Electron Microscopic Morphometry Research Articles

Parathyroid function in rats treated with growth hormone. A morphometric study.

Male rats weighing 220-250 g were injected i.m. daily with 0.1 mg commercial human growth hormone for 3 days or 12 days. The serum concentration of total calcium phosphorus and alkaline phosphatase were significantly elevated for some days and returned to normal values at the end of the test period. The parathyroid glands, as studied by light and electron microscopical morphometry showed signs of reduced activity at the 4th day and also at the 13th day: a lowered nucleo-cytoplasmic ratio and a decrease of rough endoplasmic reticulum, of Golgi apparatus and of plasmalemmal tortuosity. The findings suggest a hypercalcemic effect of growth hormone involving peripheral organs of calcium metabolism, especially kidney and bone, and a secondary suppression of parathyroid glands by hypercalcemia.20

Association of Phytochrome with Rough-surfaced Endoplasmic Reticulum Fractions from Soybean Hypocotyls

Distribution of phytochrome (as Pfr) among membranes from soybean hypocotyls (Glycine max L. cv. Wayne) was determined by the combined techniques of cell fractionation, difference spectrometry, and electron microscopic morphometry. More than 90% of the phytochrome was found in the soluble fraction. With homogenates prepared in the presence or absence of Mg(2+), the portion associated with membrane was only 6.5% and 1%, respectively. In the presence of Mg(2+), the content of particulate phytochrome correlated with the amount of endoplasmic reticulum with attached ribosomes in the fractions but not with mitochondria or other membranes (including endoplasmic reticulum membranes from which the ribosomes may have been lost during cell fractionation). In the absence of Mg(2+), phytochrome was associated with a "heavy" plasma membrane fraction. The phytochrome content was sufficiently low to be accounted for by a contamination of less than 10% by rough-surfaced fragments of endoplasmic reticulum. The findings show association of phytochrome with a particulate fraction enriched in rough-surfaced fragments of endoplasmic reticulum but do not rule out cosedimentation of some unknown or unspecific phytochrome aggregate with this fraction.

Glucose dependent alterations of mitochondrial ultrastructure and enzyme content in mouse pancreatic islets maintained in tissue culture: a morphometrical and biochemical study.

Isolated islets of Langerhans from mice were maintained in tissue culture for one week at either a high (28 mM) or a low (3.3 mM) extracellular glucose concentration. Electron microscopic morphometry by means of stereological methods revealed a much greater volume of mitochondria in islet cells cultured at low glucose than in those cultured at high glucose. The former islets also showed a higher activity of the mitochondrial marker enzyme, L-3-hydroxyacyl-CoA-dehyrogenase (E.C.1.1.1.35). These results indicate a true mitochondrial hypertrophy at the low glucose concentration. Although it is known from previous studies that the islet cell metabolism is diminished after low-glucose culture, the present observations of an increased mitochondrial volume probably do not reflect a degenerative process, but rather adaptive changes towards oxidation of energy yielding substrates other than glucose.

The aging of rat liver as revealed by electron microscopic morphometry-II. Parameters of regenerated old liver

Two thirds hepatectomy was carried out on female Wistar rats of 24–25 months of age. The regenerated livers of 5 animals each were investigated 1, 5, 10, 20 days subsequent to hepatectomy using electron microscopic morphometry. The results show that liver cells of old animals display the same degree of aging even after regeneration, i.e. the surface area and surface density of the rough endoplasmic reticulum remain reduced to the same extent, as observed in the old control group. The regeneration caused a rejuvenation only of the mitochondria showing surface area and surface density of inner membrane + cristae corresponding to the values obtained in the young (1-month-old) animals. The results seem to support the idea that the accumulation of somatic mutations such as a strong reduction of the protein synthetic apparatus, may play an important role in the aging process.

The aging of rat liver as revealed by electron microscopic morphometry—I. Basic parameters

One-, 12- and 27-month-old female rats (5 in each group) were investigated by means of point-counting morphometry at light and electron microscopic sampling levels. Morphometric parameters are given for single hepatocytes, per unit volume of tissue and as specific values, i.e. per 100 g body weight. Many liver parameters showed age-dependent changes between the young and adult or between the adult and old ages. The most important stereologic manifestation of aging was the very strong reduction of surface area, surface density and specific surface density values of rough endoplasmic reticulum between the adult and old groups. This finding explains the decreased protein synthetic activity of old rat liver. Other parameters also changed, however, e.g. in case of mitochondria the interpretation is difficult because of a considerable swelling observed only in the old animals. Lysosomal elements showed no significant age-dependent changes. Some methodical problems of morphometry are also discussed.

Is Cytochrome Oxidase a Constituent of Nuclear Membranes?

Abstract Cytochrome oxidase activities have been determined and characterized in isolated nuclei, nuclear membranes, and chromatin subfractions from rat liver and rat thymus, and compared with mitochondrial fractions isolated in parallel from the same tissues. Spectral data on these fractions were obtained at both room and low temperatures. The cytochrome oxidase activities observed in the nuclear fractions and subfractions (up to about 10% per mg of protein of that of the corresponding mitochondrial fraction) correlate well with the amounts of cardiolipin and mitochondria-type cytochromes (a, a3, b, c1) present, as well as with the activities of the mitochondrial NADH dehydrogenase complex and the mitochondrial contaminations determined by electron microscopic morphometry. It does not correlate so well with the succinate dehydrogenase activities (5 to 7% per mg of protein of that of the heavy mitochondrial membranes prepared in parallel) but this appears to be due to the higher sensitivity of this enzyme to preparative stress. When nuclear membranes are separated from membrane contaminants of the nuclear fraction, cytochrome oxidase activities (per mg of protein or phospholipid) are significantly reduced concomitant with the decreases of other mitochondrial markers. When isolated mitochondria or cytochrome oxidase solubilized from mitochondria with Triton X-100 are mixed with isolated nuclei, the oxidase activity becomes tenaciously bound to nuclear material or is recovered in the same sucrose gradient band as the nuclear membranes, respectively. The data strongly suggest that the activities observed in nuclear fractions are introduced by mitochondrial contaminants.

Structure and Function of the Stimulated Adrenal Cortex: 2. Early Effects of ACTH on the Ultrastructure of the Zona Fasciculata in Dexamethasone — Treated Rats

The effect of ACTH on the ultrastructure of the zona fasciculata of the rat adrenal cortex has been examined by electron microscopic morphometry. A detailed description of fine structural changes is presented, with special attention to the „early” (i. e., 20 minutes after ACTH application) effects. Significant changes were noted in mitochondria as well in the relative area increase of the condensed chromatin. The possible significance of these ultrastructural changes is discussed against the background of the current concepts of ACTH action on corticoid hormone production and secretion. Der ACTH-Effekt auf die Ultrastruktur der Zona fasciculata der Rattennebennierenrinde wurde mit elektronenmikroskopischer Morphometrie untersucht. Beschrieben werden Änderungen der Feinstruktur unter besonderer Berücksichtigung der frühen (20 Minuten) ACTH-Effekte. Signifikante Änderungen wurden festgestellt bei den Mitochondrien und bei dem relativen Anstieg des Flächenanteils des kondensierten Chromatins. Ursachen dieser feinstrukturellen Veränderungen werden diskutiert gegen den Hintergrund der geläufigen Vorstellungen über die ACTH-Wirkung auf die Produktion und Sekretion der Kortikoidhormone.

Nuclear membranes from mammalian liver: VI. Glucose-6-phosphatase in rat liver, a cytochemical and biochemical study

Activities of glucose-6-phosphatase (G-6-Pase) and other phosphatases were determined in nuclei, nuclear membrane and microsomal fractions and subfractions, and condensed chromatin isolated from the liver of adult, newly born and prenatal rats. The purity of the fractions was controlled by electron microscopic morphometry and by measurement of various marker enzymes. The specific G-6-Pase activity of the nuclear membranes was found to be about 60% that of the microsomes. However, when calculated on the basis of the phospholipid content, all fractions had similar activities. Determinations of G-6-Pase enrichments and recoveries were also made. The correspondence of the hydrolysing activities of glucose-6-phosphate, mannose-6-phosphate, and inorganic pyrophosphate, together with various phosphotransferases, showed the same association of the G-6-Pase with these enzymes in the nuclear envelope as in the microsomal membranes. G-6-Pase was also demonstrated in the fractions by cytochemistry, and the activity was localized alongside the cisternal surfaces of both, inner and outer, nuclear membrane. ‘Free’ inner nuclear membrane fragments contained also G-6-Pase. No activity was observed at the nuclear pore complexes. Both, nuclear and microsomal membranes revealed a parallel rapid perinatal increase of G-6-Pase activity climaxing at 23 to 28 h after birth. Triton-X-100 treatment of isolated nuclei, which was found not to selectively release outer nuclear membranes, resulted in a great decrease of G-6-Pase activity as well as in losses of membrane phospholipids. The results clarify the divergence of earlier reports concerning the presence of G-6-Pase in the perinuclear cisterna and add biochemical evidence to the morphologically derived view of the nuclear envelope as being a special form of the ER system.

Ultrastructure of the nuclear envelope of amphibian oocytes. IV. Chemical nature of the nuclear pore complex material.

In order to investigate the chemical composition of the nuclear pore complexes isolated nuclei from matureXenopus laevis oocytes were manually fractioned into nucleoplasmic aggregates and the nuclear envelopes. The whole isolation procedure takes no more than 60–90 sec, and the pore complexes of the isolated envelopes are well preserved as demonstrated by electron microscopy. Minor nucleoplasmic and cytoplasmic contaminations associated with the isolated nuclear envelopes were determined with electron microscopic morphometry and were found to be quantitatively negligible as far as their mass and nucleic acid content is concerned. The RNA content of the fractions was determined by direct phosphorus analysis after differential alkaline hydrolysis. Approximately 9% of the total nuclear RNA of the matureXenopus egg was found to be attached to the nuclear envelope. The nonmembranous elements of one pore complex contain 0.41×10−16 g RNA. This value agrees well with the content estimated from morphometric data. The RNA package density in the pore complexes (270×10−15 g/μ3) is compared with the nucleolar, nucleoplasmic and cytoplasmic RNA concentration and is discussed in context with the importance of the pore complexes for the nucleo-cytoplasmic transport of RNA-containing macromolecules.

Cytomorphometry of skeletal muscle: the influence of age and testosterone on the rat m. levator ani.

The levator ani muscle of the rat was examined by correlated light and electron microscopic morphometry. Corrections were made for shrinkage, compression, and differences in stretching. Age, castration, and subsequent testosterone treatment do not affect the fiber number, the filament lattice, and the size of the filaments and myonuclei. The fibers in intact growing males increase in width and length. The number of myonuclei rises, although relatively slower than the amount of contractile material.