extensively investigated (see review by Maqsood, 1952), but the possible

Transcription

1 390 J. Physiol. (I 955) I 27, THE EFFECTS OF SOME GONADAL HORMONES ON THYROID ACTIVITY IN THE RABBIT BY K. BROWN-GRANT From the Department of Neuroendocrinology, Maudsley Hospital, London, S.E.5 (Received 11 August 1954) The interrelationship of the thyroid gland and the gonads has been recognized for many years by clinical endocrinologists, but the experimental study of their interaction has not kept pace with clinical observation. In recent years, the effect of altered thyroid function on reproductive performance has been extensively investigated (see review by Maqsood, 1952), but the possible influence of variations in gonadal function on thyroid activity is less well understood. This paper is concerned with one aspect of the problem-the influence of various gonadal hormones on thyroid activity. MATERIALS AND METHODS Young adult male and female rabbits of mixed breeds ( kg body wt.) were used. The majority were Chinchilla rabbits. The animals were kept in a temperature-controlled animal room at 28 C and fed a pellet diet (M.R.C. diet no. 18) and tap water ad lib. All females were isolated for at least 3 weeks after arrival before any experiments were begun so as to exclude any pregnant or pseudo-pregnant animals. Thyroid activity was studied by means of the 'release curve' technique previously described (Brown-Grant, von Euler, Harris & Reichlin, 1954). Starting 48 hr after a subcutaneous injection of a tracer dose of carrier-free radio-iodine, twice daily counts were made over the thyroid region in the unanaesthetized animal. After correction for isotope decay, these counts, expressed as a percentage of the counting rate at 0 hr (48 hr after injection) were plotted semi-logarithmically against time yielding a straight line whose slope represents the rate of release of '3II-labelled thyroid hormone from the gland. Following a preliminary control period, the effect of injections of various gonadal hormones on the rate of release can be studied. After a period of hormone treatment, a further period of control observations completed the experiment. The dose of radioiodine used in most experiments was 3,c. For some of the longer experiments on the effects of treatment with various combinations of hormones a dose of 6 pc was used. For further details of the method and apparatus, the paper referred to above should be consulted. The following hormones were used in these experiments; all were administered by subcutaneous injection in a single daily dose. Actual doses are given in the relevant sections of the text. Stilboestrol, 1 or 5 mg/ml.; hexoestrol, 5 mg/ml.; progesterone, 2 or 5 mg/ml. (British Drug Houses); testosterone propionate, 5 mg/ml.; oestradiol monobenzoate, 1 mg/ml.; oestrone, 1 mg/ml.; deoxycorticosterone acetate, 5 mg/ml., and cortisone acetate, 5 mg/ml. (Organon Laboratories).

2 GONADAL HORMONES AND THYROID ACTIVITY 391 One ml. of the oily vehicle used in the preparation of the stilboestrol supplied by British Drug Houses was injected in control experiments. Thyroxine (synthetic sodium L-thyroxine, 'Eltroxin', Glaxo) was injected subcutaneously in 50,ug doses in suspension in 0-9% NaCl solution on alternate days. Thyrotrophic hormone (Armour Laboratories Ltd., Lot no. R ) was dissolved in 0-9% NaCl solution immediately before use and injected subcutaneously in doses of 200,ug U.S.P. equivalent. The anaesthetic for the operations of ovariectomy and orchidectomy was intravenous pentobarbitone sodium (Nembutal). RESULTS Oestrogentc compounds The effects of two synthetic (stilboestrol and hexoestrol) and two naturally occurring oestrogens (oestrone and oestradiol) were investigated. In all but two cases in forty-two experiments in normal and gonadectomized male and female rabbits the effect of injections of these compounds was to produce a prompt decrease and in many cases a complete inhibition of the release of thyroidal 131I. One experiment is shown in Fig. 1. The duration of the inhibitory action was estimated from the horizontal distance between the lines representing the slopes of the release curves in the preceding and subsequent control periods. Where the inhibition was not complete (reduction to a rate of 1 %/day or less) the duration of partial inhibition was expressed as the equivalent duration in hours of complete inhibition. Table 1 shows the results of forty-two experiments of this type. The drugs were given in a single subcutaneous injection in each case. The duration of the inhibition, which was established within a few hours, varied with the dose administered, the compound administered and the type of animal. Summarizing the results shown in Table 1 of forty-two experiments in twenty-three animals, stilboestrol, hexoestrol and oestradiol are roughly equipotent, while oestrone is rather less effective in this respect as judged by the effect of a 1 mg dose in normal female rabbits. The effect of varying doses of stilboestrol in normal females shows that 5 mg doses produce a more sustained' inhibition than 1 mg doses. In comparing male and female rabbits and normal and gonadectomized animals, there is no striking difference in the response to 1 mg doses of stilboestrol or oestradiol between normal and spayed females, between normal and castrate males or between male and female rabbits. Progesterone The effect of progesterone on the rate of release of thyroidal radio-iodine was investigated in eighteen experiments in fourteen rabbits. In preliminary experiments on intact does, 1 mg/day for 3 days (2 expts.), 1 mg/day for 4 days (1 expt.), 2 mg/day for 2 days (1 expt.), 2 mg/day for 3 days (1 expt.) or a single injection of 5 mg had no effect on the rate of release. In two experiments (1 mg/day for 3 days and 2 mg/day for 2 days) there was a reduction in

3 392 K. BROWN-GRANT 1 mg oestrone 1~ Rabbit K47 3tc, 9.ii Hours FiG. 1. The effect of a single injection of 1 mg of oestrone on the rate of release of thyroidal 131I in a normal female rabbit. The inhibition in this case was only partial. The method ofdetermining the duration of action in terms of hours of complete inhibition is shown. TABLE 1. The effects of injections of oestrogenic compounds in normal and castrate male and female rabbits as hours of complete inhibition produced. (P) indicates experiments in which inhibition was incomplete but the results are given as the equivalent duration of complete inhibition Compound Normal Ovariectomized Normal Castrate and dose &? C Is Stilboestrol 5 mg 60, 140, mg 70 (P) 1 mg 25, 25, 32, (P), 55, (P), 40 (P), 23 -ve, 28, ,ug 18 (P) Hexoestrol 1 mg 33 (P), 55, 65, Oestradiol 1 mg 47, 26 (P), 60 (P), 24 58, 28, 24 (P) -ve, 28 (P), 24 (P), (P), 47, 21 Oestrone 1 mg 23 (P), 16, 39 (P).57

4 GONADAL HORMONES AND THYROID ACTIVITY 393 the rate of release from 20 %/day to 15 %/day in one case and from 15 %/day to 12 %/day in the other. The dosage finally adopted was 5 mg/day for 3 days; this dose had no effect on the rate of release of two normal and three ovariectomized does, or on two normal and two castrate males. One castrate male showed a reduction in the rate of release from 32 to 27 %/day which persisted throughout the period of injections and for 72 hr after the last injection. Testosterone Fifteen experiments were performed on fourteen rabbits. In one experiment, on a normal doe, injection of 1, 2-5 and 5 mg at 2-day intervals had no effect on the rate of release. In all the other experiments 5 mg/day for 3 days was the dose employed. No effect was seen in twelve experiments (four intact does, three ovariectomized does, two intact males and three castrate males). In the other two experiments, one doe showed reduction (from 30 to 18 %/day) in the rate of release which persisted for 70 hr after the final injection, and one male rabbit showed an acceleration (from 10 to 15 %/day) which lasted for 68 hr after the final injection. Control experiments Control experiments were carried out in which 1 ml. of the oily vehicle used in the preparation of the stilboestrol injections was administered daily for 3 days. In three normal and two ovariectomized does, one normal and three castrate males, no effect on the rate of release of radio-iodine from the thyroid was seen following these control injections. The effect of various combinations of hormones The results so far described seem to indicate that progesterone and testosterone in the dosage employed have no significant effect on the thyroid activity of the rabbit under the conditions of these experiments, whereas the oestrogenic compounds studied showed a uniformly inhibitory action. The possibility, however, exists that progesterone or testosterone might be capable of modifying the effect of oestrogens on thyroid activity and this possibility was investigated in the following way. After an injection of 3 or sometimes 6 juc of 131I a 'release curve' was begun in the usual way. Following a control period, daily injections of 500 or 1000,ug of stilboestrol were begun, resulting in a marked decrease in the rate of release, usually to 1 %/day or less from initial rates of between 10 and 30 %/day. After 3 or more days, when the reduced rate of release was clearly established, additional injections of 5 mg of progesterone or testosterone per day for 3 days were begun. These injections were usually given in the evening, the stilboestrol

5 394 K. BROWN-GRANT being injected each morning as before; after 3 days the animals reverted to treatment with stilboestrol alone. The result of one such experiment is shown in Fig. 2; the addition of progesterone to the stilboestrol treatment resulted in a marked, though temporary, reversal of the inhibitory action of stilboestrol. Table 2 shows the results of eleven experiments of this type in intact female rabbits. The results were expressed in the following manner. The predicted drop in thyroidal 131J content over the period from the first injection of progesterone or testosterone to 24 hr Rabbit K26 3,uc. 20. xi i ~~~~~~~ Hours Fig. 2. The effect of progesterone (5 mg/day for 3 days) in reversing the inhibitory effect of stilboestrol treatment. S, injection of 500jsg of stilboestrol; P, injection of 5 mg of progesterone. TABLE 2. The effect of additional hormone treatment on the rate of release of thyroidal 131I in stilboestrol-inhibited normal female rabbits. Results expressed as described in text Daily dose of stilboestrol Progesterone 5 mg/day Testosterone, 5 mg/day (,ug) for 3 days for 3 days , 10*0, , 1.0, 10, , 1.0, 1.0, 1*0 after the third injection was estimated from the graph. This value was taken as one. The decrease in 131I content which actually occurred over this period was next determined from the graph. The ratio of the observed to the predicted fall was then calculated and this value was used in expressing the results. Thus a result of ten indicates a tenfold increase in the rate of release over the 72 hr period of additional treatment and a result of one indicates that the additional treatment had no effect on the rate of release. In seven experiments on seven normal female rabbits, progesterone increased the rate of release in five cases by between three- and tenfold and had no

6 GONADAL HORMONES AND THYROID ACTIVITY 395 accelerating effect in the other two. Testosterone had no effect in four experiments in four animals. In one ovariectomized rabbit, progesterone produced an acceleration of 8-0; testosterone had no effect in one case and produced an acceleration ( x 4.5) in another case. One normal male rabbit showed no effect (10) with testosterone and a second animal showed an acceleration ( x 15.0) in the rate of release following treatment with progesterone. In view of the possibility (see discussion) that these doses of stilboestrol are effective in inhibiting the release of thyroidal 1311 because of a non-specific ' stressing' effect, and because of the 'corticoid '-like activity of the progesterone which was found to reverse this inhibition, the effect of cortisone acetate and deoxycorticosterone acetate on stilboestrol inhibition of the rabbit thyroid was investigated. The experimental technique and the method of expressing the results were the same as those used in the experiments described above. In one normal female rabbit 0 5 mg DOCA a day for 2 days, 1.0 mg/day for 2 days and 5-0 mg/day for 2 days had no effect on the inhibition. In five further normal does 5.0 mg/day for 3 days had no effect. Cortisone in doses of 1 or 2 mg/day (in two doses) for 2 days had no effect in two normal does and produced an acceleration in another two animals (9 0 and 10.0); 1-0 mg produced no further acceleration than did 0 5 mg/day in these two experiments. Summarizing the results of this type of experiment, testosterone 5 mg/day for 3 days was not effective in reversing the stilboestrol inhibition; nor was deoxycorticosterone 5 mg/day for 3 days. Progesterone 5 mg/day was able to reverse the inhibition of release of thyroidal 131J produced by stilboestrol in five out of seven experiments and cortisone was effective in two of four experiments. The mechanism of oestrogenic inhibition Within the limits of a single release curve, the rate of release of thyroidal radio-iodine in the rabbit is a very direct and sensitive index of thyroid activity. It is independent of variations in renal handling of iodide and the influence of alterations in the degree of reaccumulation of 1311 from degraded thyroid hormone on the slope of the release curve is negligible (Brown-Grant, von Euler, Harris & Reichlin, 1954). The two most probable mechanisms for the inhibition of the release by oestrogens are: (1) inhibition of the secretion of TSH by the anterior pituitary; (2) a direct action on the thyroid gland to alter its response to TSH. The second of these hypotheses was tested in the following way in seven experiments on seven rabbits. At the time of the first neck count of the release curve, the rabbits were injected with 50,ug of Na-L-thyroxine subcutaneously, and these injections were continued at 48 hr intervals throughout the experiment. The inhibition of endogenous TSH secretion by the thyroxine injections resulted in a flat ( < 1 %/day) release curve.

7 396 K. BROWN-GRANT The thyroid gland is still capable of responding to exogenous TSH however, and the sensitivity of the thyroid is not significantly altered by such doses of thyroxine (Reichlin, 1954). After an initial control period, the animals were given a single subcutaneous injection of 200,ug U.S.P. equivalent of freshly prepared TSH. The result was a prompt discharge of 131J from the gland; the duration of the effect was about 18 hr and was followed by a return to the previous slow rate of release. After 2 or 3 days, treatment with oestrogen was begun. In six experiments, 5 mg of stilboestrol were injected 24 hr and again 6 hr before a second 200,ug dose of TSH. In the seventh experiment, 1 mg of T T AI >[TSH 200lig T + Rabbit K 72 3Ac,6.v. 54 T + TSH 200.g 5 5+ T % ) 12-3% fall on n o 00 0ue~~~- 146% fall 50 li Hours Fig. 3. The effect of 200,ug TSH with and without oestrogen treatment on the 131I content of the thyroid gland of the thyroxine-treated rabbit. T indicates injection of 50ug thyroxine. S indicates injection of 5 mg of stilboestrol. oestradiol was injected 36, 24 and 6 hr before the second dose of TSH. The second dose of TSH again produced a prompt discharge of radio-iodine from the thyroid. The experiment was continued for a further 2-3 days to enable the second discharge of 1311 to be accurately determined. The results of the experiment were calculated as follows. From the graph, the fall in 131J content produced by each injection of TSH was determined and expressed as a percentage of the gland content at the time of injection of TSH. The results of the seven experiments are given in Table 3; one experiment is shown in Fig. 3. There is no difference between the response to TSH alone and the response after treatment with oestrogens. DISCUSSION At least four different oestrogenic compounds will inhibit the release of 131Llabelled hormone from the thyroid gland of the rabbit. Progesterone and testosterone in the dosage employed in these experiments have no such effect.

8 GONADAL HORMONES AND THYROID ACTIVITY 397 The inhibitory effect can be demonstrated in normal and gonadectomized male and female rabbits. The inhibitory effect of oestrogens on thyroid function as judged by various criteria has been established for many years. Lederer (1946) gives an extensive review of the earlier clinical and experimental work on this subject. More recently, Money, Kraintz, Fager, Kirschner & Rawson (1951) have found a depression in the uptake of 131I by the thyroid gland of male rats treated with oestradiol, oestriol or stilboestrol. TABLE 3. The response (percentage of gland content of 131I discharged) following the injection of 200,ug TSH with and without oestrogen treatment in seven rabbits. Six rabbits were injected with 5 mg of stilboestrol 24 and 6 hr before the second dose of TSH Percentage fall in thyroid Percentage fall in thyroid 1311 content after 200,ug 131I content after 200ytg TSH following stilb- Rabbit no. TSH alone oestrol treatment * * * Average * Rabbit 7 was given 1 mg oestradiol 36, 24 and 6 hr before the second TSH injection. Two points seem to be of some importance, however, beyond the simple demonstration of a reduction in thyroid activity. One is the mechanism by which the inhibition is brought about. The failure to demonstrate an alteration in the thyroid response to TSH even with doses greater than those necessary to produce an inhibition of release, coupled with the observation (Brown- Grant, Harris & Reichlin, 1954 b) that section of the pituitary stalk abolishes the inhibitory response to the injection of 5 mg of stilboestrol, indicates that the mechanism is most probably a reduction in pituitary thyrotrophic hormone secretion. The second is the possible physiological significance of these results. The doses of the various compounds used in this work were high. McPhail (1934) found that maximal uterine development in the immature ( g body wt.) rabbit was produced by 150,ug of oestrone (cf. 1000,ug used in this work). Cameron (1947) discusses the relative potencies of various oestrogens. Stilboestrol and oc-oestradiol are of about the same potency (3 times that of oestrone), and hexoestrol at least equal to, and possibly more potent than, stilboestrol. The administration of these compounds in the same amount as oestrone would represent an even higher biological dosage. Progesterone in 5 mg doses is also at a high level; Haskins (1940) has shown that 05 or 1 mg administered systemically is sufficient to produce maximal endometrial changes in the rabbit. However, the effects of these compounds on the release

9 398 K. BROWN-GRANT curves were striking; definite effects would very likely be demonstrable with much smaller doses and a more refined technique. The possibility exists that in these experiments the effective compounds were acting not by virtue of their common oestrogenic powers, but as nonspecific stressing agents, which in general inhibit thyroid activity. It should be mentioned that tests of the effect of oestrogenic compounds in adrenalectomized rabbits would not enable this question to be settled, as stressinduced inhibition of thyroid activity can occur in the absence of the adrenals (Brown-Grant, Harris & Reichlin, 1954a). The more consistent reversal of the inhibition by progesterone than by cortisone, and the failure of testosterone or DOCA to produce this effect, suggest that the phenomenon is more likely a function of the oestrogenic potentialities of these compounds than of their adrenal activating powers. That oestrogens may possess a specific ability to reduce the secretion of pituitary thyrotrophic hormone is also suggested by the observations of Leblond, Albert & Selye (1942) who studied the effect of various steroids on the development of thyroidectomy cells in the rat pituitary. Only oestradiol and ethinyloestradiol of many gonadal and adrenal steroids studied were capable of preventing the appearance of these cells. If a depression of pituitary TSH secretion is one aspect of oestrogenic activity, as suggested by the work reported here, then it should be possible, with the newer techniques now available, to demonstrate and assess by more physiological methods than those used in this work the significance of ovarian influences upon thyroid activity. SUMMARY 1. The effect of various steroids on the thyroid activity of the rabbit has been studied using the rate of release of 1311-labelled thyroid hormone as an index of thyroid activity. 2. Oestradiol, oestrone, stilboestrol and hexoestrol decrease thyroid activity; progesterone and testosterone, in the doses employed, had no effect. 3. Progesterone consistently reverses the thyroid inhibition produced by stilboestrol. Testosterone, DOCA and cortisone do not consistently produce this effect. 4. Stilboestrol and oestradiol do not alter the response of the thyroid gland to exogenous thyrotrophic hormone; their action is probably due to a suppression of endogenous TSH secretion. 5. The possible physiological significance of these findings is discussed. It is a great pleasure to acknowledge my indebtedness to Prof. G. W. Harris in whose department these experiments were carried out. My thanks are also due to the Medical Research Council for the grant of a research studentship, during the tenure of which this work was performed.

10 GONADAL HORMONES AND THYROID ACTIVITY 399 REFERENCES BROWN-GRANT, K., VON EULER, C., HARRIS, G. W. & REICHLIN, S. (1954). The measurement and experimental modification of thyroid activity in the rabbit. J. Phy8iol. 126, BROWN-GRANT, K., HARRIS, G. W. & REICHLIN, S. (1954a). The effect of emotional and physical stress on thyroid activity in the rabbit. J. Physiol. 126, BROWN-GRANT, K., HARRIS, G. W. & REICHLIN, S. (1954b) in HARRIS, G. W. (1954). The reciprocal relationship between the thyroid and adrenocortical responses to stress. Ciba Foundation colloquia on Endocrinology. 'The human adrenal cortex' (in the Press). CAMERON, A. T. (1947). Recent Advances in Endocrinology, 6th ed., ch. VIII, pp London: Churchill. HASKINS, A. L. (1940). Modification of the intra-uterine assay method for progesterone. Endocrinology, 27, LEBLOND, C. P., ALBERT, A. S. & SELYE, H. (1942). Action of various steroids on the hypophysis of the thyroidectomized rat. Proc. Soc. exp. Biol., N. Y., 51, LEDERER, J. (1946). Les relations thyro-ovariennes, lst ed. Paris: Masson et Cie. MAQSOOD, M. (1952). Thyroid function in relation to the reproduction of mammals and birds. Biol. Rev. 27, MCPHAIL, M. K. (1934). The assay of progestin. J. Physiol. 83, MONEY, W. L., KRAINTZ, L., FAGER, J. KIRSCHNER, L. & RAWSON, R. W. (1951). The effects of various steroids on the collection of radioactive iodine by the thyroid gland of the rat. Endocrinology, 48, REICHLIN, S. (1954). Thesis for degree of Ph.D. University of London.