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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.

Pituitary, a critical component in the neuroendocrine system, plays an indispensable role in the regulation of body growth. The transcriptional factor ZBTB20 is widely expressed in brain tissues and participates in hippocampal development; however, the detailed molecular mechanism remains unknown. Therefore, the aim of this study was to investigate the effect of ZBTB20 on mouse pituitary development and related mechanisms in ZBTB20 gene knockout mice. The expressional profiles of ZBTB20 in various neuroendocrinal cells during the different developmental stages (from E10 to P0) were described by immunofluorescence staining. A ZBTB20 gene knockout mouse model was then generated. Real-time polymerase chain reaction and western blotting assays were used to detect the levels of five hormones: growth hormone (GH), prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). ZBTB20 protein expression was identified from E14 until birth. A majority of the pituitary endocrinal cells were ZBTB20-positive. In ZBTB20 knockout mice, the level of GH decreased by half and PRL expression was eliminated. No significant change was observed in the other three hormones (LH, FSH, and TSH). ZBTB20, an important transcriptional factor in pituitary development, is mainly responsible for the terminal differentiation of prolactin-secreting cells, thereby regulating the secretion of the pituitary hormones.

The purpose of this study was to determine the content of iodine and selenium in the thyroid and pituitary glands of rats under iodine-induced blockade of the thyroid gland. Electron probe microanalysis, wavelength-dispersive spectrometry, and point analysis were used in this investigation. We also determined the expression of sodium iodide symporter and caspase 32 in the thyroid and pituitary glands and the expression of thyroid-stimulating hormone in the pituitary. The samples for iodine analysis must be thoroughly dehydrated, and for this purpose, we developed a method that produced samples of constant mass with minimal loss of substrate (human thyroid gland was used for the investigation). Normal levels of iodine and selenium were found in the thyroid, pituitary, ovaries, testes hypothalamus, and pancreas of healthy rats. The levels of iodine and selenium in I- or Se-positive points and the percentage of positive points in most of these organs were similar to those of controls (basal level), except for the level of iodine in the thyroid gland and testes. Blockade of the thyroid gland changed the iodine level in iodine-positive points of the thyroid and the pituitary glands. On the sixth day of blockage, the iodine level in iodine-positive points of the thyroid gradually decreased to the basal level followed by an abrupt increase on the seventh day, implying a rebound effect. The opposite was found in the pituitary, in which the level of iodine in iodine-positive points increased during the first 6 days and then abruptly decreased on the seventh day. Expression of the thyroid-stimulating hormone in the pituitary decreased during the first 5 days but sharply increased on the sixth day, with a minimum level of iodine in the thyroid and maximum in the pituitary, before normalization of the iodine level in both glands preceding the rebound effect. The expression of sodium iodide symporter increased during the first 4 days of blockage and then decreased in both glands. The fluctuations of the thyroid-stimulating hormone in the pituitary gland reflected the changes of iodine in the thyroid gland more precisely than the changes of sodium iodide symporter. The selenium level in the selenium-positive points changed only in the pituitary, dropping to zero on the second and fifth day of the blockade. Simultaneously, the maximum induction of caspase 32 was observed in the pituitary gland. We believe that these results may help to clarify a role of the pituitary gland in the thyroid blockade.

The annual cycle of changing day length (photoperiod) is widely used by animals to synchronise their biology to environmental seasonality. In mammals, melatonin is the key hormonal relay for the photoperiodic message, governing thyroid-stimulating hormone (TSH) production in the pars tuberalis (PT) of the pituitary stalk. TSH acts on neighbouring hypothalamic cells known as tanycytes, which in turn control hypothalamic function through effects on thyroid hormone (TH) signalling, mediated by changes in expression of the type II and III deiodinases (Dio2 and Dio3, respectively). Among seasonally breeding rodents, voles of the genus Microtus are notable for a high degree of sensitivity to nutritional and social cues, which act in concert with photoperiod to control reproductive status. In the present study, we investigated whether the TSH/Dio2/Dio3 signalling pathway of female common voles (Microtus arvalis) shows a similar degree of photoperiodic sensitivity to that described in other seasonal mammal species. Additionally, we sought to determine whether the plant metabolite 6-methoxy-2-benzoxazolinone (6-MBOA), described previously as promoting reproductive activation in voles, had any influence on the TSH/Dio2/Dio3 system. Our data demonstrate a high degree of photoperiodic sensitivity in this species, with no observable effects of 6-MBOA on upstream pituitary/hypothalamic gene expression. Further studies are required to characterise how photoperiodic and nutritional signals interact to modulate hypothalamic TH signalling pathways in mammals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

We used ex vivo and in vivo experiments with Xenopus laevis tadpoles to examine the hypothesis that the set-point for negative feedback on pituitary thyroid-stimulating hormone (TSH) synthesis and secretion by thyroid hormones (THs) increases as metamorphosis progresses to allow for the previously documented concomitant increase in serum TH concentrations and pituitary TSH mRNA expression during this transformative process. First, pituitaries from climactic tadpoles were cultured for up to 96 h to characterize the ability of pituitary explants to synthesize and secrete TSHβ in the absence of hypothalamic and circulating hormones. Next, pituitary explants from tadpoles NF stages 54-66 were exposed to physiologically-relevant concentrations of THs to determine whether stage-specific differences exist in pituitary sensitivity to negative feedback by THs. Finally, in vivo exposures of tadpoles to THs were conducted to confirm the results of the ex vivo experiments. When pituitaries from climactic tadpoles were removed from the influence of endogenous hormones, TSHβ mRNA expression increased late or not at all whereas the rate of TSHβ secreted into media increased dramatically, suggesting that TSH secretion, but not TSH mRNA expression, is under the negative regulation of an endogenous signal during the climactic stages of metamorphosis. Pituitaries from pre- and prometamorphic tadpoles were more sensitive to TH-induced inhibition of TSHβ mRNA expression and secretion than pituitaries from climactic tadpoles. The observed decrease in sensitivity of pituitary TSHβ mRNA expression to negative feedback by THs from premetamorphosis to metamorphic climax was confirmed by in vivo experiments in which tadpoles were reared in water containing THs. Based on the results of this study, a model is proposed to explain the seemingly paradoxical, concurrent rise in serum TH concentrations and pituitary TSH mRNA expression during metamorphosis in larval anurans., (Published by Elsevier Inc.)

To date, there has been only one in vitro study of the relationship between neuropeptide EI (NEI) and the hypothalamic-pituitary-thyroid (HPT) axis. To investigate the possible relationship between NEI and the HPT axis, we developed a rat model of hypothyroidism and hyperthyroidism that allows us to determine whether NEI content is altered in selected brain areas after treatment, as well as whether such alterations are related to the time of day. Hypothyroidism and hyperthyroidism, induced in male rats, with 6-propyl-1-thiouracil and l-thyroxine, respectively, were confirmed by determination of triiodothyronine, total thyroxine, and thyrotropin levels. All groups were studied at the morning and the afternoon. In rats with hypothyroidism, NEI concentration, evaluated on postinduction days 7 and 24, was unchanged or slightly elevated on day 7 but was decreased on day 24. In rats with hyperthyroidism, NEI content, which was evaluated after 4 days of l-thyroxine administration, was slightly elevated, principally in the preoptic area in the morning and in the median eminence-arcuate nucleus and pineal gland in the afternoon, the morning and afternoon NEI contents being similar in the controls. These results provide the bases to pursue the study of the interaction between NEI and the HPT axis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Vitamin A (VA) deficiency (VAD) and the iodine deficiency disorders (IDD) affect > 30% of the global population and these deficiencies often coexist in vulnerable groups. VAD has multiple effects on the pituitary-thyroid axis; VA status modulates thyroid gland metabolism, peripheral metabolism of thyroid hormone, and production of thyrotropin (TSH) by the pituitary. Findings from Africa children indicate that VAD in severely-IDD-affected children increases TSH stimulation and thyroid size, and reduces risk for hypothyroidism. In children with VAD, the higher TSH concentrations in the face of higher circulating total thyroxine suggest central resistance to normal TSH suppression by thyroid hormone. In IDD- and VAD-affected children receiving iodized salt, concurrent VA supplementation improves iodine efficacy. Recent VA and iodine depletion studies in rats indicate moderate VAD alone has no measurable effect on the pituitary-thyroid axis; however, concurrent iodine deficiency (ID) and VAD produce more severe primary hypothyroidism than ID alone. Repletion studies in VA- and iodine-deficient animals suggest: 1) primary hypothyroidism in animals with concurrent moderate VAD and ID does not reduce the efficacy of high doses of oral VA; 2) VAD does not reduce the efficacy of dietary iodine to correct pituitary-thyroid axis dysfunction due to iodine deficiency; and 3) given alone, without iodine repletion, high-dose VA supplementation in combined VAD and ID may reduce thyroid hyperstimulation and reduce risk for goiter.

GATA2 is expressed in the pituitary during development and in adult gonadotropes and thyrotropes. It is proposed to be important for gonadotrope and thyrotrope cell fate choice and for TSH production. To test this idea, we produced a pituitary-specific knockout of Gata2, designed so that the DNA-binding zinc-finger region is deleted in the presence of a pituitary-specific recombinase transgene. These mice have reduced secretion of gonadotropins basally and in response to castration challenge, although the mice are fertile. GATA2 deficiency also compromises thyrotrope function. Mutants have fewer thyrotrope cells at birth, male Gata2-deficient mice exhibit growth delay from 3-9 wk of age, and adult mutants produce less TSH in response to severe hypothyroidism after radiothyroidectomy. Therefore, Gata2 appears to be dispensable for gonadotrope and thyrotrope cell fate and maintenance, but important for optimal gonadotrope and thyrotrope function. Gata2-deficient mice exhibit elevated levels of Gata3 transcripts in the pituitary gland, suggesting that GATA3 can compensate for GATA2.

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 investigated how a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects thyroid hormone regulation, especially in relation to the localization of thyroid stimulating hormone (TSH) in the pituitary and that of thyroxin (T4) of the thyroid in the rat. Female Sprague-Dawley rats were given a single oral administration of TCDD ranging from 1.0 to 4.0 microg/kg body weight (bw), and then tissue specimens were removed on day 7 post-administration. Thyroid hormone concentrations were measured in serum, and the expression of the TCDD-responsive genes, UDP-glucuronosyltransferase-1 (UGT1) and cytochrome P4501A1 (CYP1A1) were examined in the liver. TCDD administration resulted in an increase in both immunostaining intensity and the number of TSH-positive cells in the anterior pituitary. T4 was found to localize only in the follicular lumen of the thyroid in vehicle-treated control rats, while TCDD administration caused a foamy change in the colloid of some follicles, an indication of accelerating the biosynthesis of T4 in the thyroid. By morphometrical analysis, the ratio of parenchymal/lumenal area of the thyroid was found to increase in response to TCDD. TCDD treatment as low as 2.0 microg TCDD/kg bw induced a significant decrease in both serum total T4 (TT4) and free T4 (FT4) concentrations in the rats, with a significant increase in serum TSH levels in the 4.0 microg TCDD/kg bw rats. Serum total triiodothyronine (TT3) level was unchanged in all groups. The UGT1 gene was significantly induced at a TCDD dose as low as 1.0 microg/kg bw in a dose-dependent manner. TCDD concentrations in the serum, liver and adipose tissues were detected in a dose-related fashion. The present immunohistochemical results clearly support the earlier biochemical findings on the perturbation of the thyroid-pituitary axis by TCDD and suggest that UGT1 is an immediate target of a low TCDD exposure that triggers the perturbation.

Comprehensive article summarizing more than 25 years of experience with pituitary hyperplasia in surgical material. Morphologic forms of hyperplasia--diffuse and nodular--are defined and, for comparison, the normal morphology, frequency and intraglandular distribution of cell types are briefly reviewed. All cell types can give rise to hyperplasia, although their frequency, extent and clinical importance widely vary. Somatotroph hyperplasia is rare; it is limited to cases of GHRH overproduction by extrapituitary endocrine neoplasms and sporadic examples of gigantism. Prolactin cells display the highest propensity for non-neoplastic proliferation. Physiologic hyperplasia occurs in pregnancy and lactation. Pathological hyperplasia is mostly secondary to other, neoplastic or non-neoplastic, space occupying processes. Idiopathic lactotroph hyperplasia is very rare. The much-disputed corticotroph hyperplasia is infrequent cause of pituitary dependent Cushing's disease. Despite difficulties of diagnosis in fragmented biopsies, several well-documented cases prove the existence of corticotroph hyperplasia which is nearly always nodular. Thyrotroph hyperplasia, secondary to hypothyroidism, a treatable condition, is not expected to occur in surgical material, yet several cases have been identified. Operated lesions are massive nodular leading to significant pituitary enlargement thereby mimicking TSH- or PRL-producing adenoma. Hyperprolactinemia is a frequent concomitant of severe thyrotroph hyperplasia. Gonadotroph hyperplasia and proliferation of pars intermedia derived POMC cells are not likely to occur in surgical material and have no clinical significance. Adenoma formation may rarely be associated with any type of pituitary hyperplasia.

A 16-year-old Brazilian girl presented with severe growth retardation (-6.3 SDS), obesity, delayed pubertal development, facial dysmorphia, dry skin, and borderline low intelligence (IQ 89). Endocrinological evaluation showed primary hypothyroidism (no uptake of iodine-131 of the right thyroid lobe). Basal and stimulated gonadotropins were increased and ultrasonography revealed hypoplastic ovaries. The karyotype of peripheral lymphocytes was 46,X,i(Xq). The GH response in euthyroid condition after stimulation with GHRH and insulin was diminished. MRI of the pituitary region showed a suprasellar mass (12 x 15 mm) which was removed by transsphenoidal surgery because of extension to the optic chiasm. Histological examinations revealed regular pituitary tissue with hyperplasia of TSH- and FSH-producing cells. Thyroxine treatment was adjusted and GH was given. We conclude that the suprasellar mass was the consequence of long-lasting hypothalamic overstimulation with TRH and LHRH, due to gonadal and thyroid insufficiency.

We have previously isolated a cleaved prolactin variant, secreted by rat pituitary cells in culture, that stimulated [3H]thymidine incorporation into DNA in gonadotrophs and thyrotrophs when added to pituitary aggregate cell cultures of 14-day-old female rats. Using synthetic peptides homologous to the new C- and N-termini of the cleavage site, we made antisera recognizing this cleaved variant without significant cross reaction with native prolactin. Addition of these antisera to pituitary aggregate cell cultures decreased [3H]thymidine incorporation into DNA in gonadotrophs and thyrotrophs but not in the other pituitary cell types. These data are further evidence that this prolactin variant, cleaved between Tyr-145 and Leu-146, may have an important role as growth regulator of the gonadotrophs and thyrotrophs in the rat pituitary.

Since thyroid-stimulating hormone (TSH) producing cells are thought to play an essential role during metamorphosis, their development was immunohistochemically examined to reveal the appearance and topographical changes in the distribution in the hypophysis of Xenopus laevis tadpole from hatching to the end of metamorphosis. TSH-immunoreactive cells initially appeared at stage 49 (just the beginning of development of the pars nervosa in the hypophysis) as small clusters in the middle part of the pars distalis. They showed conspicuous changes in number during late premetamorphosis: their number reached a peak at stage 51, suddenly decreased at stage 52 (just before completion of the hypophysial histogenesis) followed by gradual increase until the end of metamorphosis. At later stages, they were restricted to the posterior half of the pars distalis.

The populations of cells which produce immunoreactive growth hormone (GH) and thyroid stimulating hormone (TSH) in the rat pituitary gland do not occur in fixed percentages but vary greatly under different physiological and experimental conditions. These variations can be directly correlated to the levels of stimulation and/or inhibition of the specific secretory activity. In both types of cell, sustained stimulation with trophic hormones or blockage of the feedback mechanisms induces remarkable growth in the specific cell population. Conversely, the interruption or inhibition of the stimulus thwarted the hormonal secretion and caused a massive degeneration of redundant cells. The stimulation of both GH and TSH cells is accompanied by an enhanced secretory activity as judged by their higher concentrations in serum and hypertrophy of the cytoplasmic organelles involved in synthesis and intracellular processing of the hormones. By contrast, interruption of the stimulus is followed by a variable degree of disruption of the cytoplasmic organization, including a sizable degeneration of cells. In stimulated rats, the concentrations of both GH and TSH decreased significantly in pituitary tissue due to mobilization of the hormonal stores contained in secretory granules. On the other hand, the withdrawal of stimuli blocked the hormonal release; this is reflected by the accumulation of both hormones and secretory granules in pituitary tissue. The strict correlation between the size of the GH and TSH populations with stimulation and inhibition of hormonal secretory activity reported in this investigation further supports the critical role played by the cell renewal process in endocrine secretion.

Incubation media of pituitary cells and homogenates of pituitary tumors were studied by isoelectric focusing followed by radioimmunoassay. Almost all LH-immunoreactive components detected had been observed in earlier studies on highly purified preparations. In one pituitary tumor homogenate, intact LH (LHi) was detected. The majority of LHi-components present in this tumor were relatively acidic (LH Type I) components. The corresponding beta-subunit population was in agreement with the one earlier observed for Type I LH. In all other tumor homogenates only alpha- and beta-subunits could be detected. In incubation media of pituitary cells from patients with no known endocrine disease nor a pituitary tumor highly acidic populations of alpha-subunits were detected. In only one of the incubation media significant amounts of LHi and free beta-subunits were observed. In an incubation medium of a TSH-producing pituitary tumor significant amounts of intact LH, free alpha- and beta-subunits were detected. The cell contents of this tumor contained components with lower pl-values than the medium. In general a good correlation is observed between the population of LH-components in material from individual identifiable pituitary glands and in highly purified preparations from large numbers of anonymous pituitary glands.

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.

We have previously demonstrated that TRH induces several hypophyseal cell type differentiation. In the present study, we re-evaluated the effect of GnRH on gonadotroph differentiation, and we determined whether gonadotropin is implicated in the appearance of other pituitary cell types. These effects were compared to the influence of TRH on LH and TSH cell differentiation. Pituitary primordia of 11 day-old rat embryos were cultured in a synthetic medium until the equivalent of term, and the LH and TSH cell differentiation was visualized using an immunohistochemical technique. The effect of GnRH on pituitary hormone cells was evaluated by measuring the percentage of immunoreactive area per section. GnRH, when added on the first day of culture, induced LH and TSH cell differentiation in a dose-dependent manner. GnRH was more effective than TRH in inducing gonadotropic phenotype expression. This suggests that GnRH, which evokes LH release, is the most effective factor inducing gonadotroph differentiation in early fetal life. However, GnRH was as effective as TRH in thyrotroph differentiation. Lactotrophs were detected in primordia cultured with GnRH. As lactotroph phenotype differentiation is controlled by the alpha-subunit of glycoproteic hormone, further studies are required to determine whether or not GnRH effect on lactotroph is direct. GnRH did not affect the corticotroph and somatotroph differentiation. These results confirm that hypothalamic factors such as TRH or GnRH are capable of inducing more than one pituitary cell phenotypes.

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.

To determine whether incubation of mouse thyrotropic tissue with TRH in vitro influenced the oligosaccharide structure of TSH, thyrotropic tumor tissue or pituitary tissue was incubated in vitro with [3H]mannose or with [35S]sulfate and [3H]methionine, in the absence or presence of TRH for times up to 24 h. [3H]mannose-labeled oligosaccharides from intracellular TSH and free alpha-subunits were analyzed by paper chromatography, and were predominantly Man9GlcNAc and Man8GlcNAc units both in the absence and presence of TRH. The [35S]sulfate/[3H]methionine ratio in secreted molecules was greater for TSH than for free alpha-subunits; within TSH heterodimers the ratio was greater for beta-subunits than alpha-subunits. The [35S]/[3H] ratio was not altered in TSH or free alpha-subunits by TRH. Analyses of [3H]mannose-labeled charged oligosaccharides by HPLC anion-exchange chromatography revealed similar types of oligosaccharides present on TSH subunits and free alpha-subunits (having one or two sulfate residues, one or two sialic acid residues, or both a sulfate and a sialic acid residue). These charged oligosaccharides occurred in different proportions on TSH subunits compared to free alpha-subunits, and also differed depending on whether the tissue source was tumorous or nontumorous. The proportions of oligosaccharide unit types were not altered by TRH. Thus, while this study provided information concerning the high-mannose and complex oligosaccharides of mouse TSH, there was no evidence that short incubations of tissues with TRH in vitro caused modulation of TSH oligosaccharide structures.

The adenohypophysial cell types in three species of sciaenid teleosts were identified using a combination of classical histochemical techniques and immunocytochemistry with antisera raised against piscine and human pituitary hormones. In general greater specificity for the different cell types was observed with antisera to the piscine pituitary hormones than with antisera raised against the human hormones. The distribution of the cell types did not differ significantly among the three species, Atlantic croaker (Micropogonias undulatus), spotted seatrout (Cynoscion nebulosus), and red drum (Sciaenops ocellatus), and was similar to that reported in several other teleosts. A major portion of the rostral pars distalis was composed of acidophilic prolactin cells which showed specific binding to antisera to carp, coho salmon, and chinook salmon prolactins. The acidophilic corticotrops formed cords of cells which bordered the prolactin cells and showed strong immunoreactivity with anti-human ACTH. This antiserum also cross-reacted with the lead hematoxylin-positive cells in the pars intermedia. The acidophilic growth hormone cells were restricted to the dorsal part of the proximal pars distalis in close contact with the neurohypophysis and were specifically bound by anti-carp and anti-chum salmon growth hormone. The basophilic thyrotrops were located in the dorsal-anterior part of the proximal pars distalis and showed strong immunoreactivity to antiserum to the human thyrotropin beta subunit. This antiserum also bound weakly to the basophilic gonadotrops which were concentrated in the ventral part of the proximal pars distalis and also extended posteriorly to form a border around the pars intermedia. Antiserum to croaker maturational gonadotropin, which recognizes two distinct gonadotropins in sciaendis, bound strongly and specifically to the gonadotrops. The gonadotrops were also bound specifically by antisera to the alpha and beta subunits of human luteinizing hormone. Antisera to the beta subunits of two recently isolated coho salmon gonadotropins, GTH-I and GTH-II, failed to bind the gonadotrops strongly or differentially in the three sciaenid species. Consequently, the number of gonadotropin cell types in sciaenid fishes remains unresolved.

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.

Rathke's pouches of 12- and 13-day-old rat embryos were implanted beneath the kidney capsule of adult male rats subjected to the removal of median eminence or to sham-operation. Host animals were sacrificed 28 days after grafting and the implanted pituitaries were processed for immunohistological examination. ACTH, LH-beta, FSH-beta, TSH-beta, GH and PRL immunopositive cells could be observed in fetal grafts of all experimental groups. However, the number and staining intensity of different hormone containing cells largely varied and presumably depended on the hormonal state of host animals. The results indicate that undifferentiated fetal pituitary does not require hypothalamic hypophysiotrophic neurohormones for proliferation and cytodifferentiation and that its development might be modulated by circulating trophic hormones of host animals.

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.

1. The ACTH cell type has been demonstrated for the first time in the hypophysis of the squirrels. These are large cells with homogenous cytoplasm and are more abundant in the anteromedian zone and in the peripheral portion than elsewhere. 2. A great hypertrophy and hyperplasia of the pituitary corticotrophs were observed in the experimental group at all the intervals. 3. Metopirone treated animals showed a moderate hypertrophy and degranulation of pituitary thyrotrophs. 4. By effect of metopirone, a marked regression of pituitary interstitiotrophs and total degranulation took place by 30 days treatment. The folliculotrophs did not show alterations.

The role of oestrogen in the regulation of TSH gene expression is unclear. We have examined the effect of administration of oestrogen in the rat on serum TSH, pituitary TSH content and pituitary cytoplasmic concentrations of mRNA encoding the TSH beta and alpha subunits, thus deriving measures of hormone release and synthesis. In addition, we have examined the effect of oestrogen on the binding of tri-iodothyronine (T3) to nuclear receptors in the anterior pituitary. Administration of oestrogen did not affect serum concentrations of TSH in euthyroid or untreated hypothyroid rats, but did augment the effects of T3 (1 and 2 micrograms) on serum TSH in hypothyroid animals 6 h after injection of T3. No influence of oestrogen or of thyroid status on pituitary content of TSH was seen. A marked increase in the concentrations of TSH beta and alpha mRNA in pituitary cytoplasm was found in hypothyroidism, compared with those in the euthyroid state. No effect of oestrogen on TSH mRNA was seen in euthyroid animals but concentrations of TSH beta and alpha mRNA were lower in hypothyroid animals than in vehicle-treated controls. A stimulatory influence of T3 on TSH mRNA was seen 6 h after injection of T3; this stimulation was absent in oestrogen-treated rats. No effect of oestrogen on the action of T3 was evident 72 h after beginning treatment with T3. In addition to effects on serum TSH and TSH mRNA, an increase in the number of pituitary nuclear receptors for T3 was seen after oestrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The identification of pituitary ACTH and TSH producing cells was performed by histological staining at the isoelectric points (pH levels: 4.6 and 5.2 respectively) of these hormones in cat. Intensity of stained material in the hormone producing cells was determined by microphotometric technique. By the application of hormones influencing the pituitary ACTH and TSH producing relevant changes in pituitary cells could be detected.

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.

We examined the effect of various inhibitors of oligosaccharide processing on the content and secretion of newly synthesized thyroid-stimulating hormone (TSH) from dispersed hypothyroid rodent pituitary cells. 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin, both inhibitors of glucosidases I and II, decreased intracellular TSH (to 60-76% of control) and secreted TSH (to 60-63% of control) after a 1-hour incubation (pulse) with [35S]methionine and an 8-hour incubation (chase) in isotope-free media. In contrast, deoxymannojirimycin and swainsonine, inhibitors of mannosidase I and II, respectively, increased both intracellular TSH (to 267-309% of control) and secreted TSH (to 192% of control) at 8 hours. TSH oligosaccharides synthesized in the presence of these glucosidase and mannosidase inhibitors were largely sensitive to endo-beta-N-acetylglucosaminidase H (endo H), confirming inhibition of processing. Despite differences in oligosaccharide structure, the in vitro bioactivities of these secreted TSH isoforms were nearly identical. These data confirm and extend previous work performed with 1-deoxynojirimycin suggesting that glucosylated high mannose forms of TSH are more susceptible to intracellular degradation. The novel finding that deoxymannojirimycin and swainsonine increase secreted and total TSH above control levels suggests that non-glucosylated high mannose forms as well as hybrid-type oligosaccharides may facilitate secretion and direct TSH away from a natural degradation pathway.

The prolactin-producing cells are the first hormone-producing cells of the pars distalis to be differentiated within Ambystoma gracile. They first appear when the larvae attain a length of approximately 3.0 cm snout to vent length (SVL). Thyrotropes are observed as the next chromophilic cells to appear, and they occur when the larvae are approximately 4.5 cm SVL. Both thyrotropes and lactotropes increase in numbers until metamorphosis. Gonadotropes begin to appear when larvae attain a size of 5.0 cm SVL and become extremely abundant when larvae are in excess of 7.0 cm SVL. Animals, generally, exhibit the greatest number of thyrotropes just prior to the mean size for metamorphosis, and metamorphosing animals exhibit a dramatic reduction in the number of thyrotropes. Neotenous larvae have an abundant number of thyrotropes which are mainly located along the caudal periphery of the pituitary.

Cells from the rabbit pars distalis and pars intermedia have been cultured in several ways. In clump cultures the glandular adenohypophysial cells of the primary clump remain identifiable for long periods. Both acidophil and mucoid cells can be maintained in this way for over 50 days. During this time two different modes of granule extrusion are observed, one being found only in acidophil cells and the other being exclusive to mucoid cells. From this it is concluded that specific release of secretory products from these cells can occur in the absence of hypothalmic control. A special form of collagenous fibre is produced in clump cultures. Monolayer cell cultures are derived either from isolated adenohypophysial cells or from secondarily aggregated clumps, but in either case the predominant cells are fibroblasts, which persist for over 50 days (as isolated cultures) and for over 90 days (as spreads from clumps). Rounded cells can be recognized as a separate type in isolated cell culture.

Studies of thyroid activity following hypothalamic lesions have provided indirect evidence of TSH influence on the pars distalis of birds. The present study examined the influence of hypothalamic lesions on TSH assays of the rostral and caudal lobes of the pars distalis. Lesions of the septomesencephalic tract increased TSH levels in the rostral lobe but had no influence on the caudal lobe. Lesions of the supraoptic or ventrolateral nuclei neither stimulated nor depressed TSH in either lobe of the pars distalis.

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.

A rabbit antiserum raised against human thyrotropin (TSH) beta subunit (anti-human beta-TSH) was used to identify TSH cells in the pituitaries of five species of teleosts by means of the unlabeled antibody peroxidase-antiperoxidase (PAP) technique. The anti-human beta-TSH reacted only with polygonal basophils located in the rostral pars distalis (RPD) in the pituitaries of sexually immature eel (Anguilla japonica). In the pituitaries of medaka (Oryzias latipes) and white-edged rockfish (Sebastes taczanowskii), this serum reacted specifically with only one cell-type located in the antero-dorsal proximal pars distalis (PPD) and antero-ventral PPD, respectively. In the pituitaries of two species of salmonids, sexually immature rainbow trout (Salmo gairdneri) and sexually mature chum salmon (Oncorhynchus keta), the anti-human beta-TSH bound to the polygonal basophils in the ventral PPD adjacent to the RPD. Thus, the present immunocytochemical study identified polygonal basophils located in the RPD or PPD as TSH cells in five species of teleosts.

A cytological and optical microscopic study of the anterior lobe of hypohysis in the Wistar adult rat 10, 30 or 60 minutes after intravenous injection of 50 mug of synthetic TRH discloses modifications of thyrotropic cells and prolactin cells. It's possible to recognize a quick degranulation of the prolactin cells (with a maximum at the 10th minute) and a slower degranulation of the thyrotropic cells (perceptible at the 10th minute and very pronounced at the 60th minute). The somatrotopic cells did not show any modification.

The cells that produce luteinizing hormone (LH) in the adenohypophysis of the Japanese quail were identified immunohistochemically using anti-chicken LH serum and horseradish peroxidase-labeled goat anti-rabbit gamma globulin serum. The LH cells are localized in the caudal lobe of the pars distalis. They are elongate in shape shape and are polarized toward the sinusoids, especially in their active states. Alterations in size of LH cells are directly related to changes in circulating LH levels as induced by castration or photostimulation. The LH cells identified immunohistochemically were only stained by alcian blue with periodic acid-Schiff (PAS), alcian blue and orange G. PAS-positive gonadotropic cells in the cephalic lobe were stained immunohistochemically only slightly if at all using anti-chicken LH serum and consequently may be FSH producing cells. In the cephalic lobe another type of basophilic cell was stained with alcian blue. These cells were also stained immunohistochemically with anti-chicken LH serum. These cells may possibly be identified as TSH cells due to the characteristics of the anti-chicken LH serum used in this study which cross react with LH and TSH but only slightly with FSH, and also on the basis of previous light and electron microscopic studies.

While GnRH is known to stimulate release of pituitary gonadotropins, its acute effects on the kinetics of secretion of the various hormones and subunits are not well characterized. Pulse-chase experiments were therefore performed to compare the time course of secretion of newly synthesized LH subunits and free alpha from rat pituitary quarters, and to study the effects of GnRH. After a 1-h pulse labeling with [35S]cysteine in the presence or absence of 10(-8) M GnRH, cultures were chased with excess unlabeled cysteine for 1, 2, 4, 8, or 20 h. Tissue lysates and media were immunoprecipitated sequentially with antisera to PRL, GH, LH beta and LH alpha, and the products were analyzed by gel electrophoresis. Labeled LH alpha was completely secreted by 4 h of chase without GnRH, and by 2 h with GnRH, as shown by its appearance in media and depletion from pituitary. Newly synthesized LH beta was depleted only at 8 h with GnRH from pituitary, suggesting much slower secretion. Incorporation of 35S into LH beta was approximately half that into LH alpha. Newly synthesized free alpha subunit was secreted between 4 and 20 h without GnRH, and by 8 h with GnRH. Free alpha incorporated a similar amount of radioactivity as LH beta. GnRH had no effect on the timing of the secretion of labeled total protein, PRL, GH, or TSH subunits. The amount of label incorporated during the pulse was greatest for PRL, approximately 15% of total protein label. GH incorporated approximately 20% as much label as PRL, with the glycoprotein subunits somewhat lower. The total amount of 35S incorporated into each protein studied was not affected by GnRH. The data indicate that short term in vitro GnRH exposure during a 1-h pulse labeling and chase acts to accelerate early secretion of LH alpha, LH beta and free alpha but does not affect overall protein synthesis. The peptide hormones PRL and GH incorporated the greatest amount of label and were the most briskly secreted hormones, while LH alpha led the glycoprotein subunits in the amount of incorporation and rapidity of secretion.

Poly(A)-containing RNA isolated from bovine and mouse pituitaries and a mouse pituitary thyrotropic tumor was translated in a wheat germ cell-free biosynthetic system. A precursor of the glycoprotein hormone alpha subunit, "pre-alpha," was immunoprecipitated from the translation mixtures with antiserum against bovine luteinizing hormone (LH; lutropin) alpha. The specificity of the immunoprecipitation was shown by competition with authentic bovine LHalpha and lack of competition with bovine thyroid-stimulating hormone (TSH; thyrotropin) beta. Bovine and mouse pre-alpha subunits migrated identically in sodium dodecyl sulfate gradient polyacrylamide slab gels with an apparent molecular weight of about 17,000. Pre-alpha comprised 2-3% and 20-30% of the total proteins translated with pituitary and pituitary tumor mRNA, respectively. Microanalysis of amino acid sequence of the pre-alpha subunits containing various radiolabeled amino acids gave the following partial sequence for mouse tumor pre-alpha: [Formula: see text] Met was also found in positions 1, 14, and 17 in mouse pituitary pre-alpha but only in residue 1 of the bovine pituitary pre-alpha subunit. Leu was found in identical positions in bovine pituitary pre-alpha, with an additional Leu in position 17. Leu in the common positions (12, 15, 19, and 22) has also been found in human choriogonadotropin pre-alpha subunit [Birken, S., Fetherston, J., Desmond, J., Canfield, R. & Boime, I. (1978) Biochem. Biophys. Res. Commun. 85, 1247-1253]. The data demonstrate that pituitary as well as placental glycoprotein hormone alpha subunits are synthesized with an amino-terminal hydrophobic extension, in accord with the "signal hypothesis" for secreted proteins. Furthermore, the positions of the hydrophobic amino acid Leu have been strictly conserved in pre-alpha subunits from various species and in two different tissues, the pituitary and placenta.

Studies of gene regulation are greatly facilitated by the ability to transfect DNA into cultured cells. We examined a variety of transfection techniques to optimize transient expression of the human glycoprotein hormone alpha-gene in primary pituitary cells and subsequently investigated the regulation of alpha-promoter transcription. Expression vectors driven by either the rous sarcoma virus-chloramphenicol acetyl transferase (RSVCAT) or the human alpha-gene (alpha CAT) promoters were transfected into cultures of dispersed female rat pituitary cells using calcium phosphate (CaPO4), diethylaminoethyl-dextran, lipofection, and electroporation procedures. CAT activity was optimal using the CaPO4 technique, resulting in 511 +/- 49% and 57 +/- 5% conversion/100 micrograms protein/4 h for RSVCAT and alpha CAT, respectively. Immunohistochemical analyses of alpha CAT expression using anti-CAT monoclonal antibodies demonstrated that the alpha-gene promoter is expressed in pituitary cells, predominantly if not exclusively, in gonadotropes and thyrotropes. Hormonal regulation of alpha-promoter activity was assessed using both the CAT and the luciferase (LUC) reporter systems. alpha-Promoter activity was significantly (P less than 0.001) stimulated by 8-bromo-cAMP (217% increase), GnRH (75% increase), GnRH agonist analog (141% increase), and TRH (75% increase). The expression of control plasmids (RSVLUC, TKLUC, pOLUC) was not affected by treatment with these agents. We conclude that CaPO4-mediated transfection allows analyses of transient gene expression in primary pituitary cells. The alpha-promoter directs expression specifically in pituitary cells, predominantly gonadotropes and thyrotropes. alpha-Gene transcription is stimulated by GnRH, TRH, and 8-bromo-cAMP.

A sensitive and specific radioimmunoassay has been developed for rat prolactin (rPRL), employing the double antibody solid phase technique for the separation of free and antibody-bound [125I]rPRL. The anti-serum was raised in rabbits and showed no cross-reaction with rat growth hormone (rGH), follicle stimulating hormone (rFSH), luteinizing hormone (rLH) and thyrotrophin (rTSH). The immunosorbent (sheep anti-rabbit IgG bound to cellulose) showed a surprisingly high binding of [125I]rPRL, but not of the other iodinated anterior pituitary hormones. Addition of serum to the incubation mixtures prevented the binding between [125I]rPRL and the immunosorbent. Three different clonal strains of pituitary cells have been examined for production of rPRL, rLH and rTSH, both in the basal state as well as after treatment with thyrotrophin releasing hormone (TRH) and gonadotrophin releasing hormone (LH/FSH-RH). Monolayer cultures of two of the cell strains produced and secreted rPRL spontaneously, and they showed a 2-fold increase in rPRL production after treatment with TRH (3-10(-7) mol/1). The third cell strain did not produce rPRL spontaneously, or after treatment with TRH. None of these cell strains could be stimulated to produce rTSH by treatment with TRH. Treatment of the same three cell strains with LH/FSH-RH (1.2-10(-6) mol/1) failed to induce production of rLH, and there were no changes in production of rPRL. Prostaglandins E1 and E2 (3-10(-8) mol/1) and oestradiol-17beta (10(-7)-10(-10) mol/1), however, stimulated the production of rPRL. The effects of TRH and prostaglandins E1 and E2 were observed within 24 h of treatment, while the first effect of oestradiol-17beta was seen after 3 days. These results suggest that the stimulatory effect of oestradiol-17beta on rPRL production differs from that of TRH and prostaglandins.

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.