REDUCTION O F THE THYIIUS BY (IOKXDOTROPTC HORMONE HERBERT M. E V A N S AND MIRIAN E. SIMPSON Instztute of Experimental Eiology, University of California, BerLeley The meagerness of well-established results from the numerous efforts to determine the function of the thymus is too well known f o r further comment. ( SCC,for instance, Trendelenburg's reccrit account, '34.) It is not too much to state that no investigator has as yet been able to secure specific effects from ablation of the thymus or from efforts to experimentally imitate increased thymic function. Security in the conception of the thymus as an organ of interiial secretion arid light on its role may, however, come from a study of the effect of other internal secretions on ihc thymus. The present study aims t o report one such effect-one resulting from injection of a gonadotropic hormone. Chronic injection of this hormone led to a striking reduction of thymic substance sometimes resulting in almost complete disappearance of the gland-an effect which, as will be disclosed, is probably t o be attributed t o the intermediation of malc o r female sex hormone. The years of painstaking quantitative studies devoted by August Hammar to normal arid altered thymic stnzcture established the striking and sensitive dependence of the thymus upon the maintenance of normal body weight arid satisfactory general nutrition, demonstrating as it did the 'Aided by grants from the Committee for Research i n Prohlcms of Sex of the National Research Council, and from the Rockefeller Foundation of New York City. These funds have been gcnerously augmented by the Board of Research of the University of California, Bcrkeley. The authors desire to record their appreciation of tlie invaluablc aid of Donald H. Wonder in the preparation and standardization of tlie hormones employed in these studies. 423 424 IiEBBERT & EVANS I. B E D MIHIAM E. SIMPSON rapid recession of the thymus in practically all disturbaiices of growth o r nutrition. This rclationship is so securely established that any claims f o r experimental contrivances wliich electively reduce tlie thymus cannot be accepted if impairment of body weight or of growth or other adverse effects are demonstrable. The experimental procedures herein reported were not associated with loss of body substance; they were, indeed, associated with slight gains in adults or with moderate or marked gains of weight in immature animals. The gonadotropic hormone used in these experiments is the most potent gonadotropic substance known to us, namely, that found in the placenta or in tlie blood stream of mares early in pregnancy (Cole and Hart, '30; Evans, Meyer and X i m p son, '33 ; Catclipole and Lyons, '34). The particular product used was an extract of mare's placenta and uterus at the appropriate early period of pregnancy. CIIRONIC TREATMENT O P NORilIA4L ANIMALS WIT11 EQUINE GONADOTROPIC HORMONE A. Immature females Female rats, 25 days of age, mere injected once daily snbcutaneously with 50 R.U. of gonadotropic hormone for 26 to 65 days. Normal uninjected littermate controls were sacrificed at the end of this injection period along with the injected aninials. A report has already been made of the striking hypertrophy of the ovaries and of the anterior liypophysis which characterized these injected Only the e-flfect on the thymus is considered here. As will be seen in table 1 (S25), the thymus of the controls weighed between 202 and 535 rng., averaging 264 mg., while the thymus of their littermates treated chronically with potent gonadotropic hormone were reduced to weights of only 8 t o 83 mg., average 46 mT. * The original yurposc f o r which chronic injections of this gonadotropic hormone was undertaken was t o determine the effect on the ovary. The eEeets on the ovaries and liypophyses have been reported elsenhere (Ex-ans, Simpson, and McQuecn-Williaiiis, '34 ; Evans and Simpson, '34). TABLE 1 Bcsponse of thynaus of ntniure and i n m a l u r e ittale and fentole rats t o chronzc injection of the gonndofroprc hormone s ~ c u r c dfrotn pregnant m a w s EQUINE GONADOTROPIC IIOBJIOXE 5 0 x. u. n.411~8 TYfR O F A N I X A L Fcinales immature (5%) bUB days days 25 25 25 23 23 2.5 23 65 63 65 65 65 65 65 63 65 63 61 23 26 2 .? 2.5 2J 25 _____ Average Females mature (S27) [ncreese i n ~ o d yweight Thymus ncreaee i n ~ o d ywc.ight Thymus gm. lng. gn1. mg. 139 126 164 124 96 120 21 4 14G 13-1 110 37 141 133 202 280 260 2 15 127 300 220 231 204 236 53, 130 100 98 112 129 106 118 111 94 98 142 98 111 46 4 _____ 177 177 177 176 177 177 177 ti5 6.i - 42 177 177 196 193 196 65 65 65 50 -42 196 _____ 65 65 63 65 B5 - 82 28 3 1 18 ~ 6" 6 9 0 30 20 Average 7 62 .i i 35 49 24:3 ' GO 33 44 25 8 33 83 46 03 49 57 11 65 40 32 65 63 63 65 65 20 86 Plateauci 82 10 10 10 30 186 127 b3 63 60 80 .530 122 120 Males rnature (R26) ti0 _____ 122 130 133 121 120 120 F3 6 65 65 250 301 278 334 350 "24 310 292 27 36 39 42 (1i 65 6.5 117 30 17 3.5 207 :7 i Average 10(i 132 26 33 10 ~ Svcrage 305 264 146 97 129 174 139 17 4; ti 3 hlales immature (H24) CONTROL Platcauci 191 2 7CI 272 330 265 285 400 290 196 260 347 290 290 400 201 284 This animal also did not show the characteristic hppophyseal hypertrophy seen in other members of this group. The hypophysis weighed 12.5 rng. Those nf others in the group 26, 21, 26, 26 mg. 'hTo animal is included in the table which lost ncight during t h e last 1 0 t o 20 days of injection. Animals are also excluded known t o be sick, e.g., Iv-hcezy rats, rats injured i n fighting, cte. 426 426 ZIEFCBERT M. EV.4NS A N D iLlIIlIAM E. SIMPSON B. Mature fenznles Mature females, 6 t o 64 months of age, were treated once daily subcutaneously with an identical amount of the same preparation injected into the immature females. The adult females were also sacrificed after 20 t o 65 daily injections were given. Here also, associated with the massive hyyertrophy of the ovaries3 and of the pituitary, an astonishing reduction of the thymus was encountered; at times the vestiges of thymus present were too small to give security in dissection for weighing (those cases noted in the table where the weight was 10 mg. o r less). Even the thymi of the females treated f o r the shorter periods, 20 t o 30 days, showed extreme atrophy (average 30 compared with a 127 mg. average in the controls). See table 1,S27. C. Immature males Immature males, 25 days of age, were similarly injected for 20 to 65 days. With the single exception of the animal injected only f o r 20 days, all animals showed thymi reduced f o one-fifth the weight of normal uninjccted animals of the same sex and age, 60 compared with 290 mg. See table 1,XZ-4. D. Mature males A group of adult males, 4 to 4: months old, was also treated for varying periods, 30 t o 147 days. I n this group the atrophy of the thymus was extreme, except in one animal, sacrificed after only 30 days’ injection. The average thymus weight was 37 mg. compared with 284 mg. average in control males. See table 1, S26. It is to he pointed out that at time of sacrifice all animals included in the tables were in excellent nutritive condition. All immature animals had gained weight during the injection These ovaries a s can be seen by consulting the paper dealing with this matter (Evans, Simpson and McQueen-Williams, ’34) sometimes attained a weight of 2 t o 3 grn. The original object of this experiment was to see if the ovary beearne rxhaiistrd by long continued injection mith the equine hormone. It was found, however, that they weighed between 1 and 2 gni. after more than 2 months’ injection. EEDUCTIOX' O F THYMUS BY EOBMONE 427 p e r i ~ d . ~All adults hail either gained slowly or were in growth stasis. None were included that had lost any weight during the last 10 t o 20 days of the injection period. There is no reason, therefore, for referring the thymic atrophy to malconfition of the animal. Instances were noted such as a rat injected 30 days in which the thymus weighed more than that of any other animal in the group, namely, 207 mg. yet during the full period of administration no body growth occurred in the animal, whereas others in the gronp gained as much as 20 am.yet the thymus was completely atrophic. INJECTION O F GONADECTO&IIZED RATS WITH THE EQUINE GONADOTROPIC HOJZMONF; The following experiments show that, regardless of the sex employed, gonadectoiiiy prevents this experimental reduction of the thymus by gonadotropic hormone. A. Females Females 38 days of age were castrated and immediately thereafter injected with 125 R,.U. of pregnant mare gonadotropic hormone daily for 20 days (table 2 ) . At the end of this period, they were sacrificed. Normal miinjected littermate controls were maintained and sacrificed at the same time. The thymi of the untreated controls average 393 mg. in weight, those of the ovariectoniized aiiinials treated with the hormone averaged 453 mg. The gonadotropic hormone, therefore, exerted no clear degenerative influence on the thymus in the ~ group of normal females of the absence of thc o ~ a r i e s . A same age, many of them littermates t o the above, were injected with the same hormone during the 20-day period, and their ' Comparison with normal uninjectcil animals shows, however, t h a t a dcfinitc although not serious interferenre with growth resnlted from administration of thesc substances t o the males. The administration of gonadotropic hormone from other sources (anterior hypophyses, urine of pregnancy) will u l t i m a t ~ l ybe neeessary before we can refer t h e sloww growth seen in the male t o the eff'rcts of the gonadotropic hormone itself. Gonadectoiny also prevented the hypcrtrophy of the anterior hypophysis which restilted froin the administration of' the same gonadotropic hormone (Evans and Simpson, '34). 425 HERBERT 31. EVANS A N D MIRIAM E. SIPIIPSUN thymi showed significant decrease in weight (I03 nig.) showing that the dose and period of injection were adequate. TABLE 2 Besponse of the thynws of male and female castrated ruts t o the gonadotropic hormone from p r e g m n t m u m s 1 TNBlGOIED ~ _ _ _ )URATlOb ‘PREATXENT I Castrate -~ OF NJFICTIOI xormal Normal Cwtrnte in hodv weight days Males equine hormonc 12.5 R.U daily (WE38 20 ym. mg. 50 72 76 34 40 363 157 187 10 62 80 440 426 507 445 48 44 61 70 12 -. _ 57 Avrra ge 30 -_ 10s 90 102 98 102 128 30 gm. m. my. 68 390 41 0 41 0 310 440 224 96 ~ - 99 52 62 44 - ~ 70 ___ 146 1 46 ” ,. 156 510 140 158 490 460 152 101 I ~ MLllCS ryuine hormone 2.5 R.V. d;lily (W138 I, I .ncrease -~ -___ CONTROL I-SZ 3 64 3 20 375 550 485 320 353 dvcragc - -~ E’emales equine hormone 12.5 R.lJ 38 d xily (W1238’ ~ _ _ Avcrage I ’ ~ 20 I GO 76 48 62 58 42 401 435 530 300 400 51.5 176 58 393 150 104 73 102 78 56 49.5 720 310 250 545 ~~ - 4.53 40 GO 62 48 46 ~- ~ 47 111 39 170 115 I - - B. &tales A group of six young males (39 t o 45 days of age), were castrated and injected at once -6th the same unitage of gonadotropic hormone used iii the preceding experiments. Injections were continued 20 days. As in the caw of females, the thymus did not show the characteristic atrophy after REDUCTION O F THYMUS EY H O R M O X E 429 castration, the average weight being 474 mg. as cornpared to 364 mg. in the normal males of the same agc. The nornial litterniates of these animals receiving the same dose f o r the same period of time had ihpmi reduced to a fourth of this figure (101 mg.). I n the second group of six castrate males, injections were also begun immediately after opcration, but continued 30 days, the dosage being 25 R.C. daily. Similar results were obtained, i.c., 110 reduction of the thymus ; the thymi averaged 4’73 mg., those of their normal uninjected castrate controls, 470 ing., those of their normal uninjcctcd controls, 401. mg. TREATXENT W I T H GOXADOTROPTC HORMONE 01’ M-4LES STERILE DUE TO CRYPTORCHlDIfilLl OR VlTAbIIK I3 1)EFICIESCY An attcmpt was next made to determine which tissue of the testicles, the seminiferous cpithelium or thc Ley& gtissne, is respoiisible f o r the gonadal secretion stimulated by thc gonadotropic hormone that causes the thymic atrophy. For this reason aninials were selected in which sperniatogenic tissue had been destroyed without loss of Leydig tissue. Such animals wore obtained in bmo ways, 1) by rendering them cryptorchid, and, 2) by maintaining them for a long period on a dict deficient i n vitamin E. These two types of sterile animals were then treated as above with equine gonadotropic hormone. Cryptorchid males The males were cryptorchidized by anchoring the tcsticles to the lateral body wall in a snbphenic position with two or more ligatures which passed through the mliole tuiiica albuginea. A period o€ 21 days was allowed t o elapse between operation and injection in order to allow extensive dcgeneration of the seminiferous epithelium. S f t c r this interval had elapsed, one testicle was removed and sectioned f o r information as to its condition. Ripe germ cells were no longer present in the testes; in fact, few cells other than spermatogonia and Xertoli cells remained in the tubules. Leydig tissue had suf- 430 HERBERT 1\12. EVARTS A N D MIRIAM E. SIMPSOh- fered no decrease in amount. As is well known, sex interest and the seminal vesicles of crj-ptorchidized animals remain normal-fact s which have been brought into relation with the persistence of the interstitial tissue of the testes. The animals were now injected with 50 R.U. daily of the same pregnant mare gonadotropic hormone used in the preceding studies. Injections were continued for 55 days. A t autopsy the marked enlargement of the seminal vesicles and hypertrophy of the intertubular strands of Leydig cells portrayed clear effects of the gonadotropic hornione on this portion of the testis. The testicular tubular epithelium, however, doubtless because of the coelomic location of the testis, had not been stimulated. Atrophy was even more complete than at onset of injection. There was virtually an absence of germinal tissue. Coincident with hypertrohpy of the Leydig tissue and the accessory organs of reproduction, there was a definite reduction of the thymus (table 3 ) . The thymic atrophy was, in fact, as marked in three of the fiw animals as that secured in normal males by treatment with gonadotropic hormone. The results were therefore in marked contrast to those obtained in the injected castrate males, but in agreement with the behavior of normal males. T7C'itaw? in E deficient males A further check on the striking results secured by the ireatnient of cryptorchid males was obtained by the employment of males in which the seminiferous epithelium had been gravely injured o r destroyed by tlic second contrivance, the elimination of vitamin E from the diet. Nine males were placed from the date of weaning on a dietary regimen devoid of vitamin E.s When about 7 months of age, at a time when, according l o our previous experience, a Diet contained commercial casein, 27.0; coriistarch (eooked), 35.0 ; lard, 22.0; brewers' yeast, 10.0; salts (E. V. McCollum a n d N. Simnionds, J. Biol. Chcm., vol. 33, p. 63, 1918), 4.0; cod liver oil, 2.0. This diet is niixcci togcther (except f o r the cod liver oil) and allowed t o stand 2 weeks before feeding. The cod liver oil is added the day of feeding. 431 REDUCTION O F T H Y N U S BY H O R M O X E the degeneration of the tubular epithelium sliould be almost complete, the left testis was removed from each animal, in order t o establish its condition. The testes proved t o be TABLE 3 Eflect of the gonudotropic hormone from pregnant inares on the thymus of male rats sterilized b y dietary or operative means, yet possessing normal interstitial tissue EQUIl GONAD0 OPIO OIlMONl CONTROL ~ t TYPE OF AliIYAL R.U. da uratior of ijection Cryptorchid adult males ( 5 2 1 ) - A01 days dU?/8 m. 188 176 174 185 173 185 55 1*5 55 55 32 7 34 55 S5 Fhymus Increase in body weight mu. gin. w1g. 10 52 6 34 18 249 195 162 156 167 143 85 73 30.5 80 21 8 6S -2 44 68 36 3s Average Males reared and maintained or diet deficient in vitamin E (822) 31 25 25 1.1 -6 25 216 216 216 148 1.19 148 25 26 25 2R 35 3 -5 F If Average Males reared and maintained 01 diet deficient in vitamin E, suppleniented by wheat germ oil (523) Average 128 39 64 52 72 8ri 32 0 -8 - 19 6 - 18 10 26 ~ 148 216 2116 -6 8 18 - fj 6 . i 21.5 25 25 25 21\5 315 216 216 25 25 25 26 216 215 10 87 Thp- xnus 185 180 130 160 100 163 91 129 1.55 60 85 61 - 0 24 24 20 8 26 26 83 27 164 137 83 10 10 - 18 72 0 36 28 26 13 132 - 110 195 141 214 26 16,j highly degenerate. The tubules were lined hy Sertoli cells and spermatogoiiia, with a few, frequently enlarged spermatocytes. So-called giant cells were present in the lumen. In 432 HEEtBElLT 31. EVAHS .4h7’D M I R I A X E. SIMPSON some of the aniiiials an occasional tubule contaiiied spermatids or even a few spermatozoa. The few sperm found in the testicular tubules or epididymis w7ere usually dcfornied aiid non-motile. Interstitial tissue was approximately normal in amount. As is well known (Evans and Burr, ’ W ) , these animals resemble cryptorchidized males in possessing, while sterile, normal accessory organs of reproduction and a noi-ma1 libido sexualis. Though these animals mated occasionally with estrous females, no litters resulted. These animals, whose sterility had been proved both by functional tests and by histological examination of the testis aiid epididymis, were then injected f o r periods of 25 to 35 days with equine gonadotropic hormone. At autopsy it was found that the thymus was recluced to about half the weight of that of the control uriinjected animals on the identical diet (72 mg. vs. 165 mg.) (table 3). Another group of males sterilized through lack of vitamin E mas handled exactly as was the above group, except that coincidentally with the administration of gonadotropic hormone abundant vitamin E mas fed these animals (12 drops of wheat germ oil daily). The vitamin E supplemented by the gonadotropic hormone did not, however, provoke a regrowth of the seminiferous epithelium during the 20 days of the experiment. At autopsy, the testes of these animals, just as in thc group receiving the gonadotropic liormone but not vitamin E, consisted of atrophic tubules and hypertrophied Leydig tissue. The thymi of these animals just as those i n the preceding group were reduced to half, or less than half of their previous weight (72 as compared with 165 mg.). The injected animals and their controls both increased in hodp weight slightly during this period, due to the improved condition of the animals on addition of vitamin E to the diet. This group of males was originally chosen for another use. It was known t h a t administration of high vitamin E alone ~ o i i l dnot (or rarely a t least) restore testes of males rendered sterile due t o deficiency of E. IIere it was hoped t h a t simultaneous presence of vitamin E and a potent gonadotropic liorniorie would accelerate the regeneration of the seniiniferous epithelium. No aceelrratcd regeneration was demonstrable in these experiments, but it is possible t h a t 20 day^ was inadequate time f o r repair. REDUCTTOE O F THYMUS BY H O R M O N E 433 TREATMENT O F GONADECTOMIZED MALE RATS WITH FERIALE S E X IIORMONES The experiments heretofore detailed led us naturally to the conviction that the sex hormones, male or female, were directly responsible for the reduction of the thymus which followed administration of gonadotropic hornionc. I n this way a n explanation was afforded the thymus reduction which occurred when cryptorchid or vitamin E deficient males were treated with gonadotropic hormone, but did not occur on similar injection of gonadectomized uilimals. A direct test of this hypothesis was attempted by treatment of animals with a n estrogenic hormone, amniotin and with ovarian implants containing both the estrus promoting substance and lutein hormone. Males, both castrate and iiormal, were iiijected with thew substances alone, o r coi~icidentallp received gonadotropic Iiormone. The arnniotin was given in doses of 37.5 o r 40 R.U. daily snbcntaneously. U a t u r e rat ovaries wcre used as implants, one and one-half glands heing given each 4 days. The ovaries were minced sufficiently to inject into thc muscle through a coarse cannula. As can be secn in table 4,the female sex hormor,e, amniotin, administered alone had no effect on the thymus of the castrate male, but when given simultaneously with the gonadotropic hormone had a definite thymus reducing action. I t s efiect was, however, not as extensive as was secured with treatriient of normal animals with gonadotropic hormone. Similarly, after ovarian grafts alone, the thynius of the castrates remained normal ; after combination with gonadotropic hormone, there was a slight recluetion. It is not unlikely that the administration of gonadotropic hormones to normal animals produces a greater total supply, and it would appear certain that it produces a more continuous supply of the male and female sex hormone than can be imitated by direct administration of these hormones. It woulcl appear probable that more concentrated, frequent and pros The amniotin used was kindly fiirnished by E. R. Squibb 6- Son througll the courtesy of Dr. .J. A. Morrell. TABLE 4 E f e c t of the gonadotropic hormone f r o m pregnant mares on the thymus of castrate male rats receiving female sex hormones DIIRATION OF NJECTIOE - - [nerease weight - I- -- ,~ daw Eqnine hormone 12.5 R.U. daily (W1838) 00 -_ -_ _ _ ~ Average Equine hormone 25 R.U. daily (W1381) - - 30 II _ _Average _ ~ ~ Ainniotin 37.3 KIT. daily (W1383) 20 180 50 I 455 44.5 48 440 73 426 76 34 44 -~ 53 __ 108 410 90 440 102 470 98 645 102 460 128 -_473 27 . I 1 80 64 70 12 40 I 163 1I 167 187 10 I --I I -- ___ Equine hormone 13.5 Xu. and ainniotin 40 R.U. dailj 360 270 30 _ _ _Average E:quine hormone 25 R.U.and 37.5 R.U. daily ___- ___I GO I 198 233 -Equine horinonc 23 K.TJ. daily and ovarian grafts 1.5 ovaries 1 each fourth day ___hvcrage I 1- 95 94 84-1 130 480 -301- * F o r wcights on control uninjected castrate and llormal rats, see eoritrols of 30-day injected groups, table 2. 434 BEDCCTION O F TIlYNBIUS BY H O l t N O N E 435 lorigcd injection of either male or female sex hormone monld be effective in reducing the thymus. S UMM ART 1. Chronic treatment of male or female rats, whether mature or immature, with equine gonadotropic hormone rcsults in atrophy of the thymus. 2. But when the testes o r ovaries are removed, gonadotropic treatment does not cause thymic atrophy. 3. The testicular component iiivolved in this reaction appears to be the interstitial tissue and not the germinal epithelium a s treatment with thc gonadotropic hormone after destruction of the germinal tissue by procedures which leave the interstitial tissue arid its dependent accessory organs intact (cryptorchidism or vitamin E deficiency) leads t o the same atrophic changes in the thymus as in normal males. LITERATURE CITED CATCHPOLE, H. R., AND W. R. LYONS 1934 The gonad-xtimiilating hormone of pregnant mares. Am. J. Anat., ~ o l 55, . pp. 167-218. COLE, 11. II., AND G. 13. HART 1030 The potency of blood serum of mitres i n progressive stages of pregnancy i n effecting the sexual niaturitj of the immature rat. Am. J. Phpaiol., vol. 93, pp. 37-68. EVANS, H. M., AFD G . 0. RCRR 1927 The antisterility vitamin, f a t soluble E. hlemoirs Univ. of Calif., vol. 8, p. 24. EVANS, 11. M,, K. ~ L E Y E R , A N D 31, E. YINPSON 1933 Thc growth and gonadstimulating hormones of the anterior hypophysis. RIcnioirs Unil. of Calif., vol. 11, p. 98. EVANS,H. Af., AND &I. E. SIMPSON1934 Preterition by ovariotoniy of the hypopliyseal hypertrophy following chronic administration of gonadotropic hormone. Annt. Rec., Proc. Ass. Am. Anat., vol, 38, p. 62. EVANS,H. hl., 31. E. SIMPSOF,AITD M. MCQUEEN-WILLIAMS1934 Hypertrophy of the female pituitary following injection of gonadotropic hormone. Cniv. Calif. Publ. in Anat., vol. 1, pp. 161-166. TREXDELEXBURG, P. 1934 Die Hormonc. Berliii, Julius Springer, Bd. 2, R. 443.