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.     

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     

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.     

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.     

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.     

Cytoskeletal filaments in embryonic chick myocardial cells as revealed by the quick-freeze deep-etch method combined with immunocytochemistry

Summary The three-dimensional organization of cytoskeletal filaments associated with the myofibrils and sarcolemma of the myocardial cells of early chick embryos was studied by the rapid-freeze deep-etch method combined with immunocytochemistry. In the endoplasmic region of saponin-treated myocardial cells, 12–14 nm filaments formed a loose network surrounding nascent myofibrils. These 12–14 nm filaments attached to the myofibrils and some of them converged into Z disc regions. In the non-junctional cytocortical region thinner 8–11 nm filaments composed a dense network just beneath the sarcolemma. In myofibril terminating regions at the sarcolemma, i.e., the fascia adherens, 3–5 nm cross-bridges were observed among the thin filaments. In Triton-permeabilized and myosin subfragment 1 (S1)-treated samples, subsarcolemmal 8–11 nm filaments proved to be S1-decorated actin filaments under which there was a loose network of S1-undecorated filaments. Subsarcolemmal S1-decorated actin filaments had mixed polarity and attached to the sarcolemma at one end. A loose network of S1-undecorated filaments among myofibrils in the endoplasmic region was revealed to consist of desmin-containing intermediate filaments after immuno-gold staining for desmin. These networks connecting myofibrils with sarcolemma were assumed to play an important role in integrating and transmitting the contractile force of individual myofibrils within early embryonic myocardial cells.     

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.     

Regional differences in composition of the perforatorium and outer periacrosomal layer of the rat spermatozoon as revealed by immunocytochemistry

The rat perforatorium is the part of the perinuclear theca that underlies the acrosomic system. It appears to be composed of several polypeptides. The main objective of this study was to determine the distribution of seven of these perforatorial polypeptides in the head of the rat spermatozoon. For this purpose, polyclonal antibodies were affinity purified from these polypeptides and tested 1) for their distribution on electron-microscope sections of late spermatids and spermatozoa by immunogold labeling and 2) for their specificity on Western blots of denatured perforatorial polypeptides by immunoblotting. Immunoblotting showed that all seven of the prominent perforatorial polypeptides had epitopes in common. Immunogold labeling of spermatozoa showed that antibodies against the 13, 13.4, and 16 kDa polypeptides were restricted in their localization to the thicker apical portion of the perforatorium and to the inner zone of the ventral spur. However, antibodies against the 34, 43, 57, and 63 kDa polypeptides reacted with the entire perforatorium but, in addition, reacted with the inner part of the ventral spur and with a portion of the "outer periacrosomal layer" lying between the plasma membrane and the outer acrosomal membrane. These results suggest 1) that there are regional differences in protein composition of the perforatorium, of the outer periacrosomal layer, and of the postacrosomal dense lamina; and 2) that perforatorial polypeptides may not necessarily be restricted to the subacrosomal region, but may also compose portions of the outer periacrosomal layer and postacrosomal dense lamina. Based on both immunoblotting and immunocytochemical results, using an antiactin monoclonal antibody that recognizes all known isoforms of actin, actin was not detected in the perforatorium of step 19 spermatids or spermatozoa. Actin, however, together with the seven perforatorial polypeptides tested, was present in the subacrosomal space of elongating spermatids before the process of condensation of the perforatorium takes place.     

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.     

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.     

Ultrastructural localization of Tamm-Horsfall glycoprotein (THP) in rat kidney as revealed by protein A-gold immunocytochemistry

The present study describes the intracellular distribution of Tamm-Horsfall protein (THP) in rat kidney. The localization was determined by immunoelectron microscopy using the protein A-gold technique. Various fixation and embedding protocols were evaluated for this purpose. Brief perfusion fixation (3 min) with 1% glutaraldehyde and embedding in a highly hydrophilic glycol methacrylate-polyester mixture were most appropriate for antigen-antibody recognition and structural preservation. The overall tissue distribution of THP was evaluated by indirect immunofluorescence microscopy; reaction was strong along the entire thick ascending limb of the loop of Henle (TAL) with enhanced fluorescence in the apical cytoplasm. On the electron microscopic level immunogold labelling was concentrated over numerous membrane-bound vesicles which form a compartment in the apical cytoplasm. The Golgi region was consistently labelled, whereas the plasma membranes revealed only sporadic labelling at the luminal side, and basolateral membranes were mostly unlabelled. Quantitative evaluation of the gold labelling, which was separately done for the inner stripe, outer stripe and cortical TAL, consistently showed the highest particle density in the apical cytoplasm. Middle and basal levels in the TAL cells were only moderately labelled. The results are discussed with respect to the current opinion which describes THP as a membrane glycoprotein. We speculate that the accumulation of THP in the apical vesicular compartment of TAL cells indicates a storage site of the protein, possibly prior to extrusion via exocytosis of the vesicle contents.     

Ultrastructural localization of Tamm-Horsfall glycoprotein (THP) in rat kidney as revealed by protein A-gold immunocytochemistry

Summary The present study describes the intracellular distribution of Tamm-Horsfall protein (THP) in rat kidney. The localization was determined by immunoelectron microscopy using the protein A-gold technique. Various fixation and embedding protocols were evaluated for this purpose. Brief perfusion fixation (3 min) with 1% glutaraldehyde and embedding in a highly hydrophilic glycol methacrylate-polyester mixture were most appropriate for antigen-antibody recognition and structural preservation. The overall tissue distribution of THP was evaluated by indirect immunofluorescence microscopy; reaction was strong along the entire thick ascending limb of the loop of Henle (TAL) with enhanced fluorescence in the apical cytoplasm. On the electron microscopic level immunogold labelling was concentrated over numerous membrane-bound vesicles which form a compartment in the apical cytoplasm. The Golgi region was consistently labelled, whereas the plasma membranes revealed only sporadic labelling at the luminal side, and basolateral membranes were mostly unlabelled. Quantitative evaluation of the gold labelling, which was separately done for the inner stripe, outer stripe and cortical TAL, consistently showed the highest particle density in the apical cytoplasm. Middle and basal levels in the TAL cells were only moderately labelled. The results are discussed with respect to the current opinion which describes THP as a membrane glycoprotein. We speculate that the accumulation of THP in the apical vesicular compartment of TAL cells indicates a storage site of the protein, possibly prior to extrusion via exocytosis of the vesicle contents.     

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.     

Specific distribution of messenger ribonucleic acids for 24-kilodalton proteins in the mouse epididymis as revealed by in situ hybridization: developmental expression and regulation in the adult

Specific mRNAs for 24-kDa proteins specific to the caput epididymidis were quantified by filter hybridization, and cellular distribution was assessed by in situ hybridization of tissue sections. Messenger RNAs were detectable in 10-day-old animals, rapidly increased in quantity between 15 and 20 days, and reached a maximum at 40 days of age. The marked increase in concentration of mRNAs could be associated with the increase in epididymal testosterone content. Near 26 days of age, specific perinuclear and basal localization of mRNAs occurred in the principal cells of segment I, and a wide cytoplasmic distribution was observed in segment II. In the adult, mRNA levels decreased by 50% 3 days after castration and became undetectable within 30 days. Administration of testosterone to castrated mice caused an increase in mRNA levels, which reach almost normal levels after 3 days of treatment. Nevertheless, the particular organization of segment 1 was not restored. A similar observation was made after hemicastration or ligation of the efferent duct on the operated side. If expression of the mRNAs appears to be mostly under androgenic control, other testicular factors may be involved in the regulation of mRNA distribution in segment I.     

Complex relationships between the pineal organ and the medial habenular nucleus-pretectal region of the mouse as revealed by S-antigen immunocytochemistry

Summary S-antigen-immunoreactive pinealocytes located in the deep portion of the pineal organ of inbred and wild pigmented mice give rise to long, beaded processes penetrating into the habenular and pretectal regions. In addition, the medial habenular nuclei and the pretectal area contain S-antigen-immunoreactive perikarya, which resemble pinealocytes in size, shape and immunoreactivity and are considered as pinealocyte-like epithalamic cells. Immunoblotting techniques reveal that a single protein band of approximately 48 kDa molecular weight accounts for this immunoreactivity. As shown with the use of the electron microscope, the majority of the S-antigen-immunoreactive processes is closely apposed to immunonegative neuronal profiles and perikarya of the habenular and pretectal regions. S-antigen-immunoreactive processes and perikarya of both pinealocytes of the deep pineal organ and pinealocyte-like epithalamic cells may form the postsynaptic element in conventional synapses involving axons provided with clear synaptic vesicles. Thus, certain mammalian pinealocytes may receive and transmit signals via point-to-point connections resembling neuro-neuronal contacts. These results challenge the concept that the mammalian pineal organ exerts its influence exclusively via the release of melatonin into the general circulation. Furthermore, they provide evidence (i) that neuronal circuits not involving the sympathetic system participate in the regulation of pineal functions in mammals, and (ii) that intimate histogenetic and functional relationships exist between the pineal organ and the habenular-pretectal nuclei in mammals.     

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