Radiat Environ Biophys (1986) 25:195-199 Radiation and Environmental Biophysics Springer-Verlag 1986 Change in frequency of radiation induced micronuclei during interphase of four-cell mouse embryos in vitro W.-U. Mfiller and C. Streffer Institut fiir Medizinische Strahlenphysik und Strahlenbiologie, Universit/itsklinikum Essen, Hufelandstrasse 55, D-4300 Essen 1, Federal Republic of Germany Received April 4, 1986 / Accepted April 24, 1986 Summary. Two-cell mouse embryos were X-irradiated (2 Gy) in vitro. Comparison of the frequency of micronuclei in the early interphase of the four-cell stage with that in the late interphase revealed a small, but significant loss of micronuclei during interphase: 27.9 versus 23.4 micro:. nuclei per 100 cells. Introduction Micronuclei are small DNA-positive particles in the cytoplasm which arise from the main nucleus. They can be formed by various processes. After ionizing radiation, the presumably most important one is by formation of acentric fragments, which cannot be distributed to the daughter nuclei because of a lacking centromere (Heddle and Carrano 1977). Micronuclei are also to be expected, if the centromere is present, but damaged (Brenner 1980), or if the spindle apparatus is not working properly (Yamamoto and Kikuchi 1980). All three mechanisms have in common the requirement of a mitosis between induction and expression of micronuclei. However, there are processes that might result in micronuclei without the necessity of a mitosis: after comparatively high doses of ionizing radiation (about 5 to 10 Gy) the formation of nuclear 'buds' has been reported (Wen.dt 1959), which detach from the nucleus after some time and meet all criteria of a micronucleus. With even higher doses a fragmentation of the interphase nucleus (karyorhexis) can be observed. There are no data which allow a decision whether these are high dose phenomena only, or whether some sort of' limited' fragmentation of a cell nucleus during interphase is possible also after doses in the range of a few Gray. Thus, there is a chance of micronuclei being induced and expressed during the same interphase. On the other hand, micronuclei could get lost during interphase due to, for example, enzymatic degradation. Any excessive loss or formation
196 of micronuclei during interphase would have considerable implications for those experiments using micronucleus formation as an indicator of risk exerted by presumed harmful agents. Because in this case the time point of micronucleus determination within interphase would be crucial, in particular for those agents that cause a division delay. Therefore, we studied in which way the frequency of radiation-induced micronuclei changes during interphase. We did this by comparing the number of micronuclei shortly after mitosis with the number shortly before the next mitosis, that is early and late in one and the same interphase. Mouse embryos of the two- and four-cell stage were used as in vitro test system. Mouse embryos develop rather synchronously between the oneand eight-cell stage (Streffer et al. 1980; Molls et al. 1983), so that no manipulations are required for achieving synchronism. Obviously, a synchronous cell population is of great advantage for the type of study outlined above. An additional advantage is the in vitro technique which allows the direct observation of development under a microscope. After induction of micronuclei by X-rays, we found a small, but statistically significant loss of micronuclei during interphase. Materials and methods Details of isolation, culture, and X-irradiation of embryos have been published elsewhere (Mfiller and Streffer 1984). Briefly, two-cell mouse embryos of the G2-phase were isolated about 30 h after conception, irradiated with X-rays (2 Gy) about two hours before mitosis, and checked for development to the four-cell stage every two hours. Four-cell embryos were collected and split up into two groups; one of these groups was analyzed for the number of micronuclei immediately, the other one was allowed to develop close to the mitosis to the eight-cell stage and analyzed then for the number of micronuclei. Micronuclei were determined using the fixation technique of Tarkowski (1966). The embryos were treated with 1% sodium citrate solution for about 1 rain, transferred to a glass slide, and spread by the addition of about 10 gl of fixative solution (acetic acid and ethanol 1 : 3). All particles with the following properties were counted as micronuclei: 1. stainable with ethidium bromide (detected by fluorescence at 530 nm); 2, round-shaped with distinct boundaries; 3. diameter between 5 and 20% that of the main nucleus. Results and discussion An essential point of the experimental design is, that the early group, which is tested for micronuclei, should be separated by an interval as long as possible from the late group; any change in the number of micronuclei should then best be seen. In vitro, the cell cycle of the four-cell stage lasts about twelve hours (Streffer et al. 1980). Taking into account a duration of about two hours for mitosis results in an interval of ten hours for the
interphase. However, there were two problems related to synchronism, which prevented a separation of early and late group by ten hours. On the one hand, synchronism between the embryos got worse as a consequence of the radiation event. Whereas more than 80% of the control embryos entered the four-cell stage within two to three hours, it took irradiated embryos more than ten hours. This problem was easily overcome by collecting the four-cell embryos every second hour. However, there was a certain chance that a four-cell embryo was formed immediately after observation. Therefore, the interval between early and late group had to be shortened by two hours to eight hours. On the other hand, and this problem was much more serious, synchronism within one and the same embryo deteriorated. It is well known that one of the earliest indications of differentiation is a slightly faster development of one of the two cells of the two-cell embryo (Kelly et al. 1978), so that also in controls three-cell embryos are observed for a short time (generally less than one hour). This difference in developmental speed was increased by irradiation for a considerable number of embryos. That is, many of the irradiated embryos remained as three-cell embryos for two to three hours. Thus, two of the cells were already for two to three hours in the interphase of the four-cell stage, when the other two cells entered this stage. Consequently, in order to avoid that two of the cells of the four-cell embryo already started mitosis at the time of micronucleus analysis in the late group, the time of the formation of the three-cell embryo was important and not the time of the formation of the four-cell embryo. Or, using a time of two hours for the duration of the three-cell stage, we could separate early and late group by an interval of six hours, without running into risk that some of the cells already had left the interphase of the four-cell stage. Thus, the final experimental approach looked as follows: Two-cell mouse embryos were X-irradiated in vitro (2 Gy; this dose only delays the formation of four-, eight- and sixteen-cell embryos, but does not prevent their formation); four-cell embryos were collected shortly (at most two hours) after mitosis, and partitioned into two groups; one of these groups was analyzed for the number of micronuclei immediately (early interphase), the other one six hours later (late interphase). Table 1 gives the results of six experiments performed in the way just outlined. Five of the six experiments reveal a lower frequency of micronuclei in late interphase compared with early interphase; only one experiment (no. 5) shows just the opposite. (If the totals reflect the true values, then the probability of an outcome like that of experiment 5 is greater than 0.05; thus, statistically there is no reason not to expect such a result in a series of experiments.) The summary of all six experiments points to a small, but significant loss of micronuclei during interphase: 27.9 micronuclei per 100 cells in early and 23.4 micronuclei per 100 cells in late interphase. This result does not completely rule out the possibility that micronuclei are induced and expressed within one and the same interphase, that is with- 197
198 Table 1. Micronucleus frequency in early and late interphase of the four-cell stage of mouse embryos after induction by X-rays (2 Gy) during two-cell stage. (95% confidence limits are quoted for the totals (Sachs 1984; p. 148). Figures in parentheses: Number of embryos evaluated. The frequency of micronuclei in controls is about 1 per 100 cells) Experiment Micronuclei per 100 cells Early interphase Late interphase 1 29.1 (37) 25.8 (33) 2 32.6 (66) 28.8 (53) 3 29.9 (56) 17.5 (53) 4 25.6 (42) 17.1 (38) 5 21.6 (59) 29.5 (50) 6 27.9 (57) 21.3 (61) Total 25.1 < 27.9 < 31.0 (317) 20.6 < 23.4 < 26.3 (288) (Difference between early and late interphase significant with P <0.05; (Sachs 1984; p. 152). Test compares two Poisson distributions) out the assistance of a mitosis: it could be that the loss of micronuclei is greater than estimated from the difference mentioned above, so that a potential production of micronuclei only during interphase is masked by this effect. However, preliminary experiments (unpublished) indicate that four-ceu embryos, which were X-irradiated (8 Gy) during Gl-phase, do not show micronuclei when examined in G2-phase of the same stage, that is without a mitosis between irradiation and examination. The experimental approach does not answer the question, whether there is a continuous loss of micronuclei or whether this loss is restricted to a certain time during interphase. This uncertainty prevents an extrapolation of the amount of loss per six hours (= time interval of the experiment) to the loss per ten hours (--actual length of the interphase of the four-cell stage); such an extrapolation requires a continuous loss. Thus, we did find a statistically significant loss of micronuclei during interphase of the four-cell mouse embryo; however, this loss was so small that a major confounding effect cannot be expected from this process in studies counting micronuclei at different times in one and the same interphase. However, care must be taken that counting is not performed in different interphases, because further mitoses may change the number of micronuclei due to premature chromosome condensation (Madle et al. 1976) and formation of additional micronuclei (Miiller and Streffer 1984). Acknowledgement. This work was supported by the Bundesministerium des Innern of the Federal Republic of Germany. References Brenner SL (1980) Laser microirradiation of kinetochores in mitotic PtK2 cells: chromatid separation and micronucleus formation. Cell Biophys 2:139-152
Heddle JA, Carrano AV (1977) The DNA content of micronuclei induced in mouse bone marrow by ),-irradiation : Evidence that micronuclei arise from acentric chromosomal fragments. Mutat Res 44: 63-69 Kelly SJ, Mulnard JG, Graham CF (1978) Cell division and cell allocation in early mouse development. J Embryol Exp Morphol 48:37-51 Madle S, Nowak J, Obe G (1976) Effects of inhibitors of DNA, RNA and protein synthesis on frequencies and types of premature chromosome condensation from X-ray induced micronuclei. Hum Genet 34:143-149 Molls M, Zamboglou N, Streffer C (1983) A comparison of the cell kinetics of pre-implantation mouse embryos from two different mouse strains. Cell Tissue Kinet 16:277-283 Mfiller W-U, Streffer C (1984) Distribution of micronuclei among single cells of pre-implantation mouse embryos after X-irradiation in vitro. Murat Res 125:65-70 Sachs L (1984) Angewandte Statistik. Springer, Berlin Heidelberg New York Tokyo, 6th Edn Streffer C, van Beuningen D, Molls M, Zamboglou N, Schulz S (1980) Kinetics of cell proliferation in the preimplanted mouse embryo in vivo and in vitro. Cell Tissue Kinet 13:135-143 Tarkowski AK (1966) An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 5: 394~400 Wendt E (1959) Lebendbeobachtungen an bcstrahlten Interphasekernen. Z Zellforsch 49: 677-689 Yamamoto KI, Kikuchi Y (1980) A comparison of diameters of micronuclei induced by clastogens and by spindle poisons. Murat Res 71 : 127-131 199