Hindawi Publishing Corporation
Research Letters in Biochemistry
Volume 2009, Article ID 251731, 5 pages
Metabolismof aGlycosaminoglycanduringMetamorphosis in
1Nanae Fresh Water Laboratory, Field Science Center of Northern Biosphere, Hokkaido University, Nanae, 041-1105 Hokkaido, Japan
2Department of Fisheries, Graduate School of Agriculture, Kinki University, Nara, 631-8505 Nara, Japan
3Nikko Station, National Research Institute of Fisheries Science, Fisheries Research Agency, Nikko, 321-1661 Tochigi, Japan
4Nansei Station, National Research Institute of Aquaculture, Fisheries Research Agency, Minamiise, 516-0193 Mie, Japan
5Hyogo Prefectural Fisheries Experimental Station, Akashi, 674-0093 Hyogo, Japan
Correspondence should be addressed to Yutaka Kawakami, email@example.com
Received 26 May 2009; Accepted 5 July 2009
Recommended by Robert J. Linhardt
Hyaluronan (HA) is a linear polysaccharide of high molecular weight that exists as a component of the extracellular matrix. The
larvae (leptocephali) of the Japanese conger eel (Anguilliformes: Conger myriaster) have high levels of hyaluronan (HA) which is
thought to help control body water content. We isolated glycosaminoglycans (GAGs) from Japanese conger eel leptocephali and
measured the changes in tissue HA content during metamorphosis. HA content decreased during metamorphosis. In contrast,
neutral sugar content increased during metamorphosis. We hypothesize that the leptocephali utilize a metabolic pathway that
converts HA to glucose during metamorphosis. Glucose may then be metabolized to glycogen and stored in the juvenile life-
Copyright © 2009 Yutaka Kawakami et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
A number of teleost species undergo an ontogenetic trans-
formation (metamorphosis) from the larval to juvenile form
during their transition to a new habitat [1, 2]. For example,
the morphological changes in eels (Anguilliformes: meta-
morphosis from the leptocephali to the elver)  parallel the
changes seen in many amphibians during metamorphosis.
Larvae of the super order Elopomorpha (Albuliformes,
Anguilliformes, Elopiformes, Notacanthiformes, and Sac-
copharyngiformes) are termed leptocephali. This stage is
characterized by a transparent body, small head, leaf-
like shape , and the presence of an inert gelatinous
material known as glycosaminoglycan (GAG). GAGs are
long, unbranched polysaccharides that contain repeating
disaccharide units. The role of GAGs is poorly understood
in teleosts, including Anguilliformes leptocephali.
Glass-eels of the Japanese eel (Anguilla japonica) are
known to store energy in the peritoneal cavity . It is
thought that this metabolized energy is stored during their
planktonic life-history stage. To determine whether GAGs
are an important source of energy during this period, we
measured the GAG content of Japanese conger eel lepto-
cephali during metamorphosis. In addition, we investigated
the tissue distribution and changes in hyaluronan content.
2.1. Animals. We collected fully grown leptocephali of the
Japanese conger eel from the Seto Inland Sea, Japan. The
leptocephali were reared, without feed, in a 40L tank. The
elvers and metamorphosing individuals were collected at
various stages throughout rearing and processed as outlined
below. We divided the metamorphosis from fully grown
leptocephali to elvers (juvenile stage) into five stages based
on the description of Kawakami et al.  (Figure 1).
2.2. Isolation of GAGs. We dissected the body wall of each
animal and froze the tissue in liquid nitrogen. The tissue was
then ground into a powder using a pestle and mortar. The
powder was mixed with 10mL ice-cold toluene, centrifuged
2 Research Letters in Biochemistry
Figure 1: Developmental stages of the Japanese conger eel during
metamorphosis. St.1, fully grown leptocephali; St.2, prophase of
metamorphosis; St.3, metamorphic climax; St.4, glass-eel; and St.5,
Figure 2: Cellulose acetate electrophoresis of GAGs extracted
from leptocephali and glass-eels of the Japanese conger eel before
(N) and after (T) treatment with GAG-degrading enzymes. (a)
Enzyme treatment with hyaluronidase, (b) enzyme treatment with
chondroitinase ABC. M1: lane markers for HA (hyaluronan), HS
(heparan sulfate), and C4S (chondroitin sulfate A). M2: lane
markers for KS-I (keratin sulfate), C6S (chondroitin sulfate C),
and HP (heparin). Arrows indicate HA, and asterisks indicate
Figure 3: Distribution of HA in a cross section of a fully grown
Japanese conger eel leptocephali before (N) and after (T) treatment
with hyaluronidase. HA was colored bruise blue by NBT/BCIP.
at 5000xg for 5 minutes, and the supernatant was removed.
We repeated this procedure three times. The dry residue was
mixed with 3.5mL distilled water and boiled for 3 minutes.
We then added 2mM CaCl2, 0.1M Tris-HCl (pH 8.0), and
2.3mg protease (type XIX from Aspergillus sojae) (Sigma,
St. Louis, MO) and incubated the mixture at 50◦C for 24
hours. Following this, weadded 1mg proteaseand incubated
at 50◦C for another 24 hours. This step was repeated one
additional time, and then the mixture was boiled. Following
this, we added 10U bovine pancrease DNase I (Sigma) and
incubated for 4 hours at 37◦C. We then added trichloroacetic
acid (final concentration: 10%) and incubated the mixture
overnight at 4◦C. The mixture was then centrifuged at
5000xg for 15 minutes, and the supernatant was dialyzed
against running tap water for 3 days. The final solution was
dried, and the residue was dissolved in distilled water.
We performed enzyme treatment of the GAGs as fol-
lows: approximately 20–40μg dry body weight of the GAG
samples was treated with either 100 turbidity reducing (TR)
U/mL of hyaluronidase (60◦C overnight) (Seikagaku, Tokyo,
Japan) or 5U/mL of chondroitinase ABC (Seikagaku) (37◦C
overnight). Cellulose acetate electrophoresis was performed
on Separax-SP (Fujifilm, Tokyo, Japan) under the following
conditions: 0.47M formic acid–0.1M pyridine buffer (pH
3.0) at 50V. We used the following GAG standards: chon-
droitin sulfate A (C4S), chondroitin sulfate C (C6S), heparin
sulfate (HS), heparin (HP), hyaluronan (HA), and keratan
sulfate (KS-I) (Seikagaku).
2.3. Histochemistry for HA. The whole body of fish stored
in Bouin’s solution was embedded in paraffin and sectioned
at 15–18μm intervals. The sections were then deparaf-
finized and stained with biotinylated HA binding protein
(Seikagaku) for 1 hour at RT. After staining, the slides
were washed three times in PBS for 10 minutes and then
Research Letters in Biochemistry3
μg/dry body weight (mg)
St.1St.2 St.3St.4 St.5
μg/dry body weight (mg)
μg/dry body weight (mg)
μg/dry body weight (mg)
Water contents (%)
Figure 4: Changes in HA, body water, uronic acid, amino sugar, and neutral sugar content during development of the Japanese conger
eel. HA: hyaluronan, UA: uronic acid, AS: amino sugar, NS: neutral sugar. Values represent the mean ± SEM of four independent samples.
Data from the analyses were compared using one-way ANOVA followed by the Tukey-Kramer test. Differences were considered significant
at P < .05. Asterisks indicate that the value is significantly different from the levels during stage 1.
stained with alkaline phosphatase conjugated streptavidin
(Dako Cytomation, Kyoto, Japan). After washing, the sec-
tions were developed with NBT/BCIP color reagent (Roche,
Mannheim, Germany). The control sections were treated
with 200TRU/mL hyaluronidase (Seikagaku) and incubated
at 60◦C for 4 hours.
2.4. Sugar Composition Analysis. We analyzed the sugar
composition following the methods described by Pfeiler
. Uronic acid was determined by the carbazole method
using glucuronolactone as a standard . Amino sugars
(hexosamine) were determined by the colorimetric method
using N-acetyl-D-glucosamine as a standard . Simple
sugar (nonhexosamine) concentrations were determined by
the phenol-sulfuric method using glucose as a standard
. The neutral sugar concentration was obtained by
4 Research Letters in Biochemistry
2.5. HA Analysis. A sample of the powdered body wall was
dried in a vacuum freeze drier then mixed with 1.0mL
of actinase E (Kaken Pharmaceutical, Tokyo, Japan), and
incubated at 50◦C for 24 hours. The mixture was boiled for
10 minutes and then centrifuged at 5000xg for 10 minutes.
The supernatant was then used for measurement of HA
content using an assay kit (Seikagaku).
The electrophoretic patterns of the purified GAGs are shown
in Figure 2. We observed a major band and a minor band
in both the fully grown leptocephali and the glass-eels. The
major band was broken down by hyaluronidase and chon-
droitinase ABC (Figures 2(a) and 2(b)). The minor band
was broken down by chondroitinase ABC (Figure 2(b)).
The electrophoretic pattern for the Japanese conger eel was
identical with a major HA band and a minor chondroitin
The distribution of HA in Japanese conger eel lepto-
cephali is shown in Figure 3. There is void structure with
an infill of HA. Based on our results, HA appears to be
the primary GAG in Japanese conger eel leptocephali. HA
is a linear polysaccharide that consists of D-glucuronic acid
(GlcA) and N-acetyl-D-glucosamine (GlcNAc) disaccharide
repeats , both of which are derived from glucose (GlcA:
uronic acid pathway, GlcNAc: see metabolic interrelation-
ships among the amino sugars) . Furthermore, the
addition of glucose significantly increases HA production
in rat kidney cells . The HA molecule is stabilized by
hydrogen bonds parallel with the chain axis, forming a
stiffened helical configuration, which gives the molecule an
overall expanded coil structure in solution . The coil
may be viewed as a highly hydrated sphere containing several
orders of magnitude more water relative to its molecular
weight . The water is mechanically encompassed within
the coil and not chemically bound to the polysaccha-
HA content decreased gradually throughout metamor-
phosis. The decrease was also associated with a decrease in
body water content (Figure 4). HA regulates water balance,
osmotic pressure and acts as an ion exchange resin .
Based on the relationship between GAGs and body water
content in Elopiformes , it is thought that HA is
involved in the control of body water content. Furthermore,
HA may be involved in adaptation to seawater during
the planktonic phase. In addition to the decrease in HA
content, both uronic acid, an indicator of GlcA, and amino
sugar, an indicator of GlcNAc, content decreased gradually
during metamorphosis (Figure 4). In contrast, the neutral
sugar content increased gradually during metamorphosis
(Figure 4). Neutral sugars such as glucose and/or glycogen
are one of the primary energy stores in teleosts . Given
that the leptocephali did not feed during metamorphosis,
it is unlikely that the increase in neutral sugar content was
derived exogenously. Thus, we hypothesize that HA was
metabolized to glucose, and subsequently to glycogen for
storage in the postmetamorphic juvenile.
This study was supported in part by funds from the
Environment Agency, Japan, and by a Grant from the Global
COE Program from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
 H. M. Wilbur, “Complex life cycles,” Annual Review of Ecology
and Systematics, vol. 11, pp. 67–93, 1980.
 J. H. Youson, “First metamorphosis,” The Physiology of
Developing Fish, vol. 135, pp. 135–195, 1988.
 D. G. Smith, Guide to the Leptocephali (Elopiformes, Anguil-
liformes and Notacanthiformes), NOAA Technical Report;
NMFS CIRC-424, 1979.
 W. H. Hulet and C. R. Robins, “The evolutionary significance
vol. 9, pp. 669–677, 1989.
 Y. Kawakami, N. Mochioka, R. Kimura, and A. Nakazono,
“Seasonal changes of the RNA/DNA ratio, size and lipid
contents and immigration adaptability of Japanese glass-
eels, Anguilla japonica, collected in northern Kyushu, Japan,”
Journal of Experimental Marine Biology and Ecology, vol. 238,
no. 1, pp. 1–19, 1999.
 Y. Kawakami, M. Tanda, S. Adachi, and K. Yamauchi,
“Characterization of thyroid hormone receptor α and β in
the metamorphosing Japanese conger eel, Conger myriaster,”
General and Comparative Endocrinology, vol. 132, no. 2, pp.
 E. Pfeiler, “Glycosaminoglycan breakdown during metamor-
phosis of larval bonefish Albula,” Marine Biology Letters, vol.
5, pp. 241–249, 1984.
 T. Bitter and H. M. Muir, “A modified uronic acid carbazole
reaction,” Analytical Biochemistry, vol. 4, no. 4, pp. 330–334,
 G. Blix, “The determination of hexosamines according to
Elson and Morgan,” Acta Chemica. Scandinavica, vol. 2, pp.
 M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F.
Smith, “Colorimetric method for determination of sugars and
related substances,” Analytical Chemistry, vol. 28, no. 3, pp.
 T. C. Laurent and J. R. E. Fraser, “Hyaluronan,” The FASEB
Journal, vol. 6, no. 7, pp. 2397–2404, 1992.
 K. Bender and P. A. Mayes, “The pentose phosphate pathway
& other pathways of hexose metabolism,” Harper’s Illustrated
Biochemistry, vol. 27, pp. 177–186, 2006.
 M. Takeda, T. Babazono, K. Nitta, and Y. Iwamoto, “High
glucose stimulates hyaluronan production by renal interstitial
fibroblasts through the protein kinase C and transforming
growth factor-β cascade,” Metabolism, vol. 50, no. 7, pp. 789–
 J. E. Scott, “Secondary structures in hyaluronan solutions:
chemical and biological implications,” in Proceedings of the
Ciba Foundation Symposium, vol. 143, pp. 6–14, 1989.
 T. C. Laurent, “Structure of hyaluronic acid,” Chemistry and
Molecular Biology of the Intercellular Matrix, vol. 2, pp. 703–
 R. Stern, “Hyaluronan catabolism: a new metabolic pathway,”
European Journal of Cell Biology, vol. 83, no. 7, pp. 317–325,
Research Letters in Biochemistry5 Download full-text
 E. Pfeiler, “Inshore migration, seasonal distribution and
sizes of larval bonefish, Albula, in the Gulf of California,”
Environmental Biology of Fishes, vol. 10, no. 1-2, pp. 117–122,
 M. J. Walton and C. B. Cowey, “Aspects of intermediary
metabolism in salmonid fish,” Comparative Biochemistry and
Physiology, Part B, vol. 73, no. 1, pp. 59–79, 1982.