Melatonin stimulates thyroid-stimulating hormone accumulation in the thyrotropes of the rat pars tuberalis

We have reported that the unique thyroid-stimulating hormone-immunoreactive cells (TSH cells) in the intact adult and fetal rat pars tuberalis (PT) show an intense spot-like TSH immunoreaction in the perinuclear region. The present study was designed to investigate the relationship between melatonin and these unique TSH cells. We classified TSH cells in the PT (PT-TSH cells), on the basis of immunoreactivity, into spot-like stained cells (SC) and whole cytoplasm stained cells (WC), and estimated the proportion of each TSH cell type to total cells in the experimental rats by morphometry. Chronic administration of melatonin to control rats leads to an increase of WC in number but a decrease of SC. On the other hand, the intensity of TSH immunoreactivity and the number of rat PT-TSH cells significantly decreased after pinealectomy and recovered by melatonin administration. Radioimmunoassay showed that melatonin treatment increased the TSH content in the PT. Moreover, electron microscopy showed that the number of TSH secretory granules in the PT-TSH cells increased in the melatonin-replaced rats. These results demonstrated that melatonin stimulates the accumulation of TSH in the rat PT-TSH cells via secretory granule formation and suggest that melatonin regulates TSH release from PT-TSH cells.     

Microfibrils are a major component of the mesangial matrix in the glomerulus of the rat kidney

Summary Certain secretory cells in the hypophysial pars tuberalis of the Djungarian hamster display marked circannual structural alterations. The present investigation deals with the immunohistochemical properties of this cell group. A distinct TSH-like immunoreactivity was found in secretory cells of this type in the pars tuberalis of animals exposed to long photoperiods, whereas under short photoperiods the TSH-like immunoreactivity was nearly absent. In the pars distalis, the number and distribution of TSH-positive cells did not differ significantly between animals maintained under long and under short photoperiods. LH-and FSH-positive cells could not be detected in the pars tuberalis, but they are clearly present in the pars distalis of both groups of hamsters. Our immunocytochemical results suggest that photoperiodic stimuli influence the secretory activity of TSH-like immunoreactive cells in the pars tuberalis. A connection with the neuroendrocrine-thyroid axis is discussed.The study was supported by the Deutsche Forschungsgemeinschaft (Wi 558/3-1, Pe 134/2-4)     

Immunoelectron microscopic study of the luminal release of chromogranin A from rat enterochromaffin cells

 Recently we found that raising the intraluminal pressure caused an increase in the luminal release of serotonin from enterochromaffin (EC) cells and serotonin immunoreactivity normally restricted within the secretory granules was diffusely scattered over the extragranular matrix. In the present study we investigated the intracellular localization of chromogranin A, a protein co-stored with serotonin in the EC cells, after stimulating the luminal release of serotonin. In situ vascularly and luminally perfused rat duodenum was exposed to intraluminal pressure and fixed for immunoelectron microscopic study. For immunoelectron microscopy, the pre-embedding DAB reaction for serotonin combined with the postembedding immunogold reaction for chromogranin A was used. Results showed that a number of secretory granules labeled with immunogold chromogranin A immunoreactivity located close to the apical plasma membrane. Some EC cells showed that one part of the apical cytoplasm was protruded into the lumen and a number of secretory granules with immunogold labeling were included in the protruded cytoplasm. These results suggest that EC cells may release chromogranin A into the intestinal lumen together with serotonin, by means of a different manner of secretion from that in serotonin. Received / Accepted: 9 December 1998     

Cyclical changes in the pituitary gonadotrophs (GTH cells) in relation to the testicular cycle in Puntius sarana (Hamilton)

The pituitary gland of Puntius sarana is of the leptobasic type and has three subdivisions--a proximal pars distalis (PPD), a rostral pars distalis (RPD) and a pars intermedia (PI) arranged vertically one below the other. The gonadotrophs (GTH cells) of the PPD were identified by their PAS, ATh, AF, aniline blue and alcian blue positivity and by their immunoreactivity to ovine anti-LH beta-subunit, anti-salmon GTH and anti-silver carp GTH. The testes of Puntius sarana have been classified as the "unrestricted type". The morphological, histological and histometric findings indicate that the testing passes through four distinct stages--preparatory, fully mature, spawning and spent. Spermatogenesis begins in January and continues up to July. The spawning season lasts from late July until October. Cyclical changes in the gonadotrophs correlate well with changes in the testes. The histometric data show that the GTH cells attain maximum values (size and number) during the breeding season. The peak values for testicular weight, lobule diameter, the gonadosomatic index (GSI) and the sperm concentration in the testes, and the number, size and amount of secretory material in the cytoplasm of the gonadotrophs, coincide with the time of maximum environmental temperature and the longest days.     

Localization by immunohistochemistry of renal ornithine decarboxylase in the mouse with and without testosterone treatment

The immunohistochemical distribution of renal ornithine decarboxylase was studied in male mice both with and without testosterone treatment. Testosterone (1 mg per mouse) induced a marked increase in ornithine decarboxylase activity of the mouse kidney, whereas no significant immunohistochemical difference was observed either in immunoreactivity or its localization. In intact male as well as androgen-treated mice dense ornithine decarboxylase-immunoreactive cells were observed mainly in the cortex, especially many ornithine decarboxylase-immunoreactive cells were observed in the inner portion, while a much weaker immunoreactivity was observed in the medulla. The largest number of ornithine decarboxylase-immunoreactive cells seemed to be localized in the pars recta of the proximal tubule. The immunoreactivity was not detected in all the tubular cells but scattered among them. The renal corpuscles were not immunoreactive. In each ornithine decarboxylase-immunoreactive cell, the cytoplasm showed much denser immunoreactivity than the nucleus.     

Reduced number and LH content of marginated secretory granules in pituitary gonadotropes of streptozocin-induced diabetic male rats.

The transport of secretory granules towards the subplasmalemmal (SPL) region of the luteinizing-hormone (LH) gonadotrope is controlled by the LH-releasing-hormone-dependent pathway. The SPL granules contain the most readily releasable LH. To test the effect of diabetes on both the number and LH content of marginated granules, we studied by indirect immunogold-labelling pituitaries from control and streptozocin (STZ)-treated male rats. On electron micrographs we measured the areas of the gonadotrope nucleus and cytoplasm, counted all secretory granules, and counted and measured secretory granules in selected SPL regions. Furthermore, we counted gold particles (IG) on (a) the secretory granules of the SPL regions, (b) the intergranular SPL cytoplasm and (c) the region outside the cell. Finally, in order to evaluate possible diabetes-related changes of the pituicyte cytoskeleton, we measured by densitometry actin, tubulin, vimentin, and desmin in immunocytochemically stained pituitary sections. In diabetic compared with control cells of the studied pituitary region, we observed: (a) cytoplasmic atrophy; (b) the number of secretory granules per unit area increased in the total cytoplasm, and decreased in the SPL cytoplasm (lowered regulated secretion); (c) decreased IG labelling in the SPL granules (reduced amount of hormone transported by each granule towards the cell membrane); (d) decreased IG labelling in the integranular SPL cytoplasm (reduced constitutive secretion), and (e) strongly increased actin and desmin, yet unchanged tubulin and vimentin immunoreactivity. Our data indicate that both regulated and constitutive secretion are possibly reduced in gonadotropes of diabetic male rats. The cytoskeletal alterations may also contribute to the reduced regulated secretion.     

CXCL14-like immunoreactivity in growth hormone-containing cells of urodele pituitaries

Immunohistochemical techniques were employed to investigate the distribution of a chemokine, namely, CXCL14-like immunoreactivity in the axolotl (Ambystoma mexicanum) and Japanese black salamander (Hynobius nigrescens) pituitaries. CXCL14-immunoreactive cells concentrated at an area of the pars distalis adjacent to the pars intermedia. We found that these cells correspond to the cells immunoreactive to an antibody against rat growth hormone (GH). Immunoelectron microscopy indicated that the CXCL14-like substance and GH coexisted on the secretory granules in the axolotl pituitary. Western blot analysis of axolotl pituitary extracts revealed the anti-human CXCL14 antibody labeled an approximately 16.6-kDa band that was not labeled by the anti-GH antibody. The CXCL14-like substance in the pars distalis may participate in GH functions in these species.     

Functional morphology of the pars intermedia of the rat hypophysis as revealed with the electron microscope

Summary The pars intermedia of the hypophysis of normal and experimental rats was studied by electron microscopy. Observations of the hypophysis at various intervals following formalin induced stress or adrenalectomy indicate the existence of a functional relationship between the posterior lobe, the pars intermedia, and the adrenal cortex.Glandular cells of the normal pars intermedia are divided into two types, i. e., the light and dark cells. The former type dominates in number and is characterized by a large amount of cytoplasm filled with clear vesicles 250–350 m in diameter. Dark secretory granules smaller than 300 m are few in number and restricted to the Golgi region.After a single injection of formalin, the clear vesicles of the light cell dimmish and dark secretory granules varying in opacity increase in number. Transition from dark granules to clear vesicles is suggested. Three to five days after adrenalectomy, the light cells contain an abundance of moderately dense vesicles which are smaller than the larger more electron lucent vesicles of the normal light cells. The moderately dense vesicles are about 200 m in diameter and are extremely abundant filling the entire cytoplasm of the light cells 7 days after adrenalectomy.Bundles of unmyelinated nerve fibers are often observed in the pars intermedia, and a typical neuroglandular synapse was found in the pars intermedia of a sham-operated animal suggesting neural control of the secretion process of pars intermedia cells.The author wishes to express his hearty thanks to Dr. K. Kurosumi for his guidance throughout this work.     

Postnatal development of the interstitial cells (palisade cells) of the pars intermedia in the cat pituitary gland. An immunocytochemical and ultrastructural study.

The postnatal development of the interstitial agranulated cells (so-called palisade cells) of the pars intermedia in the cat was investigated immunocytochemically and at the ultrastructural level. Since the first postnatal days, a strong vimentin immunoreactivity and a weaker S-100 protein immunoreactivity were detected in the marginal cells lining the pituitary cleft and in the interstitial bipolar cells located within the pars intermedia. No glial fibrillary acidic protein cells have been found in the pars intermedia of any of the animals studied. This immunocytochemical pattern was maintained throughout the postnatal development. Ultrastructurally these cells showed a vast number of cytoplasmic filaments and well-developed junctional complexes. Secretory granules were never seen. In older animals they lined microcavities and microchannels where they project microvilli and present pinocytotic vesicles on their apical surface. No transitional forms between these cells and granulated secretory cells were found. There is a large number of axons and synaptic endings in contact with the granulated secretory cells. From our findings we guess that palisade cells are not a glial derivative, but they may share a common origin with secretory granulated cells.     

Ultrastructural localization of thyrotropin (TSH)-like immunoreactivity in specific secretory cells of the hypophyseal pars tuberalis in the Djungarian hamster,Phodopus sungorus

Summary Specific secretory cells in the hypophyseal pars tuberalis of Djungarian hamsters maintained under different photoperiods were investigated immunocytochemically by means of the colloidal gold technique using antibodies against rat thyrotropin (TSH). Secretory cells of animals kept under long photoperiods (LD16:8) showed positive staining of secretory granules (diameters 90–130 nm), whereas other intracellular structures were free of immunoreactivity. In animals kept under short photoperiods (LD8:16) secretory cells displayed increased numbers of secretory granules, but these organelles were devoid of immunoreactivity. In contrast, immunoreactivity of thyrotropes in the pars distalis did not differ between the two groups of animals investigated. The present results confirm earlier light-microscopical studies that in the pars tuberalis specific secretory cells show TSH-like immunoreactivity; however, they differ in their reactivity pattern from classical thyrotropes in the pars distalis.     

Immunoelectron microscopic localization of epidermal growth factor in the eccrine and apocrine sweat glands.

We studied the localization of the epidermal growth factor (EGF) in eccrine and apocrine sweat glands with light microscopic and electron microscopic immunohistochemistry. Anti-human EGF (anti-hEGF) polyclonal antiserum and anti-hEGF monoclonal antibody (MAb) were used for the study. Light microscopic immunohistochemistry with monoclonal and polyclonal antibodies showed that hEGF-like immunoreactivity was strongly positive in the myoepithelial cells and weakly positive in the secretory cells of eccrine sweat glands. In apocrine sweat glands, it was strongly positive in the secretory cells as well as in the myoepithelial cells. Immunoelectron microscopy with polyclonal antibody showed that hEGF-like immunoreactivity was present in secretory granules of apocrine secretory cells. These granules had mitochondrion-like internal structure. No reactivity was observed on the eccrine secretory cells by immunoelectron microscopy. Neither dark cell granules nor mitochondria in eccrine secretory cells were labeled with anti-hEGF antibody. In both eccrine and apocrine sweat glands, hEGF-like immunoreactivity was diffusely present in the cytoplasm of myoepithelial cells. However, nuclei and mitochondria of myoepithelial cells were devoid of immunoreactivity for hEGF. Our observations indicate that apocrine sweat glands may secrete more hEGF in the sweat than eccrine sweat glands.     

Transient appearance of substance P-like immunoreactivity in the granular convoluted tubules of the male mouse submandibular gland: light- and electron-microscopic studies

The time of appearance and distribution of substance P (SP)-like immunoreactivity in the granular convoluted tubule cells of the developing male mouse submandibular glands were examined, and the subcellular localization of SP-like immunoreactivity was investiagted by electron microscopy. At 25 days of age, SP-like immunoreactivity was first detected in the supranuclear cytoplasm of the granular convoluted tubule cells, which occurred either singly or in small clusters. At 30 and 35 days of age, granular convoluted tubule cells with SP-like immunoreactivity were more numerous than in the earlier stages, as the volume ratio of the cells increased. Not all granular convoluted tubule cells demonstrated SP-like immunoreactivity. The number of cells with SP-like immunoreactivity decreased at 60 days of age, and these cells had completely disappeared at 90 days of age. Most, but not all, secretory granules in the granular convoluted tubule cells were strongly labeled with gold particles, indicating that the subcellular site of SP-like substance is in the secretory granules within the cells. The findings suggest that the physiological role of the SP-like substance secreted from the GCT cells is restricted to the early postnatal stages, and that it may be involved in the development of the oral mucosa or digestive tract as a trophic factor.     

Hypothalamic lesions stimulating growth hormone cell activity in the goldfish

Summary The cytology of the growth-hormone (GH) cells of the goldfish pituitary were examined following electrothermic lesions of the anterior praeoptic hypothalamus and telencephalon. Following lesions of the nucleus preopticus (NPO) light microscopy of the pituitary revealed a significant increase in the nuclear diameter and a degranulation of the GH cells. Lesions of the telencephalon anterior or dorsal to the NPO had no cytological effect on the GH cells. The ultrastructural appearance of the GH cells of NPO-lesioned fish was characterized by a marked degranulation of the cytoplasm and a proliferation of the rough endoplasmic reticulum indicative of enhanced secretory activity. The GH cells of the proximal pars distalis (PPD) are directly innervated by peptidergic (type A) and aminergic-like (type B) neurosecretory axons. Following lesions of the NPO, there was a marked reduction in the number of type A fibers in the PPD. These results suggest that the type A fibers innervating the GH cells originate in the NPO and act to inhibit the secretory activity of the GH cells.     

Ultrastructural localization of actin in the intermediate lobe of rat hypophysis

The localization of actin in the cells of the pars intermedia of rat hypophysis was studied by means of the polyethylene glycol (PEG) embedding and subsequent de-embedding method together with FITC and IgG-colloidal gold immunolabelling techniques. Actin immunofluorescence was detected to be punctate throughout the entire cytoplasm, except for the nuclear region. In electron microscopy actin gold-labelling was localized on portions of microtrabeculae in close association with the secretory granules, but not within the secretory granules themselves. This close association of actin with the secretory granules strongly suggests the involvement of the contractile protein in the cellular secretory process. Several restrictions due to the PEG-method itself are also discussed to interpret clearly immunoelectron microscope images from the embedment-free sections.     

Temporal sequence of cell shape changes in cultured rat sertoli cells after experimental elevation of intracellular cAMP

The ability of FSH and pharmacological agents to induce changes in the shape of cultured rat Sertoli cells has been studied by using time-lapse phase-contrast microscopy and scanning electron microscopy (SEM). Morphological studies were combined with an immunocytochemical method for the localization of cAMP in Sertoli cells and the results correlated with determinations of protein-bound cAMP in Sertoli cells. A variable number of Sertoli cells were converted from a flat, epithelial-like morphology into a stellate morphology after exposure to FSH, isobutyl-methylxanthine (MIX), dibutyryl cyclic AMP (db-cAMP) and an FSH-MIX mixture. The morphological changes followed a time- and biological agent-dependent alteration and recovery pattern. While a 120 min exposure to FSH induced shape changes in 38% of the cells, MIX, db-cAMP and FSH-MIX effected shape changes in 75 % of cells. The morphological conversion induced by MIX, db-cAMP and FSH-MIX persisted as long as these biological agents were present in the medium, whereas the effects induced by FSH alone were transient. The flat-to-stellate transition was preceded by an increase in intracellular protein-bound cAMP, a form of cyclic nucleotide which may account for cAMP immunoreactivity observed in morphologically responsive and non-responsive Sertoli cells. From these data and from previous experimental findings of androgen-binding protein (ABP) immunoreactivity in the cytoplasm of responsive and non-responsive Sertoli cells, we conclude that a surge of cAMP triggers a still undefined mechanism by which Sertoli cells modify their shape in coincidence with a progressive depletion of cytoplasmic secretory granules.     

Photoperiod-dependent changes in exocytotic activity in the hypophyseal pars tuberalis of the Djungarian hamster,Phodopus sungorus

The pars tuberalis of the hypophysis of the Djungarian hamster, Phodopus sungorus, was investigated with regard to secretory activity by applying the tannic acid-Ringer perfusion technique. Two groups were maintained under long photoperiods (16 h light: 8 h dark) or short photoperiods (8 h light: 16 h dark), respectively. Perfusion with tannic acid showed that specific pars tuberalis cells release some of their secretory granules as indicated by typical exocytotic figures. The percentage of cells displaying exocytotic activity was significantly higher in the pars tuberalis of hamsters kept under long photoperiods. The number of exocytotic figures per single cell was not increased. These results provide further evidence for a secretory activity of the pars tuberalis and support the hypothesis of its involvement as a mediator between photoperiodic stimuli and the endocrine system.     

Effect of different photoperiods on the ultrastructure of the specific secretory cells and α-subunit mRNA level in the chicken pars tuberalis

The effect of different photoperiods on the specific secretory cells of the pars tuberalis was examined in male chicks. Animals were placed in one of three different photoperiod regimens: (1) normal control (light:dark = 12 h:12 h), (2) continuous light (L:D = 24 h:0), and (3) extended darkness (L:D = 1 h:23 h). The levels of common alpha-subunit mRNA in the pars tuberalis were examined by Northern blot analysis and compared with those in the pars distalis. In chicks exposed to continuous light for 1 week, alpha-subunit mRNA level in the pars tuberalis was decreased, although the level in the pars distalis was increased. Exposure to continuous light for 30 days also induced a decrease in alpha-subunit mRNA level in the pars tuberalis. On the other hand, in chicks exposed to extended darkness for 1 week, the alpha-subunit mRNA level of the pars tuberalis was markedly increased. In situ hybridization with digoxigenin-labeled common alpha-subunit cRNA probe also showed that the hybridization signals for alpha-subunit mRNA in the pars tuberalis cells become weak under continuous light for 30 days but they are very intense under extended darkness. Thus, the synthesis of alpha-subunits in the chick pars tuberalis was inhibited by continuous light but stimulated by extended darkness. These results were confirmed by semiquantitative electron-microscopic analyses. After exposure to continuous light for 30 days, many pars tuberalis (PT)-specific cells were filled with enlarged secretory granules, showing the reduction of secretory activity. On the contrary, extended darkness for 30 days induced hypertrophy of the PT-specific cells; the areas of cytoplasm and nucleus were significantly increased. In addition, secretory granules became small in size and exocytotic features were more frequent. Mitochondria and lysosomes were also increased in number. Thus, the synthetic and secretory activities of the PT-specific cells were increased under extended darkness. The data indicate that the specific cells of the pars tuberalis are responsive to photoperiodic changes in the chick.     

Studies on secretory activity in the pars intermedia of Xenopus laevis 1: Fine structural changes related to the onset of secretory activity in vivo

Changes in the fine structural organization of the pars intermedia related to the onset of secretory activity within the gland have been studied. It is shown that during the first seven days, following the onset of secretory activity, there is an extensive membraneogenesis within the cytoplasm of the parenchymatous cells of the pars intermedia which results in the formation of a well-organized array of endoplasmic reticulum and an increased development of the Golgi complex. Simultaneously the large population of secretory granules present in the cells in the inactive condition is reduced, the granules fusing with the plasma membrane and releasing their contents into the extracellular space. During the process of intracellular reorganization, in addition to the elaboration of those components of the Golgi complex which are believed to be concerned with the formation of the secretory granules, a second distinctive cisternal element develops within the Golgi area. This component which remains confined to the Golgi area for only a short time (days 2-4) appears to be responsible for the production of membrane-bound dense bodies with a finely granular content. The dense bodies, in turn, become transformed into the larger heterogeneous structures which are a prominent feature of actively secreting pars intermedia cells.     

Thyroid medullary carcinoma of the Djungarian hamster Phodopus sungorus. Ultrastructural evidence for the production of normal and atypical intracellular granules

We have carried out an electron microscopic and immunocytochemical study of thyroid medullary carcinoma arising spontaneously in the Djungarian hamster, Phodopus sungorus. At the ultrastructural level the cytoplasm of tumor cells contained numerous round to slightly elongated, dense-cored secretory granules. The number of secretory granules differed from cell to cell in the tumor, being scanty in some cells but more or less abundant in most. Electron microscopic-immunocytochemistry demonstrated that all dense-cored secretory granules in all tumor cells exhibited calcitonin immunoreactivity. In approximately 10% of the tumor cells, unusual star-shaped secretory vesicles were also found in the cytoplasm. These vesicles contained a small, but well-defined, lucent core surrounded by a region of finely granular material of greater electron density. The outer contour of these unusual vesicles was stellate rather than smooth. They appeared to originate not from the Golgi complex, but from the rough endoplasmic reticulum. These atypical stellate vesicles did not show any calcitonin immunoreactivity. Furthermore, in a small number of tumor cells (approximately 1%) a third type of membrane enclosed structure was found. These were conspicuous rods 1-5 micron in length with tapering ends and a crystalline substructure. The presence of both normal and atypical secretory granules in some tumor cells suggests that carcinogenic transformation may interfere with the normal synthesis and assembly of secretory products by the cell.