Transplantable Luteoma in Mice and Associated Secondary Changes*

J. Furth, M.D., and H. Sobel, Ph.D.**

(From The Department of Patholo~,, Cornell UniversiD" Medical College, New York Hospital, New York 21, N. Y., and The Marine Biological Laboratory, ~/oods Hole, Massachusetts) (Received for publication October 14, 1946)

From the standpoint of oncology lutein cells are of ORIGIN AND TRANSPLANTATION OF LUTEO3,IA an imusual sort: they are not present at birth and (STRAIN IX) appcar when pituitary stimuli awaken the ovary to cyclic activity. Their mother cells are those sur- The lutcoma from which strain IX originated rounding thc ova, and following completion of a was induced by irradiation with 175 r of a 5 week cycle they gradually vanish. They are commonly re- old female mouse, an Rf/Ak hybrid. During the garded as granulosa or thcea cells modified by hv- week following irradiation, this mouse was also pophyseal hormones. This modification is profound, "painted" twice with a 1 per cent solution of me- for with it goes the acquired ability to produce thylcholanthrene in benzene (20). About one year progcstins, not elaborated by the mother cell. It after irradiation a laparotomy was performed and a has been doubtcd that they are capable of indepen- yellow, soft tumor, found at the site of the left dent existence and of giving rise to tumors. ovary, was removed with part of the uterine horn. The experiments to be described indicate that 'Iqais tumor measured 1.8 X 1.5 cm. in its two the lutcin cell can undergo a neoplastic change, and greatest diameters. The right ovary appeared normal; when this happens it becomes of a fixed type, and the left uterine horn was thickened, measuring 3 its relation to granulosa cells is no longer apparent. to 4 ram. in diameter. Two days later the mouse This neoplastic cell can retain the powcr of secret- was found dead, and bccause of advanced postmor- ing hormones--secnlingly dischargcd continuously tem decomposition it was discarded without re- and in large quantities--leading to secondary moving tissues for extensive microscopic study. A changes hitherto not dcfinitcly associated with these small nodule present in the lung proved to 1)e a cells. papillary adenoma. The very cxistcnce of luteoma is under contro- The ovarian tumor was cut into small fragments versy (6, 23 ,42, 47, '/8). The number of cascs of and injected into thc subcutaneous tissue of normal luteoma on record is small, and the authenticity of and pre-irradiated mice. many of the cases reported is uncertain. Most Table I shows that the results of the transplanta- workers suppose that luteomas are the irreversible tion expcrimcnts were successful in the original end-phase of the luteinization of granulosa cell tu- passage and that the takes varied from 0 to 100 per mors and therefore not a distinct neoplasm (36). cent in later passages. The Table indicates that the The masculinizing luteomas are generally believed luteoma is transplantable about equally to mice of to be adrenal cell rests. both sexes. In the course of successive transplantation there was a conspicuous increase in the growth The experimental production (18) and trans- rate of the tumor cells as indicated by the decrease plantation (17) of luteomas has led to a better of both the incubation and tumor-bearing periods. understanding of this hitherto debatable tumor. The cause of the failures of some attempted passages is obscure. It may have been due to poor * These investigations have been supported by The selection of tumor material; e.g., inclusion of some Donner Foundation, The National Advisory Cancer Coun- cil and The Anna Fuller Fund. necrotic parts in the inoculum, or lack of genetic * * Research Fellow of The David, Josephine and Win- uniformity of the recipient mice. The ovarian tu- field Baird Foundation. mors were originally induced in Ak/Rf hybrids and 246

TABLE I: RESULTS OF IMPLANTATION OF LUrEOMA INTO RELATED MInE

No. of Success of Incubation period, Tumor-bearing period, passage implantation days days I 4/4 5/5 83-350 (242) 116-167 (134) K 154,183 K 33,140 D 117 IIa 3/4 2/2 121-267 (198) 64-146(104) K 215-233 (224) D 129,283 IIb 3/3 3/3 45 45 D 66-124,(88) .17) 40-87 (68) #6/6 #45-58 (49) #D 40-109 (69) IIc 7/7 9/9 38-64 (53) 38-165 (71) D 71-122 (85) D 62-115 (84) K 47, 156 K 112, 123 "1/2 *2/2 "115 "81 *D 129 *D 53-79 (66) lld 2/8 6/9 125,160 (143) 77-117(109) D 22-60 (39) ilc 6/7 1/6 10-67 (33) 37 D 42-53 (48) D 43 K 73, 120 IIf 3/~ 7/8 73-95 (84) 45-72 (56) K 100, 146 D 68-110 (88) K 124 Ilia 3/6 1/7 14-29 (19) 56 K 97-118 (109) D 56 Illb 8/9 23 D 27-51 (39) K 39, 82 IIIc #2/6 #60,75 (68) K 19 D 57 IIId #0/2 #4/6 #35-69 (52) #D 68-89 (80) 4/4 58-69 (61) D 82-90 (86) IIIe 1/7 5/7 56 36-~6 (44) D 48 D 28-54 (41) K 35-146 (93) IVa 6/'8 13/14 37-110 (59) 21-74 (48) D 56 IVb 1/6 46 K 58 K 24-25 (35) V 7/7 0/4 39-82 (49) D 45-101 (73)

All reeilfiem mice were of same stock in which luteomn arose and were, about 5 to 13 weeks ohl at time of injection. The denominator intlicalvs lolal nmnlwr of mice ilqectcd with tmlmr cells and the nmnerator, the number in which the grafts took. The incub.ltion period is the inierval in clays between injection and apI)earanee of tumor. The table does not list 4 x-rayed mice inj(~,t.ed during Passage I, all of which were, grafted sueeessfully; and 10 x-rayexl mic'e of Passage lla, 4 of which were successfully grafted. A few figures on inmllmtion and tumor-b~eu'ing periods were omitted because they were not availal)le or not de, pr .~--mice subjected to gonadectomy at about the time of injection. * =these implantations were intra-splenie, all others subcutaneous. K = Killed. D =died. as recipients Ak/Rf hybrids were used. However, same passage, which averaged 198 days in males both the Ak and Rf inbred lines were broken down and 246 days in females. into sublines several years ago. In the present ex- The rationale of grafting tumor cells on x-rayed periments no special consideration was given to mice was as follows. Since the tumors arose in mice these sublines and therefore the recipient animals long after irradiation with x-rays it was thought that were related but not of a single line. these tumor cells might proliferate in such mice but The original transfer was also made into 4 female not in normal animals. Furthermore it is well known mice that had been irradiated with 350 r over the that x-rays enhance susceptibility to tumor grafts entire body 4 months prior to injection. The tunmrs (35). However, since the tumors grew readily in in these mice were palpable 85 days after trans- normal mice of both sexes no further implantations plantation, a latent period considerably shorter than were made in x-rayed mice. that in non-x-rayed young female siblings in which Metastases were not observed in mice that re- the tumor was palpable in 116 to 167 days (aveiage ceived subcutaneous implantations of the tumor, of 134). In the second passage (IIa) a luteoma was but of 3 mice in which the tumors were grafted in also grafted into six 14 months old male mice that the spleen 2 had metastases to the liver. had been irradiated 9 days before with 350 r and The interesting question arises: Why do these into four 10 month old female mice irradiated with tumor cells metatasize from one site (and thus 350 r 8 months earlier. In the former group tumors appear malignant) and not from another site (and appeared in two mice after 116 and 178 days respec- thus appear benign)? The pattern of local circula- tively; and in the latter, in two mice after 16 and tion may account for the difference, but it seems 33 days respectively. These periods are again shortei more probable that the luteoma cells degenerate than the incubation periods in normal mice of the in the lung, which is reached by cells escaping from

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1947 American Association for Cancer Research. 248 Cancer Research subcutaneous grafts, and fail to pass the puhnonary mic bordcrs of the granulosa cells are indistinct barrier; while the liver, which is in the pathway of often causing the latter to appear as crowded masses neoplastic cells escaping from the spleen, is a good of spherical, basophilic nuclei resembling those of soil for proliferation of luteoma cells. large lymphocytes with little or no cytoplasm. The The morpholo~, of the splenic grafts is similar to nuclei of the lutein cells are similar in size to those that of the subcutaneous grafts, and the splenic of granulosa cells but they are less basophilic. The grafts produce the same secondary changes. The deposition of large quantities of sudanophilic lipoids livers of mice with luteoma grafts in the spleen are goes with further enlargement of the cell, assuming slightly more congested and there are small areas a pale-staining, vaeuolated appearance in the usual of necrosis in liver cells. helnatoxylin-eosin preparation. There is a moderate The grafted tumors grew slowly but progressively: variability in the size and shape of luteoma cells regression was not observed after the transplants and flaeir nuclei, but mitotic figures are rare. The became palpable. TILe long incubation period, aver- only distinct stroma of the tumor appears to be a aging in the primary passage 242 days ill male and capillary network with vessels varying greatly in size 134 days ill female mice, is worthy of emphasis. The in different parts of the tumor. The presence of subcutaneous tulnors usually reached 4 Cln. and in a thrombi in vessels with cavernous dilatation is com- few cases 6 era. in greatest diameter without inter- mon. Areas of hemosiderosis occasionally seen, indi- fering with the health of the animals. cate antecedent hemorrhage. Small focal areas of lymphoid infiltration occur (Fig. 7) but the bulk of the tumor is free from a cellular infl-nnmatory THE MORPItOLOGY OF LIITEOMAS reaction. Areas of necrosis are common in older tu- The lutcomas arc slow-growing tumors, charac- mors with occasional loci of calcification (Fig. 2). terized in the gross by a faint yellowish hue, some- However, side bv side with such calcified areas arc what glossy appearance, and by a soft rubbery con- well preserved masses of cells with no retrogressive sistency. The deep yellow color of the induced change, or inflammatory reaction. Fig. 9 shows the luteoma was absent in transplanted luteomas. margin of a necrotic area with clefts of empty spaces The tumor cells (Figs 1, 2, 4, 6, 7) resemble resembling those left by dissolved cholesterol crys- very closely normal lutein cells (Fig. 3). q'hey are tals with no inflammatory reaction but with oc- polygonal with spherical nuclei and in contrast to casional giant cells, and adjacent typical luteoma all types of granulosa cells their cytoplasm is abun- cells. Reticulum is in general scant in luteoinas and dant and distinctly acidophilic. Cells with non- surrounds small or large groups of lutein cells. Rare- vacuolatcd, non-granular, and Inoderately eosinophi- ly, there are cyst-like spaces in the tumor containing lie cytoplasm are regarded as "healthy" (Figs. 6 and serum-like precipitate and a few red blood cells. 7). They usually contain a much smaller quantity In the course of the passages the tumor became of sudanophilie lipoids than cells with more abun- moderately anaplastie with occasional giant cells dant and vaeuolated cytoplasm which are believed having hyperchromatic nuclei in some areas and to be degenerated (Fig. 8). The ectoplasm of tile with "dwarf" cells in others, qqae growth is invasive lutein cells is usually distinct, whereas the cytoplas- (Fig. 5) but metastases did not occur.

DESCRIPTION OF FIGURES 1 TO 5 All illustrations are from strain IX mice bearing FIG. 3.--Corpus luteum of normal 4 month old mouse luteoma, unless otherwise indicated. The tissues were in diestrous. Note uniformity of size and shape of lutcin fixed in Zenker-formol solution and stained with hema- cells. Mag. X 450. toxylin and eosin. The magnifications are approximate. Fie. 4.--Transmitted luteoma (Passage 1 lc) of mouse Mc 787 9 grafted in spleen. Cells resemble closely normal FIG. 1.--Luteoma of mouse Mb 1222 from which strain lutein cells although there is a slight anaplasia. Mag. X IX originates. Both nuclei and cytoplasm of cells vary 450. greatly in size, but in general they resemble normal lutein FIG. 5.--Transmitted luteoma (Passage IIIc) of male cell. Mag. X 180. mouse Mc 1054, infiltrating abdominal muscle. Grafted Fic. 2.--An area of degeneration from same tumor with tumor measured about 3 cm. in greatest diameter wl~en deposits of dark staining material, presumably calcium, mouse was killed 4 months after implantation. Mag. X and slight fibrosis about well preserved lutein cells. Mag. 180. X 180.

CHANGES IN MICE BEARING THE TRANSPLANTED tumor (Table II). The weight increase sccms to LUTEOMA have bccn due in part to a physiological growth which in this species does not cease after puberty. The organs of tumor-bearing inicc have been In normal control female mice of this stock the compared with those of normal mice; siblings or weight has increased from 21.5 gin. at 2 months to closely related mice weaned at the same time were 32.7 gm. at 7 months. The gain in weight was some- used whenever possible. what less in mice that received implants but in The lutcoma is associated with characteristic sec- which tumors did not develop. The weight of the ondary changes in the sex organs, adrenal, thymus tumor-bearing female mice had increased to an and submaxillary gland and frequently with obesity. average of 40.1 gin. at 6 months: or to 12.1 gm. Obesity.--Mice bearing luteomas steadily gained above that of uninjected controls and 16.5 gm. above weight in spite of the progressive growth of the those in which the tmnor failed to grow. Similarly,

TABI.E II: \VEIGIIT INCREMENT IN GRAMS IN .XIICE BEARING LUTEOMA

Mice Age in months FmMALES 2 3 4 5 51/~ 6 7 8 10 11 Uninjected Number of mice 8 8 8 7 6 Extreme weights 17-23 19-27 20-29 21-29 24-30 Average weights (21.5) (24.4) (26.2) (26.9) (28) Injected Nulnber of inlce 15 12 7 7 7 negatives Extrclnc weights 16-28 21-28 21-27 20-28 22-29 Average weights (22.2) (23.9) (23.3) (23.6) (23-6) Injected Nuinber of mice 0 q 8 6 6 3 3 positives Extreme weights - 24-27 27-31 32-36 3843 50-61 52-62 Average weights - (26) (29.1) (34.7) (40.1) (56-7) (53.3 Tunmr size - -> +to++ -'to +-+-++ +++to +++to +q-+ T4-~-:- ++++ VVeight in- Over controls 1.6 2.9 7.8 12.1 crease of Over negatives 2.1 5.8 11.1 16.5 positives

MALES Uninjccted Number of mice 14 14 14 13 13 l0 3 Extreme weights 17-27 22-29 24-32 26-36 25-34 26-34 30-35 Average weights (22.1) (25.3)(28.0) (29.6)(29.9) (30.6) (32.7) Injected Number of mice 16 10 6 6 6 3 1 2 negatives Extreme weights 19.28 23-32 26-30 25-32 25-39 31.33 - 35-41 Avcrageweights (23.3) (27.0)(27.0)(28.0)(29.2) (32.3) (;1) (38) Injected Nmnber of mice 2 8 12 11 11 4 2 3 6 positives Extreme weights 2r,-26 2~-31 27-37 32-39 32-41 35-39 34-38 42-44 40-48 Average weights (25.5) (27.3) (31.6) (34.2)(36.4) (36.7) (36) (43) (44.1) Tumor size + +to++ +to +to +~-to -+-+to ++to +to +to ++++++++ ++++ ++++ ++++ ++++ ++++ "Weight in- Over controls 3.4 2.0 3.6 4.6 6.5 6.1 3.3 crease of Over negatives 2.2 0.3 4.6 6.2 7.2 4.4 5.0 6.1 positives

The values are pooled from Passages I, lI, IIe, IIf and IIIa. + ~ Tumor up to 1 era. in In'eatest diameter; ++ ~ tumor from 1 to 2 cm. in greatest diameter; +++ z tumors from to 3 em. in greatest diameter; ++++ z tumor over 3 cm. in greatest diameter.

DESCRIPTION OF FIGURES 6 TO 9 FIG. &--High magnification of large transplanted lute- with lutein cells that are vacuolated or have clear cyto- oma (Passage lid) of mouse Mc 2015 9 1 month old plasm. Mag. X 450. when implanted, 15 months old when killed. Mag. )< 900. FIG. 9.---Transmitted lutcoma (Passage I) of mouse Mc FIG. 7---Transmitted luteoma (Passage IIe) of mouse 421 8 showing necrotic area with clear spaces presumably Mc 806 8 showing mitotic figures and focal accumulations those of dissolved cholesterol crystals. There is no inflam- of lymphoid cells in tumor. Mag. X 450. matory reaction and adjacent tumor cells appear well Fro. 8.mAnother field of luteoma of mouse Mc 2015 9 preserved. Mag. X 180.

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1947 American Association for Cancer Research. 252 Cancer Research tumor-bearing male mice gained about 6 gin. more days, prepared and stained according to Papani- than the normal controls. The weight increases in colaou's technic, indicated an anestrous state. male mice may bc duc solely to the bulk of the The uterus is enlarged, usually elongated, and tumor, while the weight increment in some females measures about 3 ram. in diameter. Both lnuscula- is more than thc estimated stun of normal body lure and epithelium appear hyperplastie, while the wcight and tumor weight. Unfortunately only a few submucosa is scanty. The proliferating epithelial cells tumors were weighed. The estimated weight of large form papillary folds (Figs. 10, 11) and occasional tumors is 6 to 12 gm. downward projections into the muscularis (Fig. 12). In contrast to mice receiving tumor grafts of dif- The lilting cells are predominantly tall columnar. ferent sorts, observed by one of us in the course The nuclei are crowded, but mitoses are rare. Many of years of extensive studies of transplantable neo- glands, notably thosc of the submucosa and muscu- plasms, the lutcoma-1)caring 1nice were unusually laris are lined with cuboidal cells; some are cystically obese. Ternainally the weights tended to drop, but dilated, either devoid of or containing eosinophilic luteoma never produced eachexia. sccrction. Isolatcd glands deep down in the mus- \Vhcthcr or not lutcoma produces obesity re- cularis are more colnnlon than in the utcri of normal mains to be investigated with other strains, since mice. Occasionally they penetrate deeply and are strain IX has increased in virulence in the course seen beneath the serous coat (Fig. 12). of succcssive passages and the associated obesity be- The uterus of the normal mouse differs in ap- came less pronounced. Iu the original transfer the pearance, depending on the cyclic horlnonal stinm- tumor growth was slow, and female mice reached lation, as also indicated by the state of thc vaginal a weight of 50 to 62 gin. at 10 months. No normal epithelium. In general, the glands are small, and controls were available at that time. Subsequently the muscularis appears thinner and is seldom invad- the tumor grew faster and the weight increases were ed by glands. The stromal cells of the submncosa lcss obvious. Obcsitv was absent with progressively on the other hand arc more nunlcrous; their nuclei growing tumors. Fat was excessive not only in the are round or slightly oval. subcutaneous tissue, but also in the internal fat That the uterus is in a stimulated state in mice depots. The fat-body about the kidney was s'o mas- bearing lutcoma is clearly indicated in tumor-bear- sive that in it the atrophic adrenal could be identi- ing, spayed female mice in which the uterus is fied only with difficulty. Fat was excessive also in the noticeably thickcr than in non-tumor-bearing ovari- thoracic cavity substemally and occupied the site ectomized animals. The increase in size appears to of the atrophic thymus. The atrophic ovaries also be caused by hyperplasia of both muscularis and were embedded in much fat. endometrium. The vagina/ epit!zelitml of tumor-bearing, non- It deserves emphasis that the most characteristic castrate mice consists of 2 to 3 layers of cclls. The effect of progesterone activity, namely, swclling and lining cells are tall cuboidal or columnar with a crowding of subepithelial stromal cells (28) is absent basal nucleus and a pale pink staining bulky cyto- in luteoma-bearing mice; oil the contrary, the sub- plasm. The constant abscnce of estrogenic squamous mueosa is scant and the epithelium is hypcrplastic. metaplasia deserves emphasis. This discrepancy requires further study. The clitoris appeared larger in some tumor-bear- The ovaries of luteoma-bcaring mice are invariably ing than in normal mice. atrophic (Figs. 16, 17). Ova are present (Fig. 18) Vaginal smears of 3 ovariectomized and 3 normal and they are surrounded by the usual cells of the females bearing luteomas, taken on 5 consecutive follicles some of which contain liquor. Lutein cclls,

DESCRIPTION OF FIGURES 10 TO 14 Fro. 10.--Uterus of mouse Mc 2022 bearing transmitted FIO. 13.--Transplanted luteoma of strain III. Mouse luteoma (Passage IId). Mouse was 1 month old when Mb 1923 8 10 weeks after implantation (Passage IIIa). implanted and 4 months old when killed. There is hyper- Mag. X 450. plasia of epithelium with hillocky surface. Mag. X 90. FIG. 14.--Another field of transplanted luteoma of FIc. 11.--Higher magnification of Fig. 10. Mag. X 180. strain III. Mouse Mb 19799 (Passage Va; 10 weeks Fro. 12.--Downgrowth of endometrium of 6 months after implantation). Thick fibrous connective tissue sur- old mouse Mc 995 bearing transmitted luteoma (Passage rounds well preserved lutein cells. Mag. X 180. IIf; grafted at 6 weeks of age), penetlating muscularis and reaching the serosa Mag. X 90.

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1947 American Association for Cancer Research. 254 Cancer Research however, are invariably absent. Many ova are de- intact, but their cytoplasm is scanty. The medulla generated, and the stroma appears scant or normal often appears large but is othcrwise normal in amount (Fig. 16). (Fig. 19). In a fcw mice there is a slight or mod- In the control animals corpora lutca are conspi- erate congestion of the deeper zone of the cortex cuous (Fig. 15) occupying almost as much space and of adjacent medulla. Occasionally large, foamy as all the other ovarian clcments combined. The "brown" cells arc present in the midzonal region. cells of the corpora lutea (Fig. 3) in contrast to When the changes are advanccd, the cortex is those of the luteoma exhibit a striking uniformity in thin. All cells beyond the glomerular zone lose most size and shape and in thcir staining properties. of their cytoplasm, bccome flattened and crowded The mammary glands of tumor-bearing mice are around the mcdulla (Figs. 21, 22). There is usually as a rule fatty and atrophic. Some ducts arc dilated a sharp border between the chromaffin cells of the with pink secretion, but acini are few or are absent. medulla and thc cortical cells. Figs. 23 and 24 In an occasional nmuse there was hypcrplasia of show the characteristic appearancc .Qf the normal ducts distended with pink-staining secretion. adrenal cortex in mice of compara~ie;~a:ge. In the male sex organs of luteoma-bcaring mice The submaxillary glands of female mice bearing no conspicuous changes were noted. Spermatogenesis hlteomas havc a characteristic appearance. Cells of was not affected, and the seminal vesicles and the the secretory tubulcs, like those of normal males, prostate appeared normal. are distended with granular eosinophilic secretion Further evidence of secretion with androgenic and the nuclci are basal, while cells of acini are erect. --The fact that progesterone is androgenic smaller. In normal females there is a prcdominance (24) prompted us to compare castrate, luteoma- of aciuar cells while the secretory tubules are in bearing mice with control mice. Five pairs of tumor- the minority and their nuclei are colmnnar and are bearing and control castrated mice were examined not basal. Tim ducts are not distended with secre- 1 to 5 weeks after castration. There was marked tion. atrophy of the seminal vesicles in all controls and Kidney. No definite masculinization of the kidney in none of the mice with lutcomas. occurred in anv of the luteoma-bearing female mice In one experiment the tumor was grafted on male except onc. In this mouse, bearing the largest tumor mice castrated 3 months previously. In both animals thus far observed, the glomeruli were predominantly in which the luteoma grafts were successful, the of the male typc. Congestion of glomeruli was com- seminal vesicles were greatly enlarged. Siblings cas- mon, and the lumina of the convoluted tubules trated at the same time but not bearing the tumor frequently contained much pink-staining precipitate. were killed at the death of the tumor-bearing ani- Liver. In the liver, changes in thc parcnchyma are mals. The seminal Vesicles of these micc were pro- insignificant. There is only slight or no congestion. foundly atrophic. ha only one animal was a moderate congcstion noted Characteristic adrenal cortical atrophy.--Atrophy as seen with hypervolemia secondary to granulosa of the adrenal cortex is a regular finding with lute- cell tumors (21). A moderate leukemoid reaction omas in both sexes. The change was absent in only by myelocytes, crythroblasts and megakaryocytes is a few mice. It is associated with disappcarance of connnon. the zona reticularis and of the regular arrangement Secondary changes in pre-irradiated mice bearing of the fasciculate colunm with collapse or loss of transplanted luteomas,--In animals pre-irradiated cells of its medullary portion, and persistence of the with approximately 350 r before grafting the lute- glomerular zone (Figs. 19 to 22). Nuclei of cells omas, the changes are essentially the same as in in the atrophic portion of the gland may appear normal animals.

DESCRIPTION OF FIGURES 15 TO 18 Fro. 15.mOvary of normal mouse, 4 months old, in FIC. 17.mOvary and fallopian tube of mouse Mc 2022 diestrons, with characteristic follicles and several lutein bearing transplanted luteoma (Passage IId). Changes bodies in lower corner of field Mag. X 90. are similar to those shown in Fig. 16 (see Figs. 10 and Fxc. 16.--Ovary of mouse Mc 999, 7 months of age, II of same mouse). Mag. )< 90. bearing large transplanted lueoma (Passage IIf). Mouse FIG. 18.--Higher magnification of ovary shown in was 6 weeks of age at implantation. Note well preserved Fig. 17. Note normal appearing follicles and ova. Mag. follicles at periphery of field and degenerating ova and X 180. seemingly atrophic stroma in central part. Corpora Iutea are absence. Mag. X 90.

the lutein cells are merely lutcinizcd granulosa cells DISCUSSION (23). q'he cytoplasm of cells of hypcrnephroma is Neoplasms of lutein cells.--Thc acquisition of clear, duc to glycogen and to a lesser extent to fat, neoplastic properties by lutcin cells is of unusual while that of lutcin cells is filled with lipoids. Ac- interest, because this cell does not exist before puber- cording to Barzilai (6) lutcin cells also contain gly- ty and its normal life is linked with the ovarian cogen finely dispersed in the cytoplasm. The re- cycle. The neoplastic change endows it with height- selnblancc to Krukenbcrg tumor cells is superficial. ened growth vigor and frees it from those hormonal In the latter, clarity of cytoplasm is brought about forces that normally terminate its life. In this respect by nmcin, which is readi!v stainable with nmcicar- luteomas arc tumors ~analogous to chorionepithelio- mine. The mucin apcars to compress the nucleus mas. Both are composed of cells brought into exist- to the periphery of the cell, whereas the vesicular ence in the course of the sex cycle and have a limit- nucleus of the lutcoma is centrally placed, even ed life span, but when they become a neoplasm when sudanophilic lipoids accumulate in the cyto- they perpetuate their kind indefinitely. plasm of the cells in large quantities. The frequent occurrence of hltcoma in mice that Several cases of so-called human lutcomas were had been exposed to x-rays has already been describ- associated with masculinization, hypcrtrichosis, voice ed (18). Most x-ray-induced ovarian growths con- changes, and amenorrhca (27, 44) and these tumors tain tumor-like masses of both ]utein and granulosa are believed to bc derivatives of adrenal cell rests. cells. Both types proved transplantable and by suc- Fig. I) of t-/offman (27) showing the microscopic cessive passages of selected areas pure growths of appearance of such a neoplasm, is indistinguishable either of these cell types can be obtained. Strain IX from the luteoma described above (see also Fig. 3 here described originated from a lutein cell mass and of Furth and Buttcrworth, 18). in the course of 16 passages it never yielded granu- The morphological difference between lutcin cells losa cells. The first successfully transplanted growths and lipoid-laden cells of the adrenal cortex of mice induced by x-ray were of the granulosa cell type is slight; the lutcin cells are somewhat larger and (strain I, 19). From the third successfully trans- their cytoplasm is more bulky and eosinophilic. planted ovarian tumor (strain III), which was of a Fckete and I,ittlc (16) noted the similarity be- mixed type, two pure lines of transplantable growths were secured; a transplantable granulosa tumor that tween the prominent cells in tumors of the adrenal produced hormones and a luteoma (illustrated in cortex produced in mice by castration (49), called Figs. 13 and 14). Type A cells, and those of ovarian neoplasms. Their Figs. 1 to 4 bear a striking resemblance to granulosa Existence of luteoma in women.--Luteomas in cells of tumors induced by x-rays. Type B cells of women arc rare; they are poorly understood and the adrenal tumors (49) resemble both lipoid-laden many doubt their very existence. cells of the adrenal cortex and lutein cells. The According to Itoffman (27) only 12 cases of hu- characterization of these cells requires identification man lutcoma have thus far been recorded. Thcy of the hormones they secrete. X]lese tmnors are may be benign or malignant. ~Iqae youngest patient associated with both naascnlizing and feminizing was 15 and the oldest 65 years of age. The tumor hormones and it is possible that the Type A cell is bright canary yellow in color and the cells re- which resembles granulosa cells is the predominant semble those of a mature corpus lutcum. source of the feminizing hormone and Type B cell In order to diagnosc a human lutcoma it is of the masculizing hormone. It will still remain necessary to exclude metastatic hypcrnephroma, to be determined whether the latter is a progestin growth of adrenal cell rests, and the possibility that or a testoid substance. The isolation of transplant-

DESCRIPTION OF FIGURES 19 TO 24 FIG. 19.--Atrophy of adrenal cortex with intact medulla Fro. 22.iSimilar but less advanced changes in mouse of mouse Mc 2015 2 bearing transplanted luteoma (Pas- Me 8036 8 weeks old at implantation, bearing a large sage IId) (see Fig. 6 from same mouse). Mag. X 90. transplanted luteoma (Passage lie). Mag. X 450. FIG. 20.--Higher magnification of Fig. 19. Mag. X 180. Fro. 23.--Adrenal cortex of normal 6 months old male FIG. 21.~Still higher magnification of Fig. 19. Note mouse. Magnification same as in Figs. 21 and 22. Mag. collapse of reticular zone and of deeper part of fasciculate • 450. zone, cells of which are crowded and appear devoid of Fro. 24.--Same normal adrenal gland at lower magnifi- cytoplasm. Mag. X 450. cation (X 180) comparable to that of Fig. 20.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1947 American Association for Cancer Research. 258 Cancer Research able adrenal neoplasms composed of pure cell types similar effect (14). Desoxycorticosterone is capable might aid in the solution of this problem. of producing progestational changes in the uterus of The virilizing tumors of the ovary of women have rabbits and of cats (Burrows 10, p. 540). The been described under such names as hypcrncphroma, determination of the pregnandiol glucuronide levels hyperncphroid tumors of the ovary, interstitioma, in urine cannot be used to differentiate between a masculinovoblastoma and folliculoma lipidique as luteoma and .:t tumor arising from adrenal rests since under adrenal tumor of the ovary and luteinoma (6). both progesterone and desoxyeorticosterone are me- A unique casc described by Bingel (9) was asso- tabolized to this substance (13). ciated with polycythemia and masculinization, both Secretions of luteoma.--Complete decidualization disappcaring after rcmoval of the tumor. Expcri- of the endometrium and a daily output of 19 mgm. mental granulosa ccll tumors are often associated of pregnandiol has been associated with an ovarian with hypervolelnia which is sometimes polycythcm- tumor composed of lutein-like cells (Stewart, el. 6). ie (21). It is possiblc that this casc was a mixed Attempts to isolate hormonal steroids from luteoma- tumor as are many of the induced ovarian tumors bearing mice are in progress. (18) and probably also some human lutcomas ac- The evidence that the luteal cell secretes estrogen companied by exccssive bleeding, cnlargcmcnt of is insufficient (2, 4). None of the secondary changes breasts and other manifestations of feminizing in- induced by the experimental luteomas are caused by quences, A clcar picture of the range of changes estrogens. The weight of evidence supports the opin- by secretions of one cell type can be arrivcd at only ion that ~the ovary secretes andragenic material by obtaining a growth of one cell type in gonadcc- (I0), and that this androgenic activity is associated tomized animals. with luteinization of the ovary (26, 44). l,t is un- Identity of mouse luteoma.--To suppose that our certain whether other normal ovarian cells secrete induced luteomas originate from adrenal cell rests androgens. Progesterone is androgenic (24, 25) would imply that the ovary of every mouse contains but large amoun,ts of it,are required to produce such adrenal cell rests, which following irradiation, give an effect. In stimulating the secondary sex organs rise to tumors in every ovary at several places. In of castrated rats I m.gm. of progesterone is equiva- the adrenal gland itself tumors do not arise follow- lent to 0.03 mgm. of testosterone (33). This would ing general irradiation with x-rays. The possibility ilnply that the secreting tissue would have to syn- that luteomas are granulosa cell tumors with second- thesize enormous amounts of progesterone to pro- ary luteinization is likewise not supported by our duce an androgenic effect. On the other hand, pro- observations. None of our transplantable granulosa gesterone in the physiological state as secreted may cell tumors, grafted in hundreds of mice, have ever lye more active. changed into luteomas; nor have neoplastic lutein q~ne chemical identification of a tumor cell se- cells ever changed into granulosa cells. Such trans- creting androgenic material depends on fur,ther formations are conceivable but have thus far not knowledge of the secretions of the different lnasculin- been demonstrated. izing tumors (arrhenoblastoma adrenal cortical tu- Relation of the adrenal gland to corpus luteum.-- mor, luteol~a and tes,ticular interstitial tumor). The morphological rescmblancc bctween luteal and Inf/uence of progesterone on the adrenal gland.-- adrenal cortical cclls, notcd as early as 1906 (34) is The characteristic atrophy of the adrenal cortex in now generally recognized. The similarities and dis- luteoma-bearing mice requires an explanation. Ad- similarities in their histological and physiological ministration of adrenal cortic-d extracts causes atro- characteristics have been ably reviewed by Parkes phy of ,the adrenal cortex because of inhibition of (37), Zuckerman (50) and others. the secretion of adrenocorticotrophic hormone by Progesterone (7) and estrone (8) and several an- the pituitary, (30). During pregnancy there are de- drogenic substances have been isolated from ox ad- generative changes in the adrenal cortex (29, 45) renals (39). The ability of progesterone and desox- and similar changes are readily produced by andro- ycorticosterone to replace each other to a certain gens. Crystalline progesterone like desoxycortico- extent is well known. Pregnant cats survived adren- sterone produces atrophy of the adrenal cortex of alectomy_longer than did non-pregnant cats (Stew- rats (11) but it requires huge doses of progesterone art, cited by Parkes, 37). The conditions of preg- to produce this effect (29). nancy and of rut exert an influence in prolonging In the light of the data, the atrophy of the ad- the life of adrenalectomized dogs (40). Progesterone renal cortex in luteoma-bearing mice is best ex- prolongs the life of adrenalectomized animals (22, plained as an inhibition of the secretion of adreno- 24, 50). Luteinizing gonadotropic hormone has a corticotropin by the hypophysis by secretions of the

luteoma. The validity of this supposition remains In luteoma-bearing mice both vaginal mucosa and to be tested and the mechanism of-this inhibition ovary have a characteristic appearance. The former elucidated. is atrophic 2 to 3 cell layers thick with an inner layer The effect of progestins on the uterus.--Progest- of cuboidal cells containing basal nuclei. The ovary erone .stimulates strongly the uterus in ovariecto- is smaller than normal due to the abscnce of corpora mized mice without preliminary treatment with lutea and of lutein cells that occupy a large part of estrogens (29). The uteri of mice that had been the ovary of mature normal mice. These findings are given pellets of progesterone werc 30 to 40 per cent similiar to those produced experimentally by pro: larger than those of the controls. There was gen- gesterone. eralized stimulation of all layers. The lining cells Effect of progestins on seminal vesicles and pro- were more columnar, and there was endometrial state.--In castrated male rats the prostate and, to a proliferation with markcd "endomctrial complexity." lesser extent, the seminal vesicles could be maintain- These changcs wcre only slightly less than those cd in wcight and in secretory activity by large doses caused by normal pregnancy. Morc rcccnt studies of progesterone (24). The weight of thc prostate of (5, 28) havc shown that in castrated mice the progesterone-treated rats was 5.85 times, and that of changes induced by progcsterone in the uterus affect the selninal vesicles 2.64 times that of thc control predominantly the stroma of thc submucosa, q2ae litter mates. In female rats thcrc was enlargement of stromal cells swell, thc nuclei become rounder and the clitoris. Progcstcrone will also maintain spcrma- more vesicular and the chromatin structure becomes togcnesis in hypophysectomizcd rats but larger quan- more distinct. The changes diffcr from thosc pro- tities arc required to evoke this androgenic effcct. duccd by estrogens and may occur concurrently. In our experiments luteoma prevented the induc- The degree of change is believed to be proportional tion of atrophy of scminal vesicles by castration. to the lcvel of thc hormonal stimulus. Thc ad- This suggests either that the anaount of progcstin ministration of crystalline progcstcronc to rats in- discharged by the tumor is largc or that an andro- duced progestational changes in thc uterus which genic substance is also produccd. Progesteronc will enlarged to 1 cm. in diameter (normal about 3 repair as well as prevent changes duc to castration ram,) . (24), and this occurred in lutcoma-beariug mice The uterinc mucosa of mice with lutcoma is that had been castratcd before implantation. doubtless in a stimulatcd state; thcsc mice are anes- Effect of progesterone on the body weight, de- trous as indicatcd by the character of the vaginal position of fat, and on thymus.--In lutcoma-bcar- epithelium. But thc changes obscrvcd are not iden- ing mice atrophy of the thymus occurred in the tical with those that have bccn describcd to charac- absence of loss of weight and in thc prcsencc of terize the action of administered progcstins in mice. atrophy of the adrenal cortex. Atrophy of thc adrenal There is a conspicuous absence of stimulation of cortex has a stimulating effect on the thymus, while submucosal cells, and a marked stimulation of sur- both androgens and cstrogcns cause atrophy of this face epithelium. This discrepancy, which is as far organ. as we know the only one contradicting the thesis The gain in body weight after administration of that our luteoma secretes progcstins, remains to be progestcronc was lcss than in thc control series (11) clarified. or there was a wcight loss (29).' Our mice with In the monkey, as in our mice with luteoma, slow-growing luteomas gaincd more wcight than did there is considerable hypcrplasia of the endomet- normal control mice (particularly thc females) and rium with incrcased tortuosity of the gland with it is doubtful that this is fully accounted for by the invaginations and cvaginations (50). bulk of the tumor they carried. At the age of 10 Effect of progesterone on estrus and ovary.~ months thcse mice weighed 50 to 62 grams. Instead Large doscs of progesterone administered to mature of cachcxia the bulky growth was associated with rats do not producc mucinification or cornification of obesity, but the gain in wcight may not have bccn the vaginal epithelium (43), while combined ad- due solely to deposition of fat. Androgens cause an ministration of estrogens and progestcrone readily increase in body weight in association with nitrogen produces mucinification as illustratcd in Fig. 18 on and water retcntion (31); and progesterone causes page 476 of Allen's Sex and Internal Secretions (1). water retcntion in adrcnalcctomized animals (46). Administration of crystalline progcstcronc to rats During and following prcgnancy womcn usually causes inhibition of estrus (38), or cessation of the gain weight in exccss of thc wcight of fetus and estrous cycle with atrophy of the ovaries, which be- appcndages. Among thc possible causes of this gain come devoid of ripe corpora lutca (43). in weight are thc hormonal secretions of the corpus

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1947 American Association for Cancer Research. 260 Cancer Research luteum (41) or placenta. In mice with luteomas SUMMARY AND CONCLUSIONS obesity develops with atrophy of the adrenal cortex; Luteoma is a well defined type of neoplasm of the the latter alone has the opposite effect on body ovary which, along with granulosa cell tmnors, can weight. This phenemenon is worthy of further study. be easily induced with x-rays. Neoplastic lutein Dimorphism of the submaxillary gland.--The sex- cells do not revert to granulosa cells and neoplastic ual dimorphism of the submaxillary gland of mice granulosa cells do not change into lutein cells. was discovered by Lacassagne (32) and indepen- The induction period of luteomas is long, and dently by Fckctc (15). The female type is charac- their growth is slow but progressive, ultimately re- terized by stimulation of the terminal acinar forma- sulting in bulky masses. In the course of successive tions which become swollen, finely granular and passages they gain markedly in proliferative vigor. basophilic, while the secretory tubules arc at rest, Metastascs from transplanted subcutaneous growths their diameter being reduced and the lumina in- were not observed, but intrasplenic transplants gave creased. The male type is characterized by the re- rise to secondary !uteomas in the liver. duction in size of the acini whose cells are clear In tumor-bearing hosts of both sexes there was and vacuolated. This part appears compressed by atrophy of the adrenal cortex and of the thymus. the expanded secretory tubules, the cells of which The presence of luteoma in male mice prevented arc hypertrophied and filled so densely with granules atrophy of the seminal vesicle following castration, of varying sizes that the nucleus is flattencd against and brought about repair of castration atrophy. In the base, the cytoplasmic outline may be indistinct female mice there was uterine hyperplasia, absence and the central canals arc dilated and filled with of corpora lutea in ovaries, atrophy of vaginal epi- secretion. thelium and enlargement of the clitoris. These In female luteonm-bearing mice the appearance of changes are suggestive of secretion of progestins by the submaxillary gland resemblcd the male typc. the tmnor. Mice with transplanted luteoma, parti- Effect on the kidney and other organs.--Thc cularly the females, gain much weight and become dimorphism of the glomeruli of mice caused by obese. differences in shape of epithelial cells of the capsule It is concluded that some masculinizing human is well known (12). The majority of female luteoma- ovarian tumors, which are commonly regarded as bearing mice failed to assume the male type of derivatives of misplaced adrcnal cortical cells, arc glomcrulus. There was, however, frequently a slight lutcomas. or moderate congestion of the glomeruli, but no definite parcnchymatous damage. ACKNO\VLEDGMEN'F Other changcs produced by progestins have not Mr. R. J. Ochrig has rendered assistance in the prepara- becn adequately investigated in our tumor-bearing tion of the manuscript and in taking tile photomicro- mice. It is known that progestins produce a slight graphs; and Katie Cobbs and Thelma Weaver .Mold in carrying out the experiments. hypertrophy of the pituitary gland of mature rats (43). No marked stimulation of the mammary gland was noted and it is known that progesterone alone REFERENCES has no stimulating effect on the mammary gland of mature rats (43). The clitoris of mice is known 1. ALLEN, E. Sex and Internal Secretions. Baltimore: The Williams and Wilkins Company. 1'9,34. to enlarge following administration of progestins 2. ALLEN, E., PRATT, J. P., NEWELI,, Q.-U., and and it seemed larger in some luteoma-bearing mice BLAND, L. ]. 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J. Furth and H. Sobel

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