Columbia University, New York.)

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1 15 6I2.62I:6I2.4 THE EFFECT OF OVARIAN HORMONE ON THE PITUITARY, THYROID, AND ADRENAL GLANDS OF SPAYED FEMALE RATS. BY DOROTHY H. ANDERSEN. (From the Department of Pathology, College of Physicians and Surgeons, Columbia University, New York.) (Received July 20, 1934.) SOMETIME ago we demonstrated that the hypophysis and adrenal glands of the rat hypertrophied during cestrus [Andersen and Kennedy, 1932; Andersen, 1933]. We then attempted to produce this change by the injection of ovarian hormone (amniotin, Squibb) into spayed female rats. In these preliminary experiments the spayed controls -showed sufficiently great variation in adrenal weights to make the adrenal changes in the injected animals of no significance. Analysis of our data revealed a correlation between the interval after operation and the weight of the adrenal. In order to establish a base line we studied the changes in the adrenal, thyroid, and pituitary glands at various intervals after spaying and these data have been reported [Andersen and Kennedy, 1933]. A number of other investigators have studied the effect of ovarian hormone on these glands when injected over a long period. Leonard, Meyer and Hisaw [1931] found no change in the weights of the pituitary, thyroid, and adrenals of immature female rats which were injected with ovarian hormone for days.begiing at the age of days. They tested the sex-stimulating potency of the pituitary glands by the implant method and found it had decreased. Leiby [1933] spayed adult female rats, and after an interval of 3 weeks he injected 100 rat units of theelol daily for a week. The pituitary, adrenal, and thyroid glands were hypertrophied as compared with those of the uninjected spayed controls. Poll [1933] injected progynon, an ovarian hormone preparation, and proviron, a male sex-hormone preparation, into normal and castrated adult rats of the corresponding sex and was able to restore the wide x-zone of the adrenal which is characteristic of young animals.

2 16 D. H. ANDERSEN. Karp and Rostkiewicz [1934] produced enlarged thyroid glands which were hyperemic and contained abundant colloid by the injection of menformon, an ovarian hormone preparation, into female rabbits. When the injections were given in daily doses of 100 rat units over periods of days, the gland developed the appearance found in colloid goitre. Hohlweg [1934] injected 100 rat units of progynon daily for 6 weeks into rats. The hypophyses were enormously hypertrophied, the chief cells were much enlarged, and many contained mitotic figures. It will be noted that most of these observers used large doses of hormone over a period of one or more weeks. Hypertrophy of the adrenal was found by Leiby and by Poll; of the hypophysis, by Leiby and Hohlweg; and of the thyroid, by Leiby and by Karp and Rostkiewicz. Leonard, Meyer and Hisaw found no weight changes, but noted a decrease in the sex-stimulating potency of the hypophysis. TECHNIQUE. The present experiments were planned with the idea of reproducing as nearly as possible the conditions found at cestrus. It is known that as the interval after castration is increased, the quantity of ovarian hormone required to produce cestrus is increased, and after about 6 weeks the dosage must be high. On the other hand, the weights of the thyroid and adrenal glands do not decrease to a constant level until about 6 weeks after spaying, and the hypophysis at this time has not yet become as large as it does a few weeks later. In consideration of these facts we have uniformly spayed the animals at days of age and begun injections after an interval of days Amniotin (Squibb) dissolved in ethylene glycol was injected in doses of 5 rat units three times a day for 2 days, making a total of 30 rat units. The extract had the potency of 5 rat units per 1 c.c. The injections were given at approximately 9 a.m., 1 p.m., and 5 p.m. The rats were killed by chloroform at various intervals. Those in the group "killed 24 hours after the first injection " were killed between 9 a.m. and 12 noon, and the other groups likewise were killed during the three hours after the time named. Groups of rats were killed at intervals of 24, 36, 48, 60, 72 and 120 hours after the first injection. In order to standardize the animals as far as possible the following precautions were taken: the rats were of the same strain used in the previous experiments and were bred in the laboratory; the diet, care, and the technique of dissection and weighing were standardized and have been previously described [Andersen and Kennedy, 1932]; at autopsy the lungs and middle ears

3 OVARIAN HORMONE AND ENDOCRINE GLANDS. were inspected. In our experience the more severely infected rats have adrenal hypertrophy, but the majority of the infected rats do not. It has not yet been possible to estimate with accuracy the degree of infection which will produce adrenal hypertrophy, and therefore all rats having any evidence of infection have been discarded. Of the eighty rats used in the experiment, thirty were discarded because of infection. The first two batches of amniotin used were obtained through a pharmacy and varied greatly in potency. The first batch produced cestrous changes in the vaginal smear 48 hours after the first injection in the majority of the animals, and the animals treated with this Fre grouped in Table I under the title " Amniotin-low potency." The second batch of extract obtained from the same source did not produce vaginal smear changes in any rats and is listed as " Amniotin-non-potent." The age of these extracts was unknown, and we have since found that extracts kept in the cold for over 6 months vary in this way. The extract used in the remaining experiments has been supplied most generously by Dr A. J. Morrell of the Squibb Research Laboratories and has been uniform in potency. None over 3 months of age has been used. All rats treated with it have had an cestrous smear with an abundance of nucleated epithelial cells 48 hours after the first injection. On the following day the smear contained many cornified cells and on the fourth day a few white cells or cornified cells. The non-potent amniotin has accidentally supplied us with an additional control. Uninjected spayed animals and a small group of animals injected with an equal volume of sterile neutral broth also served as controls. Sections of the adrenals and thyroids of the animals "killed after 48 hours" and of the spayed controls were made, and stained with haematoxylin and eosin. A histological study of the hypophysis has not been undertaken. The probable error of the mean was calculated from the formula 17 P.E.mean =0-67. DATA. The essential data are presented in Table I and in Figs. 1 and 2. The spayed rats treated with potent amniotin and killed after 48 hours, that is, when the vaginal smear was cestrous in type, reproduced the picture of the cestrous weight changes found in the pituitary and adrenal glands of normal animals. The histological picture of the adrenal was also similar. PH. LXXXT1T1. 2

4 18 P D. H. ANDERSEN. P.0 ~ ~ ~ ~ ~ ~ ~~~~~0 410 n~~ ~~ X4 C4 X1 Eb tq Ce OX -0 0o0 lf tr [- *HH L g g X t O 0X X 00 X _ a*~~~ ' O O N _ X O s N> _X00 o -4 eq t---l- _ t- 0M d e *~~~~~~~~- xm t bo o = Ca go - e s X~~~~~~~(; oo J 4 g ~~~~~~-,d 4(O = t- 4 _ (o r- L_e lo--00r q 0 o~~~~~~~~~~~~~~~~~t (M q "oe 0 A) C0 oo._ W "X~ CSC O0 I 4 e C CS.O O C)r-4 O OOO 44-q O O0 O0 4 J 4* +D ~~~~~~~~~~~4 4-4 Q o 0o O 4._ F > m > m _ m _ >~~~~~~~~o4gi HQt t o.s c) o o0 o o o = - f *~ ~ ~ ~ ~ ~ ~~~~~~4 0:

5 OVARIAN HORMONE AND ENDOCRINE GLANDS. 19 The thyroid gland, which does not show oestrous changes in the normal animal, but atrophies after spaying, was restored to the condition found in the normal animal as judged by its weight and histological appearance. The mean relative weights of the glands are approximately those found in cestrous animals of the same weight: for the pituitary 65-4 mg./kg. as compared with the normal oestrous weight of 65-1 mg./kg., a figure 15 p.c. higher than in the spayed controls; for the thyroid, 104*7 mg./kg., an increase of 18 p.c. over the spayed controls, as compared with mg./kg. at normal cestrus, which is 28 p.c. higher than the spayed controls; and for the adrenals, mg./kg., as compared with 237 mg./kg. for normal cestrus, which figures are 35 and 36 p.c. respectively over the mean adrenal weight of the spayed controls [Andersenand Kennedy, 1932; Andersen, 1933]. An unexpected finding was that the time required to reach maximal weight varied for the different glands. The pituitary reached its greatest weight (within the range of experimental error) in the animals killed 24 hours after the first injection. These rats had received only 15 rat units of amniotin. The pituitary remained at about the same size during the period of observation, namely until the sixth day of the experiment. The adrenal was increased in size by 24 hours, but reached its maximum by 48 hours and then decreased rapidly in weight. The thyroid was also enlarged by 24 hours, but reached the peak of its weight by 72 hours. These differences are readily seen in the curves in Fig. 1. The various control groups showed no significant rise in pituitary weight. Strangely enough, there is a definite thyroid hypertrophy in the group treated with non-potent amniotin. It is probable that the quantity of hormone necessary to produce changes in the endocrine glands is less than that required to produce vaginal smear changes. Adrenal hypertrophy was found in all of the control groups, including the rats injected with neutral broth, which were killed between 36 and 60 hours after the first injection. That part of this hypertrophy may be a response to the toxic effect of foreign material is probable. It is, however, significant that the degree of hypertrophy varies directly with the potency of the amniotin. Sections of the adrenals of the rats treated with potent amniotin are almost indistinguishable from those found in oestrous animals which we have previously described. Mitoses are rare and the increase is in the size and lipoid content of the cortical cells. These glands differ from those of normal cestrus only in the greater congestion of the vascular bed, especially in the reticular zone (Figs. 3 and 4). The thyroid is also similar to that of a normal rat, and in marked contrast to the atrophied gland found 6 2-2

6 20 250r D. H. ANDERSEN. Ad renal 200 I Mg /Kg of body weight 100 Thyroid Pituitary WOO..' U40wooo Injections AAA AAA Hours after first injection Fig. 1. The effect of ovarian hormone (amniotin, Squibb) on the weights of the pituitary, thyroid, and adrenal glands. The dosage was 01 c.c. three times a day for 2 days. 0 1 c.c. =5 rat units.

7 250r Adrenal 200l AW M9/ 150 Thyroid Kg of body AM,' weighi loo j AW AW AW Bt 50 B AN Pituitary Injections AAA AAA Hour5 after first injection Fig. 2. The effect of non-potent or slightly potent amniotin and of neutral broth on the weights of the pituitary, thyroid, and adrenal glands. A W=weak amniotin which produced an cestrous vaginal smear in about half the injected animals. AN =amniotin which produced no vaginal smear changes. B = neutral broth. All rats were given doses of 0.1 c.c. three times a day for 2 days.

8 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~r- 0t 22 D. H. ANDERSEN. K 44R. S - \ 3B c1 R W,. X ac &IW.f2''tER 's~~~~~~~~~~~~~~~~~~~~~ *_CEr se_ x~~~~~~~~~~~~~~ \} ^ ḟfi.t w s-.. ^,~~~~~~~~A

9 OVARIAN HORMONE AND ENDOCRINE GLANDS. 23 o0 A 54CD C 0.-P o m * 4 O e C)a- 200 H 4 O P4 C4 C-a 4. ec0 dsh it 44 : 44;:-'

10 24 D. H. ANDERSEN. weeks after spaying. The colloid is darker than normal, but contains an abundance of vacuoles and the cells are large and pale. The vessels, especially the capillaries, are congested (Figs. 5 and 6). DIscusSION. It has been known for many years that the normal functioning of the reproductive tract, and especially of the ovary, is dependent on the normal functioning of the endocrine glands. The present experiments show that the normal functioning of the thyroid, adrenal, and pituitary glands, as indicated by their weight and the histological appearance of the two former ones, are equally dependent on the presence of ovarian hormone. These glands are atrophied 6 weeks after spaying and can be restored to their normal appearance within a few days by physiological doses of ovarian hormone. The rapidity of the response is of great interest. The mechanism of this effect is obscure. The ovarian hormone may affect the glands indirectly through some change in the metabolism of the body as a whole, it may stimulate each gland directly, or it may stimulate the pituitary which is known to influence the thyroid and adrenal. The latter supposition seems more probable and is now under investigation. The fact that the time required for a maximal response differs in the various glands is of interest, but does not prove that the gland which first shows evidence of change is the one which stimulates the others, for we know very little about the time factor in the response of these glands to stimuli. It is of great interest that all three glands show a definite hypertrophy within so short a time as 24 hours after the first injection. The prolonged change in the thyroid offers an explanation for the absence of oestrous changes. The effect of one cestrus is still present when the next cestrus occurs. The meaning of these rapid alterations in the weight, appearance, and, presumably, function of these glands in the animal economy is not yet clear. The recent studies of chemical and metabolic changes associated with the reproductive cycle are too numerous to be reviewed here, but they undoubtedly bear an important relation to the endocrine changes. The report of Richter and Hartman [1934] on the increased activity of spayed female rats following the injection of cestrin is of especial interest. These authors suggest that the increase in activity is due to a second ovarian hormone. From our finding that the degree of glandular hypertrophy varies roughly with the uterine and vaginal smear changes it seems probable that the same hormone affects both, but this is by no means proven.

11 OVARIAN HORMONE AND ENDOCRINE GLANDS. 25 CONCLUSIONS. 1. Injection of ovarian hormone (amniotin, Squibb) restores the atrophied pituitary, thyroid, and adrenal glands of spayed rats to the weight found at cestrus in normal animals. The histological picture of the thyroid and adrenal is similar to that of the oestrous rat. 2. These weight changes are evident within 24 hours after the injection of small doses (15 rat units) of the hormone. The maximal response of the hypophysis occurs after 24 hours, of the adrenal after 48 hours, and of the thyroid after 72 hours.. 3. A significant degree of adrenal hypertrophy may be produced by weak amniotin and neutral broth, but not by non-potent amniotin. Thyroid hypertrophy was produced by the weak and non-potent amniotin but not by neutral broth. The pituitary was not affected by either of these preparations. The generous and skilful technical assistance of Helen S. Kennedy and Elsie F. Doob is gratefully acknowledged. REFERENCES. Andersen, D. H. (1933). Proc. Soc. exp. Biol., N.Y., 30, 657. Andersen, D. H. and Kennedy, H. S. (1932). J. Phy8iol. 76, 247. Andersen, D. H. and Kennedy, H. S. (1933). Ibid. 79, 1. HohIweg, W. (1934). Klin. W8chr. 13, 92. Karp, L. and Rostkiewicz, B. (1934). Ibid. 13, 489. Leiby, G. M. (1933). Proc. Soc. exp. Biol., N.Y., 31, 15. Leonard, S. L., Meyer, R. K. and Hisaw, F. L. (1931). Endocrinology, 15, 17. Poll, H. (1933). Dtsch. med. W8chr. 59, 567. Richter, C. P. and Hartman, C. G. (1934). Amer. J. Physiol. 108, 136.