Immunolocalization of three oocyte nuclear proteins ... - Development

Development 101. 715-728 (1987) Printed in Great Britain © The Company of Biologists Limited 1987

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Immunolocalization of three oocyte nuclear proteins during oogenesis and embryogenesis in Pleurodeles

C. ABBADIE1, D. BOUCHER 1 , J. CHARLEMAGNE2 and J. C. LACROIX 1 Laboratoire de Gtnerique du Diveloppement and 2Laboratoire d'Immunologie Compare, 9 quai St Bernard, 75230 Paris Cedex 05, France

UA 1135 CNRS, University Pierre el Mane Curie.

Summary The location of three proteins of the oocyte nucleus of Pleurodeles was studied during oogenesis and embryogenesis using monoclonal antibodies A33/22, C3/l and C36/1. Immunoblotting of two-dimensional gel electophoregrams of oocyte nuclear proteins showed that these antibodies recognized proteins whose relative molecular masses and isoelectric points were 80X10 3 and 6 4, 175X103 and 5 and 270X103 and 7, respectively. In the oocyte, all three proteins were nucleoplasmic; those revealed by antibodies A33/22 and C36/1 were detected on lampbrush chromosomes: the first one on the RNP matrix of the loops, and the second one on both the loops and the chromomeres. Protein A33/22 was observed in most nuclei during embryonic, larval and adult development, except for the young embryo, before the midblastula transition. The distribution of this protein in the oocyte and its behaviour during development suggest that it might be involved in the packaging of RNAs during transcription. Antibody C3/1 recognized an oocyte nucleoplasmic protein with biochemical and biophysical

properties similar to those of protein N1-N2. After oocyte maturation, the protein moved into the cytoplasm of the animal hemisphere and, from fertilization to the midblastula stage, it shifted from the cytoplasm into the nuclei as cell division proceeded. Starting from the gastrula stage, this protein became specific to the endoderm nuclei. After hatching, it was no longer detectable. This behaviour seems to correspond to that of a nuclear protein issued from the maternal stock pile. Protein C36/1 behaved similarly during early development, but remained in most nuclei after neurulation until the adult age, with a pattern similar to that of protein A33/22. In addition, it was present on the mitotic chromosomes. Its association with mitotic as well as lampbrush chromosomes connects it with the DNP fibre proteins.

Introduction

1977; Mills, Laskey, Black & de Robertis, 1980; Kleinschmidt & Franke, 1982; Kleinschmidt et al. 1985). The oocyte also accumulates factors that might take part in the determination and/or the differentiation of the embryonic cells. This applies, for instance, to the 'substance 0' found in Ambystoma mexicanum (Briggs & Cassens, 1966) which might be essential for the normal activation, during blastulation, of the nuclear genes required for gastrulation and organogenesis (Brothers, 1976). This is also the case for two Xenopus germinal vesicle proteins, the function of which is still unknown, but which exhibit during the later stages of development a nuclear

The amphibian oocyte nucleus, or germinal vesicle, contains over 300 different proteins (Feldherr & Ogburn, 1980). Some of these are synthesized by the oocyte and stored in the nucleus for subsequent use at various stages of development. The first proteins to be described were the polymerases (Wassermann, Hollinger & Smith, 1972; Roeder, 1974; Benbow, Pestell & Ford, 1975; Fox, Breaux & Benbow, 1980) and the proteins involved in the constitution and assembly of nucleosomes, i.e. the histones, nucleoplasmin and N1-N2 proteins (Woodland & Adamson,

Key words: oocyte nuclear proteins, lampbrush chromosomes, monoclonal antibodies, amphibia, Pleurodeles waltl.

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C. Abbadie, D. Boucher, J. Charlemagne and J. C. Lacroix

histospecific location that might indicate their involvement in late differentiation events (Dreyer, Singer & Hausen, 1981; Dreyer, Scholtz & Hausen, 1982; Dreyer & Hausen, 1983). In order to isolate new germinal vesicle-derived proteins involved in the mechanisms of embryonic determination and differentiation, we built a library of monoclonal antibodies directed against nuclear antigens of Pleurodeles (Lacroix et al. 1985). In Pleurodeles, as in all urodeleans, the oocyte lampbrush chromosomes are highly developed. This allowed us to test the antibodies on lampbrush chromosome spreads, in addition to the tests usually done on oocyte sections. Therefore, we were able to select antibodies directed against nucleoplasmic antigens, as well as antibodies directed against antigens found on condensed chromatin, or antibodies directed against antigens associated with a few or all transcription units. Afterwards, the fate of the selected antigens during development was followed by immunohistology and immunoblotting techniques, with the aim of detecting tissue-specific and/or stagespecific patterns. We report here on the results obtained with three antibodies: A33/22, C3/1 and C36/1.

The thermolability of the proteins recognized by the antibodies was tested as described by Laskey, Honda, Mills & Finch (1978) and Dabauvalle & Franke (1982). Development stages were determined according to Bonnanfant-Jais & Mentre (1983) for oocytes, and to Gallien & Durocher (1957) for embryos. Results Three fusions were carried out with spleens of mice immunized against hand-isolated oocyte nuclei of Pleurodeles waltl fusions A and B) or Pleurodeles poireti (fusion C). One of the antibodies studied, A33/22, was produced by fusion A and the two others, C3/1 and C36/1, by fusion C. In all three cases, the immunoglobulins were IgGl. Antibody A33/22 crossreacted with P. poireti and, reciprocally, antibodies C3/1 and C36/1 crossreacted with P. waltl.

Materials and methods All the techniques used here for hybridoma production, ELISA tests, immunolocalization on lampbrush chromosomes, immunohistology and immunoblotting have been described elsewhere (Lacroix et al. 1985). The immunohistology technique had the two following modifications: (i) DAPI (4',6-diamidino-2-phenylindol dihydrochlorhyd; Boehringer Mannheim), a DNA-binding fluorescent dye, was used on lampbrush chromosome spreads to visualize the DNA axis, and on histological sections to make the nuclei evident (especially inside embryos with a high yolk content) and to determine the cell cycle stages. It was added to the FITC- or TRITCconjugated second antibody at a concentration of 10^/g ml"1 for lampbrush chromosome preparations, and at a concentration of 0-2ugml~' for histological sections (Dreyer el al. 1981). (ii) Fixed and dehydrated histological samples were embedded either in polyethylene-glycol 400 distearate (Steedman, 1957) (fusion point: 37°C) or in cytoparaffin (fusion point: 56°C). The two techniques differed in sensitivity, depending on the antibody used. The proteins used for immunoblotting were obtained from whole stage VI oocytes, stage VI oocytes fractionated into germinal vesicle and cytoplasm in Ringer's medium (Detlaff, Nikita & Stroeva, 1964), progesterone-maturated oocytes, cleavage embryos and old swimming tadpoles (dissected to remove cartilages). All the samples were handled as recommended by Ballantine, Woodland & Sturgess(1979).

Fig. 1. Staining of an ovary section of Pleurodeles with antibody A33/22 (A) and with DAPI (B). DAPI visualizes the chromatin in the somatic cells and the axis of the lampbrush chromosomes in the oocyte germinal vesicles. Labelling by antibody A33/22 is visible in the nucleoplasm of the three stage I oocytes and the nucleus of some somatic cells (arrow). Most other somatic cells are unstained. Bar 100j.im.

Immunolocaiization of oocyte nuclear proteins in Pleurodeles Most of our tests were conducted with P. waltl. Preliminary results obtained with antibody A33/22 have already been published (Lacroix et al. 1985). Conventionally, the antigens will be given the same reference as the antibodies that recognize them. Germinal vesicle labelling All three antibodies, used in this study, stained the oocyte nucleoplasm, but not the nucleoli. The cytoplasm was negative. The subnuclear location of the labelling differed according to the antibody and to the stage of oogenesis. Antibody A33/22 labelled the germinal

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vesicle uniformly at all stages, i.e. separate components were not distinguishable inside the nucleus (Fig. 1). On the contrary, antibodies C3/1 and C36/1 recognized intranuclear structures whose distribution and aspect varied during oogenesis. Thus, in the previtellogenic stage I oocytes, the staining was scattered in clusters (Fig. 2A), whose diameter varied from 0-5 to 5^m in proportion to the size of the oocyte. From stage II to stage V (vitellogenesis), a homogeneous staining of the nucleus was observed (Fig. 2B). As regards stage VI oocytes, some of them were uniformly labelled, as they were in the previous stages, whereas others displayed a certain degree of

Fig. 2. Immunostaining of oocyte sections with antibody C3/1 at different stages of oogenesis. Left: phase contrast; right: indirect immunofluorescence. (A,A') stage I oocyte. Staining is restricted to the germinal vesicle and is heterogeneous. Bar 50pan. (B.B') stage IV oocyte. At this stage, the germinal vesicle is uniformly labelled. Nucleoli are negative (arrow). Bar 400^m. (C,C) stage VI oocyte. Staining is essentially located near the nuclear envelope. The fluorescence of erythrocytes (arrow) is due to the autofiuorescence of the haemoglobin. Bar 400^m.

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C. Abbadie, D. Boucher, J. Charlemagne and J. C. Lacrolx

segregation of the staining at the periphery of the germinal vesicle (Fig. 2C). In addition to the germinal vesicle, antibodies C36/1 and A33/22 stained the nuclei of the epithelial cells on ovary sections, but the follicular cell nuclei were only faintly stained (Fig. 1). Antibody C3/1 did not stain any of the somatic cells (Fig. 2).

this antibody, all the normal loops were homogeneously labelled over their entire length, whatever the thickness of their matrix (Fig. 4). No change in the chromosome labelling was discernible during oogenesis. The last antibody, C3/1, did not label any chromosomal element.

Lampbrush chromosome labelling At all stages of oogenesis, antibody A33/22 labelled nearly all the normal loops of the lampbrush chromosomes, but did not label the loops with a dense matrix and the chromomeres (Fig. 3A). The staining of the normal loops was restricted to the matrices (Fig. 3B), which are structures consisting of nascent RNA and bound proteins. Particles, 2-3 pm in diameter, observed free in the nucleoplasm, were also stained by antibody A33/22. Antibody C36/1 stained the loops as well as the chromomeres on chromosomal preparations. With

Fate of the three germinal vesicle-derived antigens (1) During oocyte maturation, fertilization and cleavage stages Antibodies C3/1 and C36/1 stained the animal hemisphere cytoplasm, on histological sections of matured oocytes. The fluorescence was very intense over the animal hemisphere cortex (Fig. 5). The vegetal hemisphere was not stained. In embryos up to the midblastula stage (stage 6), we observed the same pattern of cytoplasmic labelling and, in addition, a staining of the nuclei (Fig. 6). Starting from the end of cleavage

Figs 3, 4. Labelling of the lampbrush chromosomes with antibodies A33/22 (Fig. 3) and C36/1 (Fig. 4). With A33/22, all the normal loops are stained, but the axis is negative. The intensity of the loop staining correlates with the matrix thickness (Fig. 3A). On the two giant loop pairs seen in Fig. 3B, one can clearly see that the polarized distribution of the labelling corresponds to that of the ribonucleoprotein matrix. In contrast to antibody A33/22, antibody C36/J stains the chromomeres of the axis and all the normal loops, irrespective of matrix thickness (Fig. 4B). Bar 40f.tm.

Immunolocalization of oocyte nuclear proteins in Pleurodeles (stage 7), the cytoplasmic staining disappeared; only the nuclear staining persisted. Antigen A33/22 was not detectable on sections of matured oocyte and embryos until the midblastula stage. After this stage, some staining appeared inside the nuclei. (2) During gastrulation, neurulation and tailbud stages On sections of embryos at the beginning of gastrulation (stage 8), the three antibodies yielded a nuclear staining restricted to the endoderm, i.e. ectodermal and mesodermal nuclei were negative (Fig. 7). This regional staining persisted until hatching, in the case of antibody C3/1 (Fig. 8B), whereas antibodies

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A33/22 and C36/1 again labelled the nuclei of all three embryonic layers (Fig. 8A), starting from the neurula stage (stage 12 for A33/22 and stage 18 for C36/1). (3) During late development Antibody C3/1 no longer reacted on sections of larvae after hatching. In contrast, antibodies A33/22 and C36/1 labelled most nuclei during larval development and in the adult. Cartilages and erythrocytes were always negative. In the other tissues, the labelling varied in function of the stage and type of differentiation. This was especially obvious in the central nervous system and in the gonads of the larvae. Two areas are usually

Fig. 5. Partial view of the animal hemisphere of a matured oocyte section stained by antibody C36/1. Fluorescence is located in the vitellus-free cytoplasm (arrow). It is especially intense in the cortical cytoplasm (arrowhead). Bar 100nm.

Fig. 6. Labelling of a section of a stage 2 embryo (four blastomeres) with antibody C3/1. The fluorescence is located in the nucleus (arrow) and in the cytoplasm of the animal hemisphere. The vegetal hemisphere cytoplasm is negative. Bar 350tim.

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C. Abbadie, D. Boucher, J. Charlemagne and J. C. Lacroix staining by antibody A33/22 persisted around the mitotic chromosomes, which were not themselves stained (Fig. 10). The staining by antibody C36/1 seemed to spread throughout the cell cytoplasm after the nuclear envelope had broken (Fig. 11B). In addition, the outlines of the mitotic chromosomes were stained by this antibody. This staining of the chromosomes was only perceptible at metaphase and anaphase because at prophase, the nucleoplasm remained strongly fluorescent (Fig. 11A), thus masking any staining of the condensing chromosomes. The antigens stained by antibody C36/1 quickly reentered the nuclear compartment at telophase, as soon as the karyomeres formed (Fig. 11C).

Fig. 7., Section of a young gastrula (stage 8) stained with antibody C36/1 (A) and with DAPI (B). The staining is specific for the endodermal nuclei (arrow). The ectodermic nuclei are negative (arrowhead). Bar 200/.im. distinguishable in the larval nervous tissue; a central region surrounding the lumen, which constitutes the cell proliferation area, and a more cortical region composed of cells undergoing differentiation (Straznicky & Gaze, 1972; Heaulme, 1978). Antibodies A33/22 and C36/1 stained the nuclei of the differentiation zone only. After metamorphosis, the nuclei of all nerve cells were stained. In the male and female larval gonads, the polylobate nuclei of the gonocytes were recognized by the two antibodies A33/22 and C36/1, whereas all the somatic cells of the gonads were negative. In the adult, the oocytes remained labelled throughout oogenesis, while for the male lineage the sexual cells progressively lost their fluorescence during spermatogenesis (spermatogonies and spermatocytes I were stained, but spermatocytes II, spermatids and spermatozoa were not). Evolution of the nuclear staining during the cell cycle The three antibodies gave different patterns of cell staining during mitosis. No antigen was revealed by antibody C3/1 in the mitotic cells, either in the cytoplasm or on the chromosomes (Fig. 9). The

Immunoblotting Immunoblotting of two-dimensional electrophoregrams of the germinal vesicle proteins of stage VI oocytes gave relative molecular masses of 80, 175 and 270xlO3 and isoelectric points of 6-4, 5 and 7 for the proteins recognized by antibodies A33/22 (Lacroix et al. 1985 and Fig. 12B), C3/1 (Fig. 12C) and C36/1 (Fig. 12D), respectively. When visualized on gels stained with Coomassie blue, the three proteins seemed to be abundant (Fig. 12A). Protein C3/1 was not precipitated by heating to 80°C and subsequent centrifugation at lOOOOg, but protein A33/22, used as control, precipitated during this treatment (Fig. 13). Therefore, protein C3/1 could be classified amongst the thermostable oocyte nuclear proteins. The intraoocyte location of the three proteins was visualized by one-dimensional electrophoresis of stage VI oocytes dissected into germinal vesicle and cytoplasm: the proteins A33/22 and C36/1 were found only in the nucleus (Fig. 14A,C). The protein recognized by antibody C3/1 was mainly found in the nucleus, but was also faintly detected in the cytoplasm (Fig. 14B). The immunoblots of one-dimensional electrophoresis of matured oocytes and tailbud-stage embryos (stage 26) indicated that the proteins evidenced during development were very similar to those found in the oocyte and, at least, exhibited the same MT values (Figs 14, 15). No fluorescence was detected on sections of matured oocytes and young embryos treated by antibody A33/22, although this antibody strongly reacted with the germinal vesicle on oocyte sections. Indeed, protein A33/22 was present in the matured oocytes and in the 8-cell, mid-blastula and late-

Immunolocalization of oocyte nuclear proteins in Pleurodeles blastula embryos, as revealed by immunoblotting (Figs 14 A, 16). Fig. 17 shows that, in stage 53 larva, no staining was obtained with antibody C3/1, whereas antibody A33/22 strongly stained the 80X103 band. The nSxlO^band stained faintly only when the incubation time of the nitrocellulose sheet with diaminobenzidine was greatly lengthened. Thus protein C3/1 might be absent from larval and adult somatic cells, or present at a very low level, as will be argued in the discussion section.

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Discussion Monoclonal antibodies A33/22, C3/1 and C36/1 have been used to study the fate of three oocyte nuclear proteins during embryogenesis. Our results are summarized in Table 1. The immunoblotting of oocyte proteins confirmed that the proteins stained by antibodies A33/22 and C36/1 were located only in the germinal vesicle and that a small amount of the protein recognized by antibody C3/1 was also found in the cytoplasm of the

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Fig. 8. Two consecutive sections of a stage 34 embryo stained with DAPI (A,B) and immunolabelled with antibody C3/1 (A') or with antibody C36/1 (B'). Antibody C3/1 (A') exhibits specificity for the endodermic nuclei, whereas antibody C36/1 (B') stains some nuclei in all the tissues, but not the nuclei in each tissue (this is particularly visible in the central nervous system). Bar 350 ;jm.

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C. Abbadie, D. Boucher, J. Charlemagne and J. C. Lacroix

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Figs 9-11. Staining by the three antibodies during mitosis. Fig. 9. Section of a stage 8 embryo stained with antibody C3/1 (A) and DAPI (A'). The interphase nuclei are stained (arrow), but the cell at prophase is not (arrowhead). Bar 30/im. Fig. 10. Section of a stage-33b embryo stained with antibody A33/22 (A) and DAPI (A'). The interphase nuclei are stained (arrow) in the cell at metaphase (arrowhead), the fluorescence remains located around the mitotic chromosomes, which are themselves unstained. Bar 15 j/m. Fig. 11. Sections of stage-6 embryos stained with antibody C36/1 (A-C) and with DAPI (A'-C). During interphase and prophase (A,A') staining is localized over the nucleoplasm. At metaphase (B,B'), it spreads in the whole cytoplasm; then, at telophase, it re-enters the nuclear compartment, as soon as the karyomeres reconstitute (C,C). Bar 20jrni.

Immimolocalization of oocyte nuclear proteins in Pleurodeles

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Fig. 12. Two-dimensional gel of germinal vesicle proteins of 30 stage-VI oocytes from Pleurodeles waltl (approximately 90 fig of proteins by gel) stained with Coomassie brilliant blue R250 (A) and immunoblots of the same proteins obtained after incubation with antibody A33/22 (B), antibody C3/1 (C) and antibody C36/1 (D). Arrowheads on A indicate the proteins recognized by antibodies A33/22 (middle), C3/1 (right) and C36/1 (left). Parameters of the labelled proteins are 80X103 and 6-4 for A33/22, 175xlO3 and 5 for 3/1 and 270X103 and 7 for C36/1. oocyte. The immunohistochemical staining of lampbrush chromosomes and oocyte sections disclosed that all three proteins were located inside the nucleoplasm, and that those corresponding to antibodies A33/22 and C36/1 were also found on lampbrush chromosomes. The small quantity of protein C3/1 localized to the oocyte cytoplasm by immunoblotting was not detectable on histological sections, probably because it was too diluted. The pattern of nucleoplasmic staining obtained with antibodies C3/1 and C36/1 on oocyte sections varied during oogenesis. The nature of the nucleoplasmic structures stained by these two antibodies and the significance of the staining variations observed during oogenesis are not yet clear. The three proteins were also found in the embryos by immunoblotting, as well as by immunohistology, and their possible biological functions are discussed below.

Antibody A33/22 did not stain sections of matured oocytes and young embryos, although immunoblotting indicated that the corresponding protein was actually present. Thus, the immunoblotting technique seems to be more sensitive than immunohistology. Starting from the midblastula stage, the protein recognized by A33/22 became detectable by immunohistology in the nuclei. Therefore, after maturation, the protein would be dispersed in the huge cytoplasmic volume, where it would be too diluted to be detected by indirect immunofluorescence. At the midblastula stage, the antigen would accumulate in the nuclei. Its origin, maternal or neosynthesized, is not yet established. The accumulation of this protein in the embryonic nuclei seems to be concomitant with the onset of transcription which occurs at the midblastula stage in the amphibian embryo (Bachvarova & Davidson, 1966; Newport & Kirschner, I982a,b). This suggests that protein

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C. Abbadie, D. Boucher, J. Charlemagne and J. C. Lacroix

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b

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GV O C Om GV O C Om GV O C Om

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Fig. 13. Total germinal vesicle proteins of 10 stage-VI oocytes separated in a polyacrylamide gel (lane a), then transferred onto nitrocellulose sheets, and stained with antibodies C3/1 (lane c) and A33/22 (lane e). Antibody C3/1 recognizes a 175xlO3 band, and A33/22 a 80x 103 band. Lane b: polyacrylamide gel separation of soluble proteins obtained after heat treatment at 80°C and centrifugation at 10 000 g of 10 stage VI germinal vesicles. Immunoblots of the same proteins obtained after incubation with antibodies C3/1 (lane d) and A33/22 (lane f). The protein recognized by antibody C3/1 remains in the supernatant: it behaves as protein N1-N2. Instead, protein A33/22 precipitates. Lane R: reference proteins; from bottom to top: trypsin inhibitor (201 xlO3), lactate dehydrogenase (36-5x L03), glutamate dehydrogenase (55-4X103), phosphorylase b (97-4X103), asmacroglobuline reduced (170X103) and nonreduced (340X103).

A33/22 might be involved in transcription and/or in nuclear post-transcriptional events. Three additional arguments support this hypothesis. First, cells known to have a low level of nuclear activity, such as erythrocytes, spermatids and spermatozoa, were not stained by antibody A33/22. Second, antibody A33/22 did not react with mitotic chromosomes and, third, this antibody labelled the RNP-rich matrix of the lampbrush chromosome loops. Together with the staining pattern of these loops, which exactly underlined the matrix, the staining of free RNP particles in the nucleoplasm suggests that the antigen recognized by antibody A33/22 combines with nascent RNA on the loops and then moves, in the form of free hnRNP, into the nuclear sap, where it is stored. It has already been shown that anti-RNP antisera stain the matrix of

A

B

C

Fig. 14. Distribution of the antigens recognized by antibodies A33/22 (A), C3/1 (B) and C36/1 (C) in stage VI oocytes of Pleurodeles waltl and in oocytes matured in vivo (Om). Stage VI oocytes were dissected into a nuclear fraction (GV) and a cytoplasmic fraction (C). or were not dissected (O). Ten oocytes were used in each lane. Antigens recognized by antibodies A33/22 and C36/1 are exclusively located in the nuclear fraction (A,C); antigen C3/1 is faintly visible in the cytoplasmic fraction (B). All three antigens are present in matured oocytes.

lampbrush chromosome loops (Scott & Sommerville, 1974; Sommerville, Chrichton & Malcolm, 1978; Martin & Okamura, 1981). Nevertheless, the pattern of A33/22 staining on oocyte sections does not allow us to rule out the possibility that the antigen also exists free in the nucleoplasm. The changes in staining observed during development could reflect different hnRNP concentrations in the nuclei, which, in turn, might be due to different levels of transcriptional activity and/or to differential requirement of RNP stored in the nuclear sap (Lacroix et al. 1985). These changes could be related to the mechanisms of differentiation since, in the nervous system, the nervous cells are stained only when they are differentiating, or after they have differentiated. After oocyte maturation, the protein recognized by antibody C3/1, originally nucleoplasmic in the oocyte, moves into the cytoplasm of the animal hemisphere. Just after fertilization, the protein begins to shift from the cytoplasm into the nuclei. This migration seems to go on until the midblastula stage is reached, for the cytoplasmic staining disappears only at this stage. From the gastrula stage onwards, the

Immunolocalization of oocyte nuclear proteins in Pleurodeles A

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Fig. 15. Total proteins of two tailbud-stage embryos (stage 26) separated in a polyacrylamide gel (A), then transferred on nitrocellulose sheets and stained with antibodies A33/22 (B), C3/1 (C) and C36/1 (D). The MT of the labelled proteins are respectively: 80, 175 and 270X103 - the same as those stained in the oocyte. Fig. 16. Total proteins of ten 8-cell-stage (A), mid-blastula-stage (B) and late-blastula-stage embryos (C) separated in a polyacrylamide gel, transferred on nitrocellulose sheets and labelled by antibody A33/22. The 80X103 band corresponding to protein A33/22 is detected in the three cases. Fig. 17. Total proteins of soft tissues (i.e. except cartilages) of stage 53 larvae (about 0-05 larva by lane) separated in a polyacrylamide gel (A), then transferred onto nitrocellulose sheets and stained with antibodies A33/22 (B) and C3/1 (C). The 80X103 band recognized by antibody A33/22 is strongly stained, whereas the 175X103 band corresponding to antibody C3/1 becomes faintly visible only when the revelation time is lengthened.

protein becomes specific for the endoderm nuclei; after hatching, it is no longer observed in any somatic cell (this observation is confirmed by the moresensitive immunoblotting technique). This behaviour seems to correspond to that of a nuclear protein issued from the maternal stock pile. It is worth noting that the order of disappearance of the antigen from the three embryonic tissues corresponds to the order in which the morphogenetic capacities of the nuclei are lost during development. Thus, it has been shown by nuclear transplantation that, in Pleurodeles, the ectodermal and mesodermal nuclei are severely restricted in their capacity to promote complete development starting from the neurula stage, whereas the endodermal nuclei retain this capacity until stage 27 (tailbud stage) (Aimar, 1972). Dreyer et al. (1982) found in Xenopus four germinal vesicle antigens exhibiting a behaviour looking very much like that of our antigens A33/22 and C3/1: they move in the cytoplasm of the animal hemisphere after, the germinal vesicle breakdown, then they shift from the cytoplasm into the nuclei at some time between the 32-cell and the neurula stages.

The molecular weight and isoelectric point of the protein C3/1 are similar to those reported by Dabauvalle & Franke (1982) for the protein N1-N2 of Pleurodeles. In addition, the distribution of staining by antibody C3/1 in Pleurodeles during development coincides with that given by N1-N2 in Xenopus, with the only exception that the events are delayed in Xenopus: the specificity for the endoderm occurs only at stage 40 (i.e. after hatching) and the disappearance of staining at stage 50 (Dreyer, Wang, Wedlich & Hausen, 1983; Wedlich, Dreyer & Hausen, 1985). In Pleurodeles, as in many other amphibian species, protein N1-N2 and nucleoplasmin are the only oocyte nuclear proteins to remain in solution after heating to 80°C, followed by centrifugation at 10 000 £ (Laskey et al. 1978; Dabauvalle & Franke, 1982). We report here that protein C3/1 has the same property. Therefore, there are several reasons to believe that protein C3/1 corresponds to protein N1-N2. Our antibody C3/1 did not react with larval and adult somatic cells, in immunohistology as well as in immunoblotting. However, protein Nl has been detected with a monospecific antiserum in cultured Xenopus somatic cells, although in small amounts. An immunologically

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Table 1. Biochemical parameters and main localizations of the proteins A33/22, C3/1 and C36/1

iW^xlO3) IP Lampbrush chromosomes Loops Chromomeres Nucleoplasmic particles Oocyte Germinal vesicle Cytoplasm

A33/22

C3/I

C36/1

80 6-4

175 5

270 7

++ +

-

+ + ++

++ —

++ ±

++ -

(blot only) Cleavage embryo Nuclei Cytoplasm Old blastula Nuclei Cytoplasm

± (blot only) ++ —

++ ++

++ ++

++ -

++ (except mitosis)

Nuclei of gastrula Ectoderm Mesoderm Endoderm Nuclei of tailbud embryos Ectoderm Mesoderm Endoderm Nuclei of larvae and adults

+ ± ++

± ++

+ + + +

-

± + -

-

± ++ + + + +

related protein was also found in diverse somatic cell types of several amphibians, including Pleurodeles (Krohne, 1985). These conflicting results can be explained in two ways: perhaps in our hands immunohistology and immunoblotting techniques are not sensitive enough to detect a low level of protein, or else our monoclonal antibody C3/1 recognizes an epitope specific for the oocyte protein N1-N2 that the somatic protein Nl does not share. The latter possibility is supported by the fact that protein C3/1 behaves as expected for a protein of maternal origin during development. Until the midblastula stage, the patterns of fluorescence revealed by antibodies C36/1 and C3/1 are very similar. Therefore, the protein recognized by antibody C36/1 is probably of maternal origin, until this stage. Starting from the gastrula stage, the pattern of staining by antibody C36/1 resembles that of antibody A33/22. The function of the protein recognized by antibody C36/1 remains unclear. It has two remarkable properties: first, it seems to be karyophilic, for it shifts during fertilization and segmentation into all the nuclei (even in the supernumerary male pronuclei) and also into the karyomeres, as soon as they are individualized at telophase. Second, it is located on the loops and chromomeres of the

lampbrush chromosomes, as well as on the mitotic chromosomes. This localization might connect it with the DNP fibre-proteins. We are grateful to A. M. Ohler, F. Simon, C. K. Pyne, J. Desrosiers, J. Marot and A. Fuczka for their help. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale (CRE 841013), and the Ministere de l'Education Nationale (ARU).

La localisation de trois prot£ines nucleaires de l'ovocyte de Pleurodele a 6t6 6tudi6e au cours de l'ovogenese et de l'embryogenese a l'aide des anticorps monoclonaux A33/22, C3/1 et C36/1. Sur transfert d'eiectrophorese bidimensionelle, les trois anticorps reconnaissent des prot6ines dont le poids moleculaire et le point iso61ectrique sont respectivement 80 kD et 6-4, 175kD et 5 et 270 kD et 7. Dans l'ovocyte, les trois proteines sont localises dans le nucl6oplasme; celles reconnues par les anticorps A33/22 et C36/1 sont egalement pr6sentes au niveau des chromosomes en dcouvillon: la premiere sur la matrice ribonucleoproteique des boucles, et la seconde a la fois sur les boucles et les chromomeres. La proteine A33/22 est prdsente dans la plupart des noyaux au cours du deVeloppement embryonnaire, larvaire et adulte, except^ chez le jeune embryon avant la stade miblastula (stade de la reprise de I'activite transcriptionnelle). La localisation de cette proteine dans l'ovocyte et son comportement au cours du d6veloppement indique qu'elle serait impliqude dans l'empaquetage des ARN au cours de la transcription. L'anticorps C3/1 reconnait dans l'ovocyte une prot6ine nucl6oplasmique dont les parametres biophysiques et biochimiques sont similaires a ceux des proteines N1-N2. Apres la maturation ovocytaire, la prot£ine se rdpand dans le cytoplasme de l'he'misphere animal, puis, de la f£condation jusqu'au stade mi-blastula migre dans les noyaux au fur et a mesure des divisions cellulaires. A partir du stade gastrula, elle devient spe'cifique des noyaux endodermiques. Apres l'6closion, elle n'est plus d£celable. Ce comportement correspondrait a celui d'une proteine nuc16aire provenant du stock maternel. La protdine C36/1 se comporte de la meme fagon au cours du deVeloppement pr6coce, mais elle persiste dans la majority des noyaux apres la neurulation, avec un spectre de localisation tres semblable a celui de la proteine A33/22. En outre, la proteine C36/1 est pr^sente au niveau des chromosomes mitotiques. Sa localisation sur les chromosomes mitotiques et lampbrush la rattache aux protdines composant le filament de DNP.

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(Accepted 7 August 1987)