Use of protein A-coated colloidal gold particles for immunoelectronmicroscopic localization of ACTH on ultrathin sections.

ACTH was localized in dissociated porcine adenohypophysial cells using a novel indirect EM immunocytochemical technique. Incubation of ultrathin resin sections in anti-ACTH was followed by incubation with protein A-coated colloidal gold particles. Protein A binds specifically to the Fc part of the IgG molecule, and thus the ACTH-containing secretory granules became labelled with electron-dense gold particles. With this method, the dissociated porcine ACTH cells was identified as containing numerous round or ovoid 170--300 nm secretory granules.     

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.     

The bifunctional role of pro-opiomelanocortin derivatives in the mediobasal hypothalamus of the rat

In the present study, a polyclonal antibody against pro-opiomelanocortin derivatives was characterized biochemically. Its immunoreactivity with structures of the arcuate nucleus and the median eminence was investigated by means of the immunogold method and compared with its reaction on adenohypophyseal cells with and without pre-adsorption with pro-opiomelanocortin derivatives. The antiserum detects ACTH and its fragments, in particular alpha-MSH, and beta-endorphin. In the adenohypophysis gold particles are exclusively located on small secretory granules situated in the periphery of branched cells. In the perikarya of the arcuate nucleus gold particles are observed on terminal vesicles abutting from the cis-face of the Golgi apparatus, on granules in its direct vicinity and on small dense core vesicles preferentially located in the cell periphery. Immunoreactive gold-labeled fiber profiles are found in a sub- or intra-ependymal position as well as in the nuclear neuropil proper. Here axodendritic and axosomatic synapses are observed. In both situations the gold particles are mostly restricted to the small dense core vesicles and do not decorate the synaptic vesicles. In the median eminence gold labeled fibers are detected in all layers. The labeled fibers can be closely apposed to tanycytic processes, without, however, forming special contact differentiations. In direction to the perivascular layer of the external zone the labeled profiles are more frequently arranged in groups intermingled with unlabeled fibers. The axons decorated with gold particles can be freely exposed to the perivascular space or are found as single processes in close vicinity to the capillary wall. Subsequent to preincubation of the native antiserum with ACTH1-39 and ACTH18-39 (= CLIP) neither adenohypophyseal cells nor perikarya and fibers in the arcuate nucleus nor axons in the median eminence are decorated with gold particles. Preincubation of the native antiserum with alpha-MSH or beta-endorphin does not change the immunoreaction with the small, peripherally situated granules in the branched adenohypophyseal cells. In neurons of the arcuate nucleus and in fibers of the median eminence, however, the immunoreaction is completely extinguished when the antibody is pre-incubated with alpha-MSH, whereas subsequent to preincubation with beta-endorphin only the amounts of labeled structures are reduced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Discrimination of LH,FSH, TSH and ACTH in dissociated porcine anterior pituitary cells by light and electron microscope immunocytochemistry

Summary Using the PAP unlabelled antibody method, LH, FSH, TSH and ACTH were localized at light microscope level in cultured cells dissociated from the porcine adenohypophysis. Antisera were shown to be specific for the subunits of the porcine glycoprotein hormones by radioimmunoassay and absorption studies. Using these antisera, it was found that LH and FSH were contained within the same cell, with TSH in a separate cell. When absorbed with LH, anti-porcine ACTH stained a separate distinct population of ACTH cells.Adjacent ultra-thin sections stained with anti-pLH and anti-pFSH, and examined at electron microscope level, showed that the ovoid, 150–400 nm secretory granules of the LH/FSH gonadotrophs contained both LH and FSH.The authors gratefully acknowledge the technical assistance of Carole Smith and Adrian Walsh     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.     

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.     

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.     

Intragranular colocalization of immunoreactive methionine-enkephalin and oxytocin within the nerve terminals in the posterior pituitary

To determine differential tissue antigens in the same section immunocytochemically using the electron microscope, the neurohypophysis was examined following the application of a freeze-drying tissue preparation and staining with the protein A-colloidal gold-antibody complex method (Hisano S, Adachi T, Daikoku S: J Histochem Cytochem 32:705, 1984). At the light microscopic level, colocalized immunostaining for methionine-enkephalin (ENK) and oxytocin (OXT) was found in the rat neurohypophysis under different physiological states. Small pieces of the neurohypophysial tissue were frozen and dried. The dried tissue was fixed with paraformaldehyde vapor and embedded. The ultrathin sections were stained with the antibody for ENK coupled with protein A-small colloidal gold, and antibody for OXT or vasopressin (VP) conjugated with protein A-large colloidal gold. The ultrastructures of the nerve terminals were well preserved and showed many membrane-limited secretory granules. It was possible to identify both OXT- and VP-containing nerve terminals as their secretory granules were differentially labeled with protein A-colloidal gold anti-OXT or anti-VP complex, respectively. The secretory granules, which were labeled with large gold particles for OXT, also carry small gold particles. It is evident that ENK coexists with OXT in the same granules.     

An histochemical approach to characterization of anionic constituents in mast cell secretory granules

We used cationized colloidal gold in order to investigate the distribution of anionic sites in different secretory granules of rat and mouse mast cells. The localization of the anionic sites was performed by post-embedding labeling of thin sections of rat peritoneal cells or mouse skin tissue, fixed in Karnovsky's fixative and OsO₄ and embedded in Araldite or LR white, respectively. In all cases anionic sites were demonstrated with a high density variation depending on cell type. In all mast cell secretory granules we have observed the highest density (ca. 500–900 gold particles/m²), while in other peritoneal cell granules it was about 10 times less (ca. 40–80 gold particles/m²). Pretreatment of the LR white sections with heparinase I and III resulted in a reduction of 97% and 72%, respectively, in the binding of the gold particles to the granules, indicating that the majority of the gold binding reactivity is due to heparin. Correlation of section profile area with labeling density revealed that the smaller granules were significantly more labeled when compared to the larger profiles. On the basis of these observations it seems that a post-translational change (mainly sulfation of heparin) of secretory content influences the granule anionic charge and thus may affect the intragranule buffer capacity.     

Corticotroph cells in primary cultures of rat adenohypophysis: A light and electron microscopic immunocytochemical study

Summary Monolayer cultures of trypsin-dispersed cells of the rat adenohypophysis were grown for 5 to 54 days. ACTH was localized by immunocytochemistry using an antiserum to synthetic ACTH^1–28 prepared in rabbit and sheep anti-goat immunoglobulin coupled with peroxidase. ACTH content of the culture medium was measured by radioimmunoassay.Corticotrophs were found in the cultures and ACTH in the medium at each cultivation time. The corticotrophs retained their essential morphological characteristics. Immunological staining was found in the secretory granules, some tubular or saccular structures, parts of the rough endoplasmic reticulum, and the cytoplasmic matrix. Immature secretory granules in the Golgi apparatus as well as some Golgi elements showed different degrees of immunoreactivity. In agreement with the high ACTH content of the culture medium the number, size and shape of the secretory granules, the active Golgi apparatus, the high amount of extragranular ACTH as well as pictures suggesting granule extrusion claim for a high ACTH synthesis and transport (and low ACTH storage) in the cultured corticotrophs.     

Variability in gold bead density in cells. Quantitative immunocytochemistry

Variability in gold bead distribution between individual cells was demonstrated in both pituitary melanotrophic cells immunocytochemically reacted for ACTH and in neurohypophysial terminals reacted for oxytocin-neurophysin. Gold beads were confined to the secretory granules compartment of both tissues. Density of gold beads in melanotrophic cells reacted for ACTH varied from 100-480 gold beads/microns 2. A much narrower range of gold beads distribution (460-900 gold beads/microns 2) was observed in axons of the neurohypophysis reacted with anti-oxytocin-neurophysin. These results indicate that the labeling density varies from cell to cell (as well as axon terminals) within morphologically homogeneous populations. Thus, it may reflect certain physiological differences between cells. A suggestion is being made that mean gold bead density coefficient of variation should be calculated by comparison between individual cells.     

Some improvement in tissue preparation and colloidal-gold immunolabeling for electron microscopy

The application of freeze-substitution (FS) and freeze-drying (FD) techniques and the protein A-gold-antibody complex immunocytochemical methods are described. The two tissue-preparation techniques produced excellent ultrastructure and topographical fixation of antigens when compared with conventional tissue-preparation techniques. In the FS preparation, however, occasional extragranular immunolabeling was recognized. This may suggest the leakage of antigens from the secretory granules. The FD procedure was considered the best, since such labeling was almost negligible. The protein A-gold-antibody complexes are easily prepared and label the antigens clearly. If the protein A-coated gold particles are saturated with antibodies, there is no interaction between gold particles. Thus, multiple antigens can be determined even in single secretory granules. In fact, we demonstrated intragranular colocalization of immunoreactive oxytocin, labeled with 50-nm gold particles, and immunoreactive methionine-enkephalin, labeled with 15-nm gold particles, in the axonal terminals of the FD-prepared rat neurohypophysis. This study demonstrates the value of the use of gold-antibody complexes for immunocytochemical labeling on FS- or FD-treated tissues.     

Freeze-drying technique in electron microscopic immunohistochemistry

Postembedding immunocytochemical labeling was performed on sections of rat neurohypophysis prepared by either freeze-drying, vapor fixation and Spurr resin embedding, or conventional aqueous fixation and Spurr resin embedding. Arginine vasopressin (AVP) and oxytocin (OXT) were immunolabeled with protein A-gold-anti-AVP and protein A-gold-anti-OXT complexes, respectively. The freeze-drying procedure (FD) resulted in excellent preservation of ultrastructure and greater antigenicity than the conventional procedure (Con). More gold particles were seen over secretory granules in FD sections than in Con sections. In addition, in FD sections, the gold label was restricted to secretory granules while in Con sections, both the granules and the extragranular axoplasm exhibited label. The two antigens in FD sections could be labeled simultaneously with protein A-small gold particle-anti-OXT complex and protein A-large gold particles-anti-AVP complex. In this way the two antigens were seen to be present in secretory granules within different axon terminals. Thus FD preparations should be useful for demonstrating the presence of multiple antigens in the same granules of nerve terminals.     

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.     

Ultrastructural localization of relaxin in the corpus luteum of the nonpregnant, pseudopregnant, and pregnant pig

Relaxin was localized in luteal cells of ovaries from nonpregnant, pseudopregnant, and pregnant pigs using porcine relaxin antiserum and peroxidase-antiperoxidase light microscopy immunohistochemistry. The number of immunoreactive cells seemed to increase from Days 17 to 106 of gestation. Luteal cells from pseudopregnant (Day 110) and nonpregnant (Day 14 of the estrous cycle) pigs were also positive for relaxin. However, less than 3% of the luteal cells in the nonpregnant animals were immunoreactive. Electron microscopy immunocytochemistry using porcine relaxin antiserum and goat antirabbit immunoglobulin G-colloidal gold demonstrated that relaxin was packaged in the small membrane-bound granules in luteal cells of pregnant as well as pseudopregnant and nonpregnant pigs. The intensity of labeling (number of gold particles) of the granules increased with pregnancy. There was a 10-fold increase in labeling of granules with the 10-nm versus 25-nm diameter gold. The goat antirabbit labeled with the smaller 10-nm gold particles was necessary to demonstrate the apparent low levels of relaxin in the luteal cells of the nonpregnant pigs. These data further indicate that pregnancy is not required for relaxin synthesis. However, physiologic significance of relaxin in corpora lutea of nonpregnant pigs has not been determined.     

Electron microscopic immunolocalization of seminal vesicle-specific antigen in human seminal vesicle

Seminal vesicle-specific antigen (SVSA) has been shown to be a polymorphic antigen represented by multiple immunoreactive peptides when fresh human semen is probed with monoclonal antibody (MHS-5) on Western blots. Semen samples collected directly into sodium dodecyl sulfate (SDS) demonstrate major immunoreactive peptide bands at 69-71 kDa and 58 kDa as well as a series of peptides of lower molecular mass. As semen liquefies, the higher molecular mass forms of SVSA are transformed into lower molecular mass bands, with 10-13 kDa immunoreactive peptides predominating after 8 h of liquefaction (McGee and Herr, Biol. Reprod. 37:431-439, 1987). In the present study, the 10-13 kDa form of SVSA was purified by preparative electrophoresis from SDS gels and a polyclonal antibody was generated in guinea pigs. Human seminal vesicle was fixed by immersion in combinations of glutaraldehyde and paraformaldehyde and embedded in Araldite or LR Gold. Both the guinea pig polyclonal antibody and the murine monoclonal antibody MHS-5 were employed to localize SVSA in human seminal vesicle by immunoelectron microscopy using Protein-A gold complexes. Gold particles were quantified in various subcellular compartments by a Videoplan computer. With either antibody probe, SVSA was found predominantly in the central electron-dense cores of secretory granules, with no staining evident over the electron lucent halo surrounding the granule core. With preimmune serum, the mean number of gold particles overlying secretory granules was 3/microns2; with polyclonal anti-SVSA, the mean number of particles observed over secretory granules was 182/microns2. This study represents, to our knowledge, the first fine-structural localization of a specific secretory protein to the electron-dense cores of secretory granules in principal cells of the human seminal vesicle.     

Quantitative ultrastructural immunocytochemistry using a computerized image analysis system

Secretory granules in human pituitary adenoma cells have been examined indirectly for hormone epitopes by immunogold labelling of resin-embedded ultrathin sections. The specific binding of different immunoglobulin-gold complexes to the antigrowth hormone antibodies over the secretory granules was measured using a computerized image analysis system. This facilitated the assessment of the preferential binding to the target granules of gold particles with three different average particle diameters (Au7, Au11, Au17). The time of pretreatment of sections with H2O2 or a buffer was found to influence the staining considerably. The scanning electron microscopic findings of protruded secretory granules with a mountain-like surface might be relevant to the uneven distribution of immunolabels seen over the secretory granules in the adenohypophysis.     

Localization of a 16,000-dalton fragment of the common precursor of adrenocorticotropin and beta-lipotropin in the rat and human pituitary gland

To clearly identify cells and organelles containing the common precursor (31,000 dalton) for both adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH), an immunohistochemical localization of a fragment (16,000 dalton) of the precursor that is not common to beta-LPH and ACTH was conducted in rat and human pituitary glands. With the help of specific antibodies that do not cross-react with beta-LPH and ACTH, the 16,000-dalton fragment was localized in the cells that also produce ACTH and beta-LPH in both the pars distalis and pars intermedia of the rat pituitary. At the electron microscope level, the secretory granules that contain ACTH were also stained for 16,000-dalton fragment. In the human pituitary, the 16,000-dalton fragment was also observed in all the secretory granules of lipocorticotrophs. These results suggest that, after enzymatic cleavage, fragment(s) of the common precursor and/or the whole common precursor remain packaged within the secretory granules with peptides of known activity.     

Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells

The pituitary cell line, AtT-20, synthesizes adrenocorticotropic hormone (ACTH) as a glycoprotein precursor that is cleaved into mature hormones during packaging into secretory granules. The cells also produce an endogenous leukemia virus (MuLV) that is glycosylated after translation similar to the glycosylation of the ACTH precursor. Our evidence suggests that the envelope glycoprotein and some precursor ACTH get to the cell surface in a vesicle different from the mature ACTH secretory granule. Viral glycoproteins and ACTH precursor are released from the cells much sooner after synthesis than mature ACTH. Isolated secretory granules do not contain significant amounts of the envelope glycoprotein or ACTH precursor. Exposing cells to 8Br-cAMP stimulates release of mature ACTH four to five fold, but has little effect on the release of the ACTH precursor or the viral glycoproteins. We propose that the viral glycoproteins and some of the ACTH precursor are transported by a constitutive pathway, while mature ACTH is stored in secretory granules where its release is enhanced by stimulation.     

Changes in the distribution of anionic constituents in secretory granules of mouse pancreatic acinar cells after pilocarpine-induced degranulation.

We used cationized colloidal gold (CCG) to investigate the distribution of anionic sites in different secretory granules of mouse pancreatic acinar cell regranulation. Localization of anionic sites with CCG was carried out on ultrathin sections of a mouse pancreas, fixed in Karnovsky's fixative and OsO4 and embedded in Araldite. After pilocarpine-stimulated degranulation, there was a marked diminution in the anionic charge density of immature and mature granules of the 4-hr group (approximately 43.0 gold particles/microm2) compared to the 8-hr mature granules group (approximately 64.6 gold particles/microm2). Scattergram analysis to investigate the correlation between section profile size and cationized gold labeling density revealed a reverse correlation, the small granule profiles demonstrated a higher density compared to the larger profiles of the same group. On the basis of these observations, it appears that a post-translational processing of secretory content influences the granule anionic charge and thus may affect the intragranular buffer capacity.