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Immunological studies on the 26S particles of sea urchin eggs
Copyright All rights
0 1972 by Academic Pwss, Inc. of rcprodurfion in any fovm rmwed
Experimental
IMMUNOLOGICAL
Cell Research 7.5 (1972) 385-396
STUDIES ON THE 26 S PARTICLES
OF SEA URCHIN
EGGS’
H. KONDO” and H. KOSHIHARA Zoological
Insritutr,
Faculty of Science, Tokyo Kyoiku-University,
Bunkyo-ku,
Tokyo, Japan
SUMMARY 1. 26s particles were prepared from the 105 000 g supernatant of unfertilized eggs of Anthocidaris crassispina and purified by sucrose density gradient centrifugation. By using the purified particles,
an antiserum against them was prepared with the intention of tracing and locating it by immunological methods. 2. Further distribution of the 26s particles was examined by the immunodiffusion test with the antiserum. It was found that the 26s particles were distributed independently and as a discrete component in cytoplasm of eggs, which supports our previous evidence that they are not derived from heavier granules, i.e. yolk granules. Also, the 26s particles retained the same antigenic determinant throughout sea urchin development. 3. Among several tissues from the same sea urchin, gonads from female sea urchins or perivisceral fluid irrespective of sex reacted with the antiserum against the 26s particles by forming a common conspicuous precipitation band, Furthermore, the antigenic determinants exhibiting serological similarity to the 26s particles were distributed in perivisceral fluid of other sea urchins belonging to the order of Echinoidea, whereas those in Clypeasteria did not form any crossreacted precipitation band. 4. The same antigenic determinant distributed in tissues of the same sea urchin was located in isolated particulate fractions, as prepared from perivisceral fluid or accessory cells as well as eggs. The possible biological functions of these particulate fractions are discussed.
Our previous report dealt with the centrifugal and electrophoretic characterization of the post-microsomal 26s particles of sea urchin embryos in comparison with characteristics of yolk granules from the same embryonic source [4]. The results so far obtained indicated that the 26s particles were discrete entities of the cytoplasm of sea urchin embryos and could not be regarded as being derived from heavier cytoplasmic granules, i.e. yolk granules. It was also found that the sedimentation properties of the 26s particles altered gradually in the course of developi Contributions from the Shimoda Marine Biological Station, No. 252. 2 Present address: Department of Biology, Tokyo Metropolitan Institute of Gerontology, Sakaecho, Itabashi-ku, Tokyo-173, Japan. 26
if21814
ment, and decreased quantitatively in the embryos. Further, we sought additional and decisive information on our 26s particles in the hope of confirming our earlier conclusion and to reveal those biological characteristics and function of the 26s particles, which are still unknown. In this paper, an immunological approach to our problems is described. By using an antiserum against the 26s particles purified from unfertilized eggs of Anthociduris crussispina, it was possible to follow the antigen in the course of sea urchin development and to locate it in intracellular fractions from eggs or in various tissues of adult sea urchins. Subsequently, the same antigenic determinant Exptl Cell Res 7.5 (1972)
386 H. Kondo & H. Koshihara with ammonium sulfate ad modum Malkin et al. If. The final precipitate was the purified preparation of the 26s particles, running as a single band at polyacrylamide gel electrophoresis (fig. 1). These purified 26s particles were used as the antigen for the present immunological studies.
Subcellular fractionation Unfertilized eggs of A. crassispina were homogenized in a homogenization medium containing 0.01 M Tris-HCI buffer (pH 7.2), 0.25 M sucrose, 0.15 M NaCI, 0.15 M KC1 and 0.003 M MgCl, and fractionated, as described before [4].
Polyacrylamide gel electrophoresis The method of Reisfeld et al. [6] was used. The samples were dissolved in Tris-glycine buffer solution (pH-8.6) containing 1 M sucrose, and layered on gel columns containing 5 % acrylamide [4]. Electrophoresis was conducted for 30 min at 4 mA per gel column. After electrophoresis the gel columns were stained and destained, as described in [4]. Fig. 1. Patterns of polyacrylamide gel electrophoresis of the purified 26s particles from unfertilized eggs of A. crassispina. The preparation was applied to the column by 40 pug of protein, and the electrophoresis was carried out as described in Methods.
was located in perivisceral fluid or accessory cells in gonads of the same sea urchins, and similar antigenicities were observed in perivisceral fluid of some other sea urchins. MATERIALS
AND METHODS
Materials Eggs, embryos and adults of Anthocidaris crassispina were used in the majority of these experiments. Techniques for the obtaining and handling sea urchin gametes and embryos have been described in our previous paper [4]. Several adult tissues used in immunological studies were collected from female or male sea urchins of Anthocidaris crassispina in the breeding season. Perivisceral fluid was centrifuged at 700 g for 20 min and separated into the non-cellular fraction and the cellular fraction containing planocytes and other cells. In comparative experiments, several species of Echinoidea were used: Hemicentrotus pulcherrimus, Mespilia globulus, Pseudocentrotus depressus, Clypeaster japonicus, Astriclypeus manni and Peronella japonica.
Purification
of the 26s particles
The 26s particles were prepared from a 270 000 g precipitate (27 ppt) by a sucrose density gradient centrifugation procedure, as described previously [4]. The 26s particles were further purified by salting out Exptl
Cell Res 75 (1972)
Preparation of antiserum against the 26 S particles 0.8 mg of the 26s particles prepared from unfertilized eggs of A. crassispina was suspended in 1 ml of 0.01 M phosphate buffer, pH 7.1, containing 0.145 M NaCl (PBS), and I-2 ml of the suspension was injected subcutaneously into rabbits. The injections were repeated at 7 day intervals for 4 weeks. Serum was sampled at times during the course of immunization and tested for its reactivity with the antigen by Ouchterlony’s immunodiffusion test. Satisfactory antisera were obtained through the carotid artery after 5 weeks, and stored at - 20°C until use.
Immunodiffusion
test
Ouchterlony’s immunodiffusion technique was employed. Agar was dissolved at 1 % in PBS mixed with l/l00 vol of 1 % merzonine. Wells of 9 mm diameter (in most of these experiments) or 4 mm on Ouchterlony’s agar plates were spaced out at distances of 9.5 mm or 3.5 mm, respectively. Agar plates on which antisera and antigen preparations were placed in each of the wells were incubated at 18°C. and were observed to form precipitation bands.
RESULTS Properties of the antiserum against the 26s particles The antiserum against the 26s particles was placed on Ouchterlony’s agar to react with the antigen at different concentrations from 0.8 mg/ml to 25 pg/ml (fig. 2). Only one precipitation band resulted from all these antigen concentrations. In addition, the anti-
The 26Sparticles
of sea urchin eggs
387
Fig. 2. Immunodiffusion
tests between the antiserum and the 26s particles from unfertilized eggs (a), and between the antiserum and the homogenate of unfertilized eggs (b) of A. cmssispina. (a) The central well contained the antiserum (1: 8 dilution), and the wells l-6 contained 800 pLg,400,200, 100, 50, and 25 pg of protein of the 26s particles/ml, respectively. (b) The central well contained the antsierum (1 : 8 dilution) and the well 1 contained 200 pugof protein of the 26s particles/ml. Wells 2-6 contained the homogenate of unfertilized eggs at 10 mg, 2, 500,250, and 125 pg/ml of protein respectively. Fig. 3. Immunodiffusion test of the subcellular fractions from unfertilized eggs of A. crassispina, using the antiserum against the 26s particles. The central well contained the antiserum and well 1 contained 200,~g of protein of the 26s particles/ml. Wells 2-6 contained yolk-nuclear fraction (1 500 g precipitate), mitochondrial fraction (12 000 g precipitate), microsomal fraction (105 000 g precipitate), 27 ppt (270 000 g precipitate) and 27 sup fractions (270 000 g supernatant) in order, with 1 mg/ml protein (a) or with 500 pg/ml protein (b). This subcellular fractionation was done according to the previous procedure [4].
serum was placed so as to react with the homogenate from unfertilized eggs in the agar diffusion test. As shown in fig. 2b, at the concentrations of the homogenate tested only one precipitation band was formed. This indicated that a single antigen component, presumably the 26s particles, was present in the homogenate of unfertilized eggs. Furthermore, we ascertained that the
antigen was localized only at a discrete band of the 26s particles subjected to acrylamide gel electrophoresis. The preparations corresponding to the 26s particles were isolated from morulae, gastrulae and plutei. These preparations were placed on Ouchterlony’s agar diffusion plates to react with the antiserum against the 26s particles from unfertilized eggs. They reacted Exptl Cell Res 75 (1972)
388
H. Kondo & H. Koshihara
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t
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0 10
I I
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1 I
Fig. 4. Abscissa: fraction; ordinate: OD.
(a) Sedimentation profile of the 12 000 g supernatant from unfertilized eggs of A. crussispina on a sucrose density gradient. The 12 000 g supernatant was centrifuged by layering on a sucrose density gradient (5-20 X) at 24 000 rpm for 14 h in a SW 25.1 rotor of Spinco Model L-2 Preparative Ultracentrifuge and fractionated by 1 ml. O-O, ODzso; O---O, OD,,,. (b) Immunodiffusion test between the antiserum and each fraction of the 12 000 g supernatant separated through sucrose density gradient. The central well contained the antiserum and the peripheral wells contained serially each fraction.
with the antiserum by forming a single and common precipitation band. The density of the whole precipitation band formed between the antiserum and the 26s particle preparations from various developmental stages seemed to be quite uniform. These results indicated a qualitative and quantitative similarity of antigen structure of the 26s particles throughout the developmental stages tested, whereas the sedimentation properties altered gradually during this period, as reported in [41. Distribution of the 26s particles in the sea urchin eggs As reported previously [4], the 26s particles are mostly recovered from the 105 000 g supernatant from eggs or embryos. A more Exptl Cell Res 75 (1972)
precise distribution of the 26s particles was sought among various subcellular fractions from unfertilized eggs, with the agar immunodiffusion test, using the antiserum against the 26s particles (fig. 3). At the same concentration of 1 mg or 500 ,ug/ml protein of each subcellular fraction, the 27 ppt and microsomal fractions as well as the 26s particles reacted with the antiserum to form a clear and common precipitation band. The yolknuclear and mitochondrial fractions gave a faint, but common precipitation band, suggesting that these fractions contained a small amount of the 26 S particles as a contaminant. A further fractionation by sucrose density gradient centrifugation was required to locate the antigen in the 26s particle fraction, since the differential centrifugation could not re-
The 265’ particles of sea urchin eggs 389 move contaminants including the 26s particles from the microsomal fraction, in particular. Thus, unfertilized eggs were homogenized in a 0.01 M Tris-HCl buffer solution (pH 7.2) containing 0. I5 M NaCI, 0.15 M KC1 and 0.003 M MgCl,, and centrifuged at 12 000 g for 20 min. The resultant supernatant was centrifuged by layering on the sucrose density gradient at 24 000 rpm for 14 h. After centrifugation it was fractionated as described in the caption to fig. 4. Each fraction was placed serially on an agar plate to react with the antiserum. As shown in fig. 4b, the fractions corresponding to an optical peak reacted with the antiserum by forming a single common precipitation band, whereas the other fractions including the heavier fractions (microsomes) did not form any precipitation band. This result indicates that the antigen exists in the form of 26s particles. Immunological discrimination particles from yolk granules
of the 26s
In the present experiments using the immunodiffusion tests, we examined whether yolk
Table 1. Amounts of protein solubilized from yolk-nuclear fraction of unfertilized eggs of A, crassispina by treatment with KC1 or urea solution Stepwise extraction with KCI solution ( % of the total protein)
Extraction with urea solution ( % of the total protein)
0.5 M KCI soluble 14.9 I.0 M KCI soluble 16.7 2.0 M KC1 soluble 6.2 2.0 M KCI residue 62.2
8 M urea soluble 73.1 8 M urea residue 26.9
granules contained any antigens similar to the 26 S particles. The solubility of the yolk-nuclear fraction of unfertilized eggs in KC1 solutions was so limited that less than 40 % of the total protein of the yolk-nuclear fraction was solubilized even with 2.0 M KCI solution. However, it was fairly easily solubilized in urea solution, and more than 70 y0 of the total protein could be extracted with 8 M urea solution (table 1). Yolk-nuclear fraction was first extracted with 0.5 M KCI, then the residue was extracted with 1.0 M KCI, and the residue was
Fig. 5. Immunodiffusion test between preparations of yolk granule components and the antiserum against the 26s particles from unfertilized eggsof A. crussispinabefore (a) and after dialysis against PBS of the preparations (b).(a),(b) The well 1 contained the 26s particles (200 fig protein/ml) and the central well contained theantiserum. The wells 2-6 contained washings (400 pg/ml protein), 0.5 M KCI-soluble (300 pg/ml protein), 1 M KCl-soluble (335 pg/ml protein), 2 M KCI-soluble (125 fig/ml protein) and 8 M urea-soluble component (625 pg/ml protein) of yolk-nuclear fraction, in order. (c) Wells 1 and 4 contained the 26s particles from unfertilized eggs. Wells 2 and 3 contained the 26s particles from morulae treated with 8 M urea and those subsequently dialysed against PBS, respectively. Wells 5 and 6 contained the 26s particles from gastrulae treated with 8 M urea and those subsequently dialysed against PBS. Each preparation was at a concentration of 200 pg/ml protein using the 26s particles of A. crussispina. Exptl Cell Res 75 (1972)
390
H. Kondo h H. Koshihara against the 26s particles in Ouchterlony’s agar diffusion plate (fig. 5~). The same preparations were also tested after being dialysed against PBS (fig. 5b). The antiserum also reacted by forming a faint precipitation band with non-dialysed and dialysed preparations of the yolk-nuclear fraction washings and of 0.5 M KCI-soluble component of the yolknuclear fraction. However, no precipitation band was observed between the antiserum and the 8 M urea-soluble component of the yolk-nuclear fraction which contained more than 70% of the yolk-nuclear fraction (fig. 5a, b). On the other hand, a single and common precipitation band was always formed between the antiserum and the 26s particles, irrespective of the presence of urea in the preparations, as shown in fig. 5c. These results indicate that in yolk granules, those components which react with the antiserum against the 26s particles are not present.
Fig. 6. Immunodiffusion test between the antiserum and the homogenates of several tissues from female (a) and male (b) of A. crussispina or irrespective of sex (c). Figs 6-l and 62 contained 1 mg and 250 pg of protein/ml, respectively. Each central well contained the antiserum and each of well 1 contained the 26s particles (200 pg/ml protein). (a) 1, 26s particles; 2, gonads: 3, eggs; 4, intestines; 5, perivisceral fluid (not containing any cells); 6, planocytes and other cells. (b) I, 26s particles; 2, testises; 3, sperms; 4, intestines; 5, perivisceral fluid (not containing any cells); 6, planocytes and other cells. (c) I, 4, 26s particles; 2, foot tubes; 3, siphon systems; 5, lantern muscles; 6, perivisceral fluid.
further extracted with 2.0 M KCl. These yolk-nuclear fraction components were designated as 0.5 M-, 1.0 M-, and 2.0 KCIsoluble components of the yolk-nuclear fraction. Thus, washings of the yolk-nuclear fraction with the homogenization medium and components of the yolk-nuclear fraction soluble in 0.5 M, 1.0 M, and 2.0 M KC1 solutions and in 8 M urea solution, were tested for their reaction with the antiserum Exptl Cell Res 75 (1972)
Distribution of antigenic determinants reacting with the antiserum against the 26s particles By using the immunodiffusion test, it was examined how the sameantigenic determinant with the 26s particles was distributed among various tissues in the same sea urchin, A. crassispina. The result is presented in fig. 6a-c. Among several tissues tested, the homogenate of gonads from female sea urchins or perivisceral fluid from both male and female seaurchins reacted with the antiserum to form a single and conspicuous precipitation band, which was common to that with the 26 S particles. At the higher concentration of 1 mg protein/ml, the homogenate of intestines irrespective of sex and gonads from male sea urchins, too, reacted with the antiserum by forming a faint precipitation band. Of perivisceral fluid irrespective of sex, the noncellular fraction exhibited the same antigeniticity with the 26s particles, whereas the
The 26s particles
of sea urchin eggs
391
cellular fraction including planocytes and other cells did not form any precipitation bands with the antiserum. Also, in this experiment, the other tissues, i.e. foot tubes, siphon systems or lantern muscles in the same sea urchin, did not exhibit any reactivity with the antiserum. Antigenic fluid
determinants
in perivisceral
In connection with the appearance of the antigenic component, it is interesting to examine comparatively how the antiserum against the 26s particles from unfertilized eggs of A. crassispina reacts cross-reactively with the perivisceral fluid of other echinoderms. The following results were obtained by the immunodiffusion test, as shown in fig. 7. Among several echinoderms, perivisceral fluid of P. depressus and H. pulcherrimus, respectively, included some similar antigenic determinants cross-reacting with the antiserum, as indicated by their forming a single and fair precipitation band between these and the antiserum. Similarly, that of M. globzdus reacted faintly with the antiserum. It was indicated that since these three echinoderms as well as A. crassispina belonged to the order of Echinoidea, they exhibited some serological similarity of antigenic determinants in their perivisceral fluid. Furthermore, C. japonicus, A. manni and P. japonica, belonging to another order of Clypeastroidea did not form any detectable precipitation band between their perivesceral fluid and the antiserum against the 26s particles of A. crassispina. As
observed
in
the
Ouchterlony’s
agar
plates (fig. 7), precipitin formation indicates the three echinoderms having a genetically to A. crassispina and each close relation produces some antigenic determinants in its perivisceral fluid, which seems to be partially common to that of A. crassispina unique to each of those.
and mainly
Fig. 7. Immunodiffusion test betweenthe antiserum and perivisceral fluid of severalseaurchins (u-d) or
the 26s particles from morulae of H.puZcherrimus (e). Perivisceral fluid was obtained from A. crassispina (a), H. pulcherrimus (b), M. glob&s (b), P. depressus (c), C. japonicus (c), A. manni (d) and P. japonica (d).
Each central well contained the antiserum. Wells 1 and 4 in (u-d) and I, 3 and 5 in (e) contained the 26s particles from unfertilized eggs of A. crassispinu (200 pg/ml protein). (a) 1, 4, 26s particles; 2, 5, female of A. crassispina; 3, 6 male of A. crassispina. (b), I, 4, 26 S particles; 2, 5, H. pulcherrimus; 3, 6, M. glob&s. (c) I, 4, 26s particles; 2, female of P. depressus; 3, 6, C. japonicus; 5, male of P. depressus. (d) I, 4, 26s particles; 2, 5, A. manni; 3, 6, P. japonica. (e) 1, 3, 5, 26 S particles; 2,4,6, 26 S particles of H. pulcherrimus.
On the other hand, it was in accord with the above observation that the 26s particles reacted moderately with of H. pulcherrimus the antiserum against those of A. crassispina (fig. 7e). The antiserum against the 26s particles of A. crassispina seems to contain antibodies directed against two immunologically different antigens-one appearing in the species, the other in some other. Exptl Cell Res 75 (1972)
392
H. Kondo C?H. Koshihara
L
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Fig. 8. Abscissa: fraction; ordinate: O.D. (a) Sedimentation profile of perivisceral fluid of A. crassispina by the sucrose density gradient centrifugation. Perivisceral fluid was centrifuged after layering on the sucrose density gradient at 22 000 rpm for 16 h in an SW OD,,,; O--O, OD,,,. (b) Immunodiffusion 25.1 rotor of Spinco Model L-2 Preparative Ultracentrifuge. O-O, test between the antiserum and each fraction of perivisceral fluid separated through the sucrose density gradient.
The next question is whether such antigenic determinants in perivisceral fluid behave as particles identical or similar to the 26s particles. The non-cellular fraction in perivisceral fluid was centrifuged through the sucrose gradient at 22 000 rpm for 16 h. The resultant sedimentation pattern was obtained by fractionating and measuring optically at OD,,, and OD,,, (fig. Sa). Two discrete peaks, corresponding respectively to each particulate fraction, i.e. 3-4s heavier (arrow 2) or 9-12s heavier (arrow 3) than the 26s particles of unfertilized eggs (arrow l), were observed in the profile of perivisceral fluid from A. crassispina. These particulate fractions exhibited clear reactivity with the antiserum, whereas the other fractions (excepting the former) did not react with it (fig. Sb). Whereas the heavier of these two was not observed in some experiments, the ExptI CelI Res 75 (1972)
lighter one was always observed in the profiles of perivisceral fluid, irrespective of sex. It presumably suggeststhat the lighter one is a constituent component of sea urchin perivisceral fluid. Antigenic determinants in accessory cells By further experimentation, it was found that accessory cells from gonads of female sea urchins were another source of antigenic determinants reacting with the antiserum against the 26s particles. Accessory cells were gathered without any contaminant oocytes by centrifugation of the mixed cell-suspension from gonads of premature stage of H.pulcherrimus (fig. 9). The homogenate of accessory cells was sedimented by centrifugation at 12 000 g for 20 min. The resultant supernatant was layered on the sucrose density gra-
The 26Sparticies
of sea urchin eggs
393
Fig. 9. (a) Photograph of smeared cells from immature ovary of H. pnlcherritnus. Note variety in size of premature oocytes or accessory cells. x 140. (6) Photograph of accessory cell preparation. ( x 140) Gonads including oocytes at premature stages were sliced with scissors in sea water, and then cells were pushed out gently with a loose glass homogenizer. After standing in ice-cold sea water for 30 min, the cell suspension containing accessory cells mainly, was pipetted out to prevent it from mixing with sedimented cells containing immature eggs or oocytes and broken gonad tissues. Accessory cells were sedimented by a gentle centrifugation at 200 g for 5 min, and the resultant cell fraction was twice washed with sea water by suspending and centrifuging of it. This procedure is required for complete removal of any inclusion from broken oocytes or perivisceral fluids.
Exptl Cell Res 75 (1972)
394
H. Kondo & H. Koshihara
0.8-t
10
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Fig. IO. Abscissa: fraction; ordinate: OD. 0 - 0, 280 nm; O--O, 260 nm. (a-c). Sedimentation profiles of 12 000 g supernatants from unfertilized eggs (a), accessory cells (b), and perivisceral fluid (c), using female sea urchin of H. pulcherrimus. The 12 000 g supernatant from each preparation was layered and centrifuged at 24 Ooo rpm for 14 h through the sucrose density gradient in a SW251 rotor of Spinco Model L-2. (d-f) Irmnunodiffusion test between each fraction sedimented through the sucrose density gradient and the antiserum against the 26s particles of A. crassispina. (d) supernatant from unfertilized eggs; (e) supernatant from accessory cells; (f) supernatant from perivisceral fluid.
dient and then centrifuged at 24 000 rpm for property corresponding to that of perivisceral 14 h. For a comparison among these of un- fluid, whereas the 26 S particles have sedimenfertilized eggs, perivisceral fluid and acces- tation properties clearly different from the sory cells, their sedimentation profiles are other two, as shown in fig. lOa-c. The antigenic determinant common to the presented in fig. lOa, b, c, respectively. above three particles presentsa new approach Of accessory cells, too, a unique particle exclusively includes the same antigenic de- to the location of these particles, and to elucidate their biological functions. terminant with the 26 Sparticles of unfertilized eggs. This particle also has a sedimentation Exptl CeN Res 7.5 (1972)
The 26s particles of sea urchin eggs 395 DISCUSSION Recently, several investigations have been carried out for the characterization of some discrete particles prepared from the supernatant of sea urchin eggs or embryos which exhibited similar sedimentation properties between 22 and 27s [2, 3, 4, 5, 71. Although these particles have been studied independently concerning their purification and characterization, their biological functions remain obscure. Bibring & Baxandall reported recently that the 22s protein of mitotic apparatus isolated from sea urchin eggs was not microtubule protein, and was ubiquitous in the metaphase egg fractions [l]. Our previous experiments indicated that the 26s particles seemed not to be derived from heavier granules, i.e. yolk granules, and then altered their sedimentation properties gradually in the course of sea urchin development [4]. In the present experiments, we attempted to clarify a more precise distribution of the antigenic determinant reacting with the antiserum against the 26s particles. For preparation of antiserum, the 26 S particles were purified so that a single band of them ran on gel of acrylamide by electrophoresis (fig. 1) and then its limited band corresponding to the 26s particles, exclusively, reacted with the antiserum prepared by injecting this preparation into rabbits. Thus, the antiserum used in these experiments was suitable for the study of more precise distributions among cellular fractions, or various tissues from the same sea urchin, or other echinoderms. By using the immunodiffusion test, the previous evidence that the 26s particles seem not to be derived from yolk granules was supported, as indicated by the fact that the washes or extracts of yolk granules scarcely reacted with the antiserum against the 26s particles (fig. 5). However, some subcellular
fractions prepared by differential centrifugation contained to a lesser extent the 26s particles as contaminants, for these fractions reacted moderately with the antiserum, whereas by density gradient centrifugation, the 26 S particle fractions, exclusively, reacted with the antiserum by forming a single and conspicuous precipitation band (figs 3,4). Furthermore, it was found that the same antigenie determinant with the 26s particles of unfertilized eggs was retained within the particles in the course of development notwithstanding alterations in their sedimentation properties. The same antigenic determinants were also found in perivisceral fluid (the non-cellular fraction) irrespective of sex, and localized in the unique particulate fraction in it, presumably as a constituent component (figs 7, 8). Additionally, accessory cells, too, possessed the same antigenic determinant with the antiserum, as indicated by the formation of a single and deep precipitation band common to the 26s particles, and also, their particulate fractions sedimenting through the sucrose density gradient reacted exclusively with it (fig. 10). These independent particles, variously located, possess the common antigenie determinant, although other additive components are also incorporated into the particles. In the electron microscope study of Verhey & Moyer [8] the accessory cells are seen to contain numerous spherical yolk-like inclusions. In addition to these, the accessory cells contain lipid droplets of uniform size averaging about 0.8 pm in diameter, and extensive accumulations of glycogen. Also, strands of accessory cell cytoplasm extend between the developing oocytes and the plasma membranes of the two cell types are often closely apposed. Occasionally, glycogenlike particles are observed on the outer surface of the accessory cell plasma membrane. Exptl Cell Res 75 (1972)
396 H. Kondo & H. Koshihara It is possible that direct transfer of gross material from accessory cells to oocytes or perivisceral fluid takes place. These problems should be resolved as soon as possible, for it is suggested that the particles in perivisceral fluid as well as the 26 S particles in unfertilized eggs may be supplied by their production in accessory cells of gonads. In connection with this assumption, these particles, especially in perivisceral fluid or eggs, may be consumed by other tissues or embryos and or function as a carrier transferring materials required by other tissues or oocytes. These possibilities of their biological functions are under investigation.
and also Dr Tamio Hirabayashi for advice on the immunological techniques. Finally, we wish to thank the Director and Staff of the Shimoda Marine Biological Station for the use of their facilities.
The authors wish to thank Professor Yujiro Hayashi for helpful suggestions and advice on our manuscript,
Received March 20, 1972 Revised version received May 5, 1972
Exptl Cell Res 75 (1972)
REFERENCES 1. Bibring, T & Boxandall, J, J cell biol41 (1969) 577. 2. Infante, A A & Nemer, M, J mol biol 32 (I 968) 543. 3. Kane, R E, J cell biol 32 (1967) 243. 4. Kondo, H, Exptl cell res 72 (1972) 519. 5. Malkin. L I. Manean. J & Gross. P. R. Dev biol I
I
I
12 (1965) 520. ” ’ 6. Reisfeld. R A. Lewis. V I & Williams, D E. Nature 19.5(1962) 28 I .’ 7. Stephens, R E, J cell biol 32 (1967) 255. 8. Verhey, C A & Moyer, F H, J exptl zoo1 164 (1967) 195.
0 1972 by Academic Pwss, Inc. of rcprodurfion in any fovm rmwed
Experimental
IMMUNOLOGICAL
Cell Research 7.5 (1972) 385-396
STUDIES ON THE 26 S PARTICLES
OF SEA URCHIN
EGGS’
H. KONDO” and H. KOSHIHARA Zoological
Insritutr,
Faculty of Science, Tokyo Kyoiku-University,
Bunkyo-ku,
Tokyo, Japan
SUMMARY 1. 26s particles were prepared from the 105 000 g supernatant of unfertilized eggs of Anthocidaris crassispina and purified by sucrose density gradient centrifugation. By using the purified particles,
an antiserum against them was prepared with the intention of tracing and locating it by immunological methods. 2. Further distribution of the 26s particles was examined by the immunodiffusion test with the antiserum. It was found that the 26s particles were distributed independently and as a discrete component in cytoplasm of eggs, which supports our previous evidence that they are not derived from heavier granules, i.e. yolk granules. Also, the 26s particles retained the same antigenic determinant throughout sea urchin development. 3. Among several tissues from the same sea urchin, gonads from female sea urchins or perivisceral fluid irrespective of sex reacted with the antiserum against the 26s particles by forming a common conspicuous precipitation band, Furthermore, the antigenic determinants exhibiting serological similarity to the 26s particles were distributed in perivisceral fluid of other sea urchins belonging to the order of Echinoidea, whereas those in Clypeasteria did not form any crossreacted precipitation band. 4. The same antigenic determinant distributed in tissues of the same sea urchin was located in isolated particulate fractions, as prepared from perivisceral fluid or accessory cells as well as eggs. The possible biological functions of these particulate fractions are discussed.
Our previous report dealt with the centrifugal and electrophoretic characterization of the post-microsomal 26s particles of sea urchin embryos in comparison with characteristics of yolk granules from the same embryonic source [4]. The results so far obtained indicated that the 26s particles were discrete entities of the cytoplasm of sea urchin embryos and could not be regarded as being derived from heavier cytoplasmic granules, i.e. yolk granules. It was also found that the sedimentation properties of the 26s particles altered gradually in the course of developi Contributions from the Shimoda Marine Biological Station, No. 252. 2 Present address: Department of Biology, Tokyo Metropolitan Institute of Gerontology, Sakaecho, Itabashi-ku, Tokyo-173, Japan. 26
if21814
ment, and decreased quantitatively in the embryos. Further, we sought additional and decisive information on our 26s particles in the hope of confirming our earlier conclusion and to reveal those biological characteristics and function of the 26s particles, which are still unknown. In this paper, an immunological approach to our problems is described. By using an antiserum against the 26s particles purified from unfertilized eggs of Anthociduris crussispina, it was possible to follow the antigen in the course of sea urchin development and to locate it in intracellular fractions from eggs or in various tissues of adult sea urchins. Subsequently, the same antigenic determinant Exptl Cell Res 7.5 (1972)
386 H. Kondo & H. Koshihara with ammonium sulfate ad modum Malkin et al. If. The final precipitate was the purified preparation of the 26s particles, running as a single band at polyacrylamide gel electrophoresis (fig. 1). These purified 26s particles were used as the antigen for the present immunological studies.
Subcellular fractionation Unfertilized eggs of A. crassispina were homogenized in a homogenization medium containing 0.01 M Tris-HCI buffer (pH 7.2), 0.25 M sucrose, 0.15 M NaCI, 0.15 M KC1 and 0.003 M MgCl, and fractionated, as described before [4].
Polyacrylamide gel electrophoresis The method of Reisfeld et al. [6] was used. The samples were dissolved in Tris-glycine buffer solution (pH-8.6) containing 1 M sucrose, and layered on gel columns containing 5 % acrylamide [4]. Electrophoresis was conducted for 30 min at 4 mA per gel column. After electrophoresis the gel columns were stained and destained, as described in [4]. Fig. 1. Patterns of polyacrylamide gel electrophoresis of the purified 26s particles from unfertilized eggs of A. crassispina. The preparation was applied to the column by 40 pug of protein, and the electrophoresis was carried out as described in Methods.
was located in perivisceral fluid or accessory cells in gonads of the same sea urchins, and similar antigenicities were observed in perivisceral fluid of some other sea urchins. MATERIALS
AND METHODS
Materials Eggs, embryos and adults of Anthocidaris crassispina were used in the majority of these experiments. Techniques for the obtaining and handling sea urchin gametes and embryos have been described in our previous paper [4]. Several adult tissues used in immunological studies were collected from female or male sea urchins of Anthocidaris crassispina in the breeding season. Perivisceral fluid was centrifuged at 700 g for 20 min and separated into the non-cellular fraction and the cellular fraction containing planocytes and other cells. In comparative experiments, several species of Echinoidea were used: Hemicentrotus pulcherrimus, Mespilia globulus, Pseudocentrotus depressus, Clypeaster japonicus, Astriclypeus manni and Peronella japonica.
Purification
of the 26s particles
The 26s particles were prepared from a 270 000 g precipitate (27 ppt) by a sucrose density gradient centrifugation procedure, as described previously [4]. The 26s particles were further purified by salting out Exptl
Cell Res 75 (1972)
Preparation of antiserum against the 26 S particles 0.8 mg of the 26s particles prepared from unfertilized eggs of A. crassispina was suspended in 1 ml of 0.01 M phosphate buffer, pH 7.1, containing 0.145 M NaCl (PBS), and I-2 ml of the suspension was injected subcutaneously into rabbits. The injections were repeated at 7 day intervals for 4 weeks. Serum was sampled at times during the course of immunization and tested for its reactivity with the antigen by Ouchterlony’s immunodiffusion test. Satisfactory antisera were obtained through the carotid artery after 5 weeks, and stored at - 20°C until use.
Immunodiffusion
test
Ouchterlony’s immunodiffusion technique was employed. Agar was dissolved at 1 % in PBS mixed with l/l00 vol of 1 % merzonine. Wells of 9 mm diameter (in most of these experiments) or 4 mm on Ouchterlony’s agar plates were spaced out at distances of 9.5 mm or 3.5 mm, respectively. Agar plates on which antisera and antigen preparations were placed in each of the wells were incubated at 18°C. and were observed to form precipitation bands.
RESULTS Properties of the antiserum against the 26s particles The antiserum against the 26s particles was placed on Ouchterlony’s agar to react with the antigen at different concentrations from 0.8 mg/ml to 25 pg/ml (fig. 2). Only one precipitation band resulted from all these antigen concentrations. In addition, the anti-
The 26Sparticles
of sea urchin eggs
387
Fig. 2. Immunodiffusion
tests between the antiserum and the 26s particles from unfertilized eggs (a), and between the antiserum and the homogenate of unfertilized eggs (b) of A. cmssispina. (a) The central well contained the antiserum (1: 8 dilution), and the wells l-6 contained 800 pLg,400,200, 100, 50, and 25 pg of protein of the 26s particles/ml, respectively. (b) The central well contained the antsierum (1 : 8 dilution) and the well 1 contained 200 pugof protein of the 26s particles/ml. Wells 2-6 contained the homogenate of unfertilized eggs at 10 mg, 2, 500,250, and 125 pg/ml of protein respectively. Fig. 3. Immunodiffusion test of the subcellular fractions from unfertilized eggs of A. crassispina, using the antiserum against the 26s particles. The central well contained the antiserum and well 1 contained 200,~g of protein of the 26s particles/ml. Wells 2-6 contained yolk-nuclear fraction (1 500 g precipitate), mitochondrial fraction (12 000 g precipitate), microsomal fraction (105 000 g precipitate), 27 ppt (270 000 g precipitate) and 27 sup fractions (270 000 g supernatant) in order, with 1 mg/ml protein (a) or with 500 pg/ml protein (b). This subcellular fractionation was done according to the previous procedure [4].
serum was placed so as to react with the homogenate from unfertilized eggs in the agar diffusion test. As shown in fig. 2b, at the concentrations of the homogenate tested only one precipitation band was formed. This indicated that a single antigen component, presumably the 26s particles, was present in the homogenate of unfertilized eggs. Furthermore, we ascertained that the
antigen was localized only at a discrete band of the 26s particles subjected to acrylamide gel electrophoresis. The preparations corresponding to the 26s particles were isolated from morulae, gastrulae and plutei. These preparations were placed on Ouchterlony’s agar diffusion plates to react with the antiserum against the 26s particles from unfertilized eggs. They reacted Exptl Cell Res 75 (1972)
388
H. Kondo & H. Koshihara
r 2.0
t
1.0
0 10
I I
I 1
20
27
1 I
Fig. 4. Abscissa: fraction; ordinate: OD.
(a) Sedimentation profile of the 12 000 g supernatant from unfertilized eggs of A. crussispina on a sucrose density gradient. The 12 000 g supernatant was centrifuged by layering on a sucrose density gradient (5-20 X) at 24 000 rpm for 14 h in a SW 25.1 rotor of Spinco Model L-2 Preparative Ultracentrifuge and fractionated by 1 ml. O-O, ODzso; O---O, OD,,,. (b) Immunodiffusion test between the antiserum and each fraction of the 12 000 g supernatant separated through sucrose density gradient. The central well contained the antiserum and the peripheral wells contained serially each fraction.
with the antiserum by forming a single and common precipitation band. The density of the whole precipitation band formed between the antiserum and the 26s particle preparations from various developmental stages seemed to be quite uniform. These results indicated a qualitative and quantitative similarity of antigen structure of the 26s particles throughout the developmental stages tested, whereas the sedimentation properties altered gradually during this period, as reported in [41. Distribution of the 26s particles in the sea urchin eggs As reported previously [4], the 26s particles are mostly recovered from the 105 000 g supernatant from eggs or embryos. A more Exptl Cell Res 75 (1972)
precise distribution of the 26s particles was sought among various subcellular fractions from unfertilized eggs, with the agar immunodiffusion test, using the antiserum against the 26s particles (fig. 3). At the same concentration of 1 mg or 500 ,ug/ml protein of each subcellular fraction, the 27 ppt and microsomal fractions as well as the 26s particles reacted with the antiserum to form a clear and common precipitation band. The yolknuclear and mitochondrial fractions gave a faint, but common precipitation band, suggesting that these fractions contained a small amount of the 26 S particles as a contaminant. A further fractionation by sucrose density gradient centrifugation was required to locate the antigen in the 26s particle fraction, since the differential centrifugation could not re-
The 265’ particles of sea urchin eggs 389 move contaminants including the 26s particles from the microsomal fraction, in particular. Thus, unfertilized eggs were homogenized in a 0.01 M Tris-HCl buffer solution (pH 7.2) containing 0. I5 M NaCI, 0.15 M KC1 and 0.003 M MgCl,, and centrifuged at 12 000 g for 20 min. The resultant supernatant was centrifuged by layering on the sucrose density gradient at 24 000 rpm for 14 h. After centrifugation it was fractionated as described in the caption to fig. 4. Each fraction was placed serially on an agar plate to react with the antiserum. As shown in fig. 4b, the fractions corresponding to an optical peak reacted with the antiserum by forming a single common precipitation band, whereas the other fractions including the heavier fractions (microsomes) did not form any precipitation band. This result indicates that the antigen exists in the form of 26s particles. Immunological discrimination particles from yolk granules
of the 26s
In the present experiments using the immunodiffusion tests, we examined whether yolk
Table 1. Amounts of protein solubilized from yolk-nuclear fraction of unfertilized eggs of A, crassispina by treatment with KC1 or urea solution Stepwise extraction with KCI solution ( % of the total protein)
Extraction with urea solution ( % of the total protein)
0.5 M KCI soluble 14.9 I.0 M KCI soluble 16.7 2.0 M KC1 soluble 6.2 2.0 M KCI residue 62.2
8 M urea soluble 73.1 8 M urea residue 26.9
granules contained any antigens similar to the 26 S particles. The solubility of the yolk-nuclear fraction of unfertilized eggs in KC1 solutions was so limited that less than 40 % of the total protein of the yolk-nuclear fraction was solubilized even with 2.0 M KCI solution. However, it was fairly easily solubilized in urea solution, and more than 70 y0 of the total protein could be extracted with 8 M urea solution (table 1). Yolk-nuclear fraction was first extracted with 0.5 M KCI, then the residue was extracted with 1.0 M KCI, and the residue was
Fig. 5. Immunodiffusion test between preparations of yolk granule components and the antiserum against the 26s particles from unfertilized eggsof A. crussispinabefore (a) and after dialysis against PBS of the preparations (b).(a),(b) The well 1 contained the 26s particles (200 fig protein/ml) and the central well contained theantiserum. The wells 2-6 contained washings (400 pg/ml protein), 0.5 M KCI-soluble (300 pg/ml protein), 1 M KCl-soluble (335 pg/ml protein), 2 M KCI-soluble (125 fig/ml protein) and 8 M urea-soluble component (625 pg/ml protein) of yolk-nuclear fraction, in order. (c) Wells 1 and 4 contained the 26s particles from unfertilized eggs. Wells 2 and 3 contained the 26s particles from morulae treated with 8 M urea and those subsequently dialysed against PBS, respectively. Wells 5 and 6 contained the 26s particles from gastrulae treated with 8 M urea and those subsequently dialysed against PBS. Each preparation was at a concentration of 200 pg/ml protein using the 26s particles of A. crussispina. Exptl Cell Res 75 (1972)
390
H. Kondo h H. Koshihara against the 26s particles in Ouchterlony’s agar diffusion plate (fig. 5~). The same preparations were also tested after being dialysed against PBS (fig. 5b). The antiserum also reacted by forming a faint precipitation band with non-dialysed and dialysed preparations of the yolk-nuclear fraction washings and of 0.5 M KCI-soluble component of the yolknuclear fraction. However, no precipitation band was observed between the antiserum and the 8 M urea-soluble component of the yolk-nuclear fraction which contained more than 70% of the yolk-nuclear fraction (fig. 5a, b). On the other hand, a single and common precipitation band was always formed between the antiserum and the 26s particles, irrespective of the presence of urea in the preparations, as shown in fig. 5c. These results indicate that in yolk granules, those components which react with the antiserum against the 26s particles are not present.
Fig. 6. Immunodiffusion test between the antiserum and the homogenates of several tissues from female (a) and male (b) of A. crussispina or irrespective of sex (c). Figs 6-l and 62 contained 1 mg and 250 pg of protein/ml, respectively. Each central well contained the antiserum and each of well 1 contained the 26s particles (200 pg/ml protein). (a) 1, 26s particles; 2, gonads: 3, eggs; 4, intestines; 5, perivisceral fluid (not containing any cells); 6, planocytes and other cells. (b) I, 26s particles; 2, testises; 3, sperms; 4, intestines; 5, perivisceral fluid (not containing any cells); 6, planocytes and other cells. (c) I, 4, 26s particles; 2, foot tubes; 3, siphon systems; 5, lantern muscles; 6, perivisceral fluid.
further extracted with 2.0 M KCl. These yolk-nuclear fraction components were designated as 0.5 M-, 1.0 M-, and 2.0 KCIsoluble components of the yolk-nuclear fraction. Thus, washings of the yolk-nuclear fraction with the homogenization medium and components of the yolk-nuclear fraction soluble in 0.5 M, 1.0 M, and 2.0 M KC1 solutions and in 8 M urea solution, were tested for their reaction with the antiserum Exptl Cell Res 75 (1972)
Distribution of antigenic determinants reacting with the antiserum against the 26s particles By using the immunodiffusion test, it was examined how the sameantigenic determinant with the 26s particles was distributed among various tissues in the same sea urchin, A. crassispina. The result is presented in fig. 6a-c. Among several tissues tested, the homogenate of gonads from female sea urchins or perivisceral fluid from both male and female seaurchins reacted with the antiserum to form a single and conspicuous precipitation band, which was common to that with the 26 S particles. At the higher concentration of 1 mg protein/ml, the homogenate of intestines irrespective of sex and gonads from male sea urchins, too, reacted with the antiserum by forming a faint precipitation band. Of perivisceral fluid irrespective of sex, the noncellular fraction exhibited the same antigeniticity with the 26s particles, whereas the
The 26s particles
of sea urchin eggs
391
cellular fraction including planocytes and other cells did not form any precipitation bands with the antiserum. Also, in this experiment, the other tissues, i.e. foot tubes, siphon systems or lantern muscles in the same sea urchin, did not exhibit any reactivity with the antiserum. Antigenic fluid
determinants
in perivisceral
In connection with the appearance of the antigenic component, it is interesting to examine comparatively how the antiserum against the 26s particles from unfertilized eggs of A. crassispina reacts cross-reactively with the perivisceral fluid of other echinoderms. The following results were obtained by the immunodiffusion test, as shown in fig. 7. Among several echinoderms, perivisceral fluid of P. depressus and H. pulcherrimus, respectively, included some similar antigenic determinants cross-reacting with the antiserum, as indicated by their forming a single and fair precipitation band between these and the antiserum. Similarly, that of M. globzdus reacted faintly with the antiserum. It was indicated that since these three echinoderms as well as A. crassispina belonged to the order of Echinoidea, they exhibited some serological similarity of antigenic determinants in their perivisceral fluid. Furthermore, C. japonicus, A. manni and P. japonica, belonging to another order of Clypeastroidea did not form any detectable precipitation band between their perivesceral fluid and the antiserum against the 26s particles of A. crassispina. As
observed
in
the
Ouchterlony’s
agar
plates (fig. 7), precipitin formation indicates the three echinoderms having a genetically to A. crassispina and each close relation produces some antigenic determinants in its perivisceral fluid, which seems to be partially common to that of A. crassispina unique to each of those.
and mainly
Fig. 7. Immunodiffusion test betweenthe antiserum and perivisceral fluid of severalseaurchins (u-d) or
the 26s particles from morulae of H.puZcherrimus (e). Perivisceral fluid was obtained from A. crassispina (a), H. pulcherrimus (b), M. glob&s (b), P. depressus (c), C. japonicus (c), A. manni (d) and P. japonica (d).
Each central well contained the antiserum. Wells 1 and 4 in (u-d) and I, 3 and 5 in (e) contained the 26s particles from unfertilized eggs of A. crassispinu (200 pg/ml protein). (a) 1, 4, 26s particles; 2, 5, female of A. crassispina; 3, 6 male of A. crassispina. (b), I, 4, 26 S particles; 2, 5, H. pulcherrimus; 3, 6, M. glob&s. (c) I, 4, 26s particles; 2, female of P. depressus; 3, 6, C. japonicus; 5, male of P. depressus. (d) I, 4, 26s particles; 2, 5, A. manni; 3, 6, P. japonica. (e) 1, 3, 5, 26 S particles; 2,4,6, 26 S particles of H. pulcherrimus.
On the other hand, it was in accord with the above observation that the 26s particles reacted moderately with of H. pulcherrimus the antiserum against those of A. crassispina (fig. 7e). The antiserum against the 26s particles of A. crassispina seems to contain antibodies directed against two immunologically different antigens-one appearing in the species, the other in some other. Exptl Cell Res 75 (1972)
392
H. Kondo C?H. Koshihara
L
10
I
20
27
Fig. 8. Abscissa: fraction; ordinate: O.D. (a) Sedimentation profile of perivisceral fluid of A. crassispina by the sucrose density gradient centrifugation. Perivisceral fluid was centrifuged after layering on the sucrose density gradient at 22 000 rpm for 16 h in an SW OD,,,; O--O, OD,,,. (b) Immunodiffusion 25.1 rotor of Spinco Model L-2 Preparative Ultracentrifuge. O-O, test between the antiserum and each fraction of perivisceral fluid separated through the sucrose density gradient.
The next question is whether such antigenic determinants in perivisceral fluid behave as particles identical or similar to the 26s particles. The non-cellular fraction in perivisceral fluid was centrifuged through the sucrose gradient at 22 000 rpm for 16 h. The resultant sedimentation pattern was obtained by fractionating and measuring optically at OD,,, and OD,,, (fig. Sa). Two discrete peaks, corresponding respectively to each particulate fraction, i.e. 3-4s heavier (arrow 2) or 9-12s heavier (arrow 3) than the 26s particles of unfertilized eggs (arrow l), were observed in the profile of perivisceral fluid from A. crassispina. These particulate fractions exhibited clear reactivity with the antiserum, whereas the other fractions (excepting the former) did not react with it (fig. Sb). Whereas the heavier of these two was not observed in some experiments, the ExptI CelI Res 75 (1972)
lighter one was always observed in the profiles of perivisceral fluid, irrespective of sex. It presumably suggeststhat the lighter one is a constituent component of sea urchin perivisceral fluid. Antigenic determinants in accessory cells By further experimentation, it was found that accessory cells from gonads of female sea urchins were another source of antigenic determinants reacting with the antiserum against the 26s particles. Accessory cells were gathered without any contaminant oocytes by centrifugation of the mixed cell-suspension from gonads of premature stage of H.pulcherrimus (fig. 9). The homogenate of accessory cells was sedimented by centrifugation at 12 000 g for 20 min. The resultant supernatant was layered on the sucrose density gra-
The 26Sparticies
of sea urchin eggs
393
Fig. 9. (a) Photograph of smeared cells from immature ovary of H. pnlcherritnus. Note variety in size of premature oocytes or accessory cells. x 140. (6) Photograph of accessory cell preparation. ( x 140) Gonads including oocytes at premature stages were sliced with scissors in sea water, and then cells were pushed out gently with a loose glass homogenizer. After standing in ice-cold sea water for 30 min, the cell suspension containing accessory cells mainly, was pipetted out to prevent it from mixing with sedimented cells containing immature eggs or oocytes and broken gonad tissues. Accessory cells were sedimented by a gentle centrifugation at 200 g for 5 min, and the resultant cell fraction was twice washed with sea water by suspending and centrifuging of it. This procedure is required for complete removal of any inclusion from broken oocytes or perivisceral fluids.
Exptl Cell Res 75 (1972)
394
H. Kondo & H. Koshihara
0.8-t
10
20
27
Fig. IO. Abscissa: fraction; ordinate: OD. 0 - 0, 280 nm; O--O, 260 nm. (a-c). Sedimentation profiles of 12 000 g supernatants from unfertilized eggs (a), accessory cells (b), and perivisceral fluid (c), using female sea urchin of H. pulcherrimus. The 12 000 g supernatant from each preparation was layered and centrifuged at 24 Ooo rpm for 14 h through the sucrose density gradient in a SW251 rotor of Spinco Model L-2. (d-f) Irmnunodiffusion test between each fraction sedimented through the sucrose density gradient and the antiserum against the 26s particles of A. crassispina. (d) supernatant from unfertilized eggs; (e) supernatant from accessory cells; (f) supernatant from perivisceral fluid.
dient and then centrifuged at 24 000 rpm for property corresponding to that of perivisceral 14 h. For a comparison among these of un- fluid, whereas the 26 S particles have sedimenfertilized eggs, perivisceral fluid and acces- tation properties clearly different from the sory cells, their sedimentation profiles are other two, as shown in fig. lOa-c. The antigenic determinant common to the presented in fig. lOa, b, c, respectively. above three particles presentsa new approach Of accessory cells, too, a unique particle exclusively includes the same antigenic de- to the location of these particles, and to elucidate their biological functions. terminant with the 26 Sparticles of unfertilized eggs. This particle also has a sedimentation Exptl CeN Res 7.5 (1972)
The 26s particles of sea urchin eggs 395 DISCUSSION Recently, several investigations have been carried out for the characterization of some discrete particles prepared from the supernatant of sea urchin eggs or embryos which exhibited similar sedimentation properties between 22 and 27s [2, 3, 4, 5, 71. Although these particles have been studied independently concerning their purification and characterization, their biological functions remain obscure. Bibring & Baxandall reported recently that the 22s protein of mitotic apparatus isolated from sea urchin eggs was not microtubule protein, and was ubiquitous in the metaphase egg fractions [l]. Our previous experiments indicated that the 26s particles seemed not to be derived from heavier granules, i.e. yolk granules, and then altered their sedimentation properties gradually in the course of sea urchin development [4]. In the present experiments, we attempted to clarify a more precise distribution of the antigenic determinant reacting with the antiserum against the 26s particles. For preparation of antiserum, the 26 S particles were purified so that a single band of them ran on gel of acrylamide by electrophoresis (fig. 1) and then its limited band corresponding to the 26s particles, exclusively, reacted with the antiserum prepared by injecting this preparation into rabbits. Thus, the antiserum used in these experiments was suitable for the study of more precise distributions among cellular fractions, or various tissues from the same sea urchin, or other echinoderms. By using the immunodiffusion test, the previous evidence that the 26s particles seem not to be derived from yolk granules was supported, as indicated by the fact that the washes or extracts of yolk granules scarcely reacted with the antiserum against the 26s particles (fig. 5). However, some subcellular
fractions prepared by differential centrifugation contained to a lesser extent the 26s particles as contaminants, for these fractions reacted moderately with the antiserum, whereas by density gradient centrifugation, the 26 S particle fractions, exclusively, reacted with the antiserum by forming a single and conspicuous precipitation band (figs 3,4). Furthermore, it was found that the same antigenie determinant with the 26s particles of unfertilized eggs was retained within the particles in the course of development notwithstanding alterations in their sedimentation properties. The same antigenic determinants were also found in perivisceral fluid (the non-cellular fraction) irrespective of sex, and localized in the unique particulate fraction in it, presumably as a constituent component (figs 7, 8). Additionally, accessory cells, too, possessed the same antigenic determinant with the antiserum, as indicated by the formation of a single and deep precipitation band common to the 26s particles, and also, their particulate fractions sedimenting through the sucrose density gradient reacted exclusively with it (fig. 10). These independent particles, variously located, possess the common antigenie determinant, although other additive components are also incorporated into the particles. In the electron microscope study of Verhey & Moyer [8] the accessory cells are seen to contain numerous spherical yolk-like inclusions. In addition to these, the accessory cells contain lipid droplets of uniform size averaging about 0.8 pm in diameter, and extensive accumulations of glycogen. Also, strands of accessory cell cytoplasm extend between the developing oocytes and the plasma membranes of the two cell types are often closely apposed. Occasionally, glycogenlike particles are observed on the outer surface of the accessory cell plasma membrane. Exptl Cell Res 75 (1972)
396 H. Kondo & H. Koshihara It is possible that direct transfer of gross material from accessory cells to oocytes or perivisceral fluid takes place. These problems should be resolved as soon as possible, for it is suggested that the particles in perivisceral fluid as well as the 26 S particles in unfertilized eggs may be supplied by their production in accessory cells of gonads. In connection with this assumption, these particles, especially in perivisceral fluid or eggs, may be consumed by other tissues or embryos and or function as a carrier transferring materials required by other tissues or oocytes. These possibilities of their biological functions are under investigation.
and also Dr Tamio Hirabayashi for advice on the immunological techniques. Finally, we wish to thank the Director and Staff of the Shimoda Marine Biological Station for the use of their facilities.
The authors wish to thank Professor Yujiro Hayashi for helpful suggestions and advice on our manuscript,
Received March 20, 1972 Revised version received May 5, 1972
Exptl Cell Res 75 (1972)
REFERENCES 1. Bibring, T & Boxandall, J, J cell biol41 (1969) 577. 2. Infante, A A & Nemer, M, J mol biol 32 (I 968) 543. 3. Kane, R E, J cell biol 32 (1967) 243. 4. Kondo, H, Exptl cell res 72 (1972) 519. 5. Malkin. L I. Manean. J & Gross. P. R. Dev biol I
I
I
12 (1965) 520. ” ’ 6. Reisfeld. R A. Lewis. V I & Williams, D E. Nature 19.5(1962) 28 I .’ 7. Stephens, R E, J cell biol 32 (1967) 255. 8. Verhey, C A & Moyer, F H, J exptl zoo1 164 (1967) 195.