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The coordination of pituitary development is complicated and requires input from multiple cellular processes. Recent research has provided insight into key molecular determinants that govern cell fate specification in the pituitary. Moreover, increasing research aimed to identify, characterize, and functionally describe the presumptive pituitary stem cell population has allowed for a better understanding of the processes that govern endocrine cell differentiation in the developing pituitary. The culmination of this research has led to the ability of investigators to recapitulate some of embryonic pituitary development in vitro, the first steps to developing novel regenerative therapies for pituitary diseases. In this current review, we cover the major players in pituitary stem/progenitor cell function and maintenance, and the key molecular determinants of endocrine cell specification. In addition, we discuss the contribution of peripheral hormonal regulation of pituitary gland development, an understudied area of research.

TSH is the most important biomarker in the interpretation of thyroid function in man. Its levels are determined by circulating thyroid hormone (TH) levels that feed back centrally to regulate the expression of the subunits that comprise TSH from the pituitary. The nuclear corepressor 1 (NCoR1), is a critical coregulator of the TH receptor (TR) isoforms. It has been established to play a major role in the control of TSH secretion, because mice that express a mutant NCoR1 allele (NCoRΔID) that cannot interact with the TR have normal TSH levels despite low circulating TH levels. To determine how NCoR1 controls TSH secretion, we first developed a mouse model that allowed for induction of NCoRΔID expression postnatally to rule out a developmental effect of NCoR1. Expression of NCoRΔID postnatally led to a drop in TH levels without a compensatory rise in TSH production, indicating that NCoR1 acutely controls both TH production and feedback regulation of TSH. To demonstrate that this was a cell autonomous function of NCoR1, we expressed NCoRΔID in the pituitary using a Cre driven by the glycoprotein α-subunit promoter (P-ΔID mice). Importantly, P-ΔID mice have low TH levels with decreased TSH production. Additionally, the rise in TSH during hypothyroidism is blunted in P-ΔID mice. Thus, NCoR1 plays a critical role in TH-mediated regulation of TSH in the pituitary by regulating the repressive function of the TR. Furthermore, these studies suggest that endogenous NCoR1 levels in the pituitary could establish the set point of TSH secretion.

Objective: Type 2 deiodinase gene (DIO2) polymorphisms have been associated with changes in pituitary-thyroid axis homeostasis. The -258A/G (SNP rs12885300) polymorphism has been associated with increased enzymatic activity, but data are conflicting. To characterize the effects of -258A/G polymorphism on intrathyroidal thyroxine (T(4)) to triiodothyronine (T(3)) conversion and thyroid hormone (TH) secretion pattern, we studied the effects of acute, TRH-mediated, TSH stimulation of the thyroid gland., Design: Retrospective analysis., Methods: The TH secretion in response to 500  μg i.v. TRH injection was studied in 45 healthy volunteers., Results: Twenty-six subjects (16 females and ten males, 32.8 ± 10.4 years) were homozygous for the ancestral (-258A/A) allele and 19 (11 females and eight males, 31.1 ± 10.9 years) were carriers of the (-258G/x) variant. While no differences in the peak TSH and T(3) levels were observed, carriers of the -258G/x allele showed a blunted rise in free T(4) (FT(4); P<0.01). The -258G/x92Thr/Thr haplotype, compared with the other groups, had lower TSH values at 60  min (P<0.03). No differences were observed between genotypes in baseline TH levels., Conclusions: The -258G/x DIO2 polymorphism variant is associated with a decreased rate of acute TSH-stimulated FT(4) secretion with a normal T(3) release from the thyroid gland consistent with a shift in the reaction equilibrium toward the product. These data indicate that the -258G DIO2 polymorphism causes changes in the pattern of hormone secretion. These findings are a proof of concept that common polymorphisms in DIO2 can subtly affect the circulating levels of TH and might modulate the TH homeostasis.

Objectives: The examination of potential associations between Graves' disease and thyrotropin-producing pituitary adenoma (TSHoma) after treatment using octreotide, and of the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma)., Design and Methods: A specimen of resected TSHoma tissue from our case was immunohistochemically examined for expression of somatostatin receptor 2A (SSTR2A) and PPAR gamma. Specimens of thyroid tissue from two cases with Hashimoto's thyroiditis were immunohistochemically examined for expression of SSTR2A., Results: Expression of SSTR2A and PPAR gamma was identified in TSHoma cells. SSTR2A was also expressed in lymphocytes that had infiltrated thyroid tissue in Hashimoto's thyroiditis. In previous reports, three of four patients with TSHoma displayed Graves' disease after tumor resection, and TSH is also known to play a major role in regulating immunomodulatory gene expression in thyrocytes., Conclusions: Both the immunomodulatory effects of octreotide on intrathyroidal lymphocytes and rapid reductions in TSH may contribute to the onset of Graves' disease. Patients with TSHoma-associated autoimmune thyroiditis should undergo careful follow-up for development of Graves' disease after treatment. Both octreotide and the PPAR gamma receptor-activating ligands, thiazolidinediones, may be effective for patients with TSHoma.

Objective: Local 5'-deiodination of l-thyroxine (T(4)) to active thyroid hormone 3,3',5-tri-iodothyronine (T(3)) catalyzed by the two 5'-deiodinase enzymes (D1 and D2) regulates various T(3)-dependent functions in the anterior pituitary and has been well studied in rodents. Only limited information about deiodinase expression and its cellular distribution in human anterior pituitaries is available., Design: We examined 5'-deiodinase enzyme activities in pituitary adenomas (18 non-functioning, seven TSH-producing, one GH- and TSH-producing, five GH-producing, eight prolactin (PRL)-producing, two adenomas each from patients with Cushing's disease and Nelson's syndrome) and three normal anterior pituitaries., Methods: Activities were measured as release of (125)I(-) from tyrosyl-ring labeled reverse T(3) with or without propylthiouracil, a potent inhibitor of D1 which does not influence D2 activities., Results: Most of the adenomas and normal tissues expressed both isoenzymes, with D2 activity higher than D1. In a few tissues D1 activity was higher than D2 and some tissues did not express D1 activity at all. Highest activities of both enzymes were found in TSH- and PRL-producing adenomas but absolute activities and the D1/D2 ratio were variable in the same kind of tumor in different patients., Conclusion: The finding that all examined tissues expressed 5'-deiodinase activity, most of them expressing both isoenzymes, implies that both enzymes are still active in tumors and that local deiodination is important for the function and feedback regulation of human anterior pituitary.

We have previously characterized the structure of the murine somatostatin receptor type 5 gene (sst5). Initial transient transfection studies in pituitary somatolactotropes (GH(3)) mapped the promoter activity of this gene to a region 290 bp upstream of the transcription start site. The current study identifies the sst5 promoter region critical for basal activity. A series of deletions was generated, and promoter activity was localized to a region between -83 and -19. Similar promoter deletion patterns were evident in five pituitary cell types. Seven 10-bp transversion mutations encompassing the region between -83 and -19 were generated, and functional activity was assessed. Promoter activity was reduced by the mutations spanning -67 to -47 compared with the wild-type construct. Another mutation between -26 and -17 resulted in promoter activity reduction in GH(3) cells, but not TtT-97 thyrotropes. Deoxyribonuclease I protection analysis of the sst5 promoter region between -208/+47 was performed using GH(3) and TtT-97 nuclear extracts. The most striking protected regions, located between -61 and -41 and -25 and -3, correlated with functionally important regions identified by transfection studies. In summary, the mouse sst5 gene promoter has been characterized, and functional activity and nuclear factor interactions were mapped to two specific promoter regions. The region between -67 and -47 appears to contain a nucleotide sequence critical for basal transcriptional regulation of the mouse sst5 gene in pituitary cells.

Thyroid hormone exerts its biological effect by binding to a TR. Both liganded and unliganded TRs regulate the transcription of T(3)-responsive genes. Cofactors with activating or repressing function modulate the transcriptional regulation by TRs. We showed that steroid receptor coactivator 1 (SRC-1)-deficient mice (SRC-1(-/-)) exhibit partial resistance to thyroid hormone at the level of the pituitary thyrotrophs. To determine whether SRC-1 deficiency affects globally T(3)-dependent transcriptional regulation, we studied the effects of thyroid hormone deprivation and replacement on the expression of several genes in different tissues of SRC-1(-/-) and wild-type mice (SRC-1(+/+)). Thyroid hormone deficiency was induced by a low iodine diet (LoI) supplemented with propylthiouracil (PTU) for 2 wk. L-T(3) was injected ip for the last 4 d in one group (PTU+T(3) group), and another group (PTU group) received only vehicle. Levels of mRNAs for T(3)-responsive genes were determined by Northern blotting: GH and TSH beta in pituitary; type 1 iodothyronine 5'-deiodinase, spot 14 (S14), and malic enzyme in liver; and sarcoplasmic reticulum calcium adenosine triphosphatase 2 and myosin heavy chain alpha and beta in heart. Serum parameters, TSH, total cholesterol, creatine kinase, and alkaline phosphatase (AP), were also measured. Hypothyroidism produced a comparable increase in TSH beta mRNA in both genotypes, but its suppression by L-T(3) was attenuated in SRC-1(-/-) mice. In contrast, hypothyroidism failed to reduce S14 mRNA levels in SRC-1(-/-) mice. As a consequence, the response to L-T(3) was not observed in these mice. SRC-1 deficiency had no effect on the expression of the rest of the T(3)-responsive genes examined. Of the four serum parameters, the T(3)-mediated decrease in TSH and changes in AP were attenuated in SRC-1(-/-) mice. We conclude that SRC-1 deficiency altered the expression of only some of the T(3)-responsive genes. SRC-1 appears to be involved not only in transcriptional activation by liganded TRs, but also in the suppression by liganded or unliganded TRs. Some of the effects of SRC-1 may be TR isoform specific.

Background: Cigarette smoking has been linked to thyroid disease, although studies of this problem have not shown consistent affects, with some studies linking smoking to increased thyroid hormone levels, and others to decreased thyroid hormone levels., Methods: We performed a secondary analysis of information collected from 4462 Vietnam-era male US Army veterans aged 31-49 years who participated in the Vietnam Experience Study in 1985-1986. The study group consisted of 1962 current smokers and 2406 current non-smokers who had no thyroid abnormalities on physical examination, no current use of thyroid medicine, and no history of thyroid disease., Results: We found that current smokers have higher thyroxine levels and lower thyroid stimulating hormone levels than never smokers and former smokers. The higher thyroxine levels that we detected in smokers, compared to non-smokers, diminished when we controlled for thyroxine-binding globulin and testosterone. We also found that heavy smokers had a smaller increase in thyroxine levels than did light smokers, when compared to non-smokers., Conclusions: Our findings suggest at least two distinct mechanisms for the effect of tobacco smoke on thyroid function; one related to higher levels of thyroxine-binding globulin and testosterone among smokers compared to non-smokers and another related to higher levels of thyrotoxins in tobacco smoke in heavy smokers compared to light and moderate smokers.

To obtain large amounts of hTSH and to study the role of the N-linked oligosaccharides for its biological activity, hTSH was produced using recombinant baculovirus containing the human alpha-subunit and a hTSH beta-minigene, respectively, both under the control of the polyhedrin promoter. Expression in insect cells was 800-1000 ng/ml, 30-fold higher than in our optimized mammalian transient transfection system using Chinese hamster ovary (CHO) cells (20-50 ng/ml). The in vitro activity of insect-cell expressed hTSH (IC-hTSH) was increased 5-fold compared with CHO-hTSH, judged by the ability to induce cAMP production in CHO cells stably transfected with the hTSH receptor (JP09) and the rat thyroid cell line FRTL-5, as well as growth promotion in FRTL-5 cells. Lectin binding and enzymatic desialylation studies suggested that in contrast to CHO-hTSH, IC-hTSH lacked complex-type oligosaccharides terminating with sialic acid but contained predominantly high mannose-type oligosaccharides. The in vitro activity of CHO-hTSH also increased 5- to 6-fold upon treatment of the hTSH-producing cells with the oligosaccharide processing inhibitors swainsonine and castanospermine, which inhibit formation of complex, terminally sialylated oligosaccharides, and upon enzymatic desialylation. In contrast, insect cell-expression or treatment with processing inhibitors did not affect TSH receptor binding. Despite the higher in vitro activity, IC-hTSH had a much lower in vivo activity than CHO-hTSH, due to rapid clearance from the circulation. In summary, this study shows for the first time that relatively high levels of recombinant hTSH with high in vitro bioactivity can be produced in a baculovirus system. Cell-dependent glycosylation is a major factor that determines the final in vivo biopotency of recombinant glycoproteins, a finding that should be of general relevance for all insect cell-produced glycosylated proteins. Although not suitable for clinical use, highly bioactive recombinant hTSH derived from high expression in insect cells should be useful in defining structure-function relations of hormone analogs.

Dwarf tyrosine hydroxylase-human GH (TH-hGH) transgenic mice carrying the hGH reporter gene targeted by the TH promoter express hGH in those regions of the hypothalamus responsible for regulation of pituitary GH secretion. Central expression of the hGH gene decreases GH-releasing hormone (GHRH) and increases somatostatin, which ultimately impacts on pituitary function by reducing the overall amount of GH produced. In the present study, we sought to determine if the reduction of pituitary GH in TH-hGH mice could be attributed to a decrease in somatotrope cell numbers and/or an impairment of somatotrope function. Pituitaries from TH-hGH or wild-type (WT) male and female mice were enzymatically dispersed, counted, and immunostained for GH, PRL, TSH, and ACTH. The total number of pituitary cells recovered from TH-hGH pituitaries was approximately one-half of that from WT controls. However, the proportion of cells that stained for GH and PRL were virtually identical (males, GH-TH-hGH, 58.1 +/- 1.0% [mean +/- SEM] vs. WT, 60.7 +/- 1.0%; PRL-TH-hGH, 43.4 +/- 2.2% vs. WT, 43.1 +/- 0.7%; females, GH-TH-hGH, 47.9 +/- 2.3% vs. WT, 41.5 +/- 3.5%; PRL-TH-hGH, 43.3 +/- 3.2% vs. WT, 47.1 +/- 3.3%). In contrast, percentages of both TSH- and ACTH-containing cells were increased in TH-hGH pituitaries relative to controls (males, TSH-TH-hGH, 15.1 +/- 2.3% vs. WT, 9.6 +/- 1.5%; ACTH-TH-hGH, 24.5 +/- 2.5% vs. WT, 10.9 +/- 0.9%; females: TSH-TH-hGH, 11.3 +/- 0.7% vs. WT, 7.5 +/- 0.6%; ACTH-TH-hGH, 19.8 +/- 1.6% vs. WT, 9.3 +/- 0.8%; P < 0.05). Calculation of the absolute number of each cell type per pituitary demonstrated TH-hGH mice to have about one-half the number of GH and PRL cells, whereas TSH and ACTH cell populations were comparable with that of their WT counterparts. Immunocytochemical localization of GH cells within pituitary sections from TH-hGH mice revealed that somatotropes were confined primarily to the lateral wings of the adenohypophysis, in contrast to the heterogeneous distribution of GH-immunostained cells in WT pituitaries. To assess the functional capacity of the somatotrope populations, pituitary cells from TH-hGH and WT mice were challenged with mouse GHRH (0.01-10 nM). The quantity of GH released (as assessed by both RIA and reverse hemolytic plaque assay) under basal and stimulated conditions did not differ among TH-hGH and WT pituitary cell cultures. Similarly, GHRH induced intracellular cAMP levels were comparable. These results indicate that proliferation of pituitary somatotropes and lactotropes is much more sensitive to changes in GHRH input than is the capability of developing regulated GH secretory function.

Studies of human TSH (hTSH) structure and function have been limited by difficulties in producing large quantities of recombinant hormone. We describe a system for the stable expression of high levels of recombinant human TSH (rec hTSH) using a mutant form of dihydrofolate reductase (dhfr) as an amplifiable dominant selectable marker. A vector expressing both the hTSH alpha-subunit and the mutant dhfr was cotransfected with a hTSH beta-subunit expression vector into dhfr-deficient cells. Amplification of the transfected sequences by methotrexate selection, followed by cell culture in a hollow fiber perfusion system, yielded rec hTSH production as high as 100,000 microU/ mL. Immunoradiometric assays using five different antibodies revealed no differences in the immunological activities of rec hTSH and pituitary hTSH. Bioactivity was measured in a novel TSH bioassay coupling the generation of cAMP by a transfected hTSH receptor to the cAMP-dependent regulation of a luciferase reporter gene. The ED50 for bovine TSH in this bioassay was 1.4 ng/mL (3.5 x 10(-11) mol/L). The ratio of the ED50 values for rec hTSH and pituitary hTSH was 1.0:1.1 (P = NS), indicating that the two TSHs were of equivalent potency. In conclusion, we have developed techniques for the high level production of rec hTSH that is immunologically and biologically equivalent to pituitary hTSH. The ability to produce large quantities of rec hTSH using standard laboratory techniques should facilitate future studies, such as the development of clinically useful TSH analogs.

Although the pituitary is known to produce several growth factors, their effects on pituitary cell growth and differentiation are still unclear, particularly in normal tissue. Using primary cultures of aged ewe pituitaries cultured in serum-free conditions, we studied the effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-beta (TGF beta 1), insulin, and 17 beta-estradiol (E2) on the growth, differentiation, and expression of gonadotropin subunit genes. After 72-h incubation of the monolayer (day 5) with optimal concentrations of each factor, [3H]thymidine incorporation was increased significantly (P < 0.01) over the control values by 33 +/- 8% (mean +/- SEM; n = 3; 10 nM E2), 36 +/- 10% (1 ng/ml TGF beta 1), 83 +/- 12% (10 ng/ml bFGF), and 118 +/- 12% (1 nM EGF). Insulin showed a two-phase dose-response curve, increasing [3H]thymidine uptake by 34 +/- 9% at 10 ng/ml and by 63 +/- 13% at 10 micrograms/ml. Cell counting using a Coulter counter confirmed these results. Characterization of cell types by immunocytochemistry (avidin-biotin-peroxidase complex technique) revealed that the cell cultures were predominantly gonadotrophs. However, the cultures contained cells that did not stain with any specific ovine or human antiserum against LH beta, FSH, TSH beta, PRL, GH, ACTH, or glial fibrillary acidic protein, but were of epithelial cell lineage, as shown by positive keratin staining. Treatment with EGF and bFGF increased the proportion of these undifferentiated pituitary cells and induced changes in their morphology to large cuboidal cells containing large nuclei. After treatment with E2, insulin, and TGF beta 1, pituitary cells remained differentiated, although with E2, staining for gonadotrophs was much reduced. Northern blot analysis revealed that E2 treatment for 0-48 h progressively reduced the mRNA for FSH beta (16 +/- 4.5% of control values) and LH beta (12.4 +/- 2.5%), but had little effect on the common alpha-subunit (88.4 +/- 4.6%). TGF beta 1, however, stimulated the expression of FSH beta subunit gene by 142 +/- 4.6% (P < 0.01) of the control value, but had no significant effect on LH beta and common alpha-subunit genes. Insulin, EGF, and bFGF showed no significant effect on the expression of these three subunit genes. The data define the direct effects of growth factors and E2 on the growth and differentiation of normal sheep pituitary cells and gonadotrophs in particular, which may be of relevance to the pathophysiology of the pituitary and in the multistage process of pituitary tumorigenesis.

The TSH response to TRH administration (7 micrograms/kg) was measured in 68 infants (22 premature) who had abnormal thyroid screening tests by the filter paper method and whose serum thyroid function tests were only mildly abnormal. Twenty-eight infants (12 premature) had peak TSH values of 35 mU/L or less and were considered normal (group I). Forty infants (10 premature) had peak TSH values above 35 mU/L and were considered hyperresponsive (group II). The mean age at testing, screening T4, TSH levels that prompted the testing, as well as baseline T4, T3, and free T4 at the time of TRH testing were not different between the groups. The mean (+/- SD) baseline TSH value was greater in group II (6.8 +/- 2.3 mU/L) than in group I (4.4 +/- 2.2 mU/L; P < 0.001). However, there was a great deal of overlap in the individual TSH values (group I, 0.9-10 mU/L; group II, 1.9-10.6 mU/L). Mean peak TSH levels were significantly different in the two groups (group I, 24 +/- 7.7 mU/L; group II, 60.3 +/- 26.1 mU/L; P < 0.001). During long term follow-up, all 25 group I infants available for evaluation have been confirmed as clinically and biochemically normal. No infant diagnosed as normal was later found to have evidence of hypothyroidism. Fourteen infants in group II have had evidence of thyroid dysfunction. We conclude that the TSH response to TRH stimulation is a useful tool for the evaluation of infants suspected of having primary hypothyroidism. Whether hyperresponsiveness to TRH represents a form of neonatal hypothyroidism requiring treatment remains to be determined.

Graves' disease is an organ-specific autoimmune disease, and intrathyroidal lymphocytes seem to be the major source of thyroid autoantibodies. Consequently, the intensity of the intrathyroidal lymphocytic infiltration is generally believed to reflect the activity of the autoimmune process. We, therefore, investigated the correlation of microsomal (enzyme immunoassay), thyroglobulin (RIA), and TSH receptor antibodies (RRA) with the degree of intrathyroidal infiltration by immunoglobulin G-producing plasma cells, activated T-cells, antigen-presenting cells, and the total number of lymphocytes. The immunocompetent cells were identified immunohistologically with monoclonal antibodies for immunoglobulins kappa and lambda, UCHL1, and the S100 antibody, respectively, in 26 thyroid glands of patients suffering from Graves' disease. The intensity of lymphocytic infiltration was determined by the point-counting method and by counting all lymphocytes and the labeled lymphocytes in 3 x 51 visual fields or 3 slides/thyroid gland. Microsomal antibodies correlated significantly (P = 0.001) with the total number of lymphocytes (r = 0.86), kappa (r = 0.71), lambda (r = 0.71), UCHL1 (r = 0.9), and S100 (r = 0.9) positive cells. These correlations were also significant for thyroglobulin antibodies. However, TSH receptor antibodies showed no significant correlations with any of the populations of immunocompetent cells. Patients with preoperatively undetectable TSH receptor or microsomal antibodies showed a broad variation of intrathyroidal infiltration by the immunocompetent cells investigated. Microsomal antibody titers, therefore, seem to reflect the intensity of the intrathyroidal autoimmune process in Graves' disease better than TSH receptor antibodies. However, the broad baseline variation in intrathyroidal infiltration observed with nondetectable thyroid antibodies will not always allow determination of the intensity of the intrathyroidal autoimmune process from microsomal or thyroglobulin antibody titers.

Carbohydrate structures of intrapituitary and circulating TSH were studied by Concanavalin-A (Con A) and ricin lectin chromatography under different clinical conditions. Con A permits the separation of molecules differing in the extent of their carbohydrate branching, whereas ricin gives an estimation of the degree of their sialylation. Intrapituitary TSH was more retained on Con A and less sialylated than circulating hormone, suggesting that carbohydrate chains of intrapituitary molecules are less mature than those present in the circulation. A greater proportion of TSH firmly bound to Con A, compared to control values, was found in sera from fetuses and patients with uremia, TSH-secreting adenomas, and central hypothyroidism. In primary hypothyroid patients, TSH binding to Con A was similar to that found in controls, but a greater percentage of sialylated forms was seen. In central hypothyroidism patients, TSH released in response to TRH was less sialylated. Interestingly, no sialylated TSH was found in normal fetuses. In conclusion, the present data show that both TSH carbohydrate branching and sialylation may vary in different clinical conditions. As some of the above clinical conditions are known to be accompanied by variations in the bioactivity of circulating TSH, the finding of changes in TSH carbohydrate structures further supports the view that glycosylation modulates the expression of TSH biological activity.

A 21-yr-old female with hyperthyroidism is described. Though her serum-free T3 was 17.8 pmol/L and free T4 was 60.2 pmol/L, TSH was as high as 10.7 mU/L. TRH stimulated an increase in TSH from 10.7-91.7 mU/L. T3 administration in gradually increasing doses of 100, 200, and 400 mg/day resulted in gradual reduction in serum TSH. Cranial computed tomography and magnetic resonance imaging revealed a microadenoma of the pituitary gland. Histology of the surgical specimen showed a TSH-producing adenoma with TSH cell cluster islets and decreased numbers of TSH cells in the nonneoplastic pituitary. Cultured cells from the adenoma secreted TSH spontaneously and in response to TRH. This TRH-stimulated TSH secretion was suppressed by T3 in a dose-dependent manner. One year postoperatively, neither residual tumor nor recurrence were seen by computed tomography and magnetic resonance imaging. However TSH, as well as free T3 or T4, was still high and overresponsive to TRH.

The alpha-subunit gene of the glycoprotein hormones is normally expressed in pituitary thyrotropes and gonadotropes and in placental cells. Thus, this gene must contain elements that mediate expression and hormonal responses in different cell types. The localization of DNA regions important for expression and regulation of the alpha-subunit gene in thyrotrope cells has not previously been reported. In these studies luciferase expression constructs containing 1700 basepairs of 5' flanking DNA derived from the mouse alpha-subunit gene were introduced by electroporation into freshly dispersed cells from TSH-producing mouse pituitary tumors (TtT 97). This promoter functioned with greater efficiency in thyrotropes than in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Primer extension confirmed that transcription from the alpha-subunit constructs initiated at the same site as the endogenous gene. Studies using 5' truncations showed a progressive loss of alpha-subunit promoter activity in thyrotropes between -480 and -120, with regions upstream of -254 contributing substantially to expression in thyrotrope cells. Thyroid hormone inhibited alpha-subunit promoter activity in a dose-dependent fashion, although in vivo treatment of tumors with thyroid hormone before transfection was necessary to achieve maximal inhibition. Thyroid hormone inhibition of alpha-subunit promoter activity also occurred in GH4 cells, but no effect was observed in L-cells. Studies using 5' truncations localized a region responsible for thyroid hormone inhibition between -62 and +43, encompassing the TATA sequence and the transcriptional initiation site. When this region was compared to the thyroid hormone inhibitory regions of the alpha-subunit genes from other species and the mouse TSH beta-subunit gene, a 6-basepair motif, 5' (G/A)GTG(G/A)G 3', emerged as a possible consensus sequence for a thyroid hormone inhibitory element.

The circadian pattern of serum TSH in normal children, aged 5-18 yr, is characterized by a nocturnal surge and is presumably related in some way to a biological clock within the central nervous system. To look for patients deficient in the nocturnal TSH surge, we studied 52 children with hypothalamic-pituitary disorders. Thirteen of the children were hypothyroid, as judged by subnormal serum free T4 (FT4). The hypothyroid patients had a mean nocturnal TSH surge of 22% (range, -30% to +114%), significantly less than that of normal controls (mean, 124%; 95% confidence limits, 47-300%; n = 96; P less than 0.01). Only 1 of the hypothyroid children had a value for the nocturnal TSH surge (114%) that was within the normal range. Nineteen of the 52 patients with hypothalamic-pituitary disorders had subnormal nocturnal TSH surges; their mean iodothyronine values were significantly less than those of the 33 patients with normal surges [total T4, 73 +/- 4 (mean +/- SE) vs. 109 +/- 3 nmol/L (P less than 0.01); FT4, 13 +/- 1.0 vs. 19 +/- 0.5 pmol/L (P less than 0.01)]. These data demonstrate a clear association of a deficient nocturnal TSH surge and low iodothyronine concentration in children with hypothalamic-pituitary disorders. We performed both TRH tests and nocturnal TSH surge tests in 11 of the children with central hypothyroidism; TRH was abnormal in only 2, while the nocturnal surge test was abnormal in 10 of the 11. We suggest that the nocturnal surge of TSH is important for maintenance of thyroid function and conclude that the nocturnal TSH surge is a much more sensitive test than the TSH response to TRH for the diagnosis of central hypothyroidism.

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.

Primary cell cultures of ovine pituitaries can maintain production of thyroid stimulating hormone (TSH) for as long as 24 days. These cultures responded in a normal fashion to thyroxine by decreasing TSH secretion. Addition of thyrotropin releasing hormone increased TSH secretion. Physiologic levels of estradiol-17 beta (10(-11)-10(-9)M) produced a five-fold increase in secretion of TSH and a two-fold increase in intracellular TSH concentration in cell cultures. Common estrogens, but not common progestins, androgens and glucocorticoids affected TSH production. Markedly different effects of estrogen in the pituitary on follicle stimulating, luteinizing and thyroid stimulating hormones and prolactin are discussed in terms of current models of estrogen action.

We have studied the relationship between the levels of the alpha and beta subunit of TSH and alpha and TSH-beta mRNA content in pituitaries from euthyroid, short-term, and long-term hypothyroid mice and in various mouse TSH-secreting tumors. TSH, alpha, and TSH-beta were determined by specific radioimmunoassay. alpha and TSH-beta mRNA content was measured by dot blot hybridization with labeled alpha or TSH-beta plasmid probes. Normal pituitaries contained an excess of both alpha protein and alpha mRNA compared to beta. However, in hypothyroidism there was an increase in TSH and in particular in beta such that the alpha/beta protein ratio approached 1. Similarly, a marked increase in TSH-beta mRNA led to an alpha/TSH-beta mRNA ratio of approximately 1. Ratios of alpha/TSH-beta protein and mRNA in TSH-secreting tumors varied widely. There was a fair, but imperfect, correlation between alpha and TSH-beta protein and mRNA levels. Although transcriptional control of alpha and TSH-beta production appears likely, posttranscriptional regulatory mechanisms also seem to be involved.

Primary cultures from three serially transplanted mouse pituitary thyrotropic tumors, Furth 97A, 97B, and 97C, and one newly generated tumor, NIH 101A, were shown to secrete free alpha subunits and a beta subunit (TSH-beta) in addition to intact TSH. The three glycopeptides were measured in specific, heterologous radioimmunoassays. Basal secretion of alpha subunit was found to be in excess in all four thyrotropic tumor cultures, ranging from 5.7- to 8.9-fold higher than that of intact TSH and 30- to 35-fold higher than that of free TSH-beta. In cultures from 97A, TRH (10(-7) M) stimulated the secretion of TSH, alpha subunit, and TSH-beta by 175-185% and (T4, 10(-5) M) inhibited TSH to 19%, alpha subunit to 68%, and TSH-beta to 58% of control. Secretion of TSH and its subunits in cultures from 97B was not consistently affected by TRH or T4. In cultures from 97C, TSH and alpha secretion was inhibited in a dose-dependent fashion by T3 from 2.5 x10(-9) to 1.6x10(-7) M. Gel chromatography of incubation media from tumor 97A revealed elution volumes of TSH and alpha similar to those of purified rat glycopeptides and confirmed the excess secretion of free alpha subunit. In contrast, the alpha to TSH ratios in media from cultures of pituitary cells from normal and thyroidectomized rats were 1.6 and 0.8, respectively. The total production of TSH and its subunits was measured in cultures from tumor 97A and confirmed that alpha subunit was synthesized excessively. The ratio of the plasma concentrations of alpha to TSH in tumor-bearing mice was 1.6 compared to 1.3 in thyroidectomized mice, and 0.36 in thyroidectomized rats. The alpha to TSH ratios in extracts of the corresponding thyrotropic tissues (tumor or pituitary) were 2.7, 1.7, and 0.40, respectively. These data are analogous to those reported in humans with thyrotropic and certain other pituitary tumors, and support hypothesis that the subunits of glycoprotein hormones are independently synthesized.

We examined the effect of brefeldin A (BFA), a drug that inhibits the intracellular translocation of newly synthesized glycoproteins, on the biosynthesis of TSH and free alpha-subunits by pituitary tissue from hypothyroid mice. Incubation of tissue with 5 or 10 micrograms BFA/ml for 3.5 h caused marked dilatation of rough endoplasmic reticulum (RER) and mild swelling of Golgi in all pituitary cell types. As judged by incorporation of [35S]Met into acid-insoluble radioactivity, BFA at a concentration of 5 micrograms/ml did not substantially inhibit protein synthesis, but markedly reduced protein secretion. After a 2-h pulse with [35S]Met, followed by a 4-h chase, BFA at 5 micrograms/ml reduced the release of TSH and free alpha-subunits into the medium by 94% and 99%, respectively; subunits that accumulated within cells were forms with mol wt 2000-4000 less than normal. BFA also partially inhibited the release into the medium of TSH or free alpha-subunits labeled with [3H]fucose or [35S]SO4, but this effect was less marked than that for [35S]Met-labeled subunits. Both the morphological and the isotopic data suggest that BFA blocks transport of secretory proteins between rough endoplasmic reticulum and Golgi of pituitary cells, although transport within the Golgi may also be affected to some extent.

Mouse thyrotropic tumor cells grown in primary culture were shown to synthesize TSH and proteins, as determined by the incorporation of radioactive proline into immunoprecipitable TSH and trichloroacetic acid-precipitable proteins. The net TSH content of the cells and medium determined by RIA is also increased during 24 h of incubation, and newly formed hormone is detected in the medium within 1 h after the addition of proline tracer. To study the effect of T4 and T3 on TSH synthesis, cultures were pulse-labeled with [3H]proline after they had been exposed to either T3 or T4. After 48 but not 24 h, exposure to either T3 or T4 was followed by inhibition. When studied after 48 h of incubation, T4, (10(-13) M) or T3 (10(-11) M) at the lowest concentration tested, was inhibitory to TSH synthesis. At concentrations of T4 and T3 greater than 10(-9) M, the inhibitory effects on TSH synthesis were partially reversed, suggesting a biphasic response. Incubation in TRH (10(-7) M) for 24 h led to a significant increase in TSH synthesis, total protein, acid-precipitable protein, and total DNA. The effect of TRH on TSH biosynthesis was a function of the logarithm of its concentration over the range of 10(-11)-10(-7) M. The inhibitory action of 10(-6) M T3 on TSH synthesis was reversed by exposure to 10(-10) or 10(-7) M TRH.

We have previously demonstrated differences in several cellular responses to TRH in mouse thyrotropic pituitary (TtT) cells and in rat mammotropic pituitary (GH3) cells. In this report, we further explore the mechanism of TRH action in TtT cells by measuring its effects on phosphoinositides and on cytoplasmic free Ca2+ concentration [( Ca2+]i). We demonstrate that TRH stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by a phospholipase C and elevates [Ca2+]i. Furthermore, we present evidence that hydrolysis of PtdIns(4,5)P2 is not secondary to the elevation of [Ca2+]i. TRH caused a rapid decrease in the level of PtdIns(4,5)P2 to 57% of control and stimulated an increase in inositoltriphosphate, the unique product of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2, to a peak of 280% of control. In control cells, resting [Ca2+]i was 106 +/- (SE) 27 nM, and TRH stimulated a rapid elevation to 700 +/- 210 nM. In experiments performed to determine whether PtdIns(4,5)P2 hydrolysis induced by TRH may have been caused by the elevation of [Ca2+]i, the following results were obtained: the effect of TRH to decrease the level of PtdIns(4,5)P2 was not reproduced by the calcium ionophore A23187 or by membrane depolarization with 50 mM K+; the calcium antagonist TMB-8 did not inhibit the TRH-induced decrease in PtdIns(4,5)P2; and, most importantly, inhibition by EGTA of the elevation of [Ca2+]i did not inhibit the TRH-induced decrease in PtdIns(4,5)P2. We suggest that phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 to yield inositoltriphosphate may be the initial event in TRH action in TtT cells, as in GH3 cells, that leads to elevation of [Ca2+]i and to TSH secretion.

Pituitary extracts from five patients with atrophic asymptomatic thyroiditis and from five subjects without any endocrine disease, matched for sex and age, were passed through gel filtration columns and the collected fractions were analyzed in three different radioimmunoassays (hTSH-anti-hTSH; hTSHbeta-anti-pTSH; hLHAlpha-anti-LHalpha). In the alpha subunit system, every profile showed a first peak corresponding to the elution position of the intact hTSH molecule and second peak, often more important, of free alpha chain. In the beta subunit system, a high molecular weight immunoreactive protein (big hTSHBeta) was found in seven pituitary extracts; cross-reacting intact hTSH molecule was eluted as a second peak and free Beta chain was detected in all the extracts. Free alpha chain was generally found in greater amount than free Beta. In the hTSH system, every elution pattern showed a peak of intact hTSH and another in the subunit area. In six pituitary extracts (5 thyroiditis, 1 control), a high mol wt protein was eluted (big hTSH). Its predominant presence in thyroiditis extracts seems important in view of the already described immunological heterogeneity of pituitary hTSH.

The effects of hypothalamic hypothyroidism vs. primary hypothyroidism on TSH carbohydrate structure were studied in the rat. Adult male rats with bilateral paraventricular nuclear lesions (n = 10), sham lesions (n = 10), and thyroidectomies (n = 6) were studied 2 weeks postoperatively and compared to normal animals without surgery (n = 6). Pituitaries were incubated in medium containing [3H]glucosamine for 24 h. TSH was immunoprecipitated from medium and pituitary sonicates using anti-TSH beta serum, digested with pronase to obtain TSH glycopeptides, desalted, then analyzed by Concanavalin-A (Con-A) chromatography. Compared to sham controls, hypothalamus-lesioned animals contained a greater proportion of secreted TSH glycopeptides that bound weakly to Con-A, indicating a shift from bisecting and/or multiantennary structures in control animals to biantennary and/or truncated hybrid forms in hypothalamus-lesioned animals. In contrast, thyroidectomized animals, compared to normal and lesioned animals, contained a greater proportion of secreted TSH glycopeptides that did not bind to Con-A, indicating a shift from biantennary and/or truncated hybrid forms to bisecting and/or multiantennary forms. The characteristics of the carbohydrate chains on secreted TSH differed markedly in hypothalamic vs. primary hypothyroidism despite equally low thyroid hormone levels in vivo. Thus, in addition to regulating TSH secretion, hypothalamic hormones alter TSH carbohydrate structure, which may affect its bioactivity and MCR.

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.

We have studied the effects of Brefeldin-A (BFA) on the processing of high mannose (Man) oligosaccharides of TSH. BFA is a drug that inhibits the intracellular translocation of newly synthesized glycoproteins and causes dilatation of the rough endoplasmic reticulum (RER) as well as mild swelling of the Golgi apparatus. Mouse pituitary thyrotropic tumor tissue was incubated with [3H]Man for a 2-h pulse, with and without a 3-h chase; BFA (5 micrograms/ml) was included during selected pulse and selected chase incubations. TSH and free alpha-subunits were obtained from detergent lysates of tissue by immunoprecipitation using specific antisera. Total glycoproteins were obtained by trichloroacetic acid precipitation. Endoglycosidase-H-released [3H]oligosaccharides were analyzed by paper chromatography. BFA inhibited carbohydrate processing of TSH, free alpha-subunits, and total glycoproteins, resulting in the accumulation of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2, especially during the chase period. Subcellular fractions enriched in RER, heavy (proximal) Golgi, and light (distal) Golgi were prepared by centrifugation in discontinuous sucrose gradients. [3H]Man-labeled oligosaccharides of TSH and total glycoproteins in the subcellular fractions were analyzed. In contrast to oligosaccharides with eight or nine Man residues found in control incubations, BFA caused the accumulation of oligosaccharides containing five to eight Man residues. These BFA-induced oligosaccharide alterations began in the RER and proximal Golgi with the 2-h pulse and extended into the distal Golgi during the chase incubations. Thus, BFA blocks the normal intracellular transport and processing of TSH, free alpha-subunits, and total glycoproteins within thyrotrophs, causing species with smaller than normal high Man oligosaccharides to appear in subcellular compartments as early as the RER. The translocation block between RER and Golgi produced by BFA may prevent the processing of Man8GlcNAc2 to Man5GlcNAc2 by Golgi (alpha,1-2)mannosidase I, yet the species retained within the RER may be subject to ongoing processing by endoplasmic reticulum (alpha,1-2)mannosidase, resulting in the accumulation of Man5-8GlcNAc2 within the RER.

To investigate whether TRH regulates TSH production through a pre- or posttranslational mechanism, we determined the pituitary levels of mRNAs for alpha-subunit and TSH beta in male Sprague-Dawley rats given TRH in the presence or absence of thyroid hormones, with or without hypothalamic influence. In normal rats, serum TSH increased 6-fold after a single sc injection of TRH (7 micrograms/kg BW), but the levels of mRNA for both TSH subunits did not differ from the control values. Infusion of TRH, achieved by osmotic minipumps that were implanted sc, increased serum TSH for 3 days. Conversely, the pituitary content of TSH dropped to and remained 35% of that in the controls. In these normal rats, throughout the TRH infusion, the pituitary levels of mRNA for the TSH subunits did not differ from those in the controls. Thyroidectomy increased, by 27 and 75 times, the normal levels of mRNAs for alpha and TSH beta, respectively. TRH, given either as a single injection or a 3-day infusion, did not further elevate these levels. We then studied thyroidectomized animals whose pituitaries were transplanted under their renal capsules. These pituitaries responded to TRH infusion by releasing TSH. T4 injection inhibited this response significantly, but not completely. In spite of this evidence of normal responsiveness to TRH, infusion of TRH for a week did not increase the level of mRNAs for either TSH subunit in transplanted pituitaries. We conclude that in the presence or absence of thyroid hormones, with or without concurrent hypothalamic influence, TRH did not affect rat pituitary level of mRNA for either TSH subunit despite persistent high levels of serum TSH. Therefore, TRH does not regulate TSH production through a pretranslational mechanism. Although a translational regulation cannot be completely excluded, the present data, in conjunction with previous findings, support the hypothesis that TRH regulates TSH production primarily by stimulating both posttranslational carbohydrate processing and secretion of this hormone.

Studies of TSH release and production were performed in short term monolayer cultures of transplantable, thyroid hormone responsive, thyrotropin (TSH) producing mouse pituitary tumors. These tumors contained large amounts of TSH, small amounts of growth hormone (GH) and no detectable luteinizing hormone (LH), indicating that the predominant hormone product of tumor cells was TSH. The TSH content per tumor cell was similar to that of the normal pituitary where thyrotrophs represent a small fraction of the total cells, suggesting that the TSH content per tumor cell was less than that of the normal thyrotroph. There was a time dependent release and production of TSH by tumor cells in monolayer culture. Thyrotropin releasing hormone (TRH) increased the release into the media and the production of TSH in a dose dependent manner. Maximum effects were noted at 0.2 ng/ml. Thyroid hormones and somatostatin inhibited both basal and TRH induced effects on both TSH release and production. TSH release as induced by TRH was calcium dependent. TSH release was stimulated by ouabain (10(-3)M) and potassium (57 mM), agents known to promote cellular calcium uptake in a calcium dependent manner. These studies indicate that tumor derived cells function in monolayer culture in a similar fashion to normal thyrotrophs. Studies were conducted to test the hypothesis that TRH action is mediated by adenosine 3',5' monophosphate (cAMP). Dibutyryl cAMP (6 mM) and theophylline (10 mM) increased TSH release suggesting that cAMP is involved in TSH release. However, TRH had no detectable effect on tumor cell adenylate cyclase activity or levels of cAMP. In contrast, PGE1 (1-10 mug/ml) stimulated adenylate cyclase activity and elevated cellular levels of cAMP without increasing TSH release. Thus, we are unable to confirm the postulate that cAMP is the intracellular mediator of TRH action.

The effects of hypothalamic deafferentation on TSH synthesis were studied by making cuts of 180 degrees arc in the anterior hypothalamus (n = 18) or sham cuts (n = 12) in rats. After 21 days, pituitaries were incubated with [35S]methionine (MET), [3H]glucosamine (GLCN), with or without 10(-8)M TRH for 24 h. TSH and free alpha-subunits were immunoprecipitated and analyzed by gel electrophoresis. In the deafferented group as compared to sham, MET incorporation into both subunits of secreted TSH was decreased (alpha, 96 +/- (SE) 9 X 10(3) vs. 180 +/- 20 X 10(3) dpm/mg protein; beta, 35 +/- 9 X 10(3) vs. 84 +/- 15 X 10(3) dpm/mg protein; P less than 0.05). Basal GLCN incorporation into both subunits of secreted TSH was also decreased in the deafferented group (alpha, 6.5 +/- 11 X 10(3) vs. 132 +/- 17 X 10(3) dpm/mg protein; beta, 36 +/- 8 X 10(3) vs. 101 +/- 29 X 10(3), P less than 0.05). In vitro TRH did not stimulate MET incorporation into secreted TSH in the sham controls but did in the deafferented group (alpha, 270% of basal; beta, 374% of basal; P less than 0.01). In vitro TRH increased GLCN incorporation in secreted TSH in both the sham (alpha, 253% of basal; beta, 245% of basal; P less than 0.02) and the deafferented group (alpha, 692% of basal; beta, 630% of basal; P less than 0.01). GLCN/MET ratio, reflecting relative glycosylation, did not differ for sham or deafferented groups but increased 2-fold with in vitro TRH in each group for both secreted subunits (P less than 0.01). Free alpha-synthesis and intrapituitary TSH were not altered by deafferentation or TRH. In summary, 1) anterior hypothalamic deafferentation decreases basal TSH protein and carbohydrate synthesis; 2) such deafferentation increases sensitivity to TRH stimulation of TSH synthesis, most notably apoprotein synthesis; 3) TRH increases relative glycosylation of secreted TSH in both deafferented and sham groups. These data suggest that TRH plays a significant role in regulating basal TSH protein and carbohydrate synthesis, glycosylation of TSH subunits, and subsequent bioactivity.

Ten patients (8 women, 2 men) with the "empty sella syndrome" were studied to evaluate the pituitary reserve of human thyrotropin (hTSH) and prolactin (hPRL). None of the patients had signs or symptoms of hypopituitarism or primary hypothyroidism. All patients had normal baseline thyroid function tests except for 2 patients with mild elevations in total triiodothyronine as measured by competitive protein displacement assay (T3D). Eight of ten patients had normal hTSH responses to thyrotropin releasing hormone (TRH), whereas the 2 patients with elevated T3D had blunted TRH responses. In the 4 patients studied, the metabolic clearance (MCR) and production rates (PR) of hTSH were normal. In 9 of 10 patients normal baseline serum hPRL levels were detected, and each responded to TRH. In one case serum hPRL was undetectable and failed to respond to TRH. The assessment of other anterior pituitary function revealed few minor abnormalities. In summary, like other anterior pituitary hormones, the pituitary reserve in "empty sella syndrome" of hTSH and hPRL is usually normal. When abnormalities do occur, they are attributable to other co-existent endocrine pathology.

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.