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: 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 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 [...]... Unuma et al Zinc- binding protein in the sea urchin fluid The gonads were excised and rinsed in artificial seawater A small portion of gonad was fixed in Bouin’s solution 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... Lattuca G, Parrinello N & Matranga V (199 4) Detection of vitellogenin in a subpopulation of sea urchin coelomocytes Eur J Cell Biol 64, 314–319 Zinc- binding protein in the sea urchin 20 Brooks JM & Wessel GM (200 2) The major yolk protein in sea urchins is a transferrin-like, iron binding protein Dev Biol 245, 1–12 21 Yokota Y, Unuma T, Moriyama A & Yamano K (200 3) Cleavage site of a major yolk protein. .. the labeled MYP Statistical analysis Data were expressed as the mean ± SEM Statistical analysis was performed using instat software (GraphPad Software) The normality of the distribution of data was evaluated using the Kolmogorov–Smirnov test The equality of the standard deviations of the groups was assessed with Bartlett’s test The Tukey–Kramer multiple comparisons test was performed to examine the. .. 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 chymotrypsinogen (25 kDa) were used for molecular mass estimation in gel filtration chromatography SDS ⁄ PAGE was performed using a 5–20% gradient gel... of gentle agitation on a shaker at 4 °C, the solutions were withdrawn from each cell The zinc concentration of the solutions was measured by ICP-AES The data were analyzed using prism 4 software (GraphPad Software, 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... from the intensity of the bands of standard protein using quanty one software (Bio-Rad) FEBS Journal 274 (200 7) 4985–4998 ª 2007 The Authors Journal compilation ª 2007 FEBS 4995 Zinc- binding protein in the sea urchin T Unuma et al 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... Elmer, Waltham, 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 gametogenesis, 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 Liquid chromatography PAGE and western blotting Gonads... 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 Imager FX Pro The levels of CFMYP and EGMYP in the samples were calculated from the standard curve obtained... Enfield 31 Giga Y & Ikai A (198 5) Purification of the most abundant protein in the coelomic fluid of a sea urchin which immunologically cross reacts with 23S glycoprotein in the sea urchin eggs J Biochem 98, 19–26 32 Giga Y & Ikai A (198 5) Purification and physical chemical characterization of 23S glycoprotein from sea urchin (Anthocidaris crassispina) eggs J Biochem 98, 237–243 33 Scudiero R, Capasso C, Del... USA) according to the manufacturer’s instructions Fl-CFMYP and Fl-lactoferrin were dialyzed against filtered seawater, and the protein concentration 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 depressus (15.0 ± 2.0 g; mean ± SD) at 2 mLÆ(100 g body weight )) 1 Each group of animals was stocked in an . 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 ,. 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