Regulation of the alpha and thyrotropin beta-subunit messenger ribonucleic acids by thyroid hormones

We studied the regulation of mRNAs encoding the alpha- and beta-subunits of TSH by thyroid hormones (T4 and T3) in mouse thyrotropic tumors and pituitary glands. Hypothyroid male (LAF1) mice bearing thyrotropic tumor (TtT97) were injected daily with T4 for 0, 1, 5, 12, or 33 days. After day 33, plasma levels of TSH and free (unassociated) TSH beta-subunit were reduced to less than 1% of control levels, whereas free alpha-subunit was reduced to 6% of control levels. Steady state levels of subunit mRNAs in extracts of the thyrotropic tissues were measured by blot hybridization analyses using mouse subunit-specific cloned cDNAs. Treatment of mice with T4 caused a rapid decline in the levels of tumor mRNAs for both alpha and TSH beta; after day 1, alpha and TSH beta mRNA levels decreased to 35% and 10% of control values, respectively. Levels of TSH beta mRNA were undetectable after 5 days of T4 treatment, whereas levels of alpha-subunit mRNA remained at 30-35% of control levels even after day 33. In a separate experiment, TSH beta mRNA decreased to 42% of the control level (P less than 0.05), whereas alpha-subunit mRNA remained at 64% of the control level (P = NS) 4 h after a single injection of T4. Finally, T3 also caused a rapid decrease in the levels of both subunit mRNAs in the anterior pituitary glands of hypothyroid mice, but the effect was more complete on TSH beta mRNA levels. We conclude that thyroid hormones have rapid suppressive effects on the levels of mRNAs encoding the subunits of mouse TSH in the thyrotrope. The suppressive effects of thyroid hormones occur more rapidly and are greater for TSH beta than alpha-subunit mRNAs. The parallel changes observed in the subunit mRNA levels and the plasma subunit protein levels in animals treated with thyroid hormones suggest that the changes in the plasma levels of TSH and subunits may reflect effects of thyroid hormones on TSH gene expression in addition to effects on secretion.     

Changes in tissue concentrations of thyrotropin, free thyrotropin beta, and alpha-subunits after thyroxine administration: comparison of mouse hypothyroid pituitary and thyrotropic tumors

We have previously demonstrated divergent changes in pituitary free TSH beta and alpha-subunit concentrations in hypothyroid mice during prolongation of experimental hypothyroidism and after T4 administration. This report compares the simultaneous responses of pituitary and thyrotropic tumor TSH, TSH beta, and alpha-subunit to 12 days of T4 administration in four groups of hypothyroid LAF1 mice bearing thyrotropic tumors. Half of each group received daily injections of T4 (10 micrograms/100 g BW, ip) for 12 days; the other half of each group received saline. Plasma concentrations of TSH, free TSH beta, and free alpha-subunit were suppressed by T4 administration to 0.1-1.5%, 2.0-3.9%, and 4.1-25% of control concentrations, respectively. Pituitary TSH and free TSH beta concentrations fell significantly with treatment to 24-43% and 10-28% of control concentrations, respectively. In contrast, pituitary alpha-subunit concentrations did not fall (they were 106-203% of control values), and a rise in the pituitary alpha-subunit concentration was statistically significant in one group (P less than 0.02). Thyrotropic tumor TSH and free TSH beta concentrations fell significantly with treatment to 9-31% and 8-35% of control concentrations, respectively. Tumor alpha-subunit concentrations did not fall (they were 87-195% of control values), and a 2-fold rise was statistically significant in one group (P less than 0.05). Both pituitary and tumor alpha-subunit to TSH beta molar ratios increased significantly with T4 administration in all groups. We conclude that (1) pituitary and thyrotropic tumor TSH beta concentrations fall after 12 days of T4 administration while alpha-subunit concentrations are unchanged or actually rise; (2) this divergent response is qualitatively similar in hypothyroid mouse pituitary and thyrotropic tumors; and (3) these data suggest differences between the regulation of intracellular TSH beta and alpha-subunit.     

Mouse pituitary tumor line secreting only the alpha-subunit of the glycoprotein hormones: development from a thyrotropic tumor

This report describes the conversion of a murine pituitary thyrotropic tumor (MGH 101) to a pure alpha-subunit-secreting tumor (MGH 101A) during a 6-yr period of serial transplantation. MGH 101 was a thyrotropic tumor originating from a hypothyroid mouse pituitary, growing only in hypothyroid hosts, and secreting large quantities of intact TSH and free alpha-subunit. Between the fourth and ninth transplantation generations, tumor TSH secretion declined progressively by at least 500-fold, to undetectable levels. In contrast, tumor secretion of free alpha-subunit decreased only 10-fold, and has since remained stable for nine transplantation generations. During the conversion to pure alpha-subunit secretion, MGH 101A exhibited growth in euthyroid as well as hypothyroid hosts, and increased its growth rate 2 to 3-fold. In contrast, the conventional thyrotropic tumor TtT 97 has maintained its secretion of both intact TSH and free alpha-subunit, its dependence on a hypothyroid environment, and its slower growth rate for nine generations. Gel chromatography of the media from tumor cell cultures confirmed that MGH 101A secreted only the free alpha-subunit, whereas TtT 97 secreted immunoactive TSH, TSH beta, and free alpha-subunit which eluted as separate peaks. We conclude that a dependent thyrotropic tumor has spontaneously developed into a pure alpha-subunit-secreting tumor which is independent of host thyroid function for its growth and alpha-subunit production.     

Thyrotropin (TSH)-releasing hormone regulation of TSH subunit biosynthesis and glycosylation in normal and hypothyroid rat pituitaries

The regulation of TSH apoprotein and carbohydrate biosynthesis by TRH was studied by incubating pituitaries from normal and hypothyroid (3 weeks postthyroidectomy) rats in medium containing varying doses of TRH, [14C] alanine or [35S]methionine, and [3H]glucosamine. Samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to remove LH and free LH beta, and anti-LH alpha to precipitate free alpha-subunits. Total proteins were acid precipitated. All precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In hypothyroid samples, acute TRH (6 h) stimulated [3H] glucosamine incorporation into secreted combined alpha-subunit to 204% and secreted combined beta-subunit to 227% of control values (P less than 0.01), and stimulated [14C]alanine incorporation into secreted combined alpha-subunit to 201% and secreted combined beta-subunit to 258% of control values (P less than 0.01); pituitary content was not altered by TRH. In hypothyroid incubates, the half-maximal response was 8 X 10(-10) M TRH for both labeled precursors. In contrast, in normal samples, acute TRH (6 H) did not stimulate TSH subunit carbohydrate and apoprotein synthesis, but after 24 h, TRH stimulated [3H]glucosamine incorporation into both subunits of TSH to 270% of control values (P less than 0.02), with no change in [14C]alanine incorporation. Free alpha-subunit synthesis was not altered by TRH in normal or hypothyroid incubates. The glucosamine to alanine ratio of total newly synthesized TSH, reflecting its relative glycosylation, was increased by TRH in both combined subunits in hypothyroid samples as early as 6 h (P less than 0.05) and in normal samples only at 24 h (P less than 0.01). In summary, 1) TRH in hypothyroid incubates stimulated apoprotein and carbohydrate synthesis in combined alpha- and beta-subunits, but not free alpha-subunits, at 6 and 24 h. 2) In normal pituitary incubates, TRH stimulated TSH subunit carbohydrate, but not apoprotein, synthesis only at 24 h. 3) TRH increased the relative glycosylation of TSH in hypothyroid and normal rat pituitary incubates. Such alterations in TSH glycosylation may be due to structural changes in the carbohydrate moiety and may be important for hormone release, biological activity, or clearance.     

TSH subunit gene promoters from a murine alpha-subunit producing tumor function normally

The murine thyrotropic MGH101A tumor is characterized by absent thyrotropin (TSH) beta gene expression and altered thyroid hormone (T3) regulation of the alpha-subunit. Comparison of the promoter structures of both alpha and TSH beta subunit genes from MGH101A with the promoter in expressing TtT-97 thyrotropes revealed no detectable differences. Transfection of the TSH beta promoter from MGH101A linked to luciferase showed minimal expression in primary or cloned MGH101A cells, or L-cells. However, a 6- to 10-fold increase in expression was exhibited in transfected thyrotropes. For the alpha gene, promoter activity was highest in thyrotropes and in cloned MGH101A cells, 5-fold lower in MGH101A tumors, and 10-fold lower in L-cells. Both promoters were not substantially affected by T3 treatment in MGH101A cells. In thyrotropes, promoter activity was inhibited 62.5% and 57.7% by 10 nM T3 treatment for the TSH beta and alpha genes, respectively. DNase I protection showed that factors from TtT-97 but not from MGH101A cells interacted with regions in the TSH beta promoter, while nuclear extracts from each tumor demonstrated at least one protein-DNA interaction with the alpha-subunit promoter. These studies suggest that the molecular defects in the MGH101A tumor are related to the absence of trans-acting factors and are not a result of altered primary gene structure.     

The effect of altered thyroid status on pituitary hormone messenger ribonucleic acid concentrations in the rat

To study the effects of altered thyroid status on pretranslational control of pituitary hormones, adult male rats were given propylthiouracil for 6 weeks and underwent the following studies. 1) Rats were injected with T3 at 10 micrograms/100 g BW daily for 10 days. 2) Rats were given T3 injections at 0, 0.01, 0.1, 1.0, or 10 micrograms/100 g BW for 10 days. 3) Rats were killed 0, 1, 6, or 24 h after a single injection of T3 at 10 micrograms/100 g BW or after 5 or 10 days of daily T3 injections. Pituitary mRNA concentrations of TSH beta, alpha-subunit, PRL, GH, POMC, FSH beta, and LH beta were determined for individual animals. Marked increases in TSH beta and alpha-subunit mRNAs occurred after PTU treatment, and these changes were reversed by 1.0 microgram/100 g BW T3 and within 24 h of a single T3 injection of 10 micrograms/100 g BW. Further increases in the dose or time course of T3 administration led to a relatively greater suppression of TSH beta mRNA levels than alpha-subunit mRNA levels. In contrast, GH and PRL mRNA levels were low in hypothyroid animals, and both rose toward control levels with 0.1 microgram/100 g BW T3 and by 24 h after a single T3 dose. Induction of hyperthyroidism did not further increase GH mRNA levels above control, but increased PRL mRNA levels 2-fold over control. No changes were seen in FSH beta, LH beta, or POMC mRNA levels with any treatment. Thus, studies of altered thyroid status in the rat reveal dose-response and time-course variability in the pretranslational control of TSH beta, alpha-subunit, GH, and PRL by thyroid hormone.     

Acute withdrawal of short-term or prolonged L-triiodothyronine administration to thyroidectomized rats results in similar rapid increases in TSH beta 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.     

Differential sulfation and sialylation of secreted mouse thyrotropin (TSH) subunits: regulation by TSH-releasing hormone

To determine whether sulfate and/or sialic acid are present on secreted mouse TSH, thyrotropic tumor minces and hypothyroid pituitaries were incubated with [3H]methionine and [35S]sulfate, or [35S]methionine and [3H]N-acetylmannosamine. The metabolically labeled TSH and free alpha-subunits were then analyzed by gel electrophoresis. [3H]N-Acetylmannosamine was a specific precursor (greater than 80%) for the sialic acid [3H]N-acetylneuraminic acid, as established by HPLC characterization of tritium label released by acid hydrolysis. Each of the three secreted subunits (TSH alpha, TSH beta, and free alpha) incorporated both sulfate and sialic acid. The incorporation of these labels was confirmed by the release of [35S]sulfate by endoglycosidase F and of [3H]N-acetylneuraminic acid by neuraminidase. Differential labeling of newly synthesized secreted TSH subunits was observed. In secreted TSH dimer, TSH beta incorporated 1.3 times more [35S]sulfate (P less than 0.05) and 2.5 times more [3H] N-acetylmannosamine (P less than 0.02) per carbohydrate chain than did TSH alpha. Secreted free alpha-subunit incorporated more [3H]N-acetylmannosamine, but less [35S]sulfate, then did secreted TSH alpha. To investigate the effect of TRH on TSH sulfation and sialylation, thyrotropic tumor minces and hypothyroid pituitaries were incubated with [35S]sulfate or [3H]N-acetylmannosamine, with or without 10(-7) M TRH; labeling was then normalized in each case to incorporation of [3H]mannose, a marker of the inner core sugars. TSH secreted in the presence of TRH had a lower sulfate to mannose ratio [28 +/- (+/- SE) 4% of control; P less than 0.05] and a lower sialic acid to mannose ratio (63 +/- 8% of control; P less than 0.05). TSH alpha and TSH beta were affected equally. No change was seen in the labeling of non-TSH secretory proteins. Differential glycoprotein sulfation and sialylation may, in part, explain the previously observed variability in isoelectric point, bioactivity, and MCR of TSH in different physiological states and may represent a point of regulation by TRH.     

The paraventricular nucleus of the hypothalamus has a major role in thyroid hormone feedback regulation of thyrotropin synthesis and secretion

The role of the hypothalamic paraventricular nucleus (PVN) in thyroid hormone regulation of TSH synthesis during hypothyroidism was studied in adult male rats that were normal (n = 10), had primary hypothyroidism with sham lesions in the hypothalamus (n = 17), and had primary hypothyroidism with PVN lesions (n = 14). Two and 4 weeks after initiation of treatment, plasma levels of thyroid hormones (TSH, corticosterone and PRL) and pituitary content of TSH beta and alpha-subunit mRNA were measured. TRH mRNA levels in the PVN were determined by in situ hybridization histochemistry. At 2 weeks, despite a decrease in plasma free T4 in both hypothyroid groups, plasma TSH levels increased, but to a lesser degree, in the hypothyroid PVN lesioned compared to hypothyroid sham-lesioned group (7.8 +/- 1.3 vs. 20.5 +/- 1.1 ng/dl; P less than 0.05). Similarly, at 4 weeks, the hypothyroid PVN-lesioned group demonstrated a blunted TSH response compared to the hypothyroid sham-lesioned group (6.8 +/- 0.7 vs. 24.0 +/- 1.3 ng/dl; P less than 0.05). Plasma corticosterone and PRL did not significantly differ between sham-lesioned and PVN-lesioned groups. TSH beta mRNA levels markedly increased in hypothyroid sham-lesioned rats compared to those in euthyroid controls at 2 weeks (476 +/- 21% vs. 100 +/- 39%; P less than 0.05) and 4 weeks (1680 +/- 270% vs. 100 +/- 35%; P less than 0.05). In contrast, TSH beta mRNA levels did not increase with hypothyroidism in the PVN-lesioned group compared to those in euthyroid controls at 2 weeks (140 +/- 16%, P = NS) and only partially increased at 4 weeks (507 +/- 135; P less than 0.05). alpha mRNA levels at 4 weeks markedly increased in hypothyroid sham-lesioned rats compared to those in euthyroid controls (1121 +/- 226% vs. 100 +/- 48%; P less than 0.05), but did not increase in the hypothyroid PVN-lesioned rats (61 +/- 15%; P = NS). TRH mRNA in the PVN increased in the hypothyroid sham-lesioned rats compared to those in euthyroid controls (16.6 +/- 1.3 vs. 4.8 +/- 1.2 arbitrary densitometric units; P less than 0.05), and TRH mRNA was not detectable in the PVN of hypothyroid-lesioned rats at 2 weeks. In summary, lesions in rat PVN prevented the full increase in plasma TSH, pituitary TSH beta mRNA, and alpha mRNA levels in response to hypothyroidism. Thus, factors in the PVN are important in thyroid hormone feedback regulation of both TSH synthesis and secretion.     

Discordant changes in pituitary concentrations of LH and TSH subunits following hormonal manipulation

We have recently shown that mouse pituitary concentrations of free TSH-beta and alpha-subunits respond divergently to thyroid hormone manipulations in vivo. This study contrasts the changes in free LH-beta and alpha-subunits after castration and polyestradiol phosphate (E2) administration with those in free TSH-beta and alpha-subunits after thyroidectomy and thyroxine (T4) administration. Target hormone depletion (castration or thyroidectomy) resulted in an increase in plasma concentrations of the respective tropic hormones and their subunits; target hormone administration (E2 or T4) resulted in a decrease in plasma concentrations. In contrast, pituitary subunit concentrations of LH and TSH responded discordantly to target hormone depletion or administration, and the nature of the discordance for alpha-subunit and LH-beta was opposite to that for alpha-subunit and TSH-beta. Hypogonadism caused a decrease in pituitary LH and LH-beta concentrations to 65% (P less than 0.01) and 48% (P less than 0.001) of values in control (eugonadal) mice, respectively, while alpha-subunit was unchanged. Hypothyroidism caused a two-fold (P less than 0.001) and 20-fold (P less than 0.001) increase in pituitary TSH and TSH-beta concentrations, respectively, while alpha-subunit fell to 68% (P less than 0.01) of values in control (euthyroid) mice. Administration of E2 to hypogonadal mice resulted in a two-fold increase in pituitary LH and LH-beta after 11 days, while alpha-subunit fell to 55% (P less than 0.01) of values in control (untreated hypogonadal) mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in circulating thyroid hormones and thyrotropin in euthyroid patients with Graves' disease after total thyroidectomy: comparison between responders and nonresponders to TSH-releasing hormone

Eleven euthyroid patients with severe exophthalmos of Graves' disease who had been treated with antithyroidal drugs for one to three years prior to total thyroidectomy were studied. All patients were clinically and biochemically euthyroid at the time of operation. According to their responses of TSH to TRH prior to operation, the patients were divided into two groups: (1) five responders and (2) six nonresponders. In group 1, serum TSH levels increased significantly on the third day after thyroidectomy (from 1.5 +/- 0.3 to 8.6 +/- 1.4 microU/mL: P less than 0.05); serum T4 concentrations decreased significantly and were in the hypothyroid range by the third day. In group 2, serum TSH levels rose from 0.5 +/- 0.01 to 3.2 +/- 0.5 microU/ml (P less than 0.05) on the ninth postoperative day; serum T4 concentrations decreased on the third day after operation but did not attain hypothyroid levels until the 12th day. Thus after total thyroidectomy the following are concluded: (1) serum TSH levels even in treated euthyroid patients with Graves' disease, rose more gradually in TRH-nonresponders in comparison with TRH responders; (2) the time when serum TSH elevation occurs is dependent upon serum concentrations of thyroid hormones (serum T3 and T4).     

Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing [14C]alanine and [3H] glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, [14C]alanine incorporation in combined and free beta-subunits was 26 times normal (P less than 0.001) and considerably greater than the 3.4-fold increase seen in total protein (P less than 0.05); combined and free alpha-subunits showed no specific increase in apoprotein synthesis. [3H]Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein (P less than 0.05); free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits (P less than 0.001), but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function.     

Serum thyrotropin concentrations are more highly correlated with serum triiodothyronine concentrations than with serum thyroxine concentrations in thyroid hormone-infused thyroidectomized rats

To assess the relative role of circulating T4 and T3 in the regulation of serum TSH, we have measured serum T4, T3, and TSH concentrations in normal and thyroidectomized rats, some of which were chronically infused with T3 or T4. Serum T3, T4, and TSH concentrations were measured 7 and 14 days after surgery. Some groups of infused rats were mildly hypothyroid, as judged by elevated serum TSH concentrations. At both 7 and 14 days, there was a significant inverse correlation between serum T3 and serum TSH concentrations (day 7, r = 0.65, P less than 0.01; day 14, r = 0.71, P less than 0.01). The coefficients for the inverse correlations between serum T4 and TSH concentrations were 0.37 on day 7 (P less than 0.05) and 0.37 (P less than 0.05) on day 14. Linear regression analysis was performed using TSH as the dependent variable for outcome and serum T3 and T4 concentrations as the independent predictor variables. This analysis revealed that after controlling for T3, TSH and T4 were no longer significantly correlated (P = 0.14). The correlation between T3 and TSH remained highly significant. These results suggest that in the euthyroid and mildly hypothyroid rat, serum T3 has a greater inhibitory effect on TSH secretion than does serum T4.     

Differential response to L-triiodothyronine of anterior pituitary growth hormone messenger ribonucleic acid (mRNA) and beta-thyrotropin mRNA in a hypothyroid Walker 256 carcinoma-bearing rat model of nonthyroidal disease

To continue our studies on the influence of T3 on TSH regulation in the Walker 256 carcinoma-bearing rat model of nonthyroidal disease, we measured the effect of T3 on pituitary content of beta TSH mRNA and rat (r) TSH in hypothyroid control (C) and tumor-bearing (T) rats. The effect of T3 on TSH regulation was compared to effects on GH mRNA and rGH in the same animals. mRNA content was normalized to a pool of pituitaries from euthyroid rats (= 1.0). beta TSH mRNA increased 18-fold in both hypothyroid C and T rats and then decreased similarly with increasing T3 infusion to a value of 0.1. GH mRNA content decreased to 0.11 +/- 0.01 in hypothyroid C rats, but to only 0.38 +/- 0.02 in T rats (P less than 0.001). The pituitary contents of GH mRNA and rGH in hypothyroid T rats was significantly greater than those in C rats at all T3 infusion rates. These data together with our previous report of decreased nuclear T3 in T rats suggest that regulation of beta TSH mRNA by T3 is intact in T rats, but occurs at a lower concentration of nuclear T3. In contrast, the GH mRNA response is enhanced, displaying differential regulation of these two T3-responsive gene products in this model of nonthyroidal illness.     

Effects of testosterone on gonadotropin subunit messenger ribonucleic acids in the presence or absence of gonadotropin-releasing hormone.

There is accumulating evidence that the negative feedback actions of testosterone on the pituitary may contribute to the differential regulation of FSH and LH secretion in males. In the present study we measured steady state levels of the mRNAs encoding the gonadotropin subunits in pituitary cell cultures treated with 10 nM testosterone (T) as well as in T-treated pituitary cells perifused with pulses of GnRH to explore further the direct actions of T on the pituitary. T treatment of pituitary cells in monolayer culture for 72 h increased FSH beta mRNA 1.5-fold (P less than 0.05), decreased alpha-subunit mRNA to 45% of the control level (P less than 0.05), and decreased LH beta mRNA to 75% of the control level (P less than 0.05). FSH and uncombined alpha-subunit secretion were increased and decreased by T, respectively, whereas basal LH secretion was unchanged. Treatment with 0.1 nM estradiol, a physiological concentration for males, did not change gonadotropin secretion or subunit mRNA concentrations. Between days 2 and 5 in culture in the absence of steroid treatment, steady state levels of LH beta and alpha-subunit mRNA declined (P less than 0.01) 52% and 61%, respectively, but FSH beta mRNA levels were unchanged. Pulsatile stimulation with 2.5 nM GnRH every 1 h for 10 h increased FSH beta mRNA 2.8-fold (P less than 0.05) and increased (P less than 0.05) alpha-subunit mRNA to 117% of the control level. When cell cultures were pretreated with T for 48 h and then perifused with pulses of GnRH, FSH beta, LH beta, and alpha-subunit mRNA levels were 66%, 74%, and 70% of the value during GnRH alone (P less than 0.05). T treatment also reduced (P less than 0.01) the amplitudes of FSH, LH, and alpha-subunit secretory pulses by 18%, 26%, and 41%, respectively. These data indicate that a portion of the negative feedback action of T is at the pituitary to regulate gonadotropin subunit gene expression. Our data reveal two opposing effects of T on FSH beta mRNA: a stimulatory action, which is GnRH independent, and an inhibitory effect, which is related to the actions of GnRH. These divergent actions of T represent one mechanism through which FSH and LH are differentially regulated.