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.     

Dopaminergic binding sites in human pituitary adenomas other than prolactinomas

Binding studies of [3H]-spiroperidol, a potent dopamine antagonist, were performed on dispersed cells obtained from 2 mixed PRL- and GH-secreting adenomas, 3 GH-secreting adenomas and 4 'nonsecreting' pituitary tumors. Saturable, high affinity binding sites for [3H]-spiroperidol were identified in the two adenomas of mixed PRL and GH secretion, in 2 of 3 GH-secreting adenomas and in 2 of 4 'nonsecreting' adenomas. These data indicate that dopaminergic binding sites are present in some GH-secreting adenomas in the absence of PRL hypersecretion and in some 'nonsecreting' pituitary adenomas.     

Effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) release and cell morphology in human pituitary adenoma cell cultures

Six GH adenomas and three prolactinomas were investigated by light- and electron-microscopic morphological and immunocytochemical methods and the effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) secretion was tested in vitro. The tumour cells of the acromegalic patients revealed both GH and PRL immunoreactivity while prolactinomas showed only PRL activity. All the adenomas stained immunocytochemically also for VIP. By electron microscopy, the tumours included two densely and two sparsely granulated GH, two mixed GH/PRL, and three sparsely granulated PRL adenomas. The dissociated cells were explanted, and cultured in vitro. The cultures in micro test plates were treated with VIP at different concentrations between 10(-5)-10(-12) M. GH and PRL contents in the culture media were measured by radioimmunoassay. GH release was significantly stimulated by VIP in a dose-dependent manner over the whole concentration range, while VIP was effective on the PRL release only at 10(-6)-10(-7) M concentration. The cells of a mixed adenoma were grown in Petri dishes and used for ultrastructural and immunocytochemical studies. The cytoplasmic structure of the cells treated with VIP corresponded to that of active hormone-secreting cells with large ergastoplasmic fields and Golgi zones containing secretory granules. Massive exocytotic events were encountered mainly in the GH-type cells. GH and PRL double immunocytochemistry showed the predominance of GH cells, many of them containing low amounts of PRL as well. Cells predominantly containing PRL were spread among them, they also might contain GH as well. Some of the cells contained only a single immunoreactive hormone. The intensity of gold labelling of the secretory granules appeared higher in the VIP-treated cells than in the untreated control ones which showed a cytoplasmic structure characteristic of glandular cells with low secretory activity. As all the adenoma cells both contained and reacted to VIP, our results are in agreement with an autocrine or paracrine effect of this peptide. The fine structure of the cells in the cultures treated with VIP supply an additional argument to the assumption that VIP may serve as a growth factor for these cell types.     

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.     

Subcellular distribution of secretogranins I and II in GH3 rat tumoral prolactin (PRL) cells as revealed by electron microscopic immunocytochemistry

The GH3 rat pituitary cell line which secretes prolactin (PRL) is characterized by the paucity and small size of secretory granules. We looked for the presence, in these cells and in normal PRL cells, of two acidic tyrosine-sulfated proteins which are widely distributed in dense-core secretory granules of endocrine and neuronal cells, secretogranins I and II, using immunofluorescence and electron microscope immunoperoxidase techniques. Both secretogranins were detected in secretory granules of GH3 cells and of normal cells. Moreover, with our pre-embedding approach, secretogranins were localized within some RER cisternae and within all sacules of the Golgi stacks in both PRL cell models. A few small vesicles, large dilated vacuolar or multivesicular structures, and some lysosome-like structures were also immunoreactive. Double localization of secretogranins and PRL performed on GH3 cells by immunofluorescence indicated that all cells contained secretogranins I and II, whereas only 50-70% of the cells contained PRL. Moreover, in the case of hormone treatment known to increase the number of secretory granules, most if not all mature secretory granules were immunoreactive for secretogranins, whereas in certain cells some of the granules were apparently not immunoreactive for PRL. These immunocytochemical observations show that GH3 cells, which under normal conditions form only a small number of secretory granules, produce secretogranins and package them into these granules.     

Immunogold identification of prolactin cells of goats in anoestrus, pregnancy and milk production: ultrastructural variations.

Prolactin (PRL) cells of the goat adenohypophysis have been identified by the IgG-gold procedure with anti-sheep PRL serum. The secretion of these cells show differences in size and labelling in the three reproductive stages under study. Cells containing PRL can be grouped into low secretory activity cells (PRL-I) and high secretory activity cells (PRL-II) regarding their ultrastructure and functional significance. PRL-I were the most frequent cells in animals at the anoestrus stage, presenting numerous secretory granules and scarce development of the rough endoplasmic reticulum (RER) and Golgi complex (GC). At anoestrus and pregnancy stages there are frequent granule fusions, and the hormonal content partially disappears, perhaps by digestion. PRL-II cells were the most numerous at the lactating stage, presenting a moderate number of secretory granules and well-developed GC and RER. Some PRL-II cells of lactating animals exhibiting scarce granules and numerous exocytosis suggesting a high secretory activity. In both anoestrus and pregnancy stages most granules range in diameter from 450 to 750 nm, in contrast to the lactating stage in which most granules range in diameter from 150 to 450 nm.     

Immunocytochemical localization of exocrine enzymes in rat pancreatic acinar carcinoma

Ten pancreatic secretory proteins have been demonstrated in differentiated pancreatic acinar carcinoma cells by the protein A-gold immunocytochemical approach. The high resolution of the technique has allowed for the localization of the different proteins in the cellular compartments involved in protein secretion: RER, Golgi and secretory granules. The quantitative evaluation of the labeling for amylase has demonstrated the presence of an increasing gradient in the intensity from the RER to the Golgi and to the secretory granules which may reflect the process of protein concentration along the secretory pathway. These results, together with those obtained using the pulse-labeling autoradiographic approach, demonstrate that differentiated acinar carcinoma cells are capable of processing secretory proteins. When intensities of labeling obtained for different proteins on acinar carcinoma cells were compared to those obtained on normal pancreatic acinar cells, major differences were observed for some proteins. In addition, studies performed on the pancreatic tissue of the tumor-bearing animals have shown the presence of morphological alterations in the acinar cells.     

Immunocytochemical and ultrastructural characterization of mammosomatotrope-, growth hormone-, and prolactin-cells from the gilthead sea bream (Sparus aurata l., Teleostei): an ontogenic study

Growth hormone (GH), prolactin (PRL), and mammosomatotrope (MS) cells of gilthead sea bream, Sparus aurata, a teleost fish, were studied in specimens from hatching to 15 months (adults) using conventional electron microscopy and an immunogold method using anti-tilapia GH sera and anti-chum salmon PRL serum. MS cells, immunoreactive to both anti-GH sera and anti-PRL sera, had been first identified in fish in a previous study in newly hatched larvae and in older larvae and juvenile specimens of Sparus aurata by light microscopic immunocytochemistry. In the present work, MS cells reacted positively to immunogold label only in older larvae and juveniles and their secretory granules immunoreacted with both GH and PRL antisera or with only one of them. MS cells were ultrastructurally similar to the PRL cells, with which they coincided in time. This is the first report on the ultrastructural characterization of MS cells in fish. In adults, the secretory granules of GH cells (immunoreactive to anti-GH serum) were mainly round, of variable size, and had a homogeneous, highly electron-dense content. Irregularly shaped secretory granules were also present. PRL cells (immunoreactive to anti-PRL serum) were usually observed in a follicular arrangement; they showed few, small, and mainly round secretory granules with a homogeneous and high or medium electron-dense content. Some oval or elongated secretory granules were also observed. GH and PRL cells that showed involutive features were also found. In newly hatched larvae, GH, PRL, and MS cells could not be distinguished either by their ultrastructure or by the immunogold labeling of the secretory granules. In 1-day-old larvae, presumptive GH and PRL cells were observed according to their position in the pituitary gland. In 2-day-old larvae, a few cells showed some of the ultrastructural features described for GH and PRL cells of adults. During development, the number, size, and shape of the secretory granules in both cell types clearly increased and the organelles developed gradually. Some GH cells were found undergoing mitosis.     

Immunocytochemical identification of growth hormone cells in the adenohypophysis of the golden hamster

Somatotrophs or growth hormone (GH) cells in the adenohypophysis of golden hamsters were identified by immunocytochemical staining with polyclonal rabbit anti-human GH. They were oval or columnar in shape, and had secretory granules of two size ranges, 90-150 nm and 280-320 nm, which were present in the same cells; no subtypes of GH cells were observed. Secretory granules were located in the peripheral portion of the cytoplasm or concentrated at the vascular pole of the cell. Flattened cisternae of the rough endoplasmic reticulum in parallel array and a moderately developed Golgi apparatus were often found in the cytoplasm. No sex difference was noticed in the population ratio of GH cells. Immunocytochemical staining with anti-GH or anti-prolactin (PRL) antibodies on separate adjacent sections revealed that the GH and PRL were stored in two different cell types.     

Distribution of growth hormone and prolactin in secretory granules of the normal and neoplastic human adenohypophysis

Growth hormone [GH] and prolactin [PRL] can be demonstrated simultaneously in electron micrographs by means of the double immunocytochemical labeling technique using colloidal gold particles of two different sizes. This method was used to study biopsy specimens obtained from 15 patients suffering from acromegaly, 11 patients suffering from prolactinomas, and eight biopsy specimens obtained during adenomectomy from the normal, paraadenomatous pituitary tissue. Four granule populations with different immunoreactions were found: (1) granules containing GH only, (2) granules containing PRL only, (3) mixed granules containing GH and PRL, and (4) granules displaying no immunoreactivity. The existence of mixed granules indicated that the two hormones are synthesized by the same cell and in communicating compartments of the cells; i.e., the rough-surfaced endoplasmic reticulum. The number of GH-containing granules (pure GH granules and mixed GH-PRL granules) was greater than that of PRL-containing granules (pure PRL granules and mixed PRL-GH granules) in adenomas causing acromegaly and in the normal pituitary tissue, whereas the opposite was true for prolactinomas. The number of PRL-containing granules was larger in biopsy specimens from patients who had acromegaly and hyperprolactinemia than in patients with acromegaly and normal serum PRL levels.     

The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.     

Electron microscopic immunocytochemistry of prolactin secreting cells (PRL-cells) and growth hormone secreting cells (GH-cells) in the anterior pituitary gland of partially hepatectomized rats

After 60% hepatectomy in rats, prolactin secreting cells (PRL-cells) and growth hormone secreting cells (GH-cells) of the anterior pituitary gland were distinctly identified by the protein A-gold procedure combined with electron microscopy. The animals were sacrificed by the decapitation at midnight at intervals of about 28, 76 and 124 h after the operation. The principal changes can be summarized as follows; (1) Hypertrophy of the Golgi complex, (2) Dilation of the rough endoplasmic reticulum (RER), (3) Increased numbers of secretory granules (markedly in GH-cells), and occurrence of granule extrusion or exocytosis, (4) Increased numbers of lysosomes, which were mostly seen at 124 h after the operation. These ultrastructural changes were remarkably observed in both PRL and GH-cells. Especially, the noticeable changes in PRL-cells after partial hepatectomy in the rat were new findings in this study. The above results suggest that hepatectomy induced synthesis and release of GH and PRL in cells from pars distalis.     

Regulation of chromogranin B/secretogranin I and secretogranin II storage in GH4C1 cells

GH4C1 cells are a rat pituitary tumor cell strain in which the level of cellular prolactin (PRL) and PRL-containing secretory granules can be regulated by hormone treatment. The chromogranins/secretogranins (Sg) are a family of secretory proteins which are widely distributed in the secretory granules of endocrine and neuronal cells. In the present study, we investigated in GH4C1 cell cultures the regulation of the cell content of the Sg by immunoblotting and the relationship between the storage of Sg I and Sg II and PRL by double immunocytochemistry. GH4C1 cells grown in the presence of gelded horse serum, a condition in which these cells contain a low level of secretory granules, contained low levels of PRL, Sg I, and Sg II. Treatment of GH4C1 cells with a combination of 17 beta-estradiol, insulin, and epidermal growth factor for 3 days, known to induce a marked increase in the number of secretory granules, increased the cell contents of PRL, Sg I, and Sg II. To determine whether the induction of PRL was morphologically associated with that of the Sg, the distribution of PRL and the Sg was determined by double immunofluorescence microscopy. After hormone treatment, 54% of cells showed positive PRL immunoreactivity, fluorescence being extranuclear and consistent with staining of the Golgi zone and secretory granules. Forty-six percent of PRL-positive cells stained coincidently for Sg I, while 72% of the PRL cells were also reactive with anti-Sg II. To determine whether PRL storage was associated with storage of at least one of the Sg, cells were stained with anti-PRL and anti-Sg I and anti-Sg II together. Eighty-six percent of PRL cells stained for one or the other of the Sg. Therefore, PRL storage in GH4C1 cell cultures is closely but not completely associated with the storage of Sg I and/or II.     

Lysosomes in the cessation of vitellogenin secretion by the mosquito fat body; selective degradation of Golgi complexes and secretory granules

A massive and selective degradation of Golgi complexes, secretory granules, and RER is the mechanism responsible for the rapid termination of Vg secretion by trophocytes of the mosquito fat body. These cells are involved in an intensive synthesis of a glycoprotein, vitellogenin (Vg), which is accumulated by developing oocytes as yolk protein. Previously, assays for lysosomal enzymes have demonstrated that the cessation of Vg synthesis is characterized by a sharp increase in lysosomal activity; and fluorescent microscopy has shown that, during this intense lysosomal activity, Vg concentrates in lysosomes. In this report, electron microscopy combined with cytochemistry for lysosomal enzymes and localization of Vg with colloidal gold immunocytochemistry has shown that this lysosomal activity is directed towards selective degradation of Vg and organelles associated with its synthesis and secretion. Three organelles undergo lysosomal breakdown: the Golgi complex, Vg-containing secretory granules, and RER. The degradation of Golgi complexes occurs in two steps similar to that for RER: first, the organelle is sequestered by double isolation membranes, and the resulting pre-lysosome then fuses with a primary or secondary lysosome. In contrast, mature Vg-containing secretory granules fuse with lysosomes directly. This combination of crino- and autophagy is a specific, highly intense, and precisely timed event.     

Electron microscopic autoradiographic localization of prolactin mRNA in rat pituitary

Recent immunoelectron microscopic studies have shown that immunoreactive prolactin (PRL) in rat pituitary can be detected not only in typical PRL cells, characterized by large secretory granules, but also in another type of cell, which contains small secretory granules. To determine whether or not these two cell types are involved in PRL biosynthesis, we developed a procedure to investigate PRL gene expression by using in situ hybridization at the ultrastructural level. Rat pituitary was fixed and vibratome sections were incubated with a PRL [35S]-cDNA probe and subsequently flat-embedded in Araldite. Semi-thin and ultra-thin sections were processed for autoradiography. The results indicate that only the two PRL cell types were labeled. When immunolabeling for PRL was applied to ultra-thin sections, only immunopositive cells were seen to contain silver grains. In these cells the silver grains were associated with the rough endoplasmic reticulum and nucleus. When a growth hormone (GH) [35S]-cDNA probe was used as a control, only GH-secreting cells were labeled. This study confirms that the two PRL cell types are involved in biosynthesis of PRL. Moreover, this simple in situ hybridization technique provides a new approach to accurately localize mRNA in complex tissue and to investigate the subcellular distribution of mRNA under differing experimental conditions.     

Ultrastructural immunocytochemical localization of cyclic AMP-dependent protein kinase regulatory subunits in rat parotid acinar cells

Polyclonal antibodies to types I and II regulatory (R) subunits of cyclic AMP-dependent protein kinase (cA-PK) were utilized in a post-embedding immunogold-labeling procedure to localize these proteins in rat parotid acinar cells. Both RI and RII were present in the nuclei, cytoplasm, rough endoplasmic reticulum (RER), Golgi apparatus, and secretory granules. In the nuclei, gold particles were mainly associated with the heterochromatin. In the cytoplasm, the label was principally found in areas of RER. Most gold particles were located between adjacent RER cisternae or over their membranes and attached ribosomes; occasional particles were also present over the cisternal spaces. Labeling of the Golgi apparatus was significantly greater than background, although it was slightly lower than that over the RER cisternae. In secretory granules, gold particles were present over the granule content; no preferential localization to the granule membrane was observed. Morphometric analysis revealed equivalent labeling intensities for RI and RII in the cytoplasm-RER compartment. Labeling intensities for RII in the nuclei and secretory granules were about 50% greater than in the cytoplasm-RER, and 3 to 4-fold greater than values for RI in these two compartments. Electrophoresis and autoradiography of the postnuclear parotid-tissue fraction, the contents of purified secretory granules and saliva collected from the main excretory duct, after photoaffinity labeling with [32P]-8-azido-cyclic AMP, revealed the presence of R subunits. Predominantly RII was present in the granule contents and saliva, while both RII and RI were present in the cell extracts. Additionally, R subunits were purified from saliva by affinity chromatography on agarose-hexane-cyclic AMP. These findings confirm the localization of cA-PK in parotid cell nuclei and establish the acinar secretory granules as the source of the cyclic AMP-binding proteins in saliva.     

The effects of ACTH 1-17 on GH secretion in vitro

Recently we demonstrated that ACTH 1-17 infusion in normal subjects is able to stimulate growth hormone (GH) secretion. In order to study the mechanism by which ACTH 1-17 induces this hormonal secretory pattern, we examined the effects of ACTH 1-17 addition to primary cultures of rat anterior pituitary cells and of two human pituitary adenomas (a mixed GH- and PRL-secreting adenoma and a prolactinoma) on GH and PRL secretion. Normal rat pituitary cells responded to rGRF with a dose-dependent increase of rGH: ACTH 1-17 induced a slight not significant increase of rGH secretion even at micromolar concentrations. Furthermore no additive effect of ACTH 1-17 on rGRF-stimulated GH release was observed. No significant stimulatory effect was also documented in the human tumors studied. These results suggest that the GH releasing activity of ACTH 1-17 observed in vivo is mediated via a direct action on CNS.     

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.     

Prolactin cells of female rats treated with the carcinogen 7, 12-dimethylbenz(A) anthracene (DMBA) in vivo

Pituitary prolactin (PRL) cell microadenomata developed in females of a strain of Sprague-Dawley rats (SD1) following intragastric treatment with the carcinogenic hydrocarbon 7,12-dimethylbenz(a) anthracene (DMBA). Similar treatment of another strain of Sprague-Dawley rats (SD2) resulted in mammary tumour development but no PRL cell microadenomata. In SD1 strain rats morphologically distinct populations of PRL cells appeared after DMBA treatment, one composed of cells characterised by abundant, organised but very dilated RER and with large hormone storage granules, 500-600 nm in diameter (P1). The other cell type had electron-dense cytoplasm, narrow organised arrays of RER and moderately large, pleomorphic granules (P2). Both cell types appeared active with large Golgi and prominent nucleoli. P2 cells were most numerous 2 months after DMBA treatment but had virtually disappeared at 6 months and microadenomata were common at 8 months. PRL cells of SD2 rats were uniform in morphology, characterised by only moderate accumulations of RER, pleomorphic hormone storage granules, large Golgi and prominent nucleoli, and showed no close resemblance to either P1 or P2 cells of SD1 strain rats. It is possible that the morphological variations which developed in SD1 PRL cells may represent changes in responsiveness to factors controlling PRL cell secretion and proliferation and which may be pertinent to microadenoma development.