Widespread occurrence of chromogranins/secretogranins in the matrix of secretory granules of endocrinologically silent pituitary adenomas.

To investigate the constituents of the matrix of endocrine secretory granules, we analyzed endocrinoilogically silent ("non-functioning") human pituitary adenomas for the occurrence of the chromogranins/secretogranins (granins), a protein family normally stored together with many different hormones. When five non-functioning pituitary adenomas were analyzed by immunoblotting using polyclonal and monoclonal antibodies specific for individual members of the granin family, chromogranin A was detected in four cases and chromogranin B and secretogranin II were detected in all cases. The cellular distribution of the granins and of various hormones known to be expressed in the anterior pituitary was studied by immunocytochemistry in fixed, frozen tissue sections from five additional adenomas. Of the eight hormones investigated, only thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone were detected, occurring in only two of the five adenomas. In contrast, granins were found in all five tumors. Chromogranin B and secretogranin II were detected in each of the adenomas in virtually every cell studied, whereas chromogranin A exhibited such a widespread cell distribution in only three adenomas, being focally present in one and absent from the other tumor. The subcellular localization of the granins and the three glycoprotein hormones was investigated by double immunoelectron microscopy. Chromogranin A and chromogranin B were mainly co-localized in secretory granules, whereas secretogranin II was either co-localized with the other two granins or segregated to different secretory granules. When present, glycoprotein hormones were immunodetected in both the secretory granules containing all three granins and those containing mainly secretogranin II. Our data indicate that in non-functioning pituitary adenomas chromogranin A is differentially expressed from chromogranin B and secretogranin II. Moreover, the granins appear to be the most widespread constituents of endocrine secretory granules known, forming the dense-core matrix irrespective of the presence or absence of hormones.     

Uptake of horseradish peroxidase in spontaneous adenomas of the rat pituitary

The uptake of horseradish peroxidase was investigated by electron microscopy in 9 non-tumorous adenohypophyses and 13 spontaneous pituitary adenomas of adult female Long-Evans rats. Pituitary adenoma cells retained their ability for endocytosis and exhibited more extensive deposition of horseradish peroxidase than non-tumorous adenohypophysial cells.     

Co-localization of secretogranins/chromogranins with thyrotropin and luteinizing hormone in secretory granules of cow anterior pituitary

We investigated the co-localization in secretory granules of secretogranins/chromogranins, thyrotropin, and luteinizing hormone in ultra-thin frozen sections of cow anterior pituitary by double immunoelectron microscopy, using specific antibodies and protein A-gold particles of different sizes. The distribution of secretogranin II, chromogranin A, and chromogranin B (secretogranin I) was largely similar. In cells containing secretory granules of relatively small size (100-300 nm) and low electron density (identified as thyrotrophs and gonadotrophs by immunolabeling for the respective hormone) and in cells containing both small (170-250 nm) and large (300-500 nm) secretory granules of low electron density (also identified as gonadotrophs), all three secretogranins/chromogranins were detected in most if not all granules, being co-localized with the hormone. In cells containing both relatively large (400-550 nm), electron-dense granules and small, less electron-dense secretory granules (150-300 nm), identified as somatomammotrophs by double immunolabeling for growth hormone and prolactin, all three secretogranins/chromogranins were predominantly detected in the subpopulation of small, less electron-dense granules containing neither growth hormone nor prolactin. Interestingly, this granule subpopulation of somatomammotrophs was also immunoreactive for thyrotropin and luteinizing hormone. These data show that somatomammotrophs of cow anterior pituitary are highly multihormonal, in that the same cell can produce and store in secretory granules up to four different hormones and, in addition, the three secretogranins/chromogranins. Moreover, selective localization of the secretogranins/chromogranins together with thyrotropin and luteinizing hormone in a subpopulation of secretory granules of somatomammotrophs indicates the preferential co-packaging of the secretogranins/chromogranins and these hormones during secretory granule formation.     

Double-labeling immunogold electron-microscopic study of hormonal colocalization in nontumorous and adenomatous rat pituitaries

According to the one cell, one hormone theory, the pituitary gland is composed of 5 cell types which secrete 6 hormones. Recent investigations indicate that this theory must be modified, as there are some bihormonal cells containing 2 hormones, i.e., mammosomatotrophs prolactin-growth hormone (PRL-GH). This study was undertaken in order to elucidate whether other adenohypophysial cells are capable of producing 2 hormones and to demonstrate the presence of cells coexpressing PRL-GH, PRL-thyrotropin (TSH), or TSH-GH. Sixteen nontumorous and 16 adenomatous male and female Sprague-Dawley and Long Evans rat pituitaries were removed immediately after the animals were killed and processed for transmission electron microscopy and the immunogold double-labeling technique. Coexpression of PRL-GH, PRL-TSH, and TSH-GH was found in both nontumorous and adenomatous pituitaries. Double labeling was present not only in the same cell cytoplasm but also in the same secretory granules. The question of whether these double-labeled cells represent different cell populations, transitional cell types, or precursor cells requires further investigation.     

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.     

Annulate lamellae in adenomas of human pituitary glands.

Twelve nontumorous adenohypophyses and 36 various pituitary adenomas, removed by surgery, have been investigated by electron microscopy in order to shed some light on annulate lamellae, primarily on their ultrastructural features, incidence, origin, fate and functional significance. No annulate lamellae were found in the nontumorous adenohypophyses and in 33 pituitary adenomas. They were, however, detected in two adenomas consisting of undifferentiated cells and one adenoma composed of sparsely granulated prolactin cells indicating that these unique membrane configurations cannot be regarded as an exceedingly rare finding and, furthermore, that they may be disclosed not only in undifferentiated but occasionally in highly differentiated cells. Annulate lamellae may arise from endoplasmic reticulum and/or nuclear envelope and consist of arrays of smooth walled double membrane sheets exhibiting regularly spaced interruptions as well as continuities with the endoplasmic reticulum. No relationship was established between annulate lamellae and adenohypophysial secretory activity. Our findings seem to be consistent with the view that annulate lamellae are present in those cells which have the tendency to proliferate.     

Pituitary adenomas: patterns of hPRL and hGH secretion as revealed by high resolution immunocytochemistry

Seven human pituitary adenomas obtained by transphenoidal surgery were investigated for the intracellular localization of PRL and GH, using the protein A-gold immunocytochemical technique. Among the seven cases two were prolactinomas, two were GH-secreting adenomas and three were mixed PRL and GH-secreting adenomas. When PRL or GH were revealed, immunoreactivity was found in the cellular compartments involved in protein secretion, RER, Golgi apparatus and secretory granules of corresponding secreting cells. An increasing gradient in the intensity of labeling was observed from the RER to the Golgi and to the granules which may correspond to the increasing concentration of the proteins occurring along their secretory pathway. In addition, crinophagy or destruction of secretory granules by the lysosomal system was observed for both secretory cells. Cells displaying simultaneously PRL and GH reactivity were never found, neither in pure nor in mixed adenomas demonstrating that in the different adenomas studied, secreting cells have retained their specificity and differentiation for the secretion of a single hormone.     

Pituitary adenomas: patterns of hPRL and hGH secretion as revealed by high resolution immunocytochemistry

Seven human pituitary adenomas obtained by transphenoidal surgery were investigated for the intracellular localization of PRL and GH, using the protein A-gold immunocytochemical technique. Among the seven cases two were prolactinomas, two were GH-secreting adenomas and three were mixed PRL and GH-secreting adenomas. When PRL or GH were revealed, immunoreactivity was found in the cellular compartments involved in protein secretion, RER, Golgi apparatus and secretory granules of corresponding secreting cells. An increasing gradient in the intensity of labeling was observed from the RER to the Golgi and to the granules which may correspond to the increasing concentration of the proteins occurring along their secretory pathway. In addition, crinophagy or destruction of secretory granules by the lysosomal system was observed for both secretory cells. Cells displaying simultaneously PRL and GH reactivity were never found, neither in pure nor in mixed adenomas demonstrating that in the different adenomas studied, secreting cells have retained their specificity and differentiation for the secretion of a single hormone.     

Immunocytochemical evidence for production of luteinizing hormone and follicle-stimulating hormone in separate cells in the bovine.

In all mammalian females, follicular growth and maturation are essentially dependent on the pituitary gonadotropins, FSH and LH. These glycoprotein hormones have many similarities, but their action, based on high affinity binding to specific membrane receptors, are quite different. The purpose of this study was to perform a sensitive localization of FSH and LH in secretory granules of gonadotrophs using highly specific antisera. This morphological study included light microscopy (PAP) and electron microscopy (immunogold single and double labeling) procedures. Histologically, approximatively 11.5% of cells were positive for LH, whereas only 5.4% of cells were positive for FSH. With the electron microscope, single labeling allowed identification of morphologically distinct LH-containing cells and FSH-containing cells. Double immunostaining confirmed that no cells contained both hormones. The finding that FSH and LH are produced in separate pituitary cells is in agreement with recent studies that have suggested a specific role and regulatory process for gonadotropins in the bovine species.     

Light and electron microscopic localization of ACTH and pro-opiomelanocortin-derived peptides in human developmental and neoplastic cells

Oncofetal aspects of ACTH and pro-opiomelanocortin (POMC)-derived peptides were studied immunohistochemically at the light and electron microscopic level in human fetal pituitary glands, pituitary adenomas, and small-cell carcinoma of the lung. ACTH, beta-endorphin, and gamma-MSH were localized in the same cells of both fetal and adult pituitary, as well as in the above-mentioned neoplastic tissues. However, alpha-MSH was observed only in the early fetal pituitary, its concentration decreasing with advancing gestational age. The adult pituitary contained only a few alpha-MSH-positive cells. By immunoelectron microscopy, ACTH in the adult pituitary was localized exclusively in the secretory granules. In fetal pituitary at 9 weeks' gestation, ACTH was localized in the perinuclear spaces (PNS), cisternae of rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules. The staining pattern of ACTH in these organelles varied from cell to cell. In fetal pituitaries of greater gestational ages, ACTH was localized in secretory granules. The pituitary adenomas mimicked the staining characteristics of the adult pituitary, i.e., negative or only very occasional alpha-MSH staining and localization of ACTH in the secretory granules. The ectopic ACTH-producing tumors showed a staining pattern similar to that of the early fetal pituitary, i.e., positive staining for alpha-MSH and the presence of ACTH in PNS and cisternae of RER.     

Corticotroph cells of the human pituitary in old age.

This study was undertaken, utilizing immunocytologic methods and in particular the immunoperoxidase technique, to evaluate corticotroph cell morphology and distribution in the pituitary glands of aged subjects dying of various diseases. Early histologic studies did not allow for reliable identification of the different adenohypophysial cell types. Corticotroph cell distribution within the gland is described as well as its morphological characteristics. Our findings substantiate conclusions based on biochemical investigations that corticotroph cells remain unchanged and function normally in aged subjects.     

Multiple hormone storage by cells of the human pituitary

While immunostaining serial semi-thin sections of acrylic resin-embedded normal human pituitary using antisera to human pituitary hormones, it became clear that several cells were stained by more than one antiserum. The tissue had been surgically excised from a patient with a prolactinoma. The tumor, which was immunoreactive only with antiprolactin antiserum, was distinctly different from the pieces of tissue under study which had normal pituitary architecture and demonstrated immunoreactivity with antisera against all six of the common pituitary hormones. A major immunoelectron microscopic investigation, using immunocolloidal gold and immunoperoxidase methods, revealed cells in which follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) were co-localized to the same electron-dense granules. Some similar cells also possessed electron-lucent granules immunoreactive only for anti-PRL antiserum. Adrenocorticotrophic hormone (ACTH) and PRL were also found in the same cell but were very largely localized to separate, morphologically different populations of electron-dense and -lucent storage granules. By employing double immunolabeling, a few granules in the ACTH/PRL cells were shown to be immunoreactive to both anti-ACTH and anti-PRL antisera. The possibility that the multipotential stem cells is discussed.     

Intracellular distribution of GABA in the rat anterior pituitary. An electron microscopic autoradiographic study

We studied the internalization and intracellular distribution of [3H] GABA in rat anterior pituitary cells. Electron microscopic autoradiography of anterior pituitary fragments or dispersed pituitary cells incubated with [3H] GABA showed that lactotrophs and, to a lesser extent, somatotrophs were the only cells that contained radioactive grains. Grain density analysis performed on dispersed pituitary cells after a pulse-chase experiment (10 min pulse and then change to a medium without radioactive GABA for various periods up to 2 h) revealed that GABA internalized by lactotrophs was distributed in various intracellular membranous organelles. Of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria and secretory granules had different time-dependent labeling patterns. The highest grain density values were associated with plasma membrane (at the first chase time) and the Golgi apparatus. Mitochondria and secretory granules also showed significant grain density values. A similar pattern of distribution was observed when fragments of prolactin-secreting pituitary adenomas were incubated with [3H] GABA. These results provide morphological data on the cellular specificity and intracellular distribution of GABA in anterior pituitary cells.     

Characterization of human pituitary adenomas in cell cultures by light and electron microscopic morphology and immunolabeling

The morphology and hormone production of pituitary adenoma cell cultures were compared in order to highlight their characteristic in vitro features. Cell suspensions were prepared from 494 surgical specimens. The 319 viable monolayer cultures were analyzed in detail by light microscopy and immunocytochemistry within two weeks of cultivation. Some cultures were further characterized by scanning, transmission and immunogold electron microscopy. The viability and detailed in vitro morphology of adenoma cells were found to be characteristic for the various types of pituitary tumors. The sparsely granulated growth hormone, the corticotroph and the acidophil stem cell adenomas provided the highest ratio of viable cultures. Occasionally, prolonged maintenance of cells resulted in long-term cultures. Furthermore, a variety of particular distributions of different hormone-containing granules were found in several cases. Both light microscopic and ultrastructural analyses proved that the primary cultures of adenoma cells retain their physiological features during in vitro cultivations. Our in vitro findings correlated with the routine histopathological examination. These results prove that monolayer cultures of pituitary adenoma cells can contribute to the correct diagnosis and are valid model systems for various oncological and neuroendocrinological studies.     

Leucine-enkephalin-like immunoreactivity is localized in luteinizing hormone-producing cells in the axolotl (Ambystoma mexicanum) pituitary

In this study, we used immunohistochemical techniques to determine the cell type of leucine-enkephalin (Leu-ENK)-immunoreactive cells in the axolotl (Ambystoma mexicanum) pituitary. Immunoreactive cells were scattered throughout the pars distalis except for the dorso-caudal portion. These cells were immuno-positive for luteinizing hormone (LH), but they were immuno-negative for adrenocorticotrophic, growth, and thyroid-stimulating hormones, as well as prolactin. Immunoelectron microscopy demonstrated that Leu-ENK-like substance and LH co-localized within the same secretory granules. Leu-ENK secreted from gonadotrophs may participate in LH secretion in an autocrine fashion, and/or may participate in the release of sex steroids together with LH.     

Proteolytic processing of pro-ACTH/endorphin begins in the Golgi complex of pituitary corticotropes and AtT-20 cells

The intracellular sites where proteolytic processing of pro-ACTH/endorphin or POMC take place have not yet been reliably identified. We have used affinity-purified antisera that recognize only the products of POMC processing and immunoelectron microscopy to identify the compartments of rat pituitary corticotropes and mouse AtT-20 cells in which cleavage occurs. Immunoperoxidase labeling of cryostat sections and immunogold labeling of ultrathin frozen sections were used for localization of the processing sites. By both procedures we detected processed peptides in Golgi cisternae and secretion granules. Within the Golgi, labeling was limited to the last or transmost cisterna and was most concentrated in its dilated rims which contain condensing secretory protein. No labeling of other Golgi cisternae was seen. All Golgi cisternae were labeled, however, when antisera that recognize unprocessed POMC were used for immunolabeling. We conclude that in AtT-20 and rat pituitary cells: 1) processing of POMC through at least two endo- and exoproteolytic cleavage steps and alpha-amidation of joining peptide begin in the trans Golgi subcompartment; 2) no detectable processing takes place before POMC reaches the trans Golgi cisterna; and 3) this Golgi cisterna as well as secretion granules contain the active enzymes necessary for proteolytic processing and alpha-amidation.     

Pituitary mammosomatotroph adenomas develop in old mice transgenic for growth hormone-releasing hormone

It has been shown that mice transgenic for human growth hormone-releasing hormone (GRH) develop hyperplasia of pituitary somatotrophs and mammosomatotrophs, cells capable of producing both growth hormone and prolactin, by 8 months of age. We now report for the first time that old GRH-transgenic mice, 16 to 24 months of age, develop pituitary mammosomatotroph adenomas. These findings provide conclusive evidence that protracted stimulation of secretory activity can cause proliferation, hyperplasia and adenoma of adenohypophysial cells.     

Cellular localization of orexins in human anterior pituitary

Orexins A and B are hypothalamic peptides derived from a precursor called prepro-orexin and are associated with the stimulation of food intake and arousal. There is evidence that orexins act on some pituitary functions. Since no studies have been done concerning the presence of orexins in human pituitary, it is unclear whether the local effect of these peptides is due to orexins synthesized in the pituitary or to circulating-derived orexins. To define a possible paracrine regulatory role of orexins on pituitary cell function, we have sought to characterize the expression of orexins in the human adenohypophysis as well as to identify the cell types that express these proteins. In the present study, we used immunohistochemistry and double labeling to detect the presence of orexin A and orexin B in human pituitary. Orexin A was localized in 33% of pituitary cells. With double immunofluorescence techniques we demonstrated that orexin A is present in PRL (82±5.3%), TSH (18±2.3%), GH (10±2.3%), FSH (8±2.6%), and LH (7±3.2%) cells, but not in corticotroph cells. Orexin B was found in virtually all corticotrophs cells of the anterior pituitary. These results demonstrate that lactotroph cells are the main source of orexin A and corticotroph cells of orexin B. In summary the present findings provide the first evidence that orexins A and B are expressed in specific human pituitary cell types. Our data provide the cellular basis for a paracrine role of orexins in human pituitary cell function and further our understanding regarding the mechanisms by which orexins influence neuroendocrine function.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cysteine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Localization of cellular regulatory proteins using postembedding immunogold labeling

Cyclic AMP-dependent protein kinase (cAPK) mediates the effects of catecholamines and hormones that cause elevation of intracellular cyclic AMP levels. The holoenzyme is a tetramer consisting of catalytic (C) and cyclic AMP-binding regulatory (R) subunits. The type I and type II cAPK isoenzymes are defined by R subunits (RI and RII) of differing molecular weight, primary structure, and cyclic AMP-binding properties. Postembedding immunogold labeling procedures and specific polyclonal and monoclonal antibodies to RI, RII, and C were used to study the subcellular distribution of cAPK subunits in several tissues. In the rat parotid gland, both RI and RII were present in the cytoplasm, nuclei, and secretory granules of the acinar cells, whereas secretory granules of intercalated and striated duct cells were poorly labeled. These results confirmed that the acinar secretory granules are the source of R subunits previously identified in saliva by specific photoaffinity labeling techniques. Zymogen granules of pancreatic acinar cells and secretory granules of seminal vesicle cells were labeled with antibody to RII. Pancreatic and seminal fluids were shown to contain cyclic AMP-binding proteins. The granules of several endocrine cells (pituitary, pancreatic islet, intestinal) also labeled with RII antibody. Double labeling of ovarian granulosa cells showed that both RI and C were present in the nuclei and cytoplasm. The localization of cAPK subunits revealed by postembedding immunogold labeling is consistent with the postulated regulatory functions of these proteins in gene expression, cell proliferation, exocytosis, and various metabolic events The widespread occurrence of cAPK subunits in secretory granules and their release to the extracellular environment suggests that they play an important role in secretory cell function.