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.

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.

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.

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.

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