Effects of in vitro radiocobalt irradiation of rabbit ova on subsequent development in vivo with special reference to the irradiation of maternal organism.код для вставкиСкачать
Effects of in Vitro Radiocobalt Irradiation of Rabbit Ova on Subsequent Development in Vivo with Special Reference to the Irradiation of Maternal Organism’ M. C. C H A N G AND D. M. HUNT Worcester Foundation f o r Experimental Biology, Shrewsbury, Massachusetts, a n d Department of Biology, Boston University, Boston, Massaschusetts The peculiar sensitivity of the embryo and fetus to ionizing radiations has been reviewed by O’Brien (’56) and Rugh (’59). Rugh (’59) stated that this is due to “the high preponderance of cells in transition from the primitive to the differentiated state,” and that “such interference can result from damage to cells clear back to the original fertilized egg.” In their reviews most literature cited is on lower vertebrates, rats and mice. There is practically no work done on rabbits. Previous studies (Chang et al., ’57, ’58) have shown that irradiation of rabbit spermatozoa or of unfertilized ova in vitro at 32,000 to 65,000 r has no severe damage on their capacity of fertilization, that irradiation of unfertilized ova in vitro at 800 r prohibits their subsequent normal development in vivo following fertilization, and that irradiation of two- to 4-celled fertilized ova or of spermatozoa at 800 r reduces the normal development of embryos to 1 0 % . It is obvious that irradiation of mature germ cells in vitro apparently only manifests its harmful effect on the subsequent development of ova and that fertilized ova appear to be more radioresistant than unfertilized ova. The following experiment was performed to determine (1) whether or not there is a differential radiosensitivity of fertilized rabbit ova at various stages of development before implantation, ( 2 ) whether abnormal development could be induced by irradiation of ova at various stages and ( 3 ) whether there is a differential effect of irradiation in the ova alone, recipient females alone (without irradiation of the ova), or in pregnant ani- mals at the corresponding stages for irradiation of ova and of recipient animals. METHODS A N D PROCEDURES Adult female rabbits of mixed breeds purchased from local farms were kept in separate cages in the laboratory for about three weeks before use. Four female rabbits at a time were bred twice by fertile males or were artificially inseminated. These served as donors of ova. At the same time another two rabbits were injected with sheep pituitary extract intravenously to induce ovulation and were used as recipients of ova. One, two, 4, or 6 days after insemination the donor rabbits were sacrificed and their ova were recovered either from the Fallopian tubes (one or two days after insemination) or from uteri ( 4 or 6 days after insemination) by Aushing these organs with 50% fresh rabbit serum in saline. The ova were divided into 4 groups and placed into small Pyrex tubes, 1 cm in diameter, containing 1 ml of diluted serum. These 4 tubes were then exposed simultaneously to a radiocobalt source at distances according to the dosages required. Immediately after irradiation, the ova were transplanted either into the Fallopian tube or into the uterus of a recipient animal through a flank incision under Nembutal anesthesia. As a rule, ova irradiated at one dose level were transferred into the left side and ova irradiated at another dose level were transferred into the right side of the same animal. DelThis study was supported by the Atomic Energy Commission under contract AT( 30-1)1943. 511 512 M. C . CHANG AND D. M. HUNT tailed methods for the recovery and transfer of fertilized ova have been described elsewhere (Chang, '50). The recipient rabbits were sacrificed 22-28 days after transplantation and the number of fetuses were counted, measured, and examined externally and internally. The maternal or fetal placentae in the uteri were also carefully noted to ascertain the degree of degeneration after implantation. In the study of the irradiation of recipient animals alone, the whole body of an animal was exposed to a radiocobalt source at 400 r two, 4, or 6 days after the induction of ovulation. Four to 5 hours after irradiation, non-irradiated ova of corresponding post-ovulation age were transplanted into the irradiated recipients. Ten-thousand units of penicillin was given daily for three days to prevent infection. They were examined 22 to 28 days after operation. Whole body irradiation at 400 r of another group of animals was performed two, 4 or 6 days after artificial insemination. Twenty-two to 28 days later they were sacrificed and their conceptuses were similarly examined. For the cytological study of the irradiated blastocysts, late blastocysts recovered from the uterus 6 days after mating were irradiated at 100, 1,000 and 10,000 r. Soon after irradiation one group of blastocysts were fixed, spread according to Chang ('54) and then stained by the Feulgen method and another group of blaystocysts were cultured in Carrel flasks with 50% of fresh rabbit serum for 2-3 hours and then fixed and stained. RESULTS Irradiation of ova in vitro at dose levels f r o m 50 t o 5,000 r Table 1 summarizes the results when rabbit ova, recovered one day (at 2-4 cell stage), two days (at morula stage), 4 days (at early blastocyst stage) or 6 days (at late blastocyst stage) after insemination were irradiated at 50, 100, 1,000 or 5,000 r and then transplanted into the Fallopian TABLE 1 Effects of in vitro irradiation of rabbit ova on subsequent in vivo development ( 5 0 r to 5,000r) Dosages Age of ova at irrad. r day No. of recipient rabbits No. of ova transplanted fetuses No, % Fetal placentae No. % Maternal placentae No. Total implantation % No. % ~ 50 100 1' 2 4 6 Total 5 3 3 3 14 61 21 12 21 115 28 45.9 33.4 83.45 28.8 44.4 2 0 0 7 9 3.3 0 0 33.3 7.8 1 1.6 0 0 0 0 4 19.0 5 4.3 31 7 10 6 51 7 10 17 65 50.8 33.3 83.0 80.9 56.5 12 6 3 3 3 15 77 21 16 21 135 18 10 11 6 45 23.4 47.6 68.75 28.8 33.3 2 0 0 5 7 2.6 0 0 23.8 5.2 0 0 0 1 1 0 0 0 4.8 0.7 20 10 11 12 53 26.0 47.6 68.7 57.1 39.2 3 3 3 3 12 41 22 17 25 105 5 3 0 12.2 13.6 0 2 0 0 4.9 0 0 0 0 0 0 8 3 0 3 8 7.6 2 1.9 1 2.4 0 0 0 0 3 12.0 4 3.8 14 19.5 13.6 0 12.0 13.3 3 3 3 3 12 44 24 19 26 113 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 4 6 Total 1000 13 2 4 6 Total 5000 14 2 4 6 Total 0 0 0 0 'At 45 r. At 90 r. At 800 r. 4 A t 6,500 r. These data are derived from a previous experiment (Chang et al., '58). 51n these experiments the best blastocysts were selected for transfer, see text. 0 DEVELOPMENT OF IRRADIATED OVA tubes or uteri of recipient rabbits, The recipient rabbits, were made pseudopregnant a t a stage corresponding to the age of the ova at the time of transplantation, by injection of pituitary extract one, two, 4 or 6 days previously. The efficacy of ova transplantation in the rabbit has been determined previously (Chang, ’ 5 0 ) and it is known that about 54% of one-day ova and about 42% of 4-day or 6-day ova will develop into normal young when transplanted into the proper female tract a t the corresponding stage of pseudopregnancy. From the results presented in table 1, the percentage of normal development following the irradiation of ova at 50 r was 46 in the case of one-day ova and 29 in the case of 6-day ova. The percentage of normal development following the irradiation at 100 r was 23 in the case of one-day ova and 29 in the case of 6-day ova. From these figures it appears that there may be a harmful effect even after the irradiation of ova in uitro at 50 r. Following irradiation at 100 r the percentage of normal development for one-day ova was lower (23% ) than at 50 r (46% ) but the percentages of normal development in the cases of ova of other ages were irregular; thus whether there is a differential effect between 50 r and 100 r is uncertain. As for the extremely high percentage of normal development in the case of irradiation of 4-day ova at 50 r or at 100 r, this is probably due to the fact that most of the 4-day ova shrunk during manipulation and only the best and the largest blastocysts were transferred following irradiation in this study. Following irradiation at 1,000 r , only 12 to 14% of one-day or two-day ova, but none of the 4-day or 6-day ova, developed into normal fetuses, indicating that at 1,000 r, the younger ova are slightly more radioresistant than the older ones. Degeneration after implantation, however, was observed in the irradiation of 6-day ova as shown by the presence of maternal placentae. At a dose level of 5,000 r neither normal development nor evidence of implantation was observed in any case, showing that a dose of 5,000 r is definitely lethal to rabbit ova at all stages before implantation. O/O 80 -0 0 6 0 - 513 514 M. C . CHANG A N D D. M. HUNT after insemination were irradiated at 200, 400, 600 or 800 r and then transplanted into the female tracts of rabbits pseudopregnant at the appropriate stage. Table 2 and figure 2 present the results. From the results in table 2 it can be seen that follow- ing the irradiation of ova at 200 r, there is no significant difference in the proportion of normal fetal development between ova at various stages of development. At a dose level of 400 r the proportion of normal fetal developemnt in the case of 6-day TABLE 2 Effects of in vitro irradiation of rabbit ovu o n subsequent in vivo development (200 r to 800 r ) - Dosages ~ Age of ova at irrad. Fetal placentae 9 6 8 23 62 31 39 132 22 9 14 45 35.5 29.0 35.9 34.1 7 11.3 2 6.5 3 7.7 12 9.1 2 3.2 5 16.1 9 23.1 16 12.1 31 16 26 73 7 6 9 22 51 33 47 131 15 8 24 47 29.4 24.2 51.1 35.9 2 3.9 3 9.1 9 19.2 14 10.6 3 5.9 8 24.2 3 6.4 14 10.6 20 8 5 8 21 58 28 41 127 ia 31.0 2 7.1 4 9.8 24 18.9 5 8.6 3 10.7 6 14.6 14 11.0 1 1.7 5 17.8 8 19.5 14 11.0 24 10 18 52 41.4 35.7 43.9 40.9 8 5 8 21 57 29 42 128 5 8.8 2 6.9 6 14.3 13 10.1 14 9 21 44 24.6 31.0 50.0 34.4 2DAY OVA 80 fetuses No. % ~- No. 2 4 6 400 2 4 6 Total Total 2 4 6 Total aoo No. % -. % No. % day 200 600 Total imp1ant ation No. of ova transplanted .___. 7 Maternal placentae No. of recipient rabbits 2 4 6 Total 5 2 2 9 8.8 6.9 4.8 7.0 4 7.0 17.2 13 30.9 22 17.2 5 % 6 DAY OVA 70 60- 40- 60y I 4 DAY OVA / O \ x-x I zoo I 400 normal fptuses GOO DOSAGE r. aoo I zoo I 400 600 DOSAGE r. I aoo Fig. 2 Effects of radiation dosage in vitro on subsequent development of ova. 50.0 51.6 66.8 55.3 39.2 19 57.5 36 76.4 75 57.3 515 DEVELOPMENT O F IRRADIATED OVA ova (51% ) is higher than in the case of two- to 4-day ova (24-29% ) . At a dose level of 600 r, the proportion of normal development in the case of two-day ova ( 3 1% ) is higher than in the case of 4- and 6-day ova (7-10% ) . But due to the small number of ova used and the wide variation of results between recipient animals it is uncertain whether there is a differential sensitivity of ova at a particular stage of development to a specific dose level of irradiation. Following irradiation at 800 r, the proportion of ova developing into normal fetuses ranged from 7 to 14% in all ova at various stages. Taking the results as a whole and by inspection of figure 2 it appears that there is no difference between ova at various stages of development following irradiation at dose levels from 200 r to 800 r; and that the proportion of irradiated ova developing into normal fetuses and the proportion of total ova implanted decreased together a s the dosage increased. However, the curves of percentage of total implantation and that of the percentage of normal development are relatively more close to each other in the case of two-da-y ova than in the case of 4-day and 6-day ova (fig. 2 ) . This may indicate that after irradiation younger ova would develop into normal fetuses if they were implanted while older ones might survive after im- plantation but fail to develop into normal fetuses in the end. In general the proportion of ova degenerating after implantation increased as the dose level increased. Although following a certain dose of irradiation, ova at blastocyst stage may survive for a longer time than a segmenting ova but the cellular components that determine their future development into normal fetuses are equally radiosensitive in the two-day ovum as in the 6-day ovum. Irradiation of recipient and pregnant animals as compared with the irradiation of ova alone on subsequent embryonic development In order to determine whether the irradiation of recipient females would play a role in the subsequent development of embryos, ova recovered two, 4 or 6 days after insemination were transplanted into Fallopian tubes or uteri of recipient animals that had been irradiated at 400 r on the second, 4th, or 6th day after a n ovulation-inducing injection. Another group of animals were irradiated at 400 r two, 4 or 6 days after insemination, to determine the effect of irradiation of ova in situ on their subsequent development A third group of animals, insemfnated but without irradiation served as controls. Table 3 and figure 3 present the results alongside previous data on the irradiation of ova in vitro at various stages of development at 400 r. TABLE 3 Development after irradiation at 400~: comparison o f the effects o f radiation applied to the ova in vitro, to the recipient does, and to the does while pregnant Irradiation of Age Of NO.of rabbits No' Of ?%?$' Fetal placenta Maternal placenta Total implantation ~~ No. - % No. % No. % No. % day Ova 2 4 6 Total Recirients 2 4 6 Total Pregnant 2 4 6 Total Control 7 6 9 22 51 33 47 131 15 8 24 47 29.4 24.2 51.1 35.9 2 3 9 14 3.9 9.1 19.2 10.6 3 8 3 14 5.9 24.2 6.4 10.6 20 19 36 75 39.2 57.5 76.4 57.3 4 4 4 12 35 47 37 119 15 22 11 48 42.8 46.8 29.7 40.4 4 2 2 8 11.4 4.3 5.4 6.7 2 5.7 5 10.6 11 29.7 18 15.1 21 29 24 74 60.0 61.7 64.9 62.2 3 4 4 11 47 41 48 136 7 8 8 23 14.9 19.5 16.7 16.9 4 0 4 8 8.5 0 8.3 5.9 1 0 0 1 2.1 0 0 0.7 12 8 12 32 25.6 19.5 25.0 23.6 10 115 74 64.4 2 1.7 0 0 76 66.0 516 M. C. CHANG AND D. M. H U N T 80 % 1 QO 40 20 2 DAY 4 DAY 6 DAY ALL STAGES Fig. 3 Total implantations (whole column) and degeneration after implantations (unshaded area of column) as of ova treated by irradiation a t 400 r at two, 4, and 6 days. When considering the percentages of combination of harmful effect on embrytotal implantation and normal fetal devel- onic development. Here again the results opment, the results show that following show that there is no striking differential irradiation on the second or fourth day radiosensitivity between the ova at differthese percentages are higher in the case of ent stages of development before implantathe recipient animals than in the case of tion, either when isolated and irradiated ova alone or in the pregnant animals. This in vitro or when irradiated in situ. shows that the harmful effect of irradiation Eight dead fetuses, (so-called neonatal is mainly on the ova rather than on the death), were found at the time of examinamaternal system. Following irradiation on tion among 56 “normal” fetuses following the 6th day, the percentage of total im- irradiation of two-, 4-, and 6-day ova in plantation and normal development are vitro at 100 or at 400 r. Neonatal death, higher in the case of ova alone than in the however, was not found in other cases, incase of irradiation of recipient or pregnant cluding the irradiation of recipient and animals. This indicates that on the 6th pregnant animals. There was no obvious day the harmful effect of irradiation may correlation of neonatal death with the age be greater in the maternal system rather of ova at irradiation or the dose level emthan in the embryos. In general, there is ployed. no striking difference whether the ova, Cytology of irradiated blastocysts recipients, or pregnant mothers were irradiated on the second, fourth, or on the Irradiated blastocysts were studied cytosixth day in the determination of future logically. Soon after irradiation mitosis at development of the ova; but the propor- various stages was observed in whole tions of total implantation, degeneration blastocysts - even after a dose of 10,000 after implantation, and normal develop- r. However, effects on mitotic activity bement showed a marked reduction follow- came apparent when the blastocysts were ing the irradiation of pregnant animals. cultured for 2-3 hours. Non-irradiated When the results are combined and com- blastocysts after culture showed normal pared with the non-irradiated mothers (fig. mitotic figures and no obvious fragmenta3 ) , it is obvious that the disturbance of tion of chromatin (fig. 7). After irradianormal development can be induced by ir- tion at 100 r, various phases of mitosis radiation of maternal environment without were seen, but there were many chromothe irradiation of embryo and that the ir- somal bridges (fig. 5) and a few scattered radiation of both mother and ova exert a chromatin fragments, mainly in the germ D E V E L O P M E N T O F IRRADIATED OVA 517 Figures 4-7 are Feulgen preparations of spread rabbit blastocysts photographed with a n oil immersion objective and a 1OX ocular, approx. x 750. Fig. 4 Germ disk area of a blastocyst irradiated at 1,000 r and cultured for three hours. Showing the abundance of fragmentation and condensation of chromatin without any mitotic figures. Fig. 5 Trophoblastic area of a blastocyst irradiated at 100 r and cultured for three hours. Showing chromosome bridges. Fig. 6 Germ disk area of a blastocyst irradiated at 100 r and cultured for three hours. Showing the presence of equatorial plates, and a few chromatin fragments. Fig. 7 Germ disk area of a non-irradiated blastocyst. disc area (fig. 6). In blastocysts irradiated at 1,000 and 10,000 r there were no mitotic figures, but there were many condensed chromatin fragments which gave a n intense Feulgen reaction (fig. 4 ) . DISCUSSION Fellner and Neumann ('06) observed the arrest of gestation with resorption of the embryos in rabbits irradiated during the first half of pregnancy. In the rat, Hicks ('54) reported that no damage of nervous system was observed when irradiated at 0 to 8 days but on 9th to 13th day, various malformations of the nervous system were observed. Wilson ('54) also reported a high incidence of malformations when rats were irradiated on the 9th to 1l t h day of pregnancy. In the mouse, Russell and Russell ('54) reported a high incidence of prenatal death following irradiation during the pre-implantation period, various malformations after irradiation during the major period of organo- 518 M. C . CHANG AND D. M. HUNT genesis (days 6.5 to 12.5), high incidence of neonatal death after irradiation on day 9.5 and 10.5; and the absence of gross abnormalities when mice were irradiated on day 0.5 to 5.5 or on days 13.5. Recently, however, Rugh and Grupp (’59) reported that exencephaly (cerebral hernia) may be produced by irradiation of the newly fertilized egg before any cleavages or by damage to neuroblasts at 8.5 days in the mouse. The present study revealed that in the rabbit no abnormality, or any kind of malformations externally or internally, was observed in fetuses obtained by irradiation either of ova alone or of recipient and pregnant animals, during the pre-implantation period. Certainly this does not imply that the ovum before implantation is more radioresistant than at other stages. It only shows that at this stage the ova were either killed by irradiation or developed into apparently normal fetuses. Whether or not these fetuses are physiologically normal is still to be determined. According to Rugh (’54) “the newly fertilized egg and the blastula are the most radiosensitive stages in early embryonic development of the frog, whereas the ovarian egg, the neurula and the older tadpole are the most radioresistant.” From his list of radiosensitivity, the fertilized egg is the first, the blastula is the second, the first cleavage the third, the uterine egg is the fourth, etc. It is rather surprising from the present study that the radiosensitivity of a two-celled rabbit ovum is very much the same as that of a late blastocyst which contains thousands of cells, and whose cells are differentiated into embryonic and trophoblastic portions. It has been postulated that radiation induces chromosome breaks or aberration, and thus leads to the failure of mitosis and the death of cells (Russell and Russell, ’54). From cytological examination of the irradiated blastocysts in the present study it is obvious that irradiation did not induce an instantaneous chromosomal breakage; but chromosomal abnormalities, fragmentation and condensation of chromatin show up later during culture. It seems that the effect of irradiation is on a more fundamental biological system, probably at a molecular level, which is revealed during cell division. This is especially so in the case of spermatozoa whose nucleus is at a resting stage without visible chromosomes. After irradiation the motility and fertilizing capacity of spermatozoa are not damaged but the cleavage of ova fertilized by irradiated spermatozoa is inhibited (Amoroso and Parkes, ’47; Chang et al., ’57). According to Rugh (’54) “the differentiating cell, even prior to its mitosis, is more radiosensitive than is the differentiated cell in the process of mitosis.” It is really hard to say, especially during embryonic development, which group of cells is differentiating and which group of cells is differentiated. One may say that the cells in a blastocyst are more in a differentiating stage than two blastomeres in a segmenting ovum; but their radiosensitivity in determining future development is very much the same. Furthermore, mature spermatozoa are highly differentiated cells and yet irradiation of sperm will lead to the degeneration of fetrilized ova (Chang et al., ’57). It is known that irradiation of unfertilized rabbit ova at 800 r results in a total failure of development of such ova fertilized by non-irradiated sperm (Chang et al., ’58) and that irradiation of sperm at 800 r reduces the proportion of embryonic development to 11% when non-irradiated ova were fertilized by such sperm (Chang et al.,’57). The present study has revealed that irradiation of fertilized ova at 800 r before implantation reduces the proportion of embryonic development also to 1 0 % . It seems that unfertilized ova are more radiosensitive than sperm, but once fertilized the ova at various stages before implantation are as radioresistant as sperm although their nuclear activities are quite different. One wonders whether the radiation damage to the gametes and zygotes affects the same biological system. SUMMARY Fertilized rabbit ova recovered one to 6 days after mating were irradiated in vitro from a radiocobalt source and then transplanted into recipient animals. When examined 22-28 days later 44, 33, 8 and 0% of ova irradiated respectively at 50, 100, 1,000 and 5,000 r developed into apparently normal fetuses without external or DEVELOPMENT OF IRRADIATED OVA internal malformation. No significant differential sensitivity was apparent in ova irradiated at different ages. It was found further that 34, 36, 19 and 10% of two-, 4-, and 6-day ova irradiated respectively in vitro at 200, 400, 600 and 800 r developed into “normal” fetuses. Again no malformation of fetuses and no differential radiosensitivity between ova of different ages were observed. Following whole body irradiation at 400 r, it was found that 40% of non-irradiated ova developed into normal fetuses when transplanted into recipient animals that had been irradiated (vs. 36% in the irradiation of ova alone). However, only 17% of estimated ova developed into “normal” fetuses when pregnant rabbits were irradiated two, 4 or 6 days after insemination (vs. 64% in the control). It appears that irradiation of the maternal organism influences embryonic development and that irradiation of pregnant animals exerts a combination of ill effects, on the ova and on their environment. Cytological study of irradiated blastocysts revealed no chromosomal breakage immediately after irradiation. Chromosomal abnormalities, fragmentation and condensation of chromatin were observed during the cuIture of irradiated blastocysts in accordance with the dosages applied. From this study it is concluded that ( 1 ) although 50 r may affect embryonic development, there seems to be no differential effect up to 400 r, above which greater prenatal death occurs, ( 2 ) before implantation, irradiated ova either die or develop into apparently normal fetuses and there is no evidence of differential radiosensitivity at various stages of development, 519 ( 3 ) irradiation of the maternal organism alone also affects embryonic development, and (4) radiation damage affects a fundamental biological system which leads to the nuclear damage and failure of mitosis, and the death of ova. LITERATURE CITED Amoroso, E. C., and A. S. Parkes 1947 Effects on embrvonic development of X-irradiation of rabbit spermatozoa in vitto. Proc. Roy. SOC.B, 134: 57-78. Chang, M. C. 1950 DeveIopment and fate of transferred rabbit ova or blastocyst in relation to the ovulation time of recipients. J. Exp. ZOO^., 114: 197-226. 1954 Development of parthenogenetic rabbit blastocysts induced by low temperature storage of unfertilized ova. Ibid., 125: 127-150. Chang, M. C., D. M. Hunt and E. B. Romanoff 1957 Effect of radiocobalt irradiation of rabbit spermatozoa in vicTo on fertilization and early development. Anat. Rec., 129: 211-230. 1958 Effects of radiocobalt irradiation of unfertilized or fertilized rabbit ova in vit70 on subsequent fertilization and development in viuo. Ibid., 132: 161-180. Fellner, 0. O., and F. Neumann 1906 Ueber Rontgen bestrahlung der Ovarien in der Schwangerschaft. Zentralbl. f. Gyniik. 30: 630-633. Hicks, S. P. 1954 The effects of ionizing radiation, certain hormones and radiomimetic drugs on the developing nervous system. J. Cell. and Comp. Physiol., 43: (suppl.) 151-178. O’Brien, J. P. 1956 Vertebrate radiobiology: Embryology. Ann. Rev. Nuclear Sci., 6: 423453. Rugh, R. 1954 The effect of ionizing radiations on amphibian development. J. Cell. and Comp. Physiol., 43: (suppl.) 39-76. 1959 Vertebrate radiobiology (Embryology). Ann. Rev. Nuclear Sci., 9: 493-522. Rugh, R., and E. Grupp 1959 Exencephalia following X-irradiation of the pre-implantation mammalian embryo. J. Neuropath. Exp. Neur., 18: 468-481. Russell, L. B., and W. L. Russell 1954 An analysis of the changing radiation response of the developing mouse embryo. J. Cell. and Comp. Physiol., 43: (suppl.) 103-150.