A non-autophagic pathway for diversion of ER secretory proteins to lysosomes

Intracisternal granules (ICG) develop in the rough ER of hyperstimulated thyrotrophs or thyroid hormone-secreting cells of the anterior pituitary gland. To determine the fate of these granules, we carried out morphological and immunocytochemical studies on pituitaries of thyroxine-treated, thyroidectomized rats. Under these conditions the ER of thyrotrophs is dramatically dilated and contains abundant ICG; the latter contain beta subunits of thyrotrophic hormone (TSH-beta). Based on purely morphologic criteria, intermediates were identified that appeared to represent stages in the transformation of a part rough/part smooth ER cisterna into a lysosome. Using immunocytochemical and cytochemical markers, two major types of intermediates were distinguished: type 1 lacked ribosomes but were labeled with antibodies against both ER markers (PDI, KDEL, ER membrane proteins) and a lysosomal membrane marker, lgp120. They also were reactive for the lysosomal enzyme, acid phosphatase, by enzyme cytochemistry. Type 2 intermediates were weakly reactive for ER markers and contained both lgp120 and lysosomal enzymes (cathepsin D, acid phosphatase). Taken together these results suggest that in hyperstimulated thyrotrophs part rough/part smooth ER elements containing ICG lose their ribosomes, their membrane is modified, and they sequentially acquire a lysosome-type membrane and lysosomal enzymes. The findings are compatible with the conclusion that a pathway exists by which under certain conditions, secretory proteins present in the ER as well as ER membrane and content proteins can be degraded by direct conversion of ER cisternae into lysosomes.     

An immunocytochemical study of TSH beta storage in rat thyroidectomy cells with and without D or L thyroxine treatment.

Binding sites to the beta chain of thyroid stimulating hormone (TSH) were localized in pituitaries of thyroidectomized rats. Immunocytochemical staining was observed in hypertrophied TSH cells ("thyroidectomy cells") and primarily located in dilated rough endoplasmic reticulum. Staining was also found on the few secretory granules and on some of the intracisternal granules. Some of the thyroidectomy cells stained intensely, while others exhibited very little staining. When thyroidectomized rats were treated with thyroxine 4 days before death, the TSH cells contained more secretory granules, and the intracisternal granules were larger and more numerous. L-thyroxine was 10 times as potent as D-thyroxine in promoting the build-up of granules. Both types of granules stained intensely.     

Effects of thyroidectomy, propylthiouracil, and thyroxine on pituitary content and immunocytochemical staining of thyrotropin (TSH) and thyrotropin releasing hormone (TRH)

Previous studies have demonstrated immunocytochemical staining for beta chains of thyroid stimulating hormone (TSH-beta) in rough endoplasmic reticulum of pituitary cells hypertrophied after thyroidectomy ("thyroidectomy cells") (Moriarty CG(1976): J Histochem Cytochem (24:846; Moriarty GC, Tobin RB (1976): J Histochem Cytochem 24:1140). Here we report the localization of thyrotropin releasing hormone (TRH) in serial sections of the same pituitaries to determine if it could be found at similar sites. No staining for TRH was found in hypertrophied TSH cells formed 42 days after the surgery, or after 14, 34, and 70 days of propylthiouracil (PTU) treatment. The loss in immunostaining in the PTU-treated rats was correlated with radioimmunoassay (RIA) measurements that showed a 65% reduction in anterior pituitary TRH content after 34, 70, and 98 days of PTU treatment (from 22.9--7.8 pg/mg wet wt) and a 50% reduction in TSH content after 34 days of treatment. When thyroxine was administered to hypothyroid rats for 3 days before death, our previous studies had demonstrated intense staining for TSH in granules inside the rough endoplasmic reticulum. In this study, the radioimmunoassay showed that TSH content rose dramatically in the hypothyroid animals treated with PTU for 77 days and thyroxine for 2 days before death (from 8.5--64.1 mU/mg wet wt); however, the rise in TRH content was minimal (5.8--9.8 pg/mg wet wt). The immunocytochemical stain for TRH correlated well with the RIA showing a weak reaction mainly on small granules in the cytoplasm. No reaction for TRH was found in rough endoplasmic reticulum. These results suggest that TRH and TSH storage sites are dissimilar in the hypothyroid rat. The presence of stain for TRH in granules in the cytoplasm suggests that it might play a role in the storage or packaging of TSH. Its absence in profiles of rough endoplasmic reticulum staining intensely for TSH suggests that it is not synthesized at this site. No definite conclusions about its origin can be drawn at this time.     

Immunocytochemistry of the pituitary glycoprotein hormones.

The storage sites of the pituitary glycoprotein hormones were identified with the use of electron microscopic immunocytochemical techniques and antisera to the beta (beta) chains of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH). The TSH cells in normal rats is ovoid or angular and contains small granules 60-160 nm in diameter. In TSH cells hypertrophied 45 days after thyroidectomy, staining is in globular patches in granules or diffusely distributed in the expanded profiles of dilated rough endoplasmic reticulum. The gonadotrophs (FSH and LH cells) exhibited three different morphologies. Type I cells are ovoid with a population of large granules and a population of small granules. Staining for FSHbeta or LHbeta was intense and specific only in the large granules (diameter of 400 nm or greater). Type II cells are angular or stellate and contain numerous secretory granules averaging 200-220 nm in diameter. They predominate during stages in the estrous cycle when FSH or LH secretion is high. Type III cells look like adrenocorticotropin (ACTH) cells in that they are stellate with peripherally arranged granules. They generally stain only with anti-FSHbeta and their staining can not be abolished by the addition of 100 ng ACTH. In preliminary quantitative studies of cycling females, we found that on serial sections FSH cells and LH cells show similar shifts to a more angular population of cells during stages of active secretion. However, the shifts are not in phase with one another. Furthermore, there are at least 1.5 times more FSH cells than LH cells at all stages of the cycle. Our collection of serial cells shows that some cells (usually type I or II) stain for both gonadotropic hormones, whereas others (usually type II or III) contain only one.     

Changes in thyrotroph and somatotroph cell populations induced by stimulation and inhibition of their secretory activity

Summary 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.     

Electron-microscopic study on the anterior pituitary gland of spontaneous dwarf rats

Summary The spontaneous dwarf rat is a novel experimental model animal on the study of pituitary dwarfism. The fine structure of the anterior pituitary cells was studied in the immature and mature dwarf rats. Pituitary glands were removed from 5-, 10-, 20-day-old immature dwarfs, adult (45 days-16 weeks) dwarfs and normal 3-month-old rats and processed for electron-microscopic observation. In the control animals, growth hormone cells were readily identified by their ultrastructural characteristics, such as the presence of numerous electron-dense secretory granules, 300–350 nm in diameter, well developed rough endoplasmic reticulum and a prominent Golgi complex. In contrast, growth hormone cells were not found in the anterior pituitary gland of the spontaneous dwarf rat at any age examined. Other pituitary cell types, i.e., luteinizing hormone/ follicle stimulating hormone, thyroid stimulating hormone, adrenocorticotropic hormone and prolactin cells, appeared similar in their fine structure to those found in the control rats. In the pituitary gland of dwarf rats, a number of polygonal cells were observed either with no or relatively few secretory granules. The rough endoplasmic reticulum was arranged in parallel cisternae and the Golgi complex was generally prominent in these cells. In addition, many were found to have abundant lysosomes. A few minute secretory granules were occasionally observed; however, the immunogold technique failed to localize growth hormone or prolactin in the granules. The nature of these cells remained obscure in this study. Since their incidence and fine structural features, other than the secretory granules, were quite similar to those of the growth hormone cells in normal rats, we postulate that these cells are dysfunctional growth hormone cells. These results suggest that the cause of the growth impairment in the spontaneous dwarf rat is due to a defect in the functional growth hormone cells in the pituitary gland, and since other pituitary cell types appeared normal, the disorder seems to be analogous to the isolated growth hormone deficiency in the human.     

Steroid 5α-reductase type 1 immunolocalized in the anterior pituitary of intact and castrated male rats

The localization of 5α-reductase was immunohistochemically studied in the anterior pituitary of male rats, using a polyclonal antibody against 5α-reductase rat type 1. The immunoreactive cells were concentrated in the central region and on the border of the intermediate lobe in the anterior pituitary, but not in the intermediate or posterior lobe. The immunoreaction was located mostly in the cytoplasm and occasionally in the cell nuclei. The immunoreactive cells showed alterations in size and number and in the intensity of the immunoreaction after gonadectomy. One week after castration, the cells became larger and the immunoreactivity increased. Two weeks after castration, the number of immunoreactive cells increased. Double immunostaining using antiluteinizing hormone β-subunit or anti-follicle stimulating hormone β-subunit antibody revealed that most of the cells containing 5α-reductase were gonadotrophs. Electron microscopically, the immunoreactive cells showed lamelliform rough endoplasmic reticulum and a depletion of secretory granules 1 week after castration. One week later, the rough endoplasmic reticulum was developed and dilated and the number of secretory granules increased. These results suggest that 5α-reductase is located in the gonadotrophs of rat anterior pituitary and that it is involved in the feedback regulation of gonadotropin secretion by androgens.     

The pituitary-thyroid axis in patients with pituitary disorders

The pituitary-thyroid axis of 12 patients, exposed to transsphenoidal pituitary microsurgery because of nonfunctioning adenomas (6), prolactinomas (3) and craniopharyngioma (1), or to major pituitary injury (1 apoplexy, 1 accidental injury), was controlled more than 6 months following the incidents. The patients did not receive thyroid replacement therapy and were evaluated by measurement of the serum concentration of thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), T3-resin uptake test and thyrotropin (TSH, IRMA method) before and after 200 micrograms thyrotropin releasing hormone (TRH) iv. The examination also included measurement of prolactin (PRL) and cortisol (C) in serum. Apart from 1 patient with pituitary apoplexy all had normal basal TSH levels and 9 showed a significant TSH response to TRH. Compared to 40 normal control subjects the 12 patients had significantly decreased levels of T4, T3 and rT3 (expressed in free indices), while the TSH levels showed no change. Five of the patients, studied before and following surgery, had all decreased and subnormal FT4I (free T4 index) after surgery, but unchanged FT3I and TSH. The levels of FT4I were positively correlated to both those of FT3I and FrT3I, but not to TSH. The TSH and thyroid hormone values showed no relationship to the levels of PRL or C of the patients exposed to surgery. It is concluded that the risk of hypothyroidism in patients exposed to pituitary microsurgery is not appearing from the TSH response to TRH, but from the thyroid hormone levels.     

Dominant role of thyrotropin-releasing hormone in the hypothalamic-pituitary-thyroid axis

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the beta isoforms of TH receptors (TRbeta KO), or both (double KO). TRbeta knock-out (KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.     

Localization of endothelin A receptors in the rat pituitary TSH cells: light- and electron-microscopic immunohistochemical studies

Endothelins modulate hormonal secretion in the pituitary gland. Intense signaling of endothelin A receptors (ET(A)R) has been detected by in situ hybridization, binding assay and receptor autoradiography. We used light- and electron-microscopic immunohistochemistry of ET(A)R with polyclonal antibody against a synthetic peptide corresponding to the carboxyl terminus (403-427) of human ET(A)R. Immunoreactivity was observed in 6-8% of anterior pituitary cells, which were rather large polygonal or stellate cells. These cells were often clustered. Double-staining immunofluorescence showed that the ET(A)R-positive cells immunoreacted with antibody against the beta-subunit of thyroid-stimulating hormone (TSH), but not adrenocorticotropic hormone (ACTH) or lutenizing hormone beta (LHbeta). Pre- and postembedding electron-microscopic immunohistochemistry showed that ET(A)R-positive cells had vacuolated or parallel-lined rough endoplasmic reticulum (rER) and numerous round granules in their periphery and the elongated processes. By pre-embedding immunohistochemistry, diaminobenzidine tetrahydrochloride (DAB) products were shown to be mostly located around the granules and occasionally underneath the plasma membrane. By postembedding immunohistochemistry, granules in the ET(A)R-positive cells were 90-150 nm in diameter, and colloidal gold particles due to ET(A)R were associated with about 10% of these granules. These results indicate that ET(A) receptors are associated mostly with the secretory granules of TSH cells.     

Morphological and functional changes in pituitary‐thyroid axis following prolonged exposure of female rats to constant light

Light regulates numerous physiological functions and synchronizes them with the environment, in part by adjusting secretion of different hormones. We hypothesized that constant light (CL) would disturb pituitary‐thyroid axis. Our aim was to determine morphological and functional changes in this endocrine system in such extreme conditions and, based on the obtained results, to propose the underlying mechanism(s). Starting from the thirtieth postnatal day, female Wistar rats were exposed to CL (600 lx) for the following 95 days. The controls were maintained under the regular laboratory lighting conditions. After decapitation, pituitaries and thyroids were prepared for further histomorphometric, immunohistochemical, and immunofluorescence examinations. Concentration of thyroid stimulating hormone (TSH), total T₄ and T₃ (TH) were determined. Thyroid tissue of light‐treated rats was characterized by microfollicular structure. We detected no change in total thyroid volume, localization and accumulation of thyroglobulin, thyroid peroxidase, and sodium‐iodide symporter in the follicular epithelium of CL rats. The volume of follicular epithelium and activation index were increased, while volume of the colloid and serum levels of TH decreased. In the pituitary, the relative intensity of TSH β‐immunofluorescence signal within the cytoplasm of thyrotrophs increased, but their average cell volume and the relative volume density decreased. Serum TSH was unaltered. We conclude that exposure of female rats to CL induced alterations in pituitary‐thyroid axis. Thyroid tissue was characterized by microfollicular structure. Serum TH levels were reduced without accompanying increase in serum TSH. We hypothesize that increased secretion and clearance of TH together with unchanged or even decreased hormonal synthesis, resulted in decreased serum TH levels in CL group. We assume this decrease consequently led to increased synthesis and/or accumulation of pituitary TSH. However, decreased average TSH cell volume and relative volume density, together with unchanged serum TSH, point to additional, negative regulation of thyrotrophs. J. Morphol. 275:1161–1172, 2014. © 2014 Wiley Periodicals, Inc.     

Dynamics of thyroid diseases and thyroid‐axis gland masses

Thyroid disorders are common and often require lifelong hormone replacement. Treating thyroid disorders involves a fascinating and troublesome delay, in which it takes many weeks for serum thyroid‐stimulating hormone (TSH) concentration to normalize after thyroid hormones return to normal. This delay challenges attempts to stabilize thyroid hormones in millions of patients. Despite its importance, the physiological mechanism for the delay is unclear. Here, we present data on hormone delays from Israeli medical records spanning 46 million life‐years and develop a mathematical model for dynamic compensation in the thyroid axis, which explains the delays. The delays are due to a feedback mechanism in which peripheral thyroid hormones and TSH control the growth of the thyroid and pituitary glands; enlarged or atrophied glands take many weeks to recover upon treatment due to the slow turnover of the tissues. The model explains why thyroid disorders such as Hashimoto''s thyroiditis and Graves'' disease have both subclinical and clinical states and explains the complex inverse relation between TSH and thyroid hormones. The present model may guide approaches to dynamically adjust the treatment of thyroid disorders.     

TSH and prolactin secretions in Hashimoto's thyroiditis following withdrawal of thyroid hormone therapy

Changes in the pituitary-thyroid axis in patients with Hashimoto's thyroiditis following withdrawal of thyroid suppressive therapy were analyzed. The group of patients with thyroid adenoma served as control (group I). Patients with Hashimoto's thyroiditis were divided into 2 groups on the basis of serum TSH levels 8 weeks after discontinuing the exogenous thyroid hormone (group II, less than 10 microunits/ml; group III, more than 10 microunits/ml). During treatment with L-T4(200 micrograms/day) or L-T3(50 micrograms/day), there was no significant difference in serum T4-I and T3 levels among the three groups. Following L-T4 withdrawal, basal serum TSH levels were higher at 2 to 8 weeks in groups II and III than in group I. Serum TSH response to TRH was greater at 4 to 8 weeks in groups II and III than in group I. Following L-T3 withdrawal, basal serum TSH levels were higher at 1 and 2 weeks in group II than in group I, while those of group III were consistently higher during the study. Higher TSH responses to TRH were observed at 1 to 8 weeks in groups II and III. Neither basal nor TRH-induced prolactin (PRL) secretion differed significantly among the three groups. We have demonstrated that pituitary TSH secretion in patients with Hashimoto's thyroiditis is affected more by withdrawal of thyroid hormone therapy than in patients with thyroid adenoma. In addition, the present findings suggest a difference between the sensitivity of thyrotrophs and lactotrophs in Hashimoto's thyroiditis after prolonged thyroid therapy is discontinued.     

Isolated thyrotropin deficiency due to a pituitary tumour.

Hypothyroidism due to isolated deficiency of thyrotropin (TSH) associated with an enlarged sella turcica, presumably the result of a nonfunctioning pituitary adenoma, occurred in a 58-year-old man. Low serum concentrations of TSH and thyroid hormones, together with the lack of TSH response to administration of thyroid releasing hormone, indicated a pituitary deficiency of TSH. Serum values of other pituitary hormones were normal.     

A case of the syndrome of inappropriate secretion of TSH.

A girl aged 4 years with goiter and accelerated physical and skeletal growth was found to be hyperthyroid on the basis of elevated serum thyroid hormone level, nevertheless both the basal TSH and TSH responsiveness to TRH were maintained within the normal range. Serum TSH was suppressed by exogenous T3 and dexamethasone administration, but not significantly changed after propylthiouracil (PTU) treatment. The diurnal rhythmicity of anterior pituitary hormones was preserved with the high nocturnal peak of TSH and prolactin. Clinically, neither thyrotoxic signs nor evidences of pituitary tumor were observed. Her accelerated growth and elevated thyroid hormone level appeared to be induced by inappropriate secretion of TSH. In view of the literature, this is the first case of the syndrome of inappropriate secretion of TSH excluding the neoplastic origin in Japan.     

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

On the identity of the thyrotropic cell in the red-spotted newt

Summary Although the pars distalis of the red-spotted newt has previously undergone extensive cytological examination, the identity of its thyrotropic cells has remained uncertain. From the present ultrastructural study, cells of type 3 (Masur, 1969) containing granules 150–180 nm in diameter are concluded to be the thyrotropes. Such cells were found to be present in the regions of the pars distalis where thyroidectomy cells arise after ablation of the thyroid gland. Cells of type 3 are probably identical with a population of cells containing granules which stain with silver methenamine indicating the presence of a glycoprotein such as thyroid stimulating hormone (TSH). Thyroidectomy cells containing a few residual granules 150–180 nm in diameter were occasionally found in partes distales from newts killed 3 or 7 days after ablation of the thyroid gland, and were abundant in pituitaries 21 days after thyroidectomy. Only cells of type 3 responded (by vacuolation of granules) when animals were immersed in water containing 10 g/l of thyroxine. No cells of the pars distalis showed cytological change after administration of synthetic thyrotropic releasing hormone (TRH) giving additional support to the view that this hormone has no stimulatory role in amphibians.Work supported in part by research grant 1 R01 AM 16731-01 from the Institute of Arthritis, Metabolism and Metabolic Diseases and in part by grant PCM 75-17637 from the National Science Foundation