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.     

Membrane skeleton in cultured chick cardiac myocytes revealed by high resolution immunocytochemistry

Distribution of cytoskeletal proteins with emphasis on the membrane-cytoskeleton interface was examined in cultured cardiac myocytes. Using specific antibodies recognizing α-sarcomeric actin, desmin, β-tubulin, spectrin/α-fodrin and ankyrin, respectively, the cellular localization of these cytoskeletal proteins was detected by laser scanning confocal microscopy. In addition, the fine filamentous structure of these proteins was identified by combining silver-enhanced immunogold labelling with electron microscopy. The latter technique employed the sequence of quick-freezing, deep-etching and rotary shadowing of the specimens. Conventional transmission electron microscopy of the spherical cardiac myocytes revealed a filamentous submembranous layer, approximately 100 nm thick. Specific immunolabelling of α-sarcomeric actin and spectrin/α-fodrin as well as ankyrin was seen beneath the plasmalemma. A three-dimensional meshwork of spectrin/α-fodrin was shown. Numerous desmin filaments that exhibited a tortuous course throughout the cells were also observed running in parallel with the surface in the submembranous area, whereas β-tubulin was infrequently detected in these areas. In conclusion, the present study shows that spherical cardiac myocytes contain a distinct and complex three-dimensional membrane skeleton. Major constituents of this distinct submembranous layer were spectrin/α-fodrin fibres as well as actin and desmin filaments. Accepted: 28 July 1999     

Concentration of amylase along its secretory pathway in the pancreatic acinar cell as revealed by high resolution immunocytochemistry

Summary The modified protein A-gold immunocytochemical technique was applied to the localization of amylase in rat pancreatic acinar cells. Due to the good ultrastructural preservation of the cellular organelles obtained on glutaraldehyde-fixed, osmium tetroxide-postfixed tissue, the labelling was detected with high resolution over the cisternae of the rough endoplasmic reticulum (RER), the Golgi apparatus, the condensing vacuoles, the immature pre-zymogen granules, and the mature zymogen granules. Over the Golgi area, the labelling was present over the transitional elements of the endoplasmic reticulum, some of the smooth vesicular structures at thecis- andtrans-faces and all the different Golgi cisternae. The acid phosphatase-positive rigidtrans-cisternae as well as the coated vesicles were either negative or weakly labelled. Quantitative evaluations of the degree of labelling demonstrated an increasing intensity which progresses from the RER, through the Golgi, to the zymogen granules and have identified the sites where protein concentration occurs. The results obtained have thus demonstrated that amylase is processed through the conventional RER-Golgi-granule secretory pathway in the pancreatic acinar cells. In addition a concomitance has been found between some sites where protein concentration occurs: thetrans-most Golgi cisternae, the condensing vacuoles, the pre- and the mature zymogen granules, and the presence of actin at the level of the limiting membranes of these same organelles as reported previously (Bendayan, 1983). This suggests that beside their possible role in transport and release of secretory products, contractile proteins may also be involved in the process of protein concentration.     

Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.     

Sexual difference in LH-cells of the neonatal rats as revealed by immunocytochemistry

Summary Localization and number of pituitary LH-cells were studied in neonatal male and female rats (from the birth to 12th day) applying anti-HCG serum in immunoenzymological procedures. The cells increased in number with developing age after birth. The cells in males and females were equal in number until 4 days of age, whereas thereafter the increase of the cell number in females exceeded that in males. After birth, the cells are mainly concentrated ventrally, being ventro-lateral in the anterior region but converging into the medial-ventral area in the posterior part of the gland. Some dispersion in a dorsal direction is also noted in the latter region. At birth the cells begin to appear in the dorsal area in the anterior portion, as well as in the posterior portion, particularly in the area close to the intermediate lobe and in the zone adjacent to the residual lumen. This was particularly evident in females after 4 days of age. Thus it is concluded that in rats the sexual differences in the pituitary become apparent after the 4th day of postnatal life.     

Cellular composition of the human pituitary pars tuberalis as revealed by immunocytochemistry

Summary An attempt was made to determine if any of the specialized secretory cell types common to the pars distalis also occur in the pars tuberalis of the human hypophysis. Available for study were 18 specimens of the inferior pars tuberalis, which partially surrounds the infundibular stem, and 3 specimens of the superior pars tuberalis that is attached to the median eminence. Antisera to human somatotropin, mammotropin, chorionic gonadotropin, follicle-stimulating hormone, FSH , luteinizing hormone, LH , thyrotropin, TSH , as well as to ^1–24-corticotropin, porcine ^17–39-corticotropin, and ovine LH were used with the Sternberger peroxidase-antiperoxidase immunocytochemical procedure to identify the probable cells of origin for these hormones.The evidence indicated that gonadotropic cells constitute the major portion of the parenchymal cell population in the pars tuberalis. They occurred throughout all of the pars tuberalis and were usually arranged in clusters. Somatotropic, mammotropic, corticotropic, and thyrotropic cells were rare and not found in all specimens. When present, they often formed a common group suggesting that their occurrence in the pars tuberalis resulted from displacement of primordial tissue of the pars distalis during embryogenesis.Supported in part by research grants HD-03159 and HD-08333 from the National Institute for Child Health and Human DevelopmentWe thank Dr. L.A. Sternberger for providing the PAP complex and others for antisera (Table 2) and hormones (Footnote 2) as listed     

Ultrastructure of Cryptosporidium parvum oocysts and excysting sporozoites as revealed by high resolution scanning electron microscopy

Cryptosporidium parvum oocysts isolated from calf feces were examined by scanning electron microscopy during excystation. Intact C. parvum oocysts were spheroid to ellipsoid, approximately equal to 3.5 X 4.0 micron, with length : width ratio = 1.17. The oocyst wall had a single suture at one pole, which spanned 1/3 to 1/2 the circumference of the oocyst. During excystation the suture dissolved, resulting in a slit-like opening, which the sporozoites used to exit the oocyst. Sporozoites were 3.8 X 0.6 micron and had a rough outer surface.     

Effects of actinomycin D on ribosomal RNA and protein synthesis as revealed by high resolution autoradiography.

Incorporation of tritiated amino acids and uridine was studied in untreated and actinomycin D treated HeLa cells by high resolution autoradiography. Results showed a non-selective inhibition of protein synthesis by actinomycin, as measured by the decrease in radioactive amino acid uptake. When cells pretreated with actinomycin D were incubated with radioactive amino acids and uridine, amino acid uptake in the nucleolus still occurred, while uridine uptake was almost completely eliminated. These findings suggest that in the absence of ribosomal RNA precursor synthesis, nucleolar protein synthesis continues to some extent, and that this protein is transported to the nucleolus.     

Distribution of endogenous albumin across the rat aortic wall as revealed by quantitative immunocytochemistry

Endogenous albumin was revealed over thin sections of rat aortic wall, with high resolution and specificity, by applying the protein A-gold immunocytochemical technique. Gold particles, revealing albumin antigenic sites, were observed over plasmalemmal vesicles in endothelial cells and over the interstitial space throughout the thickness of the aortic wall. The distribution of the labeling in the interstitial space varied from region to region and was associated with the collagen fibers, following the orientation of the bundles. The morphometric evaluation of this labeling demonstrated a first peak in labeling intensity in the intima followed by a steep decrease with low levels in the media, and an increasing gradient towards the adventitia. In the subendothelium, a moderate labeling was observed at the base of the endothelial cells of both aortic and capillary endothelia, followed by a decreasing gradient. Ratios between the labeling density in the intima as well as in the adventitia and that in the capillary lumen (plasma albumin) revealed different concentrations of albumin in these compartments. Endogenous albumin, under steady-state conditions, is thus unevenly distributed over the interstitial spaces across the rat aortic wall, and appears associated along the collagen fibers.     

High-level expression of the 32.5-kilodalton calelectrin in ductal epithelia as revealed by immunocytochemistry

Calelectrins are a family of antigenically related Ca2+-binding proteins that have only recently been described. They have the important property of binding to membranes only in the presence of Ca2+. We systematically studied the tissue localization of one calelectrin, the 32.5-kilodalton species, in rats using immunocytochemistry. We found that high levels were exclusively present in the epithelial cells of bile and pancreatic ducts, renal collecting ducts, bronchial epithelia, and brain ependyma. In all of these organs, the other cells were not immunoreactive. In addition, strong immunoreactivity was found in the intercalated disks of myocardial cells, and mild immunoreactivity was observed in several endocrine tissues. In contrast, the cellular distribution of the 67-kilodalton calelectrin was more diffuse, involving most parenchymal cells in addition to the already-mentioned cells. Due to the presence of high levels of 32.5-kilodalton calelectrin in some cell types, this protein may be used as a histochemical marker for differentiated ductal epithelial cells, some specialized epithelia, myocardial cells, and Paneth cells.     

High-level expression of the 32.5-kilodalton calelectrin in ductal epithelia as revealed by immunocytochemistry

Abstract. Calelectrins are a family of antigenically related Ca²⁺-binding proteins that have only recently been described. They have the important property of binding to membranes only in the presence of Ca²⁺. We systematically studied the tissue localization of one calelectrin, the 32.5-kilodalton species, in rats using immunocytochemistry. We found that high levels were exclusively present in the epithelial cells of bile and pancreatic ducts, renal collecting ducts, bronchial epithelia, and brain ependyma. In all of these organs, the other cells were not immunoreactive. In addition, strong immunoreactivity was found in the intercalated disks of myocardial cells, and mild immunoreactivity was observed in several endocrine tissues. In contrast, the cellular distribution of the 67-kilodalton calelectrin was more diffuse, involving most parenchymal cells in addition to the already-mentioned cells. Due to the presence of high levels of 32.5-kilodalton calelectrin in some cell types, this protein may be used as a histochemical marker for differentiated ductal epithelial cells, some specialized epithelia, myocardial cells, and Paneth cells.     

Albumin localization in the frog hepatocyte during vitellogenesis as revealed by protein A-gold immunocytochemistry

The protein A-gold immunocytochemical technique was used to localize albumin in the hepatocyte of the normal male American bullfrog, Rana catesbeiana, and also in the hepatocyte of this animal 8 days after treatment with estradiol-71 beta. Albumin concentration in plasma also was estimated biochemically. In the normal animal, specific immunolabeling for albumin was present in the intracellular compartments involved in protein secretion, i.e., rough endoplasmic reticulum (RER), Golgi apparatus and secretory granules, and also in lysosomes. Density of labeling increased as it progressed along the secretory pathway. In the hepatocyte of the estrogen-treated frog, specific immunolabeling for albumin was also present along the entire secretory pathway and in the lysosomes. Density of labeling over the RER was similar to that seen for this organelle in normal tissue; however, no progressive increase, but rather significant decreases, in labeling density occurred further along the secretory pathway. The biochemical data demonstrated no change in the concentration of plasma albumin in the treated frog, compared with the normal one. These observations localize albumin along its secretory pathway in frog hepatocyte and demonstrate a perturbation in its secretion in response to estrogen treatment.     

Ultrastructural localization of M-band proteins in chicken breast muscle as revealed by combined immunocytochemistry and ultramicrotomy

Cryo-ultramicrotomy and "conventional" plastic sectioning have been used in combination with extraction and immunolabeling techniques to determine the location of the two M-band proteins characterized to date, MM-creatine kinase (MM-CK: Mr, 80,000) and M-protein "myomesin" (Mr, 165,000) within the M-region of chicken pectoralis muscle. The following main results were obtained. (1) The M-band in chicken pectoralis muscle contains five major striations (M1, M4 and M4', M6 and M6' in the terminology of Sj?str?m & Squire, 1977a). (2) Extraction of the bulk of the electron-dense M-band with low ionic strength removes the M-striations M1, M4 and M4' while M6 and M6' are retained. Cross-sections through the M-region of such muscles lack primary M-bridges connecting the thick myosin filaments. (3) Labeling with antibodies against MM-CK enhances the M-striations M4 and M4'; sometimes the whole region between M4 and M4' is labeled. (4) Incubation with antibodies against myomesin results in the labeling of the whole M-band from M6 to M6'; no label is found in the rest of the bare zone outside M6 and M6'. (5) Incubation of low ionic strength extracted muscle fibers with antibodies against myomesin leads to an "incomplete" labeling of the M-band between M6 and M6'; lines M6 and M6' are sometimes seen to be enhanced presumably due to antibody labeling. From these results it is concluded that MM-CK is the major protein of the M4 and M4' (and possibly also of the M1) M-bridges. Myomesin is bound within the M-band along the thick filaments from M6 to M6'. Two hypothetical models for the possible location of myomesin are discussed. According to these models myomesin would either make up the M-filaments or be directly attached to and along the central bare zone of thick myosin filaments.     

Early stage fetal neocortex exhibits a complex ganglioside profile as revealed by high resolution tandem mass spectrometry

In this study we report on the first mass spectrometric (MS) investigation of gangliosides and preliminary assessment of the expression and structure in normal fetal neocortex in early developmental stages: 14th (Neo14) and 16th (Neo16) gestational weeks. Ganglioside analysis was carried out using a hybrid quadrupole time-of-flight (QTOF) MS with direct sample infusion by nanoelectrospray ionization (nanoESI) in the negative ion mode. Under optimized conditions a large number of glycoforms i.e. 75 in Neo14 and 71 in Neo16 mixtures were identified. The ganglioside species were found characterized by a high diversity of the ceramide constitution, an elevated sialylation degree (up to pentasialylated gangliosides-GP1) and sugar cores modified by fucosylation (Fuc) and acetylation (O-Ac). Direct comparison between Neo14 and Neo16 revealed a prominent expression of monosialylated structures in the Neo16 as well as the presence of a larger number of polysialylated species in Neo14 which constitutes a clear marker of rapid development-dependant changes in the sialylation. Also the MS screening results highlighted that presumably O-acetylation process occurs faster than fucosylation. CID MS/MS under variable collision energy applied for the first time for structural analysis of a fucosylated pentasialylated species induced an efficient fragmentation with generation of ions supporting Fuc-GP1d isomer in early stage fetal brain neocortex.     

Incorporation and fate of specific sperm plasma membrane components following insemination as revealed by ultrastructural immunocytochemistry

Studies have been carried out to 1) further characterize sperm specific plasma membrane polypeptides (33 and 35 kDa) that are recognized by a monoclonal antibody previously described (Longo, 1989) and 2) follow the incorporation and dispersal of these proteins within plasmalemmae of monospermic and polyspermic sea urchin (Arbacia punctuluta) eggs and oocytes utilizing immunocytochemical methods at the ultrastructural level of observation. Only sperm labeled when incubated with monoclonal antibody to the 33 and 35 kDa proteins followed by colloidal gold-tagged second antibody. Colloidal gold label was observed on the egg plasma membrane immediately after gamete membrane fusion; the amount and extent of label, i.e., the distance from the site of sperm incorporation, increased with time postinsemination. By 20 min postinsemination approximately one hemisphere of the inseminated egg/oocyte was associated with label. The expanding distribution of colloidal gold label on inseminated eggs and oocytes vs. time reflects the free diffusion of 33 and 35 kDa sperm surface proteins among egg/oocyte plasma membrane components. Label was also found in forming endocytotic vesicles, suggesting that sperm plasma membrane proteins may be internalized.