Nuclei during Early Development - NCBI

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May 1, 1989 - Institut Jacques Monod, Centre National de la RechercheScientifique, Tour 43,2 place Jussieu, ... and Laboratoire d'Oncologie Moleculaire, Institut National de la Sante et de la ... Institut Pasteur, 52019 Lille Cedex,2 France, and Imperial ...... 1974. The units of DNA replication in Drosophila melanogaster.
Vol. 9, No. 12


0270-7306/89/125395-09$02.00/0 Copyright C 1989, American Society for Microbiology

Translocation of a Store of Maternal Cytoplasmic c-myc Protein into Nuclei during Early Development MICHEL GUSSE,' JACQUES GHYSDAEL,2 GERARD EVAN,3 THIERRY SOUSSI,' AND MARCEL MECHALIl* Institut Jacques Monod, Centre National de la Recherche Scientifique, Tour 43, 2 place Jussieu, 75251 Paris Cedex 05,1 and Laboratoire d'Oncologie Moleculaire, Institut National de la Sante et de la Recherche Medicale-Centre National de la Recherche Scientifique, Institut Pasteur, 52019 Lille Cedex,2 France, and Imperial Cancer Research Fund Laboratories, Dominion House, London ECJA 7BE, England3 Received 1 May 1989/Accepted 3 September 1989

The c-myc proto-oncogene is expressed as a maternal protein during oogenesis in Xenopus laevis, namely, in nondividing cells. A delayed translation of c-myc mRNA accumulated in early oocytes results in the accumulation of the protein during late oogenesis. The oocyte c-myc protein is unusually stable and is located in the cytoplasm, contrasting with its features in somatic cels. A mature oocyte contains a maternal c-myc protein stockpile of 4 x 105 to 6 x 105 times the level in a somatic growing cell. This level of c-myc protein is preserved only during the cleavage stage of the embryo. Fertilization triggers its rapid migration into the nuclei of the cleaving embryo and a change in the phosphorylation state of the protein. The c-myc protein content per nucleus decreases exponentially during the cleavage stage until a stoichiometric titration by the embryonic nuclei is reached during a 0.5-h period at the midblastula stage. Most of the maternal c-myc store is degraded by the gastrula stage. These observations implicate the participation of c-myc in the events linked to early embryonic development and the midblastula transition.

achieved. We also observed a change in the phosphorylation state of the protein after fertilization. Most of this maternal store is degraded in postgastrula embryos. These data indicated an unusual stability and regulation of c-myc during the cleavage stage which implies an important function in early development and the midblastula transition (MBT).

The involvement of the proto-oncogene c-myc in cellular immortalization has been well documented and results from an abnormal regulation of the gene (see reference 10 for a review). Although c-myc expression has been well correlated with cell proliferation (10, 29), the function of the c-myc protein is not well understood. Its localization in the nucleus of somatic cells (2, 21, 22, 40), its short half-life, and its partial homology to the adenovirus Ela protein suggest an involvement in regulation of gene expression (5). Its participation in DNA synthesis has also been suggested (27, 28, 43), but these data were recently debated (19, 20, 42a). Because of similarities often observed between neoplastic cells and embryonic cells, it has been widely suggested that proto-oncogenes have major developmental functions (1, 6). However, most of the studies of c-myc expression and function in normal cells have been performed in cell culture (10). c-myc expression has been analyzed in a few cases at the RNA level during embryonic development in postgastrula embryos. A high level of c-myc expression was detected in human placenta (41) and during murine development (46). We recently analyzed c-myc expression at the RNA level during early embryonic development in Xenopus laevis, a vertebrate whose development is well characterized. c-myc is expressed as a stable maternal mRNA in oocytes and reaches a level 105-fold the level found in proliferative somatic cells (25, 30, 44). Two causes for this unusual amplification of c-myc RNA are a total uncoupling of c-myc expression from cell division and a stabilization of the RNA product during oogenesis (44). We report here the expression and fate of c-myc protein during the process of oogenesis and embryonic development. We show that a c-myc protein stockpile is accumulated in late oogenesis and maintained only during early development. The c-myc protein is stored in the cytoplasm of oocytes and is translocated into the nuclei from fertilization to the midblastula-gastrula stage, when a nearly complete titration by the nuclei is *

MATERIALS AND METHODS Xenopus cells and embryos. Collection of oocytes and eggs and [35S]methionine labeling were as previously described (44). Embryonic stages are given according to Nieuwkoop and Faber (35). Unfertilized eggs were activated in 0.2 ,ug of calcium ionophore A23187 (Sigma Chemical Co., St. Louis, Mo.) per ml for 5 min. For 32p labeling, oocytes, eggs, or embryos were microinjected with 1 ,uCi of [_y-32P]ATP in 50 nl of 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid) (pH 7.5) and incubated in lx modified Barth saline HEPES (MBSH) (18) for oocytes or 0.8x MBSH-2% Ficoll for eggs and embryos. For pulse-chase experiments, oocytes were incubated for 12 h in 2 mCi of [35S]methionine per ml and then chased for different times in MBSH containing 2 mM unlabeled methionine and 250 ,ug of cycloheximide per ml. In parallel experiments, 1.5 ,uCi of [35S]methionine was injected into the oocytes, which were further incubated for 12 to 14 h in MBSH. Then 25 nl of a solution containing 8.5 mg of cycloheximide per ml and 0.2 mM unlabeled methionine was injected, and the incubation continued for different times. Identical results were obtained in these two kinds of pulsechase experiments. Protein labeling in the Xenopuis A6 cell line was by the addition of 400 ,uCi of [35S]methionine (Dupont, NEN Research Products, Boston, Mass.) or 500 ,uCi of 32p (Commissariat a l'Energie Atomique) for 2 h. For pulse-chase analysis, cells were labeled for 1 h with 400 ,uCi of [35S]methionine per dish followed by various periods of chase in medium containing 25 mM unlabeled methionine. Isolation of nuclei of oocytes and early embryos. The cytoplasmic and germinal vesicle fractions from 50 oocytes

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FIG. 1. Quantification and stability of Xenopus c-myc protein. (A) Total proteins from 4 x 105 (lane 1) or 2 x 105 (lane 2) Xenopus A6 cells in the exponential phase of growth were fractionated on an SDS-polyacrylamide gel, together with proteins extracted from a single stage VI oocyte (lane 3). Immunoblotting was as described in Materials and Methods. Minor bands observed in A6 cell fractions are not related to c-myc as they were not abolished by preincubation of the anti-c-myc antibody with the antigenic c-myc peptide (unpublished observations). (B) Stage VI oocytes were injected with 250 ,ug of cycloheximide per ml, and proteins were extracted and analyzed by immunoblotting after 0 (lane 1), 2 (lane 2), 4 (lane 3), 6 (lane 4), and 18 (lane 5) h at 20°C. Numbers show molecular size in kilodaltons. (C) Cultures of Xenopus A6 cells were pulse-labeled for 1 h with 200 ,uCi of [35S]methionine per ml and chased with unlabeled methionine for 0 min (lane 2), 30 min (lane 3), 1 h (lane 4), 2 h (lane 5), 4 h (lane 6), and 6 h (lane 7) at 22°C. After immunoprecipitation, the products were separated on an SDS-polyacrylamide gel. Lane 1 is after immunoprecipitation with a preimune serum.

obtained after manual enucleation (18). Nuclei and strictly corresponding cytoplasmic fractions were extracted as described previously (44). For each stage of embryonic development studied, 100 dejellied embryos were pretreated by incubation in 0.8 x MBSH-2% Ficoll-250 p.g of cycloheximide per ml. After 1 h at 20°C, nuclei were prepared by slight modifications of a method previously described (15). Extraction of proteins and immunological methods. The efficiency of extraction of proteins from oocytes and embryos was assayed by different procedures (unpublished data), and the protocol previously described (44) was found to be more efficient. The anti-c-myc antibodies were two rabbit polyclonal antisera against a polypeptide domain corresponding to the last 50 residues encoded by exon 3 of human c-myc (16) and against a 12-residue peptide localized in exon 2 that is entirely conserved in c-myc proteins from different species, including Xenopus c-myc protein (34). Protein extraction and immunodetection were as described previously (44). In some experiments, the immunoblot was incubated with the anti-c-myc antibody, followed by 1251_ labeled protein A (0.2 ,uCi/ml, 37 mCi/mg). After washing, the c-myc protein was localized by autoradiography and the labeled bands were excised from the blot and counted. Specificity was controlled by incubation of the anti-c-myc antibody with the corresponding antigenic peptide (16). In situ localization. Embryos for immunohistological analysis were fixed in paraformaldehyde and embedded in esterwax (Giuvv, BDH), and 7-,um sections were prepared as described previously (25). Slides were treated for 20 min in 0.5% H202 in methanol to reduce endogenic peroxidase activity. Aspecific sites were blocked by incubation for 30 min in phosphate-buffered saline containing 1% bovine serum albumin and 0.2% Tween 20. Sections were incubated with anti-c-myc antibody overnight. Control sections were incubated with the incubation buffer only or with anti-c-myc antibody previously incubated with the corresponding antigen. After three washes in phosphate-buffered saline, slides were incubated with anti-rabbit immunoglobulin biotinylated antibody and streptavidin biotinylated horseradish peroxidase complex (Amersham Corp., Arlington Heights, Ill.). Peroxidase activity was detected by diaminobenzidin (DAB) or a DAB enhancement kit (Amersham).


RESULTS The Xenopus c-myc protein is accumulated as a stable product during oogenesis. We previously characterized Xenopus c-myc (25, 44) and showed that the c-myc mRNA encoded for a protein migrating at 60 kilodaltons on sodium dodecyl sulfate (SDS)-acrylamide gels (44; M. Gusse, unpublished data). c-myc protein was analyzed with anti-c-myc antibodies raised against c-myc peptides (Materials and Methods). As an estimate of the amount of c-myc protein stored in a mature oocyte, we compared the signal selected with one oocyte with the signal obtained with definite numbers of growing Xenopus somatic cells in culture. The amount of c-myc protein stored in one oocyte was equivalent to the amount of c-myc protein found in 4 x 105 to 6 x 105 growing somatic cells (Fig. 1A). Overexpression of the c-myc protein product was not previously reported as there is no substantial difference in the level of c-myc expression in normal versus tumor cells (10). Significantly, the high level of the c-myc protein in a mature oocyte correlated with the accumulation of c-myc RNA during oogenesis (44). As an unusual stability might explain the accumulation of c-myc protein in oocytes, its turnover was compared in oocytes and in somatic cells. Oocytes were injected with cycloheximide at concentrations (250 ,ug/ml) which completely inhibit protein synthesis (24; M. Gusse and M. Mechali, unpublished data). The c-myc protein store did not vary significantly after up to 18 h in the presence of cycloheximide (Fig. 1B). Identical results were obtained by measuring the half-life of c-myc protein after [35S]methionine pulses followed by chases (Materials and Methods). In contrast, a half-life of 20 min was measured with Xenopus A6 somatic cells (Fig. 1C). This value was as previously observed in this cell (30) or other somatic cells (22, 23). Thus, the accumulation of c-myc protein in a mature oocyte is at least in part due to its unusual stability in this germ cell. c-myc protein accumulates in late oogensis and was maintained at a high level only during the early cleavage stage in embryogenesis. Total extracts from oocytes and from embryos at different developmental stages were analyzed for their c-myc protein content by immunoblotting (Fig. 2A). Immunoblots were developed with 1251-labeled protein A to


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Embryonic Development Oogenesis FIG. 2. Expression of c-myc protein during oogenesis and embryonic development. (A) Total extracts equivalent to two oocytes or embryos were run on SDS-109o polyacrylamide gels and further processed by immunoblotting and 251I-protein A labeling. Different stages of oocyte growth are according to Dumont (14). (B) After autoradiography, the c-myc bands were excised and their radioactivity was determined by scintillation counting. The value obtained in the unfertilized egg (260,000 cpm) is 100%o. The amount of c-myc RNA per oocyte or embryo was determined from Northern (RNA) blots as previously described (17). The value obtained in the unfertilized egg (8 pg) is 100%o.

increase the sensitivity of the assay and to enable a direct quantification (Materials and Methods). Despite the high level of c-myc RNA found in young stage I and II oocytes (44), c-myc p60 was not detectable before stage III and accumulated to its maximum level in mature stage VI oocytes. We cannot formally exclude that undetected levels of c-myc protein might be expressed during early oogenesis. However, for the following reasons, we conclude that a dramatic increase in c-myc protein synthesis occurs during late oogenesis. First, total proteins accumulated in the oocyte were analyzed in these experiments, as opposed to 35S-labeled proteins, which limits the observation to the time course of labeling. Second, the elapsed time during oogenesis until stage II is reached (9 months) should be sufficient to permit translation of a specific product. When actual translation of c-myc protein was assayed by [35S]methionine labeling, synthesis of c-myc protein was not detected before stage III. Third, both c-myc RNA and protein are unusually

stable during oogenesis (44; data described above). Thus, the failure to detect c-myc protein in early oogenesis was probably not due to a rapid turnover of the protein but most likely reflected the absence of translation of the stored c-myc RNA. Maturation of the oocyte into an egg did not change the amount of c-myc protein stored. After fertilization, the store of c-myc protein remained constant during the entire cleavage stage period and was rapidly degraded from the gastrula stage. Within 2 h, the c-myc protein level fell to about 24% of its value during the cleavage stage. Figure 2B shows the level of c-myc protein compared with the level of c-myc RNA (44) during development. The accumulation of a maternal c-myc RNA pool during early oogenesis was followed by an accumulation of c-myc protein during late oogenesis. Fertilization rapidly triggered the degradation of the stored c-myc RNA, whereas the protein pool was preserved during the entire cell cleavage period, a time of transcriptional






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FIG. 3. Localization of c-myc protein in oocytes. Stage VI oocytes were enucleated, and the total extracts from 50 oocytes, 50 cytoplasms, or 50 nuclei were prepared. Extracts equivalent to 1 oocyte (lane 1), 1 cytoplasm (lane 2), 1 nucleus (lane 3), or 12 nuclei (lane 4) were run on an SDS-polyacrylamide gel. The gel was analyzed by immunoblotting followed by 125I-protein A labeling and autoradiography. Numbers on right show molecular size in kilodaltons.

quiescence (4, 37). During gastrulation, the c-myc protein level fell rapidly and reached a minimum by the tailbud stage. This level was maintained during further embryonic development, probably by the new c-myc transcription beginning with the neurula stage (44). We noted in four independent experiments that the amount of c-myc protein did not significantly change after the tailbud stage, although the c-myc RNA level increased roughly in proportion to the number of cells in the embryo. From these data, it was

apparent that both posttranscriptional and posttranslational regulation were responsible for restricting a high level of c-myc protein to the time of early embryonic development. The Xenopus c-myc protein is stored in the cytoplasm of oocytes but translocates into nuclei after fertilization. c-myc is a nuclear proto-oncogene. Its protein product has been located in the nucleus in all cell systems examined so far, including neoplastic cells (2, 21, 22, 40). The localization of c-myc protein in Xenopus oocytes was examined by two procedures. Manual enucleation showed that more than 99% of the c-myc p60 protein was in the cytoplasm (Fig. 3). A minor band migrating below c-myc p60 was often observed in Western blot (immunoblot) analysis. This band was probably not related to c-myc as it did not disappear after preincubation of the antibody with the peptide used as the antigen; it was not observed in immunoprecipitation experiments. The cellular location of c-myc protein was confirmed by immunostaining of thin sections of ovaries with a DAB silver enhancement protocol designed to increase the sensitivity of the assay. No signal was observed in young oocytes (Fig. 4A). In contrast, a strong signal was detected in the nuclei of the surrounding proliferating follicle cells (18). Stages II oocytes also did not react with anti-c-myc antibody (Fig. 4B), but in later stage V oocytes a strong signal was observed over the cytoplasm of the cell (Fig. 4C and D). The protein was not detected in the nuclei, in agreement with the immunoblot results (Fig. 3). In Fig. 4E, the anti-c-myc antibody was first preincubated with the c-myc peptide used as an antigen and then incubated with a stage V oocyte section. No signal was detected, confirming the specificity of the reaction observed in situ. In summary, both biochemical

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FIG. 4. In situ immunolocalization of c-myc protein in oocytes. Immunolocalization with c-myc antibody was done on oocyte cut sections as described in Materials and Methods. (A) Stage I oocyte surrounded by its layer of follicle cells (arrow). (B) Stage II oocyte. The nucleus (germinal vesicle) is indicating by an arrow. (C) Stage V oocyte. Arrow shows nucleus. (D) Enlargement of panel C at the cytoplasm-germinal vesicle border. The dark peroxidase reaction products are localized in the cytoplasmic fraction between the yolk platelets. The arrow shows the germinal vesicle membrane. (E) Cut section of a stage V oocyte probed with the anti-c-myc antibody previously preincubated with the polypeptide used as the antigen. The germinal vesicle (gv) and cytoplasmic yolk platelets (y) are indicated by arrows. A DAB silver enhancement kit (Amersham) was used to increase the sensitivity of the assay.


VOL. 9, 1989

and immunocytological examinations showed that c-myc protein is uniformely distributed in the cytoplasm of mature oocytes, in contrast to the result observed in other cell types. The presence of a stockpile of c-myc protein in the cytoplasm of oocytes might be due to a negtive posttranslational regulation adapted to early embryonic development. During oogenesis, the oocyte grows without division and accumulates components necessary for the cell divisions induced by fertilization (see reference 13 for a review). The stockpiling of c-myc in the cytoplasm of oocytes may be necessary to avoid activation of its function in oocyte nuclei. In such a case, c-myc protein was expected to translocate from the cytoplasm to the nuclei after fertilization. Figure SA to D shows that from fertilization c-myc protein becomes predominantly found in the nuclei. During the early cleavage stage, the egg divides every 30 min with no Gl and G2 periods, and so observation of interphase nuclei is rare. Accumulation of c-myc into nuclei was first observed during the formation of nuclei in rapidly dividing blastomeres (Fig. SA and B). At the gastrula-neurula stage, almost all the c-myc protein was located in the nuclei although the cytoplasm of epidermic cells still contained some c-myc protein (Fig. SE and F). These data strongly suggested a functional activation of c-myc by the migration of the protein from the cytoplasm to the nuclei, induced by fertilization. The observations of Fig. 5A and B also indicated that c-myc protein is gathered during the formation of the nuclei. We quantitated the c-myc protein in the cytoplasm or in the nuclei of developing embryos by cell fractionation followed by immunoblotting. As cell division occurs every 30 min during early development (37), the isolation of interphase nuclei became a major problem. To overcome this difficulty, we incubated developing embryos with cycloheximide for 1 h before extraction. This treatment arrests the embryonic cell cycle at the end of the S phase and enables nuclei to be harvested in a G2-like state (33) (Fig. 6A). Figure 6B shows the fraction of c-myc protein in the cytoplasm and nuclei of the cleaving blastomeres at different stages of early development and is the mean value of three independent experiments. The amount of c-myc protein transferred to the nuclei was not a simple function of the number of cells in the dividing embryo. Instead, most of the c-myc protein was rapidly sequestered in the nuclei, beginning with the very early cleavage divisions. Then, during the following cleavages, the proportion of c-myc protein found in the nuclei increased slowly. This nonlinear relationship between the proportion of c-myc protein found in the nucleus and the number of embryonic cells was reproducibly observed. Titration of the cytoplasmic c-myc protein pool by the replicating nuclei reached an asymptote 7 to 8 h postfertilization, a time precisely corresponding to the MBT in X.

laevis (37). In Fig.

6C, the proportion of the total


protein store per mean individual embryonic nucleus is shown at different embryonic cleavage stages. After a rapid increase, the amount of c-myc protein per nucleus decreased exponentially until an asymptotic level was reached within 30 min near the MBT stage (4,000 to 8,000 cells). Thus, the level of c-myc protein stored in the unfertilized egg is such that after a rapid saturation of the nuclei each mean individual nucleus become progressively depleted of c-myc protein until the equilibrium is reached at the MBT. Change in phosphorylation state of c-myc protein after fertilization. The presence of c-myc protein in the cytoplasm of oocytes and in the nuclei of cleaving blastomeres suggests possible posttranslational modifications responsible for the


translocation. The level of phosphorylation of c-myc protein was investigated by microinjection of [_y-32P]ATP into oocytes, eggs, or embryos (Fig. 7). Phosphorylation of c-myc protein was not detected in oocytes incubated for 6 h or for 24 h in the presence of [_y-32P]ATP. However, a signal was always detected in fertilized eggs after 6 h of incubation or in proliferating Xenopus A6 cells (Fig. 7, lanes 4 and 5). A weaker signal was also observed in unfertilized eggs activated by a calcium ionophore (A23187) which causes abortive cleavage and fewer nuclear divisions. In all cases, the phosphorylation signal was linked with proliferation. An oocyte or a cleaving embryo contains 3 to 4 mM ATP (18), a level that hinders high-specificity isotope labeling. Thus, phosphorylation occurring on a minor part of the c-myc protein would not have been detected. However, the phosphorylation signal observed in embryos, as opposed to the absence of signal in oocytes, was obtained in four independent experiments. As the amount of nucleoside triphosphate, including ATP, does not significantly change during development (11, 31), we conclude that a change in the phosphorylation state of the c-myc protein store occurs after fertilization. DISCUSSION In contrast to the correlation between cell proliferation and c-myc expression, the developmental regulation of cmyc is characterized by an unusual uncoupling of c-myc expression from cell division. This uncoupling occurs in discrete steps, beginning with the transcription of the gene in the nondividing maternal germ cell to the migration of its protein product in the nucleus after fertilization. Developmental regulation of maternal c-myc store. In the Xenopus oocyte, the combination of the stability of c-myc RNA (44) and protein (Fig.1) and the long period of oocyte growth leads to the accumulation of a large store of c-myc maternal products. The delayed translation of c-myc RNA to the late stages of oogenesis indicates that it must be sequestered and stored in a translationally inactive form during early oogenesis, as observed for other oocyte mRNAs (42). Thus, at least three factors contribute to the accumulation of the c-myc protein store: a large RNA pool, a long period of translation from stage IV to the mature oocyte, and the unusual stability of the protein product compared with that in somatic cells. A mature oocyte or an egg contains 1 x 105_ to 5 x 105-fold the content of c-myc mRNA and protein in somatic cells. As the size of an egg is around 105 times the size of a somatic cell, the concentration of c-myc mRNA is not amplified but the ratio of c-myc protein per genome number is. The amplification of the c-myc protein level in oocytes shows that c-myc expression per se is not sufficient for cell proliferation. It might be related to the accumulation of enzymes and factors needed for future embryonic cell division (13). The unusual cytoplasmic location of the maternal c-myc protein is probably not due to the lack of nuclear targeting elements. A sequence in human c-myc protein which might be necessary for its transport into the nucleus (12) is conserved in the Xenopus c-myc protein. The storage of c-myc protein in the cytoplasm might be due to a block in a posttranslational modification of the protein necessary for its transport into the nuclei. Alternatively, a positive regulation such as the sequestration of the protein in an oocyte cytoplasmic complex might prevent its migration into the nucleus. In proliferating cells, the phosphorylation of c-myc protein remains unchanged throughout the cell cycle (23). In




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FIG. 5. In situ localization of c-myc protein in embroys. Immunolocalizations with anti-c-myc antibody were done by a peroxidase-DAB reaction. (A) One blastomere of a four-cell embryo. The arrow shows the formation of the nucleus. (B) Cut section of a morula. The arrow shows an individual blastomere. (C) Young blastula. The animal part is shown. (D) Enlargment of a young blastula showing strong c-myc-positive signals in the nuclei of individual blastomeres. (E) Young gastrula. The nuclei from the layer of ectodermic cells as well as from the larger endodermic cell underneath reacted with the c-myc antibody (arrows). (F) Late gastrula. The nuclei from ectodermic, mesodermic, and endodermic cells are labeled (arrows). the oocyte, we could not detect phosphorylation of c-myc protein, in contrast to early embryos. King et al. (30) detected 32p incorporation in oocyte c-myc protein, but they could not detect oocyte c-myc protein labeled by

[35S]methionine; we have no explanation for these different results. In any case, a significant change in the phosphorylation level of the c-myc protein clearly occurs after fertilization.


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FIG. 6. Variation in the level of cytoplasmic and nuclear c-myc protein during early development. (A) Hoechst dye examination of embryo nuclei purified as described in Materials and Methods. (B) Nuclear and cytoplasmic extracts were processed from 100 embryos at each stage, and the equivalent of 10 embryos were electrophoresed in an SDS-polyacrylamide gel. After immunoblotting and 125I-protein A labeling, the bands were counted as described in the legend to Fig. 2. The values are from three independent experiments with a variation scale of 2.4 to 12% for each experimental value. The total amount of c-myc protein in the cytoplasmic and nuclear fractions was similar to the amount of c-myc protein in an unfractionated extract (4 to 10%o variation). The value at the one-cell stage was from oocytes, as an unfertilized egg does not have a nuclear membrane. Symbols: 0, percentage of radioactivity in the nuclear fraction; O, percentage of radioactivity in the cytoplasmic fraction. (C) Fraction of total c-myc per nucleus as a function of the number of cells at each developmental stage, from the oocyte to a stage 44 tadpole (106 cells). The values were directly calculated from panel B.

Fertilization activates a further step in the regulation of the rapid passage of the protein from the cytoplasm to the nuclei. A posttranslational mechanism may contribute to this transfer, which is not proportional to cell number but appears to be a hyperbolic function. As fertilization triggers cell division, it is tempting to speculate that cell division is promoted by the entry of c-myc protein into the nuclei. Moreover, the high level of the protein might explain the elevated rate of nuclear division during early development. c-myc and early development. During the early cleavage there is no significant transcription until the MBT when an c-myc:

abrupt and concerted transition in cell behavior occurs within 1 h (4, 37). The synchronous cell cleavage changes to a slower asynchronous cleavage with the first appearance of the Gi and G2 phases. At the same time, there is activation of RNA transcription and cell motility (37). The onset of this new developmental program might be due to depletion or titration of limiting DNA-binding components present in the unfertilized egg as suggested by Newport and Kirschner (38). The experiments described in this report indicate that the developmental regulation of c-myc has all the characteristics of a gene involved in the MBT. Both c-myc mRNA and











replication during early development is explained by an increase of the number of initiation points for DNA replication (8, 9), the control of MBT might be related to a component(s) controlling initiation of DNA replication. Whether c-myc is involved in such a process might deserve further investigation.


FIG. 7. Detection of 32P-labeled c-myc protein in oocytes or embryos. Oocytes, eggs, or embryos were injected with 1 ,Ci of [y-32P]ATP and precipitated with anti-c-myc antibody. The precipitates were analyzed on SDS-polyacrylamide gels. Lane 1, Oocytes incubated for 6 h; lane 2, oocytes incubated for 24 h; lane 3, activated eggs incubated for 6 h; lane 4, fertilized eggs incubated for 6 h. Xenopus A6 cells were labeled for 2 h with 500 pLCi of 32p, and the cell extract was immunoprecipitated with either a control serum (lane 5) or anti-c-myc antibody (lane 6). Numbers on right show molecular size in kilodaltons.

protein are expressed in the unfertilized egg at levels 105-fold greater than in dividing somatic cells. Thus, a store of c-myc protein is available for early embryonic development. The maternal c-myc RNA pool is degraded at fertilization, but the maternal c-myc protein pool is maintained constant during the entire cleavage period. The ratio of c-myc protein content to genomic DNA is therefore exponentially decreasing during the cleavage period. The c-myc protein is localized in the cytoplasm of the mature oocyte, but fertilization triggers its rapid accumulation in the nuclei of the dividing cells. The rapid acquisition by the nuclei of a maternal cytoplasmic protein and the high DNA-binding affinity of c-myc (45) indicate an active process of titration by the nuclei of a cytoplasmic component. Finally, most of the store of c-myc protein is degraded within 2 h after the cleavage stage. Both in vivo (17, 24, 32) and in vitro (3, 7, 26, 36) observations with Xenopus eggs showed that a large fraction of DNA injected into Xenopus eggs was immediately replicated and assembled into nuclei. In all cases, the amount of DNA which would have replicated, or nuclei which would have been formed, largely exceeds the genome equivalent duplicated during the same period of time in development. Thus, machinery for the initiation of nuclear replication is already present in the unfertilized egg and there is no progressive unmasking of the capability for nuclear replication during cell cleavage. However, a factor within the egg becomes progressively limiting for replication, from 10 to 50 ng of injected DNA, the approximate genome equivalent at the MTB (32, 39). We observed that the amount of c-myc protein entering the nuclei does not increase in proportion to cell number during the cleavage stage. Instead, a rapid entry of the majority of the c-myc protein store was observed (Fig. 6). Thus, saturation of the nuclei by c-myc protein is likely to occur rapidly after fertilization. The saturation of DNA by a component necessary for nuclear replication might permit an accelerated rate of nuclear division during the early cleavage stage. Subsequently, the amount of c-myc protein per nucleus decreases exponentially, and it is striking that the equlibrium is reached within a short time (30 min), precisely at the MBT. Since the accelerated rate of chromosomal


We thank Yannick Anddol (this laboratory) for preparations of exponential cultures of A6 cells. We also thank Peter Brooks for critical reading of this manuscript and R. Schwartzmann for the photographs. This work was supported by grants from the Association pour la Recherche sur le Cancer, INSERM, and the Ligue Nationale Franqaise contre le Cancer.

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