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Previous Article | Next Article 
Molecular and Cellular Biology, April 2002, p. 2220-2228, Vol. 22, No. 7
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.7.2220-2228.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Late Effect Studies, Radiation Biology Center,1 Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501,4 Medical Research Institute, Kanazawa Medical University, Uchinada-machi, Kahoku-gun, Ishikawa 920-02, Japan,2 Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 021153
Received 4 September 2001/ Returned for modification 5 October 2001/ Accepted 20 December 2001
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ABSTRACT |
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INTRODUCTION |
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It has been well documented that embryonic development in mouse is quite sensitive to radiation. A high level of p53 was correlated to radiation induction of apoptosis without cell cycle arrest in post-implantation stage mouse embryos (19, 23). This p53-dependent apoptosis was shown to be the major mechanism to suppress radiation-induced teratogenesis in mice (33). Earlier stages of mouse development, especially from fertilization to implantation, were shown to be highly radiosensitive (14). However, the mechanisms of the high radiosensitivity have been poorly elucidated. Mouse zygotes are known to possess an extremely high level of p53 (21). This indicates that p53 might play an important role in damage response of mouse zygotes.
One difficulty in studying the damage response is that the effect of damage itself and the effect of cellular responses are hard to distinguish, because these two always come together in irradiated cells. In a mammalian zygote, genomes of the incoming sperm and residing oocyte form two separate pronuclei. DNA synthesis occurs in two pronuclei, which thereafter merge to enter M phase. This gives a unique opportunity to analyze the mechanism of damage responses; damage can be delivered through irradiated sperm while the responses can be analyzed in damage-free female pronuclei. Indeed, our previous study clearly demonstrated that introduction of DNA damage by irradiated sperm induces genomic instability which destabilizes a maternally derived minisatellite allele in F1 mice (32).
In the present study, we have investigated the p53-dependent radioresponse in mouse zygotes fertilized by X-irradiated sperm. The results definitely demonstrate p53-dependent pronuclear cross talk in zygotes. In addition, we have found a novel function of p53 in the S-phase DNA damage checkpoint, which suppresses the rate of pronuclear DNA synthesis in mouse zygotes.
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MATERIALS AND METHODS |
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X-irradiation. Partial irradiation was performed on the testicular area of male mice, with a lead shield covering other parts of the body, at a dose rate of 1 Gy/min (250 keV; 15 mA with 1-mm-thick Al filter; Rigaku Radioflex X-ray generator). Irradiation of isolated sperm and zygotes was carried out at room temperature in microdrops of the medium submerged in liquid paraffin. Plasmid DNA was irradiated in a buffer under the X-ray generator without the filter at a dose rate of 30 Gy/min.
In vivo and in vitro fertilization. In vitro fertilization of mouse oocytes and subsequent culture were carried out as described (30, 44). Virgin ICR female mice were induced to superovulate with 5 U of pregnant mare's serum (Teikokuzoukiseiyaku Ltd., Tokyo, Japan), followed 46 to 48 h later by 5 U of human chorionic gonadotropin (Teikokuzoukiseiyaku Ltd.). Female mice were sacrificed at 15 to 16 h after human chorionic gonadotropin administration, and the oocytes were released from the ampullar portion of the oviducts into the medium submerged in equilibrated paraffin oil. Sperm was obtained from the cauda epididymides of male ICR mice and suspended in medium submerged in equilibrated paraffin oil. After capacitation by incubating for 2 h at 37°C under 5% CO2 in an air environment, sperm was added to the oocytes. Zygotes were washed 6 h later and were transferred into fresh microdroplets of culture medium.
For in vivo fertilization, females of the proestrus stage were identified by examination of vaginal smears at 5:00 p.m. These females were caged at 6:00 a.m. the next morning with males for 1 h. Successful mating were identified by the presence of a vaginal plug. Females were sacrificed at 6 h after mating, and the zygotes were collected as described above. Zygotes were washed and transferred into microdroplets of culture medium, the cumulus cells were removed by treatment with hyaluronidase (10 mg/ml; Sigma), and the zygotes were cultured further for investigation.
Plasmid constructions, microinjection, analysis of lacZ expression, and protein purification. A p53-responsive lacZ reporter plasmid was constructed. The HindIII fragment carrying 13 copies of the p53-responsive element (AGGCAAGTCCAGGCAGGCC) and the basic promoter of polyomavirus was isolated from PG13Py-lacZ plasmid (kindly provided by B. Vogelstein [22]). This was inserted in the HindIII site of plasmid pENL (kindly provided by Y. Nabeshima). The resulting plasmid, PG13-Py-NL-lacZ, carried the p53-responsive promoter upstream of the nuclear localizing lacZ.
The plasmid described above was microinjected into the female pronuclei of zygotes at 8 h postfertilization. Male and female pronuclei were distinguished by the size and the position with respect to the polar body. Expression of lacZ was examined at 20 and 24 h after fertilization by fixation with 1% glutaraldehyde and staining with 5-bromo-4-chloro-3-indolyl-ß-D-galactoside. The success rate of microinjection into female pronuclei was around 50%. Some zygotes degenerated after microinjection, possibly due to harsh treatment, and some surviving zygotes did not stain. Therefore, we used at least 30 zygotes for each microinjection experiment. When all the zygotes were devoid of staining, as it was in the case of control zygotes and p53-/- irradiated-sperm zygotes, then this was taken to represent a lack of response. When we saw staining in about 50% of the 30 zygotes, this was taken as a positive response. So, the data obtained here are qualitative rather than quantitative.
A construct was made in which human p53 cDNA was fused downstream of the glutathione S-transferase (GST) gene of pGEX plasmid (18). The plasmid was introduced into Escherichia coli, and the culture was induced with 0.1 mM isopropyl-ß-D-thiogalactopyranoside. The GST-p53 fusion protein was purified with the standard procedure using glutathione agarose beads (39). The purified protein was prepared in a buffer (17) at a concentration of 1 mg/ml and microinjected into the cytoplasm of the zygotes at 4 h after fertilization.
Analysis of pronuclear DNA synthesis. DNA synthesis and S-phase progression were monitored by pulse-labeling with 3H-labeled thymidine ([3H]TdR) (28). Briefly, zygotes were pulse-labeled for 1 h with [3H]TdR (1 µCi/ml) at 6 to 24 h after fertilization and chased with fresh medium containing 5 x 10-5 M cold TdR for another 1 h. The total amount of DNA synthesized was monitored by continuous labeling with [3H]TdR (0.1 µCi/ml) from 8 to 21 h after fertilization for in vitro-fertilized zygotes and from 8 to 18 h for in vivo-fertilized zygotes. After completion of labeling and chasing, zygotes were treated hypotonically in 0.9% sodium citrate and 3% fetal calf serum for 5 min at 37°C and fixed for 5 min in methanol-acetic acid-water (5:1:4, vol/vol/vol). The fixed zygotes were placed onto glass slides and washed with methanol-acetic acid (3:1, vol/vol). Slides were then treated with 5% trichloroacetic acid solution for 1 h at 0°C and were washed with ethanol for 5 min at room temperature. Slides were dipped in Kodak NTB2 emulsion and stored at 4°C for 2 weeks. After development, autoradiographs were stained with Giemsa stain, and the number of grains per nucleus was determined for male and female pronuclei.
Measurement of DNA content. Zygotes were washed with phosphate-buffered saline once and placed onto cover glasses. After fixation with 70% ethanol for 5 min, zygotes were stained with propidium iodide (PI) (0.1 mg/ml) in phosphate-buffered saline containing proteinase K (0.1 mg/ml) and RNase (1 mg/ml) at 37°C for 30 min. The relative intensity of PI fluorescence was measured by laser scanning cytometer (LSC 101; Olympus, Tokyo, Japan). Care was taken to compare the PI intensities of nuclei on the same slide so that the variation in the staining was minimized.
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RESULTS |
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S-phase progression of irradiated-sperm zygotes. S-phase progression in zygotes was examined by pulse-labeling with [3H]TdR of control zygotes and 6-Gy-irradiated-sperm zygotes fertilized in vitro and in vivo. Table 1 shows that pronuclear DNA synthesis was first detected at 8 h after fertilization, both in control and in irradiated-sperm zygotes in vitro. Control zygotes gave the labeling index of 23% at 8 h after fertilization, the value for irradiated sperm was 21%, and there was no arrest at the G1/S border in irradiated-sperm zygotes, even through activation of p53 was evident from Fig. 1b. In order to avoid a possible artifact of in vitro fertilization, we also investigated in vivo-fertilized zygotes recovered from natural mating. Control and irradiated-sperm zygotes recovered this way both entered S phase at 8 h after mating. The labeling index of control zygotes was 44% at 8 h after in vivo fertilization, and the value for irradiated sperm was 45% (Table 1). Direct irradiation of these zygotes with 3 Gy of X rays at 4, 6, and 8 h after mating was not effective in induction of G1/S arrest (data not shown).
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A similar analysis was performed with control and 6-Gy-irradiated-sperm zygotes fertilized in vivo. The duration of S phase in control zygotes was from 8 to 18 h after fertilization in vivo (Table 1). Some of the 6-Gy-irradiated-sperm zygotes exhibited a delay in S phase, and on average the S phase of about a half of these embryos was longer by 2 h than that of control zygotes. These results indicate that irradiation of sperm did not trigger G1/S checkpoint in mouse zygotes but retarded S phase in some.
Suppression of pronuclear DNA synthesis by irradiation to sperm. The rate of DNA synthesized was analyzed by pulse-labeling experiments. The numbers of grains were counted in male and female pronuclei. The results indicated that the rate of DNA synthesis was lower throughout S phase in both of the pronuclei of 6-Gy-irradiated-sperm zygotes fertilized in vitro (Fig. 2). A small but definite delay in S phase was evident for irradiated-sperm zygotes.
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Similar analysis was performed with p21-/- zygotes. The results show that pronuclear DNA synthesis in p21-/- irradiated-sperm zygotes was suppressed as that in wild-type irradiated-sperm zygotes was (Fig. 4). Therefore, suppression of pronuclear DNA synthesis was not dependent on p21.
Dose response of suppression of pronuclear DNA synthesis. The dose response of suppression of pronuclear DNA synthesis was analyzed. Grain count analysis was done after continuous labeling of in vivo-fertilized zygotes from 8 to 18 h after mating. Figure 5 shows the average grain count of female and male pronuclei of p53+/+ and p53-/- zygotes where the male mice were exposed to 1, 2, 3, and 6 Gy of X rays and mated with females immediately. In p53+/+ zygotes, X-irradiation of sperm at doses higher than 1 Gy suppressed grain count in both pronuclei and the grain count decreased dose dependently up to 3 Gy. In contrast, DNA synthesis was not suppressed by sperm irradiation in p53-/- zygotes. Rather, the grain count even increased in these zygotes when spermatozoa were irradiated at doses of 1, 2, and 3 Gy. This increase in p53-/- zygotes was not due to the enhanced DNA synthesis, because DNA content as assessed by the PI value was not affected by sperm irradiation (data not shown). Therefore, the increase was likely due to a decreased nucleotide pool size in irradiated-sperm p53-/- zygotes.
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M phase progression of irradiated-sperm zygotes. M phase progression was examined for in vivo fertilized zygotes by scoring two-cell-stage embryos (Fig. 7a). Control zygotes started to cleave at 17 h and all of them completed the division by 22 h. M phase progression was delayed by about 2 h in irradiated-sperm zygotes. The cleavage was little delayed in irradiated-sperm p53-/- zygotes.
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DISCUSSION |
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The p53-dependent pronuclear cross talk occurs across the cytoplasm. This is in contrast to the nuclear limited activation of the RAD9-dependent damage checkpoint in Saccharomyces cerevisiae (9). Activation of p53 by radiation involves phosphorylation at the serine 15 and serine 20 residues (31). Phosphorylation of the serine 15 site is carried out by ATM, while that of the serine 20 site is carried out by Chk2 (5, 7, 20). It is easily envisaged that DNA damage is detected during decondensation of the sperm head and activates these protein kinases. Phosphorylated p53 would then be distributed in the male pronucleus in cis and in the female pronucleus in trans during pronuclear formation.
It is interesting that the activation was also observed in both p53+/- and p53-/+ irradiated-sperm zygotes, and the latter indicates that the p53 gene product is made from a paternally inherited allele. One-cell-stage transcription of the p53 gene is consistent with the previous report (21).
Lack of G1/S arrest in irradiated-sperm zygotes.
One of the major functions of the p53-dependent damage response is to arrest the cells at the G1/S border. In our experiment, however, the onset of DNA synthesis was not affected by 6-Gy X-irradiation of sperm, even though activation of p53 was evident by the reporter experiment. p53 is known to transactivate p21, which inhibits cyclin D-CDK4/6 complexes to arrest the cells at the G1/S border (31). It was reported that embryonal stem cells and embryonal carcinoma cells lack the G1/S checkpoint even though p53 is highly activated after X-irradiation (37, 40). In a recent study on 
-irradiated F9 embryonal carcinoma cells, p53 was shown to activate transcription of the p21 gene, but a rapid degradation blocked accumulation of p21 protein, leading to elimination of the G1/S checkpoint (29). A similar mechanism may also be responsible for the lack of G1/S checkpoint in mouse zygotes.
p53 dependence of S-phase damage checkpoint in mouse zygotes. Suppression of DNA synthesis is attained by depletion of the pyrimidine nucleotide pool by treatment of the cells with N-(phosphonacetyl)-L-aspartate, and this suppression was reported to be p53 dependent (1). In this study we observed suppression of DNA synthesis in both pronuclei of irradiated-sperm mouse zygotes. The suppression was dependent on the presence of functional p53. Microinjection of the GST-p53 fusion protein restored the suppression in irradiated-sperm p53-/- zygotes. The same treatment did not affect DNA synthesis in control zygotes, showing that the fusion protein by itself had no ability to suppress DNA synthesis in the absence of radiation damage. Thus, the present report is the first to implicate p53 in the S-phase DNA damage checkpoint of mouse zygotes.
It is well known that p21 is transactivated by p53 and arrests somatic cells at the G1/S and G2/M border (6, 10, 11). Lack of G1/S arrest in zygotes may be due to the failure of accumulation of p21, as discussed in the previous section, or p21 may have a different function in mouse zygotes. In our study, suppression of DNA synthesis was observed in p21-/- irradiated-sperm zygotes, indicating that p21 is not involved in the S-phase damage checkpoint. Indeed, S-phase block by hydroxyurea results in accumulation of p53 but not accumulation of p21 (15). Detailed analysis of a series of mutant p53 proteins is in progress to elucidate the domain required for the S-phase damage checkpoint.
The S-phase damage checkpoint operates by two mechanisms: suppression of replication origin firing and suppression of replication fork movement (35). Many of the works on the S-phase DNA damage checkpoint come from the study of S. cerevisiae (34). Suppression of origin firing in hydroxyurea-treated budding yeast was shown to be dependent on Mec1 and Rad53 (36). Involvement of Rad53 in the S-phase damage checkpoint was also reported (38). In higher eukaryotes, the S-phase checkpoint and suppression of replication origin are dependent on the gene product ATM, a mammalian homologue of Mec1, and this has explained the long-known radioresistant DNA synthesis in AT cells (24). The p53 gene product was reported to have no relevance in the ATM-dependent S-phase damage response in many of the cell types studied (25, 43).
Sperm irradiation resulted in suppression of DNA synthesis throughout S phase. This suggested that replication fork movement was likely to be the target of p53-dependent suppression of DNA replication in irradiated-sperm zygotes. Replication machinery of eukaryotes is composed of DNA polymerase A and primase. Replication protein A (RPA) is also required. Mutation of any of these genes results in a defect of the S-phase damage checkpoint (13). DNA polymerase, primase, and RPA form foci in the nucleus during S phase. p53 is known to bind to RPA and suppress its ability to associate with single-stranded DNA (12). It is possible that p53 might suppress replication fork movement by binding to RPA. Indeed, colocalization of RPA and p53 was found in the S-phase nucleus of Xenopus laevis embryos after UV and -irradiation (41).
It has long been assumed that replication fork movement is retarded in irradiated cells because the progression of the replication machinery is hindered at the site of DNA damage. Our present data demonstrated that DNA synthesis was suppressed not only in the damaged male pronucleus but also in the female pronucleus which had no radiation damage. Therefore, suppression of DNA synthesis in mouse zygotes is truly a checkpoint rather than a mechanical block of replication machinery at the damaged site.
The biological function of p53-dependent S-phase damage checkpoint. In vivo fertilization gives better-synchronized zygotes than in vitro fertilization, and this is the reason for the shorter S phase in the former zygotes (Table 1). Delay in S phase can be better demonstrated for in vivo-fertilized zygotes, and about half of the in vivo-fertilized irradiated-sperm zygotes exhibited retardation of S phase by 2 h (Table 1). These delayed zygotes managed to synthesize the normal amount of DNA, while those without delay had less than the normal DNA content (Fig. 7b). Nevertheless, all zygotes went through M phase (Fig. 7a).
Mouse zygotes irradiated prior to pronuclear formation were reported to exhibit no G2/M arrest, while irradiation at later stages led to cleavage block (16). This arrest has recently been shown to correlate with the decreased activity of p34cdc2 (3). Irradiation of sperm is equivalent to irradiation of zygotes prior to pronuclear formation. DNA damage at the pre-pronuclear stage, either from sperm irradiation or from direct irradiation of zygotes, led to activation of the p53-dependent S-phase damage checkpoint in mouse zygotes.
We propose that during the p53-dependent suppression of DNA synthesis, DNA damage is modified in such a way that it is no longer capable of activating the G2/M checkpoint. In contrast to somatic cells, the irradiated-sperm zygotes which fail to synthesis full DNA content still can proceed to G2 and M (Fig. 7b). p53-/- zygotes had no S-phase damage checkpoint, and they also did not show delay in M-phase entry, which is consistent with the requirement of p53 in the G2/M checkpoint (8).
The biological function of the p53-dependent S-phase checkpoint is likely to be one of protecting early-stage mouse development. Our unpublished observation demonstrated that all zygotes produced from 6-Gy-irradiated sperm progressed at least to the eight-cell stage, and half of the fetuses were born normally. This suggests the lack of p53-dependent apoptosis in pre-implantation stage embryos. In contrast, 6-Gy-irradiated-sperm p53-/- zygotes lacking S-phase suppression had a higher frequency of micronucleus occurrence during subsequent cleavages and did not implant. Thus, the p53-dependent S-phase damage checkpoint in mouse zygotes is yet another function of p53 as a guardian of the mammalian genome.
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ACKNOWLEDGMENTS |
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This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The work was also supported by the Nuclear Safety Research Association.
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FOOTNOTES |
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REFERENCES |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Ahmadi, A., and S. C. Ng. 1999. Fertilizing ability of DNA-damaged spermatozoa. J. Exp. Zool. 284:696-704.[CrossRef][Medline]
3. Baatout, S., P. Jacquet, T. Jung, J. Hain, A. Michaux, J. Buset, C. Vandecasteele, L. de Saint-Georges, and L. Baugnet-Mahieu. 1999. Histone H1 kinase activity in one cell mouse embryos blocked in the G2 phase by X-irradiation. Anticancer Res. 19:1093-1100.[Medline]
4. Balhorn, R., B. L. Gledhill, and A. J. Wyrobek. 1977. Mouse sperm chromatin proteins: quantitative isolation and partial characterization. Biochemistry 16:4074-4080.[CrossRef][Medline]
5.
Banin, S., L. Moyal, S.-Y. Shieh, Y. Taya, C. W. Anderson, L. Chessa, N. I. Smorodinsky, C. Prives, Y. Reiss, Y. Shiloh, and Y. Ziv. 1998. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281:1674-1677.
6.
Bunz, F., A. Dutriaux, C. Lengauer, T. Waldman, S. Zhou, J. P. Brown, J. M. Sedivy, K. W. Kinzler, and B. Vogelstein. 1998. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282:1497-1501.
7.
Canman, C. E., D. S. Lim, K. A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi, E. Appella, M. B. Kastan, and J. D. Siliciano. 1998. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281:1677-1679.
8.
Chan, T. A., P. M. Hwang, H. Hermeking, K. W. Kinzler, and B. Vogelstein. 2000. Cooperative effects of genes controlling the G2/M checkpoint. Genes Dev. 14:1584-1588.
9. Demeter, J., S. E. Lee, J. E. Haber, and T. Stearns. 2000. The DNA damage checkpoint signal in budding yeast is nuclear limited. Mol. Cell 6:487-492.[CrossRef][Medline]
10. Deng, C., P. Zhang, J. W. Harper, S. J. Ellege, and P. Leder. 1995. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 82:675-684.[CrossRef][Medline]
11. Dulic, V., W. K. Kaufmann, S. J. Wilson, T. D. Tlsty, E. Lees, J. W. Harper, S. J. Ellege, and S. I. Reed. 1994. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76:1013-1023.[CrossRef][Medline]
12. Dutta, A., J. M. Ruppert, J. C. Aster, and E. Winchester. 1993. Inhibition of DNA replication factor RPA by p53. Nature 365:79-82.[CrossRef][Medline]
13. Foiani, M., A. Pellicioli, M. Lopes, C. Lucca, M. Ferrari, G. Liberi, M. M. Falconi, and P. Plevani. 2000. DNA damage checkpoints and DNA replication controls in Saccharomyces cerevisiae. Mutat. Res. 451:187-196.[Medline]
14. Goldstein, L. S., A. I. Spindle, and R. A. Pedersen. 1975. X-ray sensitivity of the preimplantation mouse embryo in vitro. Radiat. Res. 62:276-287.[Medline]
15. Gottifredi, V., S. Shieh, Y. Taya, and C. Prives. 2001. From the cover: p53 accumulates but is functionally impaired when DNA synthesis is blocked. Proc. Natl. Acad. Sci. USA 30:1036-1041.
16. Grinfeld, S., and P. Jacquet. 1987. An unusual radiation-induced G2 arrest in the zygote of the BALB/c mouse strain. Int. J. Radiat. Biol. 51:353-363.
17.
Gross, S. D., D. P. Hoffman, P. L. Fisette, P. Baas, and R. A. Anderson. 1995. A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins. J. Cell Biol. 130:711-724.
18.
Gu, J., D. Chen, J. Rosenblum, R. Rubin, and Z. M. Yuan. 2000. Identification of a sequence element from p53 that signals for Mdm2-targeted degradation. Mol. Cell. Biol. 20:1243-1253.
19.
Heyer, B. S., A. MacAuley, O. Behrendtsen, and Z. Werb. 2000. Hypersensitivity to DNA damage leads to increased apoptosis during early mouse development. Genes Dev. 14:2072-2084.
20.
Hirao, A., Y.-Y. Kong, S. Matsuoka, A. Wakeham, J. Ruland, H. Yoshida, D. Liu, S. J. Elledge, and T. W. Mak. 2000. DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science 287:1824-1827.
21. Jurisicova, A., K. E. Latham, R. F. Casper, and S. L. Varmuza. 1998. Expression and regulation of genes associated with cell death during murine preimplantation embryo development. Mol. Reprod. Dev. 51:243-253.[CrossRef][Medline]
22.
Kern, S. E., J. A. Pietenpol, S. Thiagalingam, A. Seymour, K. W. Kinzler, and B. Vogelstein. 1992. Oncogenic forms of p53 inhibit p53-regulated gene expression. Science 256:827-829.
23. Komarova, E. A., M. V. Chernov, R. Franks, K. Wang, G. Armin, C. R. Zelnick, D. M. Chin, S. S. Bacus, G. R. Stark, and A. V. Gudkov. 1997. Transgenic mice with p53-responsive lacZ: p53 activity varies dramatically during normal development and determines radiation and drug sensitivity in vivo. EMBO J. 16:1391-1400.[CrossRef][Medline]
24.
Larner, J. M., H. Lee, R. D. Little, P. A. Dijkwel, C. L. Schildkraut, and J. L. Hamlin. 1999. Radiation down-regulates replication origin activity throughout the S phase in mammalian cells. Nucleic Acids Res. 27:803-809.
25.
Lee, H., J. M. Larner, and J. L. Hamlin. 1997. A p53-independent damage-sensing mechanism that functions as a checkpoint at the G1/S transition in Chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA 94:526-531.
26. Levine, A. J. 1997. p53, the cellular gatekeeper for growth and division. Cell 88:323-331.[CrossRef][Medline]
27. Lobrich, M., P. K. Cooper, and B. Rydberg. 1996. Non-random distribution of DNA double-strand breaks induced by particle irradiation. Int. J. Radiat. Biol. 70:493-503.[CrossRef][Medline]
28. Luthardt, F. W., and R. P. Donahue. 1973. Pronuclear DNA synthesis in mouse eggs. Exp. Cell Res. 82:143-151.[CrossRef][Medline]
29. Malashicheva, A. B., T. V. Kislaykova, N. D. Aksenov, K. A. Osipov, and V. A. Pospelov. 2000. F9 embryonal carcinoma cells fail to stop at G1/S boundary of the cell cycle after gamma-irradiation due to p21WAF/CIP degradation. Oncogene 19:3858-3865.[CrossRef][Medline]
30. Matsuda, Y., T. Yamada, and I. Tobari. 1985. Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. I. Chromosome aberrations induced in sperm after X-irradiation. Mutat. Res. 148:113-117.[CrossRef][Medline]
31. May, P., and E. May. 1999. Twenty years of p53 research: structural and functional aspects of the p53 protein. Oncogene 18:7621-7636.[CrossRef][Medline]
32.
Niwa, O., and R. Kominami. 2001. Untargeted mutation of the maternally derived mouse hypervariable minisatellite allele in F1 mice born to irradiated spermatozoa. Proc. Natl. Acad. Sci. USA 98:1705-1710.
33. Norimura, T., S. Nomoto, M. Katsuki, Y. Gondo, and S. Kondo. 1996. p53-dependent apoptosis suppresses radiation-induced teratogenesis. Nat. Med. 2:577-580.[CrossRef][Medline]
34. Paulovich, A. G., and L. H. Hartwell. 1995. A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage. Cell 8:841-847.
35. Rhind, N., and P. Russell. 2000. Checkpoints: it takes more than time to heal some wounds. Curr. Biol. 10:908-911.
36. Santocanale, C., and J. F. X. Diffley. 1998. A Mec1-and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature 395:615-618.[CrossRef][Medline]
37. Schmidt-Kastner, P. K., K. Jardine, M. Cormier, and M. W. McBurney. 1998. Absence of p53-dependent cell cycle regulation in pluripotent mouse cell lines. Oncogene 16:3003-3011.[CrossRef][Medline]
38. Shirahige, K., Y. Hori, K. Shiraishi, M. Yamashita, K. Takahashi, C. Obuse, T. Tsurimoto, and H. Yoshikawa. 1998. Regulation of DNA-replication origins during cell-cycle progression. Nature 395:618-621.[CrossRef][Medline]
39. Smith, D. B., and K. S. Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31-40.[CrossRef][Medline]
40. Taga, M., K. Shiraishi, T. Shimura, N. Uematsu, T. Kato, Y. Nishimune, S. Aizawa, M. Oshimura, and O. Niwa. 2000. The effect of caffeine on p53-dependent radioresponses in undifferentiated mouse embryonal carcinoma cells after X-ray and UV-irradiations. J. Radiat. Res. 41:227-241.
41. Tchang, F., and M. Mechali. 1999. Nuclear import of p53 during Xenopus laevis early development in relation to DNA replication and DNA repair. Exp. Cell Res. 251:46-56.[CrossRef][Medline]
42. Tsukada, T., Y. Tomooka, S. Takai, Y. Ueda, S. I. Nishikawa, T. Yagi, T. Tokunaga, N. Takeda, Y. Suda, S. Abe, I. Matsuo, Y. Ikawa, and S. Aizawa. 1993. Enhanced proliferative potential in culture of cells from p53-deficient mice. Oncogene 8:3313-3322.[Medline]
43. Xie, G., R. C. Habbersett, Y. Jia, S. R. Peterson, B. E. Lehnert, E. M. Bradbury, and J. A. D'Anna. 1998. Requirements for p53 and the ATM gene product in the regulation of G1/S and S phase checkpoints. Oncogene 16:721-736.[CrossRef][Medline]
44.
Yamada, T., O. Yukawa, K. Asami, and T. Nakazawa. 1982. Effect of chronic HTO ß or 60CO radiation on preimplantation mouse development in vitro. Radiat. Res. 92:359-369.[Medline]
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) and female pronuclei (
) of control zygotes exposed to 3 Gy of X rays at 4 h after fertilization. (b) This staining was also observed in 6-Gy-irradiated-sperm zygotes, but the polar body (arrowhead) was not stained. (c) There was no staining in p53-/- irradiated-sperm zygotes. (d) Weak staining was observed in control zygotes microinjected with 3,000-Gy-irradiated reporter plasmid.
—) and female (---
---) and male pronuclei (—
---) (n = 44). (b) The PI fluorescence value in two-cell-stage embryos was measured and plotted for control embryos (open bars) and irradiated-sperm embryos (solid bars). DNA contents in irradiated-sperm early-cleaving embryos (cleaved before 19 h [n = 14]) were less than those in control early-cleaving embryos (n = 14). In contrast, irradiated-sperm late-cleaving embryos (cleaved at 21 h [n = 10]) had the same DNA content as control late-cleaving embryos (n = 14). Error bars, standard deviations.