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A protein identical to the yolk protein is stored in the testis in male red sea urchin, Pseudocentrotus depressus.

Introduction

The most abundant yolk protein found in sea urchin eggs is a glycoprotein with a molecular weight of about 180 kDa; this has been termed the major yolk protein or major yolk glycoprotein (MYP; Harrington and Easton, 1982; Kari and Rottmann, 1985; Yokota and Kato, 1988). Unlike other oviparous animals in which vitellogenin, a precursor of MYP, is female specific, sea urchins have an abundant supply of vitellogenin in the coelomic fluid of males as well as females (Harrington and Easton, 1982; Shyu et al., 1986). Shyu et al. (1986) found that vitellogenin mRNA is expressed in the intestines and gonads of both sexes in Strongylocentrotus purpuratus. This suggests the possibility that in sea urchins vitellogenin performs an unidentified function required by males in addition to its role in vitellogenesis in females.

In female sea urchins, vitellogenin is reported to be incorporated into the nutritive phagocytes (accessory cells) in the previtellogenic ovary for temporary storage, and then transported to the oocytes to be accumulated as MYP (Ozaki et al., 1986; Harrington and Ozaki, 1986). The gonads of male sea urchins also have nutritive phagocytes for nutrient storage (Walker, 1982). However, it is not clear whether the testicular nutritive phagocytes contain vitellogenin.

The main objective of this study is to examine whether male sea urchins store MYP-related proteins in their nutritive phagocytes. To achieve this, we purified MYP from unfertilized eggs of the red sea urchin, Pseudocentrotus depressus, and prepared an antiserum against MYP. We compared the distribution of MYP reactivity during gametogenesis in both sexes by immunological analysis.

Materials and Methods

Animals

Six-month-old juveniles of P. depressus, hatched and reared at the Fukuoka Prefectural Fish Farming Center, were transferred to the Coastal Station of the National Research Institute of Aquaculture. They were kept in 1000-1 tanks supplied with sand-filtered seawater at 30 1 [multiplied by] [min.sup.-1], and reared on kelp, Eisenia bicyclis. The animals used for the experiment were 2 or 3 years old.

After the peristomial membrane of the animal was removed, coelomic fluid was collected with Pasteur pipettes. Coelomocytes were removed by centrifugation at 600 x g for 5 min. The coelomic fluid was kept at -80 [degrees] C until use.

Gonads were dissected out and stored at -80 [degrees] C. Small pieces were fixed in Bouin's solution for histological observations. Paraffin sections of 6-[[micro]meter] thickness were prepared and stained with hematoxylin and eosin. The gonadal maturity of each animal was classified according to the six stages described by Fuji (1960), with some slight modifications as described previously (Unuma et al., 1996).

During the spawning season in November, eggs were obtained by coelomic injection of 20% KCl and then were stored at -80 [degrees] C.

Purification of MYP

Unfertilized eggs (3 ml) were homogenized with 15 ml of 10 mM Tris-HCl buffer containing 10 mM NaCl (TBS; pH 8.0) and 0.1 mM phenylmethylsulfonylfluoride (PMSF) using Polytron (Kinematica, Switzerland). The homogenate was centrifuged at 25,000 x g for 20 min at 4 [degrees] C, and the supernatant was applied to a HiLoad 16/10 Q Sepharose fast flow column (Pharmacia LKB Biotechnology, Sweden) equilibrated with TBS. After being washed with 40 ml of the same buffer, the retained proteins were eluted with a NaCl linear gradient from 10 mM to 1 M (200 ml in total) using FPLC (Pharmacia). The flow rate of the column was 2.0 ml [multiplied by] [min.sup.-1], and 4-ml aliquots of eluate were collected. Fractions rich in MYP were pooled, concentrated threefold using Molcut LGC (Millipore Corp., USA), and applied to a HiLoad 16/60 Superdex 200 column (Pharmacia) equilibrated with 10 mM Tris-HCl buffer containing 150 mM NaCl (pH 8.0). Proteins were eluted with the same buffer at a flow rate of 1 ml [multiplied by] [min.sup.-1], and fractions of 2 ml were collected. A gel filtration calibration kit (Pharmacia) was used to estimate molecular weight.

Electrophoresis

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 5% slab gel according to Laemmli (1970). Protein bands were visualized with Coomassie brilliant blue R-250. SDS-PAGE standards (Bio-Rad Laboratories, USA) were electrophoresed for molecular weight calibration. Precast gels (NPU-5L; Atto Corp., Japan) were used for Figures 1 and 5. These gels showed double bands in the low molecular weight end, whereas the gels prepared in our laboratory showed a single band there [ILLUSTRATION FOR FIGURE 4 OMITTED]. However, Atto Corp. assures us that there is no difference between these gels in comparisons of the middle to high molecular weight ranges.

Preparation of antiserum

The peak fraction from the Superdex 200 gel filtration was electrophoresed on SDS-PAGE under nonreducing conditions. A gel slice containing the 270-kDa band was excised. The protein was eluted from the gel by an Electro Eluter (Bio-Rad), mixed with an equal volume of Freund's complete adjuvant, and then injected subcutaneously into the back of a rabbit once a week. After 4 injections, blood was collected from an ear vein. The blood was centrifuged at 1500 x g for 20 min, and the supernatant was collected as antiserum (anti-MYP).

Immunoblotting

Gonads were homogenized with 20-fold TBS and centrifuged at 25,000 x g for 20 min at 4 [degrees] C. Supernatants were used as the gonadal extracts.

To detect the protein reactive with anti-MYP, coelomic fluids and the gonadal extracts were immunoblotted according to Towbin et al. (1979). Proteins separated by 5% SDS-PAGE were electrophoreticaily transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was immunostained using anti-MYP and POD Immunostain Set (Wako Pure Chemical Industries, Ltd., Japan).

Immunohistochemistry

To visualize MYP reactivity in the gonads, immunohistochemistry was performed on paraffin-embedded gonads sectioned at 6 [[micro]meter]. After the paraffin was removed, sections were incubated with anti-MYP (1:2000 dilution) and then treated with Histofine SAB-PO Kit (Nichirei, Japan) as described in the manufacturer's instructions. Normal rabbit serum was substituted for anti-MYP to confirm the specificity of the immunohistochemical staining.

Results

Purification of MYP

SDS-PAGE analysis under reducing conditions showed that a 170-kDa protein occurred abundantly in P. depressus eggs [ILLUSTRATION FOR FIGURE 1A OMITTED]. This protein band was reactive with periodic acid-Schiff (PAS) reagent (data not shown), similar to MYP reported for other sea urchin eggs (Harrington and Easton, 1982; Ozaki et al., 1986; Yokota and Kato, 1988). We concluded that this 170-kDa protein is the MYP of P. depressus because of the similarity in molecular weight and the PAS reactivity with other MYP. Under nonreducing conditions, a large quantity of 270-kda protein was observed instead of the 170-kDa band [ILLUSTRATION FOR FIGURE 1D OMITTED]. This suggests that intact MYP consists of subunits.

Egg extract was applied to an ion exchange column using Q Sepharose [ILLUSTRATION FOR FIGURE 2 OMITTED]. The major protein peak eluted at 280 mM NaCl was revealed to be rich in MYP by SDS-PAGE ([ILLUSTRATION FOR FIGURES 1B, E OMITTED]. After concentration, the fractions were subjected to gel filtration using Superdex 200 [ILLUSTRATION FOR FIGURE 3 OMITTED]. The major peak eluted at 600 kDa gave a homogeneous 270-kDa band on SDS-PAGE under nonreducing conditions [ILLUSTRATION FOR FIGURE 1F OMITTED]. Thus, we concluded that the MYP of P. depressus was isolated by a combination of ion exchange chromatography and gel filtration. Under reducing conditions, MYP gave a major 170-kDa band and four minor bands of about 100 kDa [ILLUSTRATION FOR FIGURE 1C OMITTED]. These minor bands are probably fragments derived from the intact MYP by proteolysis.

To raise an antiserum against MYP (anti-MYP), we electronically eluted the 270-kDa band from an SDS-PAGE gel run under nonreducing conditions, then immunized a rabbit with the isolated protein [ILLUSTRATION FOR FIGURE 1F OMITTED].

MYP-related proteins in gonads and coelomic fluids

We used SDS-PAGE and immunoblot analyses to test the gonads and coelomic fluids of Stage 1 females and males for the presence of the protein reactive with anti-MYP. A 170-kDa protein was predominant in both female and male gonadal extracts under reducing conditions [ILLUSTRATION FOR FIGURE 4B, C OMITTED]. These protein bands were immunoreactive with anti-MYP [ILLUSTRATION FOR FIGURE 4L, M OMITTED]. On the basis of the molecular weight and immunoreactivity of the major protein stored in the immature ovary and testis, we concluded that it is identical to MYP. Predominant protein bands with a slightly higher molecular weight (180 kDa) than MYP were detected in the coelomic fluid of both sexes [ILLUSTRATION FOR FIGURE 4D, E OMITTED]. In other sea urchins investigated, vitellogenin is the most abundant coelomic fluid protein; it has a molecular weight of about 200 kDa and is immunoreactive with the antiserum against MYP (Harrington and Easton, 1982; Shyu et al., 1986; Cervello et al. 1994). The protein of the predominant bands of 180 kDa in the coelomic fluids was identified as vitellogenin on the basis of its reactivity with anti-MYP [ILLUSTRATION FOR FIGURE 4N, O OMITTED] and the similarity of its molecular weight to that of vitellogenin in other sea urchins. The protein in the gonads and coelomic fluids of both sexes appears to exist as a complex - as does MYP in unfertilized eggs - because in SDS-PAGE under nonreducing conditions bands occurred at around 270 kDa [ILLUSTRATION FOR FIGURE 4F-J OMITTED].

Extracts from testes at three maturational stages (Stages 1, 3, and 4) were subjected to SDS-PAGE and immunoblotting analyses [ILLUSTRATION FOR FIGURE 5 OMITTED]. As spermatogenesis progressed, the quantity of 170-kDa protein reactive with anti-MYP decreased in the testis. Mature testis (Stage 5) contained little of this protein [ILLUSTRATION FOR FIGURE 5C, F OMITTED].

Distribution of MYP in gonads during gametogenesis

Immunolocalization of MYP in the gonads is shown in Figure 6. The follicular lumina of immature testes and ovaries (Stage 1) were filled with nutritive phagocytes [ILLUSTRATION FOR FIGURE 6A, G OMITTED]). The protein reactive with anti-MYP was stored in the nutritive phagocytes of both sexes [ILLUSTRATION FOR FIGURE 6D, J OMITTED]. With the progress of gametogenesis, testes contained peripheral lines of spermatogonia and spermatocytes, and ovaries had a row of vitellogenic oocytes. The lumen of testes later filled with spermatozoa and that of ovaries with ripe ova [ILLUSTRATION FOR FIGURE 6B, H OMITTED]. Spermatogenic cells and oocytes did not react with the anti-MYP, whereas ripe ova did [ILLUSTRATION FOR FIGURE 6E, K OMITTED]. The nutritive phagocytes had degenerated but were still reactive with anti-MYP in Stage 3 gonads [ILLUSTRATION FOR FIGURE 6E, K OMITTED]. In mature gonads (Stage 4), the gonadal lumina were filled with spermatozoa or ripe ova, and nutritive phagocytes were recognized only at the periphery of follicles [ILLUSTRATION FOR FIGURE 6C, I OMITTED]. The protein disappeared from the testis [ILLUSTRATION FOR FIGURE 6F OMITTED], but in the ovary it accumulated in ripe ova as a yolk protein [ILLUSTRATION FOR FIGURE 6L OMITTED].

Discussion

This study demonstrates that prior to gametogenesis in the red sea urchin P. depressus, a protein identical to MYP is stored abundantly in the nutritive phagocytes of males as well as those of females. However, as gametogenesis proceeds, this protein decreases in quantity in the testis while it is accumulated in ripe ova as a yolk protein in the ovary. Histological observations have led to the suggestion that nutritive phagocytes in the testis are nutrient storage sites for spermatogenesis (Walker, 1982). However, it is still unclear what kind of material functions as a nutrient storage in the testicular nutritive phagocytes. We propose that the MYP found in the testis functions as a nutrient for spermatogenesis in P. depressus. In oviparous animals, yolk protein is a nutrient source for embryogenesis and is usually found only in the female (Hara, 1987; Quinitio et al., 1989, 1990; Suzuki et al., 1992; Osada et al., 1992). P. depressus is unique in apparently using a yolk protein as a nutrient for spermatogenesis.

Vitellogenin, a precursor of MYP, is abundant in the coelomic fluid of both males and females, and its role in the male sea urchin has been discussed (Harrington and Easton, 1982; Shyu et al., 1986). Harrington and Easton (1982) postulated that vitellogenin performs an unknown physiological role required by both sexes, but related to the hermaphrodism observed in some echinoderms. Shyu et al. (1986) proposed that vitellogenin functions as an analog to the serum albumin of vertebrates, as a carrier protein, or as a store for amino acids. The possibility that male vitellogenin, like female vitellogenin, is a precursor of MYP has not been discussed because MYP storage in the testis had not been demonstrated. In female sea urchins, vitellogenin is incorporated into the nutritive phagocytes, then transported to the oocytes to be accumulated as MYP (Ozaki et al., 1986; Harrington and Ozaki, 1986). We suggest that male vitellogenin is a precursor of MYP in the testis and is incorporated into the testicular nutritive phagocytes as a nutrient source for spermatogenesis. We think that the process until the incorporation into the gonad is probably the same in both sexes, although the final site for utilization is different.

MYP of P. depressus has a molecular weight of 170 kda under reducing conditions, but vitellogenin has a slightly higher molecular weight (180 kDa). The same is true in S. purpuratus: the molecular weight of vitellogenin is 195 kDa, but that of MYP is 180 kda (Harrington and Easton, 1982; Shyu et al., 1986). Thus, a decrease in molecular weight from vitellogenins to MYPs seems to be a common phenomenon in sea urchins. It is probable that vitellogenin is slightly modified in molecular structure after its incorporation into the gonads. Shyu et al. (1986) presumed that this modification takes place in the oocytes, but we suggest that it occurs in the nutritive phagocytes of both sexes immediately after incorporation. We base our conclusion on the observation that the protein stored in the nutritive phagocytes is already modified to 170 kDa and little of the 180 kDa protein is detected in the gonads.

Acknowledgments

We are grateful to the Fukuoka Prefectural Fish Farming Center for providing the juvenile P. depressus used in this study.

Literature Cited

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Quinitio, E. T., A. Hara, K. Yamauchi, and A. Fuji. 1990. Isolation and characterization of vitellin from the ovary of Penaeus monodon. Invertebr. Reprod. Dev. 17: 221-227.

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Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350-4354.

Unuma, T., K. Konishi, H. Furuita, T. Yamamoto, and T. Akiyama. 1996. Seasonal changes in gonads of cultured and wild red sea urchin, Pseudocentrotus depressus. Suisanzoshoku 44(2): 169-175.

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Author:Unuma, Tatsuya; Suzuki, Tohru; Kurokawa, Tadahide; Yamamoto, Takeshi; Akiyama, Toshio
Publication:The Biological Bulletin
Date:Feb 1, 1998
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