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

Most of the zinc in the coelomic fluid was bound to CFMYP, whereas zinc in eggs was scarcely bound to EGMYP.. Considering the fact that the digestive tract is a major production site of M

urchin ) implications of its role as a zinc transporter for gametogenesis

Tatsuya Unuma1, Kazuo Ikeda2, Keisuke Yamano3, Akihiko Moriyama4and Hiromi Ohta5

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: unuma@fra.affrc.go.jp

(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 CFEG-MYP 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.

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 59Fe 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,

Coelomic fluid

C

E

D

0 0.2 0.4 0.6 0.8

0

50

100

150

200

250

Elution volume (ml)

Zn Fe a

0 0.4 0.8 1.2 1.6

0 20 40 60 80 100

Elution volume (ml) Egg Ovary at stage 1

0 0.6 0.8 1.2 1.6

0

20

40

60

80

100

Elution volume (ml)

Testis at stage 1

0 0.4 0.8 1.2 1.6

0 20 40 60 80 100

Elution volume (ml) b

c

d

SDS-PAGE and western analysis

250 kDa

150 kDa

100 kDa

50 kDa

25 kDa

a b c

d

a b c d

Anti-MYP

Anti-MYP CBB

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 (Vo), the column vol-ume (Vc), 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.

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

0.0

0.5

1.0

1.5

2.0

Elution volume (ml)

Coelomic fluid

A

NaCl

0 200 400 600

0.0 0.5 1.0 1.5

Elution volume (ml) 0

200 400 600

0.0 0.5 1.0 1.5

Elution volume (ml)

Native-PAGE

Ovary at stage 1

Egg

a b c

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 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.

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 non-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 (BMAX) 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 (Kd) values were estimated as 5· 10)7m for CFMYP and 3· 10)7mfor 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

0 10 20 30 40 50

1 Stage 3 0

0.05 0.1 0.15 0.2

1 Stage

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.

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)1at 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

Scatchard

Scatchard

Total binding

Nonspecific

binding

Total binding

Nonspecific

binding

0

2

4

6

8

Total Zn / MYP (molar ratio)

0 2 4 6

Bound Zn / MYP (molar ratio)

-1)

0

2

4

6

8

Total Zn / MYP (molar ratio)

0 2 4 6

Bound Zn / MYP (molar ratio)

-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).

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

15 days

Ovary

250 kDa

150 kDa

100 kDa

50 kDa

25 kDa

o t

6 days

Ovary

CFMYP EGMYP

Western blotting after immunoprecipitation

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.

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

Stage

Zn in CF

0 250 500 750 1000

Stage

CFMYP in CF

Female Male

0 50 100 150 200 250

Stage

Total Zn in the gonad

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]

a

Measured by inductively coupled plasma mass spectrometry.

b Measured by graphite furnace atomic absorption spectrometry.

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 incorporIniti-ation 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-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 molemole-cules may be

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 59Fe

[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 EG EG EG

EG EG EG EG

CF CF

CF CF

CF CF

CF CF

CF CF

CF CF

CF CF

CF CF CF

EG EG

EG

CF EG EG 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 nutrinutri-tive 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.