Báo cáo khoa học: Zinc-binding property of the major yolk protein in the sea urchin ) implications of its role as a zinc transporter for gametogenesis ppt

Zinc-binding property of the major yolk protein in the sea
urchin
)
implications of its role as a zinc transporter for
gametogenesis
Tatsuya Unuma
1
, Kazuo Ikeda
2
, Keisuke Yamano
3
, Akihiko Moriyama
4
and Hiromi Ohta
5
1 Japan Sea National Fisheries Research Institute, Fisheries Research Agency, Suido-cho, Niigata, Japan
2 Kamiura Station, National Research Institute of Aquaculture, Fisheries Research Agency, Kamiura, Oita, Japan
3 National Research Institute of Aquaculture, Fisheries Research Agency, Minami-ise, Mie, Japan
4 Graduate School of Natural Sciences, Nagoya City University, Japan
5 Department of Fisheries, School of Agriculture, Kinki University, Nara, Japan
Sea urchin gametogenesis is characterized by dynamic
interactions between the germinal and the somatic cel-
lular populations in the gonad [1–3]. Before the initia-
tion of gametogenesis, the gonads increase in size by
accumulating nutrients such as proteins, lipids, and
carbohydrates in nutritive phagocytes, somatic cells
that fill the gonadal lumina in both sexes. After game-
togenesis begins, the nutritive phagocytes gradually
decrease in size, supplying nutrients to the developing
germ cells, and finally the lumina are filled with ova
and sperm.

Major yolk protein (MYP), a glycoprotein of
170 kDa originally identified as the predominant com-
ponent of yolk granules in sea urchin eggs [4–7], plays
significant roles in gametogenesis. Unlike other ovipa-
rous animals, in which the yolk protein is female-
specific, both male and female sea urchins produce
MYP [8,9]. Before gametogenesis, MYP is synthesized
Keywords
major yolk protein; oogenesis; sea urchin;
spermatogenesis; zinc
Correspondence
T. Unuma, Japan Sea National Fisheries
Research Institute, Fisheries Research
Agency, Suido-cho, Niigata 951-8121, Japan
Fax: +81 25 2240950
Tel: +81 25 2280451
E-mail:
(Received 25 April 2007, revised 11 June
2007, accepted 27 July 2007)
doi:10.1111/j.1742-4658.2007.06014.x
Major yolk protein (MYP), a transferrin superfamily protein that forms
yolk granules in sea urchin eggs, is also contained in the coelomic fluid
and nutritive phagocytes of the gonad in both sexes. MYP in the coelo-
mic fluid (CFMYP; 180 kDa) has a higher molecular mass than MYP in
eggs (EGMYP; 170 kDa). Here we show that MYP has a zinc-binding
capacity that is diminished concomitantly with its incorporation from the
coelomic fluid into the gonad in the sea urchin Pseudocentrotus depressus.
Most of the zinc in the coelomic fluid was bound to CFMYP, whereas
zinc in eggs was scarcely bound to EGMYP. Both CFMYP and EG-
MYP were present in nutritive phagocytes, where CFMYP bound more

zinc than EGMYP. Saturation binding assays revealed that CFMYP has
more zinc-binding sites than EGMYP. Labeled CFMYP injected into the
coelom was incorporated into ovarian and testicular nutritive phagocytes
and vitellogenic oocytes, and the molecular mass of part of the incorpo-
rated CFMYP shifted to 170 kDa. Considering the fact that the digestive
tract is a major production site of MYP, we propose that CFMYP
transports zinc, essential for gametogenesis, from the digestive tract to
the ovary and testis through the coelomic fluid, after which part of the
CFMYP is processed to EGMYP with loss of zinc-binding site(s).
Abbreviations
anti-Fl, antibody to fluorescein; anti-MYP, antibody to major yolk protein; CBB, Coomassie Brilliant Blue R-350; CFMYP, coelomic fluid-type
major yolk protein; EGMYP, egg-type major yolk protein; Fl-CFMYP, coelomic fluid-type major yolk protein labeled with fluorescein;
Fl-lactoferrin, lactoferrin labeled with fluorescein; ICP-AES, inductively coupled plasma atomic emission spectrometry; MYP,
major yolk protein.
FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS 4985
mainly in the digestive tract [8] and in the nutritive
phagocytes of the ovary and testis [10], and it is accu-
mulated abundantly in the nutritive phagocytes
[9,11,12]. As gametogenesis proceeds, the stored MYP
is degraded to amino acids for the synthesis of new
proteins, nucleic acids, and other nitrogen-containing
substances that constitute eggs and sperm [12]. A smal-
ler amount of MYP is incorporated into the ova
through endocytosis via a dynamin-dependent mecha-
nism [13,14] and forms yolk granules [11]. After fertil-
ization, the MYP in the yolk granules degrades and
possibly serves as a nutrient source for the larval stage
[15] and as a cell adhesion molecule [16–18].
Another form of MYP with slightly higher molecu-
lar mass (about 180 kDa) is contained in the coelomic

fluid and is considered to be a precursor of MYP in
the gonad [4]. This higher molecular mass MYP was
formerly called sea urchin vitellogenin [8,10,19] after
the yolk protein precursor vitellogenin, which is found
in the blood of oviparous vertebrates. However, the
sequencing of MYP cDNA from Pseudocentrotus
depressus [10] and other species [20,21] has revealed
that MYP is not homologous to vertebrate vitelloge-
nin, but is slightly homologous to the transferrins, a
family of iron-binding proteins. To avoid confusion in
this article, therefore, we refer to coelomic fluid-type
MYP as CFMYP and egg-type MYP as EGMYP, and
we use MYP when the type is not to be specified. Both
CFMYP and EGMYP are products of the same gene,
as sea urchins have only one gene encoding MYP, as
has been suggested by genomic Southern blot analysis
[22,23] and confirmed recently by searching the Strong-
ylocentrotus purpuratus genome [24]. However, the
molecular differences between CFMYP and EGMYP
have yet to be fully clarified.
Transferrins perform essential roles in iron transport
and regulation, and are found widely in vertebrates
and invertebrates [25–27]. Mammalian transferrin can
also bind various trace metals, including manganese,
copper, and zinc, although the affinity of transferrin
for these trace metals is lower than that for iron
[28,29]. It was demonstrated that CFMYP purified
from the coelomic fluid of the sea urchin S. purpuratus
potentially binds
59

Fe in vitro [20]. Based on its
sequence similarity to transferrin and iron-binding
potential, MYP has been considered to be a member
of the transferrin superfamily [10,20,21,30]. However,
it is largely unknown whether and how MYP is
involved in the transport of iron and other trace met-
als in sea urchins.
In the present study, we investigated the binding of
trace metals to MYP in the coelomic fluid, gonad and
eggs of the sea urchin P. depressus, and found that
MYP has a zinc-binding capacity, which is diminished
concomitantly with its incorporation from the coelo-
mic fluid to the gonad. We propose that MYP trans-
ports zinc from the digestive tract to the ovary and
testis through the coelomic fluid to provide essential
supplies for oogenesis and spermatogenesis.
Results
Binding of trace metals to proteins
First, we investigated the binding of trace metals
(manganese, iron, copper, and zinc) to proteins in the
coelomic fluid, gonad extract and egg extract by deter-
mining their concentrations using inductively coupled
plasma atomic emission spectrometry (ICP-AES) in
the fractions separated by gel filtration chromato-
graphy (Fig. 1). Figure 1A-D shows typical elution
profiles of coelomic fluid, and extract from ovary at
stage 1 (before gametogenesis), testis at stage 1, and
eggs. In all of the samples analyzed, a large protein
peak was observed at an elution position of 72 mL
(peaks a, b, c, and d), where the estimated molecular

mass was about 600 kDa. Fractions under the bars
were pooled and subjected to SDS ⁄ PAGE and western
blot analysis using an antibody to MYP (anti-MYP),
which revealed that the main constituent of this peak
was MYP of 170–180 kDa under reducing conditions
(Fig. 1E) and MYP of about 350 kDa under nonreduc-
ing conditions (data not shown). These indicate that the
native MYP is a tetrameric molecule comprising two
disulfide-bonded dimeric subunits, consistent with other
reports [9,31,32]. When the amount of protein on wes-
tern blot analysis was decreased and the run time of the
electrophoresis was prolonged, the MYP in the ovary
and testis separated into two bands of 170 kDa and
180 kDa (Fig. 1E, right panel), indicating that ovary
and testis contain both CFMYP and EGMYP. This
means that nutritive phagocytes contain both CFMYP
and EGMYP, as, in both ovary and testis at stage 1,
MYP is not present in cells other than nutritive phago-
cytes [9].
In the coelomic fluid, zinc eluted as a single peak
coincident with CFMYP, indicating that most of the
zinc in the coelomic fluid is bound to CFMYP
(Fig. 1A). In the immature ovary (Fig. 1B) and testis
(Fig. 1C), zinc eluted as three or four major peaks.
One of the zinc peaks was coincident with the MYP
peak in both the ovary and the testis, indicating that
part of the zinc in the immature gonads is bound to
MYP. The elution patterns of zinc were similar in the
ovary and testis, but the zinc peak in the immature
ovary was larger than that in the immature testis,

Zinc-binding protein in the sea urchin T. Unuma et al.
4986 FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS
Coelomic fluid
AB
C
E
D
0
0.2
0.4
0.6
0.8
Absorbance at 280 nm
0
50
100
150
200
250
Zn, Fe (ng / ml)
30 50 70 90 110 130
Elution volume (ml)
A280
Zn
Fe
a
0
0.4
0.8
1.2

1.6
Absorbance at 280 nm
0
20
40
60
80
100
Zn, Fe (ng / ml)
30 50 70 90 110 130
Elution volume (ml)
Egg
Ovary at stage 1
0
0.6
0.8
1.2
1.6
Absorbance at 280 nm
0
20
40
60
80
100
Zn, Fe (ng / ml)
30 50 70 90 110 130
Elution volume (ml)
Testis at stage 1
0

0.4
0.8
1.2
1.6
Absorbance at 280 nm
0
20
40
60
80
100
Zn, Fe (ng / ml)
30 50 70 90 110 130
Elution volume (ml)
b
c
d
1
2
3
4
SDS-PAGE and western analysis
250 kDa
150 kDa
100 kDa
50 kDa
25 kDa
a
b
c

Vo
Vc
d
a
b
c
d
a
b
c
d
Anti-MYP
Anti-MYPCBB
Fig. 1. Survey of the binding of trace metals to proteins in the coelomic fluid, gonad extract and egg extract of P. depressus. Coelomic fluid
(A) and extracts from ovary at stage 1 (B), testis at stage 1 (C) and eggs (D) were subjected to gel filtration chromatography using a Supe-
rose 6 column. Trace metals in each fraction were determined by ICP-AES. Protein levels were monitored by the absorbance at 280 nm.
Peak fractions marked with bars were pooled and used for further experiments. Arrows in (A) indicate the void volume (V
o
), the column vol-
ume (V
c
), and the elution positions of standard proteins for molecular mass calibration (1, thyroglobulin, 669 kDa; 2, catalase, 232 kDa; 3,
BSA, 67 kDa; 4, chymotrypsinogen, 25 kDa). Arrows in (B), (C) and (D) indicate zinc-binding proteins distinct from MYP. (E) Peak fractions
were subjected to SDS ⁄ PAGE (left panel; stained with CBB) and western blot analysis (center and right panels; immunostained with anti-
MYP). (a) Coelomic fluid; (b) ovary at stage 1; (c) testis at stage 1; (d) eggs. Samples containing 1.5 lg (left panel) or 0.3 lg (center panel) of
protein were applied to each lane. When the sample amount was decreased (20 ng of protein) and the run time was prolonged, the MYP in
ovary and testis separated into two bands (right panel; lanes b and c). Molecular mass values on the left indicate the migration positions of
marker proteins.
T. Unuma et al. Zinc-binding protein in the sea urchin
FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS 4987

suggesting that MYP in the ovary binds more zinc
than that in the testis. In the ovary at stage 3
(mid-gametogenesis), zinc eluted coincidentally with
MYP, but the peak was smaller than that in the ovary
at stage 1 (data not shown). In eggs (Fig. 1D), in con-
trast, zinc did not show a clear peak coincident with
EGMYP, suggesting that zinc in the egg is scarcely
bound to EGMYP.
In ovary, testis, and egg (Fig. 1B–D), a large peak
of zinc was observed at an elution position of 86 mL
(arrows). The absorbance at 280 nm did not show a
clear peak at the same position as zinc, indicating that
the proteins binding zinc in these fractions scarcely
contained any aromatic amino acid residues. This
suggests that the proteins are metallothioneins,
well-known zinc-binding proteins that lack aromatic
residues and play an important role in zinc distribution
and storage in cells [33,34].
Manganese, iron and copper did not show any clear
peaks coincident with MYP in the samples analyzed
(data for manganese and copper not shown), indicat-
ing that MYP does not bind these trace metals at
detectable levels.
Purification of MYP
To obtain pure MYP for further experiments on the
binding of zinc to MYP, the MYP peak fractions
collected from gel filtration chromatography (bars in
Fig. 1) were subjected to ion exchange chromatogra-
phy. Figure 2A–C shows typical elution profiles of
MYP peak fractions from coelomic fluid, ovary at

stage 1, and eggs. CFMYP eluted as a single peak at
250 mm NaCl in coelomic fluid (Fig. 2A), and EG-
MYP at 150 mm NaCl in eggs (Fig. 2C). These protein
peaks revealed a single band on native PAGE (Fig. 2F,
0
200
400
600
NaCl (mM)
0.0
0.5
1.0
1.5
2.0
Absorbance at 280nm
0 5 10 15
Elution volume (ml)
Coelomic fluid
A
DE F
BC
NaCl
A280
0
200
400
600
NaCl (mM)
0.0
0.5

1.0
1.5
Absorbance at 280nm
0 5 10 15
Elution volume (ml)
0
200
400
600
NaCl (mM)
0.0
0.5
1.0
1.5
Absorbance at 280nm
0 2.5 5 7.5
Elution volume (ml)
a
c
Native-PAGE
Ovary at stage 1
a
c
b
Egg
abc
SDS-PAGE and
western blotting
b-1
b-2

b-3
b-4
b-5
0
25
50
75
100
(%)
b-1
Peak No.
CFMYP/EGMYP proportions
CFMYP
EGMYP
b-2 b-3
b-4 b-5
1
2
3
4
5
SYPRO-Ruby
Anti-MYP
Fig. 2. Purification of P. depressus MYP by ion exchange chromatography. MYP fractions obtained by gel filtration (Fig. 1) were subjected to
ion exchange chromatography using a Mono Q 5 ⁄ 50GL column. (A) Coelomic fluid. (B) Ovary at stage 1. (C) Eggs. Protein levels were moni-
tored by the absorbance at 280 nm. Fractions marked with bars were pooled and used for further experiments. (D) Peak fractions shown in
(A) (a), (B) (b-1, b-2, b-3, b-4, and b-5) and (C) (c) were subjected to SDS ⁄ PAGE (upper panel; stained with SYPRO Ruby) and western blot
analysis (lower panel; immunostained with anti-MYP). Samples containing 50 ng (SDS ⁄ PAGE) or 20 ng (western blot analysis) of protein
were applied to each lane. (E) Protein levels of the CFMYP and EGMYP bands in lanes b-1 to b-5 on SDS ⁄ PAGE gel [upper panel in (D)]
were measured by densitometry, and the proportions of CFMYP ⁄ EGMYP were calculated. The numbers of CFMYP molecules and EGMYP

molecules constituting native tetramer MYP were suggested to be different in each peak, as shown below the x-axis. (F) Pooled fractions
(a, b, and c) containing 2 lg of protein were subjected to native PAGE and stained with CBB.
Zinc-binding protein in the sea urchin T. Unuma et al.
4988 FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS
lanes a and c). We concluded that the purity of
CFMYP obtained from the coelomic fluid and
EGMYP from eggs was satisfactory for further experi-
ments. In contrast, five peaks were observed from 150
to 250 mm in the ovary at stage 1 (Fig. 2B). Each of
the peaks (b-1, b-2, b-3, b-4, and b-5) was analyzed by
SDS ⁄ PAGE and western blot analysis, and was
revealed to contain both CFMYP and EGMYP in dif-
fering ratios (Fig. 2D). The protein contents of the
CFMYP and EGMYP bands were measured for each
peak by densitometry of the SDS ⁄ PAGE gel stained
with SYPRO Ruby, and then the proportions of
CFMYP and EGMYP were calculated (Fig. 2E). The
proportions of CFMYP were higher in the latter
peaks, indicating that the numbers of CFMYP mole-
cules in the native MYP tetramer are 0, 1, 2, 3 and 4
in peaks b-1, b-2, b-3, b-4, and b-5, respectively, as
illustrated below the x-axis in Fig. 2E. Fractions from
150 to 250 mm NaCl, including these five peaks, were
pooled and subjected to native PAGE, revealing a
single band (Fig. 2F, lane b). We concluded that the
purity of the MYP in the pooled fractions from the
ovary obtained from 150 to 250 mm NaCl was
satisfactory for further experiments.
Zn/MYP molar ratio and CFMYP/MYP proportion
The elution profiles of MYP and zinc on gel filtration

chromatography of ovary (Fig. 1B) and testis (Fig. 1C)
suggested that ovary MYP binds more zinc than does
testis MYP. We therefore purified MYP from ovaries
at stage 1 and stage 3, and from testes at stage 1
and stage 3, as described above, and examined the
Zn ⁄ MYP molar ratio (Fig. 3A). In the ovaries, Zn ⁄ MYP
was 0.149 ± 0.025 at stage 1 and 0.054 ± 0.007 at
stage 3. In the testes, Zn ⁄ MYP was 0.048 ± 0.009 at
stage 1 and 0.020 ± 0.004 at stage 3.
We also measured the protein content of the
CFMYP and EGMYP bands by densitometry of the
SDS ⁄ PAGE gel of the purified MYP, and the ratio of
CFMYP to total MYP (CFMYP + EGMYP) was
calculated (Fig. 3B). In the ovaries, 43.1 ± 1.7% of
the MYP was CFMYP at stage 1 and 18.3 ± 2.6% at
stage 3. In the testes, the proportions of CFMYP were
17.7 ± 5.3% at stage 1 and 6.9 ± 3.5% at stage 3.
The higher the proportion of CFMYP, the more zinc
the MYP contained, suggesting that CFMYP binds
more zinc than does EGMYP in the gonad.
Saturation binding assay
To confirm the possibility that CFMYP has more zinc-
binding sites than EGMYP, we subjected CFMYP
purified from coelomic fluid and EGMYP from eggs
to a saturation binding assay using equilibrium dialysis
(Fig. 4). For both CFMYP (Fig. 4A) and EGMYP
(Fig. 4B), as the total Zn ⁄ MYP ratio increased, the
bound Zn ⁄ MYP ratio also increased because of non-
specific binding (left panels). To calculate specific bind-
ing, nonspecific binding was estimated and subtracted

from the total binding. A Scatchard plot was con-
ducted with the values for specific binding only. The
maximum number of binding sites (B
MAX
) was 2.6 for
CFMYP and 1.5 for EGMYP (right panels), indicating
that the numbers of zinc-binding sites are two or three
in CFMYP and one or two in EGMYP. When the
binding sites in one molecule were assumed to be iden-
tical to each other, the equilibrium dissociation cons-
tant (K
d
) values were estimated as 5 · 10
)7
m for
CFMYP and 3 · 10
)7
m for EGMYP.
Incorporation of CFMYP into the gonad
The above data led us to assume that CFMYP may be
incorporated into the gonad and processed to
EGMYP, playing a role in zinc transport. We there-
fore investigated the incorporation of labeled CFMYP
into the gonad (Fig. 5). CFMYP labeled with fluores-
cein (Fl-CFMYP) or, as a control, lactoferrin labeled
with fluorescein (Fl-lactoferrin) was injected into adult
P. depressus during the season of early gametogenesis
(early October). On western blot analysis with an anti-
body to fluorescein (anti-Fl) (Fig. 5A), Fl-CFMYP
was detected in gonad extracts both 6 and 15 days

CFMYP/MYP
Zn/MYP
AB
0
10
20
30
40
50
CFMYP/MYP (%)
1
Stage
3
0
0.05
0.1
0.15
0.2
Zn/MYP (molar ratio)
1
Sta
g
e
Testis
Ovary
3
Fig. 3. Zn ⁄ MYP molar ratio and CFMYP ⁄ MYP proportions in the
ovaries and testes of P. depressus. MYP was purified from ovaries
and testes at stages 1 and 3 by gel filtration and ion exchange
chromatography as shown in Figs 1 and 2. (A) Zinc and protein lev-

els in the purified MYP were measured by ICP-AES and the Brad-
ford method, respectively. The molar ratio of zinc to MYP (as a
monomer) was calculated. (B) Purified MYP was subjected to
SDS ⁄ PAGE, and the protein levels in the CFMYP and EGMYP
bands were measured by densitometry of the gel stained with
CYPRO Ruby. The ratios of CFMYP to total MYP (CFMYP +
EGMYP) were calculated. Values are the mean ± SEM obtained
from three individuals.
T. Unuma et al. Zinc-binding protein in the sea urchin
FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS 4989
after injection; the signals were stronger at 15 days.
Fl-lactoferrin was scarcely detected. No significant
signal was detected in noninjected animals (lane n).
These results indicate that Fl-CFMYP was selectively
incorporated into both ovaries and testes.
The bands of Fl-CFMYP on western blot analysis
appeared rather broad, probably because large
amounts of unlabeled CFMYP and EGMYP naturally
present in the gonads formed broad bands and affected
the shape of the bands of the labeled MYP. To
improve the resolution, labeled MYP was collected
from gonad extracts by immunoprecipitation using
anti-Fl, and then subjected to western blot analysis
using anti-Fl (Fig. 5B). In addition to the bands of
CFMYP, both testes and ovaries produced lower
bands with molecular masses corresponding to that of
EGMYP, suggesting that, in both the ovaries and tes-
tes, part of the incorporated CFMYP was processed to
EGMYP.
Immunohistochemistry of the gonads 15 days after

injection revealed that Fl-CFMYP was incorporated
into nutritive phagocytes in both sexes (Fig. 5C–E).
The labeled MYP was not detected in spermatogonia
(Fig. 5C) or in young oocytes with a diameter of about
30 lm (Fig. 5D), but was strongly detected in vitello-
genic oocytes with a diameter of about 60 lm
(Fig. 5E). No significant signal was detected in the
ovaries of noninjected animals (Fig. 5F). The results of
this experiment suggest that CFMYP in the coelomic
fluid is selectively incorporated into the nutritive
phagocytes of the ovary and testis and into vitellogenic
oocytes, after which part of the incorporated CFMYP
is processed to EGMYP.
CFMYP and zinc content of coelomic fluid and
gonad
As CFMYP was suggested to be a transporter of zinc
from the coelomic fluid to the gonad, we next exam-
ined changes in the concentrations of CFMYP and
zinc in the coelomic fluid and the total amount of
zinc in the gonad during gametogenesis (Fig. 6). In
female coelomic fluid, the CFMYP concentration was
517 ± 72 lgÆmL
)1
at stage 1 and reached its highest
value of 886 ± 95 lgÆmL
)1
at stage 2 (early gameto-
genesis) (Fig. 6A). There was a significant difference
(P<0.05) between stages 1 and 2. In male coelomic
fluid, the CFMYP concentration was 475 ± 131

lgÆmL
)1
at stage 1 and remained at a similar value to
stage 4 (fully mature). In female coelomic fluid, the
concentration of zinc was 135 ± 16 ngÆmL
)1
at stage 1
and reached its highest value of 285 ± 38 ngÆmL
)1
at
stage 2 (Fig. 6B). There was a significant difference
(P<0.05) between stages 1 and 2. In male coelomic
fluid, the concentration of zinc increased from
111 ± 16 ngÆmL
)1
at stage 1 to 173 ± 28 ngÆmL
)1
at
CFMYP
A
B
EGMYP
Scatchard
Scatchard
Total binding
Nonspecific
binding
Total binding
Nonspecific
binding

0 5 10 15 20 25
0
2
4
6
8
Total Zn / MYP (molar ratio)
Bound Zn / MYP (molar ratio)
0 1 2 3 4
0
2
4
6
Bound Zn / MYP (molar ratio)
Bound Zn / MYP / Free Zn (µM
-1
)
0 5 10 15 20 25
0
2
4
6
8
Total Zn / MYP (molar ratio)
Bound Zn / MYP (molar ratio)
0 1 2
0
2
4
6

Bound Zn / MYP (molar ratio)
Bound Zn / MYP / Free Zn (µM
-1
)
Fig. 4. Saturation binding assay for binding
of zinc to P. depressus MYP. CFMYP (4 l
M)
purified from coelomic fluid (A) and EGMYP
(4 l
M) from eggs (B) were subjected to
equilibrium dialysis at pH 7.6. Values for
nonspecific binding (broken line) were esti-
mated using
PRISM 4 software. Only specific
binding is shown on the Scatchard plot
(right panels).
Zinc-binding protein in the sea urchin T. Unuma et al.
4990 FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS
stage 4. The molar ratio of Zn ⁄ CFMYP in the coe-
lomic fluid showed no drastic changes in either sex,
ranging from 0.7 (testis at stage 2) to 1.2 (testis at
stage 4) (data not shown). This suggests that the
average number of zinc atoms bound by one mole-
cule of CFMYP (as a monomer) in the coelomic
fluid is 0.7–1.2, as most zinc in the coelomic fluid
appears to be bound to CFMYP, as shown in
Fig. 1A.
Changes in the total amount of zinc in the ovary or
testis of an individual with a body weight of 100 g
are shown in Fig. 6C. In females, ovary zinc was

105 lgÆ(100 g body weight)
)1
at stage 1, increased to
178 lgÆ(100 g body weight)
)1
at stage 2, and remained
at a similar value to stage 4; there were no significant
differences among the stages. In males, testis zinc was
49 lgÆ(100 g body weight)
)1
at stage 1, which was less
than half of that in ovary at stage 1, and remained at
a similar value to stage 4. Testes at stage 2 were not
analyzed because of a lack of samples. We calculated
the amount of zinc bound to MYP in the gonad by
multiplying values for the total amounts of MYP
(CFMYP + EGMYP) in the ovaries and testes pub-
lished elsewhere [12] by the Zn ⁄ MYP ratios shown in
Fig. 3A. In ovary at stage 1, the amount of MYP-
bound zinc was 34 lgÆ(100 g body weight)
)1
, which
means that 33% of ovary zinc was bound to MYP. In
testis at stage 1, the amount of MYP-bound zinc was
Western blotting
A
B
CD EF
15 days
m

l
nn
Ovary
Testis
Ovary (no injection)
250 kDa
150 kDa
100 kDa
50 kDa
25 kDa
o
t
6 days
Fl-CFMYP Fl-lactofferin
t
t
t
ooo
6 days
15 days
Ovary
CFMYP
EGMYP
Western blotting after
immunoprecipitation
mc d e
Fig. 5. Incorporation of CFMYP into the gonad of P. depressus. Fl-CFMYP or Fl-lactoferrin was injected into the coelomic cavity of P. depres-
sus, and the gonads were excised 6 and 15 days after injection. (A) Gonad extracts were subjected to western blot analysis using anti-Fl.
Five nanograms of labeled protein or extracts derived from 1 lg of gonad was applied to each lane. (l) Fl-lactoferrin; (m) Fl-CFMYP; (n) ovary
of noninjected animal; (o) ovary of injected animal; (t) testis of injected animal. (B) To remove the abundant unlabeled MYP that is naturally

present in the gonads, gonad extracts were subjected to immunoprecipitation using anti-Fl and then western blot analysis using anti-Fl. The
migration positions of CFMYP and EGMYP used as marker proteins are shown on the left. (m) Fl-CFMYP; (c) testis at stage 2; (d) ovary at
stage 2; (e) ovary at stage 2 containing larger oocytes than in (d). (C–F) Immunolocalization of labeled MYP with anti-Fl in the gonad 15 days
after injection. (C), (D) and (E) show the gonads from the same animals shown in (c), (d) and (e) in (B), respectively. (F) shows the ovary of a
noninjected animal at stage 2. np, nutritive phagocyte; sg, spermatogonium; oc, oocyte. Bar represents 100 lm. Insets are threefold magnifi-
cations of the oocytes.
T. Unuma et al. Zinc-binding protein in the sea urchin
FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS 4991
6 lgÆ(100 g body weight)
)1
, which means that 13% of
testis zinc was bound to MYP.
Concentrations of trace metals in eggs and
sperm
Concentrations of zinc, iron, copper and manganese
were measured in the eggs and sperm of P. depressus
by inductively coupled plasma mass spectrometry
(zinc, copper, manganese) or graphite furnace atomic
absorption spectrometry (iron) (Table 1). In eggs, zinc
was the most abundant of the four metals, which is
consistent with a report on the concentrations of these
metals in Xenopus laevis eggs [35]. In sperm, zinc was
the second most abundant, following iron. The zinc
level was about six times higher in eggs than in sperm.
Discussion
In the present study, we found that MYP is a zinc-
binding protein. Coelomic fluid has been considered to
be a major route for nutrient translocation in sea urch-
ins [36]. We believe that CFMYP plays a major role in
transportation of zinc from the digestive tract to the

gonad, for the following three reasons. First, most of
the zinc in the coelomic fluid is bound to CFMYP
(Fig. 1). Second, CFMYP injected into the coelomic
cavity is selectively incorporated into the gonad
(Fig. 5). Third, a major production site of MYP is the
digestive tract, and CFMYP in the coelomic fluid is
thought to be synthesized mainly in the digestive tract
[8,10]. We propose that CFMYP synthesized in the
digestive tract binds zinc derived from ingested food
and transports it to the ovary and testis through the
coelomic fluid.
Zinc is essential for cell proliferation and differentia-
tion, as many enzymes and gene regulatory proteins
require zinc for their function; moreover, zinc is a pre-
requisite for chromatin structure [35,37]. A number of
genes encoding such zinc proteins have been found in
the S. purpuratus genome, and their expression has
been investigated during embryogenesis [38,39]. In
oviparous animals, eggs must store sufficient zinc to
meet the huge demand during embryogenesis [35]. The
concentrations of zinc in both frog [35] and sea urchin
(Table 1) eggs are high compared with those of other
trace metals, which means that oogenesis in these ani-
mals requires large amounts of zinc. Sea urchin sperm
also contains considerable amounts of zinc as com-
pared with copper and manganese (Table 1), which
means that spermatogenesis also requires large
amounts of zinc, although the demand will be less for
spermatogenesis than for oogenesis. In male animals,
including sea urchins, zinc is essential for sperm motil-

ity and the acrosome reaction [40,41]. We believe that
the main purpose of CFMYP transportation of zinc to
the ovary and testis is to provide essential supplies for
oogenesis and spermatogenesis.
0
100
200
300
400
Zn (ng / ml)
1
3
Stage
Zn in CF
0
250
500
750
1000
CFMYP (µg / ml)
13
Stage
CFMYP in CF
ABC
Female
Male
2
4
24
0

50
100
150
200
250
Zn (µg / 100 g body weight)
13
Stage
Total Zn in the gonad
2
4
Fig. 6. Changes in CFMYP and zinc concentrations in the coelomic fluid and zinc content in the gonads of female and male P. depressus
during gametogenesis. (A) CFMYP concentrations in coelomic fluid measured by densitometry of SDS ⁄ PAGE gels stained with CBB. In
females, CFMYP increased significantly from stage 1 to stage 2 (P<0.05). (B) Zinc concentrations in coelomic fluid measured by ICP-AES.
In females, zinc increased significantly from stage 1 to stage 2 (P<0.05). (C) Zinc content in the gonad measured by ICP-AES. Values are
expressed as the amount of zinc per 100 g body weight. Values are the mean ± SEM obtained from six or seven individuals for the coelo-
mic fluid and four or five individuals for the gonad.
Table 1. Concentrations of trace metals in gametes of P. depres-
sus.
Eggs
[lgÆ(g wet weight)
)1
]
Sperm
[lgÆ(g wet weight)
)1
]
Zn
a
12.1 2.1

Fe
b
2.9 4.7
Cu
a
0.8 0.7
Mn
a
0.2 0.1
a
Measured by inductively coupled plasma mass spectrometry.
b
Measured by graphite furnace atomic absorption spectrometry.
Zinc-binding protein in the sea urchin T. Unuma et al.
4992 FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS
The fact that labeled CFMYP was incorporated into
nutritive phagocytes suggests that the zinc carried by
CFMYP is stored in these cells before its use in game-
togenesis (Fig. 5). The ovaries and testes analyzed at
stage 1 in this study were obtained from animals just
before the initiation of gametogenesis (late September).
Stage 1, however, continues for almost half a year
after the previous spawning season is completed [42].
Storage supplies for gametogenesis, including MYP,
accumulate gradually in the nutritive phagocytes dur-
ing this period (our unpublished data), as would zinc.
Zinc accumulation in females, however, should pro-
gress faster than that in males, as the ovary contains
about twice as much zinc as the testis at stage 1, just
before gametogenesis (Fig. 6C). After the oocytes had

developed to the vitellogenic stage in females, labeled
CFMYP was incorporated not only into the nutritive
phagocytes but also into the vitellogenic oocytes. Initi-
ation of direct incorporation of zinc into the oocytes
may accelerate zinc accumulation in the ovary, as
CFMYP and zinc concentrations in the coelomic fluid
and total zinc in the ovary were higher at and after
stage 2 than at stage 1 (Fig. 6). In males, labeled MYP
was not detected in spermatogenic cells. Other mole-
cules, such as the ZIP and CDF family proteins (which
regulate zinc uptake by and efflux from cells in organ-
isms ranging from yeasts to mammals) [43], may be
involved in zinc transport from the nutritive phago-
cytes to the spermatogenic cells, or CFMYP may
degrade immediately after its transportation of zinc
into the spermatogenic cells.
There are two transferrin-like iron-binding domains
in the sequence of MYP, one of which is split into two
portions [20,44]. However, it is unclear whether and
how these domains are related to its zinc-binding prop-
erties. In vertebrates, zinc-binding proteins in serum,
such as albumin and vitellogenin (which circulate in
the bloodstream and transport zinc between organs)
[45–47], do not have zinc-binding motifs containing
the catalytic or structural zinc commonly found in zinc
enzymes and gene regulatory proteins. Such well-
known zinc-binding motifs are also not found in the
sequence of MYP. The positions and types of zinc-
binding sites in MYP are unclear. However, we assume
that at least one zinc-binding site is located near the

N-terminus or C-terminus of CFMYP, as CFMYP has
about 10 kDa higher molecular mass and more zinc-
binding sites than EGMYP (Fig. 4); polypeptide(s)
totaling about 10 kDa should be removed from the
N-terminus and ⁄ or C-terminus with zinc-binding site(s)
when CFMYP is processed to EGMYP.
Nutritive phagocytes were found to contain both
CFMYP and EGMYP, which comprised tetramers
with CFMYP ⁄ EGMYP ratios from 0 : 4 to 4 : 0
(Fig. 2). In our previous study, MYP in nutritive
phagocytes appeared to be of a single type, because
CFMYP and EGMYP could not be divided into two
bands on SDS ⁄ PAGE, due to their similar molecular
masses [9,12]. In the present study, the two types were
separated into two bands by decreasing the amount of
protein analyzed and prolonging the electrophoresis
run time. Using this method, injection of labeled
CFMYP showed that part of the CFMYP is processed
to EGMYP after its incorporation into the gonad
(Fig. 5B). We assume that CFMYP diminishes its zinc-
binding capacity by losing at least one zinc-binding site
after its role in zinc transport is completed. We believe
that all of the CFMYP incorporated into the oocytes
is processed to EGMYP, because eggs contain only
EGMYP. In nutritive phagocytes, in contrast, part
of the incorporated CFMYP is not processed to
EGMYP, enabling it to retain zinc. Indeed, 33% of
the total zinc in the ovary at stage 1 and 13% of the
total zinc in the testis at stage 1 is still bound to MYP.
On the basis of the results of the present study and

others, we propose a model for the synthesis and accu-
mulation of MYP and its involvement in zinc trans-
port (Fig. 7). Nutrients are thought to be translocated
through the coelomic fluid in the form of free amino
acids [48] or MYP [9]. Both male and female sea urch-
ins synthesize MYP mainly in the digestive tract [8]
and nutritive phagocytes [10]. There are two possible
pathways from amino acids in the digestive tract to
MYP in the gonad. In the first, the amino acids are
transported from the digestive tract to the nutritive
phagocytes through the coelomic fluid, and then MYP
is synthesized in these cells. In this case, it is unknown
which type of MYP is synthesized (CFMYP, EGMYP,
or both). In the second, CFMYP is synthesized from
the amino acids in the digestive tract and then trans-
ported to the nutritive phagocytes. In this case,
CFMYP can carry zinc derived from ingested food to
the gonad. When CFMYP is incorporated into the
nutritive phagocytes, some of the CFMYP forming
homotetramers is processed to EGMYP, with loss of
zinc-binding site(s); the remainder retains zinc, possibly
for temporary storage. When CFMYP is incorporated
into vitellogenic oocytes, all of the CFMYP forming
homotetramers is processed to EGMYP with loss of
zinc-binding site(s). Sea urchins may use either of these
pathways, according to the demand for zinc; the latter
pathway appears to be more important in females than
in males, for the transport of larger amounts of zinc.
After its release from MYP in nutritive phagocytes
and oocytes, zinc would be bound to unknown mole-

cules involved in zinc storage. These molecules may be
T. Unuma et al. Zinc-binding protein in the sea urchin
FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS 4993
metallothioneins, zinc-binding proteins that function as
reservoirs for zinc in various animal cells [34], includ-
ing sea urchin eggs [33].
We do not exclude the possibility that MYP is also
involved in the transportation of other trace metals,
although in this study we did not obtain evidence that
MYP binds iron, copper, or manganese. The concen-
trations of these metals in the coelomic fluid is much
lower than that of zinc (about one-sixth for iron,
one-fiftieth for copper, and one-three hundredth for
manganese; our unpublished data). It is possible that
binding of these metals to MYP was not detected, due
to insufficient sensitivity of ICP-AES. Binding of iron
to CFMYP has been demonstrated in vitro using
59
Fe
[20]. If MYP has binding affinity for copper and man-
ganese as well as iron, these metals could be trans-
ported to the gonad in the same way as zinc.
Studies on MYP have clarified that it functions as a
protein reserve for oogenesis, spermatogenesis, and
early development [12,15,30]. MYP has also been pos-
tulated to act as a cell adhesion molecule during
embryogenesis [16–18]. In addition to these functions,
the present study implies that MYP has a role as a
zinc transporter for gametogenesis. In vertebrates,
vitellogenin, a precursor of yolk protein, is a zinc-bind-

ing protein that transports the zinc required for oogen-
esis to the ovary through the blood [35,45,46,49].
Vertebrate vitellogenin is thus a carrier of zinc as well
as a nutrient source for early development. Sea urchin
MYP, which is not homologous to vertebrate vitelloge-
nin, also appears to perform both of these essential
roles in reproduction.
Experimental procedures
Animals
Six-month-old juvenile P. depressus, hatched and reared at
the Fukuoka Prefectural Fish Farming Center, were trans-
ferred to the National Research Institute of Aquaculture,
raised in 1000 L tanks, and reared mainly on kelp, Eisenia
bicyclis. After about 2 years, twice per month from Septem-
ber to January 10–20 individuals (59.6 ± 4.1 mm test
diameter and 74.0 ± 13.8 g wet body weight; mean ± SD)
were randomly collected and used for the experiments.
Coelomic fluid was collected through the peristomial
membrane with Pasture pipettes and centrifuged at 500 g
for 5 min using an MC-15A centrifuge with TMA-1 rotor
(Tomy Seiko, Tokyo, Japan). The supernatant was filtered
through a 0.2 lm membrane to obtain cell-free coelomic
EG
EGEG
EG
EG
EGEG
EG
CF CF
CF CF

CF CF
CF CF
CF CF
CF CF
CF
CF
CF CF
CF
EG
EG
EG
CF
EGEG
EG
Coelomic fluid
Nutritive
phagocyte
Digestive tract
Oocyte
Amino acid
Zinc
*
*
*
X
X
X
Fig. 7. Proposed model for the synthesis and accumulation of MYP and its involvement in zinc transport in male and female sea urchins.
Two pathways are possible from amino acids in the digestive tract to MYP in the gonad. (1) Amino acids are transported to the nutritive
phagocytes and then used to produce MYP in these cells (open arrows). It is unclear which type of MYP is synthesized (CFMYP, EGMYP,

or both). (2) CFMYP is synthesized from the amino acids in the digestive tract and then transported to the gonad, playing a role as a zinc
transporter (closed arrows). When CFMYP is incorporated into the nutritive phagocytes, some of the CFMYP forming homotetramers is pro-
cessed to EGMYP, with loss of zinc-binding site(s); the remainder retains zinc. When CFMYP is incorporated into the vitellogenic oocytes,
all of the CFMYP forming homotetramers is processed to EGMYP, with loss of zinc-binding site(s). After its release from MYP in the nutri-
tive phagocytes and oocytes, zinc is bound to unknown molecules (X) involved in zinc storage. Pathways from the coelomic fluid or nutritive
phagocytes to oocytes are female-specific (arrows with an asterisk). CF, CFMYP; EG, EGMYP; small open circle, free amino acid; small
closed square, zinc; X, unknown molecule.
Zinc-binding protein in the sea urchin T. Unuma et al.
4994 FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS
fluid. The gonads were excised and rinsed in artificial sea-
water. A small portion of gonad was fixed in Bouin’s solu-
tion for histology, and the remainder was stored at ) 80 °C
for analysis for metals. Paraffin sections 6 lm thick were
prepared and stained with hematoxylin and eosin. The
gonadal maturity of each animal was classified into five
stages according to Fuji [50], with some modifications [42],
as follows: stage 1, before gametogenesis; stage 2, early
gametogenesis; stage 3, mid-gametogenesis; stage 4, fully
mature; stage 5, spent. Only gonads at stages 1–4 were used
in this study.
To obtain gametes, mature males and females were
injected with acetylcholine chloride (1 mm in seawater) and
placed in a Petri dish with the gonopore upward. Eggs and
sperm shed around the gonopore were collected with pip-
ettes and centrifuged at 5000 g for 20 s using an MC-15A
centrifuge with TMA-1 rotor (Tomy Seiko) to remove the
contaminated seawater and coelomic fluid.
Liquid chromatography
Gonads (0.5–1.0 g) or eggs (2 mL) were homogenized with
10 mL of 20 mm Tris ⁄ HCl buffer (pH 7.5) containing

100 mm NaCl (NaCl ⁄ Tris) and 1 mm phenylmethylsulfonyl-
fluoride using a Polytron homogenizer (Kinematica,
Lucerne, Switzerland). The homogenate was centrifuged at
15 000 g for 20 min at 4 °C using a RS-20IV centrifuge
and NO20N rotor (Tomy Seiko). The supernatant was fil-
tered through a 0.2 lm membrane and used as gonad or
egg extract. Cell-free coelomic fluid, gonad extract and egg
extract were subjected to gel filtration chromatography
using Superose 6 pg (Amersham Biosciences, Amersham,
UK) packed in a glass column (16 mm inner diameter,
600 mm bed height) equilibrated with NaCl ⁄ Tris. Proteins
were eluted with NaCl ⁄ Tris at a flow rate of 0.8 mLÆmin
)1
using an FPLC system (Amersham Biosciences). Aliquots
of eluate (2 mL) were collected. Fractions rich in MYP
were pooled and subjected to ion exchange chromatography
using Mono Q 5 ⁄ 50GL (Amersham Biosciences) equili-
brated with 20 mm Tris ⁄ HCl buffer (pH 7.5). The retained
proteins were eluted with an NaCl linear gradient from
0mm to 800 mm (10 or 20 mL in total) at a flow rate of
0.5 mLÆmin
)1
using the FPLC system. Aliquots of eluate
(0.5 mL) were collected. The protein concentrations of the
fractions were measured by the Bradford method [51] using
a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA)
with bovine c-globulin as a standard. Thyroglobulin
(669 kDa), catalase (232 kDa), BSA (67 kDa) and chymo-
trypsinogen (25 kDa) were used for molecular mass estima-
tion in gel filtration chromatography.

Determination of metals
Concentrations of manganese, iron, copper and zinc in the
fractions obtained by liquid chromatography were measured
by ICP-AES using model JICP-PS3000UV (Leeman Labora-
tories, Hudson, NH, USA). Coelomic fluid was diluted with
distilled water and then subjected to ICP-AES for determina-
tion of zinc. Gametes and gonads were decomposed by the
wet ashing method using concentrated nitric acid, and then
dissolved in 0.1 m nitric acid. Manganese, copper and zinc in
the nitric acid solution prepared from the gametes were
determined by inductively coupled plasma mass spectrome-
try using model SPO-9000 (Seiko Instruments, Chiba,
Japan). Iron in the nitric acid solution prepared from the
gametes was determined by graphite furnace atomic absorp-
tion spectrometry using model 4100ZL (Perkin Elmer, Wal-
tham, MA, USA). Zinc in the nitric acid solution prepared
from the gonads was determined by ICP-AES. To investigate
the accumulation of zinc in the gonads during gameto-
genesis, increases in the size of the gonads during this period
were taken into consideration. Thus, zinc levels in the
gonads were expressed as the amount per 100 g body weight.
PAGE and western blotting
SDS ⁄ PAGE was performed using a 5–20% gradient gel
under reducing conditions with dithiothreitol or under non-
reducing conditions without dithiothreitol, as described pre-
viously [9]. Native PAGE was performed using a 2–15%
gradient gel according to the Ornstein–Davis method
[52,53]. After electrophoresis, the gel was stained with Coo-
massie Brilliant Blue R-350 (CBB) or SYPRO Ruby protein
gel stain (Bio-Rad) and scanned using a Molecular Imager

FX Pro (Bio-Rad). Precision protein standards (Bio-Rad)
were used for molecular mass estimation.
After SDS ⁄ PAGE, the separated proteins were electro-
blotted onto a poly(vinylidene difluoride) membrane. The
membrane was immunoreacted with a polyclonal antibody
specific to MYP (anti-MYP) [9] and then with anti-rabbit
IgG labeled with alkaline phosphatase (Bio-Rad). The
MYP bands were visualized with Nitro Blue tetrazolium ⁄
5-bromo-4-chloroindol-2-yl phosphate (Roche, Basel,
Switzerland) as a substrate.
Measurement of MYP by densitometry
CFMYP and EGMYP contents in the MYP obtained from
the gonad and the CFMYP concentration in the coelomic
fluid were measured by densitometry of the SDS ⁄ PAGE
gel. Purified CFMYP or EGMYP, the concentration of
which had been determined by the Bradford method, was
used as the standard protein in this analysis. The samples
and serial dilutions of the standard protein were subjected
to SDS ⁄ PAGE in the same gel. The gel was stained with
CBB or SYPRO Ruby and scanned with a Molecular Ima-
ger FX Pro. The levels of CFMYP and EGMYP in the
samples were calculated from the standard curve obtained
from the intensity of the bands of standard protein using
quanty one software (Bio-Rad).
T. Unuma et al. Zinc-binding protein in the sea urchin
FEBS Journal 274 (2007) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS 4995
Equilibrium dialysis
Purified CFMYP and EGMYP were used for saturation
binding assays as previously described [54]. About 10 mg of
CFMYP and EGMYP was dialyzed twice against 1 L of

0.1 m citric acid to remove the metals bound to these pro-
teins, and then dialyzed twice against 1 L of 20 mm Hepes
(pH 7.6) containing 100 mm NaCl and 10 mm NaHCO
3
(equilibrium buffer). The protein concentration was
adjusted to 4 lm by dilution with the equilibrium buffer.
Equilibrium dialysis was performed in plastic cell compart-
ments separated with partially permeable membrane tubing
made from regenerated cellulose (Sanplatec, Osaka, Japan).
One milliliter of CFMYP or EGMYP solution (4 nmol)
and 1 mL of equilibrium buffer were pipetted on opposite
sides of the membrane. Zinc chloride (0.5 mm) dissolved in
5mm sodium citrate was added to the equilibrium buffer.
Sodium citrate (5 mm) was added to the equilibrium buffer
to adjust the concentration of citrate to 0.1 mm for all cells.
After 3 days of gentle agitation on a shaker at 4 °C, the
solutions were withdrawn from each cell. The zinc concen-
tration of the solutions was measured by ICP-AES. The
data were analyzed using prism 4 software (GraphPad Soft-
ware, San Diego, CA, USA).
Injection of labeled protein into sea urchins
Lactoferrin from bovine milk (Wako Pure Chemicals,
Osaka, Japan) was dissolved in distilled water, applied to a
Mono Q 5 ⁄ 50GL column (Amersham Biosciences) equili-
brated with 20 mm Tris ⁄ HCl buffer (pH 9.0), and then
eluted with an NaCl linear gradient from 0 mm to 400 mm.
The peak fractions eluted at 120 mm were collected as puri-
fied lactoferrin and used for further experiments. Purified
CFMYP and lactoferrin were labeled with N-hydroxysuccin-
imide-fluorescein using an EZ-Label fluorescein protein-

labeling kit (Pierce, Rockford, IL, USA) according to the
manufacturer’s instructions. Fl-CFMYP and Fl-lactoferrin
were dialyzed against filtered seawater, and the protein con-
centration was adjusted to 3 mgÆmL
)1
. The solution of
Fl-CFMYP or Fl-lactoferrin was then injected through the
peristomial membrane into the coelomic cavity of P. depres-
sus (15.0 ± 2.0 g; mean ± SD) at 2 mLÆ(100 g body
weight)
)1
. Each group of animals was stocked in an acrylic
tank (20 L) supplied with sand-filtered seawater at a rate of
0.5 LÆmin
)1
. The urchins were given an excess of dried kelp,
Laminalia sp., and allowed to feed to satiation. The water
temperature during the experimental period was about
25 °C. Six days and 15 days after injection, five animals from
each group were killed and the gonads were excised. Gonad
(0.1 g) was homogenized with 5 mL of NaCl ⁄ Tris and cen-
trifuged at 15 000 g for 20 min at 4 °C using a RS-20IV cen-
trifuge and NO20N rotor (Tomy Seiko), and the supernatant
was subjected to western blot analysis using anti-Fl (Rock-
land Incorporated, Gilbertsville, PA, USA). To purify the
labeled MYP for better resolution on western blot analysis,
small amounts of gonad extract were subjected to immuno-
precipitation using anti-Fl and Protein G Sepharose 4 Fast
Flow (Amersham Biosciences) according to the manufac-
turer’s instruction. Small pieces of gonad were fixed in Bou-

in’s solution, embedded in paraffin, sectioned at 6 lm, and
stained with hematoxylin and eosin for staging of gameto-
genesis, or used for immunohistochemistry using anti-Fl with
Nitro Blue tetrazolium ⁄ 5-bromo-4-chloroindol-2-yl phos-
phate as a substrate for localization of the labeled MYP.
Statistical analysis
Data were expressed as the mean ± SEM. Statistical analy-
sis was performed using instat software (GraphPad Soft-
ware). The normality of the distribution of data was
evaluated using the Kolmogorov–Smirnov test. The equal-
ity of the standard deviations of the groups was assessed
with Bartlett’s test. The Tukey–Kramer multiple compari-
sons test was performed to examine the differences among
stages in males and females. P-values of <0.05 were con-
sidered to be statistically significant.
Acknowledgements
We thank Atsushi Yukutake at the Fukuoka Prefec-
tural Fish Farming Center for providing us with the
sea urchin seedlings, Professor Yukio Yokota at Aichi
Prefectural University for his helpful discussion on this
study, and Ryoko Okamoto at the National Research
Institute of Aquaculture for technical assistance
throughout. This study was financially supported by
the Fisheries Research Agency.
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