TSH Cells Research Articles

Cytochemical characterization of anterior pituitary target cells for the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), using biotinylated ligands

Two novel peptides, named PACAP (pituitary adenylate cyclase activating polypeptide) containing 38 (PACAP38) and 27 residues (PACAP27) were recently isolated from ovine hypothalami. In order to investigate the pituitary cell type(s) that bear a receptor for PACAP, PACAP38 was biotinylated and used for cytochemical examination of binding. The cells were also identified by immunocytochemical methods using the antisera against each of the rat anterior pituitary hormones or an antiserum against S-100 protein, a marker for pituitary folliculo-stellate (FS) cells. Biotinylated PACAP38 (biot-PACAP) exhibited adenylate cyclase stimulating activity (ACSA) comparable to PACAP38 in rat pituitary cell cultures, and displaced the bound 125I-PACAP27 to the rat pituitary membrane preparation to the same extent as PACAP38. After 2–4 days of culture, dispersed rat pituitary cells were incubated with varying concentrations of biot-PACAP at room temperature or 4°C. The bound biot-PACAP38 was visualized by avidin-biotin-peroxidase complex (ABC) method with nickel intensification. Biot-PACAP-positive and pituitary hormone or S-100-positive cells were counted. More than 90% of S-100-positive cells bound biot-PACAP38. A considerable number of GH and PRL cells and a lesser number of ACTH cells also bound biot-PACAP38, whereas only a few identified LH, FSH, or TSH cells bound biot-PACAP38. These results suggest that FS cells are a major target cell type for PACAP. A recent study from our laboratory demonstrated that PACAP stimulated the release of interleukin (IL)-6 in rat pituitary cell cultures. FS cells are known to produce IL-6.

Differential response of amphibian PRL and TSH pituitary cells to in vitro TRH treatment

Hypophysial prolactin (PRL) and thyrotropin (TSH) cells of Rana perezi were examined after treatment in vitro with synthetic thyrotropin-releasing hormone (TRH). Ultrastructural morphometry applied to PRL and TSH immunoidentified cells estimated the volume density ( V v) of the secretory granules (SG), rough endoplasmic reticulum-(ER), and Golgi complex (GC) as well as the numerical density ( N v) of the granules. Hemipituitaries were cultured in a superfusion system with or without 100 ng TRH/ml for 4, 14, or 24 hr. PRL cells showed significant degranulation (42%) of medium size SG after 14 hr of stimulation, whereas the biosynthetic machinery (ER and GC) was significantly developed after 4 hr of TRH culture (increase of V v of GC, 1.5-fold, and ER, 1.3-fold, in comparison to the control). Most of these changes remained after 24 hr of TRH treatment. Compared with controls, TRH-treated TSH cells differed only after 24 hr when SG showed degranulation (40%), mainly of the medium size ones, and V v of GC (1.6-fold) and ER (1.3-fold) increased, These results suggest that TRH acts directly on amphibian PRL and TSH cells stimulating hormonal synthesis and release. The time courses of responses to TRH differed in that PRL cells gave an immediate response while TSH cells gave a more delayed reaction.

Pituitary adenylate cyclase-activating polypeptide stimulates calcium mobilization in amphibian pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily. Although PACAP is a potent stimulator of adenylate cyclase activity in the adenohypophysis, the precise target cells for PACAP in the anterior pituitary remain unknown. The aim of the present study was to investigate whether PACAP could stimulate calcium mobilization in individual cells of the pituitary and to determine the type of cells that responded to PACAP. Enzymatically dispersed frog distal pituitary cells were plated on photoetched coverslips and cultured for 3-7 days. The cells were loaded with the fluorescent calcium indicator indo-1, and changes in intracellular calcium concentrations ([Ca2+]i) were monitored using dual wavelength microfluorimetry. The individual cells were localized with the aid of the alpha/numeric grid of the coverslips and identified retrospectively by immunofluorescence. Approximately 45% of GH and PRL cells and 25% of ACTH and TSH cells responded to PACAP (10(-5) M) ejection by an elevation of [Ca2+]i. Only 16% of gonadotropes were stimulated by PACAP. The time course of [Ca2+]i variations showed three different patterns: transient spikes, sustained stimulations, and oscillatory responses. In addition, heterogenous responses were observed within each cell type. These data provide evidence for the involvement of calcium mobilization in the mechanism of action of PACAP on pituitary cells. The results also indicate that in frogs, PACAP may stimulate the secretory activity of GH and PRL cells and, to a lesser extent, ACTH, TSH, and gonadotrope cells.

Histopathological and biochemical effects of gossypol acetate on pituitary-gonadal axis of male albino rats

Histopathological and biochemical effects of gossypol acetate (GA) on pituitary-gonadal axis were investigated. 10 and 25 mg GA/kg were administered orally to sexually mature adult male Wistar rats for 4 and 5 weeks, respectively. STH and LTH/PRL cells showed no significant changes as compared to those of controls while TSH cells showed hypertrophy, hyperplasia and degranulation in both experimental groups. Pituitary FSH,LH/ICSH cells showed progressive regression. Gonosomatic indices of sex accessory glands at 25 mg showed significant reduction in the experimental animals as compared to those of controls. The diameter of seminiferous tubules reduced and azoospermia developed. Sertoli and Leydig cells also regressed. At 10 and 25 mg GA treatment, spermatogenesis ceased at secondary spermatocytes and spermatogonia stages,respectively. Epididymis and prostate regressed. Seminal vesicle showed no significant histological variations as compared to that of control except reduction in secretion.Biochemical observations revealed increased levels of acid phosphatase, fructose and citric acid and significant reduction in glycerylphosphoryl choline in reproductive glands of both experimental groups as compared to those of controls. Possible mechanism of antifertility action of GA is discussed.

The alpha-subunit of glycoprotein hormones exists in the prolactin secretory granules of the bullfrog (Rana catesbeiana) pituitary gland.

Our recent finding that the number of immunoreactive alpha-subunit cells was invariably greater than the total number of immunoreactive gonadotropin (GTH) and thyrotropin (TSH) cells in the bullfrog (Rana catesbeiana) pituitary gland raises the possibility that the alpha-subunit also exists in pituitary cells other than GTH and TSH cells. The present study demonstrates that there are a considerable number of immunoreactive prolactin (PRL) cells that are also stained with antibody against the alpha-subunit when adjacent sections are immunocytochemically examined. Neither immunoreactive growth hormone nor adrenocorticotropin cells are stained with the antibody against the alpha-subunit. The specificity of the antibody against the alpha-subunit and of that against PRL was demonstrated by preabsorption test, non-competitive binding test, and immunoblot analysis. Double-immunolabeling with gold particles of different sizes for the alpha-subunit and PRL revealed that most of the immunolabeled PRL-secretory granules are also labeled with the alpha-subunit antibody. The gold particles indicating the presence of the alpha-subunit were mostly found in the peripheral zone of the secretory granules.

A cell line that produces the glycoprotein hormone alpha-subunit contains specific nuclear factors similar to those present in thyrotropes.

A unique characteristics of thyrotrope-specific gene expression is the coordinated expression and regulation of the alpha- and beta-subunits of TSH. A cell line (alpha TSH) derived from the transplantable mouse thyrotropic tumor MGH101A, which no longer expresses the TSH beta-subunit gene but continues to secrete large amounts of alpha-subunit, was used as a model to study alpha-subunit gene expression independent from the TSH beta-subunit gene and was compared with the expression in TSH-secreting TtT97 tumors. Transient transfection studies showed a striking similarity in the activity of 5' deletions of the mouse alpha-subunit gene promoter in both alpha TSH and TtT97 cells and localized two regions important for expression that spanned 100 base pairs, from -480 to -417 and from -417 to -381. These regions were found to have no activity in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Analysis of the alpha-subunit 5' flanking DNA interactions with alpha TSH and TtT97 nuclear extracts showed two DNase I protected sequences, from -474 to -452 and from -447 to -400, both of which colocalized with the functionally important regions. Gel retardation analysis demonstrated the specificity of these interactions, and a similar migration of the DNA-protein complexes suggested that protein factors were similar in the two cell types. We conclude that the nuclear factors necessary for alpha-subunit expression in thyrotropes are retained in alpha TSH cells. Moreover, since alpha TSH cells do not express the TSH beta-subunit gene, the factors that determine the expression of the alpha-subunit may not be sufficient for TSH beta-subunit gene expression.

Triiodothyronine Receptor β-2 Messenger Ribonucleic Acid Expression by Somatotropes and Thyrotropes: Effect of Propylthiouracil-Induced Hypothyroidism in Rats*

mRNA for a thyroid hormone receptor isoform that is unique to the pituitary gland (TR beta-2) is down-regulated by T3. Increases in the expression of this mRNA are seen in rats rendered hypothyroid by treatment with propylthiouracil (PTU). This study used dual labeling to determine which pituitary cells expressed TR beta-2 mRNA in normal and PTU-treated rats. In situ hybridization protocols localized the mRNA (with biotinylated complementary oligonucleotide probes detected by avidin-biotin-peroxidase), and immunoperoxidase protocols identified the pituitary hormone proteins. In dispersed pituitary cells, 20 +/- 2% (average +/- SD) of cells from normal rats and 30 +/- 3% of cells from PTU-treated rats were labeled for TR beta-2 mRNA. PTU caused increases in the area of the labeled cells (from 114 +/- 11 to 225 +/- 7 microns 2), the area of the label per cell (from 27 +/- 3 to 71 +/- 11 microns 2), and label density. PTU produced increases in the percentage of TSH cells from 8 +/- 1% to 19 +/- 2%, decreases in the percentage of GH cells from 27 +/- 3% to 11 +/- 2%, and no change in other cell types. After dual labeling, 73% of cells that expressed TR beta-2 mRNA stored either TSH (35 +/- 8) or GH (38 +/- 6). Less than 10% stored other hormones. When each cell type was analyzed, 56 +/- 3% of TSH cells and 43 +/- 4% of GH cells expressed TR beta-2 mRNA. When these percentages were multiplied by the percentages of each cell type in the overall population, TSH and GH cells with TR beta-2 mRNA represented 6.8 +/- 1% and 11.6 +/- 1% of the pituitary cells, respectively. Less than 1% of all pituitary cells expressed TR beta-2 and ACTH (0.9 +/- 0.06), LH (0.8 +/- 0.1), FSH (0.8 +/- 0.1), and PRL (0.9 +/- 0.04). PTU treatment increased the percentage of TSH cells with TR beta-2 mRNA to 72 +/- 4% and decreased the percentage of GH cells with TR beta-2 mRNA to 30 +/- 3%. However, some enlarged putative TSH cells could not be identified by immunolabel because the storage levels were low. Thus, changes in TR beta-2 mRNA in hypothyroid rats may be the net result of the increase in the percentage of TSH cells, the amount of mRNA per cell (measured by area and density of label), and the decrease in the percentage of GH cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromogranin A and B in Adenomas of the Pituitary

Forty-two pituitary adenomas (10 prolactinomas; three ACTH-, nine GH-, two FSH- and two TSH-secreting adenomas; and 16 clinically nonfunctioning null cell adenomas) were investigated immunohistochemically with antibodies against chromogranin A and B as well as ACTH, GH, prolactin (PRL), FSH, LH, TSH, and alpha-HCG antibodies. For the demonstration of chromogranin B, two different antibodies were used--e.g., a polyclonal antihuman antibody and an antiserum against a synthetic peptide (DK-21, chromogranin B 306-326) present in the chromogranin B amino acid sequence. All tumors were positive for both chromogranin B antibodies. Chromogranin A was found in FSH- (two of two) and TSH- (two of two) secreting adenomas; it was also found in a focal distribution in ACTH- (one of three) and GH- (four of nine) secreting adenomas. Thirteen of 16 null cell adenomas contained chromogranin A, whereas no chromogranin A was found in prolactinomas. We conclude that null cell adenomas may arise either from FSH/LH or TSH cells (null cell adenomas with both chromogranin A and B positivity) or from ACTH, GH, or PRL cells (the respective tumors are only positive for chromogranin B). Chromogranin B may be used as a universal marker for pituitary adenomas.

Immunohistochemistry of the pituitary pars distalis of the musk shrew, Suncus murinus

Immunocharacteristics of the pituitary pars distalis cell types of the musk shrew, Suncus murinus, were studied by the unlabeled antibody enzyme technique, using peroxidaseantiperoxidase or avidin-biotin-peroxidase complex. The thyrotropin (TSH)-, gonadotropin (GTH)-, corticotropin (ACTH)-, prolactin (PRL)-, and growth hormone (GH)-secreting cells of the PD were identified on the basis of their immunoreactivity with different heterologous antisera. The TSH cells showed specific immunoreactivity with antisera against human (h) TSHβ and rat (r) TSHβ. Cells showing immunoreactivity with the antisera against hLHβ and ovine (o) LHβ were designated as GTH cells as no immunoreactivity was observed with antisera against hFSHβ and oFSHβ. The ACTH cells as well as the cells of the pars intermedia were revealed by anti-ACTH 1–24 and anti-ACTH 1–10 sera. Whereas the PRL cells were recognized by their immunoreactivity with antisera against hPRL and oPRL, the GH cells were identified with anti-hGH, anti-oGH, and anti-bovine (b) GH sera. TSH and GTH, TSH and ACTH, GTH and ACTH, ACTH and GH, ACTH and PRL, and GH and PRL cells were visualized in the same section using the dual immunoperoxidase technique. Comparison of the immunohistochemically identified cells with those described histochemically reveals several discrepancies, which expose the limitations of the latter techniques identifying adenohypophysial cells.

Effects of ovine CRF on amphibian pituitary ACTH and TSH cells in vivo: A quantitative ultrastructural study

The subcellular responses of amphibian adrenocorticotropic (ACTH) and thyrotropic (TSH) pituitary cells to the in vivo administration of ovine corticotropin-releasing factor was investigated. For this purpose, groups of six Rana perezi adult frog (three males and three females) were given daily injections of ovine CRF and sacrificed at 6 hr, 24 hr, and 4 days after the first injection. Immunogold staining, applied to ultrathin sections using antisera to human ACTH (1–39) and human β-TSH identified ACTH and TSH cells, respectively. Morphometry was used to evaluate the volume density (Vv) changes of the rough endoplasmic reticulum, Golgi complex, and secretory granules and the numerical density of the latter. CRF significantly reduced the Vv of the secretory granules in both cell types, taken as indicative of short-term enhanced hormonal release. The peptide also stimulated the development of the cellular biosynthetic machinery, although this effect was detected at an earlier stage in ACTH cells than in TSH cells. These results show for the first time the occurrence of cellular response of amphibian adrenocorticotropes and thyrotropes to CRF and suggesting that this peptide regulates ACTH and TSH production. Moreover, each type of cell differed in its sensitivity to the peptide. After long-term treatment the cytological response of ACTH cells to CRF seemed to decrease, while TSH cells remained active.

Cytochemical Studies of Responses of Corticotropes and Thyrotropes to Cold and Novel Environment Stress

Cold stress stimulates the release of both ACTH and TSH from the pituitary. More striking changes in ACTH content have been seen in the intermediate lobe after cold stress. Therefore, this study was designed to test responses of individual anterior lobe corticotropes to cold exposure. Male rats were exposed to either 30 min of cold (+3-5 C), 30 min of a novel, temperate environment (+24 C) or were unstressed (+24 C). Pituitaries were fixed and embedded in preparation for immunolabeling for ACTH or TSH-beta at the light (semithin sections) and electron microscopic levels. The semithin sections were used to measure areas of corticotropes and thyrotropes with Bioquant image analysis equipment. Separate groups of pituitaries were dissociated and the cells were cultured for 2 or 15 h. Then the cells were stimulated for 5-10 min with biotinylated analogs of corticotropin-releasing hormone (bio-CRH) or arginine vasopressin (bio-AVP) to detect the target cells cytochemically. A third group of dissociated cells were fixed for immunolabeling for ACTH, 16K fragment of pro-opiomelanocortin, beta-endorphin, or TSH-beta. Cold exposure resulted in a 1-4-fold increase in the levels of serum ACTH over that of unstressed rats. This was correlated with a 40% increase in the percentage of cells that contained 16K fragment and a 30-40% increase in percentages of cells storing ACTH or beta-endorphin. Cold stress also increased the percentage of cells that bound bio-CRH or bio-AVP by 45%. Analyses of semithin sections showed that areas of corticotropes increased by 21% following cold stress. The number of rows of immunolabeled (ACTH) secretion granules also increased in corticotropes from cold-stressed rats. Exposure to a novel environment for 30 min resulted in no significant increase in serum ACTH over that of unstressed rats. There was, however, a 20% increase in percentages of cells that stored 16K fragment, beta-endorphin, or target cells that bound bio-CRH. However, the corticotropes were not significantly larger. Many of the cells exhibited reduced numbers of immunolabeled secretory granules. Other corticotropes resembled those from cold-stressed rats. When TSH cells were studied, their percentages increased from 8 +/- 3% to 15.8 +/- 4% and their areas increased by 22% following exposure to cold. After exposure to a novel environment, percentages of cells that stored TSH-beta increased to 11 +/- 2%, however, no changes in areas of TSH cells were measured. These studies demonstrated that the anterior lobe corticotrope is clearly activated by exposure to both cold and novel environment.(ABSTRACT TRUNCATED AT 400 WORDS)

Two Types of Thyrotropes (TSH Cells) in the Adenohypophysis of the Untreated Mouse

Employing immunohistochemical techniques with OsO4 single fixation, we identified mouse pituitary thyrotropes (TSH cells). The cells stained with TSH antiserum are angular or slender with a small nucleus. These cells contain small secretory granules (about 120-200 nm in diameter) and numbers of cored vesicles, either attached to the cell membranes or lying free in a relatively electron transparent cytoplasm. In the untreated male mouse, the TSH cell modifies its morphology according to the functional phase. The first form of TSH cells [Type I] is small. The endoplasmic reticulum and the Golgi apparatus are not prominent, and secretory granules are very few. In the second hypertrophied form [Type II], the endoplasmic reticulum is very prominent and occurs as a series of grossly dilated sacs of irregular shape. The Golgi apparatus is greatly enlarged and a large number of electron-dense secretory granules and cored vesicles are observed. Type II thyrotropes are rarely encountered in normal tissue.

Identification of Lactotropes (PRL Cells), Somatotropes (GH Cells), Corticotropes (ACTH Cells) and Thyrotropes (TSH Cells) in the Pituitary Gland of the Musk Shrew, <i>Suncus murinus</i> L. (Insectivora), by Immunohistochemistry

By means of immunohistochemistry, lactotropes, somatotropes, corticotropes and thyrotropes in the pituitary of the male musk shrew, Suncus murinus L., were identified at the electron-microscopic level. Lactotropes were classified into three types: type I containing large (200-450 nm in diameter) round secretory granules, type II with medium-sized (150-250 nm in diameter) ones and type III with small (50-150 nm in diameter) ones. Somatotropes were also classified into type I somatotropes that contain large (450 nm in diameter) spherical secretory granules and type II somatotropes containing comparatively small (300 nm in diameter) round granules. Both type I and II somatotropes were small and sometimes contained rod-shaped granules. Corticotropes were round or oval cells with round secretory granules in various densities and sizes (150-500 nm in diameter) scattered all over the cytoplasm. Thyrotropes were angular or polyhedral cells containing electron-transparent round secretory granules (200-300 nm in diameter) and large irregularly shaped granules with a maximum diameter of about 1,500 nm. Each type of the cells may be distinguished by its respective ultrastructural characteristics alone without the aid of immunohistochemistry.

Immunocytochemical localization of the subunits of glycoprotein hormones (LH, FSH, and TSH) in the bullfrog pituitary gland using monoclonal antibodies and polyclonal antiserum

Using β and α subunits of bullfrog follitropin (FSH) III (p I 6.2), which were highly purified by HPLC, we generated three monoclonal antibodies (MCAs) to FSH β subunit (FSHβ) and six to FSH α subunit (FSHα). They were produced by hybridomas derived from the myeloma X63.Ag8.653 and spleen lymphocytes from mice immunized with each subunit. Non-competitive binding tests revealed that one of the MCAs against FSHβ (131731325) bound strongly to intact FSH and its β subunit, but not FSHα, lutropin (LH), LHα, and LHβ. The immunoblotting results also showed a similar immunological specificity for BF3B25. Cross-reactivity of bullfrog FSH against 131731325 was 19.4%, when compared with FSβ in the competitive inhibition assay system. On the other hand, noncompetitive binding tests and immunoblotting results showed that one of the MCAs against FSHα (B173A20) bound strongly to intact LH and FSH and their a subunits, but not their (3 subunits. The inhibition curves obtained using the a subunits of LH and FSH were similar. In the sexually mature bullfrog pituitary, immunoreactive FSH cells stained with MCA BF3B25 were distributed throughout the pars distalis, except for the rostral region, and were polygonal in shape, with well-developed cytoplasm. With respect to distribution and histological characteristics, the immunoreactive LH cells were very similar to the immunoreactive FSH cells when consecutive sections were stained with LHβ-specific MCA (BL4B11). However, immunoreactive TSH cells, revealed by anti-human TSH(3 serum, formed clusters in the ventrocentral region of the pars distalis. In young adult pituitary, almost all of the gonadotrophs showed the coexistence of FSH and LH, but some gonadotrophs contained only FSH. The number of immunoreactive α-subunit cells stained by B173A20 was always higher than the sum of the numbers of cells stained by the three β-subunit-specific antibodies.

Cytological, immunocytochemical and ultrastructural study of the adenohypophyseal pars distalis of the kid (Capra hircus): the TSH cell.

The structure and ultrastructure of the adenohypophyseal pars distalis in kids of the Murciano-granadiana breed (18 males and 12 females), aged 2-3 months and with an average carcass weight of 8 kg has been studied. Techniques of staining (Tetrachrome Herlant's, and Cleveland-Wolfe) and histochemistry (PAS, PAS-OG and BA-PAS-OG) contrasted with immunolabelling (PAP) have been used. In addition an ultrastructural study has been made and nucleus and cytoplasm, secretory granules, mitochondria and lysosomes have been measured with a semiautomatic image analyzer (IBAS-1). TSH cells are found in sagittal section in the anterior area and in an antero-caudodorsal band, and transversally in the ventral and medial region; they are 6% and their average size is 14.15 microns. Ultrastructurally they are a single cellular type with spherical granules whose size is 195 nm in males and 149 in females; these granules are characterized by a clear halo and a variable electronic density. The rough endoplasmic reticulum is found as slightly enlarged vesicles with a homogeneous and moderately electro-dense content or in concentric stratifications.

Circadian and pulsatile thyrotropin release in treated acromegalics.

We studied the 24-h TSH profiles of 16 treated male acromegalic patients (age range 26-68 yr) in clinical and biochemical remission. Eight had undergone transsphenoidal surgery, the others surgery and pituitary irradiation. Blood samples were taken at 20-min intervals; circadian rhythms were established by cosinor analysis, pulsatile release with the Cluster programme. All patients, except one irradiated subject, were euthyroid. TSH reserve was diminished preoperatively in 7 subjects and at the time of the profile study in 10 subjects, one of whom was biochemically hypothyroid. A significant circadian rhythm was present in 14 subjects and absent in the hypothyroid patient. The acrophase occurred at 2.46 +/- 0.51 h in nonirradiated patients and at 3.37 +/- 0.38 h in irradiated patients (NS). About 10 TSH pulses/24 h (range 6-13) were detected; there was no significant difference between irradiated and non-irradiated patients. With cross-correlation techniques synchronous release of TSH and PRL was demonstrated in 7 out of 8 nonirradiated patients in contrast to only 2 of the irradiated patients. This study demonstrates a qualitatively normal TSH secretion pattern for treated acromegalic patients, but the absolute TSH levels are clearly low compared with published data on normal subjects. The present findings can be explained by a diminished TSH cell mass; in addition radiation therapy causes a disturbance at the hypothalamic level, as indicated by the loss of synchronism between TSH and PRL release.

Reverse Hemolytic Plaque Assay Study of Luteinizing and Follicle-Stimulating Hormone and Thyrotropin Secretion in Diabetic Rat Pituitary Glands

Numerous studies indicate that an impaired hypothalamopituitary axis plays an important role in reproductive and thyroid disorders in diabetic humans and animal models. Yet, several questions about the pathogenesis of these diabetic complications have not been answered. To evaluate the basal secretion of single gonadotrophs and thyrotrophs in vitro, uncultured pituitary cells from control rats and 1-mo streptozocin-induced diabetic (STZ-D) rats were studied with a reverse hemolytic plaque assay and morphometry. After light-microscopy immunocytochemistry for gonadotropin and thyrotropin (TSH), we recorded the ratio of plaque-forming to non-plaque-forming cells. The area of plaques produced by luteinizing hormone (LH), follicle-stimulating hormone (FSH), and TSH cells and the area of plaque-forming and non-plaque-forming cells were clearly smaller in diabetic than control rats. The plaque area, however, was more severely reduced than the cell area. The percentage of LH-, FSH-, and TSH-immunoreactive plaque-forming cells was greatly decreased in diabetic compared with control animals. In conclusion, our findings demonstrate that the LH-, FSH-, and TSH-secreting cells of diabetic rats released less hormone and were less numerous than the corresponding cells of control rats. Thus, several pathogenetic mechanisms might be involved in reduced gonadotropin and TSH release at the cellular level: 1) anatomical lesions of organelles involved in glycoprotein hormone synthesis and secretion, possibly due to insulin deficiency; 2) decreased gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) receptors on pituitary cells; 3) inadequate GnRH and TRH stimulation; 4) high plasma corticosterone levels; or 5) a combination of points 1-4.

Postnatal development of thyrotrophs in the rat anterior pituitary as studied by immunogold electron microscopy.

Thyrotrophs (TSH cells) of the rat anterior pituitary identified by immunogold electron microscopy were classified into three subtypes according to their morphological characteristics: Immature type TSH cells are oval with a few small secretory granules (50-100 nm in diameter) and poorly developed cell organelles. These cells are frequently found in the neonatal stage between birth and 10 days of age. The intermediate type is polygonal or stellate, containing a moderate number of medium sized secretory granules (80-120 nm in diameter) and moderately or well developed cell organelles. Cells of this type are often found between 10 and 30 days of age. Mature type cells are large and polygonal in shape, and contain many large secretory granules (120-180 nm in diameter) and well developed cell organelles. Cells of the last type are frequently found at more than 30 days of age. At 45 days of age the mature type TSH cells make up about 70% of all TSH cells. The proportion of immature type cells was shown to decrease while the proportion of the mature type TSH cells increases, as the animal grows.

Characterization of Anterior Pituitary Target Cells for Arginine Vasopressin: Including Cells that Store Adrenocorticotropin, Thyrotropin-β, and Both Hormones*

Arginine vasopressin (AVP) stimulates the secretion of ACTH and TSH. Recently, affinity cytochemical techniques with a potent biotinylated analog of AVP were used to identify ACTH cells as one of the target cells. Counts showed that AVP-bound cells were 10% of the population. However, if AVP bound all corticotropes and thyrotropes, one would expect AVP to bind at least 16% of the pituitary cells. Therefore, dual cytochemical labeling protocols were used to learn if thyrotropes also bound AVP (bio-AVP). Forty-eight percent of AVP target cells contained ACTH, and 42% contained TSH beta. The percentages of AVP-bound cells were increased to 12-13% of the total pituitary cells after 1-h pretreatment in 10 nM TRH or CRH. TRH and CRH together stimulated increases to 16% of the total cells. Analysis of dual labels showed that the additional AVP-bound cells stimulated by CRH or TRH stemmed from the corticotrope or thyrotrope populations, respectively. TRH stimulated an increase in the percentage of TSH cells that bound AVP from 55% to 75%. Similarly, CRH stimulated an increase in the percentage of ACTH cells that bound AVP from 61% to 79%. In addition, the populations of cells labeled for TSH beta or ACTH antigens increased by 30% after 1 h in unlabeled AVP, supporting its direct effect on these target cells. TRH stimulated a similar increment in TSH cells. CRH pretreatment had no effect on the percentages of cells labeled for TSH or ACTH. This could be the result of loss of ACTH stores needed to identify stimulated corticotropes. Finally, analysis of the total percentages of AVP-bound TSH beta or ACTH cells suggested an overlap in the population. This stimulated the application of dual labels for ACTH and TSH beta. In populations exposed to vehicle only, 1-2% of mixed pituitary cells stored both ACTH and TSH. This unique cell type also comprised 10% of a corticotrope population enriched by counterflow centrifugation. The percentage of ACTH-TSH cells in the mixed cell population was augmented to 4.8% after 1 h in AVP. It was not affected by exposure to either TRH or CRH (or both peptides). These studies demonstrate that AVP target cells include thyrotropes, corticotropes, and unique cells that store both ACTH and TSH.

Immunohistochemical study on the development of the adenohypophysial cells in the lizard Gallotia galloti

Immunohistochemical methods have been used to study the embryonic and postnatal development of the hormone-producing cells in the adenohypophysis of the lizard Gallotia galloti. In this species, Rathke's pouch is formed between stages 30 to 32 of the embryonic development, although the first sign of immunoreactivity to antisera against adenohypophysial hormones occurs in stage 33 in the pars distalis anlage. These cells derive from the dorsal face of Rathke's pouch and are immunoreactive to anti-ACTH serum. The cytodifferentiation of ACTH and MSH cells occurs in the pars intermedia in stage 34. The TSH cells appear at stage 35 and the gonadotrope cells at stage 37. These cells derive from both the dorsal and ventral face of the Rathke's pouch. The LTH cells are revealed at stage 39 and are only originated from the dorsal face. The STH cells, which come from the dorsal as well as ventral face, are the last secretory cells differentiated just before hatching. During postnatal development an increase and also a redistribution of the immunoreactive cells occur until acquiring the adult distribution.