of Rats Injected with Radioactive Cesium 137 KITABATAKE Takashi

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1 Spermatogenesis of Rats Injected with Radioactive Cesium 137 KITABATAKE Takashi (Received in June 1961) ABSTRACT Solution of radioactive cesium, 137Cs, was injected to rats intra peritoneally, and the quantitative examination was made on its sper matogenesis. For making calculation simply and getting conclusion clearly, Germ Cell Tubulus Ratio or shortly Germ Cell Ratio method was used. Germ Cell Ratio is the proportion of the seminiferous tubules having spermatogonia, spermatocytes, spermatids and sperm cells to all seminiferous tubules on the same section of the testis. After the administration of 5, 50, and 200 micro-curies of 137Cs animals were sacrified with elapsed time. The germ cell ratio of each type of germ cells appeared significantly decreased, particularly in the 200-micro curies-injected group. Even in the 5-micro-curies-injected animals, diminution of the germ cell ratio of each type of germ cells was slight but statistically significant with exception of the sperm cells which kept the stationary level of about 90 to 100 per cent throughout observation. According to our estimation, the animals in this 5-micro curies-group received radiation dose of only less than 10 rad by in ternally administered 137Cs. Radioactive cesium, a beta and gamma-emitter, is believed to be homogeneously distributed in the soft tissue, when it is introduced into the body of animals (7). Thus this agent could be considered one of the serious sources of internal irradiation to the gonads (15). This paper will detail its hazard indicated by involution of the germ cells in rats followed radioactive cesium injection. Department of Radiology, School of Medicine, Nagoya University. Director : Prof. S. Takahashi (Read at the 18th Meeting of Japan Radiological Society, 6 Apr )

2 METHODS Male rats with the body weight of near 100 g were used. The animals were fed with the same baits since two weeks before the injection as well as that during the experiment. The feed stuff was taken as want of animals. Each animal was bred separating from the others in the fedcage, and excrement was removed from the cage as entirely as possible to prevent re-intake of radiocesium. The experiment was performed in the room temperature of 15 to 25 C. The animals increased the body weight about 10g per week up to 10th week after the injection. Solution for injection was used 137CsNO3 in solvent of HNO3. Rats were divided into three groups, A, B and C. Each group consisted of 16 male rats. Rats of the group A were injected with five micro-curies of radiocesium solution intraperitoneally, while group B 50 microcuries and group C 200 micro curies. We call these groups 5 micro-curies-group, 50 micro-curies-group, and 200 microcuries-group, respectively. Two rats in each group were sacrified under ether anaesthesia 1, 2, 3, 4, 5, 6, 7, or 10 weeks after the injection, and two testes were removed from the body. The specimens made from these testes were stained by a hematoxylin-eosin method, and studied microscopically. Definition of "Germ Cell Tubulus Ratio of the Testis" In order to estimate whether the spermatogenesis is progressed normally or not, the ratio of number of the seminiferous tubuli containing the spermatogonia, spermatocytes, spermatids and sperm cells respectively to the number of all seminiferous tubuli observed on the same section was counted. For counting, the number of the tubuli was essential, but not the number of cells. For example, it was estimated to be no difference between the tubulus con taining very few spermatogonia and the tubulus containing a number of sperma togonia. The ratio in percentage was named by us "Germ Cell Tubulus Ratio in the Testis" or shortly "Germ Cell Ratio". According to our experiences this ratio can be used as an indicator of the radiation hazard of the testis, as this ratio takes a constant value for normal adult rat (1, 10). Microscopic examination of the seminiferous tubuli revealed that the spermatogenesis was conducted by a rule. In one transverse cross section of the siminiferous tubulus one stage of the germ cells in one single phase is always existed. When the spermatogonia were in the karyo kinetic phase, fast all of spermatogonia in that tubulus were in similar or in the same phase. There were no exceptions. Thus it can be regarded that the sperma togenesis progresses in wave in developing each phase of successive stages of germ cell (1). When a number of transverse cross section of tubulus are taken at random and the germ cell ratios are counted, the duration of the phase of each stage of germ

3 cells will be surmised. If the ratio in percentage takes different value as the normal, it will mean that the wave of spermatogenesis became irregular. Thus the function of the spermatogenesis could be estimated. Usually 300 or a little more number of cross sections of the seminiferous tubuli were counted on one histological specimen. For the actual calculation the sperma tocytes in the young stage were regarded as the spermatogonia, while the pre spermatid was counted together with and into the spermatids. RESULTS Germ Cell Tubulus Ratio in Normal Rats Ten normal rats with the body weight of near 100 g were examined. On twenty histological sections made from the testes 3876 cross sections of the seminiferous tubuli including the spermatogonia were seen. The value of percentage with the confidence limits at 5 per cent level were estimated thus as 96.9±1.9%, and, of sperm cell was 98.4±1.2%, Similarly the germ cell ratio of the spermatocytes cell was 95.7±3.2,~,%, and, of spermatids was 97.1±1.9%. Five-Micro Curies Group Each two rats of this group were sacrified 1, 2, 3, 4, 5, 7, and 10 weeks after the injection, respectively, and their testes were microscoped. The average percentage of 4 testes was studied. The spermatogonia appeared slightly diminished in 2 weeks after the injection. The germ cell ratio was 68 per cent at the end of the third week, 65 per cent in the 6th week, and 92 per cent in the 10th week. To examine whether there was a statistically significant difference in these rates or not, the confidence limits were calculated. For example, the germ cell ratio of the sperma togonia at the end of 2nd week was 68 per cent, and the confidence limits at 5 per cent level were estimated. to be 68.0±24.3 per cent, that, is, 92.3; to 43.7/ As (Fig. 1) Grem cell tubulus ratio of the testis in the 5-micro-curie s-137cs-injected rats. A slight diminution of the ratio was seen with the elapsed time. compared with the germ cell rate and confidence limits of the spermatogonia in normal rats which were 96.9±3.1/ (that is 100.0/ to 93.8%), there was no overlapping area be tween two confidence limits. This indicated that the sperma togonia in the 5 micro-curies group appeared definitely di minished a few weeks after the injection of 137Cs. The germ cell ratio of the spermatocytes reached the mini mum value (73 per cent) at

4 the end of seventh week, and that of spermatids was 76 per cent in sixth week. There was also a statistically significant difference at 5 per cent level. The sperm cells showed a slight decrease of the germ cell ratio, which, however, kept near the level of 90 per cent, until the tenth week. There was no significant difference for the sperm cells. 50-Micro-Curies-Group Diminution of each type of germ cells was more exaggerated in this group than in the 5-micro-curies-group. The germ cell ratio of the spermatogonia was 72 per cent in the 4th week, 59 per cent in the 6th week, and 56 per cent in the 7th week, (Fig. 2) Germ cell ratio in the 50-micro-curies-injected rats. There is a moderate decrease of the ratio. (Fig. 3) Germ cell ratio in the 200-micro-curies-injected rats. A considerable diminution of the ratio being shown. of the spermatocytes cent in the 3rd week, was 64 per 38 per cent in the 6th week, and 40 per cent in the 7th week, and of the sper matids was 71 per cent in the 3rd week 56 per cent in the 6th week, and 64 per cent in the 7th week. These value showed a statis tical significance. The sperm cell also showed a slight ten dency toward decrease without a significant 200-Micro difference. Curies-Group In this group a considerable decay was observed. The germ cell ratio of the spermatogonia was 66 per cent in the first week, 19 per cent in the 2nd week, and near 10 to 30 per cent until the end of 10th week. Also the germ cell ratio of the spermatocytes, sperma tids and even the sperm cells was approximately 20 to 40 per cent throughout the period observed. There was no tenden cy toward recovery. CALCULATION OF INTERNAL RADIATION DOSE Assuming that radioactive cesium is distributed homogeneously in the soft tissue of animals, and that the effective half-life of 137Cs in the testis is to be 9 days (13), the

5 dose irradiated to the testis by the beta-rays of 137Cs was calculated as follows (6) : 0. _693t 73.7 CENT (1-e T ) rad. On the other hand it is generally difficult to actually measure the irradiated dose shared from gamma-rays, therefore, an approximate calculation was taken. The rat used in this study was presumed as a cylinder to be a column 4 cm in diameter and 10 cm in height. The testis was regarded to locate in 'the center of the bottom on one side. Since the gamma ray emitter was considered to uniformly distributed in this column, the dose ir radiated from the gamma-rays in the testis could be obtained as follows (3) : D= Irk 10 log tan-10 (Fig. 4) Testicular radiation dose of 200 micro -curies-injected rats. In the 50-micro-curies-injected rats the dose irradiated is estimated to be one fourth of this value (-1)n t4n (In to+1 ) n=1 n n ~'Z+1 Hence, lim D=0, and I n =f t (102+22) z dz. t--0 2 o The radiation dose and dose rate in 200-micro-curies-group thus calculated was graphed in Fig. 4. DISCUSSION It is widely known that radioactive cesium is one of the most dangerous in ternal sources for gonadal exposure (7). There were some morphological studies on the spermatogenic cells of the exposed testis to radioactive cesium (13, 15). In our opinion the morphological study has limitation to reveal minute biological reaction caused by small radiation dose. Actually, there was observed no definite difference between the testes exposed to 100 r of radiation and 150 r in the morpho logical examination (1). Even such a case it became possible to get the significant difference when the quantitative observation for the biological effect was made (10). Takahashi firstly tried to study on the roentgen testis by using this methods (11). Development of spermatogenesis would of cause become clear if the testis was examined by serial microscopic sections made of one whole testis. It was, however, not practical

6 (Fig. 5) Normal testicular histology in adult rat. (Fig. 6) Seminiferous tubulus 4 weeks after the injection of 200 micro-curies 11 Cs solution only very few spermatogonia are found in the tubulus. because of enormous time consuming. Thus only one central part section of one testis was prefered for examination. According to his report, the error of calculation caused by observation of only one section instead of the calculation of whole sections was less than 4 per cent. Takahashi reached the conclusion thus that the wave length of the spermatogenesis is 4 weeks. Asayama also applied this method to the study of roentgen testis (1). The germ cell ratio defined in this paper is a conception related to cell number in the seminiferous tubuli and their survival time. Therefore our method may be rather advanced than a simple cell number counting method dealed by Borner, Shingyoji, Tanei, etc. (2, 5, 12). Not few reports on the histological study after the cesium injection have been published. Yoshizawa and Watari described experimental results in internal cesium irradiation, in which pathological course of the testis was surveyed after the injection of 5 micro-curies per gram of rat (13, 15). However no details have been reported on the testicular change after the in troduction of radiocesium dose less than 0.1 micro-curies per gram, probably because that no remarkable changes were appeared in the testis delivered such a small dose level. Therefore our results that the spermatogonia, spermatocytes, and spermatids were significantly reduced 3 to 6 weeks after the injection of 0.05 micro-curies per gram, might be obtained only by means of quantitative study. In this case the spermatogenic function was more or less disordered even by the radiation dose less than 10 rads, which can be regarded one of new information in this field.

7 SUMMARY AND CONCLUSIONS 1. As a function test of the radiocesium injury, a quantitative observation was made on the spermatogenesis of rats. 2. For this, the idea of germ cell tubulus ratio was presented. That is defined as a ratio of a number of the transverse cross section of the seminiferous tubuli including each developmental stage of the germ cells to the number of siminiferous tubuli seen on the same histological section. 3. The germ cell ratio was considerably diminished for the group exposed to 200 micro-curies of radioactive cesium injection. No tendency toward recovery was recorded until 10 weeks after the injection. 4. In 5-micro-curies-group also the germ cell ratio except for of the sperm cells showed a slight but statistically significant decrease. In this case internal irradiation dose in the testis was estimated to be less than 10 rads. REFERENCES 1) Asayama, H. (1950) Roentgen-testis (in Japanese). Nippon Acta Radiol 10. (7) ) Borner, W. et al (1956) Die Wirkung von 180kv-Rontgenstrahlen and 6 MeV-Elektronen auf das Hodengewebe der Ratte. Strahlenther. 101: ) Iida, H. (1958) Personal communication. 4) Kohn, H. I. (1955) On the direct and indirect effect of X-ray on the testis of the rat. Radiation Res. 3 : ) Shingyoji, T. (1957) The histopathological studies of the effects upon testis, adrenal and hypophysis by X-ray irradiation on testis (in Japanese). Nippon Acta Radiol. 17: ) Okajima, S. (1959) Personal communication. 7) Report on the United Nations Scientific Committee on the Effects of Atomic Radiation, New York, ) Sirlin, J. L. and R. G. Edwards. (1957) Duration of spermatogenesis in the mouse. Nature 180: ) Spalding, J. F. (1957) Effects of rapid massive dosis of gamma-rays on the testes and germ cells of the rat. Radiation Res. 7: ) Takahashi, S. (1940) Destruction of spermatogenic cells after X-irradiation to the testes (in Japanese). Jap. J. Radiol. 7: ) Takahashi, S. Unpublished. 12) Tanei, S. (1958) Fundamental studies of sieve therapy (6) : The effect of the sieve irradiation on the testis of the rabbit (in Japanese). Nippon Acta Radiol. 17: ) Watari, T. (1958) The experimental studies of metabolism and histopathology of radio cesium in mice and rats (in Japanese). Nippon Acta Radiol. 17: ) Yamagata, N. (1959) The concentration of cesium 137 in human tissues and organs, presented at the 1st Annual Meeting of " The Japan Radiation Research Society", Sept. 1959, Tokyo. 15) Yoshizawa, Y. (1957) Studies on the metabolism of cesium 137 in animals (in Japanese). Nippon Acta radiol. 17: