Growth Promoting Effect of Hyaluronan Synthesis Promoting Substances on Japanese Eel Leptocephali
Tanaka H (2014) Growth Promoting Effect of Hyaluronan Synthesis Promoting Substances on Japanese Eel Leptocephali. PLoS
ONE 9(6): e98688. doi:10.1371/journal.pone.0098688
Growth Promoting Effect of Hyaluronan Synthesis Promoting Substances on Japanese Eel Leptocephali
Yutaka Kawakami 0
Kazuharu Nomura 0
Hideki Tanaka 0
Ted S. Acott, Casey Eye Institute, United States of America
0 Nansei Station, National Research Institute of Aquaculture, Fisheries Research Agency , Minamiise , Japan
Hyaluronans (HAs) are glycosaminoglycans produced in the bodies of Anguilliform and Elopiform leptocephali, and play a role in metabolic energy. In mammals, HA synthesis-promoting substances (HASPS) up-regulate the expression of HA synthase (HAS) and increase the amount of HA in the body. In this study, Japanese eel leptocephali were fed a HASPS containing diet. We analyzed HAS1s and HAS2 expression, HA content, and their influence on growth. HASPS extracted from Grifola frondosa promoted HAS1s and HAS2 mRNA and HA content. Other than mammals, these results are first reported in vertebrate. Moreover, HASPS extracted from G. frondosa promoted leptocephalus growth. The relationship between growth and HA in the leptocephali is not yet clear. However, based on our results we hypothesize that HA is involved in the storage of energy, which is metabolized to sugars when needed for metabolic energy.
The Japanese eel (Anguilla japonica) is an important commercial
species in Japan owing to its high market value as a food source. In
2010, successful closed-cycle breeding of the Japanese eel was
reported . However, production of artificial seeding for industry
has not been established. To culture Japanese eel, 100% natural
glass-eels, which migrate to the Japanese coast and collected in the
rivers, are used for seeding. Closed-cycle breeding fish show
improved growth and easier breeding compared with wild fish.
Kawakami et al. ,  reported that wild Japanese eel larvae,
leptocephali, start metamorphosing at 4- to 5-months-old using
otolith daily increment analysis. In contrast, cultured
leptocephalus metamorphosis begins at more than 200 days post hatching
(dph) , . The average duration from hatched larvae to
glasseel is about 299 days (minimum to maximum: 153754 days) ;
this is longer in cultured than in wild leptocephali. Low growth
rate is considered to be one of the reasons for this phenomenon. In
the ocean, Japanese leptocephali feed on readily available
particulate material originating from various sources closely linked
to ocean primary production . The artificial diet for cultured
leptocephali is based on shark eggs , . The present breeding
system, including the artificial diet, does not reflect the eels
natural environment. The annual production of glass-eel in recent
years has been less than 1,000 individuals in Japan . For
largescale glass-eel production, shortening of the breeding duration is
desirable; to do this, development of a new breeding system and/
or upgrading the present breeding system is necessary.
Hyaluronan (hyaluronic acid, HA), a high-molecular-weight
linear glycosaminoglycan (GAG) consisting of alternating
glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) residues, is a
major component of most extracellular matrices , .
Accumulation of HA is correlated with cell proliferation and
migration in several developing tissues and organs , .
Moreover, HA plays a role in tissue water homeostasis .
Anguilliform and Elopiform leptocephali produce GAGs; most of
which are HAs . In the Japanese conger eel (Conger myriaster),
about 50% of its dry body weight is HA, which degrades with body
water content during metamorphosis . It is notable that in
bonefish (Albula sp.) leptocephali some metabolic energy is
provided by GAGs during metamorphosis . In short, it may
be possible that HA in Japanese eel leptocephali also plays a role in
storing polysaccharides as glycogen. Furthermore, by enhancing
HA synthesis, it may be possible to enhance Japanese eel
In a previous study, Grifola frondosa extract enhanced hyaluronan
synthetase (HAS) and HA in human cutaneous fibroblasts in vitro
. In addition, some seaweed extracts also enhanced HAS and
HA in rat cutaneous primary cells in culture ; however, it is
not known if these extracts enhance HA synthesis in teleosts. The
aim of this study is to elucidate whether or not administration of G.
frondosa extract by feeding enhances HA synthesis and influences
the growth associated with it in Japanese eel leptocephali. First, we
cloned Japanese eel HAS genes and analyzed their function.
Second, we estimated hyaluronan synthesis enhancement by G.
frondosa extract by feeding the extract to first feeding larvae and
investigated HAS gene expression patterns. Finally, we assessed
the influence of HA accumulation on larval growth through long
term feeding experiments with G. frondosa extract.
Results and Discussion
HA is synthesized by integral plasma membrane
glycosyltransferases and is exported directly into the extracellular space. Three
distinct yet highly conserved genes encoding HAS, HAS1 ,
HAS2 , , and HAS3  were cloned. The three gene
products are similar in amino acid sequence and molecular
structural characteristics. In mammals, three HASs synthesize HA;
however, HAS activity differs between the three . The eHAS1
and eHAS2 nucleotide and deduced amino acid sequences are
shown in Figures 1 and 2. The cDNA encoding eHAS1 contains a
complete putative open reading frame of 1,701 bp, which encodes
a putative protein of 567 amino acid residues. Another type of
eHAS1, a splice variant named eHAS1L, has a 35 amino acid
insertion. The cDNA encoding eHAS2 contains an open reading
frame of 1,656 bp, encoding 552 amino acid residues. When the
amino acid sequence corresponding to the Japanese eel genes were
compared with that of other known HAS genes, the proteins
exhibited the highest homology to teleost HAS1 and HAS2
(Figure 3). Moreover, eHAS1 and eHAS2 induce HA synthesis
Figure 5 shows the changes in eHAS1 and eHAS2 mRNA
expression levels after being fed a diet both with and without
Grifola frondosa extract, which is a HA synthesis-promoting
substance (HASPS). Initially, when first-feeding larvae did not
feed, eHAS1 and eHAS2 mRNA levels were low. As for eHAS1s,
they remained the same after the eels were fed the control diet.
eHAS1 mRNA expression was elevated after they were fed the G.
frondosa extract diet, and peaked after 20 min. Between 10 min
and 2 h eHAS1 mRNA expression was higher in eels fed the G.
frondosa extract diet than in those fed the control diet. In contrast,
eHAS2 was elevated after they were fed the control diet, continued
to increase for 30 min, and subsequently decreased after 1 h. In
teleosts, the relationship between HAS synthesis and feeding is
unknown; however, from this result, it seems possible that feeding
activity and/or this diet with added maltose activates eHAS2
expression. In contrast, there was no significant difference in
eHAS2 mRNA expression between the control diet and G. frondosa
extract diets after 10, 20, or 30 min. After 1, 2, and 4 h eHAS2
mRNA expression was higher in eels fed the G. frondosa extract diet
than in those fed the control diet. The addition of G. frondosa
extract to human skin fibroblast cells in vitro activates HAS2
mRNA expression and secretes increasing quantities of HA .
In this experimental model, if we fed the G. frondosa extract diet to
first-feeding larvae, eHAS1 (containing eHAS1L) mRNA
expression was higher than in the control diet for 2 h and high eHAS2
mRNA expression continued longer than in the control diet
(Figure 5). Moreover, larval HA content was significantly
increased by G. frondosa extract (Figure 6). This is the first report
in non-mammal vertebrates that HASPS increase the amount of
HA in the body. Based on our results, we speculate that the
longrange activity of eHASs, at least eHAS1 and eHAS2, promote HA
At present, the exact substance, including HASPS extracted
from G. frondosa or seaweeds, that stimulates HA production is
unknown. The existence of glycerophospholipids in G. frondosa has
been reported previously . The addition of phosphatidylserine
and/or phosphatidylinositol to human fibroblast cells in vitro
significantly increases HAS2 mRNA expression and HA content
. It is highly possible that glycerophospholipid activates HA
synthesis in Japanese eel larvae. However, this requires further
work as the exact mechanism is unknown.
Figures 7 shows the survival rate and growth (TL and BD)
results from the larvae fed the HASPS derived from G. frondosa
extract. As for survival rates, we did not see a significant difference
between the control and the HASPS. However, TL and BD in the
HASPS experiment exhibited a significant increase compared with
those of the control [G. frondosa extract (2 mg/g) vs. control]. A
previous study, using bonefish (Albula sp.) leptocephali, looked at
energy budgets during metamorphosis, part of the energy was
provided by GAGs  and it was suggested that HA is a major
energy source . The relationships between accumulated HA
and growth in vertebrates is unknown. In this study, HASPS
enhanced leptocephalus growth; however, it is not clear whether
or not accumulated HA in the body directly enhanced
leptocephalus growth. Figure 8 shows the interrelationships in HA
metabolism . HA is synthesized in UDP-GlcNAc and
UDPGlcUA by HAS , moreover, glucose is the precursor of
UDPGlcNAc and UDP-GlcUA. We hypothesize that a reversible
relationship exists between neutral sugars and HA. In other words,
in leptocephali, HA stored energy sources are metabolized to
sugars when metabolic energy is needed. In our style of seeding
culture for Japanese eel, it is difficult for leptocephali to feed
continuously. Because the slurry-type diet causes deterioration of
the culture water , we must wash away the food immediately
after feeding. Assuming that HA is a source of stored energy, this
style of feeding may be advantageous for larval growth. In other
words, feeding style might have caused the differences in
leptocephalus growth whether or not HASPS were added.
Materials and Methods
Grifola frondosa extract
Commercially-supplied Grifola frondosa fruit bodies were
prepared and left to dry naturally. When dried, G. frondosa was mixed
with 10 times its mass of 100% ethanol and shaken overnight at
room temperature (RT). The extracted ethanol was filtered with
filter paper and freeze-dried in a freeze dryer.
Japanese eel larvae
Cultivated adult male Japanese eels (150200 g body mass)
were purchased from a commercial supplier. As for the adult
female supply, glass-eels from a commercial eel supplier were
feminized by feeding them estradiol-17b (Sigma, St. Louis, MO).
The fish were kept at the Nansei Station, National Research
Institute of Aquaculture, Fisheries Research Agency. Artificial
maturation was carried out by hormone treatment as previously
described , . Females were repeatedly injected with
salmon pituitary extract, followed by injection with
17a-hydroxyprogesterone (Sigma). Similarly, males purchased from a
commercial supplier were injected with human chorionic
gonadotropin (ASKA Pharmaceutical Co. Ltd., Tokyo, Japan). The above
hormone treatments were performed according to Kagawa et al.
. The gametes were obtained by gently stripping ovulating
females and mature males.
Larvae hatched fertilized eggs were maintained in an acrylic
tank at 25uC with running seawater. After the first-feeding [7 or 8
days post hatching (dph)], a slurry-type diet consisting of shark
eggs, soybean peptides (Fuji Oil Co. Ltd., Osaka, Japan), krill
hydrolysate (Nippon Suisan Kaisha, Ltd., Tokyo, Japan) and krill
extract ,  was given to the larvae 5 times a day at 2-h
intervals from 9 am to 5 pm. Fully-grown leptocephali were
sampled, frozen in liquid nitrogen, and stored at 280uC until
Reverse transcription (RT)-PCR and cDNA cloning of
Japanese eel hyaluronan synthase 1 and 2
Total RNA was extracted from fully-grown leptocephali using
Isogen (Nippon Gene, Tokyo, Japan). Poly (A) + RNA was
subsequently isolated from total RNA using Oligotex-dt-30
(Takara, Otsu, Japan). Isolated RNA was denatured at 70uC for
10 min, placed on ice, and reverse transcribed with M-MLV.
Second-strand cDNA was synthesized and single-strand overhands
were removed, using Takaras cDNA cloning system (Takara).
Japanese eel hyaluronan synthase 1 (eHAS1) and 2 (eHAS2)
cDNA fragments were amplified using sense and antisense
degenerate primers designed based on a consensus sequence from
the aligned deduced amino acid sequences of HAS from several
vertebrate species (Table 1). PCR was carried out in a final volume
of 50 ml containing 0.51 pg cDNA, 400 nM of each primer,
800 mM of each dNTP, and 2.5 U Ex Taq (Takara). PCR was
carried out for 35 cycles in a Thermal Cycler Dice Gradient
(Takara) under the following conditions: denaturation at 94uC for
30 s, annealing at 5055uC for 30 s, and extension at 72uC for 20
30 s. PCR products were separated by 1% agarose gel
electrophoresis, and selected bands were cut out and purified with a
QiAprep Spin Miniprep Kit (Qiagen, Venlo, the Netherlands).
Purified DNA fragments were subcloned into the plasmid vector
pGEM-T Easy (Promega, Madison, WI, USA), and positive clones
were sequenced with a Big Dye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and
an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).
59 and 39 rapid amplification of cDNA ends (RACE)-PCR
Fully-grown leptocephali were used for the construction of
cDNA for RACE-PCR with a SMART RACE cDNA
Amplification Kit (Clontech, Palo Alto, CA). For both 39-RACE and the
59-RACE, nested primers (eHAS-3RACE-S1, -S2 and
eHAS5RACE-A1 and -A2, respectively) were designed from eHAS
cDNA fragments (Table 1). Based on the eHAS cDNA fragments
amplified by 59- and 39-RACE-PCR, sense (eHAS-UTR-S) and
antisense (eHAS-UTR-A) primers were designed for the
untranslated regions (Table 1). PCR was carried out as described above.
These PCR products were sequenced following the method
described above, and eHAS cDNA sequences containing the
entire open reading frame were obtained. eHAS cDNA products
representing independent, full-length PCR clones were each
sequenced 5 times to detect PCR errors.
The GenBank accession numbers of the sequences compared
with our eHAS1s and eHAS2 sequence were: eHAS1, eHAS1L,
and eHAS2 (AB901107, AB901106, and AB823817), zebrafish
(Danio rerio) HAS1, HAS2, and HAS3 (NM_001164030,
AF190742, and AF190743), Nile Tilapia (Oreochromis niloticus)
HAS1-like, HAS2-like, and HAS3 (XM_003458416,
XM_003443695, and XM_003449190), mouse (M. musculus)
HAS1, HAS2, and HAS3 (NM_008215, NM_008216, and
A phylogenetic tree was constructed using the neighbor-joining
method . For this analysis, 1,000 bootstrap replicates were
carried out using ClustalW version 2.1 (DNA Data Bank of Japan,
Construction and transfection of eHAS1 and eHAS2
Single-stranded cDNA was prepared from the poly(A)+ -RNA
of Japanese eel tissue by the method described above. The entire
eHAS coding regions were amplified by PCR using primers that
introduced a 15 bp (59-TTTAAACTTAAGCTT-39) pcDNA3.1
(+) (Invitrogen) sequence at the 59 end (pcDNA-eHAS-S), a 15 bp
sequence (59-TGGACTAGTGGATCC-39) at the 39 end
(pcDNA-eHAS-A), and Ex-Taq (Takara) (Table 1). The eHAS
fragments were inserted into pcDNA3.1(+), which contains the
cytomegalovirus (CMV) promoter upstream of the inserted
cDNAs (pcDNA-eHAS). The BamHI and HindIII sites were
digested with an In-Fusion HD cloning Kit (Takara). The cDNA
products were sequenced and we confirmed that there were no
errors arising from the PCR.
Hepa-E1 cells, epithelial-like Japanese eel hepatocytes that have
no TH deiodinase activity , were obtained from the Institute of
Physical and Chemical Research (RIKEN) cell bank (Tsukuba,
Japan). Hepa-E1 cells were seeded in 48-well plates at densities of
0.46105 cells/well in an E-RDF medium (Kyokuto, Tokyo, Japan)
supplemented with 5% fetal bovine serum (FBS) (Sigma). After a
further 24 h of incubation, the cells were transfected with 300 ng
pcDNA-eHAS using X-treme GENE 9 DNA Transfection
Reagent (Roche, Mannheim, Germany). The transfected
HepaE1 cells were cultured for 1 day in the above medium with FBS
containing 1 mM Uridine diphosphate (UDP)-GlcUA (Nacalai
Tesque, Kyoto, Japan) and 1 mM UDP-GlcNAc (Sigma) at 28uC.
eHAS synthesized HA
HA synthesized by the eHAS transfectant was analyzed
according to Kawakami et al. . After 2 days of transfection,
the removed medium was mixed with 20 mg/ml actinase E (Kaken
Pharmaceutical, Tokyo, Japan), and incubated at 50uC for 24 h.
The mixture was boiled for 10 min and then centrifuged at
5,0006g for 10 min. The supernatant was then used for HA
content measurement using an assay kit (Funakoshi, Tokyo,
Japan). The intra-assay coefficients of variation were 0.05.0%.
Experiment 1: Culture experiment: eHAS analysis of
firstfeeding larvae fed a G. frondosa extraction diet
The first-feeding larvae (7 dph) were fed the slurry-type diet,
which consisted of 200 mg of G. frondosa extract and 2 g maltose
(Wako, Tokyo, Japan) diluted in a measuring cylinder to 10 mL
with 0.8% xanthan gum (Wako). For the control, 2 g maltose
(Wako) diluted in a measuring cylinder to 10 mL with 0.4%
xanthan gum (Wako) was supplied.
About 2,000 larvae at 7 dph, the duration of first feeding, were
moved to a 10-L acrylic tank and fed the slurry-type diet on the
bottom of the tank as described by Tanaka et al. . After 15 min
the feed was washed out. Subsequently, approximately 100200
larvae were sampled after 10, 20, 30 min, 1, 2, 4, and 8 h into
RNAlater (Ambion, Austin, TX, USA) and stored at 220uC until
Experiment 1: Real-time RT-PCR: eHAS analysis of
firstfeeding larvae fed the G. frondosa extraction diet
Real-time RT-PCR analyses were performed using a MyiQ
Real-Time Detection System (Bio-Rad, Hercules, CA, USA).
Specific primers for eHAS1 and eHAS2 were designed based on
the cDNA nucleotide sequence analysis in this study (Table 1). As
Figure 7. Effects of Grifola frondosa extract on survival rate (A), total length (TL) (B), and body depth (BD) (C) at 27 dph. Cont: control
diet (a normal slurry-type diet), Low: 0.2 mg/g G. frondosa extract in a slurry-type diet, High: 2 mg/g G. frondosa extract in a slurry-type diet. Each
value of B and C represents the mean 6 SDM. *indicates significant differences between the Control, Low, and High diets (Tukey-Kramer HSD test, B:
p,0.05, C: p,0.01).
the primers designed for eHAS1 amplify both eHAS1 and a splice
variant of eHAS1 (eHAS1L), the real-time RT-PCR method
estimates the relative abundance of mRNA for both eHAS1s.
Total RNA from 30 to 50 embryos/fish, was extracted
according to Kawakami et al. , . Synthesis of first-strand
cDNA was carried out as follows: 100 ng of isolated total RNA
was reverse-transcribed with a SuperScript VILO cDNA synthesis
Kit (Invitrogen, Carlsbad, CA, USA).
As a standard, appropriate sizes of pcDNA3.1(+) (Invitrogen)
fragments flanked by primer-binding sites were prepared by PCR
as above. PCR fragments for the standard were discriminated by
gel electrophoresis and purified with a QIAquick Gel Extraction
kit (Qiagen, Hilden, Germany). Purified PCR fragments were
quantified by UV260 absorbance and a 1/100 dilution series was
constructed (109101 copies/ml). RT-PCRs and a dilution series of
standard samples were prepared according to the manufacturers
protocol to include: 1 ng of cDNA, 5 ml of SsoAdvanced SYBR
Green Supermix (Bio-Rad), and 500 nM primers in a final volume
of 10 ml. DNA amplifications were performed in duplicate
(standards: triplicate) under the following conditions: 30 s at
95uC, followed by 40 cycles of 5 s of denaturation at 95uC, 10 s of
annealing, and extension at 60uC for 20 s. Standard templates
were used, in triplicate, to construct a standard curve ranging from
101 to 109 copies. The linear range of the curve fell within 101109
copies and the correlation coefficients of variation were greater
than 0.997 for all curves. The intra-assay coefficients of variation
were 4.822.2%. Inter-assay coefficients of variation were under
10.0% for analysis.
Primers sequences (59-39)
ATTTGCTCCACCTGGTCTGCTGGTT ATTTTGTGGCATACCCCAGGCTCAG TGCCAGTCTTATTTTGGGTGTGTCCAGT TCAACCAGCAGACCAGGTGGAGC
TGGGCTCTTGTCATCCGTAAAAATGTCGTAACAACTCCG TGTGCAATTTCCACGTTTTCACCAAGCTTAAGTTTAAACG TGGGCTCTTGTCATCCGTA TGTGCAATTTCCACGTTTTC
TTTAAACTTAAGCTTGCGCAATAAA ATACCGGTCT A
TGGACTAGTGGATCCGATAGGGTTTACAGAGGGACA Nucleotide numbers corresponding to the annealing site
26682695 bp (Fig. 1)
20742094 bp (Fig. 2)
13491373 bp (Fig. 1)
10991121 bp (Fig. 1)
11301152 bp (Fig. 1)
14561480 bp (Fig. 2)
13751399 bp (Fig. 2)
12191246 bp (Fig. 2)
14541476 bp (Fig. 2)
24862505 bp (Fig. 1)
26272646 bp (Fig. 1)
DPs (degenerate primers), primers for amplification of eHAS fragments; eHAS-UTRs (untranslated regions), sense and antisense primers for the sequencing of eHAS
containing the open reading frame; eHAS-3RACE, eHAS-5RACE, sense, and antisense primers for the sequencing of 39- and/or 59-RACE analysis of eHAS. eHAS-ST
(standard), sense, and antisense primers standard real-time RT-PCR analysis of eHAS; eHAS, sense, and antisense primers for real-time RT-PCR analysis of eHAS;
pcDNAeHAS, sense, and antisense primers for construction of eHAS expression when inserted into the pcDNA3.1(+) vector.
Experiment 2: Culture experiment: Effects of G. frondosa
extract on larval growth and HA content
For the culture experiment using first-feeding larvae (7 dph), we
prepared a slurry-type diet, as above, adding G. frondosa extract (0.2
or 2 mg/g). First-feeding larvae (7 dph), 200 per tank, were moved
to a 5-L acrylic tank at 25uC with running seawater. The above
mentioned slurry-type diet was given to the larvae 5 times a day at
2-h intervals from 9 am to 5 pm. Each experiment was duplicated
and feeding continued for 20 days (7 to 26 dph). After the culture
experiment, the total larval length (TL) and body depth (BD) were
measured, about 20 to 30 larvae per a lot were sampled, frozen in
liquid nitrogen, and stored at 280uC until required.
Experiment 2: HA analysis: Effects of G. frondosa extract
on larval growth and HA content
A sample of 20 to 30 larvae was treated according to Kawakami
et al. . Treated samples (in duplicate) were then used for HA
content measurement using an assay kit (Funakoshi). The
intraassay coefficients of variation were 0.45.5%.
eHAS1s and eHAS2 expression data were arcsine transformed
(!%). The difference in HA synthesis activity of transfected eHAS1
and eHAS2 for Hepa-E1 cells and the effect of Grifola frondosa
extract on HA content, total length, and body depth at 27 dph
were analyzed by one-way ANOVA and the Tukey-Kramer
honestly significant difference (HSD) post hoc test for individual
analyses. eHAS mRNA expression levels after being fed a
slurrytype diet with Grifola frondosa extract was subjected to
MannWhitney U tests.
We are deeply grateful to Mrs. Jyunko Kawaguchi, Mrs. Miwako Seko,
Mrs. Miki Nakamura, and Mrs. Tomoko Kobayashi at the National
Conceived and designed the experiments: YK. Performed the experiments:
YK. Analyzed the data: YK. Contributed reagents/materials/analysis
tools: YK KN HT. Wrote the paper: YK.
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