performed as previously described [19,20], and the detailed
information about the animals is given in table 1. Prior to
dissection of the pituitary gland, eels were euthanized using an
overdose of anesthetic (clove oil), followed by decapitation. Eel
pituitaries were sampled and stored in RNAlater (Ambion) at
-80 C until RNA extraction.
RNA extraction, Illumina library preparation and
Total RNA was isolated using the Qiagen miRNeasy kit
according to the manufacturers instructions (Qiagen). RNA
integrity was assessed by Agilent Bioanalyzer 2100 on a total
RNA Nano series II chip (Agilent). All RNA-seq libraries were
prepared with the Illumina mRNA-seq Sample Preparation Kit
from 10 g total RNA, according to the manufacturers
instructions (Illumina Inc.). RNA-seq paired end libraries for
silver eel 1, yellow eel, and mature eel were sequenced with a
read length of 255 nucleotides on an Illumina GAIIx
instrument, while the other silver eel samples (silver eel 2, 3
and 4) were sequenced with a read length of 251 nucleotides
on a HiSeq2000 according to the manufacturers protocol. The
Illumina pipeline was utilized for image analysis and base
Quantitative PCR (qPCR) was carried out on a LightCycler
480 Real-Time PCR system (Roche, Mannheim, Germany),
using the LightCycler 480 Master with SYBR Green (Roche).
cDNA was prepared from 1 g of DNase treated total RNA
using Superscript III reverse transcriptase (Invitrogen) and
oligo(dT) primers according to product specifications. To avoid
amplification of contaminating genomic DNA, the primers were
designed to span exon-exon boundaries, such that part of the
primer hybridizes to the 3 end of one exon and the rest of the
primer hybridizes to the 5 end of the adjacent exon. A standard
dilution curve was set up for each primer pair and the pair that
showed the best efficiency was chosen. These primer
sequences are given in table S1. Acidic ribosomal
phosphoprotein P0 (arp) shows stable expression during
different experimental treatments in eel and was used as a
reference gene to normalize the expression analysis , using
Silver eel 1
Silver eel 5
Silver eel 6
an efficiency-corrected relative quantification method .
Each sample was analyzed in duplicate and comprised 5 l
mastermix, 2 l primer mix (5 M of each or forward and
reverse), and 3 l of each 10 diluted cDNA sample in a total
volume of 10 l. The cycling parameters were 10 min
preincubation at 95 C, followed by 42 cycles of amplification at
95 C for 10 sec, 60 C for 10 sec and 72 C for 6 sec, followed
by a melting curve analysis from 65 C to 95 C. A no template
control was included on every plate to rule out nonspecific
contamination, while the melting curve analysis was included to
verify that a single specific product was measured in each run.
Reads were aligned to the draft genome of European eel 
using TopHat (version 2.0.5) . The resulting files were
filtered using SAMtools (version 0.1.18)  to exclude
secondary alignment of reads. Aligned fragments per predicted
gene were counted from SAM alignment files using the Python
package HTSeq (version 0.5.3p9) . We only considered
gene predictions which have been provisionally functionally
annotated by Blast2GO (i.e. known eel genes or gene
predictions with homologs in other species). In order to make
comparisons across samples possible, these fragment counts
need to be corrected for the total amount of sequencing
performed for each sample. As a correction scaling factor, we
employed library size estimates determined using the R/
Bioconductor (release 2.11) package DESeq . Read counts
were normalized by dividing the raw counts obtained from
HTSeq by its scale factor and by transcript length in kilobases.
Detailed read coverage for individual genes was extracted from
the TopHat alignments using SAMtools. New alignments were
generated for re-annotated genes, which were then quantified
and normalized as before, using the scaling factors determined
for the initial alignments. For each Gene Ontology category,
total expression was calculated by summing the normalized
expression of all genes annotated with that GO term (based on
Blast2GO annotations, ).
Gene expression profiling
RNA-seq reads were mapped to the European eel genome
 using TopHat . From the total number of reads that
was obtained from the silver eel samples, 9097% successfully
aligned, yielding expression values for 33649 genes with
provisional functional annotations. Details about the number of
reads and mapping for all samples are given in table S2. Gene
expression values for the different silver eel samples are
plotted against each other in Figure 1A, and correlate well
across all four samples (Spearman rank correlation 0.870.93).
One particular gene, pomc, encoding pro-opiomelanocortin, the
precursor for the peptide hormones of the melanocortin
system, stands out from the overall expression in all samples
as it is expressed at least one order of magnitude higher than
any other gene. Further highly expressed genes predominantly
encode other hormones and ribosomal proteins (Figure 1B).
Figure 1C shows the top genes by expression annotated with
the Gene Ontology category hormone activity (GO:0005184).
All silver eel gene expression values are available as table S3.
Several of the genes found to be highly expressed were
manually annotated and their sequences submitted to
GenBank (see table S4 for details).
Massive expression of pomc
Due to the striking expression of pomc we manually
improved the annotation of this gene using the RNA-seq
alignments and the gene prediction (Figure 2A). Based on the
new annotation we also recalculated the expression values,
and these are plotted on top of the new annotation in Figure
2A. In the silver eels pomc exhibits massive gene expression
levels, such that it constitutes up to 30% of the total number of
aligned RNA-seq reads mapping to the genome (table S2).
Comparison between the gene expression values for the
average of the four silver eel samples and yellow eel and
mature eel are shown in Figure S1, displaying that the pomc
gene also exhibits high expression in the two other stages. The
amino acid sequences of pomc between different Anguilla
species were compared (Figure 2B), where A. anguilla and A.
japonica display a greater similarity than A. anguilla and A.
rostrata. This is unexpected, given the evolutionary
relationships between Anguilla species . Figure 2C
illustrates the post-translational processing of the prohormone
Pomc to its bioactive hormone components, including
adrenocorticotropic hormone (ACTH), -lipotropic hormone
(LPH), -melanocyte stimulating hormone (-MSH),
corticotropin intermediate peptide (CLIP), -endorphin (-END)
and -melanocyte stimulating hormone (-MSH). The amino
acid sequences of these six bioactive hormones are 100%
identical in all three Anguilla species (Figure 2B).
Gene Ontology categorization
In order to investigate the possible involvement of other
genes in downstream processing of Pomc, we attempted to
look for overrepresentation of certain classes of genes using
gene ontology (GO) categories. Gene expression values were
summarized for each GO category (based on Blast2GO
annotations ) of the provisional annotation of the European
eel genome  (Figure S2). pomc is included in all of the top
15 highly expressed categories, stressing the dominance of
this gene in the transcriptome. Genes that belong to the
GOcategory peptide hormone processing (GO:00016486) were
investigated to look for potential important players of
downstream processing of prohormones (like Pomc) into
bioactive peptide hormones. This analysis revealed high
expression of different players involved in the post-translational
processing of Pomc , including carboxypeptidase e,
prohormone convertase 2 and neuroendocrine protein 7b2
(Figure 3). Note that the different convertases and catalysts
involved in the processing of the prohormone are included in
High expression of Pomc-processing genes
Due to the high expression of the gene encoding
neuroendocrine protein 7b2 (also called secretogranin V), we
decided to investigate if other secretogranins also exhibited
high expression. Among the genes of the granin family
proteins, known to be associated with neuroendocrine
secretion, secretogranin II and secretogranin III coding genes
were found to be highly expressed. In particular, one
secretogranin III paralogue exhibited substantial gene
expression levels (approximately 3% of total reads, see table
S2 for details). Based on alignments and gene predictions, the
highly expressed genes known to be involved in Pomc
processing were manually annotated and re-quantified (Figure
4 and table S4). In nearly all cases, the improved annotation
resulted in higher gene expression values (Figure S3). This
increase is an effect of both updated gene structure leading to
more reads aligning, and of better UTR definition leading to
shorter transcript annotations (see the definition of normalized
expression in Materials and methods). The high expression of
pomc and the genes involved in the processing of the
prohormone that were found to exhibit high expression by
RNA-seq were validated by qPCR (Figure 4), showing a good
correlation in relative expression levels between the two
This study serves as a first exploration of the complete
pituitary transcriptome of prepubertal female European silver
eel. Using the draft genome and the RNA-seq read coverage
we have improved the annotation of the most highly expressed
genes expressed in the eel pituitary.
pomc was found to be by far the most abundant transcript in
the European eel pituitary (figures 1 and 4), eclipsing the
expression of all other genes in the pituitary gland (figures 1B,
C and figure S2). In teleosts, the pituitary gland consists of
multiple hormone-producing cell types, including lactotropes,
corticotropes, somatotropes, thyrotropes, two types of
gonadotropes, melanotropes and somatolactotropes ;
however, the pomc gene is expected to be expressed only in
corticotropes and melanotropes. Since the RNA-seq analysis
was performed on complete pituitary glands, in some eels the
amount of pomc mRNA in these two cell types may very well
exceed 50% of total mRNA content. Alternatively, pomc could
be expressed in other cell types as well. It is worth noting that
the very high expression level of pomc observed may also be a
consequence of the technology employed. Using RNA-seq, the
pomc transcript was determined to be almost five hundred-fold
more abundant than the transcripts of several housekeeping
genes (e.g. b-actin). Until recently, full transcriptome profiling
was performed using microarrays, which generally detect a
much more limited range in expression levels (approximately a
1000-fold change between low and high expression values).
If we had used microarrays, we would have been unable to
detect the difference in expression level between pomc and a
housekeeping gene like b-actin: both transcripts would
probably have saturated the available probes, and would have
received the maximum high expression value. Strikingly, the
Figure 1. Gene expression in silver eels. (A): Pairwise comparisons of normalized expression values for all genes between the
four silver eel samples (for details, see Materials and methods). The red dot indicates the expression level of pro-opiomelanocortin.
Spearman rank correlations () for the different comparisons show a good correspondence between the expression values for the
different silver eel samples. (B): Top 15 expressed genes in silver eel samples, displayed as means of normalized expression
standard deviations (SD) on a log scale. (C): Top 15 expressed genes in GO category Molecular Function hormone activity (GO:
0005184), displayed as means of normalized expression SD on a log scale.
Figure 3. Expression of genes in Gene Ontology category peptide hormone processing. Expression of genes belonging to
the Gene Ontology term peptide hormone processing (GO:0016486). The four silver eel samples are grouped together to the left
(silver eel 14 from left to right), while the gene expression for the yellow eel and the mature eel is placed to the right and displays
an expression pattern similar to the silver eel samples. Genes involved in the processing of pro-opiomelanocortin are highly
expressed (red), and include carboxypeptidase e, prohormone convertase 2 and neuroendocrine protein 7b2.
top 15 of the GO categories by gene expression all include
pomc, again emphasizing the total dominance of this
prohormone in the silver eel pituitary transcriptome with
regards to gene expression.
The eels employed in this study were obtained from several
different sources (table 1), resulting in a range of external
conditions (salinity, photoperiod, season, animal handling etc.).
Nevertheless, all samples show a consistent gene expression
profile (Figure 1 and Figure S1). When examined in detail, the
expression profile for the pomc gene is similar in all four silver
eel samples (Figure 2A). The only silver eel (sample 1) that
was not obtained from a farm exhibits the highest expression of
pomc, with a slightly different coverage profile near the 5 UTR
(Figure 2A). We observed the same profile in the
(nonreplicated) yellow and mature eel samples (sequenced at the
same time as silver eel 1), suggesting that the biased coverage
profile is an artifact of different sequencing technology
generations rather than a biological difference. Because of
limited availability of high quality samples from non-silver eels,
we were unable to study ontogenetic changes in gene
expression completely throughout maturation. In addition, the
very high expression of just a single gene can make exact
comparisons of RNA-seq results for all other genes less robust
. However, in the four silver eel replicates, the levels of
pomc itself correlate positively with body weight and gonadal
development (GSI) (table 1).
The animals could have been stressed by handling,
transport, or experimental procedures prior to the pituitary
dissection, which conceivably could affect downstream gene
expression results, particularly of pomc. ACTH (corticotropin)
produced downstream of pro-opiomelanocortin is an important
player in the neuroendocrine stress response which activates
cells in the interrenal tissue to produce and release cortisol
. However, the abundance of pomc was high for all the
Figure 4. Comparison between RNA-seq and qPCR experiments of genes involved in the melanocortin
system. Quantitative PCR validation of pro-opiomelanocortin and other highly expressed genes involved in the processing of the
prohormone found by RNA-seq in the silver eel samples: pro-opiomelanocortin (pomc), prohormone convertase 2 copy 1 (pc2a),
prohormone convertase 2 copy 2 (pc2b), secretogranin II copy 1 (scg2a), secretogranin II copy 2 (scg2b), secretogranin III copy
1(scg3a), secretogranin III copy 2 (scg3b), neuroendocrine protein 7b2 copy 1(7b2a), neuroendocrine protein 7b2 copy 2 (7b2b)
and carboxypeptidase e (cpe). Note that pc2b and scg2a were studied by RNA-seq only, and not investigated by qPCR due to
limitations for designing primers from these genes where we only obtained partial sequences. Primer sequences are given in table
S1 and details about the annotation of the genes are provided in table S4. Results are presented as means standard deviations
(SD) on log scales for both methods, where the left y-axis represent the re-quantified RNA-seq normalized gene expression (n=4)
and the right y-axis represent the expression by qPCR (n=5).
samples regardless of the different sources they came from.
Farmed animals are more used to handling than wild animals,
which possibly could influence how well they cope with stress.
The transport of these animals (see Materials and methods)
may have induced additional stress, although the kinetics of
mRNA synthesis imply that by itself this interval is too brief to
result in the levels of pomc expression observed . The
silver eel that was caught in the wild exhibited the highest
pomc expression compared to the farmed silver eels, while the
artificially matured eel that received injections every seven
days for 17 weeks was the sample that exhibited the lowest
pomc expression levels of all the samples. These observations
suggest that it is unlikely that the high abundance of pomc can
be explained by induction of stress during animal handling only.
Nevertheless, a previous stressed state (either induced by
handling or by biological factors) cannot be excluded, and the
possibility should be kept in mind when interpreting the results.
It would be difficult to obtain demonstrably non-stressed eels,
which would be needed to create a baseline for a stress marker
(i.e. cortisol) that would make it possible to compare with the
levels of other animals. In fact, in the few studies in which
cortisol levels were determined in eels, these were found to be
strongly elevated, yet highly variable, in migrating silver eels
The activity of the Pomc-producing cells is regulated by
expression and cleavage of the precursor protein,
posttranslational processing of cleavage products, and release of
the end products (Figure 2C). In mammals post-translational
processing of Pomc is dependent on the proteolytic cleavage
by prohormone convertases (PC1 and PC2; for review see 39),
which are most likely also involved in the processing of fish
Pomc . PC1 mediates the initial processing of Pomc into
ACTH, -LPH and N-terminal peptide in the corticotropes of the
pituitary rostral pars distalis, while in the melanotropes located
in the pituitary pars intermedia, PC2 processes ACTH further
into -MSH and corticotropin-like intermediate peptide (CLIP),
and converts -LPH into -MSH (processed via -LPH) and
endorphin [30,41,42]. Carboxypeptidase e (CPE) catalyzes the
generation of mature -MSH from ACTH by trimming the
Cterminal, and also works as a sorting receptor of the regulated
secretory pathway by binding secretory proteins, including
Pomc [29,43]. In our data, pc1, pc2 and cpe were among the
highly expressed genes, with pc2 showing higher expression
than pc1 (Figure 3). The high expression of these genes
implies an important role for the processing of Pomc and
emphasizes that the dramatically high expression of pomc is
likely to be biologically relevant (Figure 3).
PC2 has a specific endogenous inhibitor called granin
neuroendocrine protein 7b2 (7B2), which functions as a
chaperone protein and is required for production of active PC2
enzyme [31,44]. The catalytic activity of 7B2 is regulated by
inhibiting PC2 unfolding and aggregation in the secretory
vesicle . 7B2 is a member of the granin family, which
includes biologically active peptides that are responsible for
delivery of peptides, hormones, neurotransmitters and growth
factors. These proteins are expressed in endocrine cells and
peptidergic neurons and have both constitutive and regulated
secretory pathways . Secretogranin II (Scg2) can be
proteolytically processed to generate secretoneurin [46,47]. In
goldfish gonadotropes secretoneurin has been shown to
stimulate luteinizing hormone synthesis and release . Scg3
can be cleaved to peptides in secretory vesicles . Scg3 and
CPE have been found to interact and facilitate prohormone
sorting within secretory granules . Pomc and Scg3 have
been found to coordinately increase upon stimulation of
Xenopus pituitary pars intermedia cells in vivo . In the silver
eel pituitary, several members of the granin family show high
expression levels (figures 3 and 4). In contrast to other studied
teleosts, the European eel has likely retained two scg3
paralogues after the teleost specific genome duplication. These
differ markedly in expression in the eel pituitary, indicative of
possible subfunction partitioning . One scg3 paralogue
exhibits very high expression in the silver eel pituitary (Figure
4), suggesting an important role for this granin in the secretory
pathway in eel.
The biologically active peptide hormones derived from pomc
exert a variety of physiological functions in fish, including
effects on stress, vasoregulation, thermoregulation, growth,
metabolism, metamorphosis and reproduction (for review see
53). Several studies have indicated involvement of the
melanocortin system in the regulation of energy metabolism
and food intake in fish . It has been suggested to
control the energy balance by decreasing food intake and
enhancing energy costs. -MSH has been shown to stimulate
lipase activity and increase the circulating levels of fatty acids
in rainbow trout, while trout with defective -MSH show
increased appetite, enlarged livers and accumulation of fat in
the abdominal cavity . Synchronous changes in gonadal
development and morphological characteristics (e.g. skin
coloration), have been demonstrated in European and
Japanese eel and are suggested to be hormonally regulated
[20,58]. Degeneration of the gut takes place during gonadal
maturation in European eel . Eels have an exceptionally
high fat content prior to migration , which suggests a role
for Pomc-derived peptides in fat metabolism. Adaptation to
background color is an important function found to be regulated
by -MSH (reviewed in 54), where regulation of skin
pigmentation mediated by -MSH exerts actions opposing
those of melanin-concentrating hormone (MCH) [55,61]. The
high pomc expression levels might reflect an important role for
-MSH in the changes in skin coloration occurring during
silvering in eel.
In light of these physiological adaptations mediated by the
melanocortin system, an alternative explanation of potential
stress (see discussion above) can develop, in which silver eels
naturally exhibit the characteristics of a stressed state and
therefore experience a chronic activation of the
Pomcdependent stress response. For example, the high levels of
cortisol observed in silver eels have been interpreted as being
involved in the mobilization of energy (fat stores) and the
adaptation to seawater . In addition, cortisol stimulates the
expression of the luteinizing hormone -subunit in European
eel , thus providing a more complex picture of cortisol
regulation, in which cortisol does not only negatively affect
reproduction due to stress, but can also be beneficial for the
induction of sexual maturation.
From this initial survey of the eel pituitary transcriptome it is
not possible to precisely disentangle the relative contributions
of prior biological stress-like processes and stress induced by
the experiment. This would require much more comprehensive
sampling, including additional biological states, points in time,
and well-defined stressors. Considering the dominance of
comparatively few genes in the reported trancriptomes, the lack
of detailed physiological clues in the single pomc transcript
species, and the relatively high cost of full transcriptome
sequencing, targeted proteomics techniques would be well
suited for such an experiment. A recent study utilized mass
spectrometry analyses to reveal the post-translational
processing of pomc in the pituitary of medaka (Oryzias latipes)
. The availability of the draft genome of European eel and
the detailed annotation of the central genes we supply in the
current paper makes it possible to predict the molecular
weights of all protein products, which facilitates the use of
Although more investigation is required to reveal the
mechanisms by which the melanocortin system is involved in
processes such as growth and metabolism, reproduction, water
balance, and body pigmentation in teleosts, the results
presented here support the idea that the control of this system
is a major function of the eel pituitary.
Figure S1. Gene expression in all samples. Differences in
normalized gene expression between the average expression
values for the four silver eel samples compared to the yellow
eel and mature eel samples. The red dot indicates the
expression level of pro-opiomelanocortin. Spearman rank
correlations () for the different comparisons show a good
correspondence between the expression values for the
Figure S2. Gene Ontology characterization. For each GO
category the total expression for the average of the silver eel
samples was calculated by summing the normalized
expression of all genes annotated with that GO category. The
25 most highly expressed GO categories based on expression
values in the silver eel samples are displayed (for details, see
Materials and methods), of which the top 15 categories include
pomc, underlining the dominance of this gene. The red line
corresponds to pomc gene expression alone (based on the
original annotation). The first category by expression that does
not include pomc is protein binding.
Figure S3. The importance of re-annotation of genes for
gene expression in silver eel samples. Differences in gene
expression values for the four silver eel samples, highlighting
the genes involved in the melanocortin system that were
reannotated in this study. The original gene expression values
(before re-annotation) are shown in red and the new gene
expression values (after re-annotation) are displayed in green.
The figure illustrates the high expression of the genes involved
in the melanocortin system as compared to the overall gene
expression (grey), and that re-calculation of gene expression
values after re-annotation of these genes increases their
relative gene expression in all silver eel samples.
Table S2. Alignment of RNA-Seq reads.
We are grateful to Mieke and John van Dooren (Passie voor
Vis BV) and William Swinkels (Nijvis-Holding BV) for providing
farmed eels, Bas Brittijn and Hans Jansen for animal handling
and sample preparation and Yuki Minegishi, Daniel Vodk and
Christian Tudorache for discussions.
Conceived and designed the experiments: EAW RPD EB RNL
HPS GEEJMdT KT SD FAW CVH. Performed the experiments:
EAW EB RNL DLdW CVH. Analyzed the data: EAW CVH.
Wrote the manuscript: EAW RPD EB FAW CVH.
1. Freyhof J , Kottelat M ( 2010 ) Anguilla anguilla . IUCN Red List of Threatened Species . Available: http://www.iucnredlist.org. Accessed 15 March 2013 .
2. Schmidt J ( 1923 ) Breeding places and migration of the eel . Nature 111 : 51 - 54 . doi:10.1038/111051a0.
3. Tesch FW ( 2003 ) The Eel . Blackwell Publishing House Science . 418 pp.
4. Dufour S , Lopez E , Le Men MF , Le Belle N , Baloche S et al. ( 1988 ) Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol . Gen Comp Endocrinol 70 : 20 - 30 . doi:10. 1016/0016-6480(88)90090-1. PubMed: 3286369.
5. Sbaihi M , Fouchereau-Peron M , Meunier F , Elie P , Mayer I et al. ( 2001 ) Reproductive biology of the conger eel from the south coast of Brittany, France and comparison with the European eel . J Fish Biol 59 : 302 - 318 . doi:10.1006/jfbi.2001.1642.
6. Tsukamoto K , Chow S , Otake T , Kurogi H , Mochioka N et al. ( 2011 ) Oceanic spawning ecology of freshwater eels in the western North Pacific . Nat Commun 2 : 179 . doi:10.1038/ncomms1174. PubMed: 21285957 .
7. Kanda S , Okubo K , Oka Y ( 2011 ) Differential regulation of the luteinizing hormone genes in teleosts and tetrapods due to their distinct genomic environments - Insights into gonadotropin beta subunit evolution . Gen Comp Endocrinol 173 : 253 - 258 . doi:10.1016/j.ygcen. 2011 .05.015. PubMed: 21663743 .
8. Nozaki M , Naito N , Swanson P , Miyata K , Nakai Y et al. ( 1990 ) Salmonid pituitary gonadotrophs . I. Distinct cellular distributions of 2 gonadotropins , GTH I and GTH II. Gen Comp Endocrinol 77 : 348 - 357 . doi:10.1016/ 0016 -6480(90) 90224 -A. PubMed: 2186958 .
9. Weltzien FA , Kobayashi T , Andersson E , Norberg B , Andersen O ( 2003 ) Molecular characterization and expression of FSH beta, LH beta, and common alpha-subunit in male Atlantic halibut (Hippoglossus hippoglossus) . Gen Comp Endocrinol 131 : 87 - 96 . doi:10.1016/ S0016-6480(02)00606-8 . PubMed: 12679085 .
10. Zhang C , Forlano PM , Cone RD ( 2012 ) AgRP and POMC neurons are hypophysiotropic and coordinately regulate multiple endocrine axes in a larval teleost . Cell Metab 15 : 256 - 264 . doi:10.1016/j.cmet. 2011 .12.014. PubMed: 22245570 .
11. Cloonan N , Forrest AR , Kolle G , Gardiner BB , Faulkner GJ et al. ( 2008 ) Stem cell transcriptome profiling via massive-scale mRNA sequencing . Nat Methods 5 : 613 - 619 . doi:10.1038/nmeth.1223. PubMed: 18516046 .
12. Nagalakshmi U , Wang Z , Waern K , Shou C , Raha D et al. ( 2008 ) The transcriptional landscape of the yeast genome defined by RNA sequencing . Science 320 : 1344 - 1349 . doi:10.1126/science.1158441. PubMed: 18451266 .
13. Li B , Ruotti V , Stewart RM , Thomson JA , Dewey CN ( 2010 ) RNA-Seq gene expression estimation with read mapping uncertainty . Bioinformatics 26 : 493 - 500 . doi:10.1093/bioinformatics/btp692. PubMed: 20022975 .
14. Mortazavi A , Williams BA , Mccue K , Schaeffer L , Wold B ( 2008 ) Mapping and quantifying mammalian transcriptomes by RNA-Seq . Nat Methods 5 : 621 - 628 . doi:10.1038/nmeth.1226. PubMed: 18516045 .
15. Henkel CV , Burgerhout E , de Wijze DL , Dirks RP , Minegishi Y et al. ( 2012 ) Primitive duplicate Hox clusters in the European eel's genome . PLOS ONE 7: e32231. doi:10.1371/journal.pone.0032231. PubMed: 22384188.
16. Henkel CV , Dirks RP , de Wijze DL , Minegishi Y , Aoyama J et al. ( 2012 ) First draft genome sequence of the Japanese eel, Anguilla japonica . Gene 511 : 195 - 201 . doi:10.1016/j.gene. 2012 .09.064. PubMed: 23026207 .
17. Stanke M , Diekhans M , Baertsch R , Haussler D ( 2008 ) Using native and syntenically mapped cDNA alignments to improve de novo gene finding . Bioinformatics 24 : 637 - 644 . doi:10.1093/bioinformatics/btn013. PubMed: 18218656 .
18. Burgerhout E , Brittijn SA , Kurwie T , Decker P , Dirks RP et al. ( 2011 ) First artificial hybrid of the eel species Anguilla australis and Anguilla anguilla . BMC Dev Biol 11 : 16 . doi:10.1186/ 1471 - 213X - 11 -16. PubMed: 21396126 .
19. Pankhurst NW ( 1982 ) Relation of visual changes to the onset of sexual maturation in the European eel Anguilla anguilla . J Fish Biol 21 : 127 - 140 . doi:10.1111/j.1095- 8649 . 1982 .tb03994.x.
20. Durif C , Dufour S , Elie P ( 2005 ) The silvering process of Anguilla anguilla: a new classification from the yellow resident to the silver migrating stage . J Fish Biol 66 : 1025 - 1043 . doi:10.1111/j. 0022- 1112 . 2005 .00662.x.
21. Aroua S , Weltzien FA , Le Belle N , Dufour S ( 2007 ) Development of real-time RT-PCR assays for eel gonadotropins and their application to the comparison of in vivo and in vitro effects of sex steroids . Gen Comp Endocrinol 153 : 333 - 343 . doi:10.1016/j.ygcen. 2007 .02.027. PubMed: 17418843 .
22. Weltzien FA , Pasqualini C , Vernier P , Dufour S ( 2005 ) A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase . Gen Comp Endocrinol 142 : 134 - 142 . doi:10.1016/j.ygcen. 2004 .12.019. PubMed: 15862557 .
23. Trapnell C , Pachter L , Salzberg SL ( 2009 ) TopHat: discovering splice junctions with RNA-Seq . Bioinformatics 25 : 1105 - 1111 . doi:10.1093/ bioinformatics/btp120. PubMed: 19289445 .
24. Li H , Handsaker B , Wysoker A , Fennell T , Ruan J et al. ( 2009 ) The Sequence Alignment/Map format and SAMtools . Bioinformatics 25 : 2078 - 2079 . doi:10.1093/bioinformatics/btp352. PubMed: 19505943 .
25. Anders S ( 2012 ) HTSeq, version 0.5.3p Genome Biology Unit9; Heidelberg EMBL . Available: http://www-huber.embl.de/users/anders/ HTSeq/doc/overview.html. Accessed 15 March 2013 .
26. Anders S , Huber W ( 2010 ) Differential expression analysis for sequence count data . Genome Biol 11 : R106 -. PubMed: 20979621.
27. Conesa A , Gotz S , Garcia-Gomez JM , Terol J , Talon M et al. ( 2005 ) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research . Bioinformatics 21 : 3674 - 3676 . doi: 10.1093/bioinformatics/bti610. PubMed: 16081474 .
28. Minegishi Y , Aoyama J , Inoue JG , Miya M , Nishida M et al. ( 2005 ) Molecular phylogeny and evolution of the freshwater eels genus Anguilla based on the whole mitochondrial genome sequences . Mol Phylogenet Evol 34 : 134 - 146 . doi:10.1016/j.ympev. 2004 .09.003. PubMed: 15579387 .
29. Cool DR , Loh YP ( 1998 ) Carboxypeptidase E is a sorting receptor for prohormones: binding and kinetic studies . Mol Cell Endocrinol 139 : 7 - 13 . doi:10.1016/ S0303-7207(98)00081-1 . PubMed: 9705069 .
30. Benjannet S , Rondeau N , Day R , Chrtien M , Seidah NG ( 1991 ) PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues . Proc Natl Acad Sci U S A 88 : 3564 - 3568 . doi:10.1073/pnas.88.9.3564. PubMed: 2023902 .
31. Mbikay M , Seidah NG , Chrtien M ( 2001 ) Neuroendocrine secretory protein 7B2: structure, expression and functions . Biochem J 357 : 329 - 342 . doi:10.1042/ 0264 - 6021 : 3570329 . PubMed: 11439082 .
32. Lee SN , Lindberg I ( 2008 ) 7B2 prevents unfolding and aggregation of prohormone convertase 2 . Endocrinology 149 : 4116 - 4127 . doi: 10.1210/en.2008- 0064 . PubMed: 18467442 .
33. Weltzien FA , Andersson E , Andersen O , Shalchian-Tabrizi K , Norberg B ( 2004 ) The brain-pituitary-gonad axis in male teleosts, with special emphasis on flatfish (Pleuronectiformes) . Comp Biochem Physiol A Mol Integr Physiol 137 : 447 - 477 . doi:10.1016/j.cbpb. 2003 .11.007. PubMed: 15123185 .
34. Bullard JH , Purdom E , Hansen KD , Dudoit S ( 2010 ) Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments . BMC Bioinformatics 11 : 94. doi: 10.1186/ 1471 - 2105 - 11 -94. PubMed: 20167110 .
35. Wendelaar Bonga SE ( 1997 ) The stress response in fish . Physiol Rev 77 : 591 - 625 . PubMed: 9234959 .
36. Ardehali MB , Lis JT ( 2009 ) Tracking rates of transcription and splicing in vivo . Nat Struct Mol Biol 16 : 1123 - 1124 . doi:10.1038/ nsmb1109- 1123 . PubMed: 19888309 .
37. Van Ginneken V , Durif C , Balm SP , Boot R , Verstegen MWA et al. ( 2007 ) Silvering of European eel (Anguilla anguilla L.): seasonal changes of morphological and metabolic parameters . Anim Biol 57 : 63 - 77 . doi:10.1163/157075607780002014.
38. Palstra A , Van Ginneken V , Van den Thillart G ( 2009 ) Effects of swimming on silvering and maturation of the European eel , Anguilla anguilla L. In: G Van den ThillartS DufourJC Rankin. Spawning migration of the European eel . Springer Verlag. pp. 229 - 251 .
39. Seidah NG , Chrtien M ( 1997 ) Eukaryotic protein processing: endoproteolysis of precursor proteins . Curr Opin Biotechnol 8 : 602 - 607 . doi:10.1016/ S0958-1669(97)80036-5 . PubMed: 9353231 .
40. Lee J , Danielson H , Sollars C , Alrubaian J , Balm P et al. ( 1999 ) Cloning of a neoteleost (Oreochromis mossambicus) proopiomelanocortin (POMC) cDNA reveals a deletion of the gamma-melanotropin region and most of the joining peptide region: implications for POMC processing . Peptides 20 : 1391 - 1399 . doi:10.1016/ S0196-9781(99)00148-5 . PubMed: 10698113 .
41. Pritchard LE , Turnbull AV , White A ( 2002 ) Pro-opiomelanocortin processing in the hypothalamus: impact on melanocortin signalling and obesity . J Endocrinol 172 : 411 - 421 . doi:10.1677/joe.0.1720411. PubMed: 11874690 .
42. Tanaka S ( 2003 ) Comparative aspects of intracellular proteolytic processing of peptide hormone precursors: studies of proopiomelanocortin processing . Zool Sci 20 : 1183 - 1198 . doi:10.2108/ zsj.20.1183. PubMed: 14569141 .
43. Cool DR , Normant E , Shen FS , Chen HC , Pannell L et al. ( 1997 ) Carboxypeptidase E is a regulated secretory pathway sorting receptor: Genetic obliteration leads to endocrine disorders in Cpe(fat) mice . Cell 88 : 73 - 83 . doi:10.1016/ S0092-8674(00)81860-7 . PubMed: 9019408 .
44. Lindberg I , Van den Hurk WH , Bui C , Batie CJ ( 1995 ) Enzymatic characterization of immunopurified prohormone convertase 2: potent inhibition by a 7B2 peptide fragment . Biochemistry 34 : 5486 - 5493 . doi: 10.1021/bi00016a020. PubMed: 7727407 .
45. Arvan P , Kuliawat R , Prabakaran D , Zavacki AM , Elahi D et al. ( 1991 ) Protein discharge from immature secretory granules displays both regulated and constitutive characteristics . J Biol Chem 266 : 14171 - 14174 . PubMed: 1860833 .
46. Kirchmair R , Hogue-Angeletti R , Gutierrez J , Fischer-Colbrie R , Winkler H ( 1993 ) Secretoneurin - a neuropeptide generated in brain, adrenal medulla and other endocrine tissues by proteolytic processing of secretogranin II (chromogranin C) . Neuroscience 53 : 359 - 365 . doi: 10.1016/ 0306 -4522(93) 90200 - Y . PubMed: 8492910 .
47. Fischer-Colbrie R , Laslop A , Kirchmair R ( 1995 ) Secretogranin-II - molecular properties, regulation of biosynthesis and processing to the neuropeptide secretoneurin . Prog Neurobiol 46 : 49 - 70 . doi: 10.1016/ 0301 -0082(94)00060-U. PubMed: 7568909 .
48. Zhao E , Basak A , Trudeau VL ( 2006 ) Secretoneurin stimulates goldfish pituitary luteinizing hormone production . Neuropeptides 40 : 275 - 282 . doi:10.1016/j.npep. 2006 .05.002. PubMed: 16806466 .
49. Holthuis JCM , Jansen EJR , Martens GJM ( 1996 ) Secretogranin III is a sulfated protein undergoing proteolytic processing in the regulated secretory pathway . J Biol Chem 271 : 17755 - 17760 . doi:10.1074/jbc. 271.30.17755. PubMed: 8663421 .
50. Hosaka M , Watanabe T , Sakai Y , Kato T , Takeuchi T ( 2005 ) Interaction between secretogranin III and carboxypeptidase E facilitates prohormone sorting within secretory granules . J Cell Sci 118 : 4785 - 4795 . doi:10.1242/jcs.02608. PubMed: 16219686 .
51. Holthuis JCM , Martens GJM ( 1996 ) The neuroendocrine proteins secretogranin II and III are regionally conserved and coordinately expressed with proopiomelanocortin in Xenopus intermediate pituitary . J Neurochem 66 : 2248 - 2256 . PubMed: 8632145 .
52. Postlethwait J , Amores A , Cresko W , Singer A , Yan YL ( 2004 ) Subfunction partitioning, the teleost radiation and the annotation of the human genome . Trends Genet 20 : 481 - 490 . doi:10.1016/j.tig. 2004 .08.001. PubMed: 15363902 .
53. Metz JR , Peters JJM , Flik G ( 2006 ) Molecular biology and physiology of the melanocortin system in fish: a review . Gen Comp Endocrinol 148 : 150 - 162 . doi:10.1016/j.ygcen. 2006 .03.001. PubMed: 16620815 .
54. Volkoff H , Canosa LF , Unniappan S , Cerd-Reverter JM , Bernier NJ et al. ( 2005 ) Neuropeptides and the control of food intake in fish . Gen Comp Endocrinol 142 : 3 - 19 . doi:10.1016/j.ygcen. 2004 .11.001. PubMed: 15862543 .
55. Takahashi A , Kawauchi H ( 2006 ) Evolution of melanocortin systems in fish . Gen Comp Endocrinol 148 : 85 - 94 . doi:10.1016/j.ygcen. 2005 .09.020. PubMed: 16289182 .
56. Coll AP , Loraine Tung YC ( 2009 ) Pro-opiomelanocortin (POMC)- derived peptides and the regulation of energy homeostasis . Mol Cell Endocrinol 300 : 147 - 151 . doi:10.1016/j.mce. 2008 .09.007. PubMed: 18840502 .
57. Yada T , Morlyania S , Moriyama S , Suzuki Y , Azuma T et al. ( 2002 ) Relationships between obesity and metabolic hormones in the 'cobalt' variant of rainbow trout . Gen Comp Endocrinol 128 : 3643 .
58. Han YS , Liao IC , Huang YS , He JT , Chang CW et al. ( 2003 ) Synchronous changes of morphology and gonadal development of silvering Japanese eel Anguilla japonica . Aquaculture 219 : 783 - 796 . doi:10.1016/S0044-8486(02)00578- 1 .
59. Pankhurst NW , Sorensen PW ( 1984 ) Degeneration of the alimentary tract in sexually maturing European Anguilla anguilla (L.) and American eels Anguilla rostrata (LeSueur) . Can J Zool 62 : 1143 - 1149 . doi: 10.1139/z84- 165 .
60. Heinsbroek LTN , Van Hooff PLA , Swinkels W , Tanck MWT , Schrama JW et al. ( 2007 ) Effects of feed composition on life history developments in feed intake, metabolism, growth and body composition of European eel, Anguilla anguilla . Aquaculture 267 : 175 - 187 . doi: 10.1016/j.aquaculture. 2007 .03.028.
61. Mizusawa K , Kobayashi Y , Sunuma T , Asahida T , Saito Y et al. ( 2011 ) Inhibiting roles of melanin-concentrating hormone for skin pigment dispersion in barfin flounder, Verasper moseri . Gen Comp Endocrinol 171 : 75 - 81 . doi:10.1016/j.ygcen. 2010 .12.008. PubMed: 21185295 .
62. Rousseau K , Aroua S , Schmitz M , Elie P , Dufour S ( 2009 ) Silvering: metamorphosis or puberty? In: G Van den ThillartS DufourJC Rankin. Spawning migration of the European eel . Springer Verlag. pp. 39 - 63 .
63. Huang YS , Rousseau K , Sbaihi M , Le Belle N , Schmitz M et al. ( 1999 ) Cortisol selectively stimulates pituitary gonadotropin beta-subunit in a primitive teleost, Anguilla anguilla . Endocrinology 140 : 1228 - 1235 . doi: 10.1210/en.140.3.1228. PubMed: 10067848 .
64. Yasuda A , Tatsu Y , Kawata Y , Akizawa T , Shigeri Y ( 2011 ) Posttranslational modifications of pro-opiomelanocrtin related hormones in medaka pituitary based on mass spectrometric analyses . Peptides 32 : 2127 - 2130 . doi:10.1016/j.peptides. 2011 .08.016. PubMed: 21889556 .
65. Nakanishi S , Inoue A , Kita T , Nakamura M , Chang AC et al. ( 1979 ) Nucleotide sequence of cloned cDNA for bovine corticotropin-betalipotropin precursor . Nature 278 : 423 - 427 . doi:10.1038/278423a0. PubMed: 221818 .
66. Alrubaian J , Sollars C , Danielson PB , Dores RM ( 2003 ) Evaluating the radiation of the POMC gene in teleosts: characterization of American eel POMC . Gen Comp Endocrinol 132 : 384 - 390 . doi:10.1016/ S0016-6480(03)00119-9 . PubMed: 12849961 .
67. Cone RD ( 2006 ) Studies on the physiological functions of the melanocortin system . Endocr Rev 27 : 736 - 749 . PubMed: 17077189 .