_ H.325:352322;:2:22.23 mmm ?HYRO§D rflCK 4*! ON THE @933 far The Saga-so :35 M. '. I .19. l 5 3d nu.» 5r... ~ 1 O .\'_ ‘i V - .‘wtt..~."- ‘ "‘u‘r s O {I C 4-. ‘ . fir. J I ~— .. .I- ‘4--‘ a. , -‘ . ~ I I o 3 v r r 'F" "1"" v " c ' o 7‘ 9 'AJ‘“ ‘ ¢ x H: 'X‘ ‘5'- \_ ~4— 3 Q . Vt: "“L‘s.’ ‘ ’ '- L J g I ". "‘ . ' ‘1 q . Lw.‘ D 05‘ . - I ‘L- It [5" . — L...3-}_ . ,- s ‘,.‘ \V N- A" ‘0 u 4‘ ’ 's J- b <—. Q _~. 8;: - ’ .o . New" «‘J . 1—“, ' ' s. \- x .‘ I Y ---\ "JIfih-d' ‘F‘jx ,_ .. V ~ ‘A. l ~U r J n ’ | V V J J ' . ,l‘, t' s; ‘ :37. ‘r z ’ O I D D ., 1s. .- ‘3“ .r— _ “' _k, .. i \ 1- j 5". ' QW;‘4~3 . . '_ . ’. q 3.. 3).!- .4 i .53 -- Af’ 1 '3 ,. 1 h \ o" I ~ I q.‘ o " '- a . .03.- u.‘ 4‘. . v Y‘J' '.—_4 4' 3 \ MW"! 4.. Date a" 22) /¢;fl a, . .. - fl 7 I. ‘ -' .N.af [‘1‘"..;. ’ This is to certify that the thesis entitled The Effects of Estrogen on the Chick Thyroid presented by David I. Epstein has been accepted towards fulfillment of the requirements for m.s. dafimgh.Ph siology Mf/szz Major professor . o I... __-a_v THE EFFECTS OF ESTROGEN ON THE CHICK THYROID By DAVID IRVING §£§TEIN A THESIS Submitted to the school of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology and Pharmacology 1950 INTROWCTION . o o e O o o e o o o o o o e o o e 0 EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . RESULTS . o o o e o o o e e o o o e o o APPWDIX O O O O O O O 0 O O O O C O 0 TABLE OF CONTENTS Effect of Estrogen on Thyroid Size . . . . . . . The Effect of Estrogen on the Uptake of Radio- Iodine by the Thyroid Gland. . . . . . . . . . . The Effect of Estrogen on Turnover Time of Radio- IOdlnemthCThymid.eoooooooeoo.. The Effect of Estrogen on the Daily Rate of Iodine outPUtvbyth-O'rhymld000.000.00.00 Thyroid Secretion Rates as Calculated by the Method of Homer; Reineké‘anclo'l‘urner (19411) . Effect of Estrogen on Body Weights . . . . . . . . Effect of Estrogen on Comb Growth . . . . . . . . Summary of Effects of Estrogen on the Chick. t . . DISCUSSION 0 O O 0 O O O O O O O O O O O O O O O O 0 SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . . REFERENCES CITED . . . . . . . . . . . . . . . F‘J‘ fit?! a Page oooouw 15 19 25 28 33 37 #4 47 51 DEDICATION The writer affectionately dedicates this work to the memory of his beloved parents, Benjamin and Sena Irma Epstein, who continue to be a constant source of inspiration to their son. His greatest ambition is to accomplish all the great things of which they had thought him capable. It is his fervent wish that this work is a step in that direction. ACKNOWLEDGEMENTS The writer wishes to eXprsss his sincere appreciation to Dr. Lester I. Wolterink, Associate Professor of Physiology, Michigan State College, for his invaluable guidance, encouragement and advice through- out the course of this work and in the preparation of this manuscript; to Dr. E. P. Reineke, Department of Physiology, for his many valuable suggestions and for supplying the thyroxine solutions used in these experiments; to Dr. J. Meites, Department of Physiology, for his many valuable suggestions; and, last but not least, to Dr. M. M. Butt, Pakistan Government Scholar, whose many thoughtful questions and suggestions aided the writer greatly in the earlier phases of the work. INTRODUCTION It has been shown by Andrews and Sohnetzler (1946), 'Muhrer (1947) , Roman 9.31.211. (1947) , and many other workers, that feeding thiourea or its derivatives will increase the fat content of many domesticated animals including swine and.fowl. The goitrogens depress thyroid activity which causes a decrease in the metabolic rate, which, in turn, is associated with the deposition of fat. It has also been shown that feeding natural or syn- thetic estrogens may cause fat deposition in birds. Lorenz (1943 and 1945), Thayer‘gt_a;‘ (1945), Glazener and Jun (1946), Thayer and Davis (1948), Davis and Thayer (1948), and Quisenberry and.xrueger (1948), to name but a few, have obtained relatively good deposition of abdominal fat. However, Sykes gtgal, (1945) did not get as pronounced results as did any of the above investi- gators. Neither did Davidson g§_alg (1946), studying the administration of synthetic estrogens tc.turkeys. They found.an increase in plasma fat but usually a decrease in abdominal fat. The best fat depositions in birds have been obtained.in the Oklahoma and California exper- iments. Large deposits of abdominal fat in turkeys were obtained when the birds were reared out-of-doors in areas with rather warm mean temperatures (Davis and Thayer, ibid.). In contrast, turkeys reared.out-of-doors in the northern portion of Michigan with much colder mean f! (2) temperatures did not show the increase in abdominal fat with estrogen (Davidson m, 1946). Apparently a difference in climate may have something to do with the laying down of fat. One immediately suspects the thyroid gland. If thicuracil, which depresses the thyroid, also induces fat deposition, 9. relative thyroid stimulatign induced by a colder environment might prevent the fatten- ing effect of estrogen. Unpublished data (Wolterink, Davidson and Epstein) on turkey hens at the end their first laying period indicate that the thyroid activity, per unit body weight, of these birds.during the cold spring months is as great as that found in young chicks. Similar results were found by Blakely. £51. (1949) in five-week-old poults. Whether or not climate, m, is responsible for the decreased fattening results in turkeys is unknown. It becomes desirable, however, to reinvestigate the effect of estrogen administration under different conditions of thyroid activity. The existing literature is primarily concerned with the effects of thyroidectomy or thyroid administration on the hypophysis, the gonads themselves, or the hvpophyseal-gonadal axis. However, the consequences of different thyroid status on effects produced by the secre- tions of the ovary have been studied by Fleischmann (1946) in the chick. He showed that the inhibiting effect of , . thyroxine is confined to the metabolic changes induced by I estrogen, whereas the morphological structural changes (3) brought about by estrogen on secondary sex characters are not effected. His work demonstrated that thyroxine was able to neutralize the ability of the estrogen to increase serum calcium, inorganic phosphate, protein phosphate and cholesterol, yet it does not inhibit the growth of the oviduct induced by estrogen. This is an extension of the earlier work of Van Horn (1933) and Meyer and Wertz (1938) who hypothesized that excess thyroxins may, by increasing metabolism, cause a more rapid elimination of estrogen from the body. Heyer and Wertz. showed that in rats, with a dose level of thyroxins small enough to barely decrease the body weight in five days, the threshold for estrogen was raised, _e_,_g,_, it required more estrogen to induce an estrus-type vaginal smear than was required in the normal intact animal. The technique of thyroxine inhibition of estrogen-induced estrus-type vaginal smear in the thyroidectomized rat has been used by Barker _e_t__a_l_‘ (1950) in studying thyroxins analogues. The effects of estrogen administration on the hypophyseal-thvroidal axis is not as well understood. leyer M, (1930) , Leonard (1933), Severinghaus (1934), and Wolfe and Chadwick (1936) showed that estrogen injec- tion into rats caused either a decrease in the gonad- stimulating potency of the anterior pituitary or that the basOphil cells were degranulated by the estrogen. Since thyrotrophin is secreted by the basophils (Griesbach and (4) and Purves, 1945), it would seem logical to assume that the estrogen would depress the secretion of that hormone. In assaying for the thyrotrophic hormone, Meites and Turner (1948) actually found such a decrease in the amount of that hormone in the pituitary of estrogen-treated rabbits. The precise effect of estrogen on the pituitary cytolog as affected by thyroidectomy has been studied most recently by Baker and Everett (1947). Their results are somewhat inconclusive due to incomplete removal of thyroid remnants and.to the short time following thyroidectamy in which the thyroid effects might have become apparent. It is sufficient to point out that the precise effects are not only controversial, but apparently depend upon a combination of the effects of estrogen dose levels and total time of administration. Baker and Everett do conclude that 'the stimulating effect of estrogen on the hypophvsis is not mediated through the thyroid gland.‘ The present experiments were designed '. to determine 'whether or not exogenous estrogen administered" to intact immature chicks with relatively nonfunctional gonads would alter thyroid secretion rate. In securing these data, information was also obtained on the effect of thyroxine administration on the estrogen-induced slowing of the iodine turnover time in the thyroids of thiouracil-treated animals. (5) EXPERIMENTAL PROCEDURE In this work we conducted seven experiments. In EXp. l, 2 and 7 we used Rhode Island Reds, while in the interven- ing ones we used Barred Rocks. All chicks were pullorum- tested and came from a hatchery in Southern Michigan at the age of one day. The birds were caged in a commercial chick battery, and.were fed.ad libitum. All groups contained both males and females. All chicks were started on an Arcady Chick Ration” for the two weeks after hatching prior to the beginning of the experiments. They were then leg-banded and.Weighed to the nearest gram. Groups as nearly equal as possible in average weights were set up, and the experiments were continued for two weeks. The general plan was to set up a conventional thyroid secretion rate assay by the method of Mixner, Reineke and Turner (1944). One half of the chicks were placed on a normal ration and assayed. The other half was usually given estrogen in the feed. Due to limited space, it was not possible to use as many thyroxins dosage groups as might have been desirable. The exact conditions for each experiment may be noted from the tables. In addition, the turnover of radio-iodine (I 131) was used as a check on the thyroid activity in the last four eXperiments. At the end of the assay period, the animals were sacrificed and their weights and sex recorded. Gonads, combs and thyroids Were removed and weighed on a Roller e Arcady Milling Company, Chicago, Illinois. (6) Smith balance to the nearest tenth of a milligram. Organ weights were expressed both in actual weight and as weight per hundred grams of body weight. The thyroids of the animals receiving radio-active iodine were placed on capper discs and dried. They were then counted with a thin mica end window Geigerbmuller counter. I Unless otherwise stated, thiouracil was administered in the feed at a dose level of 0.1%.. This dose level proved to be too low to completely suppress thyroid activity. However, it is the dose level employed by Mixner et 3;, (1944) and Schultze and Turner (1945). Thyroxine solutions were prepared from crystalline thyroxins isolated by Dr. E. P. Reineke from iodinated casein. These were injected subcutaneously in the indi- cated doses daily for the two weeks of the experimental period. The synthetic estrogen used, 3,4-dianisylhexene-3: was injected in a corn oil suspension in the first two experiments. In the rest of the experiments it was given in the feed at a dose level of 0.01%. Radio-active iodine (I 131) was obtained from Oak Ridge in a weak bisulphite medium. In eXperiment #3 it was given to the chicks in the drinking water. However, in the succeeding experiments approximately 0.2 of a micro- curie was injected subcutaneously. e The dimethyl ether of stilbesterol, Breon. is (7) Turnover times for the output of iodine for the thyroid glands~are the times in which the radio-iodine content has been reduced to one half its value from the start of the period. These were computed from counts obtained at twenty-four hours and a later time (between forty-eight and seventy hours) after the injection of the radio-active tracer. An exponential rate of loss is assumed. The method is essentially that derived.by [eating and Albert (1949). (8) RESULTS Effect of Estrogen gnLThyrcid Sig; Table 1 summarizes data obtained in six eXperdments in which a low estrogen dosage rate was employed. The average thyroid size of normal fouruweek-old Rhode Island Red chicks was 9.5:O.4 milligrams per hundred grams body weight. In comparable chicks given estrogen the thyroid weight averaged lO.2tO.2 milligrams per hundred grams body weight. The difference between the two groups was not significant. In Barred Hodks of the same age, the control thyroids taveraged 6.6t0.l milligrams per hundred grams body weight, while the thyroids of the estrogen-treated birds averaged 6.0:O.1. Estrogen treatment in Barred Rocks evidently decreased the average thyroid.weights of the groups used in these experiments when the weighted average was computed from individual group averages. However, the actual difference in weights was only about ten per cent, and there was. no significant difference in any of the groups from which the over-all average was com- puted. Thus, the effect of this estrogen dose level in reducing thyroid weights in Barred Rooks was probably of little importance. If real, the difference would indicate that estrogen slightly depressed thyrotrophin secretion by the pituitary. (9) thyroid Table 1 also lists the/weights of similar animals receiving radio-iodine. In EXP. 7 with Rhode Island Reds no significant difference in thyroid size was produced by the amount of radio-iodine injected. In Exp. 3 (Barred Rocks), radio-iodine apparently decreased thyroid size, whereas in Exp. 5 (Barred.Rocks) radio-iodine apparently increased thyroid size. No significant differ- ence was noted in either Exp. 4 or 6. From the weighted means of all the experiments with Barred Rocks, it is‘ impossible to conclude that radio-iodine treatment had any effect on the thyroid weights. We will now discuss the effect of estrogen adminis- tration on thyroid.size in birds which received thiouracil in the assay procedure. Results for groups which received 0.5 gamma of thyroxine per day will be omitted. As reported by Schultze and Turner (1945), a low dose of thyroxine in a thyroid assay gives a very low thyroid weight, a result which has been observed by numerous other investigators. Since the cause of this phenomenon is obscure, data in which this effect plays a part cannot be discussed.profitably at the present time. Data for this dose level may be found, however, in the appendix and wherever the thyroid weight itself is not critical to the discussion. Although there is considerable variation between experiments in the average thyroid weights of groups on the same thyroxins dose level, the average thyroid weight of all groups on the same dose level shows a very interest- ing picture. The average of the thyroid weights in Exp. 4, 5 and 6 (Barred.Rocks) for the chicks at a one gamma thyroxins dose level is 28.5tl.4-milligrams thyroid per hundred grams body weight for the estrogen-treated ‘birds as compared with 28.9tl.l milligrams thyroid per hundred grams body weight for the controls. This indicates that estrogen seems to have no effect on the thyroid weight when one gamma of thyroxine is given simultaneously with the thiouracil. However, when one gives two gamma of thyroxine, a different picture results. The controls (i‘g‘, groups receiving.0.x% thiouracil plus two gamma of thyroxine per day) average 30.0i:l.2 milligrams thyroid per hundred grams body weight as compared With 18.81.- 1.4 milligrams per hundred grams body weight for similar groups receiving estrogen. This reduction in thyroid size might indicate that at this level of thyroxine dosage, the adminis- tration of estrogen may have reduced the thyrotrophin secretion of the pituitary (either by stimulating the output of thyroxine from the thyroid and thus depressing pituitary thyrotrophin secretion indirectly or by direct depression of the pituitary output of thyrotrophin). If estrogen had.no effect on thyrotrophin secretion, the reduced thyroid size could be accounted for only if the sensitivity of the thyroid to an unchanged thyrotrophin output was decreased. (11) We have only one group of animals with which to compare the thyroid weights from chicks receiving four gamma of thyroxine. As in the animals receiving two gamma of thyroxine, the control thyroids were larger than the ones treated with estrogen, l6.l::l.9 as compared with ll.21:l.0 milligrams per hundred grams body weight. This again indicates the possibility of mechanisms similar to those mentioned above. whether or not thyrotrOphin secretion was effected by the conditions imposed in these treatments was not determined directly. As will be shown subsequently, a direct check on the thyroid output of radio-iodine did indicate that in the smaller thyroids (produced by estrogen), the turnover rate of iodine was increased. Thus, a more rapid thyroid activity may have resulted in a reduced thyrotrophin secretion which, in turn, may have been reflected in the reduced thyroid size. once: 05 Ho hogan uncommon i cocoon 05 no.“ mmoo. 0 3 use: pend on» soaps 50.0 some 3300." new someones 05 33235 3S 3 I. 633355.03” non mu dag moopcoautmowoapmu new a.“ cocoon ”flamenco .39 a ma season woman s $m1 hmmowlmi hmmwmmuwl .mp4 .. 30.0 3475 .o Edema 903.0 0.0.36 . 50.0 01?: .m Emunéa 5.0“..06 Edema .. 30.0 T013 .e 0.0“: 0.836 . 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L c. | o O O o o b o O O 0 9 9 I I 0 O O O O 6 . O . I O O o 4 I O .53: 05 no mosh..— cndcfiuam * .andohpnuwwoapmm no.“ 3 3303 “30.3900 hon a." .895: pmfig t 3B3 . .. $8.0 main” 33mg .. «Hod DTNH 33H 33.: “86 3.3 .m ESE Eom mama - . _ 1: ‘ ammnzmm mum mgfiqagv 538., 3ng $8 ufixofifia anon 2305mm Enfim .oz .mxm agazoufllm malls The Effect of Estro an on the U take of In normal chicks, estrogen had no effect on the uptake of the administered radio-iodine (Table 3). In the groups on thiouracil and graded thyroxins levels, estrogen did modify radio-iodine uptake. Exp. #,5 and 6 were run on Barred Rocks, while Exp. 7 was on R. I. Reds. When one gamma of thyroxine was used it was found that the thyroids of the R. I. Reds receiving estrogen picked up more iodine than did.their controls, whereas in two of the three experiments run on Barred Rocks the control thyroids accumulated.more radio-iodine at twenty-four hours. when two and four gamma of thyroxine were given to the thiouracil-treated groups, both breeds showed a greater ‘uptake of radio-iodine with estrogen administration. .Apparently in Barred Rocks estrogen increases iodine uptake by the thiouracil-treated.thyroid.only when a dose of thyroxine above one gamma is administered. these results indicate that estrogen administered to thiouracil-treated chicks producssan.effect on uptake Opposite to its effect on thyroid size. Thus, despite the decrease in thyroid weight produced by estrogen (Table 2), the synthetic female hormone produced an actual increase in iodine uptake. If estrogen first decreased thyrotrOphin secretion, it is difficult to see how iodine uptake could have been increased (l6) subsequently. On the other hand, if estrogen first stimulated the thyroid, reduced thyrotrophin secretion by the anterior pituitary would normally follow. .30: on.» no Aoanm undoudpm a. 2n 0.33. 5 noon on. has macaw node a.“ woman. no Ace—5n one e m: .032. .0 m8” .ougm .o é. 5n .oflbafi §.ou¢mo.m .w aw...” «sod oodnorwda .m anioflen .o a mom .032. .o .e Puma: waonhfi Efioomxmagoo zmwomam m manoH .mxm enema .3532 no 2548 Samba. 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In Exp. 4, 5 and.6 (Barred Rocks) the groups receiving estrogen require from fifteen to more than 370 hours longer for half of the thyroid iodine to be discharged than do their normal controls. This indicates that in the dose level employed here, estrogen causes a depression of the tnyroid iodine turnover in intact animals. Exp. 7 employed.R. I. Reds. In this eXperiment the control birds had an infinite turnover time. That is, in forty-eight hours no iodine loss from the thyroid occurred. There was no apparent reason for these presumably normal ‘birds having completely inactive thyroids. The estrogen- treated group in this experiment had a turnover time of 75.3:5.8 hours. In view of the aberrant control values, the effect of estrogen cannot be determined in this case. Vhen the chicks were p1aced.under the influence of thiouracil, the thyroxins dose level again seemed to determine what the estrogen effect would be. When a low dose of exogenous thyroxine was administered to chicks receiving thiouracil and estrogen, turnover time for the radio-iodine was longer than in their non-estrogen-treated controls. This was true for the 0.5 and 1.0 gamma level of thyroxine. However, when 2.0 and 4.0 gamma of thyroxine were administered, A(2o) the iodine turnover times in the thyroids of the chicks receiving estrogen were shorter than those in non-estrogen- treated.birds. It is apparent that in hypothyroid animals ti.g., in birds whose thyroids have been depressed by thiouracil and in which the exogenous/Egggoifiggl is low) estrogen depresses the thyroid gland. However, in an animal on a higher dose of thyroxine (a relatively hyperthyroid animal) the estrogen seems to stimulate thyroidal turnover. If the thiouracil were completely blocking the synthesis of thyroxine, the data would suggest that in this case exogenous estrogen acts prhmarily on the turnover of the inorganic iodine which is returning to the blood stream. This might or might not be under direct pituitary control. However, the dose or thiouracil used.(0.l% in the feed) apparently did.not completely suppress thyroxins secretion in the two week period or these eXperiments. This is shown by the effect of thyroxine on the thiouracil but non-estrogen- treated birds (Table 6). The greatest slowing of iodine turnover occurred only when extra thyroxins was administered. In the presence of estrogen, the additional thyroxine.. induced slowing wag, in fact,reversed. These considerations do not provide direct proof for the localization of the estrogen effect in a single locus in the thyroid gland itself. They are in line, however, with the hypothesis that there is at least some initial effect I. (21) of estrogen in the thiouracil-treated birds in stimulating thyroxins output. Only in this case would it be logical to expect to get the observed depression of thyroid weight. If the initial effect of estrogen were to increase pituitary thyrotrcphin secretion, the increased iodine turn- over might be explained, but not the decreased.thyroid weight. The third possibility, that estrogen reduced the thy- roid sensitivity to thyrotrophin, should.mean that both tmyroid weight and iodine should.be concurrently decreased. The observation is that although.thyroid weight was decreased, iodine turnover was increased by estrogen in these thiouracil- treated birds. We come to the tentative conclusion that in thiouracil- treated chicks, if sufficient exogenous thyroxins is administered, there is at least some direct stimulation of thyroxine outpuvggggggfgsgsenof whatever other effects the estrogen might have at other points in the thyroid- pituitary... gonadal axis. In normal birds, and in birds on thiouracil and low thyroxins dosages, estrogen depressed the iodine turnover with no evidence of change in thyroid size. The pituitary could not have been extensively involved, even secondarily, in the normal birds since even iodine uptake was unaffected. . The net result of these data is to emphasize the point that the effect of estrogen may be either to depress the thyroid or to stimulate it. Depression was observed in normal or relatively hypothyroid animals. Stimulation was (22) observed only where higher thyroxins levels were maintained. In the birds on these experiments, no evidence was found to indicate that the primary effect of estrogen was solely on the pituitary, although the pituitary was probably involved secondarily. It is always possible that estrogen administration produced.minor effects by direct action on either the pituitary cytology (which was not studied) or on other points in the axis. Such effects, if present, were not large enough to result in changes demonstrable by the techniques used here. use: on... no hogan 0.30530 .0 0.000.00 .0000000 .0 0.000.400 04000.00 .0 0.30.40 0. négda .0 0.00: 0.00 .4 0.300 0.330 mmmmmmwm 000000 [40mm mmmmmnmmmmmm 00 00000000 000 20 000000-00040 00 0000 00000000 00 20000000 no 000000.. N 0004a :03— 05 00 00.0.0.0 0.000030 00 £0000”? 0.500.000 3000 0 0 0.0000 00 0.000000 «050 0.0 0 0000.000 06000.00 90.000 0.0 .0. 0.000.00 3000000 350 0.0 0.000200 0.0.3.00 3000 0.0 . 0.30.00 0.80.00 «500 0.0 0 300 on 00000.00 000.00 0.0 0.30.00 0.30.00 3.50 0.0 . 0.000.00 0 0.0.00.3 3000 0.0 0 A 50030 0.0 n.mm H013 300000.033 v daauw o...“ A 0030.0 0.0 .0 ‘ 0.00.00 0.30;“ 00000 00.000000400000000 , . 00000 00.000.000-000 0000 000000000 .000 . 000000000 .0. .00. d0<0.0.0. 00“ 000000000 .000. 00..I0.0I.0I.|000000||0 .00. 00000000040 .00 0000. 00000000. 00 00000000 .00 000000 0 00000 (25) _The Effect of Estrogen on the Daily B§5g4gg_lodine Output WWW The data cited in the previous section may also be computed in terms of the daily output of iodine from the thyroid gland. This is conveniently expressed as per cent or the iodine or the thyroidelninatd in twenty-four hours per hundred grams or body weight.' In the four experiments on normal birds (Table 7), the estrogen caused a decreased output as compared with the;5 control groups. Thus, in the dose level administered, estrogen depressed the thyroid out- put of iodine. In one case (EXp. 7, R. I. Reds) a net uptake, rather than an output, or iodine was found forty-eight hours after the injection or radio-iodine in the estrogen-treated birds as compared with 19.d% output for the normal controls. As reported previously, no apparent reason can be given for this aberrant result. Again the rate or daily output is correlated with the thyroxins dose rates in the groups receiVing thiouracil and thyroxins in addition to the estrogen (Table 8). In the thiouracil-treated birds receiving one ug of thyroxine per day, the daily output from the thyroid glands is less in the estrogen groups. Conversely, in the animals receiving 2.0 and 4.0 ug of thyroxine, the amount or iodine eliminated per_twenty-rour hours is increased by estrogen administration. 1L "1 moaned—smog 49.3500 figmama ao.ma Jameson azanoa .s and: 8.4.28 8889mm «5.3 amazon H.233 .8 «Han. 85.28 E858 no.3 8828 sang .n udn . .828 8888 «Tea .8538 szS .e 88m em and nmifiaqm @238 928 mum agéma .mxm mafia 3&0: 50 950mm #N mum mnzdfiu nHOmHmB Hma .mo mag mgozma HZHQOH HE zo zawamymm .mo Bowman H and“. an.mm mo.pe mm.m mm.mm mo.m¢ aw.on some on mm.mm no.m¢ an.¢n manm madamalzmwomamm 8.3 §.an ac.mo modpcauasood a¢.>H ao.o¢ an.¢> .>m we mF.mn :? o a «a .3 o o daadw o.¢ camuw o.m casdw o.H .> meadw o.¢ cascw o.m daamw o.H .o «meow o.m ossdw o.a «snow m.o .m Acsssw o.m vcaasw 0..” Assad» m6 .¢ q. mamHm EGEBmHIzoz H.048 magma. .53 MZHNQNNSH 924 AHOdeOHnH. aaH>HHOfim manm so 88: em sluice n33: Illa 353. .3; so my? $858 n Hands manoahmma zo ascomama mo sommmm ( 28) Thyroid Secretion Rates as Calculated by the Method of Mixner;_Reinpke d: Turn r h Computation of thyroid secretion rates invelves plotting the thyroid weights of birds on thiouracil against the thyroxins dosage. Since the lower thyroxins dosages used.in these eXperiments give aberrant thyroid weights (as mentioned above» the calculation of secretion rates had to be made on the basis of only the 2.0 and #.0 ug thyroxins dose level. The data are, therefore, subject to considerable error. Nevertheless, the values so computed are listed in Table 9. They are in general agreement with those found by Boone eta (1950). The data obtained.show no clear evidence for any effect of estrogen on the thyroid secretion rate. In view of the rest of the data and the inadequacies in these direct assays, it seems impossible to credit these results for more than an estimate of the general level of thyroid secretion. The average thyroid secretion rate was 2.1 gamma per day per hundred grams body weight. In view of this magnitude of thyroxine secretion rate, four gamma of exogenous thyroxins per day should definitely produce a hyperthyroid animal. "-11.24% C TABLE 2 THYROID SECRETION RATES AS COMPUTED FROM THYROID WEIGHTS PER HUNDRED GRAMS BODY WEIGHT E32,: OONLR_O_L_S ESTROGEN-TREATED BIRDS 40 1.8 118/633 5. 2.7 us/daJ 6. 2.2 ug/day 7. 1.9 “8/53! 1.9 ug/dey (30) Effect of Estrogen on Body Weights In all of the groups under observation, both control and eXperimental, there seems to be no change in body weight when the animals are treated with estrogen (Tables 10 and 11). Thayer, Jaap and Penquite (1945) found an increase in the body weights of cocks on an estrogen diet, while Davidson fl. (1946) found that the weights of young turkeys was usually decreased by the administration of estrogen. £35.23 do echo €558 .. w.oaua.mam o.maa.es~ o.maus.oem w-m-m .a m.>um.mmm w.mnm.amm o.m“m.aem mausauefl .o a.aaan.mma m.s~ua.mmm b.8“s.smm wua-aa .m ~.nmo.mmm H.>Ho.oflm a.oaam.moa oumaoa .e c.3909“ infidaam E3368 naumauwa .n o.amoa.ama. o.mua.wmm eauma .m .H.omum.omm ad .a 05.5 |mdmazoo nmmmmmuammunuammw. 338 58 so amew..§..-.w..nlnom zo ficomaau limo sinuses. _ on uqmH m.0HHo.¢om huuxw: m . mHo.o mam . >.mHn.mom o.mHo.mom Hau\wn H Acoouv «Ho.o mum H m.nmum.m>H m.HHHm.>nm .LaHoaonmoanp .. 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Anna H mama“ TABLE 14 SUMMARY OF THE EFFECTS OF ESTROGEN ON THE CHICK 1. ON THYROIQ SIZE a. Normal Animals - estrogen has no effect b. Thiouracil-trgated Animals (1) Low dose of thyroxine - estrogen has no effect (2) Two and four gamma of thyroxine - estrogen depresses size of glands 2. ON UPTAKE OF RADIO-IODINE a. Eggmal_§gimalg - estrogen has no effect b. Thiouracil-treated Animals (1) Low dose of thyroxine - estrogen generally depresses uptake (2) Two and four gamma of thyroxine - estrogen stimulates uptake 3. on'movzn TLMES OF RADIO-IODINE a. Nbrm Animals - estrogen causes a longer turn- over thme (reduced daily output) b. iour oil-treated in s (1) Low dose of thyroxine - estrogen causes a longer turnover time (reduced daily ‘output) (2) Two and four gamma of thyroxine - estrogen causes a shorter turnover time (increased daily output) 4. ON COMB SIZE In all cases estrogen depresses the size of the combs. 5. ON BODY WEIGHTS In all cases estrogen has no effect on the body weights. «my», P (38) DISCUSSION The earliest intimation of the interrelationship between the thyroid and the ovary seems to be the discovery by the Chinese ' that women are more prone to goiter than men. Salter (1940) points out that the ratio of goiters goiters in in women to’men decreases progressively as sex becomes less important in the life cycle, becoming equal between the sexes after the menopause. Much work has been done on the relationship of these two glands of internal secretion, but many of the results are of a very contro- versial nature. Kunde et a1, (1931) found that when estrin was administered to dogs for seventeen weeks, there was hyperplasia of the thyroid. Pincus and.Werthessen (1933) found similar results in rate if the estrin were administered to rats for five to ten days, but they found involution of the thyroid if they administered the hormone for twenty or more days. Karp and.KostkieWicz (1933). working with rabbits, and Benazzi (1933) . working with mice, found that folliculin caused an increase in thyroid 3126. An increase in thyroid weight might indicate that the thyrotrophin secretion is either directly increased by estrogen or indirectly stimulated by decreasing the secretion of thyroxine. The possibility also exists that the estrogen directly increases the sensitivity of the Szvfli1\ 9m 11d (39) thyroid to thyrotrophin. Conversely, a decrease in thyroid weight might indicate that the thyrotrophin is being depressed. Again this might be a direct action on the anterior pituitary, or it might be an indirect consequence of a primary thyroid depression. As reported in the introduction,.most of the work on pituitary cytology indicates that estrogen causes degranulation of the basophil cells which produce the thyrotrOphin. This cytologic picture is usually interpreted as being sufficient to .indicate a depression of the secretion of that hormone, a result in agreement with the depression of pituitary thyrotrOphin on direct assay as reported by Meites and Turner (Lyig). The increased thyroid weights induced by estrogen, as reported by the investigators cited above, are difficult to explain if thyrotrophin is reduced. If, on the other hand, estrogen first supresses the thyroid output of thyroxine, as is suggested by the chick data reported herein, pituitary thyrotrOphin should be increased and, given sufficient time, thyroid enlargement should result, as was reported by Kunde et al., Pincus and Werthessen, and Karp and Kostkiewicz (ibid.). Such enlargement was not observed in the chicks in these experiments,however. The data from thiouracil-treated birds given two or four gamma of thyroxine indicated that estrogen does not always suppress thyroid turnover, but may, in fact, stimulate it if circulating thyroxins is present in sufficient amounts. In that case estrogen should depress thyrotrophin as suggested by the evidence of pituitary cytology. In either event, the decisive factor, or factors, apparently lies in some combination of the ratio of estrogen to cir- culating thyroid hormone and the duratiOn of treatments. Data from experiments set up with these points in mind are urgently needed. If we were to base our conclusions solely on thyroid size, we should be forced to conclude that estrogen adminis- tration to normal and hypothyroid chicks has no effect on the thyroid gland. Also, estrogen administration to chicks made somewhat hyperthyroid by thyroxins increases thyroid activity. Data obtained from the uptake and turnover of radio- iodine give a more sensitive criterion of thyroid activity. We found that in normal chicks estrogen had no effect on the twenty-four hour uptake of radio-iodine. Similar results were found in rats by Paschkis et a1, (1948) at four hours after iodine administration. Since Morton et a1, (l9#2) have shown that the uptake of iodine by the thyroid gland is controlled by thyrotrophin, we might readily assume that in our experiments, two weeks of estrogen administration had no effect on the secretion of thyrotrophin. In chicks on the thiouracil assay, estrogen caused less radio-iodine to be taken up when a low dose of thyroxine ‘Was administered daily. This Would indicate a depression of thyrotrOphin secretion. Yet, it is difficult to see (41) how a decreased thyrotrophin secretion could have caused a decreased uptake of iodine and still not decrease the size of the thyroid gland, that is, in view of the observation of Keating et alI (1945) thatthyrotrophin first influences thyroid.Weight and only later influences uptake of radio-iodine. When two or four gamma of thyroxine were administered to the chicks on the thiouracil assay, estrogen stimulated the thyroid to take up more of the injected radio-iodine. If the stimulation were the result of increased pituitary thyrotrophin secretion, an increased size of the thyroid should have been observed. The data give no indication there Was any direct effect of the estrogen on the secretion of thyrotrophin by the anterior pituitary (unless it be concurrently assumed, in disagreement with.Keating et al,, that under the influence of thyrotrOphin altered radio-iodine uptake can occur without change in thyroid size). A Turning our attention to the turnover of radio-iodine, we find that estrogen increased the turnover time in normal chicks. This indicates that the estrogen is depressing the activity of the thyroid by decreasing the output of iodine. From the present data this is the first indication we have of any effect of estrogen on the thyroids of normal animals. We had no indication of this from the_size of the glands, ‘or from the uptake of radio-iodine. This must mean that, in normal chicks at least, estrogen depresses (42) the thyroid directly. If it did exert its effect through the pituitary, even secondarily, we should have expected to see some change in the size of the thyroid.snd.in the 'uptake of radio-iodine. However, such was not Observed. In the chicks on the thiouracil assay, we find that when a low dose of thyroxine was administered, estrogen again depressed thyroidal activity, indicated by a longer turnover time in the estrogen-treated.birds than in their controls. This depression was reflected in the decreased iodine uptake of these birds. However, we found no change in the size of their thyroids. If Ieating and his co-workers are correct, this again indicates that thyrotrophin secretion could not have been extensively involved. They showed the differential effect of thyrotrophin on thyroid size as compared to radio-iodine uptake in three days. Surely, in the two weeks of our experiment, the thyroids of the chicks should have had enough time to react to altered thyrotrOphin levels if such had.been induced by estrogen. Therefore, since we found no change in the thyroid size and a decrease in the uptake of iodine, we must presume that there is no effect on the pituitary thyrotrOphin. This is in direct contrast to the effect of estrogen on gonadotrophin. As pointed out earlier, the depression of comb size by estrogen is clear evidence of the depression of the secretion of gonadotrophin. fiqvk °U( (43) The conclusions reached here add direct evidence for the thyroid-pituitary mechanisms in the modifications of . those metabolic effects of estrogen cited by Fleischmann (1946). He found that the ability of the estrogen to increase serum calcium, inorganic phosphate, protein phosphate and cholesterol was neutralized by the administration of equal parts of thyroxine. It is thus apparent that the fattening of birds by estrogen must be profoundly influenced by the level of thyroid activity. The data of the present experiments directly demonstrate that estrogen can primarily alter thyroid activity. \w-r’ SUMMARY AND CONCLUSIONS 1. Thiouracil administrated orally as 0.1% of the feed did not completely surpress iodine turnover in the thy- roid. 2. Estrogen had.no effect on the thyroid size of normal chicks. 3. Radio-iodine injections (0.2 microcuries) had no effect on the size of the thyroid glands. 4. In chicks receiving thiouracil and one gamma of thyroxine a day, estrogen had no effect on the thyroid size. 5. In chicks receiving thiouracil and either two or four gamma of thyroxine per day, estrogen caused a decrease in the size of the thyroid glands. 6. In normal chicks, estrogen had no effect on the uptake of radio-iodine by the thyroid. 7. In chicks receiving thiouracil and one gamma of thyroxine per day, the Rhode Island.Reds picked up more radio-iodine under the influence of estrogen than did their controls, while in the Barred Rocks the estrogen administrar tion caused less iodine to be taken up. In chicks receiving thiouracil and two or four gamma of thyroxine per day, estrogen caused a greater uptake of radio-iodine in both breeds. 8. Estrogen caused a depression of thyroxine turn- over in normal animals. (45) '9. When the chicks were placed under the influence of thiouracil, the thyroxins dose level seemed to determine what the estrogen effect would be. When a low dose of exogenous thyroxins was administered to chicks receiving thiouracil and estrogen, turnover time for the radio- iodine was longer in these birds than in the non-estrogen- treated controls. However, when two or four gamma of thy- roxine were administered to comparable animals, the turn- over time was shorter in the estrogen-treated animals than in their controls. 10. If sufficient exogenous thyroxine is administered, estrogen stimulates thyroxins output by a direct action on the thyroid.gland. With low thyroxins levels, estrogen depresses iodine turnover'by the thyroid gland. 11. Changes in pituitary thyrotrophin output, as nearly as can be determined from these data, were the secondary results of the alteration in the thyroid as induced by estrogen. 12. The direct effect of estrogen on the pituitary or on the sensivity of the thyroid to thyrotrophin could not be demonstrated from these data. 13. Estrogen was not observed to have any effect on body weight under these conditions of ad libitum feeding. l4. Comb weight was depressed by estrogen but not by radio-iodine in the normal birds. 15. The depression of comb weight induced by estrogen ‘was not modified either by thiouracil or by exogenous thyroxins. (46) Since comb weight is controlled by pituitary gonadotrOphin, no pronounced effect of thyroxine depression or excessive thyroxins on gonadotrophin secretion could be demonstrated from these data. (47) REFERENCES CITED 1. Andrews, F. N. and Schnetzler, E. E., 1946. The influence of thiouracil on growth and fattening in broilers. Poult. Sci. 25: 124. 2. Baker, B. L. and Everett, N. B., 1947. The effect of diethylstilbesterol on the anterior hypOphysis of thyroidectomized rats. Endocrinology 41: 144. 3. Barker, S. B., Dirks, H. B., Jr., Klitgaard, H. E., lawzonek, S. and Wang, S. C., 1950. Anti-thyroxine effects of thyroxine analogues. Fed. Proc. 9: 8 (abstract). 4. Benazzi, M., 1933. The follicular hormone inhibits f ‘ _ 5‘?” 738°(dft’e3fid’ £18113?” 151i. ggiggafif‘figggfa "3301. 5961'. 5. Blakely, R. M., Anderson, R. W. and MacBregor, H. I., 1949. Determination of thyroxine secretion rate of turkey poults. Poult. Sci. 28: 757 (abstract). 6. Boas, N. P. and Ludwig, A. W., 1950. The mechanism of estrogen inhibition of comb growth in the cockerel, with histologic observations. Endocrinology 46: 299. 7. Boone, M. A., Davidson, J. A., and Reineke, E. P., 1950. Thyroid studies in fast and slow-feathering Rhode Island Red chicks. Poult. Sci. 29: 195. 8. Davidson, J. A., Wolterink, L. E., Epstein, D. I. (Unpublished data). E. P 9. Davidson, J'. A., lolterink, L. F., and Reineke/1946.’ Some effects of high dosages of synthetic estrogens in young turkeys. Poult. Sci. 25: 400. (abstract). 10. Davis, G. T. and Thayer, R. 11., 1948. Finishing market turkeys with estrogens. Poult. Sci. 27: 79. 11. Dempsey, E. I. and Astwood, E. B., 1943. Determination of the rate of thyroid hormone secretion at various environmental temperatures. Endocrinolog .32: 509. 12. Fleischmann, W., 1946. Effect of thyroxine on estrogen- induced changes in the fowl. Fed. Proc. Part 2 5: 28. (abstract) 13. Glazener, E. W. and Jull, N. A., 1946. Effect of thiouracil, dessicated thyroid and stilbesterol 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. (48) derivatives on various glands, body Weight and dressing appearance of chickens. Poult. Sci. 25: 236. Griesback, W. E. and.Purvis, H. D., 1945. The significance of the basophil changes in the pituitary accompanying various forms of thyroxine deficiency. Brit. Jour. Exp. Path. 26: 13. Earp, L. and.Kostkiewicz, 1933. Experimental Colloidal Goiter produced by folliculin. Compt. Rend. Soc. de Biol. 144: 1339. Resting, R. F. and Albert, A., 1949. The metabolism of iodine in man as disclosed with the use of radio- idoine. Recent Progress in Hormone Research. Vol. 4: 429. Ed.: G. Pincus. Academic Press Keating, F. R., Rawson, R. N., Peacock, W. and Evans, R. D., 1945. The collection and loss of radio- active iodine compared.With anatomic changes induced in the thyroid of the chick by the injection of thyrotrophic hormone. Endocrinology 36: 137. Kunde, M. M., D'Amour, F. E., Gustavson, R. G. and Carlson, A. J., 1931. The effect of estrin adminis- tration on the reproductive and blood vascular systems: the thyroid, thymus, hypOphysis, adrenals, kidneys, liver and spleen. Amer. Jour. Physiol. 96: 677. Leonard, S. L., 1933. Differential effect of prolan in decreasing the potency of the hypOphysis in normal and castrate rats. Anat. Record 57: 45. Lorenz, F. N., 1943. Fattening cockerels by stilbesterol administration. Poult. Sci. 24: 94. Lorenz, F. N., 1945. The influence of diethystilbesterol on fat deposition and meat quality in chickens. Poult. Sci. 24: 128. McMillen, W. N., Reineke, E. P., Bratzler, L. J. and Francis, M. J., 1947. The effect of thiouracil on efficiency of gains and carcass quality in swine. Jour. An. Sci. 6. \ Meites, J. and Turner, C. N., 1948. Studies concern- ing the induction and maintenance of lactation. I. The mechanism controlling the initiation of lactation at parturition. Bull. Univ. of Missouri 415. Meyer, R. E., Leonard, S. C., Hisaw, F. L. and Martin, S. J., 1930. Effect of oestrin on gonad stimulating pOWer of the hypOphysis. Proc. Soc. Exp. Biol. and Med. 27: 702. ' Meyer, A s and Wertz A 1938 Infl ’0 h . . , . . uence of hyroid Mggmo%§:on8g§trin action. Proc. Soc. EXp. B101. and 26.- 27. 28. 29. 31. 32. 33. 34. 35. 36. (49) Mixner, J. P., Reineke, E. P. and Turner, C. N., 1944. Effect of thiouracil and thiourea on the thyroid gland of the chick. Endocrinology 34: 168. Morton, M. C., Perlman, 1., Anderson, E. and Chaikoff, I. L., 1942. Effects of hypOphysectomy on the determination of labeled thyroxine and diiodotyrosine in the thyroid gland and plasma. Endocrinology 30: 495. Muhrer, M. C., Warner, D. R., Palmer, J. and Hogan, A. G., 1947. Effects of thiouracil and protamone on graving swine. Jour. Animal Sci. 6: 489. Paschkis, K. E., Cantarow, A. and.Peacock, W. C., 1948. The influence of estrogen on thyroid function as measured by uptake of radio—active iodine. Proc. Soc. Exp. Biol. and Med. 68: 485. Pincus, G. and Werthessen, N., 1933. Continued injection of oestrin into young rats. Amer. Jour. Physiol. 103: 631. Quisenberry, J. H. and.Krueger, W. F., 1948. Effects of feeding various combinations of protamone, oestrogens and thiouracil on growth and fattening of broilers and fryers. Poult. Sci. 47: 681. Salter, W. T., 1940. “The Endocrine Function of Iodine Oxford Univ. Press. Schultze, A. B. and Turner, C. F., 1945. The determination of thyroxine secretion by certain domestic animals. Bull. Univ. of Missouri 392. Severinghaus, A. E., 1934. Changes in the hypOphysis of adult male and female rats after pregnancy urine extract injections. Proc. Soc. EXP. Biol. and.Med. 31: 593. Sykes, J. F., Davidson, J. A. and Barrett, F. N., 1945. The effect of feeding diethylstilbesterol to cockerels.‘ Poult. Sci. 24: 542. Thayer, R. H. and Davis, G. T., 1948. Use of estrogens in turkey broiler production. Poult. Sci. 27: 172. 37. 38. 39. (so) Thayer, R. B., Jaap, R. G. and Penquite, R., 1945. Fattening chickens by feeding estrogens. Poult. Sci. 24: 483. Van Horn, w. M., 1933. The relation of the thyroid to the hypOphysis and ovary. Endocrinology 17: 1520 Wolf, J. M. and Chadwick, C. 8., 1936. Quantitive studies on the structural changes induced in the anterior hypOphysis by injection of oestrin. Endocrinology 20: 503. APPENDIX Complete data for each experiment are found on two successive pages. Body and organ weights are listed on the first page and radio-iodine data are listed on the second page. The headings on each page give the experiment number and the breed of the chicks used. EXPERIMENT 1 RHODE ISLAND REDS .nso: on» no gonna ohmonmam % oasmoaaonm anchopmopmoa amawaaaa: a omawonhga hm amnwoaoaz o.H p.395 Emmandoa as Sausages mic AnowoApomv onoxonadmhnmaa mamawaaads o.m onaxoahna E. SEES: 04 2334.5 0.8.66.3 as Ceasefire 3.0 onaxoahna hm amawoaoa: 0.H o.wflm.mn m.wmud.¢ma Eb HHomASOAAH RH.0 he 36G 03 dflfln Oz meOHHem * HemNflmooNN a DHO-HHQOU ”58.45: .Eooawaa .5 code: name a: 328 a: 860 .9: 30mg .93 Moon mMono .0: 5354mm; EXPERIMENT 2 RHODE ISLAND REDS .303 05% HO hog-Ha Ugaflm * onoNothmdmmg e canon; h b mfimhwgoga ¢ 0.0.8.00 an: 011nm .EN 2 0 505830 3.0 . agnohnna h m namnwohoda o.m 58.0.? 0.3.0.3 Héflofimm m m doetsoafl 3.0 easncanonm h o." 23.390303. 0.0.00 .111. 0.93.3 4.03908 2 a. 3.90233 04 h m emowgpmm mdflm .nw 1.3m 3: owamflnfioa a m oesawoaoa: o.m . .— 0 38 oz fifiwadoa n.m«m.0a a. 994.08 a «a 38.280 4.2000H\02 .fioooqims .30 coda: 94mm . 93 92.8.0 .9: 8200 .5: 5659 .93 Noon mMono .oz , .agadmms. 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