TSHβ mRNA Research Articles

Starvation-induced changes in the hypothalamic content of prothyrotrophin-releasing hormone (proTRH) mRNA and the hypothalamic release of proTRH-derived peptides: role of the adrenal gland.

The purpose of this study was to investigate the mechanisms involved in the reduced thyroid function in starved, young female rats. Food deprivation for 3 days reduced the hypothalamic content of prothyrotrophin-releasing hormone (proTRH) mRNA, the amount of proTRH-derived peptides (TRH and proTRH160-169) in the paraventricular nucleus, the release of proTRH-derived peptides into hypophysial portal blood and the pituitary levels of TSH beta mRNA. Plasma TSH was either not affected or slightly reduced by starvation, but food deprivation induced marked increases in plasma corticosterone and decreases in plasma thyroid hormones. Refeeding after starvation normalized these parameters. Since the molar ratio of TRH and proTRH160-169 in hypophysial portal blood was not affected by food deprivation, it seems unlikely that proTRH processing is altered by starvation. The median eminence content of pGlu-His-Pro-Gly (TRH-Gly, a presumed immediate precursor of TRH), proTRH160-169 or TRH were not affected by food deprivation. Since median eminence TRH-Gly levels were very low compared with other proTRH-derived peptides it is unlikely that alpha-amidation is a rate-limiting step in hypothalamic TRH synthesis. Possible negative effects of the increased corticosterone levels during starvation on proTRH and TSH synthesis were studied in adrenalectomized rats which were treated with corticosterone in their drinking water (0.2 mg/ml). In this way, the starvation-induced increase in plasma corticosterone could be prevented. Although plasma levels of thyroid hormones remained reduced, food deprivation no longer had negative effects on hypothalamic proTRH mRNA, pituitary TSH beta mRNA and plasma TSH in starved adrenalectomized rats. Thus, high levels of corticosteroids seem to exert negative effects on the synthesis and release of proTRH and TSH. This conclusion is corroborated by the observation that TRH release into hypophysial portal blood became reduced after administration of the synthetic glucocorticosteroid dexamethasone. On the basis of these results, it is suggested that the reduced thyroid function during starvation is due to a reduced synthesis and release of TRH and TSH. Furthermore, the reduced TRH and TSH synthesis during food deprivation are probably caused by the starvation-induced enhanced adrenal secretion of corticosterone.

In Situ Hybridization Detection of TSH? Subunit Gene Expression in the Serum-Free Primary Culture of the Adult Rat Pituitary

This study was designed to construct the primary culture system to detect the change in TSH beta subunit (TSH beta) gene expression in individual cells. Adult, male Wistar rats were sacrificed by transcardial perfusion of 0.25% trypsin solution under pentobarbital anesthesia (50 mg/kg body weight). Their anterior pituitaries were removed, dispersed and cultured for 1, 2, 3, or 6 days with or without 1 nM triiodothyronine (T3) under the serum-free condition. In some cultures, TRH was added to a final concentration of 1 microM on 6, 12 or 24 h before fixation. Then the culture media were removed to measure TSH concentration. Cells were fixed with paraformaldehyde and hybridized with 35S-labeled RNA probe complementary to TSH beta mRNA. Emulsion autoradiography was subsequently performed. T3 treatment markedly suppressed relative cellular levels of TSH beta mRNA on 2, 3 and 6 days after the onset of culture (day 2, 3 and 6) and suppressed TSH secretion on day 3 and 6. TRH treatment increased TSH beta mRNA on 12 and 24 h after the treatment on day 2 and 3 but did not increase TSH beta mRNA on day 6. TSH concentration in the culture medium was increased by TRH treatment on 6, 12 and 24 h after the treatment on day 2, on 12 h and 24 h on day 3, and 24 h on day 6. On day 2 and 3, although T3 treatment suppressed basal level of TSH beta mRNA, TRH-induced increase in TSH beta mRNA was not suppressed by T3 treatment. These results show that the thyroid hormone and TRH regulate TSH beta gene expression independently. Our culture system may provide a useful model to examine the action of individual substances on a specific subpopulation of the anterior pituitary cells.

Diisopropylfluorophosphate (DFP) reduces serum prolactin, thyrotropin, luteinizing hormone, and growth hormone and increases adrenocorticotropin and corticosterone in rats: Involvement of dopaminergic and somatostatinergic as well as cholinergic pathways

Cholinergic mechanisms have been implicated in the regulation of anterior pituitary hormone secretion. The present study was designed to determine the effect of a single injection of an organophosphate acetylcholinesterase inhibitor, diisopropylfluorophosphate (DFP), on anterior pituitary function in male rats. DFP increased serum ACTH (2.7-fold) and corticosterone (9.1-fold), while suppressing TSH, PRL, LH, and GH by up to 95%. The earliest response was at 1 hr, with a duration of at least 18 hr for TSH and LH. Responses were similar in adrenalectomized animals. After DFP, responses to hypothalamic releasing factors were normal for TSH, GH, and ACTH, but significantly blunted for PRL and LH. TSH suppression was partially prevented by combined therapy with a nicotinic (mecamylamine) and a muscarinic (atropine) antagonist. TSH suppression was partially reversed by immunoneutralization with somatostatin antibody, and PRL suppression was completely prevented by a dopamine antagonist (haloperidol). Atropine alone prevented the effects on corticosterone. TSH pituitary content and TSH-β mRNA were reduced by 37 and 22%, respectively, by DFP. In contrast, PRL mRNA was unchanged but PRL content was increased 3-fold. We conclude that cholinesterase inhibition evokes a multiplicity of effects on anterior pituitary function. There is a hierarchy of responses, with corticosterone being the most and TSH the least sensitive. There is evidence for inhibition at both the hypothalamic and pituitary levels, involving both nicotinic and muscarinic receptors. Although cholinesterase inhibition is the proximate event, other neurotransmitter pathways involved in TSH and PRL suppression are somatostatin and dopamine, respectively.

Serum Luteinizing Hormone, Prolactin, and Thyrotropin and Their Pituitary Subunit mRNA Levels during Proestrus in the Syrian Hamster

mRNA levels for alpha, luteinizing hormone beta (LH beta), and prolactin (Prl) were examined during the hamster estrous cycle, with sampling most frequent (1-hour intervals) on the afternoon of proestrus. These transcripts encode the peptide subunits for the pituitary hormones LH and Prl which are necessary for reproductive function. Serum hormone levels of LH and Prl, analyzed by 24-hour periodic regression, exhibited a 24-hour periodicity on proestrus characterized by a large surge peaking at about 18.00 h. Combining the data for non-proestrous days of the cycle disclosed a rhythm with similar timing for LH and Prl. Thyroid-stimulating hormone (TSH) and TSH beta RNA profiles during hamster proestrus are reported for the first time. Serum TSH exhibited a pronounced peak coincident with that of the other hormones on proestrus. Because of variations at other times on the day of proestrus, however, a 24-hour periodicity was not manifested by regressional analysis. Combined non-proestrous serum TSH data also revealed no consistently timed regressional 24-hour periodicity. During proestrus, pituitary mRNA values for alpha, LH beta, and Prl simultaneously exhibited a rise from the lowest to the highest of all proestrous values in the 3-5 h prior to the time of the pre-ovulatory peak of circulating hormone concentrations. RNA for TSH beta exhibited an earlier, broader peak on proestrus. Periodic regression indicated a significant 24-hour rhythm for alpha mRNA in data pooled from non-proestrous days (acrophase 05.00 h) and for TSH beta mRNA on proestrus (acrophase 04.54 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of α and thyrotrophin-β subunit mRNA levels by androgens in the female rat

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.

Testosterone Increases TSH-β mRNA, and Modulates α-Subunit mRNA Differentially in Mouse Thyrotropic Tumor and Castrate Rat Pituitary

TSH, LH and FSH, the three pituitary glycoprotein hormones, are each composed of a common alpha-subunit and a hormone specific beta-subunit. Testosterone is known to regulate all three intact hormones differently in the rodent. However, there is only one gene encoding the common alpha-subunit. In order to elucidate the effects of testosterone on TSH subunit synthesis and its regulation of the common alpha-subunit, two in vivo models were studied: castrate rat pituitary was used as a gonadotropin-enriched tissue; and mouse thyrotropic tumor was used as a thyrotropin-enriched tissue. Male castrate rats were treated with testosterone propionate, 500 micrograms/100 g BW, sc, for 11 days. Testosterone increased plasma TSH to 131% of control values (P less than 0.02), while plasma LH fell to undetectable levels, and plasma alpha-subunit fell to 14% of control values (P less than 0.001). Testosterone increased TSH-beta mRNA to 237% of control values (P less than 0.02), while alpha-subunit mRNA fell to 20% of control values (P less than 0.001). Hypothyroid mice bearing thyrotropic tumors were treated with testosterone propionate, 150 micrograms/100 g BW, sc, for 11 days. In this model plasma TSH-beta and alpha-subunit concentrations are 1000-fold higher than in non-tumor bearing animals, and the contribution of pituitary gonadotropes to plasma subunit concentrations is negligible. "Total" TSH-beta and alpha-subunit concentrations were estimated as one-half of intact TSH plus the respective free subunit concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute withdrawal of short-term or prolonged L-triiodothyronine administration to thyroidectomized rats results in similar rapid increases in TSHβ mRNA

In man, following the treatment of hyperthyroidism, or the withdrawal of prolonged suppressive thyroid hormone therapy, recovery from thyrotrope suppression may not occur until thyroid hormone concentrations have been subnormal for several weeks. Short-term studies in rodents have demonstrated a rapid increase in TSH synthesis after the acute withdrawal of thyroid hormones. We treated thyroidectomized rats with supraphysiologic doses of 1-triiodothyronine (T3) for 67 days, then abruptly withdrew treatment and compared the time course of thyrotrope recovery to that in animals given T3 for only 10 days. Increases in TSH beta mRNA after abruptly stopping T3 were qualitatively similar in both groups. After short-term T3 administration, TSH beta mRNA was detectable on the second day after stopping T3 administration and rose an additional 10-fold by day 5. After prolonged T3 administration, TSH beta mRNA was barely detectable on the second day after stopping T3 administration, clearly detectable on the third day, and increased an additional 20-fold by day 7. Compared to animals who received T3 for only 10 days, suppression of the thyrotrope by T3 administration for 67 days resulted in a nonsignificant delay in TSH beta mRNA synthesis of 24 h or less following the abrupt withdrawal of thyroid hormone. It is possible that differences in rat and human thyrotrope responsiveness account for the apparently different biologic behavior.

Effects of irradiation and semistarvation on rat thyrotropin beta subunitmessenger ribonucleic acid, pituitary thyrotropin content, and thyroid hormone levels

The effect of radiation-induced anorexia on serum thyrotropin (TSH), pituitary TSH-β mRNA, pituitary TSH content, serum thyroxine (T 4), and serum 3,5,3′-triiodothyronine (T 3) was investigated using feed-matched controls. Rats received 10 Gy gamma whole-body irradiation and were examined 1–3 days postirradiation. Feed- matched and untreated controls were also studied. The average food intake of the irradiated and feed-matched groups was approximately 18% of the untreated controls. Over the three day period both the irradiated and feed-matched groups lost a significant amount of body weight. The serum T 4 levels of both the irradiated and feed-matched groups were not significantly different from each other, but were significantly depressed when compared to the untreated control group. The serum TSH and T 3 were, however, significantly greater in the irradiated than the feed-matched groups at day 3 posttreatment. To determine if the difference in the serum TSH level between the two groups was due to a pretranslational alteration in TSH production, we measured the TSH-β mRNA using an RNA blot hybridization assay. We found that the TSH-β mRNA level was the same in the irradiated and feed-matched groups, suggesting that the mechanism responsible for the radiation-induced increase in the serum TSH level is post- transcriptional. Pituitary TSH content in the irradiated rats was significantly less than in pair-fed controls, suggesting that irradiation may permit enhanced secretion of stored hormone.

A study of pituitary thyrotropin, its subunits, and messenger ribonucleic acids in nonthyroidal illness

We studied serum TSH, pituitary TSH and α and β-subunits of TSH and their messenger ribonucleic acids (mRNAs) in three models of nonthyroidal illness (NTI) in the rat, ie diabetes mellitus (1 wk after 65 μg streptozotocin/g BW IP), turpentine oil-injection (8 to 48 hours after after a dose of 5 μl/g bw SC), and complete fasting for 72 hours. Euthyroid, hypothyroid (two months after thyroidectomy) and hyperthyroid rats (30 μgT 4/d × 7, SC) were also studied for comparison. Pituitary TSH, α and β subunits and serum TSH, T 4, and T 3 were measured by RIA. Pituitary mRNAs coding for common δ and TSH-β subunits were determined by cytoplasmic dot hybridization technique using specific [ 32P]-cDNA probes. In all NTI models there were significant decreases in serum levels of TSH, T 4, and T 3, but no significant changes were observed in the pituitary content of TSH, and α and TSH-β subunits. Hypothyroid rats had an increase in serum TSH, pituitary TSH, and pituitary TSH-β subunit and a decrease in pituitary α subunit. On the other hand, hyperthyroid rats showed a decrease in serum TSH, pituitary TSH, and pituitary TSH-β subunit, while there was no change in the α subunit. A significant reduction in the pituitary TSH-β mRNA levels was observed in all NT1 models and hyperthyroidism, while TSH-β mRNA was increased in thyroidectomized rats. α-mRNA was increased only in the pituitary of hypothyroid rats; there was no appreciable change in the pituitary α-mRNA in the various other pituitary groups. The kinetics of changes were studied in turpentine oil-injected rats and showed a significant reduction in serum TSH at four and eight hours after the injection, whereas TSH-β mRNA levels were decreased significantly at eight hours but not at 12 hours or later. We also studied 5′-monodeiodination of 126I-T 4 (to T 3) in pituitaries of all three experimental models and control rats; there was no significant difference. Our data suggest that NTI is associated with a decrease in the pituitary content of the mRNA for TSH-β subunit that may be transient and may contribute to fall in serum TSH in chronic NTI. Since pituitary TSH content was normal, a reduction in TSH release may be a more important factor than reduced TSH biosynthesis in determining fall in serum TSH at an early stage of NTI. Low serum TSH in NTI is not a result of an increase in the conversion of T 4 to T 3 in the pituitary.

A comparison of changes in plasma thyrotropin β- and α-subunits, and mouse thyrotropic tumor thyrotropin β- and α-subunit mRNA concentrations after in vivo dexamethasone or T 3 administration

Dexamethasone, like T 3, inhibits the production of TSH. T 3 inhibits TSH synthesis by reducing transcription of the genes encoding TSH-β and α-subunits. Little information is available concerning the effects of dexamethasone on the individual subunits, or the combined effects of dexamethasone with T 3. In a preliminary study, hypothyroid mice bearing the thyrotropic tumor TtT 97 were treated with 25, 250, or 500 μg dexamethasone ip daily for ten days. Plasma levels of TSH and its subunits were unchanged after 25 μg of dexamethasone and maximally suppressed after 250 μg. Plasma TSH was reduced to 51% ( P < .02), free TSH-β to 54% ( P < .01), and α-subunit to 62% ( P < .001) of control values. In two similar experiments hypothyroid mice bearing TtT 97 were treated with dexamethasone (250 μg), T 3 (0.5 or 1 μg), or both dexamethasone with T 3 for 10 days. Total α-subunit and TSH-β were calculated by adding 1 2 TSH + free subunit concentrations. In the experiment using 1 μg of T 3, total plasma α-subunit was reduced by dexamethasone to 72%, by T 3 to 45% ( P < .02), and by combined treatment to 23% ( P < .01) of control values. In the experiment using 0.5 μg of T 3, total plasma α-subunit was reduced by dexamethasone to 66% ( P < .05), by T 3 to 67% ( P < .05), and by combined treatment to 46% ( P < .02) of control values. In the studies using the 1 μg or 0.5 μg dose of T 3 respectively, total plasma TSH-β was reduced by dexamethasone to 57% ( P < .05) or 48%, by T 3 to 11% ( P < .01) or 19% ( P < .05), and by combined treatment to 14% ( P < .01) or 16% ( P < .02) of control values. TSH-β and α-subunit mRNA were measured by blot hybridization of total RNA in thyrotropic tissue using cloned subunit specific cDNAs. There was no change in TSH-β or α-subunit mRNA after dexamethasone; α-subunit mRNA was unchanged after 0.5 μg T 3, however, 1 μg T 3 reduced steady-state α-subunit mRNA to 26% of controls ( P < .01). After 0.5 μg T 3 TSH-β mRNA was suppressed to 11% ( P < .01) of controls, and 1.0 μg T 3 reduced TSH-β mRNA to barely detectable levels. Although both dexamethasone and T 3 reduce plasma TSH and subunits, dexamethasone does not act like T 3 in reducing steady-state levels of TSHβ and α-subunit mRNAs in the mouse thyrotropic tumor. Additionally, at the doses studied, the suppressive effects of dexamethasone and T 3 on plasma total α-subunit were additive.

Hormonal Regulation of Thyrotropin Alpha and Beta Subunit mRNAs

We have examined the effects of 3,5 3'-triiodo-L-thyronine (T3), dexamethasone, bromocriptine, thyrotropin releasing hormone (TRH) and estrogen on the levels of pituitary alpha and TSH-beta protein and mRNA levels in hypothyroid mice. After 3 days of treatment with T3 (0.5 micrograms/100 g body weight) serum TSH, alpha and TSH-beta levels were 77%, 79% and 44% of control, respectively. Pituitary alpha and TSH-beta mRNA content was estimated by dot blot hybridization of total RNA with 32P-labelled alpha and TSH-beta plasmid probes. There was no change in alpha mRNA after 3 days of T3 treatment but TSH-beta mRNA had decreased to 60% of control. With T3 at 2 micrograms/100 g body weight for 3 days, TSH protein was 27% of control and TSH-beta was undetectable, but there was no change in alpha. TSH-beta mRNA was decreased to 40% of control at 1 day and was barely detectable at 3 days, whereas alpha mRNA was 70% of control at 1 day and 42% at 3 days. Dexamethasone and bromocriptine caused no consistent change in pituitary levels of alpha and TSH-beta mRNA. Treatment with TRH caused small increases in serum TSH and in both alpha and TSH-beta mRNA levels. Estrogen treatment increased serum TSH and subunit levels and TSH-beta mRNA, but not alpha. We conclude that thyroid hormones decrease alpha and beta subunit mRNA levels discordantly in both the hypothyroid pituitary and in thyrotropic tumors and that the suppressive effect of thyroid hormone is the major regulator of TSH.

Pure α-subunit producing tumor derived from a thyrotropic tumor: Impaired regulation of a-subunit and its mRNA by thyroid hormone

We have recently described a mouse pituitary tumor line which produces only the α-subunit of the glycoprotein hormones. This tumor line may be a useful animal model to study autonomous pituitary tumors which secrete only α-subunit. Our pure α-subunit producing tumor was derived from a thyrotropic tumor which secreted intact TSH as well as free α-subunit. Our current studies compare the regulation of α-subunit biosynthesis in a conventional thyrotropic tumor and the α-subunit producing tumor. Thyroxine or triiodothyronine administration to mice bearing the a-subunit producing tumor resulted in no change in plasma α-subunit concentration, and a 10–19% reduction in tumor α-subunit mRNA concentration that was not statistically significant. In contrast, thyroxine administration to mice bearing the thyrotropic tumor resulted in an 81% reduction in plasma α-subunit concentration, and a 75% reduction in tumor α-subunit mRNA concentration (P < 0.01). Other studies using a cDNA specific for thyrotropin-β (TSHβ) failed to detect TSHβ mRNA in the α-subunit producing tumor, while TSHβ mRNA was easily detected in the conventional thyrotropic tumor. We conclude that during the development of the a-subunit producing tumor from a thyrotropic tumor, loss of TSHβ mRNA was also associated with an impaired capacity for thyroid hormone to decrease concentrations of α-subunit mRNA.