/. Embryo!, exp. Morph. Vol. 29, 3, pp. 601-615, 1973 601 Printed in Great Britain Abnormal development of pre-implantation mouse embryos grown in vitro with [3H]thymidine By M. H. L. SNOW1 From the Institute of Animal Genetics, Edinburgh SUMMARY Mouse embryos were grown in vitro from the 2-cell stage to blastocysts in the presence of [3H]thymidine. Methyl-T-thymidine and thymidine-6-T(n) were used and both forms found to be lethal at concentrations above 01 ^Ci/ml. Both forms of [3H]Tdr at concentrations between 0-01 and 01 /tCi/ml caused a highly significant (P < 0001) reduction in blastocyst cell number. The reduction in cell number, which was positively correlated with specific activity and tritium concentration, was associated with cell damage typical of radiation damage caused by tritium disintegration. Thymidine-6-T(n) also significantly reduced the number of 2-cell embryos forming blastocysts whereas methyl-T-Tdr did not. This difference in effect is assumed to be caused by contamination of one form of [3H]Tdr with a by-product of the tritiation process. A study of the cleavage stages showed that almost all the reduction in cell numbers could be accounted for by selective cell death occurring at the 16-cell stage. Cells which survive that stage cleave at a normal rate. The cells that are most susceptible to [3H]Tdr damage were found to normally contribute to the inner cell mass. The [3H]Tdr-resistant cells form the trophoblast. It is possible to grow blastocysts in [3H]Tdr such that they contain no inner cell mass but are composed entirely of trophoblast. Comparatively short (12 h) incubation with [3H]Tdr at any stage prior to the 16-cell stage will cause this damage. Possible reasons for this differential effect are discussed, and also compared with damage caused by X-irradiation. INTRODUCTION It is well documented that thymidine can inhibit cell proliferation almost completely if supplied in excessive concentrations. In murine tissue cultures the range of concentrations which is inhibitory is similar for most cell types, interference with growth first appearing between 10~5 and 10~4 M and being complete above 10"4 M (see Cleaver, 1967, for discussion and references). Ten Broeck (1968) has shown that cleavage-stage mouse embryos tolerate a greater range of thymidine concentrations in vitro but that development is impaired above 10 4 M. The use of isotopically labelled thymidine, potentially more informative, carries the added hazard of possible radiobiological effects. Although the majority of studies involving labelled thymidine utilize low concentrations and/ or short incubation times there are many investigations in which large quantities of labelled nucleoside and long incubation times would be advantageous. It is 1 Author's address: Institute of Animal Genetics, West Mains Road, Edinburgh, EH9 3JN, U.K. 39-2 602 M. H. L. SNOW in these experiments in particular that radiation damage may become a problem. The effects of radioactive disintegrations of 3 H or 14C in labelled thymidine may cause growth delay (Post & Hoffman, 1965,1967,1968,1971), chromosomal aberrations (Wimber, 1959; Brewen & Olivieri, 1966), mutations (Bateman & Chandley, 1962) and cell death (Whitmore & Gulyas, 1966; Apelgot, 1966). Further description, references and discussion can be found in Cleaver (1967). Nucleic acid synthesis has been studied in cleavage stages of mouse embryos (Mintz, 1964, 1965; Monesi & Salfi, 1967; Ellem & Gwatkin, 1968; Woodland & Graham, 1969; Piko, 1970). These investigations have involved short incubations, in the region of 6 h or less, with labelled uridine or labelled thymidine and have studied a variety of cell stages during cleavage. Notwithstanding the extensive literature describing radiobiological effects of isotopically labelled nucleosides, none of these studies investigated the normality of the embryos with respect to the long-term effect of their various experimental procedures. Some unpublished data gathered in this laboratory in 1966 by Caroline Naysmith indicate that long term culture with [3H]thymidine can be damaging to mouse embryos. The following report describes experiments designed initially to corroborate Naysmith's observations and which were subsequently extended to a detailed examination of the effects of long- and short-duration incubations of [3H]thymidine upon cleaving mouse embryos. MATERIALS AND METHODS All mice belonged to the randomly bred Q strain and were housed at 22 + 1 X under a natural diurnal light cycle. Two-cell embryos were flushed from the oviducts 36-38 h post-ovulation, i.e. between 1500 and 1600h on the second day of pregnancy (vaginal plug found on the first day), and cultured in microdrops under liquid paraffin (Brinster, 1963). They were cultured in Brinster's (1965) medium modified according to Bowman & McLaren (1970), incubated at 36 °C and gassed with 10% CO2 in air. [3H]thymidine ([3H]Tdr) in solution in distilled water, was added to the medium to give a range of tritium concentrations. The maximum dilution of the culture medium was 1 %, which has no discernible effect upon embryo growth. [3H]Tdr was obtained from the RadioChemical Centre, Amersham, namely Tdr-6-T(n) at specific activities of 5-0 and 21-6 Ci/mM and methyl-T-Tdr of specific activities 17-4 Ci/mM and 27-0 Ci/mM. At concentrations of 1-0 ju Ci/ml these yield total thymidine concentrations of 2xlO~ 7 , 5xlO~ 8 , 6 x l 0 ~ s and 4 X 1 0 ~ 8 M respectively. Unlabelled thymidine was obtained from the Sigma Chemical Co. Ltd. The effects of experimental conditions were measured in two ways. First, the number of 2-cell embryos producing expanded blastocysts after 68-70 h culture was taken as an indication of the developmental potential of the 2-cell embryo. Secondly, as an indication of the normality of development, the number of normal cells in these blastocysts was ascertained by counting normal Effect of [3H]thymidine on mouse embryos 603 Table 1. The effect of Tdr-6-T(n) (sp. act. 5-0 Cijnm) and methyl-T-Tdr (sp. act. 17-4 Ci/nnr) upon the development of blastocysts from 2-cell embryos (Data pooled from 5 replicate experiments.) Thymidine-6-T(n) Methyl-T-thymidine r Treatment C/'Ci/ml) 0 001 0025 005 01 Mean angular response (±S.E.) Blastocyst mean cell no. (±S.E.) Mean angular response (±S.E.) Blastocyst mean cell no. (±S.E.) 58-9 ±8-2 51-3 ±3-9 43-6± 12-4 38-0 ±2-4 13-7± 5-8 51-7± 5-7 32-9 + 3-2 21-0±3-4 160 ±0-98 120 ±2-4 67-5 ±6-5 690 ±3-2 68-7 ±6-1 78-4 ± 6 0 60-7 ±3-6 61-9 ±6-4 40-3 ± 4 0 33-1 ±3-9 30-3 ±2-5 16-9 ±2-5 nuclei in preparations fixed with 3:1 ethanol/acetic acid and air-dried according to the technique described by Tarkowski (1966). For expanded blastocysts the hypotonic treatment was carried out in 0-75 % sodium citrate for about 10 min. Preparations were stained either with lacto-acetic orcein or Giemsa. RESULTS Naysmith's unpublished data showed that 2-cell embryos cultured in 0-1 /*Ci/ ml Tdr-6-T(n) (specific activity 28-0 Ci/mM) fail to complete more than one cleavage. Of 262 2-cell embryos about 90 % cleaved once but none progressed farther. At concentrations between 005 and 0-025 /^Ci/ml some 75 % of embryos formed morphologically normal morulae but only 15 % expanded into blastocysts compared with control figures of 68 %. The cell number of the blastocysts was not determined. Tdr-6-T(n), specific activity 5-0 Ci/mM and methyl-T-Tdr, specific activity 17-4 Ci/mM, were used to corroborate Naysmith's data. Each precursor was tested in five replicate experiments comparing development in medium containing 0, 001, 0-025, 0-05 and 0-1 /iCi/ml [3H]Tdr. The data were submitted to an analysis of variance. There was no significant variation (P> 0-2) between replicates and the data were therefore pooled. They are shown in Table 1. It is clear from these data that both forms of [3H]Tdr, when used in this range of concentrations, exert a harmful effect upon the development of 2-cell embryos. Fewer embryos expand to form blastocysts in Tdr-6-T(n) and those forming blastocysts have a reduced cell number. The regressions for both criteria against concentration are highly significant (P < 0-001). Methyl-T-Tdr does not interfere with blastocyst formation but the regression of blastocyst cell number against concentration is again highly significant (P < 0-001). These data are plotted graphically in Fig. 1. There is a significant difference between the effect 604 M. H. L. SNOW 1-90 - 0 001 0025 1 50 005 fi Ci/ml [3H]thymidine Fig. 1. The growth of 2-cell embryos in [3H]Tdr. x, • , Methyl-T-Tdr; O, A, thymidine-6-T(n). , Regression of the number of 2-cell embryos forming blastocysts; —, regression of blastocyst cell number. of methyl-T-Tdr and Tdr-6-T(n) on blastocyst formation (P < 0-01) but not for the effect upon cell number. A possible cause for the low degree of blastocyst formation in Tdr-6-T(n) lay in the difference in the specific activities of the two forms of [3H]Tdr. This possibility was tested by varying the specific activity of the nucleoside and also by testing the effect of unlabelled thymidine in this culture system. The specific activity was altered by varying the concentration of unlabelled thymidine in the culture drops while maintaining the tritium concentration at a constant level. The results of these investigations are shown in Table 2. It is immediately clear that lowering the specific activity of methyl-T-Tdr does not reduce the number of blastocysts produced from 2-cell embryos over the range 270000-027/^Ci/mM. It was also found that using high specific activity (21-6 Ci/mM) Tdr-6-T(n) does not significantly increase the yield of blastocysts. From Table 2 it can be seen that high concentrations of thymidine do not interfere with blastocyst formation, but do result in depression in cell number. The data in Table 2 indicate that specific activity has an important bearing upon the depression in cell number. This suggests that the mechanism causing the reduction in cell number is associated with cell bound or incorporated [3H]Tdr. It seems that lowering the specific activity causes a lessening of the radiation toxicity until a point is reached where thymidine poisoning becomes the major consideration. This is clear in Fig. 2, where data from Table 2 and some unpublished data of Naysmith's have been plotted graphically. Apart from the reduction in the number of blastocysts formed in Tdr-6-T(n), Effect of [3H]thymidine on mouse embryos 605 Table 2. The effect of specific activity of methyl-T-Tdr and of unlabelled thymidine upon the development of blastocysts from 2-cell embryos Methyl-T-Tdr (6 replicates) Unlabelled Tdr (3 replicates) •\ (M) Sp. act. (mCi/mM) Mean angular response (±S.E.) 0 2x 10 ° 2x 10 s 2x10 7 2x10 ° 2 x 10 r> 2x10 * 2x 10 3 .—. 27000 27000 27000 2700 2-70 0-27 0027 73-8 + 5-5 62-4 ±4-2 76-0 ±4-7 72-6 ± 9 0 70-1 ±1-5 63-5 ±3-7 68-8 ±3-3 621 ±4-6 Tdr cone. Blastocyst mean cell no. (±S.E.) Mean angular response (±S.E.) Blastocyst mean cell no. (±S.E.) 60-3 ±7-4 37-4 ±3-2 40-6 ±1-4 43-0 ±2-0 48-2 ±5-2 35-6±2-6 30-5 ±0-7 251+0-5 71-1+6-2 — 450±4-3 — 69-8 ± 5 1 71-5 ±3-7 72-4 ±2-7 74-3 ±2-7 41-8 ±8-9 • — • 390±l-9 — 39-5 ±2-7 30-2 ±2-8 23-3 ±1-6 25-2 ±1-6 Thymidine concentration ( M ) 100 io io -•' io 6 io 5 io 10 0-27 0027 90 80 70 60 50 40 27 000 2700 270 270 2-70 Specific activity (mCi/mM) Fig. 2. The effect of unlabelled thymidine concentration (O—O) and of varying the specific activity of methyl-T-Tdr at 0 0 5 /<Ci/ml ( x — x ) . 606 M. H. L. SNOW Effect of^H]thymidine on mouse embryos 607 most of the embryos grown in concentrations as high as 0-025 /tCi/ml Tdr-6-T(n) and 0-05//Ci/ml methyl-T-Tdr look normal when viewed with a dissecting microscope (Fig. 3). None show a conspicuous inner cell mass after 68 h in culture but this feature is not uncommon in untreated cultured blastocysts. Some cultured blastocysts were transferred to pseudopregnant foster mothers to investigate their further development. Fifty-three control blastocysts, 44 grown in 0-025 //Ci/ml and 83 grown in 0-05 //Ci/ml methyl-T-Tdr yielded 36, 24 and 55 implantation sites respectively. Sections through uteri fixed about 24 h after transfer show blastocysts in the process of implanting. Fig. 4 shows a section through an untreated blastocyst apparently with a small inner cell mass, but one which is actually composed of 43 cells. (The number of cells was determined from camera lucida drawings of serially sectioned blastocysts.) Sections cut through [3H]Tdr treated material clearly show that about half the blastocysts grown in 0-025 //Ci/ml methyl-T-Tdr have an inner cell mass reduced to two or three cells (instead of about one-third to one-half of the number of cells in the blastocyst). The remainder of the blastocysts and about 80 % of those grown in 0-05 //Ci/ml have no inner cell mass (Figs. 5, 6). Air-dried preparations of blastocysts reveal many abnormal, misshapen nuclei, and some nuclear fragments, broken chromosomes and fragmented metaphase plates, in those cultured in 0-05 //Ci/ml methyl-T-Tdr (Figs. 7, 8). An analysis of well-displayed, complete and partial metaphase plates from this material shows a large increase in the number of chromosome breaks (Fig. 9) and gaps in [3H]Tdr-treated embryos. In a total of 1212 chromosomes from untreated blastocysts only 1 break and 1 gap were encountered, whereas in 818 chromosomes from 0-05 //Ci/ml [3H]Tdr-cultured blastocysts 19 breaks and 8 gaps were found. It should be noted, however, that these chromosome aberrations may be the result of the airdrying technique upon blastocysts which are damaged in other respects. They FIGURES 3-10 Fig. 3. Blastocysts grown in 005/iCi/ml methyl-T-Tdr. Fig. 4. Section through an untreated, cultured blastocyst apparently with a small inner cell mass, but one which actually consisted of 43 cells. Blastocyst transferred to a pseudo-pregnant female. Scale as in Fig. 3. Figs. 5, 6. Section through [3H]Tdr-treated (005/tCi/ml) blastocysts showing the absence of any inner cell mass. Blastocysts transferred to pseudo-pregnant females. Scale as in Fig. 3. Fig. 7. Part of an air-dried preparation of an untreated blastocyst. Fig. 8. An air-dried preparation of a blastocyst grown in 005 /tCi/ml methyl-T-Tdr. Note the misshapen nuclei, numerous nuclear fragments (arrows) and 'pulverized' metaphase plates (P). Scale as in Fig. 3. Fig. 9. A partial metaphase plate from a preparation similar to that in Fig. 8. Note the broken chromosomes (arrow). Fig. 10. An embryo at the '16-cell' stage grown in 005/*Ci/ml methyl-T-Tdr. Lobed nuclei and nuclear fragments are already evident. Scale as in Fig. 3. 608 M. H. L. SNOW Table 3. The course of development of 2-cell embryos grown in vitro with 0-05 juCi/ml methyl-T-Tdr for various periods of time (Data pooled from 12 experiments (cell numbers ±S.E.).) Time in methyl-T-Tdr (h) • A 0-12 Time Control 0-24 0-60 0-36 0-84 A A A A. in f i ^ r f A K ( ^ cul- No. Mean No. Mean No. Mean No. Mean No. Mean No. Mean cell cell ememture ememcell emcell cell cell em(h) bryos no. bryos no. bryos no. bryos no. bryos no. bryos no. 0 165 12 161 24 42 36 48 60 48 34 61 20 40 6-3 ±0-8 15-4 ±0-7 24-6 ±10 520 66 66 30 10 15-6 ±1-3 350 — ±4-9 — — 20 40 61 ±0-8 120 ±11 15-1 ±1-9 27-2 ±2-6 — 22 73-4 66 66 30 — — — — ±0-9 10 11-9 10 ±11 10 ±2-9 72 20 40 61 10 10 66 64 60 — 20 40 6-6 ±0-7 13-6 ±1-4 14-4 ±2-3 27-5 ±1-7 __ —. — — •— 20 10 10 66 66 66 20 10 20 40 6-6 ±0-7 13 6 ±1-4 15-7 199 160 — 20 40 44 15 6 ±0-9 15-3 ±0-4 26-6 ±1-8 37-4 27 ±2-1 10 23-7 ±1-7 51 51 ±3-5 84 20 113-3 ±7-1 ±3-7 — 21 65-6 ±10-2 therefore may not be present in situ. Blastocysts grown in 0-1 /*Ci/ml [3H]Tdr are usually small, always misshapen with no inner cell mass and yield badly damaged nuclei in air-dried preparations. In contrast, blastocysts with cell numbers reduced by growth in unlabelled thymidine usually have a conspicuous inner cell mass and do not show damaged nuclei or chromosomes when air-dried. The mitotic index was taken as an indication of cleavage rate in the blastocysts. In methyl-T-Tdr this figure did not differ significantly from the control value of 0-023 at any concentration. In Tdr-6-T(n) there was a significant reduction in mitotic index in concentrations of 0-05 and 0-1 /^Ci/ml (0-015 and 0-013 respectively). This suggests that concentrations of Tdr-6-T(n) above 0-05 /<Ci/ml reduce cleavage rate by prolonging interphase or by arresting the cell cycle in interphase. In an investigation into the manner in which the cell number is reduced, 2-cell embryos were grown to blastocysts in 0-05 /*Ci/ml methyl-T-Tdr (a concentration that effectively destroys the inner cell mass and halves cell number). Samples of embryos were taken at various intervals and their cell numbers determined. Analysis of ten experiments indicates that development under Effect of[3H]thymidine on mouse embryos 609 20 1-9 1-8 1-7 1-6 1-5 J-4 1-3 1-2 H 10 30 40 50 60 70 Time in culture (h) 80 90 Fig. 11. The course of development of 2-cell embryos in 005/tCi/ml methyl-T-Tdr. O—O, Control; x — x , [3H]Tdr. control and experimental conditions is synchronous up to the 16-cell stage but at that point embryos in [3H]Tdr fail to increase their cell number for some 1012 h. It is during this period that fragmented and lobed nuclei can first be detected cytologically (Fig. 10). Many (up to 50%) of the nuclei and mitotic figures seen in these preparations are so abnormal in appearance that it is extremely doubtful if the cells from which they came would have developed normally. Air-dried preparations of damaged embryos at subsequent stages in development (e.g. Fig. 8) contain evidence of some fragmented nuclei and 'pulverized chromosomes' (see Ikeucki, Weinfeld & Sandberg, 1972) from dead cells. When normal embryos are at the 32-cell stage these experimentally damaged embryos contain between 16 and 18 cells. Thereafter the experimental embryos cleave at a rate very similar to controls. The developmental progress of these [3H]Tdr-damaged embryos is consistent with the theory that half of the cells of a 16-cell embryo die but that the remaining eight blastomeres cleave at their normal rate, uninterrupted in any way. These results are shown in Table 3 and Fig. 11. A possible explanation for cell death occurring at the 16-cell stage was accumulation of [3H]Tdr within the cell during the preceding two cleavages. Three experiments were carried out with 8-cell embryos grown in 0-05 /tCi/ml methylT-Tdr. In all three experiments a sharp reduction in cell number was again observed to occur at the 16- to 32-cell stage, although these embryos have undergone only one DNA replication phase and not two as in the previous experiments. 610 M. H. L. SNOW 1-8 1-7 1-6 1-5 1-4 1-3 g, 1-2 11 10 0-9 0-8 0-7 20 30 40 Time in culture (h) 50 60 Fig. 12. Effect of shorter incubations in 005 /iCi/ml methyl-T-Tdr. x — x , Control; O—O, 0-12 h; # — • , 0-24 h; A—A, 0-36 h; A—A, 0-60 h. One further experiment was done in which 2-cell embryos were grown in 0-05 /tCi/ml methyl-T-Tdr for 12, 24 or 36 h, then transferred to fresh medium not containing [3H]Tdr. The results of this experiment are also shown in Table 3 and in Fig. 12. There is clear evidence for interference with development at the 16-cell stage even in embryos exposed to [3H]Tdr for only 12 h (a time which included the 4-cell S phase). The decline in cell number is more marked in those embryos grown for 24, 36 or 60 h in [3H]Tdr. X-irradiation can cause chromosome damage and cell death. For comparison with the [3H]Tdr data 101 8- or 16-cell embryos were irradiated with 50, 100, 200 or 400 R of X-rays (R = 2-58 x 10~4 Ckg" 1 ). Blastocyst formation after these treatments was unaffected (in total 73/101 formed blastocysts). Cell number was slightly reduced in most cases but the reduction does not appear to be dose-dependent over the range investigated (mean cell number of controls 51-1 ±3-2, X-irradiated 38-5 ±3-3). Nor were cells lost preferentially from the inner cell mass. A number of subnuclei (nuclear fragments) were invariably found in air-dried preparations but no chromosome damage was observed in the metaphase plates present. Effect of[3H]thymidine on mouse embryos 611 DISCUSSION It is clear from these data that the interference with embryo development by [ H]Tdr, at low concentrations of thymidine, is primarily a radiobiological effect associated with tritium concentration. The increased incidence of chromosome damage, and the fact that cell death at the 16-cell stage can be induced by a 12 h incubation in [3H]Tdr some time prior to that stage, support the contention that incorporated [3H]Tdr is the important factor. This conclusion is reinforced by the correlation between specific activity and the reduction in cell number in the blastocyst (see Table 2). The data show clearly that radiation damage causing cell death is alleviated by competition from unlabelled thymidine. Unlabelled thymidine becomes toxic at concentrations above 10~ 5 M (Table 2) but blastocysts grown in such concentrations (and up to 10~3 M) do not show evidence of cell death, nor do they lose their inner cell mass. In these cases cleavage rate appears to have been depressed giving rise to small but morphologically normal blastocysts. In apparent contradiction to the bulk of the data are the observations on blastocysts development in Tdr-6-T(n). Although the blastocyst cell number is not significantly different from that observed in the similar concentrations of methyl-T-Tdr, the number of 2-cell embryos actually forming blastocysts is greatly reduced with increasing Tdr-6-T(n) concentration. Two possible explanations seem likely. First that the position of the isotope in the thymidine molecule has a specific biological effect and secondly that some other product in Tdr-6-T(n), either a by-product of synthesis or a breakdown product, is toxic. It is apparent, from the data presented above, that the interference with blastocyst formation by Tdr-6-T(n) is not correlated with specific activity, which indicates that this effect at least is not due to [3H]Tdr incorporated into DNA. Nor can it be incorporated into any other molecule since that incorporation should also be affected by changes in specific activity. Although the effects of [3H]Tdr can be simulated by 3H-labelled water provided the concentration and specific activity within the nucleus are comparable (Wimber, 1964; Bond & Feinendegen, 1965), it is difficult to envisage an isotope position effect that would act independently of specific activity. With regard to toxic substances other than Tdr-6-T(n), radiolytic degradation products can immediately be ruled out. All stocks of [3H]Tdr were kept refrigerated at 2 °C, under conditions in which radiolytic breakdown was some 1-2% per month (Evans, 1966). The process is autocatalytic with thymine a major product. However, degradation products of all kinds should increase in concentration with time. New stocks of Tdr-6-T(n) and stocks some 9 months old gave similar results. The older stocks may have been more than 20% degraded and to contain 10-15 times the concentration of degradation products. Substances other than [3H]Tdr introduced during the tritiation process cannot be similarly eliminated. Both methyl-T-Tdr and Tdr-6-T(n) are produced 3 612 M. H. L. SNOW 3 enzymically from [ H]thymine, and although the chemical analyses carried out by the Radiochemical Centre, Amersham, indicate a high degree of purity they do not rule out the possibility of a finite amount of contaminant related to tritium concentration being present in one form of [3H]Tdr and not the other. Such a contaminant would act on a system other than the thymidine incorporation pathway and be independent of thymidine concentration (and hence specific activity). This would appear to be the most likely reason for the difference in the effect on blastocyst formation between the two forms of [3H]Tdr. The contaminant may be in Tdr-6-T(n) and may act to prevent the trophoblast from transporting water into a blastocoele. Alternatively, there may be some contaminant in methyl-T-Tdr which serves to alleviate this aspect of [3H]Tdr poisoning. Whatever the mechanism may be that differentiates between two populations of cells in this system, it is noteworthy that the effect of [3H]Tdr should be expressed particularly at the 16-cell stage. Autoradiographs of air-dried blastocysts grown in 0001 /tCi/ml methyl-T-Tdr show a number of nuclei within the blastocyst much more heavily labelled than the remainder. In extreme cases there may be a fourfold difference in the number of silver grains over these nuclei. Such a difference can be explained in two ways. First there could be an acceleration in DNA replication in the more heavily labelled cells such that their S phases (or at least one S phase) are shorter. They consequently could utilize more [3H]Tdr while the precursor pool is large. This phenomenon could occur without alteration to the total cell cycle time. Callan (1972) has recently shown, in some Amphibia, that variations between tissues and species in S phase duration are associated not with an altered speed of DNA replication (in //m/min) but with a change in the length of replicon available for duplication resulting in an increase in the number of replicating units present in the genome at any one time. Such a variation in replicon length could explain the difference in labelling index of some cells reported here and by Graham (1971), and have an important bearing upon differentiation processes. Secondly, the more heavily labelled cells could have undergone a greater number of S phases. Graham (1971) has reported a similar difference in the labelling index of nuclei within a blastocyst and locates the more heavily labelled nuclei within the inner cell mass. It seems likely that the cells damaged by [3H]Tdr represent one or other of these classes since autoradiographs of blastocysts with reduced cell number show more uniformly labelled nuclei. The reduction of inner cell mass in blastocysts with reduced cell numbers is in itself not surprising. Tarkowski (1959) and Tarkowski & Wroblewska (1967) demonstrate that blastocysts developing from single blastomeres of the 2- and 4-cell embryo range in appearance from 'normal' with as few as eight cells, to 'blastocysts' composed entirely of 'trophoblast', although containing 20 or more cells. These studies indicate that this is not a regular feature but that it is unusual for blastocysts produced from half or quarter embryos to lack entirely Effect of [3H]thymidine on mouse embryos 613 an inner cell mass. Blastocysts grown in 0-05 /iCi/ml methyl-T-Tdr totally lack an inner cell mass in about 80 % of cases even though many of these are composed of 30-40 cells and some contain over 50. It seems most likely that the cells of these 'blastocysts' are unable to form inner cell mass. An analysis of the further development of these radiation-damaged blastocysts has been carried out and shows quite clearly that the cells are capable of further differentiation, but only as invasive trophoblast cells with no trace of foetal tissue (Snow 1973). Preimplantation mouse embryos have previously been found to be sensitive to low doses of X-irradiation in vivo. Russell (1950, 1954, 1956) established that irradiation of embryos between sperm penetration and implantation resulted in more embryonic mortality than irradiation of subsequent stages. Russell & Montgomery (1965) and Russell (1965) demonstrate times of high sensitivity to X-rays (5-200 R) during the first 2 days post ovulation. The general conclusion from these papers is that most of the embryonic mortality induced by X-rays occurs at or after implantation. It is, however, worth while considering some of the associated phenomena described by Russell (1965). Irradiation of early embryos in vivo during the sensitive periods resulted in a high proportion of embryos with cells containing 'subnuclei'. These subnuclei appear very similar to the nuclear fragments found in air-dried preparations of [3H]Tdr-treated blastocysts. It seems probable that these subnuclei contain chromatin (they are labelled in autoradiographs) and may be associated with radiation-induced chromosome l o s s - a phenomenon discussed by Russell (1961) and Russell & Saylors (1963). It is possible that the scarcity of complete metaphase plates after [3H]Tdr treatment is associated with chromosome loss. In comparison to [3H]Tdr-damaged blastocysts X-irradiated embryos appear little damaged and certainly have not lost a large number of cells from their inner cell mass. 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