TSH Molecules Research Articles

Enhanced Photon Emission of Chemiluminescent Luminophore for Ultra-Fast and Semi-Automatic Immunoassay toward Single Molecule Detection.

Optical single molecule detection is normally achieved via amplifying the total emission of photons of luminophores and is strongly anticipated to extend the commercialized application of chemiluminescence (CL). To overcome the limited CL photons of molecule luminophores, herein, a nanocrystal (NC) luminophore self-amplified strategy is proposed to repetitively excite CL luminophores for amplifying the total CL photons per luminophore, which can be exploited to perform CL immunoassays (CLIAs) toward single molecule detection via employing KMnO4 as the CL triggering agent and the dual-stabilizer-capped CdTe NCs as the CL luminophore. KMnO4 can oxidize the S element from each stabilizer of mercaptopropionic acid (MPA) and release enough energy to excite the CdTe core for flash CL. The substantial MPA around each CdTe core enables every CdTe luminophore to be repetitively excited and give off amplified total CL photons in a self-enhanced way. The CL of CdTe NCs/KMnO4 can release all photons rapidly, and the collection of all these photons can be utilized to determine the model analyte of thyroid-stimulating hormone antigen (TSH) with a limit of detection of 5 ag/mL (S/N = 3), which is corresponding to about 2-4 TSH molecules in a 20 μL sample. The whole immunologic operating process can be terminated within 6 min. This strategy of repetitively breaking the CL reaction involving chemical bonds within one luminophore is promising for semi-automatic as well as fully automatic single molecule detection and extends the commercialized application of CL immunodiagnosis.

A Million Reasons for Hyperthyroidism. Reporting a Case of Thyrotoxicosis in a Setting of Metastatic Choriocarcinoma

Background: Human chorionic gonadotropin (hCG) induced transient hyperthyroidism is often seen during pregnancy. Other rare causes of beta hCG induced hyperthyroidism include trophoblastic tumors (hydatidiform mole and choriocarcinoma) and in men, germ cell tumors. The mechanism for hCG induced thyrotoxicosis lies in the structural similarity between TSH and hCG molecules leading to the direct stimulation of the TSH receptors. In regard to treatment, while gestational thyrotoxicosis is usually mild, self-limited and does not need medications, the hyperthyroidism of trophoblastic tumors may require antithyroid medications in addition to treating the underlying tumor. Thionamide use is reserved for moderate to severe cases of hyperthyroidism and for presurgical optimization. In our case report, we present a 22-year-old African American female with choriocarcinoma induced thyrotoxicosis requiring thionamide therapy. Clinical Case: A 22-year-old African American female presented to our emergency room after a witnessed generalized tonic-clonic seizure. Her brain CT scan showed a 4 CM mass concerning for AVM malformation with a subsequent brain MRI confirming parenchymal hematoma with surrounding vasogenic edema. Past medical history was significant for molar pregnancy managed with dilation and curettage followed by laparotomy to remove the pelvic mass. Given her history of molar pregnancy, pelvic ultrasound was performed which showed complex heterologous structure of uterine origin concerning for malignancy. Additional imaging studies of the lung, brain, abdomen and pelvis were performed, which showed possible metastasis to the lung, adnexa, and to the brain. Her clinical exam showed heart rate of 130 beats/min with a normal rhythm, fine tremors, aphasia, and a decrease grip strength in her right upper extremity. Her thyroid gland was slightly enlarged with no tenderness. Laboratory tests showed hCG of >1,000,000 mIU/mL, TSH of < 0.01mcU/mL, free T4 of 5.1ng/dL. Her TPO and TSI were negative. With a diagnosis of hyperthyroidism due to trophoblastic disease, she was treated with methimazole and propranolol resulting in rapid clinical and biochemical improvement. Later, left frontal craniotomy and hematoma evacuation was performed with histology confirming the diagnosis of metastatic choriocarcinoma. She was later transferred to a specialized center where she received chemotherapy. Her hCG subsequently dropped down to 699 mIU/mL. Conclusion: We report a rare case of metastatic choriocarcinoma causing symptomatic hyperthyroidism. The diagnosis is made by excluding other common causes of hyperthyroidism and it should be considered in females of childbearing age. Methimazole is an effective agent to control the hyperthyroidism while waiting for treatment of the underlying cancer.

Unique Pattern of N-Glycosylation, Sialylation, and Sulfonation on TSH Molecules in Serum of Children Up to 18 Months.

N-glycosylation and glycan composition of human TSH molecules modulate the biological properties of TSH in different physiological and clinical situations. The degree of sialylation of serum TSH was reported to be very low in normal third-trimester fetuses compared with normal adults. The circulating TSH glycoforms and their glycan compositions in young children have hitherto not been determined. To characterize N-glycosylation and glycan composition of circulating TSH molecules in young children. Serum samples were obtained from euthyroid individuals: 33 children, age 2 weeks to 3 years, and 264 adults. The di-glycosylated TSH and tri-glycosylated TSH glycoforms were determined and characterized with respect to sialylation and sulfonation. The TSH N-glycosylation was also examined in pituitary extracts of 75 individuals. In children up to 18 months of age, most TSH molecules were low-N-glycosylated, high-sulfonated, and low-sialylated compared with older children and adults. The degree of N-glycosylation was similar in serum and pituitary extracts up to 3 months of age and after that was higher in serum than in pituitary extracts. Children up to age 18 months had low-sialylated TSH molecules, similar to those reported for third-trimester fetuses. Most TSH molecules in young children were of smaller size and less negatively charged, favoring transport into their target tissues. The low sialylation favors a high biopotency at thyroid and extrathyroidal TSH receptors. A delayed development of the liver SO3-N-acetylgalactosamine receptor function after birth is a likely explanation of the highly sulfonated TSH molecules in serum samples of infants.

Effects of subacute hypothyroidism on metabolism and growth-related molecules.

Thyroid hormones are crucial hormones that primarily regulate the metabolism of entire body cells. In this study, Sprague-Dawley rats were grouped into sham thyroidectomy (Sham Tx), thyroidectomy (Tx), Tx with thyroxine replacement (Tx + T4), and PTU injection (PTU) groups. Metabolic parameters were measured by means of metabolic cages for 14 days. After 14 days, the rats were sacrificed while the levels of plasma or serum TSH and growth-related molecules, such as active and total ghrelin, GH, and IGF-1, were assayed. The results revealed that hypothyroid rats tended to eat less food and experienced substantial body weight gain, whereas the rats with T4 replacement tended to eat more food while continuing to lose weight. In hypothyroid rats, the growth-related molecules, such as active ghrelin and total ghrelin secretion, were enhanced, and the ghrelin receptors were also up-regulated. However, circulating GH levels were not elevated and IGF-1 secretion was inhibited in hypothyroid rats. In the Tx + T4 group, the changes of active ghrelin, total ghrelin, GHS-R expression, and IGF-1 were reversed, whereas the GH secretion was higher than that of the Sham Tx group and hypothyroid groups. This study resulted in the novel finding that the ghrelin/GHS-R axis and GH/IGF-1 axis are interrupted in hypothyroid rats.

Microarray Analysis of Thyroid Stimulating Hormone, Insulin-Like Growth Factor-1, and Insulin-Induced Gene Expression in FRTL-5 Thyroid Cells

To determine which genes are regulated by thyroid stimulating hormone (thyrotropin, TSH), insulin and insulin-like growth factor-1 (IGF-1) in the rat thyroid, we used the microarray technology and observed the changes in gene expression. The expressions of genes for bone morphogenetic protein 6, the glucagon receptor, and cyclin D1 were increased by both TSH and IGF-1; for cytochrome P450, 2c37, the expression was decreased by both. Genes for cholecystokinin, glucuronidase, beta, demethyl-Q 7, and cytochrome c oxidase, subunit VIIIa, were up-regulated; the genes for ribosomal protein L37 and ribosomal protein L4 were down-regulated by TSH and insulin. However, there was no gene observed to be regulated by all three: TSH, IGF-1, and insulin molecules studied. These findings suggest that TSH, IGF-1, and insulin stimulate different signal pathways, which can interact with one another to regulate the proliferation of thyrocytes, and thereby provide additional influence on the process of cellular proliferation.

Investigating the paradox of hypothyroidism and increased serum thyrotropin (TSH) levels in Sheehan's syndrome: characterization of TSH carbohydrate content and bioactivity.

Serum TSH levels are often paradoxically elevated in patients with hypothyroidism due to Sheehan's syndrome. To investigate this apparent discrepancy, the biological activity and glycosylation of serum TSH were studied in 9 untreated patients with Sheehan's syndrome and 11 normal controls. TSH bioassay was based on cAMP generation, measured by RIA, in a culture system of CHO cells transfected with recombinant human TSH receptor. The oligosaccharide branching of TSH was studied by Con A lectin affinity chromatography, which discriminates TSH isoforms according to their mannose content, and the sialic acid content of TSH was studied by Ricinus communis affinity chromatography in combination with enzymatic removal of sialic acid with neuraminidase treatment. TSH bioactivity was expressed as the ratio between biological and immunofluorometric assays (B/I). Bioactive TSH concentrations were calculated by multiplying serum TSH intrinsic bioactivity by serum immunoreactive TSH concentration (B/I x I). Serum free T(4) (FT(4)) levels were lower in patients than in controls (3.7 +/- 0.4 vs. 14.0 +/- 0.9 pmol/L, respectively; P < 0.0001). Circulating immunoreactive TSH was higher in patients with Sheehan's syndrome than in controls (3.8 +/- 0.8 vs. 1.8 +/- 0.2 mU/L, respectively; P = 0.01). In contrast, TSH B/I was significantly decreased in Sheehan's patients compared with controls (0.6 +/- 0.4 vs. 1.7 +/- 0.8, respectively; P = 0.003). However, the resultant bioactive TSH concentrations in serum of Sheehan's patients were not significantly different from control values (2.1 +/- 0.6 vs. 3.0 +/- 0.4; P = 0.25). A significant correlation was found between the bioactive TSH concentrations and serum FT(4) levels in patients with Sheehan's syndrome (r = 0.66; P = 0.05), but not between serum immunoreactive TSH and FT(4) levels (r = 0.21; P = 0.59) or between intrinsic TSH bioactivity and FT(4) levels (r = 0.56; P = 0.12). The Con A chromatography of serum TSH showed a similar distribution (0.3 < P < 0.5) of unbound, weakly bound, and firmly bound TSH in Sheehan's patients (16%, 38%, and 47%, respectively) and controls (15%, 34%, and 52%, respectively). The ricin chromatography of serum TSH showed a higher proportion of sialylated TSH molecules in Sheehan's patients than in controls (55% vs. 29%; P = 0.02). These results show that circulating TSH in Sheehan's syndrome, albeit increased, has decreased biological activity. The relevance of this finding is supported by the direct correlation between bioactive serum TSH concentrations and circulating FT(4). The reduced intrinsic TSH bioactivity in pituitary hypothyroidism of Sheehan's syndrome results from increased sialylation of TSH.

Molecular Cloning and Sequence Analysis of the cDNA for Thyroid-Stimulating Hormone β Subunit of Muscovy Duck

The cDNA-encoding thyroid-stimulating hormone β subunit (TSHβ) of the Muscovy duck (Cairina moschata) was cloned and sequenced to investigate the phylogenic diversity and evolution of TSH molecules. Oligonucleotide primers were designed and used for reverse transcription and polymerase chain reaction amplification of a TSHβ cDNA fragment from the total cellular RNA of pituitary glands. The remaining sequence was completed by rapid amplification of cDNA ends. The nucleotide sequence of Muscovy duck TSHβ cDNA includes 66 bp of 5′-untranslated region (UTR), 402 bp of coding region, and 82 bp of 3′-UTR, followed by an 18-bp poly(A)+ tract. The total number of amino acids deduced from the cDNA of duck TSHβ is 134, with a signal peptide of 19 amino acids and a mature protein of 115 amino acids. The homologies of the amino acid sequence of Muscovy duck TSHβ compared with those of chicken, quail, mammals, amphibian (frog), and teleosts are 97, 98, 68–69, 56, and 42–47%, respectively. To test for tissue specificity of the duck TSHβ cDNA, total cellular RNA samples from brain, liver, pituitary gland, testis, and thyroid gland were analyzed by Northern blotting. A band, approximately 700 bases, was found in the pituitary gland alone. The pituitary tissue fragments were treated for 24 h in serum-free medium containing thyrotropin-releasing hormone (TRH), triiodothyronine (T3), or thyroxine (T4). TRH increased and T3 and T4 decreased TSHβ mRNA levels. This study demonstrated that the amino acid sequence of TSHβ subunits is highly conserved among birds, exhibiting a high degree of inter-order homology, and that hypothalamic TRH upregulates the TSHβ mRNA expression in duck.

Circulating thyrotropin bioactivity in sporadic central hypothyroidism.

The etiopathogenesis of sporadic central hypothyroidism (CH) involves pituitary and hypothalamic lesions. Pituitary CH (pCH) implies a diminished number of functioning thyrotropes, accounting for the quantitative impairment of TSH secretion. Hypothalamic CH (hCH) is characterized by normal or even increased TSH concentrations and qualitative abnormalities of TSH secretion, including a decreased bioactivity of circulating TSH. However, controversy still exists about the actual occurrence of bioinactive TSH among CH patients, and no data are available in pCH. Therefore, we studied 41 CH patients with different hypothalamic-pituitary disorders. Immunoreactive TSH (TSH-I) ranged from 0.08-11.1 mU/L (normal, 0.24-4.0), free T4 (FT4) ranged from 0.6-8.8 pmol/L (normal, 9-18), and FT3 ranged from 1.2-5.4 pmol/L (normal, 4-8). A blunted TSH response to TRH (<4 mU/L), indicating prevalent pCH, was found in 56% of the patients, and a net TSH-I increment > or =4 mU/L, indicating prevalent hCH, was found in the remaining 44%. Net TSH-I increments showed significant correlation with basal FT4 (P < 0.02), indicating the relevance of pituitary TSH reserve in the pathogenesis of CH. Circulating TSH was immunoconcentrated and tested in bioassay and in ricin affinity chromatography. The ratio between biological (B) and immunological (I) activities of circulating TSH was reduced (n = 25; TSH B/I, 0.38+/-0.19) compared to the values recorded in normal subjects (n = 26; TSH B/I, 1.53+/-0.54; P < 0.001) and primary hypothyroid patients (n = 24; TSH B/I, 0.74+/-0.31; P < 0.001), but no difference between pCH (n = 9; 0.36+/-0.16) and hCH (n = 16; 0.39+/-0.20) was seen. TSH B/I values in CH patients showed a limited overlap with normal values (20%) and a highly significant correlation with the FT3 response to endogenous TRH-stimulated TSH (P < 0.005). The elevated sialylation degree of TSH molecules may explain part of these findings. In conclusion, the secretion of TSH molecules with reduced bioactivity is a common alteration in the patients with hypothalamic-pituitary lesions, contributing along with the impairment of pituitary TSH reserve to the pathogenesis of CH.

Hypothalamic thyrotropin-releasing hormone and thyrotropin biological activity.

Hypothalamic thyrotropin-releasing hormone (TRH) is the main positive regulator of thyrotropin (TSH) secretion. TRH action and the negative feedback of thyroid hormone are integrated in order to guarantee appropriate thyroid stimulation. TRH action affects various steps of the biosynthetic process within thyrotrophs, with major effects on the posttranslational maturation of TSH oligosaccharide chains, and is necessary for the secretion of the glycoprotein hormone with full biological activity. Since the first description in 1979 of some patients with central hypothyroidism of hypothalamic origin associated with the secretion of TSH molecules with conserved immunoreactivity but decreased bioactivity, a large body of evidence has accumulated in more recent years showing that changes of the oligosaccharide chains have a great impact on the biological properties of circulating TSH and occur in various in vivo situations. These findings have lead to the new concept of a qualitative regulation of TSH secretion. This can be achieved mainly through the transcriptional and posttranscriptional regulation of the complex enzymatic machinery devoted to the processing of the three oligosaccharide chains linked to specific asparagine residues of TSH heterodimer. Data obtained in several physiological and pathological conditions, which are characterized by an increased or diminished TRH action, indicate that both qualitative and quantitative regulations cooperate within thyrotrophs in order to adjust thyroid-stimulating activity to the temporary needs.

Thyrotropic action of human chorionic gonadotropin.

Hyperthyroidism or increased thyroid function has been reported in many patients with trophoblastic tumors. In these cases, greatly increased human chorionic gonadotropin (hCG) levels and suppressed TSH levels suggest that hCG has thyrotropic activity. Recent investigations have clarified the structural homology not only in the hCG and TSH molecules but also in their receptors, and this homology suggests the basis for the reactivity of hCG with the TSH receptor. The clinical significance of the thyrotropic action of hCG is now also recognized in normal pregnancy and hyperemesis gravidarum. Highly purified hLH binds to recombinant hTSH receptor and is about 10 times as potent as purified hCG in increasing cAMP. The beta-subunits of hCG and hLH share 85% sequence identity in their first 114 amino acids but differ in the carboxy-terminal peptide because hCG beta contains a 31-amino acid extension (beta-CTP). A recombinant mutant hCG that lacks beta-CTP showed almost identical potency to LH on stimulation of recombinant hTSH receptor. If intact hCG were as potent as hLH in regard to its thyrotropic activity, most pregnant women would become thyrotoxic. One of the roles of the beta-CTP may be to prevent overt hyperthyroidism in the first trimester of pregnancy when a large amount of hCG is produced by the placenta. Nicked hCG preparations, obtained from patients with trophoblastic disease or by enzymatic digestion of intact hCG, showed approximately 1.5- to 2-fold stimulation of recombinant hTSH receptor compared with intact hCG. This suggests that the thyrotropic activity of hCG may be influenced by the metabolism of the hCG molecule itself. Deglycosylation and/or desialylation of hCG enhances its thyrotropic potency. Basic hCG isoforms with lower sialic acid content extracted from hydatidiform moles were more potent in activating adenylate cyclase, and showed high bioactivity/immunoactivity (B/I) ratio in CHO cells expressing human TSH receptors. This is consistent with the finding that the beta-CTP truncated hCG with higher thyrotropic potency is substantially deglycosylated and desialylated in the beta-subunit relative to intact hCG because all four O-linked glycosylation sites occur within the missing C-terminal extension. The desialylated hCG variant also interacts directly with recombinant hTSH receptors transfected into human thyroid cancer cells. There is thyroid-stimulating activity in sera of normal pregnant women, and this correlates with serum hCG levels. The thyroid gland of normal pregnant women may be stimulated by hCG to secrete slightly excessive quantities of T4 and induce a slight suppression of TSH, perhaps being about 1 mU/L less than nongravid levels, but not high enough to induce overt hyperthyroidism. Maternal thyroid glands may secrete more thyroid hormone during early pregnancy in response to the thyrotropic activity of hCG that overrides the normal operation of the hypothalamic-pituitary-thyroid feedback system. Biochemical hyperthyroidism associated with hyperemesis gravidarum has been attributed to hCG. In patients with hyperemesis gravidarum, thyrotropic in serum correlated with hCG immunoreactivity, and the severity of vomiting as indicated by clinical and biochemical parameters correlated with the degree of thyroid stimulation. To understand the thyrotropic action of hCG, it is necessary to know whether hCG activates the same domain of the TSH receptor as does TSH. The identification of the molecular structure of the hCG isoform with the highest thyrotropic potency will resolve the enigma of gestational thyrotoxicosis and the hyperthyroidism associated with trophoblastic disease and hCG-producing tumors.

Circadian variations of thyrotropin bioactivity in normal subjects and patients with primary hypothyroidism.

Circadian rhythm of TSH secretion is characterized by a pronounced nocturnal increment that is not followed by the expected rise of circulating thyroid hormone levels. These findings suggest that the nocturnal TSH surge may be constituted by molecules with reduced bioactivity. We, therefore, investigated TSH bioactivity (measured as cAMP accumulation in FRTL-5 cells) and its carbohydrate structure (by Concanavalin A affinity chromatography) in different blood pools taken during the day and night from seven normal subjects and from one patient with mild (mPH) and five with severe primary hypothyroidism (sPH). Patients with sPH were also studied during low dose L-T4 treatment. Cosinor analysis showed a significant TSH circadian rhythm in the control group and in L-T4-treated sPH patients. The nocturnal TSH surge was not followed by any increase in free thyroid hormone levels. In normal subjects, the daytime ratio of TSH bioactivity to immunoreactivity (TSH B/I) was higher than the nocturnal one [1.4 +/- 0.6 (+/- SD) vs. 1.1 +/- 0.6; P < 0.02]. The same pattern was observed in the only mPH patient (1.0 +/- 0.2 vs. 0.7 +/- 0.1; P < 0.01), but not in the sPH patients (0.8 +/- 0.3 vs. 0.7 +/- 0.1; P = 0.3). L-T4 administration to sPH patients caused the daytime TSH B/I to increase and restored the day/night difference in the TSH B/I (1.0 +/- 0.3 vs. 0.8 +/- 0.3; P < 0.02). Concanavalin A chromatography showed that a higher percentage of less mature forms of TSH are secreted during the night. These data indicate that TSH molecules secreted during the night are less bioactive and differently glycosylated than those circulating in the same individual during the day, thus explaining why thyroid hormone levels do not rise after the nocturnal TSH surge. In sPH patients, the TSH circadian rhythm is restored during L-T4 administration, and day/night differences in TSH B/I similar to those recorded in normal subjects are observed.

Ricin and lentil lectin-affinity chromatography reveals oligosaccharide heterogeneity of thyrotropin secreted by 12 human pituitary tumors

Some patients with thyrotropin (TSH)-producing pituitary tumors are more hyperthyroid than others despite similar TSH levels in serum, suggesting that qualitatively different TSH molecules with differing bioactivities may be secreted by different tumors. We used ricin and lentil lectin-affinity chromatography to test whether the TSH oligosaccharides varied among 12 patients with TSH-producing tumors. We found that each tumor secreted heterogeneous isoforms of TSH that differed in their extents of exposed galactose (Gal) residues, and their degrees of sialylation and core fucosylation. These biochemical parameters also varied markedly for TSH secreted by different tumors. Isoforms appeared to reflect poor sialyltransferase activity in two tumors and efficient sialyltransferase in the remainder. TSH secreted by tumors was more fucosylated than TSH secreted by control euthyroid persons. There was an inverse relationship between the sialylation and fucosylation of tumor TSH. No simple relationship between TSH oligosaccharide structures and bioactivity was evident, although mixtures of isoforms having the least and most sialylated TSH seemed to be the most bioactive clinically. In three patients from whom serum and medium TSH were both available, TSH in serum was more sialylated than TSH secreted by the tumor in vitro, perhaps reflecting slow clearance of sialylated isoforms form the circulation. Core fucosylation of serum TSH was less than that of medium TSH. These data prove that human tumors secrete TSH with heterogeneous oligosaccharide structures.

The glycoprotein hormones: recent studies of structure‐function relationships

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common alpha subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are alpha 26 to 46 and alpha 75-92. Two synthetic disulfide loop peptides from the hCG beta subunit beta 38-57 and beta 93-100 also block binding of hCG to its receptor. In addition, the beta 38-57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the alpha and beta subunits, and that the alpha subunit sites are similar for several hormones.

Effect of thyrotropin-releasing hormone on the carbohydrate structure of secreted mouse thyrotropin. Analysis by lectin affinity chromatography.

Thyrotropin (TSH) is a glycoprotein hormone whose secretion from the anterior pituitary is regulated, in part, by the hypothalamic tripeptide thyrotropin-releasing hormone (TRH). We have used serial lectin affinity analysis to explore whether TRH, in addition to promoting TSH secretion, alters the carbohydrate structure of secreted TSH. Hypothyroid mouse hemipituitaries were incubated in medium containing [3H] mannose, [3H]glucosamine, or [3H]fucose either with or without 10(-7) M TRH. TSH was immunoprecipitated, proteolytically digested into glycopeptides, and chromatographed on serial lectin-Sepharose columns. Under basal conditions, 37% of secreted [3H]mannose-labeled TSH glycopeptides failed to bind to concanavalin A (ConA)-Sepharose, 55% bound and eluted with 10 mM alpha-methylglucoside, and 8% bound and eluted with 500 mM alpha-methylmannoside. Approximately 35% of glycopeptides not binding to ConA-Sepharose were bound by pea lectin-Sepharose, suggesting the presence of certain core fucosylated triantennary complex oligosaccharides. TRH caused a 2-fold increase in secretion of [3H]mannose-labeled TSH glycopeptides due almost exclusively to a specific increase in structures that bound to ConA-Sepharose and eluted with 10mM alpha-methylglucoside, corresponding to biantennary complex or unusual hybrid species. There was no change in the distribution of intrapituitary TSH glycopeptides with TRH. Acid hydrolysis of secreted proteins showed little metabolism of the tritiated sugar precursors, except for a 20% conversion of [3H]mannose to [3H]fucose. Moreover, ConA-Sepharose chromatography of secreted [3H]glucosamine- and [3H]fucose-labeled TSH glycopeptides showed similar increases in ConA-Sepharose binding with TRH as noted with [3H]mannose labeling. Subsequent lectin analysis of secreted [3H] mannose-labeled TSH glycopeptides on erythroagglutinating phytohemagglutinin-Sepharose and leukoagglutinating phytohemagglutinin-Sepharose disclosed no significant differences in TRH-treated versus control samples. These data suggest that secreted mouse TSH has greater carbohydrate heterogeneity than has been recognized previously. In addition, TRH in vitro promotes the secretion of specific TSH molecules apparently enriched in biantennary complex or unusual hybrid oligosaccharides.

Further studies on the covalent crosslinking of thyrotropin to its receptor: Evidence that both the α and β subunits of thyrotropin are crosslinked to the receptor

Highly purified α and β-subunits of thyrotropin were individually radioiodinated and, subsequently, recombined with their unlabeled complementary subunits. This procedure resulted in the formation of [ 125I]thyrotropin(TSH) hybrid molecules which were labeled on only one hormone subunit. Characterization of the binding properties of these two hybrid molecules demonstrated that both yielded nonlinear Scatchard plots with K d and B max values similar to those obtained with radioiodinated native TSH and that both were capable of interaction with the high- and low-affinity binding components of the TSH receptor. The recombined [ 125I]TSH molecules were then crosslinked to the TSH receptor using disuccinimidyl suberate. Following electrophoresis and autoradiography, two labeled TSH-receptor complexes with M r of 68,000 and 80,000 were observed. These two complexes exhibited hormone specificity and electrophoretic mobility identical to those previously observed using native [ 125I]TSH. Crosslinking with increasing concentrations of disuccinimidyl suberate suggested that the formation of the 68,000 and 80,000 complexes was sequential with the 68,000 appearing before the 80,000. Furthermore, the two bands were labeled regardless of which TSH subunit of the hybrid TSH was radioiodinated. These data strongly suggest that the 68,000 and 80,000 TSH-receptor complexes are the result of crosslinking to the TSH α-β dimer and not to one subunit in the case of the 68,000 complex and to the TSH α-β dimer in the case of the 80,000 complex, as had been hypothesized previously.

Thyrotropin interaction with high-density lipoproteins

Human high-density lipoproteins (HDL), but not other lipoprotein classes, bind bovine thyrotropin (TSH) with moderately high affinity. Binding of 125I-labeled HDL to TSH has been measured in a solid-phase assay; it is saturable and can be displaced by unlabeled HDL but not by other lipoproteins or bovine serum albumin. The interaction of HDL with TSH has been studied by fluorescence spectroscopy: HDL specifically modifies the fluorescence properties of the biologically active damsyl derivative (DNS, (5-dimethyl-aminonaphtalene-1-sulfonyl) chloride) of TSH (DNS-TSH) causing a 12 nm shift to lower wavelength of the emission maximum, a two-fold increase of the quantum yield and a significant increase of fluorescence polarization. The primary site of TSH binding on the HDL particle is likely to be located on its protein moieties, since other lipoprotein classes, which share similar lipids with HDL, do not bind TSH. 125I-labeled apolipoprotein A-I binds TSH in the solid-phase assay and titration of DNS-TSH with apolipoprotein A-I causes perturbations nearly identical to those observed with intact HDL. One HDL particle has at least 12 binding sites for TSH with an association constant, K = 10 7 M −1 whereas one apolipoprotein A-I molecule binds one or two TSH molecules with an association constant slightly lower than that for HDL ( K = 10 6 M −1). The lipid moieties of HDL also appears to be perturbed by the interaction with TSH.

Solubilized human thyrotrophin receptors behave as one class of high-affinity binding sites

Evidence is presented justifying the conclusion that, in contrast to the dextran-coated charcoal technique, the widely used technique of separating bound and free TSH with polyethylene glycol is inadequate and yields inaccurate results. Optimum values for the concentration of Triton X-100, pH, salts, temperature and time of incubation were established for the TSH-TSH receptor interaction. According to Scatchard analysis, soluble TSH receptors behaved as one class of binding sites. The affinity constant for this class of binding sites ( K a 1.3 × 10 9 M −1) is identical to that for the high-affinity binding sites found in human thyroid membranes ( K a 1.2 × 10 9 M −1). No low-affinity binding sites could be detected after solubilization of membrane receptors. Chromatography experiments on Sepharose CL-6B indicated that, in excess TSH, each micelle containing TSH receptors (molecular weight 150000) binds 4 [ 125I]TSH molecules. These data, together with the absence of low-affinity binding sites, led to the hypothesis that high-affinity TSH binding sites may be formed by the clustering of 4 low-affinity binding sites. Cross-reactivity experiments showed that both α- and β-subunits are involved in the binding of TSH to its receptor; the TSH β-subunit showed an increased cross-reactivity with soluble receptors.

Two Avian Luteinizing Hormone Radioimmunoassay Procedures Compared by Measurement of Changes During the Ovulatory Cycle of Turkey and Broiler hens

Two radioimmunoassay (RIA) procedures for measuring avian luteinizing hormone (LH) were compared using divided aliquots of plasma samples collected during the ovulatory cycles of turkey and broiler breeder hens in three separate experiments. The RIA systems compared were the homologous turkey LH assay of Wentworth et al. (1976) and the homologous chicken LH assay of Follett et al. (1972). Both assays provided the same assessment of circulating LH changes. The preovulatory surge of LH was observed 6 hr before ovulation in turkeys and 4 hr before ovulation in chickens. In addition, both assays detected an increase in chicken LH after ovulation, followed by a gradual decline prior to the next preovulatory surge. The correlation between RIA were highly significant (P<.001) in each experiment.The crossreaction of purified ostrich LH, FSH, and TSH and of the α and β subunits of turkey LH was assessed in each assay system. Ostrich LH, FSH, and TSH crossreacted in the chicken LH assay in a dose-related manner: ostrich LH showed the highest activity. Only ostrich TSH produced a response in the turkey assay that was dose related. The α subunit, but not the β subunit, of turkey LH was quite potent in the chicken LH RIA, but neither subunit competed as well as the native hormone in the turkey RIA. Thus, the two LH antisera recognize different portions of the LH molecule. The chicken antiserum apparently recognizes a site on the α subunit that may be at least partially masked in the FSH and TSH molecules. The turkey antiserum recognizes a site on the native LH molecule that is altered when the subunits are separated.

Direct visualization of membrane clustering and endocytosis of thyrotropin into cultured thyroid cells

We prepared a highly fluorescent conjugate of thyrotropin (TSH) which retains ∼25% of the binding affinity of native TSH towards TSH receptor and ∼25% of the potency of the native hormone in stimulating the accumulation of cAMP in thyroid cells. Using an image-intensified microscopy system, we observed that our fluorescent TSH bound specifically to diffusely distributed membrane receptors on live rat or bovine embryo thyroid cells grown in culture. At 37°C the fluorescent hormone formed visible patches which were internalized and subsequently degraded. Hence, TSH, like other polypeptide hormones, is internalized by a process of receptor-mediated endocytosis. The addition of bivalent antibodies against the fluorophore rhodamine, to thyroid cells that were previously exposed to sub-optimal concentrations of rhodamine TSH, elicited maximal stimulation of the thyroid adenyl cyclase. Monovalent Fab' fragments did not enhance the response of rhodamine TSH. Therefore we conclude that enhanced surface clustering of TSH molecules increases their capacity to stimulate the adenyl cyclase of thyroid cells.

The thyrotropic cells of the guinea pig pituitary. Electron microscopic study after characterization by immunocytochemical means.

Three different immunocytoenzymatic techniques were used to identify and characterize the thyrotropic cells in the pituitary of normal guinea pigs at the ultrastructural level (superimposition technique, immunocytochemical technique using P.A.P. and indirect immunohistoenzymatic method before embedding). These cells are characterized by a dark cytoplasm with granules ranging from 1500 to 2000 A in diameter. The appearance of these granules is very variable: some display a marked electron density and are homogeneous but some have a less marked electron density with a more electron dense peripherally situated region. The TSH molecules are essentially confined to the granules but when the immunocytochemical reactions are carried out before embedding, positive staining is also seen in the cytoplasm and the outer surface of most of the rough endoplasmic reticulum membranes. These results are discussed.