EFFECT OF IRRADIATION IN INFANCY ON THE MOUSE OVARY

EFFECT OF IRRADIATION IN INFANCY ON THE MOUSE OVARY A QUANTITATIVE STUDY OF OOCYTE SENSITIVITY HANNAH PETERS and EMILIA LEVY Einsen Laboratory, Copenhagen, Denmark (Received 13th June 1963) Summary. The effect on the ovary of a single dose of 20 r X-ray given at different ages early in life varies according to the age of the animal at the time of radiation. This has been investigated by determining the number of surviving 24 hr after radiation, and at varying intervals after irradiation but at a constant age of the animal, i.e. 49 days. The sensitivity of the ovary depends on the response of the small as well as on the response ofthe growing and large. The sensitivity of these two groups differs. Further, a variation within the two groups is noted, which is age dependent. Fifty per cent ofsmall survive 24 hr after radiation on the day of birth whereas radiation after this age leaves only between 1 and 9 % of these cells intact. The number of in the ovary at the time the animal enters maturity is 85 % of the normal number after irradiation at birth, but only 1 % after irradiation at the age of 3 weeks. The changing radiation sensitivity is discussed in relation to certain morphological changes in the developing ovary. INTRODUCTION The marked radiation sensitivity of ovaries of infant mice has been known for some time (Brambell, Parkes & Fielding, 1927; Russell, Russell, Steele & Phipps, 1959; Peters, 1961 ; Oakberg, 1962). That this sensitivity is not constant, but varies considerably in early life, has recently been shown by a marked variation in the subsequent reproductive behaviour of mice irradiated at different ages during infancy and early maturity (Peters & Levy, 1963 a, b). The present work was undertaken to determine quantitatively the effect on the ovary of a single dose of 20 r X-ray given at different ages early in life. To investigate the immediate effect, oocyte survival was determined at a constant time (24 hr) after radiation. The status of the ovary was also investigated quantitatively at varying times after radiation, but at a fixed time of the animal's fife. For this purpose, the oocyte population present in the ovary at the onset of maturity (7 weeks) was determined. * Fellow of the International Atomic Energy Agency. ß* 37

_ 38 Hannah Peters and Emilia Levy MATERIAL AND METHODS EXPERIMENTAL ANIMALS The mice were females of the Street strain; their ages ranged from Day 0 (day of birth) to 51 days. RADIATION Radiation was given as a single dose of 20 r X-ray ( 175 kv, 8 ma, half value 1 2 mm Cu, distance 57 cm, dose rate 30 r/min) to the whole body except for the head, which was shielded. Eight groups of mice of different ages were irradiated : new-born mice and groups that were 7, 14, 21, 28, 35, 42 and 49 days old. HISTOLOGICAL PREPARATIONS The ovaries of the control and irradiated mice were fixed in either Carnoy's or Bouin's solution, serially sectioned at 5 µ and stained with haematoxylin and eosin. CLASSIFICATION OF OOCYTES AND FOLLICLES The were divided into two main groups: (1) The small, i.e. 20 µ or less in diameter. (2) The growing and large, i.e. those germ cells that have begun to grow and have a diameter of more than 20 µ, as well as those that have reached their maximum size of 70 µ. For the follicles the following classification was used : Type 1 : oocyte without accompanying follicle cells ; Type 2 : oocyte with a few follicle cells ; Type 3a : small oocyte with a complete ring offollicle cells, usually flat ; Type 3b : growing oocyte with a complete ring of round follicle cells ; Type 4 : oocyte surrounded by two rows of follicle cells ; Type 5 : oocyte surrounded by many rows of follicle cells; Type 6: follicle with beginning antrum formation; Type 7: follicle with multiple antra; Type 8: corona formed, last stage of follicle development. ESTIMATION OF THE NUMBER OF OOCYTES The total number of was computed by the method described by Mandi & Zuckerman (1951). A separate count was made (1) of the small (in Type 1 to 3a follicles) and (2) of the growing and large (in Type 3b to 8 follicles). The small were counted in every tenth section, using the nucleolus as marker. Abercrombie's (1946) correction factor was used for overcounting due to the size of the marker (2 µ). The number of small (S) was therefore determined as: _ S Number counted 10 x-r-r-.-?-:-:-~-; ; = _ thickness of section,, thickness ol section+size ot nucleolus The growing and large were sampled in every fifth section. All counts were made on one ovary only and doubled to express the total number of difference between the two ovaries, as Jones (1957) found no significant in several strains of mice. Apart from the determination of total numbers of, partial counting

Irradiation of mouse 39 was done in a few instances. In these cases germ cells were counted in twentyfive non-adjacent middle sections of the ovary. These were then compared to counts obtained in the same way in corresponding normal ovaries. In two instances small in irradiated ovaries were determined per 100 growing follicles. A corresponding count was made in a normal ovary of the same age. RESULTS NORMAL OVARIES The number of present in normal ovaries during the first 7 weeks of life has been determined in twenty-one animals (Table 1 ). Table 1 control animals Total count Age (days) Small Growing + large Total Age (days) Partial count 12514 13284 10572 12514 13284 10572 9114 8442 278 198 9392 8640 13 7328 6358 7 670 8078 7028 16 7114 4886 942 900 8056 5786 16 Small : 1252 per 100 growing follicles 19 Small : 1013 per 100 growing follicles 20 8228 6858 420 756 8648 7614 21 4672 3772 7114 464 600 928 5136 4372 8042 22 Small : 630 in 25 sections Growing + large : 69 in 25 sections 28 00 6210 3628 756 720 600 5756 6930 4228 28 Small : 589 in 25 sections Growing + large : 6 in 25 sections 35 4714 5384 728 524 5442 5908 42 42 Small : 417 in 25 sections Growing+large : 15 in 25 sections Small : 406 in 25 sections Growing + large : 21 in 25 sections 49 3672 3686 492 478 4164 4164 The Street mouse is born with 12,000, this number decreases expo At the time of birth all the nentially, and is halved in 32 days (Text-fig. 1 ). are small. The growing ones first appear after 3 days. Their number

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Irradiation of mouse 41 increases rapidly (Text-fig. 1) from about 240 on the 7th day to 920 on the 16th day, the highest number reached at any time. Before the end of the 3rd week their number is already reduced by a phase during which the rate of destruction exceeds that of production. After this they comprise 11 % of au, a proportion which remains constant until maturity is reached. Total Growing * large Text-fig. 1. 35 49 Age (days) Number of in mouse ovaries of normal mice in relation to age. IRRADIATED OVARIES The number of in irradiated ovaries was determined in thirty-three animals 24 hr after irradiation (Table 2). The response of the small 24 hr after radiation at different ages is not uniform. Radiation on the day of Table 3 surviving as a proportion of the normal complement Age at radiation (days) 0 7 14 21 28 35 42 49 Small (%) 52 61 1 3 5 5 9 Survival after 24 hr Growing and large (%) 52 36 42 Total (%) 52 14 6 7 9 12 Survival at the age of 49 days Small (%) 85 2 1 5 6 Growing and large (%) 90 30 14 7 39 61 Total (%) 85 5 3 1 10 12 12 birth leaves half of the small intact, but only between 1 after treatment at any of the later ages tested (Table 3). The time of greatest sensitivity is at the beginning or the end of the 3rd week of life, when 1 % and 9 % survive

42 Hannah Peters and Emilia Levy survive 24 hr after irradiation. Radiation after the 3rd week permits an in creasing proportion of small to survive, so that treatment at the age of 7 weeks leaves 9 % of a normal small oocyte complement surviving on the next day. The immediate (24 hr) radiation effect on the group of growing and large was determined in mice irradiated at 3, 4, 5 and 7 weeks. The number of in follicles of Types 3b to 8 was reduced in all groups, 36 to % of a normal complement surviving 24 hr after radiation (Table 3). In an attempt to detect a possible difference in the radiation response between growing and that have already reached their maximum size, differential survival counts were made. The response of the growing oocyte in follicles of Types 3b and 4 was distinguished from that offully grown in follicles oftypes 5 to 8 (Table 4). This could be done in animals aged 28 days or over. Half of the Table 4 oocyte survival in relation to follicular development Age at radiation (days) 21 28 35 49 Survival after 24 hr Growing (in Type 3b to 4 follicles) (%) 27 31 Large (in Type 5 to 8 follicles) (%) 55 Survival at the age o/"49 days Growing (in Type 3b to 4 follicles) (%) 1 14 26 Large (in Type 5 to 8 follicles) (%) 27 118 157 fully grown survived 24 hr in the three groups tested, whereas the survival rate of growing increased from 27 % after radiation on the 28th day to % after treatment at the age of 49 days. In 21-day-old animals this determination could not be made with any degree of accuracy, as the distribution of growing and large at this age varies widely in normal animals. A count distinguishing between growing and large was therefore only done when this group reached the age of 49 days (Table 4). At that time 1 % of the growing and 27 % of the large were found to survive, suggesting a very high sensitivity and disappearance rate for the growing after radiation at the age of 21 days. AT THE AGE OF MATURITY The condition of the ovaries at the time the animal becomes mature was with which evaluated quantitatively in order to determine the number of the animals of the different irradiated groups actually entered reproductive life. The time chosen as the age of early maturity was 49 days, because at this age ovulation is established in most control animals of this strain. Animals irradiated on the day of birth enter maturity with 85 % of a normal complement of (100% representing the number of in ovaries of 49-day-old control animals). Radiation at all later ages effects a drastic reduction of the

Irradiation of mouse 43 total oocyte number by the time the animal enters reproductive life. Five, 3 and 1 % of all survive radiation given at the age of 1, 2 and 3 weeks, respectively. Ovaries irradiated after this age enter maturity with 10 to 12% of a normal complement of (Table 3). The survival of small follows closely the survival curve of the total number of (Text-fig. 2). However, the group of growing and large shows a different distribution. Animals irradiated at birth have 90 % of the expected number; after treatment at the age of 1 week 30 % remain, whereas radiation at the beginning or end of the 3rd week of life leaves 14 and 7 % of growing and large intact by the time the animal reaches maturity. Animals irradiated when more than 3 weeks old enter reproductive life with 40 % or more of the usual number ofgrowing and large. C R.0 7 21 35 Age on day of radiation (days) Text-fig. 2. Oocytes surviving at the age of 49 days after radiation on different days after birth. C = Control.. Total ; O, small ; D, large. 49 DISCUSSION From the survival rate of 24 hr after radiation, and the numbers of present in the ovary at the time of maturity, it can be deduced that the response of to radiation varies with the age of the animal at the time of treatment. Radiation at birth causes least disturbance ; although % of the germ cell population is immediately destroyed, the surviving ones develop in such a way that 85 % of a normal complement still populate the ovary by the time maturity is reached. Radiation at all later ages, however, reduces the oocyte number present at the beginning of maturity to 12 % or less. To discuss this marked variation in response we shall try to correlate it with some morphological developments in the ovary which occur during infancy and puberty, being aware, however, that these are not the only ones that influence

44 Hannah Peters and Emilia Levy the radiation sensitivity. The hormonal changes and influences are not dis cussed in this presentation. The period of lowest sensitivity (day of birth) coincides with an ovary con sisting entirely of small. The transformation of the small to the large has not started yet, and the immediate radiation effect is therefore only dependent on the response of the small. At birth the small are still in transitory stages of the meiotic prophase. About half of them are in pachytene, these are highly radiation sensitive and quickly destroyed (Peters, 1961); half are in early diplotene, a stage apparently not significantly dis turbed by 20 r of X-ray. This particular composition of the ovary at birth, consisting partly of highly sensitive pachytene and partly of resistant (to X-ray) 20 r early diplotene stages, permits % ofthe total oocyte number to survive 24 hr, enough to populate the ovary with an oocyte complement, which is only slightly below normal by the time the animal enters reproductive life. The effect of radiation on the ovary at all later ages must, however, depend on the response not only of the small but also of the growing and large. The small ofovaries treated at the age of 1 week or later show a distinct increase in sensitivity; only 1 to 9 % of their number survive 24 hr compared to % surviving radiation on the day of birth. This change in response can be correlated with a concurrent change in the morphology of the oocyte nuclei. These develop beyond the transitory stages ofmeiosis through late diplotene to the 'stationary' phase (stationary only in the sense, that with present cytological methods no well-defined morphological changes can be found) of dictyotene, stages which prove to be highly radiation sensitive. In addition to the change in the radiation sensitivity of the small oocyte a varying effect on the growing and large oocyte determines the response of the ovary to radiation given after the neonatal period. If the total number of present at the beginning of maturity is taken as a measure of damage done, it is shown that radiation given between the 1st and 3rd week of life causes the severest damage. This high sensitivity coincides with the period of ovarian development when the transformation of small to large ones is at its height, and when growing are present in the largest numbers. The growing oocyte seems to be particularly sensitive during this phase of fast transformation ; this is suggested by the fact that the number of growing found at the age of maturity is least in animals irradiated at the age of 3 weeks. Radiation in the subsequent weeks permits a larger proportion of growing to reach the maturing ovary, suggesting a gradual decrease in the radiation sensitivity of these cells with advancing age. The reason for the high sensitivity of the growing oocyte is not known. Morphologically, the nuclei in the dictyotene phase are indistinguishable from nuclei of less sensitive in later stages. However, in this period of growth a variety of processes are beginning in these cells, e.g. augmentation of the cytoplasm, the formation of microvilli, the formation of the zona pellucida and the transport of material between the follicle cell and the ovum (Chiquoine, 1960). Which of these processes are interfered with by radiation is yet unknown. It seems, however, that with further growth of the cells and advancing age of the animal towards maturity the group of growing and large becomes

Irradiation of mouse 45 less vulnerable. Yet the overall sensitivity of the ovary considerable, measured by the total number of surviving animal reaches maturity. to radiation remains at the time the ACKNOWLEDGMENTS The authors wish to thank Mr J. Ambrosen, Radiophysical Laboratory, Radiumstationen, for his supervision of the radiation used in these experiments. Thanks are also due to Miss A. Knudsen, Miss H. Lorenzen, Miss K. Storm and Mrs I. Aaberg for their technical assistance. REFERENCES Abercrombie, M. (1946) Estimation of nuclear population from microtome sections. Anat. Ree. 94, 239. Brambell, F. W. R., Parkes,. S. & Fielding, U. (1927) Changes in the ovary of the mouse following exposure to X-rays. Part I. Irradiation at three weeks old. Proc. roy. Soc. B, 101, 29. Chiquoine, A. D. (1960) The development of the zona pellucida of the mammalian ovum. Amer. J. Anat. 106, 149. Jones, E. C. (1957) The aging ovary. Thesis, University of Birmingham. Mandl, A. & Zuckerman, A. (1951) The relation of age to numbers of. J. Endocrin. 7, 190. Oakberg, E. F. (1962) Gamma-ray sensitivity of of immature mice. Proc. Soc. exp. Biol., N.T. 109, 763. Peters, H. (1961) Radiation sensitivity of at different stages of development in the immature mouse. Rad. Res. 15, 582. Peters, H. & Levy, E. (1963a) The effect of radiation in infancy on the fertility of female mice. Rad. Res. 18,421. Peters, H. & Levy, E. ( 1963b) Radiation sensitivity ofthe mouse ovary. Fertility after radiation during infancy and early maturity. Proc. Joint Meet. Netherlands Radiobiol. Soc. and Brit. Assoc. Rad. Res. (Abstract). Int. J. Rad. Biol. (In press). Russell, W. L., Russell, L. B., Steele, M. H. & Phipps, E. L. (1959) Extreme sensitivity of an immature stage of the mouse ovary to sterilization by irradiation. Science, 129, 1288.