An electron and optical microscopic study of juxtaposed odontogenic keratocyst and carcinoma.

Odontogenic keratocyst and squamous cell carcinoma commonly occur within the oral cavity; however, the juxtaposition of these lesions is rare. The light microscopic and ultrastructural features of such an event are reported. Although some morphologic similarities between the cyst and tumor were observed, definitive evidence of a common origin was not obtained.

Use of the protein A-gold immunocytochemical and enzyme-gold cytochemical techniques in studies of vitellogenesis.

Vitellogenesis in the frog hepatocyte was investigated by applying the protein A-gold immunocytochemical and RNase-gold cytochemical techniques in conjunction with morphometric and biochemical analyses. The morphometric studies demonstrated that the surface density of rough endoplasmic reticulum (RER) and nucleolar size increased more than fourfold and 1.25-fold, respectively, while the nuclear size and the mitochondrial compartment size remained constant following estrogen treatment. Concurrently, liver RNA concentration increased 2.5-fold while protein and DNA concentrations did not change. In addition, total plasma protein more than doubled, with vitellogenin accounting for 40% of the final volume. The secretory proteins vitellogenin and protein-RcX (a nonvitellogenin, estrogen-induced plasma protein of unknown function, found in the plasma of Rana catesbeiana) were detected immunocytochemically in the RER, Golgi apparatus, and secretory granules in hepatocytes only of estrogen-treated frogs. Lysosomes also were labeled. These observations established that protein-RcX was synthesized and secreted by the hepatocyte in parallel with vitellogenin and that both of these export proteins were confined to the secretory pathway and lysosomes. Quantitation of labeling density indicated that the concentration of vitellogenin increased as it progressed along the secretory vector. Albumin was detected immunocytochemically also within these same hepatocyte entities from both untreated and treated animals. In the untreated animals, albumin concentration also increased progressively along the secretory vector. A marked alteration of albumin processing was observed following estrogen treatment. While albumin concentration in the RER was unchanged, its concentrations within the Golgi apparatus and secretory granules were lower than those observed in the RER or in counterpart compartments under control conditions. RNase-gold cytochemistry for total RNA demonstrated a 1.5-fold increase in labeling density over the nucleolus but no change in RER labeling following estrogen treatment. These labeling data, in combination with the morphometric data, suggest an increase of approximately 80% in the total amount of RNA in the nucleolus and 430% in the RER in response to estrogen. This review thus illustrates the significant contributions which can be made by gold-probe techniques, alone or in combination with morphometric and biochemical techniques, to investigations of the intracellular processing of secretory proteins.

Immunocytochemical localization of the Ca2+-ATPase polypeptide in human platelets.

Specific polyclonal antibodies raised against purified human platelet Ca2+-ATPase were used with protein A-gold immunocytochemistry to localize this protein in human platelets. Immunolabeling specifically detected Ca2+-ATPase over the surface connected membrane system (SCS) in sections of paraformaldehyde-fixed, Lowicryl-embedded platelets. The maximum density of label, determined by quantitative morphometric techniques, was observed over electron-dense regions within the SCS which may represent specialized structures for uptake and release of Ca2+. Less intense immunolabeling was observed over cytosol and may represent localization over the dense tubular system (DTS) which was not readily visualized under the processing procedures employed.

A correlated morphometric and cytochemical study on hepatocyte nucleolar size and RNA distribution during vitellogenesis.

The enzyme-gold cytochemical technique was used to label RNA in the nucleolus and rough endoplasmic reticulum (RER) of the hepatocytes of normal, male American bullfrogs (Rana catesbeiana) and in bullfrogs eight days following treatment with estradiol-17 beta. Concurrently, stereology was applied to quantitate: (1) the density of RNA labelling, and (2) changes in the size of the nucleus and nucleolus in response to estrogen treatment. In the hepatocytes from untreated frogs, specific labelling for RNA was present over the fibrillar and, to a greater extent, the granular portions of the nucleolus, and, to the greatest extent, over the RER. Following estrogen treatment, the density of RNA labelling increased over both parts of the nucleolus but was unchanged over the RER. The size of the nucleolus enlarged in response to estrogen: in combination with the increase in its RNA labelling, this suggested an increase of about 80% in the total amount of RNA in the nucleolus. Previous data on enlargement of the RER compartment, along with the present data on RNA labelling of RER, suggested that the total amount of this nucleic acid increased about 430% in this entity, in response to estrogen. However, the density of RNA labelling over the RER appears to be constant in spite of changes in the amount of RER.

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.

A review of the study of protein secretion applying the protein A-gold immunocytochemical approach.

Exocrine and endocrine types of secretion were investigated in various cells by applying the protein A-gold immunocytochemical approach. Several proteins secreted by rat pancreatic and parotid acinar cells, mouse ameloblasts, rat pancreatic B cells and lymph-node plasma cells, and frog hepatocytes were studied using specific antibodies. While light microscope immunohistochemistry has allowed for good topographical identification of positive cells in tissues, the protein A-gold approach used at the electron microscope level has demonstrated the presence of specific antigenic sites in particular cellular compartments. All secretory proteins studied were detected in the rough endoplasmic reticulum, the Golgi apparatus, and the secretory granules of the corresponding secreting cells. In addition, some of the proteins were also found in lysosome-like structures. When good ultrastructural preservation of the cellular organelles was achieved, the labeling was revealed with very high resolution and precise localization. In such cases, we found labeling over transitional elements of the endoplasmic reticulum and in smooth vesicles in the Golgi area. The Golgi apparatus was subdivided into three compartments according to differences in labeling: the cisternae on the cisside, those of the trans-side and the trans-most rigid one. Quantitative evaluations of the intensities of labeling have allowed for 1) demonstration of the high specificity of the different labelings; 2) revelation of the existence of a gradient of increasing intensity that follows precisely the progress of the proteins along their secretory pathway; and 3) identification of intracellular sites where increments of protein antigenicity occur. Furthermore, they have revealed the existence of alterations in protein processing that occurred under experimental and pathological conditions. Double-labeling approaches were performed to demonstrate two different antigenic sites on the same tissue section by applying protein A-gold complexes formed by gold particles of different sizes. Protein A-gold immunocytochemistry has also been combined with cytochemical and radioautographic techniques. This review thus demonstrates that high-resolution quantitative immunocytochemistry can contribute significantly to the investigation of the intracellular processing of secretory proteins. It also illustrates the potential and versatility of the protein A-gold technique, which in combination with other procedures constitutes a powerful method in cell biology.

Immunocytochemical localization of a non-vitellogenin, estrogen-induced plasma protein in the hepatocyte of the American bullfrog, Rana catesbeiana.

Recently, a non-vitellogenin, estrogen-induced frog plasma protein of unknown function and site of synthesis, which has been given the temporary name, protein-RcX, was isolated and partially characterized by Mitchell et al. In the present study, the protein A-gold immunocytochemical technique was applied to investigate its site of secretion; this was found to be the hepatocyte of the estradiol-17 beta-treated adult, male American bullfrog, Rana catesbeiana. Specific immunolabeling for protein-RcX was present over those intracellular compartments involved in protein secretion, i.e., the rough endoplasmic reticulum, Golgi apparatus and secretory granules, in addition, lysosomes were also labeled. No specific labeling for this protein was observed on hepatocytes of normal, non-estrogen treated, adult, male bullfrogs. Further, the labeling was abolished when plasma containing protein-RcX was added to the antibody prior to incubation but remained when purified vitellogenin was added. These observations support the hypothesis that protein-RcX is a non-vitellogenin, estrogen-induced plasma protein which is synthesized and secreted in parallel with vitellogenin by the hepatocyte of the estrogen-treated frog.

The intracellular pathway of vitellogenin secretion in the frog hepatocyte as revealed by protein A-gold immunocytochemistry.

The protein A-gold immunocytochemical technique was applied to the localization of vitellogenin in the hepatocyte of the bullfrog, Rana catesbeiana, eight days after treatment with estradiol-17 beta. Specific labeling was present in cellular compartments involved in protein secretion and was shown to progress in sequence through RER, Golgi apparatus, immature secretory granules, and mature secretory granules. Labeling intensities were quantitated and the values ranged from 34.6 to 172 gold particles/micron 2. In contrast, low background labeling was observed over mitochondria, nuclei, lipid droplets, and bile canaliculi. These observations support the hypothesis that vitellogenin synthesis and secretion in the frog hepatocyte lies exclusively along the RER-Golgi-granule secretory pathway. In addition to the cellular compartments involved in protein secretion, labeling was found over the majority of the lysosomes. The intensity of lysosomal labeling was intermediate between that of RER and Golgi apparatus. This labeling of lysosomes may be a consequence of the high blood plasma concentrations of vitellogenin that occur in the frog model, or to the well-known crinophagy phenomenon present in secretory cells.

A correlated morphometric and biochemical study of estrogen-induced vitellogenesis in male Rana pipiens.

Adult male Rana pipiens were administered estradiol-17 beta to induce vitellogenesis. Liver and blood were taken from control animals and experimental animals on Days 1, 2, 4, 8, 12, 16, and 120 following hormone treatment. Stereological analysis of livers showed that mitochondrial structural parameters remained constant while rough endoplasmic reticulum (RER) parameters increased significantly by 4 days and to more than four times control, or 120-day levels, by 8-16 days. Liver RNA concentration increased 2.5-fold and in parallel with RER, while liver protein and DNA concentrations did not change. Increases in total plasma protein and plasma vitellogenin (Vg) lagged behind increases in liver RER and RNA. Of the total plasma protein, Vg constituted 6% by 4 days, 40% by 12-16 days and less than 2% by 120 days. The half-life of plasma Vg was estimated to be no greater than 22 days. These studies provide the first quantitative correlations between ultrastructural and biochemical changes occurring in frog tissues following estrogen administration.

Biochemical and morphometric studies of heart, liver and skeletal muscle from the hibernating, arousing and aroused big brown bat, Eptesicus fuscus.

Heart, liver, pectoralis major, and plasma of hibernating, arousing and aroused bats were studied. The activities of four mitochondrial enzymes and three morphometric parameters of mitochondria did not change in the heart. Mitochondrial enzyme activities in the liver and pectoralis major did not change. Lactate dehydrogenase activity and isoenzyme content in heart, liver and pectoralis major did not change. Heart lipid content determined morphometrically decreased transiently after 30 min arousal from hibernation. Plasma free fatty acid concentration increased significantly by 7.5 min and peaked at 15 min after arousal from hibernation. Concentrations of heart free fatty acids, triglycerides, glycerol, and cholesterol and liver triglycerides did not change.

A morphometric study of the density of mitochondrial cristae in heart and liver of aging mice.

Mitochondria in sections of left ventricular wall and liver from C57BL/6J mice, 9, 18, and 36 mo. old, were analyzed using stereological procedures. At 9 mo. of age mu2 cristae surface per mu3 cytoplasm(Sv-c/c was fivefold greater in heart compared with liver reflecting the larger values in the former for both mu2 cristae surface per mu3 mitochondrion (Sv-c/m) and mu3 mitochondria per mu3 cytoplasm (Vv-m):Sv-c/c = Sv-c/m. Vv-m. At 36 mo. in both tissues, Sv-c/c had decreased to 65% of the earlier value. This was due to a decrease in Vv-m alone in heart and both Vv-m and Sv-c/m in liver. In both tissues the number of mitochondria per mu3 cytoplasm (Nv-m) also decreased with age while mu3 organelle average volume (vm) remained constant, supporting previous observations: Vv-m = Nv-m-vm. These data support the views: different tissues in the same organism age independently; and mitochondrial inner and outer membranes have a semi-independent existence.

A morphometric study of age-dependent changes in mitochondrial population of mouse liver and heart.

Mitochondria in sections of liver and heart (wall of left ventricle) from C57BL/6J mice, 8, 30 and 43 to 44 mo. old, were analyzed, using sterological techniques. The mitochondrial volume density decreased with age in both tissues: in liver the value estimated as 43 to 44 mo. of age was 65% the value estimated at 8 mo. of age; in the heart the 43 to 44 mo. value was 84% of the 8-mo. value. In both tissues, the decrease in mitochondrial volume density was the result of a decrease in the mitochondrial numerical density; the average volume of the mitochondrion remained constant throughout life.