THE ANTERIOR PITUITARY GLAND I N TUMOR-BEARING RATS

JACOB HEIMAN, M.D.

(From the Institute of Cancer Research, Coluinbia University, Francis Carter Wood, Director)

Since 1927 a large number of rats with spontaneous neoplasms, trans- planted tumors, and tumors induced by antuitrin-S and theelin have been under observation by the writer (1). Of late the gonads and pituitary gland have been particularly studied with the object of ascertaining whether a rela- tionship exists between the growing tumor and morphologic changes in the glandular organs of the host. Since hormonal assay in laboratory animals is difficult, the attempt has been made to discover, also, whether the physiological activity of a gland is linked with changes in its cellular morphology. The hypophysis, because of its complex structure, lends itself well to this purpose.

REVIEW OF THE LITERATURE The literature recording attempts to discover some correlation between

the anterior pituitary lobe and tumor growth may be briefly reviewed as it deals with ( a ) the normal pituitary; ( b ) changes in the gland in normal ani- mals injected with hormones; (c ) changes in the gland in animals with spon- taneous, induced, or transplanted tumors; (d ) hypophysectomy and tumor growth; ( e ) gonadectomy and tumor growth.

The Normal Pituitary: There are many detailed reports concerning the gross and minute anatomy of the pituitary in the rat and mouse, giving its size and weight in relation to other organs. Bailey (2) mentions the close resemblance between the structure of the pituitary in man and in laboratory animals, even to the finer details. Hatai (3), in studies on the albino rat, concluded that increase in body weight is accompanied by a proportionate increase in the weight of the pituitary gland, but that this increase is greater in females than in males. Addison and Adams (4) found the female pituitary to be twice as heavy as that of the male. Rasmussen ( 5 ) also states that the gland is heavier in the female. He observed that as body weight increased the anterior or glandular lobe showed a greater increase than the posterior lobe or pars nervosa. Andersen (6) gives the weight of the pituitary in the female rat as between 7.7 and 12.3 mg. Olga Fischer (5) measured the gland in rats and found the average transverse diameter in females to be 4.2 mm., and in males 4.0 mm., while the sagittal diameter in females was 2.32 mm. and in males 2.25 mm.

Wolfe and Cleveland (8) describe the cyclic histological variations in the anterior pituitary gland of the albino rat. Erdheim (9) , as is well known, has demonstrated an increase of acidophilic cells in pregnancy, and Martins and de Mello (10) have recorded an increase of basophilic cells in the an- terior lobe in castrated animals.

423

424 JACOB HEIMAN

The percentages of the different cells in the anterior lobe of the pituitary show wide fluctuations, which make the establishment of normal standards difficult. Rasmussen (11) found the following percentages in man: in the male, chromophobes 52 per cent, acidophils 37 per cent, basophils 11 per cent; in the non-pregnant female chromophobes 50 per cent with a coefficient of variation of 14 per cent, eosinophils 44 per cent with a variation of 19 per cent, basophils 7 per cent with a variation of 42 per cent. Wolfe (12) also found fairly large variations in the level of chromophobes. He obtained the following differential count in rats: chromophobes 62 per cent ( 5 5 to 73), eosinophils 34 per cent (30 to 40), and basophils 4 per cent (3 to 6). Baso- phils occur in greater number in the young. Severinghaus and his associates (13) have thoroughly reviewed the present knowledge of the pituitary, em- phasizing the uncertainties encountered in studying the normal gland.

Changes in the Pituitary in Normal Animals Injected with Hormones: Changes in the pituitary following hormone injection include (I) those pro- duced by gonadotropic hormones and (11) those produced by the administra- tion of the estrogenic hormone.

I. Over fifteen years ago Evans and Long (14) studied the effect of intraperitoneal injections of the anterior lobe of the pituitary upon growth and maturity and the estrous cycle in rats, but made no mention of changes in the pituitary gland. In 193 1 B. Zondek (15) reported that injection of anterior pituitary substance in immature animals was followed by changes .in the anterior lobe of the hypophysis which could be considered as a maturation of the gland. Collip, Selye, and Thomson (16) observed that prolonged administration of the gonadotropic hormone caused enlargement of the pitui- tary, but only in females, Wolfe (17), studying the effects of the same sub- stance in immature and mature females, reported that the changes induced consisted in loss of granules in both eosinophils and basophils. As a result of similar experiments Collip, Selye, and Bachman (18) concluded that after repeated injections an anti-gonadotropic substance develops, a view which corresponds with the recent concept of the presence of anti-hormones in the blood. Severinghaus (19) found that injection of the gonadotropic hormone was followed by a depletion of basophilic granules but an increase in the number of basophil cells and a corresponding decrease in chromophobes. Fluhmann (20) observed an increase in weight of the pituitary in female rats, and in the number of basophils and of castration cells. Similar changes were observed by him after injections of gonadotropic substance from the blood of pregnant women, with a decrease in eosinophils and an increase in chronio- phobes. Nelson ( 2 1) concluded from his experiments with antuitrin-S that in castrates no effect is exerted on the pituitary gland, which would indicate that castration prevents degranulation in the injected animals. Wolfe (22) gave daily injections for 140 days of anterior pituitary-like extract of preg- nancy urine to immature female rats and observed enlargement of the pituitary and a definite morphologic reaction in the anterior lobe. After prolonged in- jections the gland returns to normal size and degranulation of the chromophile cells occurs.

A summary of these scattered observations shows the following effects on the pituitary gland of the injection of the gonadotropic hormone.

THE ANTERIOR PITUITARY GLAND IN TUMOR-BEARING RATS 425

(1) In immature animals, maturation of the pituitary. (2) In castrated animals, no influence on the pituitary. (3) In normal animals of both sexes, increase in weight and appearance

(4) In immature rats, a decrease in eosinophils; enlargement and de-

( 5 ) Depletion of basophilic granules with an increase in the basophil cells

(6) In females, increase in weight of the pituitary (8.5 to 26 mg.). ( 7 ) With prolonged injections, no effect except possible increase in

(8) Increase in basophils and chromophobes; diminution in acidophils.

11. The literature abounds with reports on the influence of estrogenic substance (estrin, theelin, amniotin, synthetic estrogenic hormone, etc.) on the glandular portion of the pituitary. In 1932 Spencer and D’Amour (23) re- ported that no histologic changes were observed in the pituitary following sub- cutaneous injection of 20 rat units of estrin daily for fifty-four days. Moore and Price (24) , on the contrary, concluded that the presence of sufficient amounts of gonadal hormones of either sex lowers hypophyseal activity. In 1934, Nelson (25) reported that estrin, injected subcutaneously, diminished the number of basophils in the pituitary, and caused the disappearance of castration cells in previously castrated animals of both sexes. He obtained a greater reaction in the female than in the male rat, with an increase in chromophobe celIs. Andersen (26) reported that in castrated rats amniotin caused a restoration of the atrophied pituitary to the original weight as ob- served in normal animals in estrus. Collip et al ( 2 7 ) found that the ad- ministration of large doses of estrin to female rats led to hypertrophy of the anterior lobe within a few days, an effect which was less marked in males and in castrates.

Crooke and Korenchevsky (28) in 1935 found that estrin, in normal males, increased the weight of the pituitary, but caused no histologic changes, while in female and male castrates the normal histology was restored, as noted by Andersen. Still later (1936) McEuen et al (29) reported on some effects of prolonged administration of estrin. In 6 castrated female rats the pituitary was enlarged, and in 3 females adenomata developed. In 5 normal males and one castrate male there was also an increase in size, and another castrate male developed a cystic adenoma. B. Zondek (30) in 1936 concluded that after prolonged administration to mice of follicular hormone (estrin), the function of the anterior lobe is inhibited, rendering inactive the growth and sex hor- mones. The gland becomes enlarged and there is an increase in weight in males, while in females tumors appear. Cramer and Horning (31) in 1936 reported that prolonged application of estrin to mice resulted in functional inactivity, producing a systemic condition resembling that following hypo- physectomy. Morphologically a hyperplasia of the anterior lobe was ob- served with diminished chromophils and an increase in chromophobe cells.

In an early report (1934) Burrows (32) described the effect produced by applying estrin to the skin of mice, but did not mention the pituitary. I n a

of castration cells.

granulation of basophils ; increase in small granular basophilic cells.

and a decrease in the chromophobe cells.

chromophobes.

426 JACOB HEIMAN

later report (1936) he recorded the occurrence of pituitary hyperplasia in one male mouse out of 567 treated with estrin.

The action of synthetic estrogenic hormone on the anterior pituitary of castrated females is discussed by Wolfe (33). Structural changes which occur in the pituitary of castrates were prevented and degranulation of baso- phils and eosinophils occurred, with an increase in chromophobes ; eosinophils were reduced to 24 per cent as compared to 34 per cent in normal uninjected rats and 41 per cent in uninjected castrates; basophils were reduced to 2 per cent, the figures for normal controls and uninjected castrates being 4 per cent and 13 per cent respectively; chromophobes increased to 74 per cent as compared to 62 per cent and 44 per cent in normal animals and uninjected castrates. The average weight of the pituitary in the injected castrates was 14 mg. as compared to 10 mg. in the normal controls and 9.7 mg. in uninjected castrates. Wolfe and Hamilton (34) produced similar changes with estrin, i f . , a diminution of the total number of eosinophils and basophils, with de- granulation, and an increase in size and number of chromophobes.

Fevold et a1 (35) reported that estrin caused an increased secretion of the luteinizing hormone as evidenced by early and rapid luteinization.

Wolfe and Chadwick (36) carried out quantitative studies on the effect of estrin on the pituitary and reported an increase in the weight of the gland. Ellison and Burch (37) also noted an increase in the size of the pituitary after administration of synthetic estrogenic substance.

Fluhmann and Kulchar (38) observed no changes in the pituitary in fe. males receiving prolonged injections of estrin, and concluded that estrin is not the factor concerned in the formation of castration cells. In immature female rats receiving estrin injections Wolfe and Chadwick (39) recorded changes in the pituitary similar to those occurring in pregnancy, while in fe- male castrates no changes were noted. Halpern and D’Amour (40) state that the administration of estrin leads to an increase of 100 to 200 per cent in the weight of the hypophysis, that it causes hypertrophy and hyperplasia of the chromophobe cells, with increased mitosis, enlargement of the Golgi apparatus and the nucleolus, and an increase in the size and number of the mitochondria; the basophils are depleted of granules and in spayed females castration cells disappear. The explanation of these effects is that estrin causes a release of gonadotropic hormones; when the secretory capacity of the gland is exceeded by the demand, compensatory hyperplasia of the chief or primary cells results. Lipschutz (41) also reported an increase in the weight of the pituitary in male rats after folliculin.

The following effects on the pituitary have thus been recorded as occurring after injection of estrogenic hormones:

1. In young rats, no histologic changes. 2. A decrease in hypophyseal activity, when sufficient amounts are given. 3. Increase in weight of the gland, marked in females, less so in males. 4. Decrease in basophils. 5. In castrates, a disappearance of acidophils. 6. An increase in chromophobes. 7. A greater reaction in the female than in the male.

THE ANTERIOR PITUITARY GLAND IN TUMOR-BEARING RATS 427

8. In castrated rats restoration of the atrophied pituitary to normal weight. 9. In large doses, hypertrophy of the anterior lobe within a few days in fe-

males; less marked enlargement in males and castrates; in castrate males and females, a retufn to normal histology.

10. Following prolonged administration, enlargement of the pituitary in nor- mal and castrated males and females, with production of adenomata.

11. Following prolonged administration, inhibition of the activity of the anterior lobe and of the growth and sex hormones, with enlargement in the male and the production of tumors in the female.

12. A functional condition following prolonged application resembling that after hypophysec tomy.

13. Hyperplasia in the mouse. 14. Prevention of structural changes in the pituitary of castrated rats; de-

granulation of acidophils and basophils, with increase in chromophobes. 15. No difference between control and injected castrates, indicating that estrin

is not a factor concerned in the formation of castration cells.

The Pituitary in Tumor-bearing Animals: The relationship between in- ternal secretions and tumor growth was studied by Leo Loeb (42) and his coworkers over twenty years ago. Their observations, however, were limited to the ovary, no mention being made of the pituitary. Later, Cori (43) re- ported on ovariectomy and the spontaneous occurrence of cancer in mice, but he also seems to have made no studies of the pituitary. Gardner et a1 (44) described a primary tumor occurring in the pituitary of a mouse (chromo- phobe adenoma) associated with the presence of six mammary tumors and two granulosa-cell tumors of the ovaries.

As early as 1915 Robertson and Burnett (45) reported that the growth rate of the Flexner-Jobling rat carcinoma was greatly increased both by an emulsion of the anterior lobe of the pituitary and by cholesterol, and called attention to the presence in both these substances of the hydroxybenzol radi- cal. Their observations so far as cholesterol is concerned have not been confirmed.

Karlefors (46) in 1920 reported that in the presence of cancer in man the chromophobe cells of the pituitary were increased in size, and Rossler (47) in 1929 reported an increase in the eosinophils in the presence of chorionepi- thelioma. Novak and Koff (48) in 1930 made similar observations in pa- tients with hydatidiform mole and chorionepithelioma. Guyer and Claus (49) described changes in the anterior pituitary in rats with Flexner- Jobling rat carcinoma. The gland was found to resemble the pituitary in the cas- trated animals with increase in basophils, vacuolization of the basophile cells and hyperactivity. These investigators concluded that the changes are brought about by the influence of the growing tumor on the gland. When the Flexner-Jobling carcinoma is transplanted to the uterus there is an increase in acidophile cells in the pituitary, resembling that observed in pregnancy or following estrin injections. This change is less marked in castrated females. Wyeth (50) reports an increase in the eosinophile cells in the presence of human cancer. Even with general cachexia, the weight increased (0.60 to 1.80 gm.).

The gland is also heavier than normal.

428 JACOB H E I M A N

McEuen and his co-workers (51) maintained that changes in the pituitary are not related to tumor growth itself but partly at least to decomposition of tissue in the necrotic center of the tumor. In rats bearing Walker tumor 256, they found vacuolated cells in the anterior lobe of the pituitary. Engel (52) reports that the growth of transplanted Ehrlich mouse carcinoma was slightly stimulated by the growth hormone, but somewhat inhibited by the gonado- tropic hormone.

In mice injected with estrin Cramer and Horning (53) observed enlarge- ment of the pituitary and three pituitary adenomata associated with mammary cancer. Oberling (54) obtained four hypophyseal adenomata in 100 rats inoculated with mammary fibroadenomata. He believes that the relationship is more than casual. He also observed 4 hypophyseal tumors among 7 cas- trated male rats in which ovarian implantation was done. MendelCeff (55 ) concludes that hormonal activity increases during the evolution of a tumor, and that the hypophyseal growth hormone is responsible for the continued proliferation of a neoplasm once this has been initiated. Twort and Twort (56) report that in mice with malignant growths both the area and weight of the pituitary are less than in animals with benign tumors. Witherspoon (57) describes the clinical relationship of benign and malignant tumors to estro- genic, carcinogenic and anterior pituitary growth principles. Malavazos and Goldman (58) also attempt in their review to establish a relationship between endocrines and neoplasms.

In a comprehensive analysis of pituitaries examined at necropsy Close (59) observed an increased incidence of adenomas among persons with cancer of various organs, particularly carcinoma of other glands, as the prostate and pancreas, and carcinoma of the rectum and bronchi.

There appear to be no reports in the literature dealing with pituitary changes in tumor-bearing animals following hormone injection, though there are numerous studies of the influence of hormones on tumor growth (60-64).

Hypophysectomy and Tumor Growth: Samuels and Ball (65) concluded that the pituitary is a factor in tumor growth since hypophysectomy retards the growth rate of Walker carcinoma in rats and also slows the rate of growth of subcutaneous tumors induced by dibenzanthracene. Lacassagne (66) ob- served no effect on transplanted Brown-Pearce carcinoma following destruction of the hypophysis in the rabbit.

Influence of Castration upon the Pituitary: The influence of castration upon the pituitary is summarized by Severinghaus et al (13). In a compre- hensive review Koch (67) states that gonadectomy leads to hypertrophy of the pituitary with an increase of basophilic cells followed by vacuolization and development of signet ring or castration cells. A physiological increase of the sex hormones occurs. No study seems to have been made, however, of histologic changes in the pituitary of tumor-bearing castrates.

EXPERIMENTAL STUDIES An attempt at establishing a relationship between the pituitary and tumor

growth is obviously beset with difficulties. As has been pointed out, the vari- ations in size and shape of the normal rat pituitary are gr'eat and the cell

THE ANTERIOR PITUITARY GLAND IN TUMOR-REARING RATS 429

percentages show wide differences within normal limits. Furthermore, the introduction into the histologic picture of alien cells such as castration cells and degranulated typ'es makes quantitative cell studies difficult. From a large number of observations, however, certain deductions based on differential counts may be hazarded.

In the writer's studies, begun in 1932, white rats with spontaneous, trans- planted, and induced neoplasms, benign or malignant, have been divided into two groups, of which one received injections of gonadotropic or estrogenic hormones, while the other did not. Rats immune to tumor growth and tumor- bearing castrates were included.

The animals were distributed as follows: 1. Rats with spontaneous fibroadenomata ................................... 3 adult females 2. Rats immune or resistant to tumor growth . . . . . . . . . . . . . . . . 6 adult females

3 adult males 3. Rats with transplanted fibroadenomata . . . . . . . . . . . . . . .16 adult females

2 adult males 4. Rats with transplanted fibroadenomata injcctcd with antuitrin-G . . . . . . . . . . . 1 adult female

2 adult males 5. Rats with transplanted fibroadenomata injected with antuitrin-S . . . . . . . . . . . 2 adult females

2 adult males 6. Rats with transplanted fibroadenomata injected with theelin . . . . . . . . . . . . . . . . 1 adult female

2 adult males 7. Rats with transplanted fibroadenomata injected with antuitrin-G and theelin . . 4 adult females

2 adult males 8. Rats with transplanted fibroadenomata injcctcd with antuitrin-S and thcelin . . 2 adult females 9. Rats with fibroadenomata induced by antuitrin-S and theelin ............... 5 adult females

10. Rats with fibroadenomata induced by antuitrin-G and progynon-B .......... 4 adult females 11. Castrates immune to transplanted tumors ................................. 2 adult males 12. Castrates susceptible to transplanted tumors . . . . . . . . . . . . . . . . . 13. Rats immune to tumor, injected with antuitrin-G and theelin . . . . . . . . . . . . . . . 1 adult male 14. Castrated rats susceptible to tumor, injected with antuitrin-S or G or theelin . . 1 adult female

3 adult males ........ 6 adult females

8 adult males 16. Rats fed with 2-amino-5-azo-toluene .................................... 2 adult females

3 adult males 17. Rats with tumors R 8 ; R 39; R 256 .................................... 3 adult males

15. Rats with induced rat sarcoma or with benzpyrene tumors . . . .

Those pituitaries which appeared on gross examination to be enlarged were weighed and measured. The glands were fixed in formalin or Zenker solu- tion, and serial sections between 2 and 4 microns in thickness were stained with hematoxylin and eosin, with Dominici or with Masson triple stain.

The differential counts were made as nearly as possible in accordance with the method employed by Kasmussen. The accompanying tables show the de- tailed studies on each rat, and the averages obtained from several counts in animals with similar tumors or like injections. In order to minimize error, counts on the same slide were repeated at intervals. For purposes of ab- breviation, the different cells are designated as follows:

* Acidophile or eosinophile cells ........................... Basophile or cyanophile cells ............................ Chromophobe or chief cells . . . . . . . . .................... c cells Degranulated cells ...................................... Castration cells . . . . . . . . . . . ................................... e cells

The pituitaries which on gross examination appeared enlarged weighed

430 JACOB HEIMAN

T A ~ L E I : The Pituitary iic Rats with Spontaneous Fibroadenoma of the Breast (Non-Castrated) ___- _____. ___________- - __ .- -~

No. of rats, Age of Cell sex, and age Injection or Control tumor percentages Remarks

1 F.20 nios. Ant. G: I 1 injections, 10 r.u., 85 days a. 0 Vascular 43 days b. 12.0 chromophobe

c. 75.0 adenoma d. 13.0

1 F.17 nios. Control 90 days a. 10.0 Chromophobe adenoma b. 5.0

c. 85.0

1 F.20 mos. Control ? a. 6.0 b. 22.4 c. 41.6 tl. 30.0

TADLE I1 : The Pituitary i n Non-Tumor (Immune) Rats (Non-Custrated) _ . _ _ ~

No. of rats, Cell sex, and age Injection or Control percentages Remarks

1 1:. 16 mos.

2 I;. 18 nios.

1 17. 64 mos.

1 1;. 13 nios.

Control

Control

Control

Control

a. 41.0 Size ++++ b. 6.0 Littered c. 53.0

a. 42.0 Size + b. 4.0 No litters c. 54.0

a. 27.0 b. 4.0 c. 69.0

a. 31.0 b. 4.0 c. 65.0

1 1;. 12 1110s. Control a. 32.0 b. 11.0 c. 57.0

~

between 8 and 2 2 mg. For those which weighed more than 10 mg. the size is designated in the charts in the column headed Remarks as ++++. About 44 per cent of the uninj’ected animals with benign fibroadenomata of the breast showed an increase in the pituitary weight (Table 111). One animal immune to tumor, which had borne several litters, also showed an appreciable increase in the w’eight of the pituitary (Table 11).

Normal rats injected with large doses of estrogenic hormone showed a very marked increase in size and weight of the pituitary. This is in con- formity with the findings of Collip, Halpern and D’Amour, and others, that the administration of estrin leads to increase in the weight of the hypophysis. A similar increase in size and weight of the gland appeared in tumor-bearing rats receiving large hormone injections (Table V I I ) . In 2 old rats with spon- taneous fibroadenomata of the breast, the pituitary show’ed chromophobe

TABLE I1 I : The Pituitary in Rats with Transplaitled Fihroadenomata (Tumor 342) , Not Injected

Cell percentages

No. of rats, Tumor and sex, and age age

1 F., 12 nios. Fibroma 5; nios.

Remarks

a. 30 b. 4 c. 66

Vascular + + + +

1 F., 16 mos. Fibroadenoma 4 nios.

a. 33 b. 6 c. 61

a. 29 b. 3 c. 65 d. 3

Fibroadenoma 43 mos.

1 F., 11 mos.

1 F., 15 mos.

1 F., 16 mos.

a. 33 b. 5 c. 62

Fibroadenoma 3B mos.

Size ++

Fibroma 2 mos.

a. 34 b. 6 c. 60

Vascular + + + + Size ++++

1 F., 6 mos. Fibroaclenoma 2 mos.

Size ++++ a. 30 b. 12 c. 53 d. 5

a. 36 b. 6 c. 58

1 M., 6 mos.

1 F., 6 mos.

Sarcoma 2 mos.

Size ++++ Vascular + + + +

Fibroma 2 mos.

a. 29 b. 5.5 c. 65.5

Size ++++ Vascular + + + +

1 F., 5 mos.

1 M., 6 mos.

Fibroadenoma 2 mos.

Size ++++ Vascular + + + + Vascular ++ Fibroma

4 mos. a. 30.5 b. 4.0 c. 65.5

1 F., 15 mos. Sarcoma a. 10 3 : mos. . b. 4

c. 78 d. 8

Vascular ++

1 F., 61, nios.

1 F., 18 mos.

Fibroma 5 mos.

a. 40 b. 4 c. 56

a. 36 b. 6 c. 58

Vascular + + + +

Sarcoma 8 mos.

Size ++++

1 F., 6 mos. Fibroadenoma a. 32.1 90 days b. 11.6

c. 56.3 [Cant . on p . 4321-

431

432 JACOB HEIMAN

TABLE III-Continued

No. of rats, Tumor and Cell sex, and age age percentages Remarks

1 F., 8 mos. Fibroadenoma a. 30 5 mos. b. 1

c. 47 d. 22

1 F., 7 mos. Fibroadenoma a. 28.9 4 mos. b. 20.5

c. 42.6 d. 8.0

1 F., 13 mos. Fibroadenoma a. 16 Chromophobe 7 mos. c. 56 adenoma

d. 28

1 F., 5 mos. Fi broadenoma 2 mos.

adenomata, as was observed by Oberling (Table I ) . Among 2 2 rats with induced and transplanted fibroadenomata, 3 chromophobe tumors were ob- served (Tables I11 and VIII). In normal rats injected with large doses of estrogenic hormone, pituitary adenomata were more frequent (Table IX) . This is in.conformity with the findings in mice reported by Cramer and others.

Tumor-bearing castrates not receiving injections showed a marked re- duction in castration cells similar to the reduction occurring in immune and control rats injected with estrogenic substance (Tables VII, IX-MI) . Im- mune castrates, on the contrary, when uninjected, showed a high percentage of castration cells (62 per cent). Tumor-bearing castrates injected with estrogenic hormone, like those uninjected, showed few or no castration cells but exhibited a marked increase in degranulated cells, up to 95 per cent (Table XIII) . Where antuitrin-S or growth hormone was combined with theelin or progynon in an empirically balanced dose, a neutralizing effect manifested itself in restoration of the count to normal in all animals, control, tumor-bearing, and castrated.

Rats immune to tumor growth showed a normal differential count, but rats with spontaneous, induced, or transplanted benign tumors showed an in- crease of the chromophobes as high as 85 per cent. Tumor-bearing rats in- jected with antuitrin-S or theelin separately did not show as great an increase in chromophobes as animals receiving the two hormones in unbalanced dosage (Tables IV-VII) .

Rats bearing benzpyrene tumors or induced sarcoma, and those receiving 2-amino-5-azo-toluene, showed a moderate increase in chromophobes and in degranulated cells (Tables XIV-XV). Rats less than six months old, with sarcoma or carcinoma, showed a high normal acidophil count, while older rats showed an increase in chromophobes (Table XVI).

Certain of our conclusions and observations are in conformity with those reported in the literature. For example, small doses of antuitrin-S or G or theelin, singly or in combination, failed to produce convincing changes in the

TABLE IV: The Pituitary in Rats with Transplanted Tumor Injected with Antuitrin-G (Non-Castrated)

No. of rats, Number of injections, dose, Tumor and Cell sex, and age and duration age percentages Remarks

1 F. 13 mos. 5 injections, 10 r.u., Fibroadenoma a. 24.0 Vascular + + + + 40 days 40 days b. 4.0

Sarcoma c. 72.0

1 M. 64 mos. 9 injections, 5 r.u., Fibroma a. 24.0 Vascular + + + + 42 clays 136 days b. 6.0

c. 70.0

1 M. 9 mos. 5 injections, 5 r.u., Fibroma a. 16.4 22 days 116 days b. 5.3

c . 78.3

TABLE V: The Piluitary in Rals with Transplanted Tumor Injected with A ntuitrin-S ( Non- Castrated)

No. of rats, Number of injections, dose, Tumor and Cell sex, and age and duration age percentages Remarks '

1 M. 61 nios. 5 injections, 50 r.u., Fibroma 22 days 120 days

1 F. 15 mos. 8 injections, 100 r.u., Fibroadenoma 49 days 97 days

1 F. 15 mos. 8 injections, 100 r.u., Fibroma 56 days 104 days

1 M. 7 rnos. 9 injections, 100 r.u., Fibroma 42 days

a. 20.9 Vascular + + + + b. 4.0 c. 75.1

a. 16.0 Size ++-I-+ b. 6.0 Vascular ++++ c. 78.0

a. 18.0 Vascular + + + + b. 7.0 Size + c. 75.0

TABLE VI: The Pituitary in Rats with Transplanted Tumor Injected with Theelin (Non-Castrated)

No. of rats, Number of injections, dose, Tumor and Cell sex, and age and duration age percentages Remarks

1 M. 18 mos.

1 M. 6 mos.

1 F. 64 mos.

24 injections, 25 r.u., Sarcoma a. 9.0

c. 86.0 179 days b. 5.0 ,

5 injections, 65 r.u., Fibroma a. 36.0 Vascular ++++ 120 days b. 6.0

c. 58.0

9 injections, 65 r.u., Fibroma a. 12.0 Vascular ++++ 135 days b. 10.0

c. 78.0

433

TAIII.B VII : The Pituitary in Rats wilk Trunsplantahle Tumors Injected with Antwitrin-G and Theelin and with Antuitrin-S and Theelin

~~ _______.______ ___ __.~_____ ._____ ~.________ No. of rats, Injections: number, dose, Tumor and Cell sex, antl age and duration age percentages Remarks

-

1 F., 7: nios. Ant.-(;: 18 inj. , 2 r.u., 48 days Imniune t o Theelin: 5 r.u. tumor

1 I T . , 7; mas. Ant.-(;: 21 inj., 2 r.u., 68 days Sarcoma Theelin: 5 r.u. 68 days

1 M., 10 nios. Ant.&: 33 inj., 10 r.u., 252 days 1:ibronia Theelin: 25 r.u.

1 M., 10: rnos. Ant.-G: 35 inj., 2 r.u., 121 days Sarcoma Theelin: 5 r.u.

1 F., 12 mos. Ant.-G: 15 inj., 10 r.u., 39 days Sarcoma Theelin : 50 r.u.

1 F., 18 mos. Ant.-

T H E ANTERIOR PITUITARY GLAND I N TUMOR-BEARING RATS

TABLE IX: The Pituitary in Rats Injected with Antuitrin-G, Theelin, or Progynon-B

435

No. of rats, sex, and age

1 F., 7 mos -

1 M., 16mos

1 F., 16 mos

1 F., 18 mos.

1 M., 18 mos.

1 F., 15 mos.

1 F., 6 mos. (virgin)

1 F., 19 mos.

1 M., 5 mos.

Injections

Ant.-G: 11 inj., 2 r.u., 20 days Theelin: 25 r.u.

Ant.-G: 5 inj., 1 r.u., 34 days Prog.-B: 600 r.u.

Ant.-G: 5 inj., 1 r.u., 34 days Prog. - B : 600 r.u.

Ant.-G: 11 inj., 2 r.u., 36 days Prog.-B : 600 r.u.

Ant.&: 11 inj., 2 r.u., 36 days Prog.-B: 600 r.u.

Ant.-G: 22 inj., 10 r.u., 123 days Prog.-B: 400 r.u.

Ant.-G: 78 inj., 10 r.u., 170 days Theelin: 50 r.u.

Ant.-S: 37 inj., 16 r.u., 269 days Prog.-B: 5000 r.u.

Ant.-G: 4 inj., 10 r.u., 13 days

Tumor

None

None

Induced adenoma of breast

Induced fibroade- noma of breast

Subcut. cysts

Induced adenoma breast

Hyper- plasia mammary glands

Subcut. cysts

None

Cell percent

ages

a. 30.1 b. 11.8 c. 58.1

a. 17.5 b. 1.8 c. 78.5 d. 2.2

a. 13.0 b. 1.4 c. 76.8 d. 8.8

a. 12.0 b. 6.9 c. 81.1

a. 6.5 b. 1.5 c. 47.5 d. 44.5

a. 8.7 b. 5.3 c. 86.0

a. 30.0 b. 8.0 c. 42.3 d. 19.7

a. 0.6 c. 99.4

a. 23.8 b. 2.1 c. 74.1

Remarks

Theelin neutralizes Ant.&

Size ++++ Chromophobe

adenoma

Size ++++ Chromophobe

adenoma

Size ++++ Vascular + + + -t Chromophobe

adenoma

Size + + + + Vascular + + + + Adenoma

Size ++++ Chromophobe

adenoma

Ant.-G neutralizes t heelin

Chromophobe ad e n o m a

Thick pars Vascular + + + + Size ++++

percentage ratio of the various cells in normal animals. Prolonged injections of antuitrin-S evoked no response, while increasing doses over a short period produced an enlargement of the pituitary and an increase of chromophobes and of degranulated dells. Large doses of estrogenic hormone over a short period of time prevented the formation of castration cells and caused an in- crease of chromophobes, leading to the formation of pituitary adenomata. Many of these changes which occurred in normal animals following injection appeared also in uninjected animals bearing benign transplanted breast tu- mors, as increase of the chromophobes, increase in the size of the gland, and the appearance of pituitary adenomata.

An observation of significance is the fact that castrated rats immune to benign tumor growth show a normal cell count with the pr'esence of castra-

436 JACOB HEIMAN

TABLE X: ThP Pituitary in Castrated Rats I m m u n e to Transplanted Tumor, Uninjected _ _ ~ . - - __ _. -.

Cell Remarks No. of rats, sex, and age percentages

1 M., 18 mos.

1 M., 18 mos.

a. 13.8 b. 3.3 c. 64.2 d. 10.1 e. 8.6

a. 2.6 c. 9.2 d. 6.0 e. 82.2

Size ++++

Castration cells

TAULE XI : The Pituitary in Castrated Ruts Susceptible to Transplanted Tumor, Uninjected

No. of rats, Tumor and Cell sex, and age age percentages Remarks

1 M., 20 mos. Fibroma a. 23.5 Few castration cells after tumor growth 56 clays b. 16.5

c. 52.1 e. 7.9

1 M., 15 mos. Sarcoma a. 8.8 No castration cells after tumor growth 139 days b. 12.5

c. 68.1 d. 10.1 e. 0.5

1 M., 18 mas. Fibroadenoma a. 26.8 49 days b. 10.1

c. 46.4 e. 16.7

T A m B XII: The Pituitary in Casfrated Rat Immune to Transplanted Tumor and in Custrated Control Non-Tumor Rat , Injected

No. of rats, Cell age, and sex Inject ion percentages Remarks

1 M., 13 m o b . (in1 ni une) Theelin: 50 r.u. b. 32.5 Size ++++

___-_ . ._____ ___ Ant.-G: 77 inj., 10 r.u., 183 days a. 28.6 Vascular + + + +

c. 38.9 No castration cells

1 M . , 17 mos. Ant.&: 90 inj., 10 r.u., 480 days a. 2.0 No castration cells (control) l’rogynon 13: 1000 r.u. c . 5.0

d. 93.0

tion cells, while castrates with growing tumors show few or no castration cells. In the castrated tumor-bearing group (Table XI I I ) the injection of antuitrin- G or antuitrin-S in addition to theelin tends to keep the cell count within nor- mal limits, suggesting a neutralizing effect, but the injection of theelin alone in a tumor-bearing castrate produced 95 per cent of degranulated cells in the anterior lobe. In the attempt to analyze these findings allowance must be made for certain unexplained factors. Among these are the degree of endog-

T H E ANTERIOR PITUITARY GLAND I N TUMOR-BEARING RATS 437

TABLE XI1 I : The Pituitary in Castrated Rats Susceptible to Transplanted Tumor, Injected with Anluitrin-G, Antuilrin-S, and Theelin

- -.

No. of rats, Tumor Cell age, and sex Injections and age percentages Remarks

1 M., 11 mos. Ant.-G: 15 inj., 10 r.u., 84 days Sarcoma Theelin : 50 r.u. 101 days

1 M., 17 mos. Theelin: 12 inj., 100 r.u., 124 (lays Sarcoma 134 days

I M., 11 mos. A n t . 3 : 11 inj., 5 r.u.. 45 days Sarcoma Theelin : 5 r.u. 45 days

1 F., 11 mos. An t .3 : 12 inj., 5 r.u., 52 days Sarcoma Theelin: 5 r.u.,

a. 7.7 b. 14.2 c. 57.1 d. 18.5 e. 2.5

a. 0.5 b. 1.3 c. 3.1 d. 95.1

a. 30.0 b. 12.1 c. 36.2 d. 18.4 e. 3.3

a. 12.9 b. 8.1 c. 14.9 d. 60.2 e. 4.0

Few castration cells

No castration cells

Few castration cells

I;cw castration cells

enous hormonal activity and the interplay of the pituitary and ovarian secre- tions of the host, acting as stimulating, depressing, or neutralizing factors. The same holds true of the exogenous hormones introduced by injection.

In determining the relationship of the anterior pituitary to tumor growth certain facts must be taken into consideration. The first is that the unex- plained increase of benign tumor growth in castrated males bears a con- stant relation to the disappearance of castration cells in these animals. A possible explanation is that the transplanted tumor elaborates a substance which closely resembles the estrogenic hormones. I t is probable that all tumors induce the production of small amounts of estrogenic substances. Numerous reports are available dealing with the injection of estrogenic hor- mones in normal castrated rats, with consequent prevention of the appearance of castration cells. In our series of castrated females, breast tumors grew very poorly or not at all. Hence no interpretation can be complete without further study of the pituitary in castrated tumor-bearing females. On the other hand, the increase of the chromophobes in normal tumor-bearing females is more marked than in normal tumor-bearing males.

The relationship of the pituitary to malignant growth, if such relationship exists, does not find satisfactory explanation in the studies set forth in the Tables XIV-XVI. An increase in chromophobes is evident in old rats but not in young ones.

SUMMARY

1. In the pituitary glands of male and female rats bearing spontaneous, induced, or transplanted fibroadenomata there is a decrease in the percentage

438 JACOB HEIMAN

TABLE XIV: The Pituitary in Rats with Benepyrene Tumors and Induced Rat Sarcoma

No. of rats, Cell sex, and age Tumor and age percentages Remarks

1 M., 6 mos.

1 M., 6 mos.

1 M., 6 mos.

2 F., 6 mos.

1 F., 6 mos.

1 M., 9 mos.

1 M., 12 mos.

1 F., 12 mos.

1 F., 12 mos.

1 F., 6 mos.

Induced sarcoma 32 days

Induced sarcoma 32 days

Induced sarcoma

Benzpyrene sarcoma

Benzpyrene sarcoma

Benzpyrene thread sarcoma

Renzpyrene sarcoma 297 days

Renzpyrene sarcoma 262 days

No tumor following benzpyrene in lard

Controls (no tumor)

a, 17.0 b. 0.5 c. 82.5

a. 24.1 c. 58.9 d. 17.0

a. 22.9 c. 77.1

a. 18.2 b. 3.1 c. 78.7

a. 10.0 b. 2.8 c. 87.2

a. 2.5 c. 97.5

a. 12.0 b. 2.0 c. 68.0 d. 18.0

a. 18.0 b. 4.0 c. 75.0 d. 3.0

a. 32.0 b. 11.0 c. 57.0

a. 42.3 b. 8.7 c. 49.0

Subcut. benzpyrene tumor

Hyperplasia of pars intermedia

Adenoma of pituitary

Degranulation in various stages

Normal count

Normal count

of acidophils (5.3 per cent, 9 per cent, 9.8 per cent respectively) ; an increase in the small chromophobe cells (60.1 per cent, 67.2 per cent, 75.4 per cent), and an increase in large degranulated cells, possibly basophils (3.6 per cent, 6.9 per cent, 14.0 per cent). In some of the sections of pituitary glands from tumor-bearing rats a thickened pars intermedia with an increase in basophils is evident.

2. In the series here observed hormonal activity of the anterior pituitary could not be definitely gauged by morphologic changes.

3 . Castration cells were present in the pituitary in negligible numbers (8.7 per cent) in tumor-bearing male and female castrates and were absent altogether in tumor-bearing male castrates injected with estrogenic hormone.

4. Normal and tumor-bearing rats injected with the growth hormone showed a marked increase in chromophobes (73.4 per cent) in the pituitary and a negligible number of basophils.

T H E ANTERIOR PITUITARY GLAND IN TUMOR-BEARING RATS 439

TABLE XV: The Pituitary i n Rats Fed with Z-Amino-5 Azotoluene

No. of rats, Cell sex, and age percentages

1 M., 18mos.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. 8.3 c. 91.7

1 M., 18 m o s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. 4.5 c. 51.9 d. 43.6

1 M., 18mos.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. 27.2 b. 20.9 c. 51.9

1 F., 18mos.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. 8.4 b. 40.7 c. 50.9

1 F., 18mos.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. 20.6 b. 1.8 c. 73.3 d. 4.3

TABLE XVI: The Pituitary in Rats with Tumors R.S., R 256, and R 39

No. of rats, Tumor and Cell sex, and age age percentages

1 M., 5 mos.

1 M., 6 mos.

1 M.

Sarcoma R.S. 21 days

Carcinoma R 256 28 days

Sarcoma R 39 34 days

a. 36.1 b. 8.1 c. 55.0 d. 0.8

a. 42.7 b. 6.4 c. 48.8 d. 2.1

a. .21.8 b. 0.8 c. 77.4

5. Tumor bearing castrates injected with antuitrin-G and theelin showed few or no castration cells and more degranulated cells than uninjected animals (3.5 per cent castration cells, 47.5 per cent degranulated cells).

6. Male and female animals fed with 2-amino-5-azo-toluene for over one year showed a marked increase in chromophobes, a negligible number of basophils (0.5 per cent), a reduced percentage of eosinophils (0.9 per cent), and an increase in degranulated cells.

7. Animals with induced subcutaneous benzpyrene tumors and with in- duced rat sarcoma showed changes similar to those described above, but with the appearance, in addition, of large degranulated basophils (3.6 per cent).

8. Tumor-bearing female rats did not produce litters after the appearance of the tumors. Whether this is associated with the reduction or absence of beta or gonadotropic cells is to be investigated.

440 JACOB HEIMAN

TABLE XVII: Average DiJerential Cell Count of Anterior Pituitary i n Various Groups - .-

Series

Spontaneous fihroadenoma Rats immune to tumor growth Rats with transplantable tumor

Ant.-(; in rats with tumor 342 Ant.-C and Theelin in rats with

Ant.-G in rats immune to tumor

Ant.-S in rats with tumor 342 Theelin in rats with tumor 342 Ant.-S and Theelin in rats with

Rats with induced tumor T 2011 Ant.-G in normal male, no tumor Ant.-(; and Theelin in normal rats,

small doses Ant.-G and Progynon in normal

rats, large doses Castrates immune to tumor 342,

uninjected Castrates positive to tumor 342,

uninjected Castrates immune to tunior 342,

injected with Progynon Castrates immune to tumor 342,

injected with Ant.-G and Theelin Castrates positive t o tumor 342,

injected with Ant.-Gand Theelin Castrates positive t o tumor 342,

injected with Theelin alone Castrates positive to tumor 342,

injected with Ant.-S and Theelin male and female

Rats with IRS or/and benzpyrene tumors

Rats fed with 2-amino 5-azotolu- ene over one year

342

tumor 342

342

tumor 342

___ -

No. of rats

3 4

17 3

5

1 3 3

2 4 1

1

5

2

3

1

1

1

1

--

2

10

4

5.3 36.2

29.8 21.5

13.7

34.0 18.3 19

7 9

23.7

30.1

12.6

8.2

19.4

2.0

28.6

7.7

0.5

21.5

15.6

12

- - 3aso- phils (b)

13.5 5.8

6.5 5.1

9.2

8 5.6 7

4 8.7 2.1

11.8

3.6

1.7

13.0

0

32.5

14.2

1.3

-_

10.1

1.5

15.4

Ihromo- phobes

(C)

67.2 58.0

60.1 73.4

76.1

58.0 76.1 74.0

89.0 75.4 74.2

58.1

73.0

36.7

55.5

5.0

38.9

57.1

3.1

25.5

79.3

61.6

)egranulated cells (4 14.0 0

3.6

1 .o 0 0 0

0 6.9 0

0

10.8

8.0

3.4

93.0

0

18.5

95.1

39.3

3.6

11

Iastration cells (el

45.4

8.7

0

0

2.5

0

3.6

0

0

9. The combined injection of antuitrin-G and theelin in small doses tends to restore the cell count to normal-acidophils 30; basophils 12 ; chromophobes 58-suggesting that the growth hormone may neutralize the action of the estrogenic hormone.

10. The combined injection of antuitrin-G and theelin in large doses in- creases the chromophobe cells to 73 per cent and the degranulated cells to 10.8 per cent.

11. Rats immune to tumor growth, whether benign or malignant, showed a normal differential cell count: acidophil cells 36.2 ; basophil 5.8; chromo- phobe 58.

12. Tumor-bearing animals invariably had a chromophobe cell count of over 70 per cent, a percentage which appears also in normal animals injected with estrogenic hormones.

THE ANTERIOR PITUITARY GLAND IN TUMOR-BEARING RATS 441

13. Tumor-bearing rats injected with antuitrin-G and antuitrin-S or theelin, alone or in combination, showed an increase in chromophobe cells: 73.4 per cent, 76.1 per cent, 89 per cent.

NOTE: The author is indebted to Parke, Davis & Co. for the antuitrin-G, antuitrin-S, and theelin, and to Schering Corporation for the progynon, used in these experiments.

BIBLIOGRAPHY

1. HEIMAN, J.: Am. J. Cancer 22 : 497, 1934.

2. BAILEY, P.: In: E. V. Cowdry, Special Cytology, New Uork, P. Hoeber, 1928. 3. HATAI, S.: Am. J. Anat. 15 : 87, 1913-14. 4. ADDISON, W. H., AND ADAMS, M.: Anat. Rec. 33: 11, 1926. 5. RASMUSSEN, A. T.: Anat. Rec. 42: 60, 1929. 6. ANDERSEN, D.: Proc. SOC. Exper. Biol. & Med. 30: 657, 1932-33. 7. FISCHER, 0.: Virchows Arch. f . path. Anat. 259: 9, 1926. 8. WOLFE, J. M., AND CLEVELAND, R.: Anat. Rec. 55: 233, 1933. 9. ERDHEIM, J., AND STUMME, E.: Beitr. z. path. Anat. u. z. allg. Path. 46: 1, 1909.

HEIMAN, J., AND KREHBIEL, 0. F.: Am. J. Cancer 2 7 : 450, 1936.

10. MARTINS, T., AND DE MELLO, R. F.: Compt. rend. SOC. de biol. 118: 916, 1935. 11. RASMUSSEN, A. T.: Proc. SOC. Exper. Biol. & Med. 26: 424, 1929; Am. J. Path. 9: 459,

12. WOLFE, J. M.: Anat. Rec. 61: 321, 1935. 13. SEVERINGHAUS, A. E., ENGLE, E. T., AND SMITH, P. E., in Sex and Internal Secretions,

14. EVANS, H. M., AND LONG, J. A.: Anat. Rcc. 21: 62, 1921. 15. ZONDEK, B., AND BERBLINGER, W.: Klin. Wchnschr. 10: 1061, 1931. 16. COLLIP, J. B., SELYE, H., THOMSON, D. L., AND WILLIAMSON, J. E.: Proc. soc. Exp.

17. WOLFE, J. M.: Proc. SOC. Exper. Biol. & Med. 31: 812, 1934. 18. BACHMAN, C., COLLIP, J. B., AND SELYE, H.: Proc. SOC. Expcr. Biol. & Med. 32: 544,

19. SEVERINGHAUS, A. E.: Proc. SOC. Exper. Biol. & Med. 31: 593, 1934. 20. FLUHMANN, C. F.: Proc. SOC. Exper. Biol. & Med. 34: 691, 1936. 21. NELSON, W. 0.: Proc. SOC. Exper. Biol. & Med. 32 : 452, 1934. 22. WOLFE, J. M.: Anat. Rec. 63: 3, 1935. 23. SPENCER, J., AND D’AMOUR, F. E.: Am. J. Anat. 50: 129, 1932. 24. MOORE, C. R., AND PRICE, D.: Am. J. Anat. 50: 13, 1932. 25. NELSON, W. 0.: Proc. SOC. Exper. Biol. & Med. 31: 1192, 1934. 26. ANDERSEN, D. H.: J. Physiol. 83: 15, 1934. 27. SELYE, H., COLLIP, J. B., AND THOMSON, D. L.: Proc. SOC. Exper. Biol. & Med. 32 :

28. CROOKE, A. C., AND KORENCHEVSKY, V.: Proc. Roy. SOC. Med. 28: 1266, 1935. 20. MCEUEN, C. S., SELYE, H., AND COLLIP, J. B.: Lancet 1: 775, 1936. 30. ZONDEK, B.: Lancet 1: 776, 1936.

32. BURROWS, H.: Am. J. Cancer 28: 741, 1936.

33. WOLFE, J. M.: Am. J. Physiol. 115: 665, 1936. 34. WOLFE, J. M., AND HAMILTON, J. B.: Proc. SOC. Expcr. Biol. & Med. 36: 307, 1937. 35. FEVOLD, H. L., HISAW, F. L., AND GREEP, R.: Am. J. Physiol. 114: 508, 1936. 36. WOLFE, J. M., AND CHADWICK, c. s.: Endocrinol. 20: 503, 1936. 37. ELLISON, E. T., AND BURCH, J. c.: Endocrinol. 20: 746, 1936. 38. FLUHMANN, C. F., AND KULCHAR, G. V.: Proc. soc. Exper. Biol. & Med. 28: 417,

39. WOLFE, J. M., A N D CHADWICK, C. S.: Proc. soc. Exper. Biol. & Med. 34: 56, 1936.

1933.

ed. by Edgar Allen, Williams and Wilkins, Baltimore, 1932, p. 805.

Biol. & Med. 30: 590, 1933.

1934. I . ,

1377, 1935.

31. CRAMER, w . , AND HORNING, E. s.: Lancet 1: 1056, 1936. BURROWS, H., AND KENNAWAY, N. M.: Am. J. Cancer 20: 48, 1934.

193e1.

442 JACOB HEIMAN

40. HALPERN, S. R., AND D’AMOUR, F. E.: Am. J. Physiol. 115: 229, 1936. 41. LIPSCHUTZ, A., AND VILLAGRAN, G.: Compt. rend. SOC. de biol. 121: 203, 1936. 42. LOEB, L.: J. M. Research 40: 477, 1919.

43. CORI, C. F.: J. Exper. Med. 45: 983, 1927. 44. GARDNER, W. U., STRONG, L. C., AND SMITH, G. M.: Am. J. Cancer 26: 541, 1936. 45. ROBERTSON, T. B., AND BURNETT, T. C.: J. Exper. Med. 21: 280, 1915. J. Cancer Res.

46. KARLEFORS, J.: Ztschr. f . Krebsforsch. 17: 195, 1920. 47. ROSSLER, H.: Ztschr. f . Geburtsh. u. Gynak. 96: 516, 1929. 48. NOVAK, E., AND KOFF, A. K.: Am. J. Obst. & Gynec. 20: 481, 1930. 49. GUYER, M. F., AND CLAUS, P. E.: Anat. Rec. 52: 225, 1932; 56: 373, 1933. 50. WYETH, G. A,: Endocrinol. 18: 59, 1934. 51. MCEUEN, C. S., SELYE, H., AND THOMSON, D. L.: Brit. J. Exper. Path. 1 5 : 221, 1934. 52. ENGEL, P.: Ztschr. f . Krebsforsch. 41: 281, 1934.

54. OBERLING, C., G U ~ R I N , M., AND G U ~ R I N , P.: Compt. rend. SOC. de biol. 123: 1152, 1936. 55. MENDEL~EFF, P.: Cancer, Bruxelles 12: 287, 1935. 56. TWORT, J. M., AND TWORT, c. C.: Am. J. Cancer 29: 78, 1937. 57. WITHERSPOON, J. T.: Am. J. Obst. & Gynec. 31: 173, 1936. 58. MALAVAZOS, A., AND GOLDMAN, L. M.: Urol. & Cutan. Rev. 40: 338, 1936. 59. CLOSE, H. G.: Lancet 1: 732, 1934. GO. ZONDEK, H., ZONDEK, B., AND HARTOCH, W.: Klin. Wchnschr. 11: 1785, 1932. 61. MOLLER, H.: Frankfurt. Ztschr. f . Path. 45: 571, 1933. 62. GRUHZIT, 0. M.: Arch. Path. 16: 303, 1933. 63. BISCHOFF, F., MAXWELL, L. C., AND ULLMANN, H. J.: Am. J. Cancer 21 : 329, 1934. 64. SUGIURA, K., AND BENEDICT, S. R.: Am. J. Cancer 18: 583, 1933. 65. SAMUELS, L. T., AND BALL, H. A,: Am. J. Cancer 23: 801, 1935.

66. LACASSAGNE, A.: Compt. rend. SOC. de biol. 120: 833, 1935. 67. KOCH, F. C.: Physiol. Rev. 17 : 153, 1937.

LATHROP, A. E. C., AND LOEB, L.: J. Cancer Res. 1: 1, 1916.

3: 75, 1918.

53. CRAMER, w., AND HORNING, E. s.: Lancet 1: 247, 1936.

BALL, H. A., AND SAMUELS, L. T.: Am. J. Cancer 26: 547, 1936.