George H. Herbener

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.

Quantitative comparison of the mitochondrial populations in the livers of newborn and weanling rats.

Abstract Mitochondria in sections of liver samples from newborn and weanling rats were analyzed by quantitative electron microscopy. Specific activities of the mitochondrial enzymes, NADH-cytochrome c reductase, succinate-cytochrome c reductase and cytochrome oxidase, were determined in similar samples. In the liver parenchymal cell of the weanling, compared to that of the newborn, the number of mitochondria per unit volume of cytoplasm was 2.6 times larger, while the volume of the average mitochondrion was 0.58 times smaller, resulting in a net increase of 1.4 in the relative size of the mitochondrial compartment. The decrease in volume of the average mitochondrion was the result of a decrease in the number of large mitochondria and an increase in the number of small mitochondria. These data indicate a mitochondrial biogenesis in rat liver during postnatal development, the magnitude of which exceeds the overall growth of the organ parenchyma during this period. The increase in the size of the mitochondrial compartment was paralleled by increases of 18–21 times in the specific activities of the mitochondrial enzymes.

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.

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.

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.

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.

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 Microcomputer Program for Evaluating Sampling Error: an Application to Stereological Methods for Electron Microscopy

In situations where there is a need to minimize sampling error or sample size, the coefficient of variation (CV) may be used to evaluate sampling error as a function of the number of observations or subjects in a sample. For example, CV is useful for estimating the minimum number of electron micrographs (Nmin) required to obtain a representative field sample for stereological analysis. To facilitate the determination of Nmin, we have written a program (COEFficient) for DOS microcomputers which calculates CVs. COEF assists the user in reducing error to that which solely reflects biological variability, thereby minimizing the time and cost of subsequent analyses.

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

SummaryThe 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-17β. 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.

Uterine Phosphamidase in Sex Hormone-Treated and Deciduomata-Bearing Ovariectomized Rats.

Summary The sites of phosphamidase were localized histochemically and amount of enzyme activity measured quantitatively in the uteri of untreated, estrogen- and progesterone-treated, and deciduomata-bearing ovariectomized rats. Enzyme activity was found in the lumenal and glandular epithelia of the endometria of all the animals studied. In addition, phosphamidase was present in the differentiating stromal cells in the uteri in which the deciduomal response had been elicited. Estrogen in high dosage and progesterone depressed the enzyme activity compared with that in untreated castrates. During the development of deciduomata the enzyme level rose to over 5 times that in animals treated with progesterone but not traumatized.