injection. golden hamsters, and also established that pentobarbitone blockade of (Received 18 August 1969)

Transcription

1 J. Phy8iol. (1970), 206, pp With 1 text-figure Printed in Great Britain INFLUENCE OF OESTROGEN ON THYROID FUNCTION IN THE EWE BY IAN R. FALCONER From the Department of Applied Biochemistry and Nutrition, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough, Leicestershire (Received 18 August 1969) SUMMARY 1. Thyroid activation at oestrus occurs in several species, which may be related to oestrogen secretion occuirrinig at the same time. In the ewe this relationship has been examined by mneasuring thyroid activity before and after oestrogen administration. 2. Measurements have been made of thyroid 1311 release rate and proteinbound 131I concentration in the plasma in normal and ovariectomized ewes and of protein-bound 1251 in plasma from thyroid vein blood in ewes with exteriorized thyroid glands. 3. No evidence ot an activation of the thyroid by oestrogen was observed in immature ewes or adult oestrous cycle, anoestrous or ovariectomized ewes, given oestrune or oestradiol dibenzoate in doses of pg by injection. 4. It was concluded that oestrogen may not be responsible for thyroid activation ubserved in normal oestrous ewes. INTRODUCTION An increase in the activity of the thyroid gland near to the time of ovulation has been recorded in several mammalian species. The work of Brown-Grant (1966) demonstrated this phenomenon in rats, mice and golden hamsters, and also established that pentobarbitone blockade of ovulation prevented the normal increase in thyroid activity during oestrus in rats. A similar marked activation of the thyroid gland occurs at oestrus in the ewe (Robertson & Falconer, 1961), which is preceded by a rapid rise in the concentration of oestrogen in the blood (Moore, Barratt, Brown, Schindler, Smith & Smyth, 1969). The effect of oestrogen on thyroid function in the rat has been examined extensively (Feldman, 1956; D'Angelo & Fisher, 1969), with the general conclusion that oestrogen in physiological amounts will increase the

2 472 IAN R. FALCONER activity of the thyroid. There is, however, no direct evidence that circulating oestrogen is the normal activator of the thyroid during the period of oestrus. It is therefore of value to examine the relationship between oestrogen and thyroid activity in species other than rodents, which also show an activation of the thyroid at normal oestrus. METHODS Animals. The ewes were kept inside throughout the experiments, in a room with a controlled minimum temperature of 100 C. During the experiments, the results of which are shown in Table 3, the animals were held in metabolism cages in a controlled temperature room at 15 +±10 C. The ewes were fed a constant diet at a quantity sufficient to maintain normal growth. Oestrus was detected by the presence of a vasectomized ram, and also by observation of the external genitalia. As the ewe is a seasonally breeding animal which undergoes a period of anoestrus during the summer, experiments were carried out at different times of the year so that the effects of oestrogen could be examined during a variety of reproductive states. Four adult ewes were ovariectomized to provide animals with minimal concentrations of endogenous circulating oestrogens. Immature pre-pubertal ewes were also investigated. Measurement of thyroid activity. (1) By release of 131I from the thyroid. Directional scintillation counting was used to measure the radioactivity of the thyroid gland after intramuscular injection of 50 jug 1311-, as described by Robertson & Falconer (1961 a). The measurements so obtained were subjected to regression analysis to obtain the slope of best fit to the data, and statistical comparison of these regressions was carried out using an English Electric KDF 9 computer. (2) By measurement of the concentration of protein-bound 131I (PB1311) in the sera, as also described by Robertson & Falconer (1961 a). Samples obtained during the 2 days before oestrogen treatment were compared with samples obtained during the 2 days after oestrogen treatment. The difference between the mean values for PB'31I before and after treatment were calculated with the fiducial limits for P = 0-05, using an Olivetti Programma 101 desk computer. (3) By measurement of the concentration of PB 125J in sera obtained from thyroid vein blood of undisturbed conscious sheep previously injected with 50 /C6c 125I-. This technique employed ewes bearing permanently exteriorized thyroid glands, as described by Falconer (1963a, 1967). Statistical comparisons were made between sera obtained in the 5 hr immediately following oestrogen injection as compared with sera obtained in the period of 5-11 hr after injection. Measurements were also made on sera obtained before oestrogen injection, and on sera obtained from samples of normal jugular blood at all stages of the experiment. Hormone treatment. Oestradiol dibenzoate (Nutritional Biochemical Corporation, Ohio) was administered as a solution in arachis oil by intramuscular injection into the hind leg. Oestrone (British Drug Houses, Poole) was administered as a solution in 60% propylene glycol/water by intramuscular injection into the hind leg or by intravenous injection into the jugular vein. The minimum dose of 25,ug oestrogen used in these experiments was sufficient to induce behavioural oestrus within 10 hr under the conditions of these experiments. The observed vulval enlargement persisted for hr after oestrogen injection, and presented an appearance similar to that of a ewe in normal oestrus.

3 OESTROGEN AND THYROID 4'i3 RESULTS The effect of oestrogen on the release of radioactive 131I from the thyroid gland of ewes The results of the oestrogen treatments on thyroid 131I release are shown in Table 1, with the differences between the rates of release of radioactivity obtained by subtracting the rate after treatment from the rate before treatment. Thus a positive difference between rates represents an increase in thyroid hormone output and a negative difference between rates a decrease in hormone output. The control anoestrous and ovariectomized animals showed a small increase in hormone output after the control injection, whereas the two control oestrous cycle animals showed a small decrease in hormone output. The only statistically significant alteration in hormone output after oestrogen treatment was shown by an anoestrous ewe administered 50,ug oestrone, which demonstrated a decrease in hormone output. It can therefore be concluded that there is no evidence of an increased release of hormone from the thyroid due to oestrogen treatment in these experiments. The effect of oestrogen on the concentration of PB 1311 in sera from ewes The anoestrous and ovariectomized animals used in the above experiments were also subjected to regular blood sampling, and the results of measurements of PB 131J in sera from these and other sheep are shown in Table 2. It is apparent from these results that there is no evidence of an increase in circulating thyroid hormone due to oestrogen, as no significant increases in PB 131I concentration occurred in the anoestrous or ovariectomized sheep given oestrogen. The effect of oestrogen on the concentration of PB 131I in plasma from thyroid vein blood of ewes with permanently exteriorized thyroid glands This experiment was carried out during the oestrous cycle of ewes which had been transferred during the autumn into a controlled environment with a constant daylength of 12 hr. The measurements of PB 125I were obtained from these animals as a sequence at approximately 20 min intervals and have, for the sake of brief presentation, been grouped into measurements made between the time of injection and + 5 hr, and between + 5 and + 11 hr. Examination of the detailed data (Fig. 1) showed a general decrease in PB 125I during the first 5 hr after injection; this commonly occurs in these experimental conditions during the recovery from the initial thyroid activation due to the stress of cannulation.

4 474 IAN R. FALCONER o; X U: ~ ~ ~~~~~* * < rn 3-3 _ > es~~~~t-t- > b oo C 12 - OC12~ ce ~~~~C E t N ~~~~~+l+l +l +l+l +l +l+l+l+l+l+l S Q G e : e c _ c C> 0 0 o. Q e 0o_om _ e;;~~~~~~~~~~~~~ -- C> Cq M - - b o o _ o o o b' 0 0 X O O c DH II,, I +I+ +I +ll+ll +I +I+I I;=H NO-nOe- + O1 D1-4 ;. 0-0 Cd.,)-4 - =~~~~~~~~~(D C.z M t- 2 r12-4 ce c o- S o_: o o Xo o -1 - O O00 O O- o o o o o o o oooo o Q;; So; t o~~~~~~~~~ ;~~~~~~~~' 4; H l+ l+ Qo etn- ^U +l 00 o _ o * o o b o o Ca0 5 O.= O O Q t N e < e > n 0 0 0I 0 5 S0 *,, a O O t d 5~~~~~~~~~T ~~~~~~~0 ~ ~ ~ U e;~~c2 ooooo 0 oo t e,xcx 4E - $ Ca cw O :5 ~~~~~~~~~~- 4-; o; 0 'N~~~~~~~~~~~~~~- r-- ;- Q4 4 UUd5>.. OOO

5 OESTROGEN AND THYROID 475 o~ ~~ C C)d _; o ~~~~~~~~~~~~~~~o_* c 0c C) * :D _4 > *_; +l +l +l +l +l +l +l +l +l -H- O*k~~~~~~~O >4-~ ;4 c ~~~ =~~~~~~~~~~~~~~~~~~~~~~~~~C C) ( a 0 ~-;' -D z O O -t ~ z _C) E ) \,:~~~~ i \ O O-5 3 o 30a;;;I n _ n_ b; ;0 c cs ) ~ 00 o 3o coozqc; ~~ Ci2Ct2 ~ C C L ;;o = H 2 ~~~~~~~~~~~~~~~~~~~~> > > > Hc/ OO

6 476 IAN R. FALCONER The data presented in Table 3 show a consistent decrease in thyroid hormone secretion after oestrogen treatment in eight out of nine experiments. There is therefore no evidence of any activation of the thyroid gland during the period of 11 hr after oestrogen injection. m E 010 -A 008 : E o8o 006 I '- o Time (hr) Fig. 1. The effect of an intramuscular injection of 100 jg oestradiol dibenzoate on the concentration of protein-bound (PB) 125I in plasma from the thyroid vein blood of an undisturbed conscious ewe. TABLE 3. Effect of oestrogen treatment on the concentration of protein-bound radioactive 125I (PB1251) in plasma from thyroid vein blood of undisturbed conscious ewes bearing exteriorized thyroid glands. The results are expressed as in Table 2. All ewes were treated with oestradiol during the oestrous cycle Dose of oestradiol (/sg) t I I I I I I I I I Mean PB125I during the 5 hr following injection Number of measurements Mean PB1251 from 5 to 11 hr following injection * P < Number of measurements Difference between means Fiducial limits at P = * * * * ± * * DISCUSSION The changes in gonadotrophin secretion during oestrus and ovulation in the sheep have been studied by Robertson and co-workers, who have shown with precision the timing of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release from the pituitary.

7 OESTROGEN AND THYROID 477 The release of FSH from the sheep pituitary preceded both the onset of oestrus and the initiation of LH release by about 12 hr. The lowest pituitary FSH content occurred at 6 hr after the onset of oestrus, which also coincided with the lowest pituitary LH content. The release of LH occurred over a much shorter time than of FSH, between 0 and 6 hr after the onset of oestrus (Robertson & Rakha, 1966). Ovulation in the ewe occurs between 22 and 30 hr after the onset of oestrus (Cole & Miller, 1935), which is some 20 hr after the pituitary release of LH. Measurements of circulating oestrogens in the ewe have shown a peak just before the onset of oestrus, during the period when FSH but not LH is being released from the pituitary (Moore et al. 1969). The observations of increased thyroid activity in the ewe at oestrus were not as precise with respect to the time of onset of oestrus as were the studies of Robertson & Rakha (1966), but close examination indicated several examples of acceleration of thyroid activity at or near the onset of oestrus (Falconer, 1960; Robertson & Falconer, 1961 b). It would therefore appear that the activation of the thyroid during oestrus is approximately coincident with the period of rapid release of gonadotrophins and of high oestrogen concentrations. Oestrogen can be used to stimulate both oestrus and ovulation in the sheep. In ovariectomized ewes a single injection of 60,tg oestradiol dibenzoate will induce behavioural oestrus in 75 % of ewes treated (Robinson, 1967). Intact oestrous cycle ewes and anoestrous ewes pretreated with progesterone can be brought into oestrus with 10-50,ug oestradiol, and a proportion will ovulate (Brown, Catt, Cumming, Goding, Kaltenback & Mole, 1969). Ovulation in the sheep in normal oestrus follows a large release of pituitary LH, which reaches a peak concentration in blood a few hours after the onset of oestrus. The intramuscular injection of 10 or 50,tg oestradiol was shown to cause a similar LH release, with a peak some 12 hr after oestrogen administration (Brown et al. 1969). It was therefore concluded that the increased circulating oestrogen which preceded oestrus in the sheep caused the release of LH which resulted in ovulation. In the ovariectomized rat the chronic injection of physiological doses of oestrogen has been shown to cause an increase in pituitary TSH secretion, compared with the reduced TSH secretion observed in ovariectomized rats not given steroid treatment (D'Angelo & Fisher, 1969). The significance of oestrogen as a normal stimulator of thyroid activity in the oestrus rat is however still in doubt. The experiments of Brown-Grant (1966) support the view that it is the neuro-endocrine changes at the hypothalamic or pituitary level which lead to ovulation that are responsible for the increased thyroid activity at oestrus in the rat, and not changes in ovarian

8 478 IAN B. FALCONER steroid secretion. An increase in blood TSH at the beginning of oestrus in the rat was observed by Boccabella & Alger (1967), demonstrating that the activation of the thyroid at normal oestrus was due to a pituitary release of TSH. The experiments reported in this paper did not show any activation of the ewes' thyroid by oestrogen, whether the experimental animal was prepubertal, anoestrous, ovariectomized or undergoing oestrous cycles. The dosages of oestrogen used were from 25 to 1000,g, with the majority of the experiments carried out with doses of 50,tg. This quantity of oestrogen will cause behavioural oestrus in the majority of animals, and can be expected to cause ovulation in a proportion of the oestrous cycle ewes treated. It therefore appears that oestrogen, in a dose which will cause LH release from the pituitary of the ewe, does not also cause TSH release. According to the work of Brown et al. (1969), LH release begins about 6 hr after oestrogen injection, and reaches a peak after hr in most cases. The injection of TSH in the ewe with an exteriorized thyroid will cause a detectable increase in PB 1251 in thyroid vein blood within 30 min of administration (Falconer, 1968) so that any release of TSH concurrently with LH would have easily been detectable in the experiments summarized in Table 3. Comparison of PB 131J concentration in thyroid vein and whole jugular blood showed secretion of labelled hormone from the tlhyroid before and after oestrogen administration, with no changes attributable to the oestrogen. It would appear likely that the activation of the thyroid gland at oestrus in the ewe occurs independently of oestrogen secretion, and is due to a release of pituitary TSH mediated by the hypothalamus. The functional significance of thyroid activation at oestrus does not lie in any necessity for thyroid secretion during mating or conception (Falconer, 1963b), but is probably related to the increased physical activity observed at oestrus in the ewe. I wish to thank the Wellcome Trust for their financial support during part of this study, Miss Kathleen Smith for her careful measurements of hormone secretion in the exteriorized thyroid ewes, and Mr J. Jones for his technical assistance. REFERENCES BOCCABELLA, A. & ALGER, E. A. (1967). Quantitative variations in serum thyrotrophin levels during the estrous cycle of the rat. Endocrinology 81, BROWN, J. B., CATT, K. J., CUMMING, I. A., GODING, J. R., KALTENBACK, C. C. & MOLE, B. J. (1969). The release of luteinizing hormone in the ewe following oestradiol administration. J. Physiol. 201, 98-lOOP.

9 OESTROGEN AND THYROID 479 BROWN-GRANT, K. (1966). The relatioinship between ovulation and the changes in thyroid gland activity that occur during the oestrous cycle in rats, mice and hamsters. J. Physiol. 184, COLE, H. H. & MILLER, R. F. (1935). Changes in the reproductive organs of the ewe with some data bearing on their control. Am. J. Anat. 57, D'ANGELO, S. A. & FISHER, J. S. (1969). Iinfluence of estrogen on the pituitarythyroid system of the female rat: mechanisms and loci of action. Entdocrinology 84, FALCONER, I. R. (1960). Aspects of thyroid function relating to growth and reproduction in the eve. Ph.D. thesis, University of Aberdeen. FALCONER, I. R. (1963a). The exteriorization of the thyroid gland and measureinent of its function. J. Endocr. 26, FALCONER, I. R. (1963b). The effect of thyroid deficiency on the oestrous cycle and conception in the ewe. J. Endocr. 27, FALCONER, I. R. (1967). The effect of adrenaline and noradrenaline oin hormone secretion and blood flow from the thyroid veiin in sheep with exteriorized thyroids. J. Physiol. 188, FALCONER, I. R. (1968). The effect of vasopressin on hormone secretion and blood flow from the thyroid vein in sheep with exteriorized thyroids. J. Physiol. 199, FELDMAN, J. D. (1956). Effect of estrus and estrogen on thyroid uptake of I131 in rats. Endocrinology 58, MOORE, M. W., BARRATT, S., BROWN, J. B., SCHINDLER, I., SMITH, M. A. & SMYTH, B. (1969). Oestrogen and progesteroine content of ovarian vein blood of the ewe during the oestrous cycle. J. Endocr. 44, ROBERTSON, H. A. & FALCONER, I. R. (1961a). The estimation of thyroid activity: an evaluation of certain parameters. J. Endocr. 21, ROBERTSON, H. A. & FALCONER, I. R. (1961 b). Reproduction and thyroid activity. J. Endocr. 22, ROBERTSON, H. A. & RAKHA, A. M. (1966). The sequence, time and duration of the release of follicle-stimulating hormone and luteinizing hormonie in relation to oestrus and ovulation in the sheep. J. Endocr. 35, ROBINSON, T. J. (1967). Control of the ovarian cycle in the sheep. In Reproduction in the Female Mlammal, ed. LAMMIING, G. E. & AmOROSO, E. C., pp London: BUtterworths.