Monoclonal antibody production against a subcellular fraction of vegetal pole cytoplasm containing the germ plasm of Xenopus 2-cell eggs

Cell Differentiation and Development, Elsevier Scientific Publishers Ireland,

CELDIF

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21 (1989) 163-174 Ltd.

00601

Monoclonal antibody production against a subcellular fraction of vegetal pole cytoplasm containing the germ plasm of Xenopus 2-cell eggs Sakiko Nakazato and Kohji Ikenishi Department

of Biology, Faculty of Science, Osaka City University, Osaka 558, Japan (Accepted

11 April 1989)

In attempts to understand the molecular nature of substances localized in the germ plasm (GP), monoclonal antibodies against a subcellular fraction of vegetal pole cytoplasm containing the GP of Xenopus 2-cell eggs were produced. The monoclonal antibody produced by the hybridoma cell line (designated as no . 48) reacted specifically with the GP of embryos at the cleavage stages. Beyond the gastrula stage, the antibody reacted with not only the GP of germ line cells but also the cytoplasm of somatic cells. By immunoblotting analysis with two-dimensional (2-D) gel the antibody vvas shown to react with two protein spots of approx. 40 kDa, with a p1 of 6.0-6.5. Monoclonal antibody; Germ plasm; Germ line cell; Presumptive

Introduction

In the development of some types of animals, a localized cytoplasm plays an important role in the differentiation of certain cell types (Davidson, 1976). In anuran amphibians it is well known that germ plasm (GP) (Bounoure, 1934) located in the vegetal cortical region of fertilized eggs is indispensable for the formation of primordial germ cells (PGCs), i.e. a few cells, containing the GP at the end of the cleavage, proliferate, migrate with development, and finally differentiate into PGCs in the genital ridges at the tadpole stage (Bounoure, 1934; Blackler, 1958; Ikenishi and Kotani, 1975;

Correspondence address: K. Ike&hi, Department of Biology, Faculty of Science, Osaka City University, Sugimoto 3-3-138, Sumiyoshi, Osaka 558, Japan. 0922-3371/89/$03.50

0 1989 Elsevier Scientific

Publishers

Ireland,

PGC; PGC; Xenopus Levis

Whitington and Dixon, 1975; Kamimura et al., 1976). Smith (1966) showed in Rana pipiens embryos that the wave-length of a UV light most effective in reducing the number of PGCs formed at the tadpole stage was 253.7 nm when the vegetal hemisphere of the fertilized eggs was irradiated. Wakahara (1977, 1978) demonstrated in Rana chensinensis and Xenopus faevis that neither heat nor pronase treatment abolished PGC-forming activity in subcellular fractions of vegetal pole cytoplasm. From these facts it is suggested that nucleic acid, probably mRNA, rather than protein in the vegetal pole region, plays an important role in the differentiation of PGCs. On the other hand, any work on the nature of the substances localized in the GP has not yet been reported, except for cytochemical detections of RNA in the GP of Rana temporaria (Blackler, Ltd.

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1958) and Xenopz4.sZueuis (Czolowska, 1969), and in the germinal granules restricted to the GP in Rana pipiens (Mahowald and Hennen, 1971). A monoclonal antibody (mAb) specific for the GP will give us an opportunity to study the substances, principally proteins, which are unique to the GP. It may also be possible to obtain information on the RNA which is responsible for the differentiation of PGCs, as mentioned above, because informational RNAs produced during oogenesis of amphibians are known to be stored in an inactive form such as ribonucleoprotein particles (Davidson, 1976). In the present study, we tried to raise mAb against the GP, using a 20000 x g pellet made from the vegetal pole cytoplasm containing the GP of Xenopus 2-cell eggs as an antigen, instead of the GP itself. This is because it is difficult to collect the GP only from the eggs, and the pellet is known to have a PGC-forming activity (Wakahara, 1978) and a PGC-inducing activity in somatic blastomeres (Ike&hi et al., 1986). Only one mAb (designated as no. 48 antibody) specific for the GP in squash preparations of 32-cell embryos was obtained. Distribution of the corresponding antigen was investigated in polyester wax preparations of embryos of stages l-46. The antigen was found to be restricted to the GP at the cleavage stages. From the gastrula stage onward, it was demonstrated in the juxtanuclear location of the germ line cells, presumptive PGCs (pPGCs) and PGCs. In addition, the antigen was noticed in the somatic cells from the gastrula stage onward. Antigens identified with no. 48 antibody were also clarified by immunoblotting to be two acidic proteins of approx. 40 kDa. Materials and Methods Fertilized Xenopus eggs were obtained as described earlier (Kotani et al., 1973) and staged after Nieuwkoop and Faber (1967). The eggs were treated with thioglycollate solution to remove the jelly coat, as described elsewhere (Ikenishi, 1982). Preparation of the 20 000 X g pellet

The cytoplasm of vegetal pole sixths of frozen 2-cell eggs (stage 2) was collected in the same

manner as described by Moen and Namenwirth (1977), since the GP is located in the vegetal cortical region of the eggs. The rest of the frozen eggs, which never contain the GP, were assumed to be the animal hemisphere cytoplasm. Both 20000 X g pellets from the vegetal pole and the animal hemisphere cytoplasms were prepared according to the method of Wakahara (1978) by differential centrifugation. The pellets were used for the following monoclonal and polyclonal antibody production. Rabbit antiserum for immunoabsorption

The 20000 x g pellet from the animal hemisphere cytoplasm of 250 eggs was dissolved in 2.5 ml phosphate buffered saline (PBS: 1.5 mM KH,PO,, 8.1 mM Na,HPO,, 2.7 mM KC1 and 136 mM NaCl), which was emulsified with an equal volume of Freund’s complete adjuvant. The emulsion was injected subcutaneously into a male rabbit. The pellet prepared from the 250 eggs in 2.5 ml PBS was then injected without the adjuvant on the 9th day after the first injection. Finally, the pellet obtained from 200 eggs in PBS was injected on the 18th day after the second injection. The next day the animal was sacrificed and blood was collected from the heart. The antiserum was recovered from the blood, which was placed at 4 o C overnight and kept at - 20 o C until used. Partial purification of antigen for mAb production

Two ml of the above-mentioned rabbit antiserum was added to 2 ml Protein A-Sepharose CL4B beads (Pharmacia, Sweden) equilibrated with PBS and kept at 4O C overnight. To remove some of the proteins common to the animal hemisphere, the 20000 x g pellet from the vegetal pole cytoplasm of 3000 eggs at stage 2 was mixed with the beads with which the antibodies in the serum were conjugated and stored at 4 o C overnight. The mixture was centrifuged at 3000 rpm for 10 min, and the supematant was again immunoabsorbed with the beads in the same manner. The final supematant was used as an antigen for mAb production. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was used to compare the supematant to the nonabsorbed pellet. Most

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of the bands in the nonabsorbed samples were faint in intensity or disappeared from the supernatant (figure not shown). Immunization

The supernatant was condensed in collodion bags (Sartorius, Gottingen, F.R.G.) to reach 0.6 ml in volume. The condensed supernatant (0.2 ml), which corresponds to the antigen from 1000 eggs, was mixed well with an equal volume of the complete adjuvant and divided into two parts, each of which was injected intraperitoneally into two 7-week-old male mice (BALB/cAuNCrj, Charles River, Japan, Inc., Kanagawa). One month later, these mice were intraperitoneally injected with the supematant (0.1 ml, corresponding to the antigen from 500 eggs) without the adjuvant. A booster injection of the supematant (0.1 ml) without the adjuvant was given one month after the second injection, and the mice were sacrificed 3 days later to obtain splenocytes for fusion. Cell fusion Cell fusion was carried

out as described by Kiihler and Milstein (1976). The culture supernatants of hybridoma cells were screened by indirect immunofluorescent staining of squash preparations of both the isolated blastomeres with and without the GP from a 32-cell (stage 6) embryo for examination of antibody production. The cells producing antibodies were cultured in plastic Petri dishes (Falcon 3001) and kept in liquid nitrogen if necessary. Cloning was carried out by the limiting dilution method, i.e., the cells were suspended in the cloning medium (which was RPM1 1640 HT medium containing 10% cultured supematant of myeloma (P3Ul)cells) containing 15% fetal bovine serum, and poured into a 96-well plate at the rate of 0.5 cells per well. Mass production and purification of mA b

After two successive cloning procedures by the limiting dilution method, hybridoma cells were injected intraperitoneally into a 7-week-old male mouse (BALB/cAuNCrj) which had been treated with 2,6,10,14-tetramethylpentadecane (pristane: Wako Pure Chemicals Industries Ltd., Osaka, Japan). Ascites fluid of the mouse was collected

on the 10th day and precipitated with ammonium sulfate (50% saturation) at 4°C overnight. The precipitate, dissolved in 1 ml PBS, was eluted in a Bio-Gel A-1.5m (Bio-Rad) column, equilibrated with PBS. Fractions exhibiting a peak in UV absorbance at 280 nm were collected and used for identification of the corresponding antigen in polyester wax preparations of embryos at the various stages described below. The identification of the class and subclass of the mAb was determined by the Monoclonal Typing Kit (ICN ImmunoBiologicals, U.K.). ImmunoJluorescent staining

For squash preparations, four vegetal pole blastomeres of a stage-6 embryo, which are known to contain the GP, were isolated in Sorensen phosphate buffer as described previously (Ikenishi, 1982). Similarly, blastomeres without the GP were also isolated from the animal hemisphere of the embryo. They were placed onto 3% gelatin-coated glass slides, covered with a SIGMACOTE-coated (Sigma, MO, U.S.A.) cover glass, and clipped at one end. The preparations were made permeable by freezing rapidly in liquid nitrogen. Taking the slides out of the liquid nitrogen, the cover glasses were immediately removed, and the slides fixed in 95% ethanol at -20°C for 20 min and air-dried at room temperature (RT, 22-24 o C) for 30 min. Embryos at stages 1, 2, 4, 6, 9, 10.5, 12, 17, 23, 28, 33/34, 35/36, 40 and 46 were used for polyester wax preparations. Eight-pm thick sections mounted on albumin-coated glass slides were airdried at RT and kept at 4O C until used. Prior to the immunological reaction, the slides were immersed in 95% ethanol at RT for 5 min to remove the wax, and rinsed with PBS for 15 min. Squashed and polyester wax preparations were incubated with hybridoma culture supematants and the antibody (no. 48), purified from the ascites fluid, respectively, at RT for 1 h. After washing in PBS for 30 min, the preparations were incubated at RT for 1 h in fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse IgG (Cappel Laboratories, PA, U.S.A.) and washed by soaking in PBS for 30 min. After addition of glycerine buffer (nonfluorescent glycerine: PBS = 9 : l), the preparations were examined and photographed

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under an Olympus microscope model BH equipped with epifluorescence optics. Two-dimensional

(2-D) PAGE

The protein samples from Xenopus stage-2 embryos, animal cap (ectodermal cells) of stage 10.5 embryos, stage-23 embryos deprived of endoderm cell mass in which the pPGCs reside and stage-46 tadpoles, deprived of gonads, were prepared and separated by 2-D PAGE in the same manner as in the previous study (Ikenishi and Tsuzaki, 1988). That is, the samples (250 pg protein equivalent each) obtained by freezing and thawing of whole embryos, or partial embryos, were run first on a nonequilibrium pH gradient gel and then on a 7.5% slab gel. After the electrophoreses, the gels were used for the following immunoblotting or silver staining by Morrissey’s (1981) method. Immunoblotting

Proteins separated by 2-D PAGE were blotted onto nylon membranes (Biodyne A, Nippon Pall Co. Ltd., Tokyo) essentially as described by Towbin et al. (1979). The membranes were soaked in 2.5% skimmed milk at 37” C for 1 h and then incubated with the no. 48 antibody at 4” C overnight. As a control, mouse IgM antibody standard (Tago Inc., CA, U.S.A.) or other mouse mAb of the IgM type was used in the present study because no. 48 antibody is a mAb of the IgM type (data not shown). The membranes were washed three times with PBS and then incubated with HRP-labeled goat anti-mouse IgG + IgM antibody (Tago Inc., CA, U.S.A.) at RT for 2 h. After

washing with PBS three times for 15 min each, HRP reaction was carried out by an ordinary method, using o-dianisidine (Wako Pure Chemical Industries, Ltd., Osaka) as a substrate.

Results As the result of four series of cell fusion experiments, 830 hybridoma cell lines were obtained. Each of the culture supernatants from these cell lines was screened by indirect immunofluorescent staining for squash preparations of the isolated blastomeres with and without the GP from a stage-6 embryo. The supematants from 162 of the 830 cell lines reacted with the preparations of both types of blastomeres. Only one supematant from a hybridoma cell line, designated as no. 48, reacted specifically with a yolk-free cytoplasm of the blastomeres with the GP (Fig. l), while it did not react with the blastomeres without the GP, notwithstanding an existence of yolk-free cytoplasm (Fig. 2). The rest did not produce any detectable antibody for antigens of Xenopus. The supematant of no. 48 was further examined immunohistologically with polyester wax preparations of stage-6 embryos in which the location of the corresponding antigen could be easily determined. It reacted specifically with a granular, yolk-free cytoplasm, the GP, in the vegetal pole region of the embryos (Fig. 3). With no. 48 antibody, purified from the mouse ascites fluid mentioned above, distribution of the corresponding antigen was investigated in polyes-

Figs. la, 2a, 3a, 5a, 6a, 6b, I, 8 and 9 are phase-contrast micrographs. Figs. lb, 2b, 3b, 4, 5b, 6c, lo-14 and insets of Figs. l-9 are immunofluorescect micrographs. In polyester wax sections the yolk platelets exhibit a certain autofluorescence. However, it is easy to discriminate the positive reaction from the autofluorescence by color, i.e., the former is greenish and the latter yellowish. bp, blastopore; da, dorsal aorta; dm, dorsal mesentety; ep, epiderm; n, notochord; s, somite; sp, spinal cord; vp, vegetal pole; Wd, Wolffian duct. Fig. la. A squash preparation of the isolated vegetal pole blastomeres containing the GP from a 32-1x11 (stage 6) embryo of Xenopus. An island of yolk-free cytoplasm, filled with granular elements, probably the GP, is seen against many yolk platelets. Bar = 20 pm. b. The same preparation as that in Fig. la. Only the island is stained. Fig. 2a. A squash preparation of isolated animal blastomeres, which never contain the GP from a stage-6 embryo. Many islets of yolk-free cytoplasm are scattered among the yolk platelets which are variable in size and much smaller than those in Fig. la. Bar = 20 pm. b. The same preparation as that in Fig. 2a. The islets as well as other cytoplasm are not stained. Fig. 3a. A polyester wax section parallel to the animal-vegetal axis of a stage-6 embryo. An island of the GP (arrow) is located at the vegetal pole region. Bar = 40 pm. b. The same section as that in Fig. 3a. The island is stained.

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ter wax preparations of embryos extending from the fertilized to the tadpole stage. The antigen was detected in the GP and never recognized in other cytoplasmic regions at the cleavage stages (Figs. 3-5). From the gastrula to the younger tadpole stage (stage 40), it was found distinctly in the juxta-nuclear position of certain endodermal cells whose location in the endodermal cell mass was similar to that of the pPGCs of the corresponding stages in the previous studies (Ikenishi and Kotani, 1975; Whitington and Dixon, 1975; Kamimura et al., 1976). That is, the antigen was found in a few endodermal cells which were located close to the vegetal surface at the early gastrula stage (stage 10.5) (Fig. 6), and in those situated under the archenteron floor at the late gastrula stage (stage 12). It was noted in the endodermal cells beneath the gut cavity at the mid neurula (stage 17) (Fig. 7) and in the cells located at the central part of the posterior endoderm cell mass at the late neurula (stage 23) (Fig. 8) and the mid-tailbud (stage 28). It was found in the cells primarily in the lateral or dorsal part but rarely in the central part of the endoderm cell mass of the late tailbud (stage 33/34) and the hatching stage (stage 35/36) (Fig. 9), and in the cells mostly in the uppermost part of the endoderm cell mass at the younger tadpole stage (stage 40) (Fig. 10). However, the fluorescent area, which was recognized around the nucleus of those endoderm cells, decreased with development (Figs. 6-10). At the feeding tadpole stage (stage 46), the antigen was found in the cytoplasm of the PGCs in the genital ridges (Fig. 11).

In addition to the localization of the antigen in certain endodermal cells, probably pPGCs and PGCs, the antigen was recognized not only in the ectodermal or mesodermal derivatives from the gastrula stage onward but also in the endoderrnal derivatives from stage 23 onward; the antigen was first noticed in the ectoderm and mesoderm cells at stage 10.5 (Fig. 12). It appeared in epidermal cells from stage 17 onward, and in the spinal cord, notochord, somite and the periphery of endoderma1 cells from stage 23 onward (Figs. 13 and 14). The intensity of the fluorescence in the periphery of the endoderm cells, however, was considerably weaker than that of the juxta-nuclear position of certain endodermal cells or the pPGCs described above. The antigen was also present in the Wolffian duct cells from stage 33/34 onward (Figs. 10 and 11) and in dorsal mesentery cells at stage 46 (Fig. 11). Above all, an increase in the intensity of fluorescence was noticeable in the somite as development proceeded (Figs. 10 and 13). In order to determine the relationship between the antigen in the GP of cleaving embryos, recognized by the no. 48 antibody, and that in the somatic cells of embryos beyond the gastrula stage, immunoblotting analyses with 2-D gel were carried out in protein samples from stage-2 embryos, animal ectodermal cells of stage-lo.5 embryos, and partial embryos deprived of germ line cells at stages 23 and 46. No. 48 antibody, being of the IgM type, always reacted with the two protein spots of approx. 40 kDa with a p1 of 6.0-6.5, and a spot of approx. 47 kDa with a p1 of approx. 4.6 in all samples, while other mAbs of the IgM type

Fig. 4. A polyester wax section parallel to the animal-vegetal axis of a fertilized egg. Many islets which are situated in the vegetal cortical region are stained. Bar = 30 pm. Fig. Sa. A hemisection of an embryo of the late blastula (stage 9). The GP is present in a group of cells located close to the vegetal surface (rectangle). Animal pole to the top. Bar = 200 pm. b. Higher magnification of the rectangle in Fig. 5a. The GP in three cells (arrows) are stained. Bar = 30 pm. Fig. 6a. A median section of an embryo of the early gastrula (stage 10.5). Rectangle A shows the position of the endodermal cells containing the granular cytoplasm which are located close to the vegetal surface. Rectangle B shows a position shown in Fig. 12. Bar = 200 pm. b. Higher magnification of the rectangle A in Fig. 6a. Two endodermal cells having a granular cytoplasm, probably pPGCs (arrows) are seen. This type of granular cytoplasm is never recognized at the juxta-nuclear location of the somatic endodermal cells (arrowheads). Bar = 40 pm. c. The same region of the embryo in Fig. 6b. Only the granular cytoplasm in the two endodermaf cells are stained. The position of the nucleus seems to be empty. vp, vegetal pole; bp, blastopore.

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used as a control reacted only with the 47 kDa spot (Fig. 15). Accordingly, it is reasonable to think that the reaction of the no. 48 antibody to the 40 kDa protein spots is specific.

Discussion

In the present study, only one mAb specific for the GP in Xenopus stage-6 embryos was obtained. Distribution of the corresponding antigen was investigated in embryos extending from the fertilized to the tadpole stage. The antigen was found only in the GP at the cleavage stages (Figs. 3-5). From the gastrula to the younger tadpole stage (stage 40), it was highly concentrated in certain endodermal cells, probably the pPGCs (Figs. 6-10). The localization of the endodermal cells in the embryos described here was in good agreement with that of the pPGCs of the corresponding stages in the previous study (Ike&hi and Kotani, 1975). In addition, the location of the antigen in the endodermal cells was consistent with a juxtanuclear location of a granular cytoplasm or the GP in the pPGCs. It was also suggested that a decrease in the fluorescent area around the nucleus in the endodermal cells with development seems to be related to a decrease in the volume of the GP in pPGCs, resulting from a distribution of the GP into two daughter pPGCs at divisions beyond the gastrula stage (Whitington and Dixon, 1975;

Kamimura et al., 1976). At the feeding tadpole stage (stage 46) the antigen was detected in the PGCs in the genital ridges (Fig. 11). Likewise, the antigen was demonstrated in the germ line cells in all embryos of stages l-46 examined. Therefore, the present study has added new evidence at the molecular level, for the continuity of the GP in germ line cells. It is quite reasonable that the germ line cells, the pPGCs and PGCs, in embryos from the gastrula stage onward had the same antigen as that in the GP of the cleavage stage embryos: with light microscopic observations the pPGCs and PGCs are confirmed to inherit the GP from the GP-bearing cells at the cleavage stage (Bounoure, 1934; Blackler, 1958; Whitington and Dixon, 1975; Kamimura et al., 1976), and, on the basis of electron microscopic observations, they are also confirmed to inherit the germinal granule, which is restricted to the GP or its derivatives (Ikenishi and Kotani, 1975). Unexpectedly, the somatic cells were also recognized by the no. 48 antibody from the gastrula stage onward (Figs. 10-14). It is also suggested that the antibody specific for P-granules of germ line cells at early developmental stages cross-reacted with the muscle cells at later developmental stages in the nematode, Caenorhabditis elegans (Miwa, J., personal communication), and that the antibody specific for polar granules in Drosophila cross-reacted with somatic nuclei at later developmental stages (Hay et al., 1988). Ad-

Fig. 7. A frontal section just below the archenteron floor of an embryo of the mid neurula (stage 17). The cell containing the corresponding antigen to the no. 48 antibody is located in the posterior half of the endodermal cell mass (rectangle). Anterior to the left. Bar = 200 pm. Inset: higher magnification of the rectangle. A perinuclear region of the endodermal cell is stained. Bar = 20 pm. Fig. 8. A transverse section of an embryo of the late neurula (stage 23) at the level of a rather posterior part of the endodermal cell mass. The endodermal cell containing the antigen is present in the central part of the endodermal cell mass along the dorsoventral axis (rectangle A). Rectangles B and C show positions of Figs. 13 and 14, respectively. Bar = 200 pm. Inset: higher magnification of rectangle A. An area surrounding the nucleus of the endodermal cell is intensely stained. Bar = 20 pm. Fig. 9. A transverse section through the presumptive mesonephros region of a hatched tadpole (stage 35/36). Usually the endodermal cells containing the antigen are situated in the dorsal or lateral part of the endodermal cell mass at this stage. Rarely is the cell still located in the central region of the mass along the dorsoventral axis (rectangle). Bar = 100 pm. Inset: higher magnification of the rectangle. A perinuclear region of the endodermal cell is stained. Bar = 20 pm. Fig. 10. A transverse section through the presumptive mesonephros region of the right half of a younger tadpole (stage 40). A perinuclear region of the endodermal cell which is located in the uppermost part of the endodermal cell mass (arrow) is stained. The somites and the Wolffian duct cells are heavily stained. The periphery of the somatic endoderm cells are also stained. Bar = 40 pm. da, dorsal aorta; s, somite; Wd, Wolffian duct.

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Fig. 11. A transverse section of a feeding tadpole (stage 46). The cytoplasm of the PGC (arrow) in the genital ridges is stained. The granular cytoplasm in the pPGCs becomes smaller with development so that the positive area in the PGC is quite small. The dorsal mesentery cells (dm) and the Wolffian duct cells (Wd) are also stained. Bar = 20 pm. Fig. 12. A higher magnification of Fig. 6a, rectangle B, at the animal cap of the embryo of the early gastrula. Almost all ectodermal cells are stained. Bar = 40 pm. Fig. 13. A higher magnification of Fig. 8, rectangle B, at the dorsal region of the embryo of the late neurula. The epiderm (ep), the spinal cord (sp), the somite (s) and the notochord (n) are stained. Bar = 40 pm. Fig. 14. A higher magnification of Fig. 8, rectangle C, at the ventral region of the embryo of the late neurula. Not only the ectodermal and mesodermal derivatives but also the periphery of the somatic endodermal cells (arrows) are stained. Bar = 40 pm.

ditionally, in the latter it was shown by immunoblotting that the antibody recognized the same protein band in the protein samples from both the pole cell-enriched fraction and the latestage embryos, in which many somatic nuclei were

reacted with the antibody. This points to the possibility that the pertinent antigen in the polar granules is identical to that in the somatic nuclei. In the present study, immunoblotting analyses with 2-D PAGE showed that two acidic proteins

Mr x 103

Fig, 15. Silver stain (a) or immunoblots (b-d) of 2-D PAGE of protein samples (250 pg protein equivalent each) from Xenopur embryos. a. Stage-2 embryos. Two protein spots of approx. 40 kDa with a pI of 6.0-6.5 (arrows) and one spot of approx. 47 kDa with a pI approx. 4.6 (arrowhead) were recognized with no. 48 antibody in the following immunoblots. b. Stage-2 embryos: the two 40-kDa and the 47-kDa protein spots are reacted with no. 48 antibody. c. The animal ectodermal cells of the stage-lo.5 embryos. Two 40-kDa spots and a 47-kDa spot, which are recognized in the stage-2 embryos, are reacted with no. 48 antibody. d. Stage-2 embryos with the antibody of mouse IgM standard as a control. Only the 47-kDa protein spot has reacted.

in the samples from the stage-2 embryos, which were recognized specifically by the no. 48 antibody, were also detected in those of animal ectodermal cells from the gastrula embryos, and those of the neurulae and tadpoles deprived of germ line cells (Fig. 15). This means that the antigen in the GP of stage-2 embryos is considered to be identical to that of the somatic cells in embryos from the gastrula stage onward, because the antibody reacted only with the GP in cleaving embryos. Therefore, the same antigen is thought to appear in the somatic cells beyond the gastrula stage due

to the de novo synthesis. Accordingly, it is safe to conclude that the no. 48 antibody is specific for the GP of cleavage stage embryos. It has been demonstrated in Xenopz~ that tubulin and vimentin are present in the GP as well as in other cytoplasm at early cleavage stages, using anti-tubulin (Wylie et al., 1986) and antivimentin (Tang et al., 1988) antibodies. In those studies, tubulin and vimentin were identified as a 55-kDa protein and a 55-kDa protein with a pI of 5.6, respectively, by immunoblotting. In the present study, no. 48 antibody reacted specifically

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with the GP in cleaving embryos and recognized the two 40-kDa proteins with slightly different pIs of 6.0-6.5. As the functional significance of the two proteins remains obscure, they are considered to be the first members of macromolecules restricted to the GP at the cleavage stages, judging from the above-mentioned immunohistochemical and molecular characteristics.

Acknowledgments

We are grateful to Dr. M. Kotani, Department of Natural Science, Osaka Women’s University, and Dr. M. Furusawa, Deputy Director of the Research Institute, Daiichi Seiyaku Co. Ltd., for their critical reading of the manuscript. Thanks are also expressed to Dr. Y. Yamaguchi, Mr. K. Murakami and Mr. Y. Saeki for their technical advice in monoclonal antibody production, and to Mr. T. Okuda for his assistance in preparing the antigen for the monoclonal antibody. We also wish to thank Drs. T. Yamamoto and N. Minamiura of our department for offering facilities for electrophoresis of antigens, and Dr. K. Ito and Mr. Y. Tsuzaki for useful suggestions regarding electrophoresis.

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