Concentrations of Triiodothyronine, Growth Hormone, Luteinizing Hormone in the Plasma of Thyroidectomised (Gal/us domesticus) S.HARVEY, Wolfson
R.J. STERLING,* AND H. KLANDORP
of Hull, Hull, Centre, Roslin,
North Humberside Mdlothian EH25
HU6 7RX, 9PS, England
Accepted July 28, 1982 Surgical thyroidectomy increased (P < 0.05) the basal concentrations of growth hormone (GH) and luteinizing hormone (LH) in the plasma of lO- to 12-week-old domestic fowl. The administration of thyrotrophin releasing hormone (TRH) (100 H, SC) increased (P < 0.01) the GH concentration in both intact and thyroidectomised bids. The magnitude of the TRH-induced increase in GH level was greater (P < 6.01) in thyroidectomised birds than in intact controls. Although TRH had no effect on LH secretion in the controls, it induced a small (P < 0.05) rise in the plasma LH level in thyroidectomised birds. In both the intact and thyroidectomised birds the LH concentration was enhanced (P < 0.05) following the administration of LH-releasing hormone (LH-RH) (20 pg, SC). The increase in the LH level by LH-RH in the thyroidectomised bids was greater (P < 0.001) than that in the intact controls. Plasma GH concentrations were unaffected by LH-RH treatment. These results suggest that thyroid hormones inhibit the secretion of LH and GH in birds. In thyroidectomised birds low levels of immunoreactive triiodothyronine (T&like material were measurable in the circulation, despite the absence of regenerated thyroid tissue. The administration of TRH (100 pg, SC) did not enhance the plasma level of this material in thyroidectomised birds, whereas plasma T, concentrations were enhanced in intact bids following TRH treatment. These results suggest that the T, immunoreactive substance in thyroidectomised birds is extrathyroidal in origin.
In mammalian species hypothyroidism (induced by propylthiouracil or thyroidectomy) has been associated with deficiencies in growth hormone (GH) and luteinizing hormone (LH) secretion. In hypothyroid animals ovarian function is impaired and the secretion of gonadotrophins is suppressed (Davis and Borger, 1973; Bruni et al., 1975). The synthesis and pituitary storage of GH is also reduced in hypothyroid animals (Peake et al., 1973; Wilkins et al., 1974), and the circulating GH level is lowered (Davis and Borger, 1973; Takeuchi et al., 1978; Coiro et al., 1979). These effects of hypothyroidism are reversed by the administration of thyroxine (T3 (Wilkins et al., 1974; Hervas et al., 1976; Coiro et al., 1979), which enhances pituitary GH synthesis and plasma GH levels in euthyroid
animals (Figurova et al., 1975; Hervas et al., 1975). In marked contrast hypothyroidism in birds appears to stimulate the secretion of GH and LH. Elevated circulating GH levels have been demonstrated in hypothyroid chicks suffering from autoimmune thyroidites (Scanes et al., 1976) and plasma GH and LH levels are high in birds made hypothyroid by goitrogen treatment (Chiasson et al., 1979). Thyroidectomy has also been reported to stimulate gonadal activity in birds (Thapliyal and Pandha, 1967; Singh and Parshad, 197%), possibly as a result of increased gonadotrophin secretion. In the present study the effect of surgical thyroidectomy on the basal and stimulated levels of GH and LH in the plasma of immature domestic fowl has therefore been 275 001~6480/83 $1.50 [email protected]
0 1983 by Academic Press, Inc. AU rights of reproduction in any form reserved.
examined. In view of reports on triiodothyronine (TJ- and TJike material in the circulation of thyroidectomised birds (Mellen and Wentworth 1962; Peczely and Daniel, 1979; Davison et al., 1980; Peczely et al., 1980; Klandorf et al., 1981), the possibility that this may be of extrathyroidal origin has also been investigated. MATERIALS
One-day-old broiler cockerels were reared with food and water available ad libirum and were kept under continuous light until 4 weeks of age and thereafter under a 14L:lOD photoperiod. At 4 weeks of age 11 birds were surgically thyroidectomised under sodium pentabarbitone anaesthesia and at 6 and 8 weeks of age each bird was subcutaneously (SC) injected (in the nape of the neck) with 1 and 2 mCi, respectively, of radioactive iodine (NarSII, Radiochemical Centre, Amersham) to destroy any remaining thyroid tissue (Mellen and Wentworth, 1962; Davison er al., 1980). The success of surgery was subsequently confirmed at autopsy by gross visual observation and in most cases by histological examination of the thyroid region. At 10 weeks of age the thyroidectomised birds were SCinjected with 0.9% saline (0.5 ml) or thyrotrophin releasing hormone (TRH, 100 pg) and blood samples were taken by brachial vein venepuncture immediately before and 30,60, and 90 min after injection. The blood samples were centrifuged and the plasma separated and stored at -20” to await analysis. Two weeks later groups of intact and thyroidectomised birds were SC injected with saline (0.5 ml) or with LH-releasing hormone (LH-RH, 20 cog)or TRH (100 pg) and blood samples were taken as before. The order of the treatments were randomized and the experiments were carried out on consecutive days. Plasma analysis. All the plasma from both experiments were assayed in single hormone assays to eliminate interassay variation, Plasma concentrations of T, were determined by a solid phase radioimmunoassay (Seth et al., 1976). Plasma free of T, was included in the assay standards to equalise protein concentrations between incubations containing samples and standards. The minimum detectable dose for the T, assay was 0.13 rig/ml and the assay has an intraassay coefficient of variation of 5.9%. Concentrations of plasma GH and LH were determined by specific homologous radioimmunoassays for chicken GH (Harvey and Scanes, 1977) and LH (Follett et al., 1972). The LH standard and ‘251-labelled tracer were a preparation of chicken LH (fraction AE 1) purified to minimise thyroid stimulating hormone contamination (Scanes and Follett, 1972). The GH and LH assays had intraas-
AND KLANDORF say coefftcients of variation of 5.1 and 3.1%, respectively. Statistical differences in the results were determined by paired or unpaired Students t test wherever appropriate.
At 10 weeks of age levels of T, were measurable in the circulation of the thyroidectomised birds, although the concentrations (0.98 + (SEM) 0.10 (n = 10) rig/ml) were much lower (P < 0.001) than those in intact birds sampled at the same time (2.82 + 0.16 (N = 8) &ml). The administration of TRH to the thyroidectomised birds had no effect on the plasma T, concentration (Table I), but markedly (16.dfold, P < 0.001) increased the GH level 30 min after injection. TRH treatment also resulted in a small increase in the LH level 30 min after injection (P < 0.05 in comparison with saline controls) . The administration of TRH to the thyroidectomised birds at 12 weeks of age similarly had no effect on the levels of plasma T, but again resulted in increased LH (P < 0.01) and GH (P < 0.001) concentrations 30 min after injection (Fig. 1). TRH treatment increased GH levels in intact birds (P < 0.01) and also enhanced the T, concentration 60 and 90 min after injection but had no effect on the level of plasma LH (Fig. 1). The GH response to TRH in the thyroidectomised birds was greater (P < 0.01) than that in the intact controls. The mean basal plasma GH level in the thyroidectomised birds prior to treatment with saline, TRH, or LH-RH (41.1 & 10.0 (n = 27) @ml) was also higher (P > 0.01) than the GH concentration in the controls (9.0 & 0.9 (n = 18) @ml). The mean LH concentration in the thyroidectomised birds prior to treatment (7.15 + 0.45 (n = 27) &ml) was also higher (P < 0.05) than that in the controls (5.43 +- 0.52 (n = 18) @ml). In both the intact and thyroidectomised birds the administration of LH-RH had no
TABLE 1 INFLUENCE OF SUBCUTANEOUS INJECTIONS OF THYROTROPIN RELEASING HORMONE (TRH) CONCENTRATIONS (&ml) OF PLASMA TRIIODOTHYRONINE (T3), LUTEINIZING HORMONE (LH), GROWTH HORMONE (GH) IN IMMATURE, IO-WEEK-OLD THYROIDECTOMISED COCKEREL~ Minutes Hormone
a * t $
saline (0.5 ml) TN--I (100 /.d
0.56 t 0.52 k
0.49 + 0.05 0.48 c 0.09
0.49 2 0.06 0.46 t 0.06
saline (0.5 ml) TRH (100 /a)
6.28 f 8.92 2
6.15 r 0.59 6.82 k 0.46
5.69 t 0.53 5.94 + 0.40
saline (0.5 ml) TRH (100 pg)
lo? 481 k
26 + 19 29 k 13
The birds were surgically thyroidectomised at 4 weeks of age. Significantly different from pretreatment levels, P < 0.001. Significantly different from control levels, P < 0.05. Means ? SEM (n = 8).
effect on plasma GH or T, concentrations (Fig. 1). In both groups LH-RH induced an increase (P < 0.05) in the LH level 30 min after injection, the increase in the thyroidectomised birds being greater (P < 0.001) than that in the controls. DISCUSSION
Although immunoreactive T, was measurable in the circulation of the thyroidectomised birds, the removal of the thyroid gland undoubtedly induced severe hypothyroidism. The finding of a low level of T,-like material in the blood of thyroidectomised chicks is in agreement with other studies in gallinaceous birds in which T,and T,-like activity has been demonstrated (see Introduction). This immunoreactive material may indicate the presence of accessory T,- and T,-producing cell populations, despite the absence of regenerated thyroid tissue (Peczely et al., 1980). It is, however, possible that this material may represent a nonhormonal fraction. Although immunoreactive T4 and T, is reported to be absent from the plasma of thyroidectomised ducks, nonhormonal protein bound iodine is present (Astier and
Newcomer 1978; Astier et al., 1978). The demonstration of nonhormonal protein bound iodine in the chicken has also been reported (Davison, 1978). In thyroidectomised birds this protein may be an iodinated albumin (thyralbumin) (Shulman et al., 1957; Stanburg and Janssen, 1962), which could extrathyroidally replace thyroglobulin as the precursor for thyroid hormone synthesis. In intact birds TRH stimulation markedly increases the concentrations of plasma T, (Fig. 1; Klandorfet al., 1978). The results of the present study clearly demonstrate that the concentration of immunoreactive T,-like material in thyroidectomised birds is not responsive to TRH stimulation. Similarly while photostimulation markedly affects blood T, and T, levels in intact Japanese quail, photostimulation does not affect the concentration of thyroid hormone-like material (Peczely et al., 1980) in thyroidectomised quail. These results suggest that the origin of this material is extrathyroidal. In immature domestic fowl TRH is a potent stimulus for GH secretion (Harvey et al., 1978, 1981). The increase in GH secre-
Time after injection
FIG. 1. Effect of subcutaneous injections of saline (0,0.5 ml), TRH (m, 100 pg), and LH-RH (A, 20 pg) on the concentrations (&ml) of triiodothyronine, growth hormone, and luteinizing hormone in the plasma of intact (left) and thyroidectomised (right) 12-week-old chickens. Means * SEMs. The number of samples at each point varied between 6 and 9.
tion in hypothyroid (Scanes et al., 1976; Chiasson et al., 1979) and thyroidectomised (Fig. 1) birds may therefore indicate it is physiologically involved in the control of GH secretion. Increased GH secretion in thyroidectomised birds may therefore result from increased endogenous TRH release, TSH secretion being enhanced in thyroidectomised mammals (Davis and Borger, 1973; Takeuchi et al., 1978). The
exaggerated GH response to TRH and the higher basal GH level in thyroidectomised birds agrees with similar findings in hypothyroid rats (Kato et al., 1975; Chihara et al., 1976) but contrasts with other mammalian studies (see Introduction). In intact birds the GH response to TRH diminishes during sexual maturation and is virtually absent in adults (Harvey et al., 1981). As thyroidectomy may block the sexual maturation of some birds (Jallageas and Assenmacher, 1973; Chaturvedi and Thapliyal, 1979) it is possible that their greater responsiveness to TRH stimulation may reflect a delay in their maturation. Indeed while adult birds are unresponsive to TRH, GH secretion in adult (although sexually immature) thyroidectomised fowl may remain responsive to TRH stimulation (Harvey, 1983). In immature thyroidectomised fowl the basal concentration of plasma LH was elevated, in agreement with unpublished findings in thyroidectomised canaries (by Nicholls and Storey, cited by Follett et al., 1978). Thyroidectomy has similarly been reported to enhance gonadal steroid secretion in birds (Peczely and Daniel 1979; Peczely et al., 1980). These results suggest that the thyroid hormones inhibit LH secretion in birds and indeed their administration has been shown to impair gonadal activity (van Tienhoven, 1961). These findings differ from those in mammalian species in which thyroidectomised and hypothyroid animals have reduced gonadotrophin secretion (Davis and Borger, 1973; Bruni et al., 1975). The influence of the thyroid gland on the pituitary-gonadal axis of birds is, however, complex as the thyroid hormones have been thought to stimulate gonadal development in some avian studies (e.g., Thapliyal and Pandha, 1967; Singh and Parshad, 1978). In view of the stimulatory effect of TRH on gonadal growth and testicular 32P uptake (Wentworth et al., 1976), it has been suggested that TSH may have some go-
GH AND LH IN THYROIDECTOMISED
nadotrophic activity in birds. TRH has been found to marginally increase in viva LH secretion in turkeys and quail (Wentworth et al., 1976; Follett et al., 1978) and the in vitro release of LH from chicken pituitaries (Bonney and Cunningham, 1977). In the present study although TRH had no effect on plasma LH levels in intact birds it slightly increased the concentration in thyroidectomised fowl. This may indicate that in the absence of thyroid hormones TRH can affect LH secretion in chickens, but it is possible that these results may be due to the incomplete specificity of the LH radioimmunoassay (Follett, et al., 1978; Sharp et al., 1979). Thyrotrophin secretion would be expected to be increased in thyroidectomised birds (Davis and Borger, 1973; Takeuchi et al., 1978) and as in mammalian species the TSH response to exogenous TRH might also be exaggerated (Chihara et al., 1976). As very high levels of TSH may be measurable in the LH radioimmunoassay it is possible that the high basal LH level in the thyroidectomised birds and the response to TRH may be due to TSH cross-reaction in the immunoassay. The stimulatory effect of LHRH on avian LH secretion is now well documented (e.g., Wentworth et al., 1976; Follett et al., 1978). The augmentation of the LH response to LH-RH in the thyroidectomised birds may have been due to the removal of inhibitory thyroidal influences on the hypothalamicpituitary -gonadal axis. The exaggerated LH response to LHRH in the thyroidectomised birds is unlikely to be due to an increase in immunoreactive TSH secretion as LHRH had no effect on the T, concentration in intact chickens (Fig. 1) nor on plasma T, levels in geese (Campbell and Leatherland, 1979). Although LH-RH stimulates GH release in human patients with active acromegaly (Rubin et al., 1973) it had no effect on GH secretion in the present study. In summary, these results demonstrate that basal and stimulated levels of GH and
LH are enhanced in thyroidectomised chickens and that the T,-like material in the plasma of thyroidectomised birds may be of extrathyroidal origin. ACKNOWLEDGMENTS The authors thank Professor B. K. Follett, Dr. C. G. Scanes, and Dr. J. Beckett for reagents used in the radioimmunoassays and Dr. P. J. Sharp for his advice and interest in this work. This work was supported in part by a grant from the Science Research Council (CHUB 47782).
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