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Administration of a goitrogen (methimazole) and a low iodine diet to rats over a two-week period resulted in hypothyroidism and thyroid hyperplasia compared with controls (control: total serum thyroxine (T4) 66 +/- 4 nmol/l, thyroid weight 5 +/- 1 mg/100 g body weight; experimental: T4 undetectable, thyroid weight 27 +/- 4 mg/100 g body weight after 2 weeks of treatment; mean +/- S.D., n = 10). Immunohistochemistry carried out using a specific endothelial cell marker, CD31, and morphometric analysis (point counting of immunopositive cells) revealed that the progression of goitre in the rat thyroid is accompanied by an increase in capillary endothelial cell growth (neovascularisation). Fibroblast growth factor-2 (FGF-2) immunohistochemistry revealed widespread staining for the protein in the follicular cells of control glands. Less intense staining was found in the stroma and follicular cell nuclei. During hyperplasia and subsequent neovascularisation there was a progressive increase in the FGF-2 immunoreactivity at all locations during the two-week treatment period. Thrombospondin-1 (TSP1) immunoreactivity in the control rat thyroid was found in the stroma and in the endothelial cells, while weak follicular cell staining was also present. In the goitrous rat thyroid the TSP immunoreactivity was present after 1 week of treatment in the endothelial cells and most follicular cells, whilst stroma localisation was weak. After week 2 of treatment the endothelial cell and stromal localisation was no longer apparent, although a follicular localisation was still present. Transforming growth factor-beta 1 (TGF beta 1) immunoreactivity was present in the cytoplasm of a minority of the follicular cells in control rat thyroids, while their nuclei were unstained. In the goitrous rat thyroid an increase intensity of staining for TGF beta 1 was seen in all follicular cells, many of which now also demonstrated immuno-positive nuclei, within one week of goitrogen administration. These results show that in the hyperplastic thyroid increases in FGF-2 and TGF beta 1, and decreases in TSP1 accompany angiogenesis. These factors may interact in an autocrine/paracrine relationship to stimulate the neovascularisation that occurs during goitre formation.

Thyroid and steroid hormones act by similar mechanisms to influence gene expression in the anterior pituitary gland. The genes encoding the common alpha and TSH-beta glycoprotein subunits are known to be regulated by thyroid hormones; we report here the effects of androgen administration on levels of alpha and TSH-beta mRNA in pituitary cytoplasm in the euthyroid and hypothyroid female rat. Dihydrotestosterone (DHT) suppressed both alpha and TSH-beta mRNAs to levels lower than those found in untreated animals; a similar reduction was seen in hypothyroid animals treated with DHT. A biphasic response of TSH-beta mRNA was seen following administration of tri-iodothyronine (T3) to hypothyroid rats, with early stimulation followed by later inhibition; these changes were also evident after administration of T3 to androgen-treated animals, although mRNA levels were again suppressed. The effects of testosterone were similar to those of DHT. In contrast to the changes in mRNA levels, androgen administration did not lead to significant alterations in serum TSH concentrations or pituitary TSH content. These results indicate that, like thyroid hormones, androgens suppress both alpha and TSH-beta subunit mRNA levels in the female rat. Androgens, however, exert differential effects on TSH synthesis and release which contrast with those of thyroid hormones.

Basal and thyroid hormone-regulated expression of constructs containing varying lengths of 5' flanking sequence of the human glycoprotein hormone alpha subunit gene ligated to the reporter gene encoding chloramphenicol acetyl transferase (CAT) were examined in transfected pituitary GH3 cells. CAT gene expression was quantified by measurement of CAT enzyme activity. In addition, an RNase protection assay was used to confirm changes in CAT mRNA levels and to verify regulation of transcription from the authentic alpha gene start site. The pattern of basal alpha promoter activity indicated the presence of inhibitory DNA elements between -98 and -172 and upstream of -846 relative to the transcriptional start site, as well as a stimulatory element between -346 and -846. Each of the transfected constructs exhibited tri-iodothyronine inhibition of alpha promoter activity, suggesting the presence of an inhibitory thyroid hormone response element within 98 bp of the alpha gene transcriptional start site.

Thyroid hormones may directly regulate gene expression in the anterior pituitary. In order to examine this possibility we have studied the effect of hypothyroidism in the rat on pituitary cytoplasmic concentrations of messenger RNA (mRNA) encoding thyrotrophin (TSH) beta and alpha subunits, prolactin and GH. We demonstrated a marked increase in TSH beta and alpha subunit mRNA, accompanied by a decrease in GH mRNA, in the hypothyroid state, changes largely reversed by thyroid hormone replacement. We have thus shown a direct influence of thyroid status on the pretranslational events occurring in pituitary hormone synthesis. The simultaneous rise in cytoplasmic TSH beta and alpha mRNA levels and fall in GH mRNA in hypothyroidism suggests that thyroid status exerts a differential effect on the expression of these genes.

We have investigated the use of a multiwire camera for the rapid direct quantitation of 3H labelled cDNA/mRNA hybrids. Growth hormone (GH) complementary DNA (cDNA) probes were labelled with either 3H or 32P and used in dot-blot hybridization assays of pituitary cytoplasmic samples. The results were assessed either by counting in the multiwire camera (3H) or by autoradiography and scanning densitometry (32P). Results obtained by both methods were comparable over a similar range of pituitary cytoplasmic dilutions. The use of a multiwire camera may have several advantages over conventional methods for the detection of radioisotopically labelled cDNA/mRNA hybrids and has great potential for use in this field of endocrine research.

Interactions between DNA sequences and nuclear proteins are important in the regulation of gene expression. We have applied a gel retardation method to examine the binding of nuclear proteins from the rat pituitary gland to regulatory sequences of the rat TSH beta-subunit gene. Binding between nuclear protein and DNA is demonstrated by an alteration in electrophoretic mobility of the DNA. A 0.4 M NaCl fraction of pituitary nuclear proteins bound to a fragment of DNA containing the promoter region of the TSH-beta gene, but not to plasmid DNA or insulin cDNA of comparable size. This nuclear protein fraction also bound to fragments of DNA containing sequences from the 5'-flanking region of the gene; binding of nuclear proteins was evident as far as 1 kb 5' to the structural gene. Each of these regions of the TSH-beta gene formed a number of distinct complexes with the nuclear protein extract, distinguished by their differing binding affinities in the presence of poly(dI-dC), and by their electrophoretic mobility. These findings suggest that transcriptional regulation of the rat TSH-beta gene may be mediated by interaction of a number of nuclear proteins with regulatory sequences of DNA up to 1 kb upstream of the transcriptional start site of the gene.

The effect of tri-iodothyronine (T3) treatment on myocardial levels of alpha and beta myosin heavy chain (MHC) mRNAs in the rat was defined in vivo and in vitro. Dose-response experiments were performed in intact hypothyroid and euthyroid rats; in addition, studies in vitro examined the effect of T3 on MHC mRNAs in neonatal cardiac myocytes in primary culture. Specific alpha and beta MHC mRNAs were determined by Northern blot and dot hybridization to oligonucleotide probes complementary to the 3' untranslated regions of the MHC genes. An increase in myocardial beta MHC mRNA was demonstrated in hypothyroidism, accompanied by a reduction in alpha MHC mRNA. Marked differences in the sensitivity of alpha and beta MHC mRNAs to T3 replacement were found; a dose-dependent increase in alpha mRNA was evident at 6 h after T3 treatment, in the absence of consistent effects on beta mRNA, whereas 72 h after T3 replacement was commenced, stimulatory effects of T3 on alpha MHC mRNA, evident at all doses, were accompanied by a dose-dependent inhibition of beta MHC mRNA. No effect of thyroid status on actin mRNA was found, indicating the specificity of MHC gene regulation. T3 treatment of cardiac myocytes in vitro exerted similar actions on MHC mRNAs to those found in vivo, with a more marked influence on alpha than beta MHC mRNA. These studies of the action of T3 in vivo and in vitro have thus demonstrated specific effects of T3 on pretranslational regulation of the alpha and beta MHC genes, influences which differ not only in terms of stimulation or inhibition, but also in magnitude of effect.

We have reported previously the effect of thyroid status in vivo on pituitary cytoplasmic concentrations of messenger RNA (mRNA) encoding the thyrotrophin (TSH) beta-subunit (Franklyn, Lynam, Docherty et al, 1985). Studies in vitro of the regulation of TSH beta gene transcription have been confined to thyrotrophic tumour cells. We now report the demonstration of TSH beta-subunit mRNA in non-tumorous rat pituitary cells in primary culture. Treatment of cells with thyrotrophin-releasing hormone (TRH) and with forskolin resulted in a marked increase in cellular concentration of TSH beta-mRNA. These results suggest that TRH exerts a direct effect on the pretranslational events involved in TSH synthesis and further that the adenylate cyclase system may be involved in the regulation of synthesis. We have thus described a novel system for the study of TSH beta-subunit gene expression in normal rat pituitary cells in vitro.