OBSERVATIONS ON THE HISTOGENESIS OF OVARIAN TUMORS PRODUCED IN MICE BY X-RAYS J. S. BUTTERWORTH, M.D. (Frorr~the Urfiarinzerzt of Pathology, Cornrll Ultiver,\ity Mrrlicul College, NFW York) The occurrence of ovarian tumors in x-rayed mice has been described in a previous paper (1). These tumors were an incidental finding in a colony of 2068 nice, of which 775 were irradiated to increase the incidence of leu- kemia. The ovarian tumors were 15 times more frequent in the irradiated stock than in the control stock (2). In general the tumors were of two types: granulosa-cell tumors, including luteomata, and tubular adenomata.' All of them occurred in senile mice and, with the exception of the tubular adenomata, the same types were found in smaller numbers in the non-irradiated control mice. The present paper deals with the histogenesis of these tumors, which, like the histogenesis of the components of the normal ovary, has long been a perplexing problem. Granulosa-cell Tumors: Considerable interest has recently been shown in granulosa-cell tumors of the ovary because of their endocrine activity. Von Kahlden (4) and Mengershausen (5) during the last decade of the nineteenth century were among the first to describe this type of tumor. Although it is a rather uncommon tumor in women, Bland and Goldstein (6) have recently reviewed the literature of the subject up to 1935, and found reports of 248 cases. Many different names have been applied to the growth, but the term granulosa-cell tumor, supplied by Werdt (7), is now in general use. The endocrine significance of the granulosa-cell tumor was first recognized by Schroder (8), Neumann (9), and Robert Meyer (lo), who reported cases showing cystic hyperplasia of the uterine mucosa, hypertrophy of the uterus, metrorrhagia, and other signs of excessive hormone production. Hormones have been demonstrated experimentally by injecting extracts of the tumors into immature or ovariectomized mice ( 11, 12, 13). The human granulosa-cell tumors are comn~onlysupposed to be derived from embryonic rests of granulosa cells, as first suggested by Robert Meyer in 1918 (14). His concept is based upon the observation that mature follicle cells proliferate only in association with ova, and upon the finding of nests of granulosa cells (Robert Meyer, 12) and of a small granulosa-cell tumor (Te Linde, 15) in the medulla of adult ovaries. Granulosa cells located in this I This investigation has been supported by a grant from the International Cancer Research Foundation. 2 These tumors differ from the aclenoma tubulare ovarii testiculare first described by Pick (3) in that the cells are smaller and low-cuboidal in type, with scanty cytoplasm, and bear 110 re- semblance to the tubules of the testes. 86 J. S. BUTTERWORTH region are believed by most authors to be embryonic rests and not derivatives of adult follicle cells. The occurrence of a large proportion of these tumors after the menopause, when normal follicles are absent, is often cited in sup- port of this theory. Robinson (16), however, has taken exception to this theory and believes that granulosa-cell tumors develop from the epithelium of the ovarian follicles. In an ovary with a granulosa-cell tumor he describes, in the part not occupied by the tumor, follicles of which granulosa cells were undergoing proliferation and invading the stroma. Ewing and Schroder concur in this view. For further reference to literature on granulosa-cell tumors the reader is referred to the paper by Bland and Goldstein (6). The luteoma is a rare type of ovarian tumor. In 1921 Glynn (17) col- lected 14 cases of lutein-cell tumors which had been reported as " ovarian hypernephromata." He believes that there has been no verified example of hypernephroma in the ovary. Novak and Te Linde (18) accept most of Glynn's cases as true luteomata. Ewing (19) states that the peculiar structure of the tumor makes its origin from lutein cells probable, although no positive proof of the relation has been furnished. Novak and Brawner (20) and others have recently suggested that luteomata arise from lutein transformation of granulosa-cell tumors. The similarity of the human and mouse granulosa-cell tumors has recently been pointed out by Dr. H. F. Traut (2 1). Eflect of X-Rays on the Ovary: The effect of x-rays on the ovaries of both immature and adult mice has been carefully studied by Brambell, Field- ing and Parkes (22-26). Their work, however, covered a period of only four or five months after irradiation and no mention is made of the changes in the ovaries of senile mice irradiated early in life. When the mice were irradiated at three weeks of age (puberty occurs at six to seven weeks of age in the mouse) with a sterilizing dose of x-rays, Bram- bell, Fielding and Parkes (22) found that the graafian follicles underwent complete atrophy and were usually entirely reabsorbed. In a few cases some of the larger follicles were invaded by growth of the theca interna and mem- brana granulosa and eventually formed corpora lutea atretica which persisted indefinitely but seemed to have no physiological function. As these degenerative processes occurred, the germinal epithelium prolif. erated, forming a parenchymatous tissue which was a usual constituent of the ovary sterilized before puberty. In some cases this tissue became luteinized so that the entire ovary resembled a single large corpus luteum and inhibition of the estrous cycle occurred. Occasionally a second proliferation occurred from the germinal epithelium which formed cords of cells resembling anovular follicles . These authors also report three cases in which another kind of proliferation of the germinal epithelium occurred and resulted in the formation of epithelial canals, each of which, growing into the ovary, carried with it a layer of the tunica albuginea cells as a sheath. These canals were found in mice 98, 98 and 195 days after irradiation. The authors offer no explanation for these " anomalous " structures. Irradiation of the adult mouse (24) led to approximately the same histo- logical changes, but in this case the tissues forming the cortex of the ovary at the time of irradiation constituted the bulk of the ovary after irradiation. All ova disappeared and the ovary ultimately consisted of tissue derived from cells of anovular follicles formed by disintegration of the ovum and prolifera- tion of the membrana granulosa of primordial and mature follicles. These tis- sues seldom underwent lutein transformation in the adult irradiated ovaries. Parkes (27) has found that the estrous cycle persists in animals with ir- radiated ovaries, even after complete disappearance of all normal follicles. This is attributed to the elaboration of estrus-producing hormones by the cells that proliferate in the sterilized immature animals shortly after irradiation, and by the cells derived from follicles in the sterilized adult animals. The material used in this investigation was from three series of experi- ments : (1) The ovarian tumors previously reported (1) were studied from the point of view of histogenesis. (2) Thirty-nine female mice were irradiated at about sixty days of age and some of them were killed; the ovaries of others were removed by biopsy, at intervals from 3 days to 412 days after irradiation. As controls, 10 female mice of the same strains and the same age were kept under identical conditions. Part were killed; the ovaries of the others were removed by biopsy, at intervals of 35 to 320 days after irradiation. These irradiated and control female mice were never in contact with male mice. (3) Fifty-two female mice irradiated at ages varying from 30 to 130 days, and 15 non-irradiated female mice of various strains were killed or died at intervals from 17 to 745 days after irradiation and were used to study the later effects of irradiation. The tissues were ~reservedin Kaiserling's fixative and were stained with hematoxylin and eosin. In a few cases sections were stained for fat with Sudan 111. In several instances interrupted serial sections of the ovaries were made, and the hypophyses, adrenals, uterine horns, and vaginas of many animals were sectioned. The mice were irradiated over the entire body in groups of 15 to 25. The irradiation factors were as follows: 190 kv., 30 ma., 50 cm. distance, 0.5 mm Cu + 1 mm. Al. Three of the mice in this series were exposed to gamma rays emanating from 0.1 mg. of radium. These mice were exposed from the ages of 47, 60, and 64 days to the ages of 377, 676, and 495 days respectively, according to a technic previously described (28). In Table I all irradiated mice in the second and third series are grouped in order of the time which elapsed between the first irradiation and the ex- amination of the ovaries. Many of the mice were exposed to x-rays more than once and each figure in the fourth column indicates a separate dose. Multiple doses were given a month or more apart. The last column in the table indi- J. S. BUTTERWORTH TABLEI Mice Ex@osed to X-Rays Time in days between Age in days Doses of Degree of Mouse irradiation and at first x-rays in proliferation No. examination of irradiation r units of germinal the ovaries epithelium Af 644 200 0 Ak 375 200 0 Af 665 200 0 Af 669 200 0 Af 656 200 0 Slb 181 50-50-50 0 Arb 157 50 X 4 0 Af 666 200-300 0 Af 677 200-250 0 Af 640 200-400 0 Ak 370 200-300 0 Af 679 200-300 0 Af 633 200-400 0 Ak 366 200-400 f Af 674 200-400 0 Ak 383 200-300 0 Af 680 200-300 0 Rg 326 400 0 Rg 337 400 0 Rg 329 400 0 Rg 338 400 f Af 665 200-400 0 Rg 336 400 0 Rg 345 400 0 Af 655 200400 0 Ak 388 200-300 + A 9238 400 +f Ak 372 200-300 0 Rg 334 400 + Af 649 200-400 + Ak 379 200-400 + Rg 331 400 + Ak 375 200-300 + Af 653 200-400 + Rg 332 400 + Af 674 200-400 ++++ Af 681 200-400 f Rg 333 400 0 Af 670 200400 + Aqe 17 200-250 ++ Aqe 20 200-250 +++ Af 671 200-400 ++ Af 644 200-400 + Rfb 124 400 ++ Rgc 38 200-300 + Rfb 89 450 + cates the extent of tubular down-growth of the germinal epithelium: + indi- cates a thickened epithelium with no definite down-growths; + indicates short down-growths; $ + and ++ longer down-growths ; ++ + $.