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Effect of Low-Dosage X-irradiation upon Pituitary Gland and Ovaries of the Rhesus Monkey
Effect of Low-Dosage X-irradiation upon Pituitary Gland and Ovaries of the Rhesus Monkey Gertrude J. Vermande-Van Eck, M.D.
THE THERAPEUTIC usE OF SMALL nosEs of roentgen irradiation to the ovaries of the pituitary gland of amenorrheal women is somewhat controversial. Both the consistency of the immediate symptomatic improvement of the patient and the possible effects of irradiation on subsequent generations have been the subject of a number of publications. In general, gynecologists have been impressed by the results of such treatment, and geneticists have been skeptical. The radiation effect on tissues and organs was reviewed by Bloom and Bloom in 1954. Early studies were done by Desjardin, 4 ·" who concluded that cells cannot be stimulated in the sense that they acquire a greater ability to function normally; any such apparent stimulation is only a transient phase, invariably followed by more or less pronounced functional or organic deterioration. Ewing remarked that the initial hyperemia after x-irradiation was always followed by induration and relative anemia; the hyperemia was .an inflammatory process, with vasodilatation, exudation of serum, leukoFrom the Departments of Anatomy, and Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Conn. This investigation was supported by grants from the USPHS. The author expresses gratitude to Dr. William U. Gardner, Chairman of the Department of Anatomy, for his guidance and stimulating comments; to Dr. Chu H. Chang, Assistant Professor of Radiology, for his help in the radiological part of this study; to Dr. C. Lee Buxton, Chairman of the Department of Obstetrics and Gynecology, for his suggestions in the preparation of this paper; and to Mr. Edward Lopes for his technical assistance. Received for publication March 24, 1958. 190
Vol. 10, No. 2, 1959
EFFECT OF X-RAYS ON PITUITARY AND OVARIES
191
cytes, and red blood cells. Lacassagne and Gricouroff described the reaction of connective tissue to irradiation in an early phase as a type of inflammation, followed by sclerosis; collagenous and elastic fibers became swollen and hyalinized, and the collagen had an altered staining capacity. Shields mentioned the vasodilatation in the capillary loops of the skin as a reversible process in the lower dosage range; the period of vasodilatation lasted several days or weeks after the initial erythema, during which the dilator stimuli produced a greater effect on the vessels than the constrictor stimuli. The action of x-rays on the ovaries of lower mammals has been studied extensively and so far only destructive action has been found, even after low dosages, in such studies as those of Geller in rabbits and Schugt in mice, in which destruction of the follicle was apparent after a dosage of 21 r, and the studies of Drips and Ford in rats. The last authors 1emarked that in the first days after irradiation a marked hyperemia was present, together with degeneration of all large follicles. Studies of low-dosage irradiation to the pituitary in experimental animals are rather frequent and all had negative results as far as changes in the ovaries were concerned. 2 • 3 • 8 ' 11 • 14 It is known that high dosages of x-rays are needed to affect the pituitary. Combined ovarian and pituitary irradiation on rabbits by Hodes and Israel did not induce ovulation. The summarized experimental evidence shows that ovarian irradiation only is destructive and that pituitary irradiation, unless massive doses are used, has no influence on the ovary. What then is the paradoxical effect resulting in instigation of menstruation and ovulation in the human female? We have approached this problem by administering human dosage to the female monkey and by following the morphologic and physiologic changes that occurred after treatment. Our experiments were designed to reveal something of the unknown mechanism of the action of x-rays on ovarian function, as well as the nature of such modifications, for once we know the mode of action of x-irradiation we might be able to achieve the same effect by a less harmful method.
METHODS AND MATERIALS In the first experiment we took advantage of the natural anovulatory period of monkeys during the summer months. Twelve adult females were
192
VERMANDE-VAN ECK
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selected for this purpose (4 of these were used as controls). Another anovulatory period occurs in the adolescent monkey during the first months after the menarche, and this was used in a second group of 18 monkeys. Both groups received x-irradiation to the ovaries and/ or pituitary; factors were 250 kv., 15 rnA., 50 em. target distance, half value layer of 3 mm. Cu. The field entrance to the ovaries was 10 x 10 em. from an abdominal port, and to the pituitary a circular port of 4 em. diameter, alternately from the left and right side. Three irradiations of 33.3 r each were given at weekly intervals to the ovaries and/ or the pituitary; the organ dose was 100 r total to ovaries and to pituitary. The irradiation was, if possible, started during the first half of the menstrual cycle. Records were kept of sex skin changes and vaginal lavages. In the adult animals the ovaries and uterus were palpated daily per rectum in order to detect the ovulation directly. The ovaries and uteri were removed 1-6 weeks after the last irradiation. The tissues were fixed in Bouin's solution, cut in serial sections, and stained with hematoxylineosin or VanGieson stain. Two groups of monkeys (adult and adolescent) received x-irradiation to both ovaries and the pituitary, a third group only to the ovaries, and a fourth group only to the pituitary. The ovaries were studied for the distribution of follicles in all stages of development. The technique of Green and Zuckerman was used with the following classifications: 1. Primordial ova with complete cover of flattened cells, or incomplete
2. 3. 4. 5. 6. 7.
epithelial cover. Ova completely surrounded by I layer of cuboidal cells. Ova completely surrounded by 2layers of cuboidal cells. Ova completely surrounded by 3 layers of cuboidal cells. Ova completely surrounded by 4-5layers of cuboidal cells. Ova completely surrounded by 6 or more layers of cuboidal cells. Follicles with antra.
Where counts were made of primordial ovocytes and the ovocytes in the small follicles, every fortieth section was counted. For the larger follicles ( 6 and 7) every section was screened. Ovocytes were counted only when the nucleolus was visible. A correction factor for the thickness of the section was applied. 1 Special attention was paid to the atresia of the ovocytes, the occurrence of degeneration of the follicles, of polyovular follicles, and the vascular supply.
Vol. 10, No. 2, 1959
TABLE 1.
EFFECT OF X-RAYS ON PITUITARY AND OVARIES
Changes in Ovaries of Rhesus Monkeys after Low Dosage X-irradiation
Mon- Age key (yrs.) Controls
Group
70 77 I
90 96 69
82 85 86 88 89 91 93 Group II
115 116 114
124 125 Group III
126 100 101
109 112 83
123 Group IV
193
104 105 106 113 127
3 3 3 3 3 3~
3 3~
3 3~
3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
In~Degen. X-rayed teroal foU. 100 r (wks.) Grp. 6 Grp. 7 Corp. atr. None None None None ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. & pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ovaries ovaries ovaries ovaries ovaries ovaries pituitary pituitary pituitary pituitary pituitary
3 3 1 1 3 3 6 1
4 4 4 4 4 4 4 4 4 4 4 4
5 4 4 4 4
50 36 16
39
33
47
68 63
32
60
40 27
54 50 51
33 37 33
15 61 25
60
58 72 64 73 81
20
70 40 70
20
60
20 12 15 20
37 30
25
10
10 3
70 40 40 36
Few Few Average Few Many Many Few Many Many Many Many Many Many Many Many Many Many Many Many Many Many Average Many Many Few Few Few Few Few
Polyov. foll. C. L. None Few None None Few Few Few Few Many Many Many Few Many Many Few Many Many Few Many Many Few Few None None None None None None None
None 1 None 1 1
!Old None None None 1 1
None 1 None None None None None None None None None None None None None None None None
Vessels Average Average Small Average Wide Wide Wide Wide Wide Wide Average Wide Wide Wide Wide Wide Wide Wide Wide Wide Wide Wide Average Wide Average Average Average Average Average
OBSERVATIONS
Group 1: Adult Monkeys, Irradiation to Ovaries and Pituitary Twelve adult monkeys (M), 3-3* years ·old (estimated by Dr. G. Van Wagenen, according to dentition, sitting height, and growth pattern of hand and foot) were observed for several months. During the summer the animals had regular menstrual cycles but ovulations were not detected. In the late summer irradiation was given to ovaries and pituitary. The size of the ovaries and uterus was followed by rectal palpations. Ovulations were felt in Ms 89 and 91; M69 was not palpated because of an anal stricture. In the other monkeys no ovulation was felt. Laparotomy was done 1--6 weeks after the last irradiation. All ovaries, macroscopically, had small and medium sized follicles. Ms 89, 91, and 69 had recent corpora lutea; M82 had an old small
Fig. 1. Section of ovary of adult monkey (86) after x-irradiation of ovaries and pituitary. Ovary removed one week after the last irradiation. The large follicles are in atresia; the smaller follicles seem unchanged. Young corpora atretica are scattered around. Wide vessels throughout the organ (X 16). Fig. 2. Ovary of adolescent monkey (115), x-rayed to ovaries and pituitary. The ovary was removed 4 weeks after the last irradiation. Follicle with antrum, containing many ova (X 115). Fig. 3. Ovary of monkey (100) x-rayed to the ovaries only and removed 4 weeks after the last irradiation, showing many corpora atretica. The small follicles are intact ( X 35). Fig. 4. Vessels around follicle in normal monkey ovary ( X 115) .
Vol. 10, No. 2, 1959
EFFECT OF X-RAYS ON PITUITARY AND OVARIES
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corpus luteum. Two of the 4 control monkeys ovulated during the same period. At microscopic examination an intense degeneration of the large follicles had occurred in all x-rayed ovaries, visible in the excessive number of young corpora atretica in the centrum and the high percentage of atretic follicles with antra (Fig. 1). Most of the nonatretic follicles with antra were small and often had only 2 layers of granulosa cells. They were apparently formed from small follicles in which cavitation had occurred prematurely; they were follicles with antra by definition only. Otherwise the smaller follicles seemed unaffected by the irradiation. There was an increased number of polyovular follicles, up to 31 ova in one follicle. The vascularization was intense, with wide vessels from the hilus and branches up to the periphery. Only M91 had no dilated vessels. Group II: Adolescent Monkeys, Irradiation to Ovaries and Pituitary Six adolescent monkeys about 2 years old, weighing 2500--3350 Gm., were observed for 2-4 months. Two monkeys developed sex skin during the observation period. Ovulation had not occurred according to the cornification curve of the vaginal lavages. On Day 10 of the cycle, if known, x-irradiation was begun and 100 r total were given to both the ovaries and the pituitary during the next 3 weeks. At the end of the fourth week, after the last irradiation, laparotomy was performed. The ovaries appeared macroscopically normal; one ovary of Mll5 contained a corpus luteum. Microscopically there was a large increase in the number of corpora atretica. The follicles with antra had an increased atresia; the smaller follicles seemed not influenced by the irradiation. Polyovular follicles were present with some follicles containing up to 11 ova (Fig. 2). The vessels were very wide (Figs. 5, 6, and 7). The monkeys that had developed sex skin before the experiment showed no increase after the irradiation. .The uteri had only slight estrogenic activity and straight glandular canals with high epithelial cells. No evidence of hormonal stimulation by the irradiation could be found. Group Ill: Adolescent Monkeys, Irradiation to Ovaries Five monkeys about 2 years old, weighing 287~350 Gm., were observed for several months and then x-irradiated with a 100 r tissue dose to the ovaries. During the observation period 3 monkeys developed moderate adolescent sex skin but had not yet ovulated, according to the vaginal lavages. Laparotomies were done 4 weeks after the last irradiation. On
Fig. 5. Ovary of adolescent monkey (125) removed 4 weeks after the last irradiaFig. 6. Detail of tion, showing large vessels around growing follicles (X 35). Fig. 5. ( X 115). Fig. 7. Adolescent monkey ( 115) x-rayed to the ovaries and pituitary. Ovary removed 4 weeks after the last irradiation. The section shows part of a corpus luteum with rich vascularization around it (X 35). Fig. 8. Adolescent monkey (102) x-rayed with one dose of 33.3 r to the ovaries only. Ovary removed on fourth day. Wide vessels around a degenerating follicle (X 115).
Vol. 10, No. 2, 1959
EFFECT OF X-RAYS ON PITUITARY AND OVARIES
197
macroscopic examination the ovaries had a normal appearance, follicles were visible, and there were no corpora lutea. Microscopically there were, :as in the previous groups, an excessive number of young corpora atretica .(Fig. 3). Counts were made of all follicles; their distribution over the different stages of development was normal (Fig. 11). There was an increased atresia in the follicles with antra (Fig. 12), but the atresia of the primary follicles was within normal limits. The vessels were widened. Many polyovular follicles were present, and the ovocytes in these follicles were generally small and underwent atresia in the stage of the primary follicle. Ovulation had not occurred in any of the ovaries. The uteri showed slight estrogenic activity. •Group IV: Adolescent Monkeys, Irradiation to Pituitary
Five animals, about 2 years old, weighing 2300-2925 Gm., were observed for 3-6 months. During this period adolescent sex skin developed and men.arche was observed in 2 of them. There had not yet been an ovulation. The :animals were x-rayed with a total dose of 100 r to the pituitary; 4-5 weeks .after the last treatment the ovaries and uteri were removed for microscopic .study. The ovaries were normal macroscopically and microscopically; they had follicles in all stages, no corpora lutea, and normal vascularization. The total number of ovocytes fell within normal limits and the distribution curve of the different groups of follicles was the same as in untreated monkeys (see Fig. 11). There was no stimulation of growth at any particular stage of the follicles, and there was a normal rate of atresia in all stages (see Fig. 12). The uteri were small and showed slight estrogenic activity. DISCUSSION
The first experiment on 12 adult monkeys gave information about the destructive action of this small amount of x-ray on the large follicles. During the months preceding the treatment, 11 of the 12 monkeys had not ovulated. Five monkeys started ovulation soon afterward, treated or nontreated. The experiment was therefore inconclusive so far as stimulation to ovulate was concerned. It is extremely difficult to find a group of anovulatory monkeys. We took the natural anovulatory period that occurs during the summer months, but this time is short for the experiment, and in our colony, in air-conditioned quarters with the same atmospheric conditions the year around, we occa-
198
VERMANDE-VAN ECK
Fertility & Sterility
sionally find monkeys ovulating during the summer. Another natural anovulatory period occurs during the first months after the menarche. For this reason we used adolescent monkeys for the rest of the experiment, most of them shortly after the first bleeding. Of the 6 monkeys of Group II, with irradiation to both the ovaries and the pituitary, the same destruction to the large follicles that was found in the adult animals was observable; there was no evidence of hormonal stimulation. There was, however, a marked hyperemia of the organ (Figs. 5 and 6). The monkeys treated only to the ovari~s showed essentially the same results, destruction of large follicles and vasodilatation. The ovaries of animals treated only to the pituitary showed no changes by microscopic examination. In the rhesus monkey, as in lower mammals, the most sensitive tissue in the ovary to x-irradiation is the large maturing follicle. The intense destruction of these follicles was striking in all x-rayed ovaries; one dose of 33.3 r was sufficient to bring it about (Figs. 9 and 10). In comparing normal and x-rayed monkeys with regard to the number of ovocytes divided over their different stages of development, 17 there appeared to be no difference (see Fig. 11 ) . Thus the irradiation neither induced a wave of development of primordial ovocytes, nor enhanced growth of the
Fig. 9. Adolescent monkey ( 102) 4 days after irradiation with one dose of 33.3 r to the ovaries. Extensive degeneration in large follicles (X 16). Fig. 10. Detail of Fig_ 9 (X 115).
A
Vol. 10, No. 2, 1959
EFFECT OF X-RAYS ON PITUITARY AND OVARIES
100
•
199
NORMAL
D PITUITARY
X-RAYED
~OVARY
..."',_
.,,.. g
80
eo
0
.J
,_c
e.
...
40
0
.. 20
GROUPS
Fig. ll. Distribution of ovocytes of the rhesus monkey according to the size of the follicle. Comparison of normal monkeys with monkeys with irradiation of the pituitary glands and monkeys with ovarian irradiation.
medium-sized follicles. The only change in development was the occurrence of cavities in young follicles, which often had only 2 layers of epithelial cells. In other words, the factors governing the growth of the follicle were not influenced by irradiation either to the ovary itself or to the pituitary, but irradiation to the ovary caused premature cavitation. In comparing normal and x-rayed monkeys with regard to the percentage of atretic follicles in their different states of development (see Fig. 12) there appeared to be an increased atresia after irradiation to the ovary, especially marked in the larger follicles. Group 7 in the chart represents follicles with 100
•
NORMAL
D PITUITARY ill OVARY
80
.....
......~
.."'
ISl PIT. 121 PIT.
a OY. a OY.
X-RAYED II
ADULT
II
80
c at 40
20
GROUPS
2
:s-s
•
7
Fig. 12. The percentages of atretic ova in the rhesus monkey. Distribution over groups according to size of follicle. Comparison of normal monkeys with monkeys x-rayed to the pituitary gland, to the ovaries, or both.
200
VERMANDE-VAN ECK
Fertility & Sterility
antra and includes small follicles with premature cavitation. Without these small follicles the column for atresia would reach nearly 100 per cent. The presence of the many young corpora atretica in the x-rayed ovaries indicated that the atresia started shortly after the irradiation and was still continuous 4 weeks later. This implies that the damage by irradiation is done not only to the large follicle but also to the ovocyte at a younger stage, and becomes evident later in the development. A discussion of the effect of x-rays on tissues in general and their effect in relation to the normal physiological changes in the ovary seems warranted at this point. In the normal ovary, growing young follicles are surrounded by capillaries. During the development of the follicle more and wider vessels can be observed surrounding it; large vessels from the medulla divide at the outside of the theca (see Fig. 4) and envelop the follicle in a network of capillaries. This intense blood supply is essential for its rapid growth and maturation. In the x-rayed ovary we see ( 1) a destruction of the large follicles and ( 2) a dilatation of the vessels from the third day until 4-6 weeks after the irradiation (see Figs. 5-8). This hyperemia is similar to that in other x-rayed tissues. Mter 6 weeks the hyperemia has passed off and an increase in connective tissue becomes prevalent. In the x-rayed ovary it is probably this short period of hyperemia that evokes the growth of new follicles after the large follicles have died because of their enhanced sensitivity to the x-rays. So in spite of the destruction and because of their temporarily increased nourishment, the smaller follicles will grow and, if not damaged, will be able to mature. The occurrence of polyovular follicles can possibly be explained in this way. The experiment could not show statistically that irradiation resulted in ovulation. We saw maturation of follicles in some of the adult animals but are inclined to say "in spite of the irradiation," since the irradiation, even at these low dosages, appeared to be so destructive to the large follicles. We could not evoke by x-rays maturation of the follicles in adolescent animals that normally would not yet have ovulated. It appears that the hyperemia cannot induce ovulation when the necessary pituitary gonadotropins are lacking. Irradiation of the pituitary with low dosage had no influence on this process at all. How may we apply this knowledge to the human female? It is likely that in women x-rayed with low dosages the same morphological changes that
.,.
Vol. 10, No. 2, 1959
EFFECT OF X-RAYS ON PITUITARY AND OVARIES
201
we saw in the rhesus monkey will occur. The hyperemia due to the irradiation may be sufficient in many cases to mature growing follicles, and in spite of the damaging influence on the large follicles, which degenerate, one of the smaller follicles will come to ovulation. The anovulatory cycle has been interrupted, and normal ovarian function may continue. In postulating these processes that probably occur in the ovary after x-irradiation, with its destructive as well as beneficial influence, it seems indicated to direct part of our research in this field to new methods of achieving hyperemia in the ovaries of anovulatory women, thus avoiding the hazards of x-rays. SUMMARY The purpose of the experiment was to reveal something of the unknown mechanism of the action of x-rays on ovarian function, as well as the nature of such modifications. Twelve adult and 18 adolescent monkeys were used. They received x-irradiation ( 100 r tissue doses) to the ovaries or pituitary gland. One group received 100 r to both ovaries and pituitary, one group to the ovaries only, and one group to the pituitary only. The main results of ovarian irradiation were an intensive destruction of the large follicles and hyperemia of the organ. The occurrence of ovulation was not statistically increased in adult animals, and occurred in only one adolescent animal. The irradiation of the pituitary gland had no effect on the ovary. Implications of these findings are discussed. REFERENCES
..
1. ABERCROMBIE, M. Estimation of nuclear population from microtome sections. Anat. Rec. 94:238, 1946. 2. BAIDINS, A., CLAESSON, L., and WESTMAN, A. Ueber der Einfluss der Roentgenbestrahlung auf die gonadotrope Funktion der Hypophyse. Gynaecologia 122: 347, 1946. 3. BLOOM, W., and BLooM, M.A. Histological changes after irradiation. Radiation · Biology I: 1091, 1954. 4. DESJARDIN, A. U. Stimulation and immunity in radiotherapy. ].A.M.A. 87:1537, 1926. 5. DESJARDIN, A. U. Radiosensitiveness of cells and tissues and some medical implications. Arch. Surg. 25:926, 1932. 6. DRIPS, D. G., and FoRD, F. A. The study of the effect of Roentgen rays on the estrual cycle and the ovaries of the white rat. Surg. Gynec. & Obst. 55:596, 1932. 7. EwiNG, J. Tissue reactions to radiation. Am.]. Roentgenol. 15:93, 1926. 8. FREED, J. M., et al. Effect of low dosage roentgen radiation on the gonadotropic fooction of the hypophysis of the mature and immature female albino rat. ]. Clin. Endocrinol. 8:461, 1948.
VERMANDE-VAN ECK
Fertility & Sterility
9; GELLER, F. C. Ueber die Wirkung schwacher Eierstockbestrahlung auf Grund tierexperimenteller Untersuchungen. Strahlentherapie 19:22, 1925. 10. GREEN, S. H., and ZucKERMAN, S. The number of oocytes in the mature rhesus monkey. ]. Endocrinol. 7:194, 1950. 11. HARTMAN, C. G., and SMITH, C. Non-effect of irradiation of the hypophysis in sterile monkey female. Proc. Soc. Exper. Biol. & Med. 39:330, 1938. 12. HoDES, E. G., and IsRAEL, S. L. Quoted by Freed, 1948. 13. LACASSAGNE, A., and GRICOUROFF, G. Actions des radiations sur les tissus. Paris, Masson, 1941. 14. KoTZ, J., ELWARD, J. F., and PARKER, E. Non-harmful effects of irradiation of the pituitary region in rabbits. Am.]. Roentgenol. 46:543, 1941. 15. ScHUGT, P. Untersuchungen tiber die Wirkung abgestuften Dosen von Roentgenstrahlen verschiedener Wellenlange auf die Struktur und Funktion der Ovarien. Strahlentherapie 27:603, 1927. 16. SHIELDS, W. Effects of radiation on normal tissues. Arch. Path. 34:1070, 1942. 17. 'V£RMANDE-VAN EcK, G. J. Neo-ovogenesis in the adult rhesus monkey. Anat. Rec. 125:201, 1956.
Pan-Pacific Surgical Association The Eighth Congress of the Pan-Pacific Surgical Association will be held in Honolulu, Hawaii, September 28 through October 5, 1960. All members of the profession are cordially invited to attend and are urged to make arrangements as soon as possible if they wish to be assured of adequate facilities. An outstanding scientific program by leading surgeons promises to be of interest to all doctors. Nine surgical specialty sections will be held simultaneously. Further information. and. brochures may be obtained by .wr,iting to Dr. F. J. Pinkerton, Director General of the Pan-Pacific Surgical Association, Suite·230, Alexander Young Building, Honolulu 13, Hawaii.
THE THERAPEUTIC usE OF SMALL nosEs of roentgen irradiation to the ovaries of the pituitary gland of amenorrheal women is somewhat controversial. Both the consistency of the immediate symptomatic improvement of the patient and the possible effects of irradiation on subsequent generations have been the subject of a number of publications. In general, gynecologists have been impressed by the results of such treatment, and geneticists have been skeptical. The radiation effect on tissues and organs was reviewed by Bloom and Bloom in 1954. Early studies were done by Desjardin, 4 ·" who concluded that cells cannot be stimulated in the sense that they acquire a greater ability to function normally; any such apparent stimulation is only a transient phase, invariably followed by more or less pronounced functional or organic deterioration. Ewing remarked that the initial hyperemia after x-irradiation was always followed by induration and relative anemia; the hyperemia was .an inflammatory process, with vasodilatation, exudation of serum, leukoFrom the Departments of Anatomy, and Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Conn. This investigation was supported by grants from the USPHS. The author expresses gratitude to Dr. William U. Gardner, Chairman of the Department of Anatomy, for his guidance and stimulating comments; to Dr. Chu H. Chang, Assistant Professor of Radiology, for his help in the radiological part of this study; to Dr. C. Lee Buxton, Chairman of the Department of Obstetrics and Gynecology, for his suggestions in the preparation of this paper; and to Mr. Edward Lopes for his technical assistance. Received for publication March 24, 1958. 190
Vol. 10, No. 2, 1959
EFFECT OF X-RAYS ON PITUITARY AND OVARIES
191
cytes, and red blood cells. Lacassagne and Gricouroff described the reaction of connective tissue to irradiation in an early phase as a type of inflammation, followed by sclerosis; collagenous and elastic fibers became swollen and hyalinized, and the collagen had an altered staining capacity. Shields mentioned the vasodilatation in the capillary loops of the skin as a reversible process in the lower dosage range; the period of vasodilatation lasted several days or weeks after the initial erythema, during which the dilator stimuli produced a greater effect on the vessels than the constrictor stimuli. The action of x-rays on the ovaries of lower mammals has been studied extensively and so far only destructive action has been found, even after low dosages, in such studies as those of Geller in rabbits and Schugt in mice, in which destruction of the follicle was apparent after a dosage of 21 r, and the studies of Drips and Ford in rats. The last authors 1emarked that in the first days after irradiation a marked hyperemia was present, together with degeneration of all large follicles. Studies of low-dosage irradiation to the pituitary in experimental animals are rather frequent and all had negative results as far as changes in the ovaries were concerned. 2 • 3 • 8 ' 11 • 14 It is known that high dosages of x-rays are needed to affect the pituitary. Combined ovarian and pituitary irradiation on rabbits by Hodes and Israel did not induce ovulation. The summarized experimental evidence shows that ovarian irradiation only is destructive and that pituitary irradiation, unless massive doses are used, has no influence on the ovary. What then is the paradoxical effect resulting in instigation of menstruation and ovulation in the human female? We have approached this problem by administering human dosage to the female monkey and by following the morphologic and physiologic changes that occurred after treatment. Our experiments were designed to reveal something of the unknown mechanism of the action of x-rays on ovarian function, as well as the nature of such modifications, for once we know the mode of action of x-irradiation we might be able to achieve the same effect by a less harmful method.
METHODS AND MATERIALS In the first experiment we took advantage of the natural anovulatory period of monkeys during the summer months. Twelve adult females were
192
VERMANDE-VAN ECK
Fertility & Sterility
selected for this purpose (4 of these were used as controls). Another anovulatory period occurs in the adolescent monkey during the first months after the menarche, and this was used in a second group of 18 monkeys. Both groups received x-irradiation to the ovaries and/ or pituitary; factors were 250 kv., 15 rnA., 50 em. target distance, half value layer of 3 mm. Cu. The field entrance to the ovaries was 10 x 10 em. from an abdominal port, and to the pituitary a circular port of 4 em. diameter, alternately from the left and right side. Three irradiations of 33.3 r each were given at weekly intervals to the ovaries and/ or the pituitary; the organ dose was 100 r total to ovaries and to pituitary. The irradiation was, if possible, started during the first half of the menstrual cycle. Records were kept of sex skin changes and vaginal lavages. In the adult animals the ovaries and uterus were palpated daily per rectum in order to detect the ovulation directly. The ovaries and uteri were removed 1-6 weeks after the last irradiation. The tissues were fixed in Bouin's solution, cut in serial sections, and stained with hematoxylineosin or VanGieson stain. Two groups of monkeys (adult and adolescent) received x-irradiation to both ovaries and the pituitary, a third group only to the ovaries, and a fourth group only to the pituitary. The ovaries were studied for the distribution of follicles in all stages of development. The technique of Green and Zuckerman was used with the following classifications: 1. Primordial ova with complete cover of flattened cells, or incomplete
2. 3. 4. 5. 6. 7.
epithelial cover. Ova completely surrounded by I layer of cuboidal cells. Ova completely surrounded by 2layers of cuboidal cells. Ova completely surrounded by 3 layers of cuboidal cells. Ova completely surrounded by 4-5layers of cuboidal cells. Ova completely surrounded by 6 or more layers of cuboidal cells. Follicles with antra.
Where counts were made of primordial ovocytes and the ovocytes in the small follicles, every fortieth section was counted. For the larger follicles ( 6 and 7) every section was screened. Ovocytes were counted only when the nucleolus was visible. A correction factor for the thickness of the section was applied. 1 Special attention was paid to the atresia of the ovocytes, the occurrence of degeneration of the follicles, of polyovular follicles, and the vascular supply.
Vol. 10, No. 2, 1959
TABLE 1.
EFFECT OF X-RAYS ON PITUITARY AND OVARIES
Changes in Ovaries of Rhesus Monkeys after Low Dosage X-irradiation
Mon- Age key (yrs.) Controls
Group
70 77 I
90 96 69
82 85 86 88 89 91 93 Group II
115 116 114
124 125 Group III
126 100 101
109 112 83
123 Group IV
193
104 105 106 113 127
3 3 3 3 3 3~
3 3~
3 3~
3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
In~Degen. X-rayed teroal foU. 100 r (wks.) Grp. 6 Grp. 7 Corp. atr. None None None None ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. & pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ov. &pit. ovaries ovaries ovaries ovaries ovaries ovaries pituitary pituitary pituitary pituitary pituitary
3 3 1 1 3 3 6 1
4 4 4 4 4 4 4 4 4 4 4 4
5 4 4 4 4
50 36 16
39
33
47
68 63
32
60
40 27
54 50 51
33 37 33
15 61 25
60
58 72 64 73 81
20
70 40 70
20
60
20 12 15 20
37 30
25
10
10 3
70 40 40 36
Few Few Average Few Many Many Few Many Many Many Many Many Many Many Many Many Many Many Many Many Many Average Many Many Few Few Few Few Few
Polyov. foll. C. L. None Few None None Few Few Few Few Many Many Many Few Many Many Few Many Many Few Many Many Few Few None None None None None None None
None 1 None 1 1
!Old None None None 1 1
None 1 None None None None None None None None None None None None None None None None
Vessels Average Average Small Average Wide Wide Wide Wide Wide Wide Average Wide Wide Wide Wide Wide Wide Wide Wide Wide Wide Wide Average Wide Average Average Average Average Average
OBSERVATIONS
Group 1: Adult Monkeys, Irradiation to Ovaries and Pituitary Twelve adult monkeys (M), 3-3* years ·old (estimated by Dr. G. Van Wagenen, according to dentition, sitting height, and growth pattern of hand and foot) were observed for several months. During the summer the animals had regular menstrual cycles but ovulations were not detected. In the late summer irradiation was given to ovaries and pituitary. The size of the ovaries and uterus was followed by rectal palpations. Ovulations were felt in Ms 89 and 91; M69 was not palpated because of an anal stricture. In the other monkeys no ovulation was felt. Laparotomy was done 1--6 weeks after the last irradiation. All ovaries, macroscopically, had small and medium sized follicles. Ms 89, 91, and 69 had recent corpora lutea; M82 had an old small
Fig. 1. Section of ovary of adult monkey (86) after x-irradiation of ovaries and pituitary. Ovary removed one week after the last irradiation. The large follicles are in atresia; the smaller follicles seem unchanged. Young corpora atretica are scattered around. Wide vessels throughout the organ (X 16). Fig. 2. Ovary of adolescent monkey (115), x-rayed to ovaries and pituitary. The ovary was removed 4 weeks after the last irradiation. Follicle with antrum, containing many ova (X 115). Fig. 3. Ovary of monkey (100) x-rayed to the ovaries only and removed 4 weeks after the last irradiation, showing many corpora atretica. The small follicles are intact ( X 35). Fig. 4. Vessels around follicle in normal monkey ovary ( X 115) .
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corpus luteum. Two of the 4 control monkeys ovulated during the same period. At microscopic examination an intense degeneration of the large follicles had occurred in all x-rayed ovaries, visible in the excessive number of young corpora atretica in the centrum and the high percentage of atretic follicles with antra (Fig. 1). Most of the nonatretic follicles with antra were small and often had only 2 layers of granulosa cells. They were apparently formed from small follicles in which cavitation had occurred prematurely; they were follicles with antra by definition only. Otherwise the smaller follicles seemed unaffected by the irradiation. There was an increased number of polyovular follicles, up to 31 ova in one follicle. The vascularization was intense, with wide vessels from the hilus and branches up to the periphery. Only M91 had no dilated vessels. Group II: Adolescent Monkeys, Irradiation to Ovaries and Pituitary Six adolescent monkeys about 2 years old, weighing 2500--3350 Gm., were observed for 2-4 months. Two monkeys developed sex skin during the observation period. Ovulation had not occurred according to the cornification curve of the vaginal lavages. On Day 10 of the cycle, if known, x-irradiation was begun and 100 r total were given to both the ovaries and the pituitary during the next 3 weeks. At the end of the fourth week, after the last irradiation, laparotomy was performed. The ovaries appeared macroscopically normal; one ovary of Mll5 contained a corpus luteum. Microscopically there was a large increase in the number of corpora atretica. The follicles with antra had an increased atresia; the smaller follicles seemed not influenced by the irradiation. Polyovular follicles were present with some follicles containing up to 11 ova (Fig. 2). The vessels were very wide (Figs. 5, 6, and 7). The monkeys that had developed sex skin before the experiment showed no increase after the irradiation. .The uteri had only slight estrogenic activity and straight glandular canals with high epithelial cells. No evidence of hormonal stimulation by the irradiation could be found. Group Ill: Adolescent Monkeys, Irradiation to Ovaries Five monkeys about 2 years old, weighing 287~350 Gm., were observed for several months and then x-irradiated with a 100 r tissue dose to the ovaries. During the observation period 3 monkeys developed moderate adolescent sex skin but had not yet ovulated, according to the vaginal lavages. Laparotomies were done 4 weeks after the last irradiation. On
Fig. 5. Ovary of adolescent monkey (125) removed 4 weeks after the last irradiaFig. 6. Detail of tion, showing large vessels around growing follicles (X 35). Fig. 5. ( X 115). Fig. 7. Adolescent monkey ( 115) x-rayed to the ovaries and pituitary. Ovary removed 4 weeks after the last irradiation. The section shows part of a corpus luteum with rich vascularization around it (X 35). Fig. 8. Adolescent monkey (102) x-rayed with one dose of 33.3 r to the ovaries only. Ovary removed on fourth day. Wide vessels around a degenerating follicle (X 115).
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macroscopic examination the ovaries had a normal appearance, follicles were visible, and there were no corpora lutea. Microscopically there were, :as in the previous groups, an excessive number of young corpora atretica .(Fig. 3). Counts were made of all follicles; their distribution over the different stages of development was normal (Fig. 11). There was an increased atresia in the follicles with antra (Fig. 12), but the atresia of the primary follicles was within normal limits. The vessels were widened. Many polyovular follicles were present, and the ovocytes in these follicles were generally small and underwent atresia in the stage of the primary follicle. Ovulation had not occurred in any of the ovaries. The uteri showed slight estrogenic activity. •Group IV: Adolescent Monkeys, Irradiation to Pituitary
Five animals, about 2 years old, weighing 2300-2925 Gm., were observed for 3-6 months. During this period adolescent sex skin developed and men.arche was observed in 2 of them. There had not yet been an ovulation. The :animals were x-rayed with a total dose of 100 r to the pituitary; 4-5 weeks .after the last treatment the ovaries and uteri were removed for microscopic .study. The ovaries were normal macroscopically and microscopically; they had follicles in all stages, no corpora lutea, and normal vascularization. The total number of ovocytes fell within normal limits and the distribution curve of the different groups of follicles was the same as in untreated monkeys (see Fig. 11). There was no stimulation of growth at any particular stage of the follicles, and there was a normal rate of atresia in all stages (see Fig. 12). The uteri were small and showed slight estrogenic activity. DISCUSSION
The first experiment on 12 adult monkeys gave information about the destructive action of this small amount of x-ray on the large follicles. During the months preceding the treatment, 11 of the 12 monkeys had not ovulated. Five monkeys started ovulation soon afterward, treated or nontreated. The experiment was therefore inconclusive so far as stimulation to ovulate was concerned. It is extremely difficult to find a group of anovulatory monkeys. We took the natural anovulatory period that occurs during the summer months, but this time is short for the experiment, and in our colony, in air-conditioned quarters with the same atmospheric conditions the year around, we occa-
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sionally find monkeys ovulating during the summer. Another natural anovulatory period occurs during the first months after the menarche. For this reason we used adolescent monkeys for the rest of the experiment, most of them shortly after the first bleeding. Of the 6 monkeys of Group II, with irradiation to both the ovaries and the pituitary, the same destruction to the large follicles that was found in the adult animals was observable; there was no evidence of hormonal stimulation. There was, however, a marked hyperemia of the organ (Figs. 5 and 6). The monkeys treated only to the ovari~s showed essentially the same results, destruction of large follicles and vasodilatation. The ovaries of animals treated only to the pituitary showed no changes by microscopic examination. In the rhesus monkey, as in lower mammals, the most sensitive tissue in the ovary to x-irradiation is the large maturing follicle. The intense destruction of these follicles was striking in all x-rayed ovaries; one dose of 33.3 r was sufficient to bring it about (Figs. 9 and 10). In comparing normal and x-rayed monkeys with regard to the number of ovocytes divided over their different stages of development, 17 there appeared to be no difference (see Fig. 11 ) . Thus the irradiation neither induced a wave of development of primordial ovocytes, nor enhanced growth of the
Fig. 9. Adolescent monkey ( 102) 4 days after irradiation with one dose of 33.3 r to the ovaries. Extensive degeneration in large follicles (X 16). Fig. 10. Detail of Fig_ 9 (X 115).
A
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•
199
NORMAL
D PITUITARY
X-RAYED
~OVARY
..."',_
.,,.. g
80
eo
0
.J
,_c
e.
...
40
0
.. 20
GROUPS
Fig. ll. Distribution of ovocytes of the rhesus monkey according to the size of the follicle. Comparison of normal monkeys with monkeys with irradiation of the pituitary glands and monkeys with ovarian irradiation.
medium-sized follicles. The only change in development was the occurrence of cavities in young follicles, which often had only 2 layers of epithelial cells. In other words, the factors governing the growth of the follicle were not influenced by irradiation either to the ovary itself or to the pituitary, but irradiation to the ovary caused premature cavitation. In comparing normal and x-rayed monkeys with regard to the percentage of atretic follicles in their different states of development (see Fig. 12) there appeared to be an increased atresia after irradiation to the ovary, especially marked in the larger follicles. Group 7 in the chart represents follicles with 100
•
NORMAL
D PITUITARY ill OVARY
80
.....
......~
.."'
ISl PIT. 121 PIT.
a OY. a OY.
X-RAYED II
ADULT
II
80
c at 40
20
GROUPS
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:s-s
•
7
Fig. 12. The percentages of atretic ova in the rhesus monkey. Distribution over groups according to size of follicle. Comparison of normal monkeys with monkeys x-rayed to the pituitary gland, to the ovaries, or both.
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antra and includes small follicles with premature cavitation. Without these small follicles the column for atresia would reach nearly 100 per cent. The presence of the many young corpora atretica in the x-rayed ovaries indicated that the atresia started shortly after the irradiation and was still continuous 4 weeks later. This implies that the damage by irradiation is done not only to the large follicle but also to the ovocyte at a younger stage, and becomes evident later in the development. A discussion of the effect of x-rays on tissues in general and their effect in relation to the normal physiological changes in the ovary seems warranted at this point. In the normal ovary, growing young follicles are surrounded by capillaries. During the development of the follicle more and wider vessels can be observed surrounding it; large vessels from the medulla divide at the outside of the theca (see Fig. 4) and envelop the follicle in a network of capillaries. This intense blood supply is essential for its rapid growth and maturation. In the x-rayed ovary we see ( 1) a destruction of the large follicles and ( 2) a dilatation of the vessels from the third day until 4-6 weeks after the irradiation (see Figs. 5-8). This hyperemia is similar to that in other x-rayed tissues. Mter 6 weeks the hyperemia has passed off and an increase in connective tissue becomes prevalent. In the x-rayed ovary it is probably this short period of hyperemia that evokes the growth of new follicles after the large follicles have died because of their enhanced sensitivity to the x-rays. So in spite of the destruction and because of their temporarily increased nourishment, the smaller follicles will grow and, if not damaged, will be able to mature. The occurrence of polyovular follicles can possibly be explained in this way. The experiment could not show statistically that irradiation resulted in ovulation. We saw maturation of follicles in some of the adult animals but are inclined to say "in spite of the irradiation," since the irradiation, even at these low dosages, appeared to be so destructive to the large follicles. We could not evoke by x-rays maturation of the follicles in adolescent animals that normally would not yet have ovulated. It appears that the hyperemia cannot induce ovulation when the necessary pituitary gonadotropins are lacking. Irradiation of the pituitary with low dosage had no influence on this process at all. How may we apply this knowledge to the human female? It is likely that in women x-rayed with low dosages the same morphological changes that
.,.
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we saw in the rhesus monkey will occur. The hyperemia due to the irradiation may be sufficient in many cases to mature growing follicles, and in spite of the damaging influence on the large follicles, which degenerate, one of the smaller follicles will come to ovulation. The anovulatory cycle has been interrupted, and normal ovarian function may continue. In postulating these processes that probably occur in the ovary after x-irradiation, with its destructive as well as beneficial influence, it seems indicated to direct part of our research in this field to new methods of achieving hyperemia in the ovaries of anovulatory women, thus avoiding the hazards of x-rays. SUMMARY The purpose of the experiment was to reveal something of the unknown mechanism of the action of x-rays on ovarian function, as well as the nature of such modifications. Twelve adult and 18 adolescent monkeys were used. They received x-irradiation ( 100 r tissue doses) to the ovaries or pituitary gland. One group received 100 r to both ovaries and pituitary, one group to the ovaries only, and one group to the pituitary only. The main results of ovarian irradiation were an intensive destruction of the large follicles and hyperemia of the organ. The occurrence of ovulation was not statistically increased in adult animals, and occurred in only one adolescent animal. The irradiation of the pituitary gland had no effect on the ovary. Implications of these findings are discussed. REFERENCES
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1. ABERCROMBIE, M. Estimation of nuclear population from microtome sections. Anat. Rec. 94:238, 1946. 2. BAIDINS, A., CLAESSON, L., and WESTMAN, A. Ueber der Einfluss der Roentgenbestrahlung auf die gonadotrope Funktion der Hypophyse. Gynaecologia 122: 347, 1946. 3. BLOOM, W., and BLooM, M.A. Histological changes after irradiation. Radiation · Biology I: 1091, 1954. 4. DESJARDIN, A. U. Stimulation and immunity in radiotherapy. ].A.M.A. 87:1537, 1926. 5. DESJARDIN, A. U. Radiosensitiveness of cells and tissues and some medical implications. Arch. Surg. 25:926, 1932. 6. DRIPS, D. G., and FoRD, F. A. The study of the effect of Roentgen rays on the estrual cycle and the ovaries of the white rat. Surg. Gynec. & Obst. 55:596, 1932. 7. EwiNG, J. Tissue reactions to radiation. Am.]. Roentgenol. 15:93, 1926. 8. FREED, J. M., et al. Effect of low dosage roentgen radiation on the gonadotropic fooction of the hypophysis of the mature and immature female albino rat. ]. Clin. Endocrinol. 8:461, 1948.
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9; GELLER, F. C. Ueber die Wirkung schwacher Eierstockbestrahlung auf Grund tierexperimenteller Untersuchungen. Strahlentherapie 19:22, 1925. 10. GREEN, S. H., and ZucKERMAN, S. The number of oocytes in the mature rhesus monkey. ]. Endocrinol. 7:194, 1950. 11. HARTMAN, C. G., and SMITH, C. Non-effect of irradiation of the hypophysis in sterile monkey female. Proc. Soc. Exper. Biol. & Med. 39:330, 1938. 12. HoDES, E. G., and IsRAEL, S. L. Quoted by Freed, 1948. 13. LACASSAGNE, A., and GRICOUROFF, G. Actions des radiations sur les tissus. Paris, Masson, 1941. 14. KoTZ, J., ELWARD, J. F., and PARKER, E. Non-harmful effects of irradiation of the pituitary region in rabbits. Am.]. Roentgenol. 46:543, 1941. 15. ScHUGT, P. Untersuchungen tiber die Wirkung abgestuften Dosen von Roentgenstrahlen verschiedener Wellenlange auf die Struktur und Funktion der Ovarien. Strahlentherapie 27:603, 1927. 16. SHIELDS, W. Effects of radiation on normal tissues. Arch. Path. 34:1070, 1942. 17. 'V£RMANDE-VAN EcK, G. J. Neo-ovogenesis in the adult rhesus monkey. Anat. Rec. 125:201, 1956.
Pan-Pacific Surgical Association The Eighth Congress of the Pan-Pacific Surgical Association will be held in Honolulu, Hawaii, September 28 through October 5, 1960. All members of the profession are cordially invited to attend and are urged to make arrangements as soon as possible if they wish to be assured of adequate facilities. An outstanding scientific program by leading surgeons promises to be of interest to all doctors. Nine surgical specialty sections will be held simultaneously. Further information. and. brochures may be obtained by .wr,iting to Dr. F. J. Pinkerton, Director General of the Pan-Pacific Surgical Association, Suite·230, Alexander Young Building, Honolulu 13, Hawaii.