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SUMMARY

A radioimmunoassay technique for measuring the thyrotrophin (TSH) concentration of sheep and cattle plasma is described. The sensitivity of the assay allowed the measurement of 1–50 ng TSH/ml unextracted plasma. Cross-reaction with ovine luteinizing hormone, prolactin and growth hormone was very low. The average recovery of added TSH was 103 ± 4·1 (s.e.m.)% and the between-assay coefficient of variation was 13·8%.

The normal plasma TSH levels of sheep and cattle were approximately 2·5 ng/ml (5 mu. bovine TSH/100 ml). Foetal sheep had plasma TSH concentrations of approximately 3·2 ng/ml during the last 20 days of gestation. Levels of TSH in the circulation decreased abruptly after hypophysectomy of the foetal lamb and a decline in the plasma thyroxine (T₄) concentrations was also apparent within 24 h of the operation. However, thyroidectomy of adult and foetal sheep did not increase plasma TSH concentrations until almost all the T₄ had been cleared from the circulation.

The injection of T₄ into thyroidectomized sheep rapidly reduced plasma TSH concentrations to normal values. However, the continued injection of T₄ did not further reduce TSH concentration. The injection of T₄ or triiodothyronine into normal sheep was also without effect on plasma TSH concentrations.

SUMMARY

The changes in plasma concentration and anterior pituitary content of thyrotrophic hormone (TSH) after thyroidectomy in the rat were followed by means of an in vitro assay method. A triphasic change in plasma TSH was demonstrated which consisted of a threefold rise by the 2nd day, a fall to the normal range by the 5th day, and a gradual rise from the 10th day, which continued for at least 6 weeks.

The initial rise in plasma TSH was accompanied by a fall in the pituitary TSH store after 5 days to 7 % of the normal value; the store then remained low for the next 6 weeks.

Thyroxine (as measured by protein-bound ¹³¹I) disappeared from the circulation after thyroidectomy at a rate compatible with the observed rate of rise of plasma TSH; 20 % remained after 2 days.

The replacement of thyroid hormone by exogenous thyroxine in thyroidectomized rats resulted in 4 days in a decline in plasma TSH concentration to a subnormal value. During the next 3 days the blood level of TSH rose to 400 % of the control level. Thyroxine administration caused a large increase in the pituitary store of TSH.

The results indicate that circulating thyroxine influences primarily the rate of release of TSH from the anterior pituitary, and that a slower effect on the synthesis of TSH may be indirect and secondary to changes in the TSH content of the pituitary gland.

Slice preparations of normal human thyroid tissue have been used to investigate the effect of normal immunoglobulin G (IgG) on thyrotrophin (TSH)-induced accumulation of cyclic AMP. Incubation of slices in the presence of both TSH and normal IgG for 20 min reduced the stimulation of cyclic AMP accumulation elicited by TSH alone by approximately 30%. However, preincubation of slices with IgG for 100 min before addition of TSH virtually abolished the response to TSH. The latter effect of normal IgG was reversible, and removal of IgG before exposure to TSH allowed an unimpaired cyclic AMP response to TSH. The implications of these observations with respect to the application of this system to the functional bio-detection of thyroid-stimulating antibodies in IgG fractions from thyrotoxic sera are discussed.

ABSTRACT

Confluent monolayer cultures of human thyroid cells secreted low levels of immunoassayable triiodothyronine (T₃) and this process could be stimulated by TSH in a concentration-dependent manner. The characteristics of the response to TSH were related to the age of the thyroid cell culture both in terms of the relative sensitivity to TSH and the quantity of T₃ released. Cells which had been in culture for 2–3 days (primary cultures) secreted high levels of T₃ under unstimulated and TSH-stimulated conditions with a median effective dose (ED₅₀) for TSH of 0·030 mu. TSH/ml. However, cells which had been subcultured and consequently had been in culture for a longer period of 6–7 days secreted lower levels of T₃ under basal and stimulated conditions. This was approximately 30% of that released from primary cultures with an ED₅₀ for TSH of 0·1 mu. TSH/ml. Reorganization of human thyroid cells into follicular structures was seen during growth with TSH but these cultures showed little response to subsequent acute stimulation by TSH; the return of a diminished, less sensitive response to TSH was seen after a recovery period of 8 h. The time-course of T₃ release was dependent on the TSH concentration with low TSH concentrations stimulating T₃ secretion after increased incubation periods. Human thyroid cells had lost the ability to concentrate and organify free iodide after several days in culture but were still secreting T₃. This indicates the presence of intracellular stores of T₃ which are released on stimulation with TSH, rather than new synthesis of T₃.

J. Endocr. (1985) 104, 285–290

ABSTRACT

Thyrotrophin (TSH) is the conditional growth factor of thyroid epithelial cells. Abnormalities in TSH-receptor binding such as a low receptor number or low binding affinity may be a marker of thyroid carcinoma or metastases, or may exhibit a relationship with the functional variability of such tissues. The dog was used as a model to characterize TSH-receptor binding in normal thyroid tissues, naturally occurring thyroid neoplasms and distant metastases. In normal dog thyroid tissues, specific ¹²⁵I-labelled TSH binding ranged from 2·7 to 15·5%, and low cross-reactivity with bovine LH (0·023%) was observed. One class of TSH-binding sites was found in eight normal thyroid tissues and 22 thyroid carcinomas; two normal thyroid tissues and one tumour exhibited two classes of binding sites. The concentration of binding sites was lower in the five carcinomas with reduced pertechnetate uptake (0·09 pmol/mg protein) than in the five thyroid neoplasms with increased uptake (0·19 pmol/mg) (P= 0·055). Compared with the original carcinoma tissues, TSH binding revealed a reduced binding affinity in eight out of eleven metastases. Two metastases showed a complete absence of TSH binding, suggesting that they were not dependent on TSH for growth. We conclude that one class of TSH-binding site is predominant in normal dog thyroid tissues and dog thyroid carcinomas. TSH could therefore contribute, at least in theory, to further growth of primary dog thyroid carcinomas. Secondly, assays measuring TSH binding may not be able to discriminate between malignant and benign dog thyroid tumours. TSH receptor number or affinity may be related to the functional variability of thyroid neoplasms. The absence of TSH binding in some metastases demonstrated that this characteristic can be acquired during the natural history of a differentiated thyroid carcinoma.

Journal of Endocrinology (1992) 132, 461–468

ABSTRACT

Previous studies have shown that freezing and thawing of human thyroid homogenates releases a water-soluble substance which reversibly binds to TSH-receptor antibodies. This substance has been designated long-acting thyroid stimulator absorbing activity (LAA). We now describe a new method for measuring LAA based on the TSH-receptor assay and application of the technique to the study of LAA.

Our results indicate that LAA is a heat-labile glycoprotein which co-elutes with haemoglobin on gel filtration. Furthermore, LAA is retarded by columns of Sepharose–TSH but not by Sepharose coupled to human chorionic gonadotrophin, normal immunoglobulin G or bovine serum albumin, suggesting that LAA contains a binding site for TSH as well as for TSH-receptor antibodies. It would seem therefore that LAA is a water-soluble fragment of the TSH receptor possibly resulting from proteolytic cleavage of the receptor at a site close to the cell surface.

J. Endocr. (1984) 100, 113–118